Journal of Medicinal Plants Research

Volume 6 Number 29 ISSN 1996-0875 1 August, 2012

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Editors
Prof. Akah Peter Achunike Editor-in-chief Department of Pharmacology & Toxicology University of Nigeria, Nsukka Nigeria Prof. Parveen Bansal Department of Biochemistry Postgraduate Institute of Medical Education and Research Chandigarh India. Dr. Ravichandran Veerasamy AIMST University Faculty of Pharmacy, AIMST University, Semeling 08100, Kedah, Malaysia. Dr. Sayeed Ahmad Herbal Medicine Laboratory, Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi, 110062, India. Dr. Cheng Tan Department of Dermatology, first Affiliated Hospital of Nanjing Univeristy of Traditional Chinese Medicine. 155 Hanzhong Road, Nanjing, Jiangsu Province, China. 210029 Dr. Naseem Ahmad Young Scientist (DST, FAST TRACK Scheme) Plant Biotechnology Laboratory Department of Botany Aligarh Muslim University Aligarh- 202 002,(UP) India. Dr. Isiaka A. Ogunwande Dept. Of Chemistry, Lagos State University, Ojo, Lagos, Nigeria.

Associate Editors Dr. Ugur Cakilcioglu Elazg Directorate of National Education Turkey. Dr. Jianxin Chen Information Center, Beijing University of Chinese Medicine, Beijing, China 100029, China. Dr. Hassan Sher Department of Botany and Microbiology, College of Science, King Saud University, Riyadh Kingdom of Saudi Arabia. Dr. Jin Tao Professor and Dong-Wu Scholar, Department of Neurobiology, Medical College of Soochow University, 199 Ren-Ai Road, Dushu Lake Campus, Suzhou Industrial Park, Suzhou 215123, P.R.China. Dr. Pongsak Rattanachaikunsopon Department of Biological Science, Faculty of Science, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand.

Editorial Board
Prof Hatil Hashim EL-Kamali Omdurman Islamic University, Botany Department, Sudan. Prof. Dr. Muradiye Nacak Department of Pharmacology, Faculty of Medicine, Gaziantep University, Turkey. Dr. Sadiq Azam Department of Biotechnology, Abdul Wali Khan University Mardan, Pakistan. Kongyun Wu Department of Biology and Environment Engineering, Guiyang College, China. Prof Swati Sen Mandi Division of plant Biology, Bose Institute India. Dr. Ujjwal Kumar De Indian Vetreinary Research Institute, Izatnagar, Bareilly, UP-243122 Veterinary Medicine, India. Dr. Arash Kheradmand Lorestan University, Iran. Prof Dr Cemit Karakurt Pediatrics and Pediatric Cardiology Inonu University Faculty of Medicine, Turkey. Samuel Adelani Babarinde Department of Crop and Environmental Protection, Ladoke Akintola University of Technology, Ogbomoso Nigeria. Dr.Wafaa Ibrahim Rasheed Professor of Medical Biochemistry National Research Center Cairo Egypt.

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Fees and Charges: Authors are required to pay a $600 handling fee. Publication of an article in the Journal of Medicinal Plant Research is not contingent upon the author's ability to pay the charges. Neither is acceptance to pay the handling fee a guarantee that the paper will be accepted for publication. Authors may still request (in advance) that the editorial office waive some of the handling fee under special circumstances. Copyright: 2012, Academic Journals. All rights Reserved. In accessing this journal, you agree that you will access the contents for your own personal use but not for any commercial use. Any use and or copies of this Journal in whole or in part must include the customary bibliographic citation, including author attribution, date and article title. Submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, or thesis) that it is not under consideration for publication elsewhere; that if and when the manuscript is accepted for publication, the authors agree to automatic transfer of the copyright to the publisher. Disclaimer of Warranties In no event shall Academic Journals be liable for any special, incidental, indirect, or consequential damages of any kind arising out of or in connection with the use of the articles or other material derived from the JMPR, whether or not advised of the possibility of damage, and on any theory of liability. This publication is provided "as is" without warranty of any kind, either expressed or implied, including, but not limited to, the implied warranties of merchantability, fitness for a particular purpose, or non-infringement. Descriptions of, or references to, products or publications does not imply endorsement of that product or publication. While every effort is made by Academic Journals to see that no inaccurate or misleading data, opinion or statements appear in this publication, they wish to make it clear that the data and opinions appearing in the articles and advertisements herein are the responsibility of the contributor or advertiser concerned. Academic Journals makes no warranty of any kind, either express or implied, regarding the quality, accuracy, availability, or validity of the data or information in this publication or of any other publication to which it may be linked.

International Journal of Medicine and Medical Sciences

Journal of Medicinal Plants Research
Volume 6 Number 29

Table of Contents:

1 August, 2012

ences
ARTICLES
Review Overview of commonly used Chinese herbs Pornanong Aramwit and Suntchai Wirotsaengthong 4501

Research Articles Essential oil content and composition of Mentha longifolia (L.) Hudson grown wild in Iran Zarir Saeidi, Hadi babaahmadi, Keramat Allah Saeidi, Amin Salehi, Ramin Saleh Jouneghani, Hosain Amirshekari and Abdallah Taghipour

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Quantification of -, - and -mangostin in Garcinia mangostana fruit rind extracts by a reverse phase high performance liquid chromatography Abdalrahim F. A. Aisha, Khalid M. Abu-Salah, Mohammad J. Siddiqui, Zhari Ismail and Amin Malik Shah Abdul Majid

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Genetic identification and relationship analysis of medicinal Limonium by rDNA ITS sequence, single nucleotide polymorphism (SNP) and amplification refractory mutation system (ARMS) Ge Ding, Daizhen Zhang, Yanqiu Yu, Beibei Zhang and Lingling Zhao

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Ethnomedicinal uses of plant resources in Gilgit-Baltistan of Pakistan Arshad Ali Shedayi and Bibi Gulshan

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Table of Contents:

Volume 6

Number 16

30 April, 2012

ences
ARTICLES
Short Communication Efficacy of chloroform, ethanol and water extracts of medicinal plants, Malva sylvestris and Malva neglecta on some bacterial and fungal contaminants of wound infections Payman Zare, Razzagh Mahmoudi, Sina Shadfar, Ali Ehsani, Yasaman Afrazeh, Anoosha Saeedan, Farzad Niyazpour, and Babak Seyed Pourmand

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Journal of Medicinal Plants Research Vol. 6(29), pp. 4505-4521, 1 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.481 ISSN 1996-0875 2012 Academic Journals

Review

Overview of commonly used Chinese herbs

Pornanong Aramwit1* and Suntchai Wirotsaengthong2
1

Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand. 2 Department of Medicine, Taksin Hospital, Khlong San, Bangkok, Thailand.
Accepted 9 May, 2011

There are several Chinese herbs used as food supplement or as an alternative medicine. Fructus lycii, Panax ginseng, Cervi cornu pantotrichum, Herba epimedii and Angelica sinensis are among the most commonly prescribed. All of them have been claimed for various indications which are mainly due to their variety of active ingredients or chemical compositions. Even though Chinese herbs have been used and claimed for their efficacy for decades, some toxicity has been found, thus the need to be cautious. In this review, we describe the most common indications of F. lycii, P. ginseng, C. cornu pantotrichum, H. epimedii and A. sinensis and also report their toxicity, if any have been found. Key word: Chinese herbs, Fructus lycii, Panax ginseng, Cervi cornu pantotrichum, Herba epimedii, Angelica sinensis

INTRODUCTION Traditional medicine involving Chinese herbs is currently gaining a lot of attention due to its quite promising effect, as well as its better clearance (Schulz, 2006), resulting in less accumulation and lower toxicity. Since several active compounds have been found in some herbs, certain herbal medicine products can possess many biological activities. The use of Chinese herbs not only depends on its active ingredients and clinical results, but also on peoples belief. Many Chinese herbs have become very popular and have been used as alternative medicine for certain diseases, such as cancer, because of their effectiveness and lower adverse reactions compared to modern medicine (Schulz, 2006; Yi et al., 2010). Even though many herbs have been recognized for their efficacy, some toxicity can be found, especially with their impurities. Quality control of active ingredients and impurities are necessary for herbal medicine in order to obtain the optimum action. Some of the commonly used Chinese herbs will now be described in detail. FRUCTUS LYCII Fructus lycii, also called gouqizi or Chinese wolfberry in Chinese pharmacopeia, has been used for thousands of years as traditional Chinese herbal medicine. The standard species of this fruit are Lycium barbarum L. and L. chinense mill. belonging to the Solanaceae family (Chen and Chen, 2004). F. lycii is an oblong, orange to dark red berry and possesses a bitter to sweet taste (Wu, 2005). Traditional and current uses In Chinese pharmacopeia, F. lycii is one of the ingredients in Chinese medicine formula. It is used for nourishing the blood in the liver and kidney, and helping to re-balance Yin and Yan in the body. The ancient Chinese subscribe to a concept called Yin Yang which is a belief that there exist two complementary forces in the universe. One is Yang which represents everything

*Corresponding author. Email: aramwit@gmail.com. Tel: + (66)089-921-7255. Fax: + (66)02-218-8403.

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positive or masculine and the other is Yin which is charac-terized as negative or feminine. One is not better than the other. Instead they are both necessary and a balance of both is highly desirable (Fong, 2010). F. lycii has been used in Chinese medicine for treating dizziness, diminished visual acuity, dryness or tearing from exposure to wind, soreness and weakness of the lower back and knees, nocturnal emission, night sweating, blurry vision, tinnitus and impotence (Chen and Chen, 2004; Wu, 2005). It also possesses antioxidant, anti-aging, anti-tumor, immune-stimulatory, hypoglycemic, hypolipidemic, hepatoprotective and cytoprotective activity (Potterat, 2009).

Sources and chemical composition There are various components found in F. lycii such as polysaccharides, which are the major constituents in this fruit, carotenoids and avonoids. L. barbarum polysaccharides (LBP) consist of a complex mixture of highly branched and only partly characterized polysaccharides and proteoglycans. Luo et al. (2001) tested the anti-fatigue dose of LBP in mice and found that 10 mg/kg/day was the best amount for remarkable adaptability to exercise load, enhanced resistance and accelerated elimination of fatigue. A second major substance is carotenoid, whose expression increases during the ripening process. Zeaxanthin dipalmitate is the predominant constituent from this compound group. In addition, -cryptoxanthin palmitate, zeaxanthin monopalmitate, and small amounts of free zeaxanthin and -carotene are also present. This fruit contains some vitamins such as riboflavin, thiamin and ascorbic acid. Flavonoids, such as aglycones myricetin, quercetin and kaempferol, are other important classes of compounds in this fruit. Essential oil and fatty acids are also found in this fruit. Hexadecanoic acid, linoleic acid, -elemene, myristic acid and ethyl hexadecanoate have been identified as the main constituents. F. lycii also contains free amino acids with proline as the major constituent, taurine, -aminobutyric acid and betaine (trimethylglycine). Some miscellaneous compounds such as -sitosterol and its glucoside daucosterol, scopoletin, p-coumaric acid, the dopamine derivative lyciumide A and L-monomethyl succinate are also found in this fruit (Bensky et al., 2004; Chen and Chen, 2004; Potterat, 2009).

flavonoids. Both compounds show three types of antioxidant activities: Radical-scavenging activity, reducing capacity and metal chelating activity (Le et al., 2007; Li and Zhou, 2007a, b). Qian et al. (2004) studied the effect of flavonoids in the polar extract of L. chinense where the extract exhibited free radical scavenging activity (Qian, 2004). LBP has been observed to decrease DNA damage in peripheral lymphocytes in noninsulin dependent diabetes mellitus rats via a decrease in oxidative stress. Therefore, LBP can control blood glucose and modulate glucose metabolism (Wu et al., 2006). In addition, LBP has been shown to increase glycogen level and antioxidant enzyme activities, and decrease malondialdehyde (MDA) level and creatine kinase activities in rats undergoing an exhaustive exercise program. This demonstrates that LBP administration can signicantly decrease the oxidative stress induced by exhaustive exercise (Niu et al., 2008). In aged mice, oral administration of LBP over a period of 30 days significantly restored marker enzyme (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px)) activity in aged mice compared to control (Li et al., 2007a). The extract of L. barbarum has been demonstrated to significantly reduce blood glucose levels and serum total cholesterol (TC) and triglyceride (TG) concentrations. In addition, the extract could increase high density lipoprotein cholesterol (HDLc) levels. The results suggested that LPB containing several monosaccharides and 17 amino acids were major bioactive constituents of hypoglycemic effect. Polysaccharides and vitamin antioxidants could be the possible active inducers of the hypolipidemic effect (Luo et al., 2004). Amagase et al. (2009) investigated the effects of L. barbarum in a 30-day randomized, doubleblind, placebo-controlled clinical study. L. barbarum increased antioxidant efficacies in humans by stimulating endogenous factors. They suggested that long-term use of L. barbarum might help prevent or reduce free radicalrelated conditions (Amagase et al., 2009).

Hepatoprotective effects The study of Kim et al. (1997) found that a chloroform/methanol extract of the fruit contained two cerebrosides showing significant protection against carbon tetrachloride (CCl4)-induced injury in primary cultured rat hepatocytes. However, the mechanism of action was not determined (Kim et al., 1997a). Ha et al. (2005) studied the protective effects of L. chinense fruit on carbon tetrachloride-induced hepatotoxicity in rats. Pretreatment with the fruit before the injection of carbon tetrachloride significantly reduced the elevation of serum liver enzyme levels [alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP)]. The mechanism of action was free radical scavenging, which could act against pathological alterations

Pharmacological and toxicological effects Antioxidation The antioxidant activity of F. lycii has been investigated in various in vitro and in vivo studies. The components that show antioxidant activity are the polysaccharides and

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caused by the administration of carbon tetrachloride (Ha et al., 2005). In 2010, the mechanism of the hepatoprotective effect of L. chinense Miller fruits was investigated in primary human liver cell culture. The results showed that Lycium alcoholic extract protected liver cells against hydrogen peroxide-induced liver cell damage. The underlying mechanism appeared to involve antioxidative properties and a decrease in the expression of CYP2E1 (Pan et al., 2010). Zeaxanthin and zeaxanthin dipalmitate have been demonstrated to display anti-hepatotoxic activity by reducing hepatic fibrosis induced by bile duct ligation/scission in rats at a dose of 25 mg/kg through antioxidative activity (Kim et al., 1997b). Furthermore, the three pyrrole derivatives of the ethyl acetate fraction of the fruit have also shown hepatoprotective effects comparable to silybin during carbon tetrachloride-induced toxicity in rat hepatocytes (Chin et al., 2003).

QGY7703 cell growth with cycle arrest in the S phase and apoptosis induction (Zhang et al., 2005b). In addition, LBP inhibited proliferation and stimulated p53-mediated apoptosis in rat and human hepatocellular carcinoma cells (Chao et al., 2006). In 2009, Li et al. studied the effect of L. barbarum on the human breast cancer cell line MCF-7. The results showed that an aqueous extract was shown to inhibit the growth of the estrogen receptor-positive human breast cancer cell line MCF-7 by altering estradiol cellular metabolism (Li et al., 2009).

Neuroprotective effects There are few studies on this field and the data are mainly in vitro studies by the same authors. Pretreatment with an aqueous extract of L. barbarum has been demonstrated to significantly reduce the release of lactate dehydrogenase (LDH), an enzyme that catalyzes the conversion of lactate to pyruvate which is an important step in energy production in cells, during Abeta peptide-induced toxicity in a primary neuron cell line. Moreover, it has been shown to attenuate Abeta peptideactivated caspase 3-like activity and elicit a typical dosedependent neuroprotective effect. The underlying mechanism appears to involve inhibition of the A-triggered cJun N-terminal kinase (JNK) signaling pathway (Yu et al., 2005) which is a member of mitogen-activated protein kinase family participating in many of the signaling pathways associated with stress (Kwon et al., 2011). There are many studies indicating that oxidative stress can induce cell damage and has been implicated in a variety of neurodegenerative disorders (Behl, 1999; Olanow, 1993). The extract from L. barbarum protected against DTT-induced toxicity in neurons via attenuated DTT-induced PERK phosphorylation that increased with age (Yu et al., 2007). The mechanisms of the cytoprotective effects were independent of the antioxidative effects (Yu et al., 2006). Furthermore, pretreatment of LBP protected neurons effectively against A-induced apoptosis by reducing the activity of both caspase-2 and -3. LBP can also inhibit phosphorylation of double-stranded RNA-dependent protein kinase (PKR). Oral administration of L. barbarum in Sprague-Dawley rats significantly reduced the loss of retinal ganglion cells in the retina, although elevated intraocular pressure was not significantly altered. This was the first in vivo report showing the therapeutic function of L. barbarum against neurodegeneration in the retina of a rat ocular hypertension model (Chan et al., 2007). Treating male infertility L. barbarum fruits have been used as a traditional remedy for male infertility in China. There are currently few studies on the fertility-facilitating effects of F. lycii.

Immune modulation The polysaccharideprotein complex from LBP are the bioactive components in immune modulation; whereas, carotenoids and avonoids have not shown any involvement in this process (Chang and So, 2007). Previous studies have demonstrated that LBP can enhance immune function and induce lymphocyte proliferation and cytokine production (Gan, 2003; Gan, 2004). Gan et al. (2003) studied the effects of LBP on the expression of interleukin-2 (IL-2) and tumor necrosis factor- in human peripheral blood mononuclear cells. After LBP addition to the primary cell culture, LBP increased the expression of IL-2 and tumor necrosis factor- at both mRNA and protein levels in a dosedependent manner (Gan, 2003). Gan et al. (2004) investigated the effect of LBP on the immune system of S180-bearing mice and observed that LBP could significantly inhibit the growth of transplantable sarcoma S180 and increase macrophage phagocytosis, spleen lymphocyte proliferation, cytotoxic T-lymphocyte (CTL) activity and IL-2 mRNA expression, but these effects were not dose-dependent (Gan, 2004).

Antitumor activity If LBP can increase IL-2 and tumor necrosis factor- level, two cytokines that can inhibit tumor growth, LBP might also contribute to antitumor activity. As the study of Gan et al. (2004) showed, LBP could significantly inhibit the growth of transplantable sarcoma S180 in S180bearing mice (Gan, 2004). Zhang et al. (2005) studied the effect of LBP on the proliferation rate, cell cycle distribution and apoptosis in the human hepatoma QGY7703 cell line. LBP displayed an inhibitory effect on

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Polysaccharides are the most important functional constituent of F. lycii. There was a study that showed Fructus lycii polysaccharides inhibiting time-and hyperthermia-induced structural damage in murine seminiferous epithelium and delaying apoptosis in this system (Wang et al., 2002). The major cause of structural degradation and apoptosis in hyperthermic testes is oxidative stress. F. lycii polysaccharides were shown to protect against this testicular degeneration via an antioxidant effect (Wang et al., 2002). LBP provided a protective effect against the testicular tissue damage induced by heat-exposure and chemical (hydrogen peroxide). LBP has also been demonstrated to increase testis and epididymis weights, improve SOD activity, and raise sexual hormone levels in damaged rat testes. In addition, LBP has been shown to improve reproductive function in hemicastrated male rats, increase sexual hormone level, raise accessory sexual organ weights, and improve sperm quantity and quality (Luo et al., 2006). Product availability and quality control There are two related species of this fruit, L. barbarum L. and L. chinense Mill. L. barbarum L. is a deciduous shrub one to three meters high and widely distributed in warm regions of the world, particularly in the Mediterranean area, southwest and central Asia. It is also cultivated in North America and Australia. L. chinense is smaller than L. barbarum L. and mainly distributed in East Asia, and grown particularly in South China, Korea and Japan. However, the fruits of the Lycium species possess a highly similar anatomy and tissue structure. The chemical components of fruits from both species appear similar (Potterat, 2009). The differences in composition and properties among different F. lycii from different geographic region, due to soil and climate, are observed historically, which lead to the differences in their functions (Lu et al., 2008). In order to discriminate F. lycii from different geographic regions, several analytical methods have been applied to detect a few active components such as high performance liquid chromatography (HPLC) fingerprint, thin layer chromatography (TLC) and colorimeter. However, since there are many active components and being that it is inappropriate to select only several specific components as essential criteria (Lu et al., 2008), two-dimensional near-infrared correlation spectroscopy has been used to successfully control the quality of F. lycii without losing the original natural instinct and compati-bility of traditional Chinese herbal medicine (Hua et al., 2003; Lu et al., 2008). Moreover, the polysaccharides of L. barbarum, the major component in this fruit, were also analyzed by preparative high performance size exclusion chromatography (HPSEC). After protein hydrolysis, two major fractions were obtained with a molecular weight of 79250

and 24470 amu, respectively (Wang et al., 2009a).

Reported toxicity The median lethal dose (LD50) of a water extract of F. lycii has been reported as 8.32 g/kg by subcutaneous application in mice (Chang et al., 2001). Acute overdose of F. lycii is characterized by tremor, dyspnea, tearing, gastrointestinal disturbances, and dermal redness and itching (Chen and Chen, 2004).

PANAX GINSENG P. ginseng L. is a perennial herb that grows in the mountainous forests of eastern Asia, the United States and Canada. The use of this herb in Chinese traditional medicine stems from the doctrine of signature concept, because the root of the ginseng looks like human and is therefore used to treat all male diseases. Traditional and current uses Ginseng is used as a tonic to invigorate a humans physical, mental and sexual capability. According to traditional Chinese medicine philosophy, predomination of yin generates cold and obstructs yang-qi, clinically leading to cold symptoms such as chills, dispiritedness, pale complexion, cold limbs, loose stool, clear urine, whitish tongue coating, deep and slow pulse; while predomination of yang leads to heat, so usual clinical manifestation is fever, accompanied by profuse sweating, thirst, reddish complexion, reddish tongue with yellowish coating, full and large or rapid pulse; or accompanied by pathogenic heat disturbing the interior with the symptoms of dysphoria, insomnia, mania, dry feces and scanty brownish urine (Guilin Sino-Western, 2003). Because of that concept, two types of ginsengs have been used in opposite effect. P. ginseng (Korean ginseng) is a hot or yang tonic used to treat cold diseases, whereas P. quinquefolium (American ginseng) is cool or yin and is used to treat hot symptoms, such as stress, insomnia, palpitations and headache. It is also said to possess antistress activity (also means an adaptogen) (Awang, 1998), improve glycemic control, stimulate immune functions, treat toxic hepatitis, improve athletic performance, enhance longevity, and relieve symptoms associated with cancer, aging and senility such as asthenia, atherosclerosis, blood and bleeding disorders. Ginseng is also widely used as an aphrodisiac. Sources and chemical composition The term ginseng can refer to the species of the genus

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Panax (Korean, Chinese, Japanese or American ginseng), as well as to Eleutherococcus senticosus (Siberian or Russian ginseng). The distinction between the species is important because there are many differences in habitual and harvest characteristics in addition to chemical compositions that can cause different therapeutic and toxic effects. Ginseng and ginsenoside (termed saponin) have been identified as active ingredients of ginseng (Hu et al., 2008; Rhim et al., 2002). In this review, the term ginseng will be used to refer to P. ginseng (also called Korean ginseng or Chinese ginseng), while Japanese ginseng and American ginseng will be used to refer to P. pseudoginseng and P. quinquefolius, respectively. Others are less important than these three species and will not be included in the scope of this review. Pharmacological and toxicological effects Endocrine effects Ginseng has been used in various types of endocrine disorders, especially in diabetes mellitus, because of its hypoglycemic effects by possibly accelerating hepatic lipogenesis and increasing glycogen synthesis and storage (Oshima et al., 1985; Yokozawa et al., 1975). In a small human study of 36 newly diagnosed type II diabetes patients, ginseng at a dose of 200 mg daily was shown to have a statistically significant benefit on glycosylated hemoglobin compared to 100 mg of ginseng or placebo daily after 8 weeks of therapy (6 versus 6.5% for the 200 mg ginseng extract powder and 100 mg ginseng extract powder or placebo group, respectively). Patients who received 100 mg of ginseng had a smaller mean fasting blood glucose level than those who took 200 mg of ginseng or placebo; the mean fasting blood glucose was 7.7 mmol/L for the 100 mg ginseng extract powder group, 7.4 mmol/L for the 200 mg ginseng extract powder group, and 8.3 mmol/L for the placebo group at the end of the study (Sotaniemi et al., 1995).

small but statistically significant improvement in the index of memory quality as compared to placebo (Wesnes et al., 2000). Attention-deficit hyperactivity disorder (ADHD) patients may partly benefit from ginseng products due to data from the study of Lyon and colleagues. This was a pilot study to evaluate the effects of a combination product containing both American ginseng and ginkgo for the treatment of ADHD. The investigators reported improvement in 31 to 67% of the subjects depending on the type of outcome measured. However, no placebo group was included so it is difficult to ascertain if the effect was caused by the herbal treatment or not (Lyon et al., 2001).

Cardiovascular effects In animal models, ginsenoside Rb1 decreases blood pressure by relaxing vascular smooth muscle (Kaku et al., 1975). Administration of ginseng at a dose of 4.5 g/day was studied in a small number of human subjects, which demonstrated its hypotensive effect in two patients from a total of 26 subjects (7.7%) (Han et al., 1998). An in vitro study using a crude extract of Ginseng saponins showed the relaxation effect on rabbit corpus carvernosum smooth muscle, suggesting that some components of ginseng may be a nitric oxide donor (Kim et al., 1998). This finding might provide scientific evidence for traditional claims that ginseng enhances sexual potency, and supports results of another study that showed increased penile rigidity and girth compared to placebo or trazodone in patients with erectile dysfunction (Choi et al., 1995). Yamamoto and colleagues used the ginseng powder in their pilot clinical study to demonstrate the effects of ginseng on human lipid profiles. Red ginseng powder was shown to decrease triglyceride concentration as well as increase high-density lipoprotein (HDL)-cholesterol concentration (p < 0.05 compared with baseline for both triglyceride and HDL levels) (Yamamoto et al., 1983). In another study, ginseng also showed antiplatelet activity by regulating the levels of cyclic guanosine monophosphate (cGMP) and thromboxane A2 (Park et al., 1995).

Neurological effects Ginseng products that are available on the market have been reported to have stimulatory effects on the central nervous system (CNS) in both animal models and humans (Siegel, 1979; Takagi et al., 1972). Ginsenoside Rg1 inhibits neuronal apoptosis in vitro while ginsenoside Rb1 reverses short-term memory loss in rats (Awang, 1998; Li et al., 1997; Takagi et al., 1972). It is believed that ginseng may play an important role in the treatment modality of human dementia. Wesnes and colleagues studied the memory-enhancing effect of either ginseng or Ginkgo biloba in healthy middle-aged volunteers and found that administration of either agent resulted in a

Product availability and quality control Two commercial forms of ginseng are available. White ginseng consists of dried root and red ginseng is prepared by fresh, unpeeled root steaming before drying. White and red ginseng contain different amount of active ingredient. Red ginseng contains 2.5 times higher concentration of protopanaxadiol class ginsenosides, the more cytotoxic and efficient cellular uptake on breast cancer cell line, than white ginseng while protopanaxatriol class ginsenosides, the less active compound, were only

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present in white ginseng (Lee et al., 2011). The greater cellular uptake of protopanaxadiol class ginsenosides resulted in more substantial antiproliferative activity in human breast cancer cells. Many formulations of ginseng are available including tablets, capsules, soft capsules, powders, tinctures, teas, candy, fresh or dry slices, and whole fresh or dry root. There are also a variety of products that claim to contain ginseng such as cigarettes, toothpastes, cosmetics, soaps, beverages, gum and coffee. These products have varying amounts of ginseng, quality of formulation, price and shelf-life. In general, tinctures are more expensive but last for years while powder capsules are cheaper but have a short shelf-life of only one year. The most important aspect of the quality control process of these ginseng products is based on standardization of the ginsenosides, the purported active ingredients. The amount of ginsenosides in ginseng products, available on the market worldwide, varies widely among brands and often differs from the content stated on the label or lacks consistency between batches. Ginseng commonly can be taken as pure compound (P. ginseng from Now Foods (Bloomingdale, IL, USA), contained 1.04 g of P. ginseng/capsule) or combination with other components such as Ginseng Power Max from Action Labs (Anaheim, CA, USA) contained red Chinese ginseng 350 mg, Korean ginseng 350 mg, Peruvian Maca (Lepidium spp.) 175 mg, Eleuthero (E. senticosus) 150 mg, American ginseng 150 mg and Rhodiola (Rhodiola rosea) 50 mg. There are other types of ginseng on the market including Siberian, Brazilian and Indian ginseng as intentional or unintentional adulterant to ginseng herbal products. These are not of the genus Panax and do not contain ginsenosides. For quality assessment, ginsenosides need to be determined. Ginsenosides can be simply classified as neutral and acidic saponins. Major neutral saponins are ginsenosides Rg1, Re, Rf, Rb1, Rb2, Rc, and Rd; the acidic ginsenosides include four malonyl derivatives of the ginsenosides Rb1, Rb2, Rc, and Rd and ginsenoside R0 (Yamaguchi et al., 1988). Numerous analytical techniques, including TLC, gas chromatography (GC), HPLC coupled with ultraviolet detection (HPLC-UV), evaporative light scattering detection (HPLC-ELSD), or MS (LC-MS), capillary electrophoresis (CE), near infrared spectroscopy (NIR), and enzyme immunoassay (EIA) have been used to quantitate ginsenosides (Fuzzati, 2004). Of these techniques, HPLC-UV is the most frequently used and considered a valuable tool for the quality assessment of ginseng products (Kim et al., 2007).

persons who had been using ginseng products for at least a month. Most subjects experienced CNS arousal and excitation at an average dose of 3 g of ginseng root. Fourteen people experienced GAS, which was composed of hypertension, nervousness, sleeplessness, morning diarrhea, and skin eruptions. Five of the 14 subjects also had edema while 4 reported depersonalization and confusion at a dose of 15 g, and depression at a dose greater than 15 g. The CNS effects appeared to be dosedependent (Siegel, 1979) and most users reported that dosing titration was required to minimize nervousness and tremor. There are several case reports that show some adverse reactions from ginseng products on other organ systems such as hypertension, Steven-Johnson syndrome, mastalgia, abnormal vaginal bleeding, and masculinization of the fetus in mothers using ginseng products while pregnant.

CERVI CORNU PANTOTRICHUM C. cornu pantotrichum or Lurong is pilose deer antler, which is the one of most popular components of traditional Chinese herbal medicine. Young pilose antlers of the male C. nippon Temminck or C. elaphus Linnaeus (Family Cervidae), which are the standard species, are used. The horn is cut into slices or ground into powder for use. There are three other products derived from deer velvet and antlers: deer antler (C. cornu), deer antler glue (C. cornu colla) and degelatinated deer antler powder (C. cornu degelatinatum). Even though the four products have the same source, their therapeutic functions are different (Bensky et al., 2004; Liu et al., 2010; Wu, 2005). It has been used for thousands of years in treating neurosis, enriching vital energy, nursing the blood, strengthening the kidney and prolonging life (Won, 1994). Traditional and current uses In Chinese medicine formula, Cervi cornu pantotrichum is used to treat kidney yang deficiency with impotence, infertility, anemia due to blood and kidney yin deficiency, strengthen the tendon and bones, and heal chronic Yin sore and boils (Bensky et al., 2004; Chen and Chen, 2004). It is currently used in the effective remedy for erectile dysfunction, acute and chronic arthritis, osteoporosis, and fracture in animal model or human clinical trials (Chen and Lin, 2008; Ghosh et al., 2001; Kim et al., 2008; Low and Tan, 2007; Wang et al., 2007d). Sources and chemical composition There are two different species of deer used for their velvet in China: the sika deer (C. nippon) and red deer

Reported toxicity The term ginseng abuse syndrome (GAS) was coined from the result of Siegels study that included 133

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(C. elaphus). Velvet from the sika deer is known in Chinese as either plum blossom deer velvet or simply blossom deer velvet, which is a yellow-haired velvet covering the young antlers. Velvet from red deer is known as horse deer velvet, which is a greenish blue color. The therapeutic action of the two species is similar. However, deer velvet is collected from deers all over the world at present, including North America, Europe, Australia and New Zealand (Bensky et al., 2004). C. cornu pantotrichum is the deer antler velvet that is collected from the early stage of antler growth and is formed from cartilage that later rapidly converts to bone, after which all blood supply is lost. Antlers are deciduous every spring in male deer while new antlers grow on the base of the previous years antlers. The antlers are removed safely by surgery after the new antlers have grown for 55 to 60 days (Bensky et al., 2004). Of all the products derived from deer velvet and antlers, C. cornu pantotrichum has the strongest therapeutic effect (Bensky et al., 2004). All the products have similar components but their therapeutic functions are different. In C. cornu pantotrichum, the following can be found: proteins, amino acids, nine kinds of fatty acids including oleic acid, linoleic acid, linolenic acid, ten kinds of phospholipids, endocrine hormones and polysaccharides. Furthermore, inorganic elements such as Ca, Na, Zn, Fe, Mg and Si have also been found (Liu et al., 2010). There are additional chemical compounds found in C. cornu pantotrichum such as pantocrine, lysophosphatidylcholine, ganglioside, putrescine, spermidine, spermine, growth factors, condrotin sulfate, androgen, estradiol, estrone, ceramide, lecithin, cephalin and sphingomyelin (Chen and Chen, 2004; Lee et al., 2004). In addition, the velvet and antlers have a high content of keratin. It has been demonstrated from the protein analysis of velvet antlers that keratin may be used as an index of standardization of velvet antler components because keratin is common to each specic species (Won, 1994).

administration of the extract does not inhibit morphineinduced analgesia and morphine-induced hyperactivity (Kim et al., 1999). Bone proliferation and growth A number of growth factors has been identified in deer antler extract, including bone morphogenetic proteins that induce the formation of bone and cartilage (Feng et al., 1995; Feng et al., 1997) and broblast growth factors that affect the multiple-step progress of osteoblast differentiation and mineralization (Sim et al., 2001). In 2005, there was a study that found that deer antler aquaacupuncture (DAA) possessed anti-bone resorption activity in adjuvant-induced arthritic rats (Kim et al., 2005). When cervus and cucumis polypeptides were injected into the radius fracture of rabbits at different concentrations, the expression level of vascular endothelial growth factor in the high dose group was higher than that in the low dose group. In addition, fractures in the high dose group had all completely healed and the bone was of higher strength compared to the low dose and control groups (Wang et al., 2007d). In addition, chloroform extract of deer antler also inhibited osteoclast differentiation in mouse bone marrow cultures stimulated by receptor activator of NF-B ligand and macrophage-colony stimulating factor. The extract suppressed the activation of extracellular signal-regulated kinase, protein kinase B and inhibitor of kappa B in osteoclast precursor cells (Li et al., 2007b). These results were consistent with the study of Kim et al. (2005). Chen et al. (2008) reported that the pilose antler polypeptides also improved proliferation of rat chondrocytes in vitro (Chen and Lin, 2008). After induction of avascular necrosis of the femoral head by corticosteroids in rats, oral administration of water extract of deer antler reduced the degree of necrosis. The extract also promoted osteoblastic proliferation through regulating cell cycle progression (Shi et al., 2010).

Pharmacological and toxicological effects Anti-narcotic Oral administration of water extract of velvet antler from C. elaphus prior to morphine treatment can develop morphine-induced conditioned place preference and postsynaptic dopamine (DA) receptor super-sensitivity in mice. These results suggested that velvet antler recovered the dysfunction in the dopaminergic system produced by morphine (Kim and Lim, 1999). The repeated administration of water extract of velvet antler can also inhibit analgesic tolerance, physical dependence, and reverse tolerance caused by the repeated administration of morphine. However, a single

Anti-arthritis C. cornu pantotrichum has been shown to exert an inhibitory effect on the production of IL-1 and tumor necrosis factor- (TNF-) from macrophages in mice that have been stimulated with bacterial lipopolysaccharides. In the in vitro study, it also strongly inhibited T-cell activation, including blastogenesis and cytokine production, stimulated by antigens. The function of Bcells is important in the severity and length of arthritis and therefore, the effect of the extract on B-cells function was examined. The results showed that intraperitoneal injection of the extract inhibited antibody production in rats (Kim et al., 2003). In 2004, Kim et al. reported that the C. cornu pantotrichum from C. korean TEMMINCK

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var. mantchuricus Swinhoe (Nokyong in Korean) suppressed the development of arthritis, consistent with the results from the previous study (Kim et al., 2004). Moreover, when the extract treatment and the initial or booster immunization started, the progression of arthritis and the immune response to collagen were inhibited (Kang et al., 2006). The inhibitory effect of the extract on arthritis correlated with the effects of transforming growth factor- (TGF-), which has potent immunosuppressive effects of cytokine. The results suggested that the extract and TGF-3 exerted inhibitory effects on the development of an induced arthritic condition in a similar pattern in rats. The extract has also been shown to control inammatory proteins and protect cartilage (Kim et al., 2008). Product availability and quality control In Chinese pharmacopeia, the quality criteria for C. cornu pantotrichum from sika deer include a thick, round appearance with a full end part as well as a reddish brown surface with soft, yellowish red hairs. Good quality C. cornu pantotrichum from red deer consists of full and lightweight antler, with grayish black or grayish yellow hairs. Beside C. cornu pantotrichum from sika and red deer, deer velvet is collected from deers in many countries as afore-described. Because of the high price of this medicine, many counterfeit versions are found on the market. The adulterants include slices of old deer antlers that have been artificially coated with fine hairs. These fakes are usually easy to recognize (Bensky et al., 2004). For chemical composition, C. cornu pantotrichum contains proteins, amino acids, 9 kinds of fatty acids include oleic acid, linoleic acid, linolenic acid, 10 kinds of phospholipids, endocrine, and polysaccharide which are considered as active ingredients (Wang et al., 2007b; Yan et al., 2004). Reported toxicity The median lethal dose (LD50) of pantocrine, a liquid alcoholic extract from antlers in the velvet of the deer cervus, in mice has been reported as 34 ml/kg via intravenous injection, 104 ml/kg via intraperitoneal injection, 114 ml/kg via subcutaneous injection, 97.8 ml/kg via intramuscular injection and 117 ml/kg via oral ingestion. Overdose symptoms of C. cornu pantotrichum are characterized by tremor, dyspnea, tearing, gastrointestinal disturbances, dermal redness and itching (Chen and Chen, 2004). HERBA EPIMEDII Herba epimedii or Yin yang huo in Chinese pharmacopeia

is one of the most popular components of traditional Chinese herbal medicine. It is made from the aerial parts of Epimedium L. (Berberidaceae) including the herbs multiple species: Epimedium brevicornum Maxim., E. sagittatum Maxim., E. pubescens Maxim., E. wushanense T. S. Ying and E. koreanum Nakai (Bensky et al., 2004; Wu, 2005). They have been commonly used in the treatment of cardiovascular diseases and other chronic illnesses (infertility, amnesia and asthenia, impotence and senile functional diseases) in China for over 2,000 years (Meng, 2005). Traditional and current uses In Chinese medicine formula, H. epimedii is the herb used to reinforce the kidney yang, strengthen the tendon and bones and expel wind-dampness (Bensky et al., 2004; Chen and Chen, 2004). It is widely used in the effective remedy for cardiovascular diseases, osteoporosis and for improving sexual and neurological functions (Sze et al., 2010). Sources and chemical composition H. epimedii is also grown as a herb for various medicinal purposes in Japan, Korea and the Mediterranean region (Zhang et al., 2008c). In China, it is mainly produced in the provinces of Shananxi, Sichuan, Hubei, Shanxi and Guangxi. This herb is collected in the spring and autumn, then dried in sunlight and sliced and used unprepared or stir-baked with sheep fat (Wu, 2005). There are several compounds in this plant including lignans, avonoids, avonol glycosides, terpene glycosides and phenolic carboxylic acids (Guo et al., Ito et al., 1988; Li et al., 1995a, 1995b, 1996; Matsushita et al., 1990; Mizuno et al., 1987; Wang et al., 2007a). The major effective medicinal compounds are the flavonoids and more than 60 kinds have been identified. The major constituents of flavonoids are epimedin A, B, C and icariin, which possess important pharmacological activities (Zhang et al., 2008c). Epimedin A, B, C and icariin are usually used as the marker compounds for evaluating H. epimedii by HPLC and capillary zone electrophoresis (Zhang et al., 2008a). In addition, icariin has been used to monitor the quality of this herb as defined by the Committee of China Pharmacopoeia (Committee of China Pharmacopoeia, 2005). Pharmacological and toxicological effects Antioxidation H. epimedii contains antioxidant constituents such as total flavonoids, icariin, polysaccharides and vitamin C.

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These compounds exert antioxidant activity in different organs. The total flavonoids of this herb contribute to antiinflammatory effects, which are believed to be related to their antioxidant activities. They also inhibit prostaglandin E and malondialdehyde, the metabolic product of lipid peroxidation, and enhance the activity of free-radical scavenging enzyme (Sze et al., 2010). Icariin has been reported to protect against oxidative-induced hemolysis (Liu et al., 2004). Icariin protected against oxidative damage of DNA in a concentration-dependent manner, as demonstrated by measuring the formation of carbonyl compounds that can react with thiobarbituric acid (TBA) to form thiobarbituric acid reactive substances (TBARS) (Zhao et al., 2007). The possible mechanism of antioxidative activity of icariin might be the stabilization function derived from intramolecular hydrogen bonds when the hydrogen atom in the OH group of icariin is subtracted by a radical (Zai-Qun, 2006). Meanwhile, the antioxidative activity of polysaccharides has been shown to be due to an increase in the activities of key intracellular antioxidant enzymes, such as superoxide dismutase and glutathione peroxidase. In addition, vitamin C found in H. epimedii can contribute to the antioxidant properties of H. epimedii (Sze et al., 2010).

another in vivo study, the ethanolic E. brevicornum extracts increased estrogen receptor activity in rat after oral administration (Yap et al., 2007). Among the major Epimedium species, specimens of E. koreanum, E. pubescens and E. brevicornum have exhibited high estrogen receptor and activities (Shen et al., 2007).

Osteoprotective effect H. epimedii has been one of the most frequently used herbs in formulas to be prescribed for the treatment of osteoporosis for a long time in China. There are various studies about the effect of this herb on the process of bone metabolism and bone formation in Chinese language journals. Results show that H. epimedii stimulates osteoblastic proliferation, inhibits osteoclastic resorption and increases mineral content (Wong and Rabie, 2006). Meng (2005) studied the effects of H. epimedii in an osteoblast model. The ethanolic extract and N-butanol extract of H. epimedii promoted the proliferative activity of osteoblasts, with icariin eliciting the most significant effect on osteoblast proliferation (Meng, 2005). An in vivo study demonstrated that the extract of H. epimedii prevented ovariectomized-induced bone loss by increasing bone turnover rate and restoring the loss of trabecular bone architecture in ovariectomized rats. In addition, in vitro data showed that the mechanisms were mediated by its direct action of stimulating osteoblast activities and inhibiting osteoclastogenesis (Xie et al., 2005). Icariin has been investigated for its antiosteoporotic activity in an ovariectomized rat model of osteoporosis where it was shown to effectively prevent bone loss due to estrogen deciency (Nian et al., 2009). The mechanism of icariin enhancing bone formation is mediated via the induction of bone morphogenetic protein-2 and nitric oxide synthesis. It also regulates transcription factor gene expression for bone formation such as the Cbfa1/ Runx2, OPG and RANKL genes (Hsieh et al., 2010). Icaritin, the metabolite of icariin, can enhance the differentiation and proliferation of osteoblasts, facilitate matrix calcification, as well as inhibit osteoclastic differentiation and activity. Interestingly, the effects of icaritin are more potent than icariin (Huang et al., 2007). Total flavonoids of H. epimedii have also been reported to promote the differentiation of primary rat osteoblasts by increasing alkaline phosphatase activity (Zhang et al., 2008a). In human mesenchymal stem cells, the flavonoids of H. epimedii were reported to promote osteogenesis, facilitate the process of matrix calcication and inhibit osteoclastic differentiation (Zhang et al., 2009). The flavonoids of H. epimedii can enhance the mRNA expression of BMP-2, BMP-4, Runx2, betacatenin and cyclin D1, regulators in the BMP and Wnt/catenin signaling pathways (Zhang et al., 2010). Zhang et al.

Estrogen-like activity H. epimedii has been applied as a tonic for the reproductive system such as increasing libido, treating impotence and infertility (Bensky et al., 2004). It also has been used for estrogen hormone replacement therapy (Zhang et al., 2005a). Icariin, icaritin and desmethylicaritin, the flavonoids of H. epimedii, were investigated for their estrogen-like activities using the modified MCF-7 cell proliferation assay. The results showed that only icaritin and desmethylicaritin possessed estrogen-like activities and that the activities were mediated by the estrogen receptor (Wang, 2004; Wang and Lou, 2004). However, icariin can be metabolized to icaritin and desmethylicaritin by human intestinal bacteria in vitro. All preparations of H. epimedii are taken orally; therefore, inactive icariin first metabolizes to estrogenic icaritin and desmethylicaritin in vivo and then generates effects as selective estrogen receptor modulators to exert pharmacological activities (Ye and Lou, 2005). However, minor bioactive flavonoid aglycones such as apigenin, kaempferol, luteolin and quercetin have been found to significantly exert estrogenic effects whereas the major flavonoids, icariin and epimedin A, B, C, do not increase estrogenic activity (Shen et al., 2007). The ethanol extract of H. epimedii was investigated for estrogenic activity, which partially demonstrated it (Zhang et al., 2005a). In addition, the polyphenolic extract of the leaves of E. brevicornum was found to exhibit significant estrogenic activity in a recombinant yeast cell assay and the Ishikawa Var-I assay (Denaeyer et al., 2005). In

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(2008) investigated the effect of epimedium-derived avonoids (EPF) on both skeletal and nonskeletal factors related to hip fracture risk in late postmenopausal women in a 24-month randomized, double-blind and placebocontrolled trial. The results showed that EPF had beneficial effects on both skeletal and non-skeletal factors related to hip fracture risk in late postmenopausal women by significantly increased muscle force coefficient of lower limb and dynamic balance coefficient after 24 months of EPF administration(Zhang et al., 2008b).

Treatment of erectile dysfunction H. epimedii has been used in sexual disorders such as impotence, lack of sexual desire, incomplete erection, premature ejaculation, spermatorrhea, low sperm count, soreness and weakness of the lower back and knees, and infertility (Chen and Chen, 2004). This herb contains a phosphodiesterase type 5 (PDE5) inhibitory compound, icariin, which has the same target as sildenal (Lin et al., 2003). The PDE5 inhibitory effects of icariin have been reported to be greater than those of zaprinast. In addition, icariin can enhance cGMP levels in cavernous smooth muscle cells treated with sodium nitroprusside (Ning et al., 2006). The effect of icariin on erectile function was investigated by using castrated rats in an in vivo study. Oral administration of icariin for 4 weeks improved erectile function of castrated rats, which correlated with an increased percentage of smooth muscle in trabecular tissue, and the mRNA and protein expression of nitric oxide synthase, but had no influence on serum testosterone levels (Liu et al., 2005). Intracavernous administration of E. brevicornum extract enhanced penile erection in rats, with nitric oxide activity considered to be the mechanism (Chen and Chiu, 2006). Moreover, the extract affects sympathetic modulatory activities of the heart but do not increase the sympathetic control of the cardiovascular system or blood pressure (Han et al., 2007). Makarova et al. (2007) studied the effect of lipidbased suspension of E. koreanum Nakai extract on sexual behavior in rats and found that the extract improved erectile function of aged rats (Makarova et al., 2007). PDE5 inhibitory effects of icariin can also be implicated in the therapeutic activity of cardiovascular diseases, pulmonary hypertension, female sexual dysfunction, premature ejaculation, stroke, leukemia and renal failure (Rahimi et al., 2010).

icariin possessed potent antidepressant-like properties that were mediated by a decrease in brain monoamine oxidase (MAO) A and B activities, as well as serum corticotropin-releasing factor (CRF) levels (Pan et al., 2005). In 2007, the effects of icariin on a chronic mild stress (CMS) model of depression in male Wistar rats were studied. Icariin exhibited potent antidepressant-like activities and was found to improve the abnormalities in the hypothalamicpituitaryadrenal axis functions (Pan et al., 2007). Icariin can also target the interaction of the limbic-hypothalamic-pituitary-adrenal (LHPA) stress circuit and serotonergic function in CMS rats (Pan et al., 2010).

Anticancer There are a few in vitro studies on this field. The aqueous extract of E. sagittatum showed strong anti-angiogenic activity both in chick embryo chorioallantoic membrane and bovine aortic endothelial cell models, useful in suppressing tumor growth (Wang, 2004). Icariside II, the flavonoid glycoside in E. koreanum, decreases the protein level of Hypoxia-inducible factor-1 (HIF-1) and leads to suppression of hypoxia-induced responses such as angiogenesis, glucose metabolism and metastasis in human osteosarcoma (HOS) cells. Icariside II can potentially treat cancers that involve overexpression of HIF-1 which is normally found in ovarian, breast or colon cancer (Choi et al., 2008). Moreover, icariin was investigated for its anticancer effect on a human gastric cancer cell line and the results showed that icariin suppressed tumor cell invasion and migration via the Rac1-dependent VASP pathway and may be a potential anti-cancer drug (Wang et al., 2010).

Immunological effects H. epimedii has been studied for its immunostimulating effects in mice. Polysaccharides, flavonoids and icariin of H. epimedii have been found to enhance the immune response in mice after oral administration of sheep red blood cells. They increased T-cell proliferation and phagocytic activity of macrophages (Xiao et al., 1993). The aqueous extract of H. epimedii was effective on Th1 and Th2 cells, as exhibited by the enhancement of IgG1, IgG2a and IgM levels, and cytokines in mice after immunization with ovalbumin (Kim et al., 2001).

Antidepressant Cardiovascular effects There are few studies on this field and the data are from the same authors. The behavioral and neuroendocrinological effects of icariin were studied in rats by using the forced swimming test and the tail suspension test. After oral administration for 21 consecutive days, Icariin from H. epimedii inhibits H2O2-induced nitric oxide (NO) decrease by inhibiting NO degradation. Nitric oxide from endothelial cells possesses antioxidant and antiapoptosis activity. Therefore, icariin may be useful in

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preventing endothelial cell damage induced by reactive oxygen species (Wang and Huang, 2005). In addition, icariin can enhance the expression of eNOS gene and up-regulate the production of NO (Xu and Huang, 2007). The H. epimedii extract has also been reported to inhibit the activity of S-adenosyl-L-homocysteine hydrolase, an enzyme that converts methionine to homocysteine and causes various diseases such as cardiovascular disease (Zhang et al., 2005c).

significant difference in icariin content between freshly prepared samples and those stored (Quan et al., 2010; Sheng et al., 2008). In Chinese pharmacopeia, the quality criteria of H. epimedii consist of many leaves and only a few stalks. The leaves should be yellowish green and unfragmented (Bensky et al., 2004).

Reported toxicity The median lethal dose (LD50) of H. epimedii extract in mice has been reported as 36 g/kg via intraperitoneal injection. Overdose symptoms of H. epimedii are characterized by dilation of pupils, increased physical movement, mild spasms and cramps. Respiratory depression can be found in severe cases (Chen and Chen, 2004). However, no toxicity has been found via oral administration in mice (Kim et al., 2001).

Neurological effects Icariin has protective effects against oxygen and glucose deprivation (OGD)-induced neuron injury. This might be related to its antiapoptotic effect, antioxidative stress and the stabilization of intracellular calcium concentration (Li et al., 2005). The mechanism underlying this neurological protective effect involves the enhanced expression of Sirtuin type 1 (SIRT1), a member of a highly conserved gene family that increases DNA stability and prolonged survival in yeast and higher organisms, as well as mammals. In addition, the mechanism is partially involved with the activation of the mitogen activated protein kinase (MAPK/P38) pathway (Wang et al., 2009b). Icariin can protect against brain dysfunction induced by lipopolysaccharide in rats, and improve spatial learning and memory abilities by decreasing expressions of TNF-, IL1 and COX-2 in the hippocampus (Guo et al., 2010). Icariin can also reduce the degeneration of cortical neurons induced by LPS-activated microglia by blocking the TAK1/IKK/NF-B and JNK/p38 MAPK pathways (Zeng et al., 2010a). In an Alzheimers disease model, icariin was shown to improve memory loss after chronic D-galactose administration in rats is as preferred as aging and age-associated neurodegeneration model (Li et al., 2010). Moreover, icariin also exhibits a signicant neuroprotective effect on neurons affected by b-amyloid proteins by inhibiting tau protein hyperphosphorylation (Zeng et al., 2010b).

ANGELICA SINENSIS (DONG QUAI) A. sinensis (Dong quai) is a fragrant, perennial herb commonly found in China, Japan and Korea, and has various names including Chinese Angelica, dang gui (Chinese), toki (Japanese), tanggwi (Korean), and kinesisk kvan (Danish). Dong quai is a member of the Umbelliferae family, which produces white flowers that bloom in umbrella-like clusters. In traditional Chinese medicine, Dong quai is used as dried root for treating several disorders. Its flavor is a combination of bitter, sweet and pungent tastes. Its overall effect is warming in nature and is suitable for strengthening heart, lung and liver meridians, as well as lubricating the bowel. Dong quai is considered a blood tonic and has been used by generations of women for health concerns such as menstrual pain, menstrual cycle regulation, and peri- and post-menopausal symptoms. Traditional and current uses

Product availability and quality control Many Epimedium species are used in Chinese medicine (Quan et al., 2010). They can vary in their icariin and flavonoid contents according to the species, harvest season, storage, geographical origin or other growing conditions (Zhang et al., 2008c). However, the quality of medicinal preparations is not assured because raw and processed materials can be mixed from different species (Sheng et al., 2008). Based on the standard criteria by the Chinese Pharmacopoeia (2005), flavonoid content as determined by UV is required to be more than 5.0% and icariin content (determined by HPLC) more than 0.5% (Committee of China Pharmacopoeia, 2005). Epimedium herbs can be stored long term because there is no

According to traditional Chinese medicine, Dong quai possesses sweet, acrid, warm, and pungent properties and enters the heart, liver and spleen channels. These properties make dong quai the most appropriate herb for harmonizing the blood. It has been used as a blood tonic to invigorate blood circulation and treat heart and liver blood deficiencies including anemia, pale complexions, brittle nails, dry hair, dizziness, blurred vision, palpitation and abdominal pain related to blood deficiency, and coolness. The combination of dong quai with other herbs, such as astragali roots, is useful in various blood disorders. Dong quai has also been used in gastrointestinal disorders to moisten or lubricate the bowel for unblocking. For female health, Dong quai plays several important roles especially

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in the postpartum period, menstrual disorders, amenorrhea, menopausal symptoms and even during pregnancy if used with caution. Dong quai is also used as an aphrodisiac to enhance sexual performance. Sources and chemical composition Dong quai grows at high altitudes in the cold damp mountains of China, Korea and Japan. The yellowishbrown thick-branched roots of Dong quai are the parts used as medicinal herb. Dong quai root contains 0.4 to 0.7% volatile oil, the key components of which are ligustilide, n-butylidenephthalide, n-butylphthalide, ferulic acid, nicotinic acid and succinic acid (Zhu, 1987). Significant amounts of vitamin A and carotenoids (0.675%), vitamin B12 (0.25 to 0.40 mcg/100 g), vitamin E, ascorbic acid, folinic acid, biotin, various phytosterols (e.g., beta-sitosterol), calcium, magnesium and other essential macrominerals are also found in Dong quai roots (Zhu, 1987). Other constituents of this medicinal herb include Nvalerophenone-O-carboxylic acid, delta-2,4dihydrophthalic anhydride, uracil, adenine, carvacrol, safrole, isosafrole, sesquiterpenes, beta-cadinene, ndodecanol, n-tetradecanol, palmitic acid, angelic acid, myristic acid, sucrose (40%) and a polysaccharide with a molecular weight of approximately 3,000 (Zhu, 1987). Natural coumarin derivatives have been attributed to dong quai, but reports differ regarding which ones are really present. The coumarin derivatives include angelol, angelicone, bergapten, oxypeucedanin, osthole, psoralen and 7-desmethylsuberosin (Zhu, 1987). Pharmacological and toxicological effects Dong quai has various clinical implications due to its varied pharmacologically active constituents.

and promoting endothelial nitric oxide synthase (eNOS) expression (Hou et al., 2004). Coumarins and coumarin derivatives, natural anticoagulants in Angelica spp., have been associated with both the bioactivity and toxicity of the plants; however, dong quai (A. sinensis) contains a lower coumarin content compared to other closely related species. Ferulic acid, one of the constituents of Dong quai, can inhibit the polymerization of platelets in blood circulation. It retards the platelet release of 5-hydroxytryptamine (5-HT) and adenosine diphosphate (ADP) (Zhu, 1987). Both ferulic acid and an aqueous extract of Dong quai have been found to inhibit platelet aggregation and serotonin release. These might be the mechanisms underlying the cardio-protective activity aforementioned.

Immune support and hematopoiesis Lymphocyte proliferation assays indicate dong quai consistently exerts an immunostimulatory effect (Wilasrusmee et al., 2002a, b). A high molecular weight polysaccharide found in Dong quai has demonstrated immunostimulating activity and a blood tonifying effect by inducing hematopoiesis in the bone marrow. This is accomplished, in part, by either direct or indirect stimulation of macrophages, fibroblasts, erythrocytes, granulocytes and lymphocytes, and can induce an increased secretion of human growth factors from muscle tissue. Hematopoiesis is further supported by the presence of significant amounts of vitamin B12, folinic acid and biotin in Dong quai.

Nephroprotective effects A herbal preparation of Dong quai has long been used in China to treat nephrotic syndrome, as it has been thought to elicit antifibrotic effects. In a rat model study, chronic nephritic rats induced by puromycin were treated with either a Dong quai and other herb (Astragalus) mixture (3 ml/day) or enalapril (10 mg/kg). The normal control group received saline and another group received puromycin but no treatment. After 12 weeks, the untreated rats showed marked renal fibrosis. However, Dong quai significantly retarded the progression of renal fibrosis and deterioration of renal histological damage, with effects comparable to those induced by enalapril (Wang et al., 2004).

Cardiovascular effects Dong quai has demonstrated quinidine-like activity on the heart. It can prolong the refractory period, lower blood pressure, and correct experimental atrial fibrillation induced by atropine, pituitrin, strophanthin, acetylcholine or electrical stimulation. Dong quai can dilate the coronary vessels, increase coronary flow, and reduce respiratory rate. An animal study using a water-based extract of Dong quai demonstrated a marked protective effect against myocardial dysfunction and myocardial injury induced by ischemia. A histological study has also demonstrated that a preparation of Dong quai and ligusticum significantly protected human umbilical vein endothelial cells against hydrogen peroxide damage, primarily by inhibiting reactive oxygen species formation

Antispasmodic activity Ligustilide, butylidenephthalide and butylphthalide have been found to have antispasmodic activity against rat uterine contractions and in other smooth muscle systems. The components are characterized as non-specific

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antispasmodics with the mechanism involved different from that used by papaverine, an opium alkaloid used primarily in the treatment of visceral spasm, vasospasm (Ko, 1980). Gynecological effects Administration of Dong quai is associated with both stimulating and relaxing effects on the uterine smooth muscle because there are two general components of Dong quai that affect uterine smooth muscle in opposite ways. The antispasmodic component of the herb is attributed to the volatile oils, such as ligustilide, butylidenephthalide and butylphthalide. As a balance, the uterine stimulating aspect is caused by the water and alcohol extract, nonvolatile constituents of the herb (Zhu, 1987). A small clinical study showed that when the uterus is in a relaxation state, Dong quai can induce contraction, while when it is in a contraction state, Dong quai promotes relaxation. Animal experiments in vivo have demonstrated increased excitability of the uterus, where the contractive rhythm of uterine smooth muscle changed from fast, weak and irregular to slower, stronger and more coordinated, depending on the uterine tone. This is believed to be the pharmacological basis for using dong quai during dysmenorrhea, which is not related to its estrogenic activity (Hirata et al., 1997). One of the most common applications for Dong quai is for relieving vasomotor symptoms associated with the menopause. Such symptoms include hot flushes, skin flushing, perspiration and chills. The mechanism of action, however, is still unclear. In a randomized, doubleblind, placebo-controlled clinical trial, 71 postmenopausal women received either Dong quai root (4.5 g) or placebo daily for 24 weeks (Hirata et al., 1997). There were no differences in vasomotor symptoms between the two groups and there appeared to be no estrogen-like effects on vaginal epithelial tissue. However, this lack of effect could be attributed to using Dong quai alone since traditional Chinese practitioners never prescribe it alone, but rather in combination with several other herbs. The researchers chose to study Dong quai alone because many women in the United States who take it to relieve menopausal symptoms purchase the herb over-thecounter as a single entity. Women should be discouraged from using Dong quai alone for relieving menopausal complaints. A herbal mixture containing A. sinensis root, Paeonia lactiflora root, Ligusticum rhizome, Atractylodes rhizome, Alismatis rhizome and Sclerotium poria has been reported to reduce menopausal disturbances, including vasomotor symptoms by 70% (Hirata et al., 1997). Product availability and quality control Dong quai is available in several forms, and dosages

vary accordingly. Typical oral dosages are as follows: dried root, 3 to 15 g daily by decoction; powdered root, 1 to 2 g 3 times daily; tea, 1 cup 1 to 3 times daily (1 g per cup); tincture (1:2), 4 to 8 ml (1 to 2 teaspoonful) per day; and capsules/tablets, 500 mg 1 to 6 times daily. HPLC has been used to analyze chemical constituents of A. sinensis. By comparing the retention times, UV spectra, mass spectra and molecular weights of detected compounds with those published in literature, 15 constituents of A. sinensis could be tentatively identified and used for assessment of its quality (Wang et al., 2007c). Reported toxicity Dong quai has very low toxicity. The LD50 of a concentrated (8:1 to 16:1) Dong quai extract in mice is at 100 g/kg body weight. Intravenous administration of the essential oil to animals at doses of 1 ml/kg can cause a drop in blood pressure and depression of respiration. Overdose of Dong quai is characterized by fatigue, drowsiness, itching, dyspepsia and abdominal pain. Long-term ingestion at a dosage of 6 g/kg displays no abnormalities in physical activities, food intake body weight, urine examination or hematological examinations. There is a case report of a man who developed gynecomastia after taking Dong quai capsules daily for approximately one month (Goh and Loh, 2001). The patient discontinued the Dong quai pills and his gynecomastia regressed completely when examined three months later. It is important to note that the pills in question were not properly analyzed to confirm or refute the purity of the product. Consequently, the authors could not rule out presence of a pharmacologically active contaminant that may have contributed to the patients condition (Kiong, 2001). Although no reported side effects have occurred with the use of authentic dong quai, various sources continue to warn of potential photosensitivity reactions due to the presence of psoralen and bergapten. Both psoralen and bergapten are furanocoumarins widely studied for their photosensitizing properties. Other related species of Angelica (e.g., A. gigas, A. dahurica and A. pubescens) pose a greater risk than Dong quai due to their higher furanocoumarin content. Dong quai is contraindicated in pregnancy, particularly in the first trimester, due to potential uterine stimulant and relaxant effects.

Pharmacokinetic properties and drug interactions Data from an in vitro study has demonstrated the inducing property of Dong quai on the cytochrome P450 isoenzyme system, but report of a pharmacokinetic interaction between Dong quai and warfarin in an animal model does not support this interaction. Single subcutaneous doses of warfarin (2 mg/kg) were administered

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with or without oral Dong quai extract (2 g/kg, twice daily for three days). The Dong quai treatment did not affect prothrombin time on its own, but significantly lowered the value three days after co-administration with warfarin. No significant variation in the pharmacokinetic parameters of warfarin after Dong quai treatment was found for either single-dose administration or steady-state concentrations of warfarin (Heck et al., 2000). In a case report, a 46-year-old woman who had been taking 5 mg/day warfarin for nearly two years and had an international normalized ratio (INR) stabilized at 2 to 3 experienced an increase in her INR to 4.9 over the course of approximately two months. Changes in medication regimen, diet, alcohol consumption or other lifestyle factors that may have affected the INR were ruled out. However, the patient stated that for the previous four weeks she had been taking Dong quai for perimenopausal symptoms as recommended by a herbalist and had forgotten to mention this earlier. The dosage was one 565-mg tablet once to twice daily. The patient was instructed to discontinue Dong quai, and within four weeks her INR returned to the therapeutic range of 2.48. In view of this information, caution is advised for patients receiving chronic treatment with warfarin. Dong quai may potentiate the therapeutic and adverse effects associated with antiplatelet medications.

CONCLUSION Chinese herbal medicine is widely used worldwide. Both beneficial activities and toxicities have been reported, and their uses should therefore be carefully considered. Because of their well-known benefits, combination of several herbal medicines has been registered as food supplements in many countries.

ACKNOWLEDGEMENTS This research was supported by the National Research University Project of CHE and the Ratchadaphiseksomphot Endowment Fund (HR001B).
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Journal of Medicinal Plants Research Vol. 6(29), pp. 4522-4525,1 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.933 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

Essential oil content and composition of Mentha longifolia (L.) Hudson grown wild in Iran
Zarir Saeidi1, Hadi babaahmadi2*, Keramat Allah Saeidi3, Amin Salehi4, Ramin Saleh Jouneghani3, Hosain Amirshekari5 and Abdallah Taghipour2
Agricultural and Natural Resources Research Center, Chaharmahal va Bakhtiari, Iran. 2 Jahad Daneshgahi Center, Charmahal and Bakhtiari, Iran. 3 Department of Horticulture Science, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran. 4 Department of Agronomy, Faculty of Agriculture, Yasuj Universiy, Iran. 5 Department of Agronomy, Faculty of Agriculture, Shahed Universiy, Iran.
Accepted 29 August, 2011
1

Essential oil content and composition of Mentha longifolia (L.) Huds. collected from five regions in south-west of Iran were determined. The essential oil isolated by hydro-distillation for 3 h using a Clevenjer-type apparatus and the oil was analyzed by GC and GC-MS. Essential oil content was varying from 1.39 to 4.05%. From 23 identified compounds, the major compounds were; Piperitenone oxide (7.41 to 59.67%), Pulegone (3.61 to 49.43%), 1,8Cineole (7.25 to 24.66%), -Terpineol (2 to 6%) and pinene (1.32 to 4.19%). Key words: Mentha longifolia, essential oil, Pulegone, Piperitenone oxide, Iran. INTRODUCTION Iran is considered as one of the major centers of plant biodiversity in the word. Irans natural environment is very diverse, ranging from tropical to cold temperate. This ecological diversity has contributed not only to a high genetic diversity, but also allowed the successful introduction and cultivation of a great number of plant taxa. Menthe longifolia, a perennial essential oil bearing plant that belonging to the Lamiaceae family, grows as wild in various regions of Iran (Omidbaigi, 2005; Mozaffarian, 1996). M. longifolia (L.) Huds. is used in Iranian traditional medicine as a stomach pain-relieving agent, antispasmodic, digestive and carminative (Zargari, 1990). Aerial part of the M. longifolia containing essential oils that has medicinal effects. The essential oil of Mentha species was reported to have fungicidal, antiinflammatory, antimicrobial and antioxidant activities (Mimica-Dukic et al., 2003; Gulluce et al., 2007; Mkaddem et al., 2009). The essential oil content of this medicinal plant, depending on the climatic and geographical factors (Hajlaoue et al., 2008), is varied between 0.9 to 1.8% (Younis and Beshir, 2004). Main identified compounds of the M. longifolia oil were: Dihydrocarvone (23.64%), Piperitone (17.33%) and Cisdihydrocarvone (15.68%) (Dzamic et al., 2010). Ciscarveol (53 to 78%) was reported as the major constitute of Iranian samples (Zenali et al., 2005), whereas, the main compounds in the collected samples from Sudan and India were Carvone (67.3 to 78.9%) and Piperitenone oxide (54.23%) (Younis and Beshir, 2004; Singh et al., 2008). Hafedh et al. (2010) reported that the most important components of essential oil from M. longifolia were: Menthol (32.51%), Menthone (20.71%) and Pulegone (17.76%). Lawrence (1998) and Pino et al. (1990) showed that Pulegone was the main constituent of M. longifolia essential oil, and its percentage ranged from 25 to 92%. Maffei (1998) reported that essential oil of a new chemotype of M. longifolia, growing wild in the Piedmont valley (Italy), was rich in Piperitenone oxide (77.43%). The aim of the present investigation was to study essential oil content and its composition in M. longifolia grown wild in different regions of south-west in Iran.
MATERIALS AND METHODS Collection of samples

*Corresponding author. E-mail: hadi_babaahmadi@yahoo.com

Aerial parts of M. longifolia were collected at the full flowering stage

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Table 1. Environmental conditions in the studied regions during the flowering season of M. longifolia.

Region Felard Lordegan Semirom Shahrekord Yasuj

Minimum temperature (C) 11 13 7 6.8 10.1

July Maximum temperature (C) 39.9 39 35 36.4 38.8

Humidity (%) 38.5 34.5 39.2 42.5 39.5

Minimum temperature (C) 11.2 13.8 7.2 7.4 10.7

August Maximum temperature (C) 38.4 38.6 32.5 33.8 35

Humidity (%) 37.5 28 38.7 43.5 38.2

Table 2. Geographical factors in the studied regions in the South-West of Iran.

Geographical factors Elevation (m) Longitude (E) Latitude (N)

Felard 1740 51 15 31 17

Lordegan 1577 50 49 31 30

Regions Semirom Shahrekord 2150 51 21 31 17 2064 50 55 32 18

Yasuj 1831 51 03 30 41

from provinces of Charmahal and Bakhtiari (Felard, Lordegan and Shahrekourd regions), Isfahan (Semirom region) and Kohgiluye and Boyer Ahmad (Yasuj region) in South-west of Iran (Tables 1 and 2). Extraction of the essential oils Shade dried samples of M. longifolia (90 g three times) were subjected to hydro distillation for 3 h using a Clevenjer-type apparatus to produce oil according to the method recommended by the European pharmacopoeia. The oil was dried over anhydrous sodium sulfate and stored in sealed vial at low temperature before analysis. Gas chromatography analysis GC analysis was performed using a Shimadzu GC-9A gas chromatography equipped with a DB-5 fused silica column (30 m x 0.25 mm, film thickness 0.25 mm). Oven temperature was held at 40C for 5 min and then programmed to 250C at a rate of 3C/min. Injector and detector (FID) temperature were 260C; helium was used as carrier gas with a liner velocity of 32 cm/s, split ratio 1/60. Percentages were calculated by electronic integration of FID peak areas without the use of response factor correction. Essential oil sample was diluted with normal hexan and 0.1 ml was injected into the oven. Gas chromatography-mass spectrometry (GC-MS) analysis GC-MS analysis was carried out on a Varian 3400 GC-MS system equipped with a DB-5 fused silica column (30 m x 0.25 mm i.d.). Oven temperature was 40 to 240C at a rate of 4C, transfer line temperature 260C, carrier gas helium with a linear velocity of 31.5 cm/s, split ratio 1/60, Ionization energy 70 e V; scan time 1 s and mass range 40 to 300 amu.

RESULTS AND DISCUSSION Essential oil content of aerial parts was given in Table 3.

The highest and lowest essential oil content of M. longifolia was found to be 4.05% v/w (Felard in Charmahal and Bakhtiari Province) and 1.39% v/w (Semirom in Isfahan Province), respectively. In a study by Younis and Beshir (2004), content of essential oil in aerial parts of M. longifolia ranged between 0.9 to 1.8%. In another study, the content of essential oil of M. longifolia was 1.55 to1.64% (Abbaszadeh, 2009). Differences in the results can be explained by differences in the environmental conditions of regions under study which affect on essential oil content of M. longifolia leaves. The chemical composition of the essential oil presented in Table 3. From 23 identified compounds, the major compounds and their percentage were: Piperitenone oxide (7.41 to 59.67%), Pulegone (3.61 to 49.43%), 1,8 Cineole (7.25 to 24.66%), -Terpineol (2 to 6%) and pinene (1.32 to 4.19%) (Table 2). The highest and lowest Piperitenone oxide content were found to be 59.67% (Shahrek ord, Charmahal and Bakhtiari Province) and 7.41% (Felard, Charmahal and Bakhtiari Province), respectively. The highest and lowest of Pulegone content was found to be 49.61 (Lordegan, Charmahal and Bakhtiari Province) and 3.43% (Yasouj, Kohgiluye and Boyer Ahmad Province), respectively. Previous studies showed that M. longifolia essential oil is a good source of Mihydrocarvone, Piperitone, Cis-dihydrocarvone, Ciscarveol, Piperitenone oxide, Menthone and Pulegone (Dzamic et al., 2010; Zenali et al., 2005; Younis and Beshir, 2004; Singh et al., 2008; Hafedh et al., 2010; Lawrence, 1998; Pino et al., 1996; Maffei, 1998). Bisio et al. (1999) claimed that ecological factors (climatic and soil conditions) have strong influence on the essential oil content, we found significant variation in the composition of M. longifolia essential oils obtained from five distinct regions of Iran.

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Table 3. Essential oil content and composition of M. longifolia.

1

Compounds (%) Camphene Sabinene -pinene -Myrcene 3- octanol 1, 8 - Cineole 1,3-cyclohexadiene 3-cyclohexen-1-ol isopulegol borneol Cis-isopulegone -Terpineol 1-methoxy-4- (1-methylethenyl) pulegone 2-cyclohexen-1-one 6-Octen-1-ol Biocyclo [2.2.1]heptan-2-ol 1,6,octaden-1-ol Camphen Piperitenone oxid Cis-jasmone Trans-caryophyllene biocyclo germacrene Oil yield (%)
1

RI

941.57 968.27 970.24 989.05 994.09 1027.32 1136.28 1095.2 1151.34 1157.11 1169.89 1185.56 1220.21 1243.69 1265.21 1279.24 1281.52 1296.02 1310.5 1363.82 1393.08 1414.11 1490.75

Felard 0.76 0.15 2.06 0.71 0.12 13. 07 0.09 4.52 2.97 0.20 0.10 2.00 2.71 32.07 2.33 0.60 0.30 28.07 0.12 0.78 4.05

Lordegan 1.04 0.47 4.19 0.80 0.33 24.66 3.91 0.13 0.11 5.75 49.61 0.42 0.48 0.11 7.41 0.2 2.44

Regions Yasouj Shahrekord 0.98 0.26 0.24 2.96 1.02 0.73 0.72 7.25 13.41 0.11 0.17 0.18 0.55 1.07 0.48 0.09 3.26 2.12 8.63 3.43 7.60 7.37 5.14 4.85 3.14 1.44 1.73 54.18 59.67 0.23 0.03 0.53 1.18 0.13 1.53 1.70

Semirom 0.4 3.37 0.28 0.16 7.73 0.21 1.06 0.31 6.00 1.09 31.08 0.14 5.17 1.33 37.96 0.97 1.02 1.39

RI: retention index.

Differences between essential oil content and composition of collected M. longifolia leaves can be explained as the results of environmental and geographical factors (temperature, rainfall, altitude, hours of sunshine, etc.). In conclusion, our study has shown that the chemical composition of the essential oil obtained from the leaves and fruits of M. longifolia collected from three different regions of Iran have different qualitative and quantitative properties.
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Zenali H, Arzani A, Razmjoo K, Rezaee MB (2005). Evaluation of oil compositions of Iranian mints (Mentha ssp). J. Essent. Oil Res. 17:56-159.

Journal of Medicinal Plants Research Vol. 6(29), pp.4526-4534, 1 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1253 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

Quantification of -, - and -mangostin in Garcinia mangostana fruit rind extracts by a reverse phase high performance liquid chromatography
Abdalrahim F. A. Aisha 1, Khalid M. Abu-Salah2, Mohammad J. Siddiqui3, Zhari Ismail1 and Amin Malik Shah Abdul Majid1*
1 2

School of Pharmaceutical Sciences, University of Science Malaysia (USM), Minden 11800, Pulau Pinang, Malaysia. Biochemistry Department and King Abdulla Institute for Nanotechnology, King Saud University (KSU), Riyadh 11451, Saudi Arabia. 3 International Medical University (IMU), Kuala Lumpur 57000, Malaysia.
Accepted 1 February, 2012

Garcinia mangostana fruit rinds contain high concentration of xanthones such as -, and -mangostin. The -mangostin-rich extracts have been used widely in nutritional supplements, herbal cosmetics and pharmaceutical preparations. This study aims to develop a reverse phase HPLC method for the quantification of -, - and -mangostin in G. mangostana fruit rind extracts. The method was validated at 244, 254, 316 and 320 nm. Selectivity was determined by comparing the retention time and the UV-Vis spectra of -, - and -mangostin in G. mangostana extracts with those of the reference compounds. Linearity was in the range 0.2 to 200 g/ml at R2 > 0.9999. The intraday and interday precision was determined as a relative standard deviation, and was found to be (0.4 0.4) and (0.3 0.3)%, respectively. The percentage recovery was in the range (96.3 2.5) to (100.5 3.4)%. The limits of detection and quantification were in the range 0.06 to 0.12 and 0.14 to 0.37 g/ml, respectively. The reported method was applied for the determination of -, - and -mangostin concentration in 7 extracts of G. mangostana fruit rinds, and it could be considered as an important analytical tool for quality control, stability studies, pharmacokinetics, and standardization purposes. Key words: Alpha-, beta-, and gamma-mangostin, Garcinia mangostana, mangosteen.

INTRODUCTION Garcinia mangostana L., (Guttiferae) or mangosteen is a tropical tree cultivated for centuries in the tropical rainforests of Southeast Asia and many other countries (Ji et al., 2007). The pericarps of the fruit have been used in folk medicine by Southeast Asians in the treatment of several human diseases such as skin and wound infections (Harborne et al., 1999). Mangosteen is also used worldwide as an ingredient of several commercial products including nutritional supplements, herbal cosmetics and pharmaceutical preparations (Ji et al., 2007). Previous phytochemical studies on G. mangostana reported this plant as one of the richest sources of xanthones, where more than 50 xanthones have been isolated such as -, - and -mangostin (Figure 1) (Ee et al., 2006; Peres et al., 2000; PedrazaChaverri et al., 2008; Zhang et al., 2010). G. mangostana is gaining more and more interest due to the remarkable pharmacological effects of the fruit rind xanthones such as analgesic (Cui et al., 2010), antioxidant (Jung et al., 2006), anti-tumor (Doi et al., 2009; Akao et al., 2008), anti-inflammatory (Chen et al., 2008; Tewtrakul et al., 2009), anti-allergy (Nakatani et al., 2002), anti-bacterial (Sakagami et al., 2005; Chomnawang et al., 2009), anti-tuberculosis (Suksamrarn et al., 2003), anti-fungal (Kaomongkolgit et al., 2009), anti-viral activities (Chen et al., 1996) and enhancement of the immune system (Tang et al., 2009). Due to the increasing interest in G. mangostana, reliable procedures are needed for the quantitative

*Corresponding author. E-mail: aminmalikshah@gmail.com. Tel: +6046534582. Fax: +6046534582.

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Chemicals and reagents The reference compounds were obtained ChromaDex, USA. The solvents were of HPLC or analytical grades, and were acquired from Merck. The reverse phase Nucleosil C18 column (5 m, 4.6 250 mm) was purchased from Macherey-Nagel, USA.

Extraction Three extracts of the G. mangostana fruit rind powder were prepared including methanolic, 75% ethanolic and toluene extracts. The methanolic extract was prepared at room temperature at 5:1 (solvent: solid) ratio for 48 h, and the 75% ethanolic and the toluene extracts were prepared at 60C for 48 h. Methanol was evaporated to dryness at 50C using a rotary evaporator, and the crude extract was extracted sequentially (3 100 ml, 10 min each) with petroleum ether, chloroform, ethyl acetate and methanol. The ethanolic and toluene extracts were concentrated, at 55 to 60C, until a yellow precipitate starts to form. Subsequently, the concentrated solutions were kept at 2 to 8C for 24 h. A yellow mass was formed in both extracts and was collected and dried at 50C. This material was considered for the 75% ethanolic and toluene extracts.

Instrumentation and HPLC conditions The instrument consisting of Dionex-Ultimate 3000 Rapid Separation LC (RSLC) system, was equipped with a auto sampler, quaternary pump, degasser, column oven, and a DAD detector. The chromatographic analysis was carried out using a reverse phase Nucleosil C18 column (5 m, 4.6 250 mm). The column temperature was set at 30C, the mobile phase was consisting of A (acetonitrile) and B (0.1% H3 PO4 in water), the elution program was isocratic at 95% (A) and 5% (B) for 10 min, the flow rate was maintained at 1 ml/min, and the injection volume was 10 l. The spectral data from the DAD-3000RS Diode Array Detector was collected at 244, 254, 316 and 320 nm, and data acquisition was performed by Chromeleon software version 6.8.

Figure 1. Chemical structure of -, - and -mangostin.

analysis of its bioactive ingredients. Few analytical methods have been reported for the standardization of G. mangostana extracts (Walker, 2007; Yodhnu et al., 2009; Pothitirat and Gritsanapan, 2009; Li et al., 2011). Though these methods are validated and reliable, they are either time consuming or have been developed for analysis of -mangostin only. This study was conducted in order to develop and validate a new HPLC method for the quantification of the major xanthone components of G. mangostana including -, and -mangostin. The effect of the wavelength on the validation parameters was also investigated.

Preparation of the standard mixture Stock solution of the standard mixture consisting of -, - and mangostin reference compounds was prepared at 2 mg/ml in HPLC grade methanol. The solution was further diluted to obtain 200, 100, 25, 10, 5, 1 and 0.2 g/ml. The sample extracts were prepared at 1 mg/ml in the same solvent, and were further diluted to obtain 200 g/ml. The stock solutions were filtered through 0.45 m syringe filters.

Method validation The described method was validated according to the ICH guidelines (ICH, 1997). The following validation characteristics were evaluated: selectivity, linearity, precision, accuracy and the limits of detection and quantification (LOD and LOQ). Linearity Linearity was determined by injecting 10 l of the standard mixture in a concentration range of 0.2 to 200 g/ml. The calibration curves were obtained for each individual compound by plotting the peak area versus the concentration. Regression analysis was performed in order to determine the linearity, in terms of R2, of the calibration

MATERIALS AND METHODS Preparation of raw material Ripened G. mangostana fruit was obtained from a local fruit farm at the Island of Penang, Malaysia, on June 2009. Taxonomic authentication was performed by Taxonomist, USM. A voucher specimen (No: 11155) was deposited at the Herbarium at School of Biological Sciences, USM, Malaysia. The fruit rinds were separated from the edible part, chopped using an electric grinder, and dried at 45 to 50C for 24 h.

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Figure 2. UV-Vis spectra of the reference compounds. -Mangostin (A), -mangostin (B), and -mangostin (C). The spectra were collected by scanning the peaks at their start, apex and end.

graphs.

deviation method (ICH, 1997), using the following formula: LOD = (3.3 ) / S, and LOQ = (10 ) / S,

Selectivity The selectivity of the method was determined by comparing the retention time and the ultraviolet-visible (UV-Vis) spectra of -, and -mangostin obtained in the sample extracts with those of the reference compounds. Where: : is the standard deviation of the Y intercept of the linear regression equations. S: is the slope of the linear regression equations. Determination of -, - and -mangostin concentration in G. mangostana extracts Ten microliters of G. mangostana extracts were injected at 200 g/ml, and the peak area corresponding to -, - and -mangostin was recorded. The concentration of the marker compounds in the samples was calculated by applying the linear regression equations of the standard calibration curves. The results are presented as a wt/wt percentage using the formula: Wt/wt percentage = (the found concentration / 200 g/ml) 100 (n = 3).

Precision The peak area and the retention time were considered as the parameters for the precision analysis. The standard mixture was analyzed at 7 concentration points in the range 0.2 to 200 g/ml. The intraday and interday precision was determined in terms of the relative standard deviation (%RSD), (n = 5).

Accuracy Accuracy was determined as a percentage recovery of -, - and mangostin added to the toluene extract at 10 g/ml. The recovery was studied at a concentration of 1, 5, 10 and 25 g/ml of each individual reference compound. The peak area of the compounds in the toluene extract (B), the individual reference compounds (C) and their combinations (A) was recorded. The percentage recovery was calculated as the following: Percentage recovery = ((A B) / C) 100 The results are presented average SD (n = 3).

Statistical analysis Statistical calculations were carried out using the SPSS 16.0 software package. The students t-test or One-way ANOVA analysis was applied, and differences were considered significant at P < 0.01.

RESULTS The UV-Vis spectra of -, - and -mangostin reference compounds showed peaks at 244 and 316 nm, in addition to 259 nm in -mangostin (Figure 2). In this

Limits of detection and quantification The LOD and LOQ were calculated through the slope and standard

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study, peak detection was carried out at 244 and 316 nm, and at two commonly used wavelengths 254 and 320 nm. Selectivity

significant effect of the wavelength on the percentage recovery of the reference compounds, and hence on the methods accuracy, P values > 0.01.

LOD and LOQ The selectivity of the method was determined firstly by comparing the retention time of -, - and -mangostin obtained in the sample extracts with those of the reference compounds. The retention time of -, - and mangostin reference compounds were 4.72 0.001, 6.97 0.001 and 3.97 0.002 min, respectively. The retention time of the same compounds in the G. mangostana extracts was 4.71 0.001, 6.93 0.01 and 3.97 0.001 min, respectively. The UV-Vis spectra of the target compounds were recorded in same manner of the reference compounds. These spectra were also used, as a second parameter, for the selectivity evaluation. Eight compounds were detected, and showed distinct UV-Vis spectra. The spectra of -, -, and -mangostin detected in the fruit rind extracts were found to match those of the reference compounds (Figure 3). Taken together, these findings confirm the selectivity of the developed method. Linearity Linearity of the developed method was presented in terms of regression coefficient (R2) and was > 0.9999 in the 3 reference compounds at the 4 studied wavelengths. Precision The precision was presented in terms of the %RSD of the retention time and the peak area (n = 5) of the standard mixture. -, - and -mangostin reference compounds were eluted at 4.72, 6.97 and 3.98 min, respectively. The %RSD was less than 0.003% which indicated good reproducibility of the retention time. The %RSD of the peak area, as a second parameter, was also calculated in the range 0.2 to 200 g/ml. Table 1 depicts the average %RSD values of the 3 reference compounds. Statistical analysis by One-way ANOVA indicates no significant effect of the wavelength on the methods precision, P > 0.01. Likewise, analysis by Students t-test indicates no significant difference between the intraday and interday precision, P > 0.01. Accuracy and recovery The accuracy of the method was evaluated by performing a recovery study at 4 concentrations: 1, 5, 10 and 25 g/ml. The results are presented as average percentage recovery SD (Table 2). Statistical analysis was performed by One-way ANOVA and indicates no Analysis of an enriched mixture of xanthones A fraction obtained by column chromatography from the toluene extract was enriched with -, -, and mangostin, and was analyzed for separation of the compounds. The HPLC chromatogram is shown in Figure 5, which shows the presence of 10 peaks. DISCUSSION AND CONCLUSION - and -mangostin were selected as reference compounds because they constitute the major xanthone components in G. mangostana extracts. -mangostin is one of the most hydrophobic xanthones in G. mangostana, and hence the compound has been selected as a marker for the end of elution. The use of acetonitrile at 95% and the 0.1% H3PO4 at 5% resulted in short elution time (<10 min), while maintaining high resolution. The wide concentration range (0.2 to 200 g/ml) gives flexibility to the developed method. Consequently, it can be applied in analysis of samples containing high concentration of the target compounds The linear regression equations of the reference compounds along with the LOD and LOQ values are presented in Table 3. Statistical analysis by One-way ANOVA indicates no significant effect of the wavelength on the LOD and LOQ, P > 0.01. Concentration of -, - and -mangostin in G. mangostana extracts Seven extracts from G. mangostana fruit rinds were analyzed (Figure 4). The concentration of -, - and mangostin in the extracts was calculated by applying the linear regression equations of the reference compounds, and the results are presented as average (wt/wt)% SD. No significant difference in the concentration of the marker compounds was found when the same samples were analyzed at 244, 254, 316 and 320 nm, P values > 0.01. The results showed that - and -mangostin are the main xanthone components of the extracts, with much less amount of -mangostin. The highest concentration of -, and -mangostin was obtained in the toluene extract, whereas the highest -mangostin content was achieved in the petroleum ether sub-extract of the methanolic extract (Table 4).

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Figure 3. UV-Vis spectra of 8 xanthones from G. mangostana extracts. The compounds are labeled with the name and the retention time. The spectra were collected by scanning the peaks at their start, apex and end.

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Table 1. Precision analysis of the HPLC method. The relative standard deviation of the peak area was calculated in the intraday and interday data, and the results are presented as average SD (n = 5). P value was calculated by two tests: One-way ANOVA to study the effect of the wavelength on the precision, and Students t-test to study the difference between the intraday and interday data. Differences are considered significant at P < 0.01.

Compounds Intraday data -mangostin -mangostin -mangostin Interday data -mangostin -mangostin -mangostin

244 0.23 0.14 0.21 0.22 0.17 0.08

Wavelength (nm) 254 316 0.27 0.20 0.31 0.30 0.19 0.09 0.59 0.78 0.67 0.53 0.46 0.38

320 0.40 0.63 0.61 0.71 0.51 0.48

P value (One-way ANOVA)

0.54 0.55 0.20

0.17 0.13 0.27 0.33 0.18 0.10

0.20 0.15 0.20 0.08 0.23 0.18

0.26 0.16 0.27 0.11 0.54 0.42

0.33 0.28 0.57 0.64 0.34 0.14

0.03 0.04 0.04

P value (student t-test) -mangostin 0.41 -mangostin 0.73 -mangostin 0.93

0.47 0.37 0.95

0.31 0.10 0.69

0.79 0.91 0.40

Table 2. Accuracy of the HPLC method in the concentration range 1 to 25 g/ml. The study was performed at 4 wavelengths and the results are shown as average percentage recovery of the reference compounds SD, (n = 3). P value was calculated by One-way ANOVA.

Compounds -mangostin -mangostin -mangostin

244 nm 99.0 2.6 97.8 1.7 100 2.9

Concentration (g/ml) 254 nm 316 nm 320 nm 98.3 1.7 97.8 1.3 97.8 1.3 97.0 1.2 97.3 1.5 96.3 2.5 100.0 2.9 99.8 2.5 100.5 3.4

P value 0.41 0.38 0.53

Table 3. Summary of the calibration data of the reference compounds. The regression equation is (y = a x + b), where (a) is the slope and (b) is the y intercept. The data are presented as average SD (n = 5).

Wavelength (nm) -mangostin 244 254 316 320 -mangostin 244 254 316 320 -mangostin 244 254 316 320

a 0.90 0.004 0.78 0.003 0.64 0.003 0.59 0.002

b 0.33 0.021 0.33 0.020 0.24 0.015 0.22 0.014

LOD (g/ml) 0.07 0.01 0.09 0.01 0.08 0.03 0.08 0.02

LOQ (g/ml) 0.21 0.02 0.26 0.04 0.24 0.08 0.23 0.05

1.00 0.99 1.00 1.00

0.87 0.003 0.78 0.004 0.62 0.003 0.57 0.002

0.40 0.022 0.37 0.017 0.28 0.015 0.24 0.012

0.08 0.02 0.07 0.01 0.08 0.02 0.07 0.03

0.25 0.06 0.21 0.04 0.24 0.05 0.21 0.07

1.00 1.00 1.00 1.00

0.75 0.003 0.75 0.003 0.55 0.002 0.52 0.002

0.31 0.019 0.30 0.018 0.20 0.015 0.18 0.012

0.08 0.01 0.08 0.03 0.09 0.03 0.08 0.01

0.25 0.03 0.24 0.08 0.27 0.10 0.23 0.03

1.00 1.00 1.00 1.00

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Figure 4. HPLC chromatograms of G. mangostana extracts at 244 nm. Standard mixture of -, -, and -mangostin (A), toluene extracts (B), methanolic extract (C), and the sub-extracts of the methanolic extract; petroleum ether (D), chloroform (E), ethyl acetate (F), methanol (G). The 75% ethanolic extract (H).

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Table 4. -, - and -mangostin content in G. mangostana fruit rind extracts. Results are depicted as average (wt/wt)% SD (n = 3).

Extracts Toluene Methanol a Petroleum ether b Chloroform Ethyl acetatec d Methanol 75% Ethanol
ad

-mangostin 72.0 0.2 33.0 0.1 51.0 0.1 46.0 0.8 42.0 0.7 7.0 0.1 53.0 0.3

-mangostin 0.9 0.01 1.3 0.01 5.0 0.01 1.9 0.02 1.7 0.02 0.4 0.01 1.4 0.01

-mangostin 16.9 0.04 7.3 0.02 0.7 0.02 10.1 0.20 9.3 0.20 1.8 0.02 11.0 0.07

refer to sub-extracts of the G. mangostana methanolic extract.

Figure 5. HPLC chromatogram of the enriched mixture of known and unknown xanthones. -, -, and -mangostin were eluted at 3.97, 4.71 and 6.93 min, respectively. The other peaks refer to unknown xanthones.

such as G. mangostana extracts and its commercial products. The method can also be used in pharmacokinetics, where low concentration of the marker compounds in the blood or animal tissues is expected. The use of different wavelengths also gives flexibility to the developed method especially when used in cosmetics, pharmaceutical preparations or in pharmacokinetic studies where interference from the additives or blood components is expected, so the wavelength without or with the minimal noise can be selected. Analysis of the enriched mixture of different xanthones showed the presence of 10 isolated peaks. Consequently, and providing the availability of the reference compounds, it can be concluded that our method can be used for quantification of 10 xanthones in G. mangostana extracts. It is noteworthy to mention that the reported method was applied in the drug release

studies of 2 nanoparticle formulations prepared from mangostin and the toluene extract of G. mangostana (Aisha et al., 2012). The method was also used to study the accumulation of -mangostin in the gastrointestinal tract wall of mice after long term treatment with the formulated and non-formulated toluene extract (unpublished data). Overall, we report a novel reverse phase HPLC method for the quantification of -, - and mangostin in G. mangostana extracts. The reported method was found to be rapid, selective, precise, accurate and with high sensitivity. This method provides several advantages over the previously published methods as it required less elution time and hence less solvent, more flexibility of the wavelength, and the number of marker compounds. We believe that this method may have an important contribution to the research on G. mangostana, and could be considered as an analytical tool for quality control assurance in

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cosmetics and mangostana.

pharmaceutical

industry

of

G.

ACKNOWLEDGEMENTS This work was supported by the Malaysian Ministry of Science and Technology (MOSTI) Science fund (305/PFARMASI/613219) and by the Malaysian Ministry of Higher Education (FRGS-MOHE 203/PFARMASI/61154). This work was also funded by the research chair of "Drug Targeting and Treatment of Cancer use Nanoparticles "at King Saud University, Riyadh, Saudi Arabia. The first author would like to acknowledge with thanks the University of Science Malaysia for providing fellowship during the academic year 2010/2011. Finally, we would like to thank Mr. Shanmugan A/C Vellosamy, School of Biological Sciences, for authentication of the plant samples.
REFERENCES Aisha AF, Ismail Z, Abu-Salah KM, Majid AM (2012). Solid dispersions of alpha-mangostin improve its aqueous solubility through selfassembly of nanomicelles. J. Pharm. Sci. 101(2):815-825. Akao Y, Nakagawa Y, Iinuma M, Nozawa Y (2008). Anti-cancer effects of xanthones from pericarps of mangosteen. Int. J. Mol. Sci. 9(3):355370. Chen LG, Yang LL, Wang CC (2008). Anti-inflammatory activity of mangostins from Garcinia mangostana. Food Chem. Toxicol. 46(2):688-693. Chen SX, Wan M, Loh BN (1996). Active constituents against HIV-1 protease from Garcinia mangostana. Planta Med. 62(4):381-382. Chomnawang MT, Surassmo S, Wongsariya K , Bunyapraphatsara N (2009). Antibacterial activity of Thai medicinal plants against methicillin-resistant Staphylococcus aureus. Fitoterapia 80(2):102104. Cui J, Hu W, Cai Z, Liu Y, Li S, Tao W , Xiang H (2010). New medicinal properties of mangostins: analgesic activity and pharmacological characterization of active ingredients from the fruit hull of Garcinia mangostana L. Pharmacol. Biochem. Behav. 95(2):166-172. Doi H, Shibata MA, Shibata E, Morimoto J, Akao Y, Iinuma M, Tanigawa N, Otsuki Y (2009). Panaxanthone isolated from pericarp of Garcinia mangostana L. suppresses tumor growth and metastasis of a mouse model of mammary cancer. Anticancer Res. 29(7):2485295. Ee GC, Daud S, Taufiq-Yap YH, Ismail NH, Rahmani M (2006). Xanthones from Garcinia mangostana (Guttiferae). Nat. Prod. Res. 20(12):1067-1073. Harborne JB, Baxter H, Moss GP (1999). Phytochemical dictionary: a handbook of bioactive compounds from plants, CRC.

ICH (1997). Guidance for Industry, Q2B: Validation of Analytical Procedures: Methodology. USA: International Conference on Harmonisation. Ji X, Avula B, Khan IA (2007). Quantitative and qualitative determination of six xanthones in Garcinia mangostana L. by LC-PDA and LC-ESIMS. J. Pharm. Biomed. Anal. 43(4):1270-1276. Jung HA, Su BN, Keller WJ, Mehta RG, Kinghorn AD (2006). Antioxidant xanthones from the pericarp of Garcinia mangostana (Mangosteen). J. Agric. Food Chem. 54(6):2077-2082. Kaomongkolgit R, Jamdee K, Chaisomboon N (2009). Antifungal activity of alpha-mangostin against Candida albicans. J. Oral. Sci. 51(3):401406. Li L, Brunner I, Han AR, Hamburger M, Kinghorn AD, Frye R, Butterweck V (2011). Pharmacokinetics of alpha-mangostin in rats after intravenous and oral application. Mol. Nutr. Food Res. 55(Suppl. 1): S67-S74. Nakatani K, Atsumi M, Arakawa T, Oosawa K, Shimura S, Nakahata N, Ohizumi Y (2002). Inhibitions of histamine release and prostaglandin E2 synthesis by mangosteen, a Thai medicinal plant. Biol. Pharm. Bull. 25(9):1137-1141. Pedraza-Chaverri J, Cardenas-Rodriguez N, Orozco-Ibarra M, PerezRojas JM (2008). Medicinal properties of mangosteen (Garcinia mangostana). Food Chem. Toxicol. 46(10):3227-3239. Peres V, Nagem TJ, de Oliveira FF (2000). Tetraoxygenated naturally occurring xanthones. Phytochemistry 55(7):683-710. Pothitirat W, Gritsanapan W (2009). HPLC quantitative analysis method for the determination of -mangostin in mangosteen fruit rind extract. Thai J. Agric. Sci. 42(1):7-12. Sakagami Y, Iinuma M, Piyasena KG, Dharmaratne HR (2005). Antibacterial activity of alpha-mangostin against vancomycin resistant Enterococci (VRE) and synergism with antibiotics. Phytomedicine 12(3):203-208. Suksamrarn S, Suwannapoch N, Phakhodee W, Thanuhiranlert J, Ratananukul P, Chimnoi N, Suksamrarn A (2003). Antimycobacterial activity of prenylated xanthones from the fruits of Garcinia mangostana. Chem. Pharm. Bull. (Tokyo), 51(7):857-859. Tang YP, Li PG, Kondo M, Ji HP, Kou Y, Ou B (2009). Effect of a mangosteen dietary supplement on human immune function: a randomized, double-blind, placebo-controlled trial. J. Med. Food 12(4):755-763. Tewtrakul S, Wattanapiromsakul C, Mahabusarakam W (2009). Effects of compounds from Garcinia mangostana on inflammatory mediators in RAW264.7 macrophage cells. J. Ethnopharmacol. 121(3):379-382. Walker EB (2007). HPLC analysis of selected xanthones in mangosteen fruit. J. Separ. Sci. 30(9):1229-1234. Yodhnu S, Sirikatitham A, Wattanapiromsakul C (2009). Validation of LC for the determination of alpha-mangostin in mangosteen peel extract: a tool for quality assessment of Garcinia mangostana L. J. Chromatogr. Sci. 47(3):185-189. Zhang Y, Song Z, Hao J, Qiu S, Xu Z (2010). Two new prenylated xanthones and a new prenylated tetrahydroxanthone from the pericarp of Garcinia mangostana. Fitoterapia 81(6):595-599.

Journal of Medicinal Plants Research Vol. 6(29), pp. 4535-4539 , 1 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR12.492 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

Genetic identification and relationship analysis of medicinal Limonium by rDNA ITS sequence, single nucleotide polymorphism (SNP) and amplification refractory mutation system (ARMS)
Ge Ding1, Daizhen Zhang2*, Yanqiu Yu2, Beibei Zhang1 and Lingling Zhao1
2

Chemical and Biological Engineering College, Yancheng Institute of Technology, Jiangsu Yancheng 224003 China. Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, Yancheng Teachers University, Jiangsu Yancheng 224051 China.
Accepted 12 July, 2012

The quality of crude drug is important for ensuring consumers safety and efficacy. Limonium sinense (Girard) Kuntze has been used in traditional medicine in China for several thousand years. But identifying L. sinense and the adulterants based only on its morphological and chemical appearance is generally difficult. In order to establish an effective method, single nucleotide polymorphism (SNP) of rDNA ITS region was analyzed. A pair of amplification refractory mutation system (ARMS) diagnostic primers based on SNP was designed. Thirty dried samples were also identified by the diagnostic primers in the study. Moreover, the result of interspecies relationship showed that adulterants were clustered with L. sinense in NJ tree. It demonstrated that SNP sites and ARMS were simple and effective in medicinal drug identification and the rDNA ITS region can be used for genetic relationships analysis of Limonium. Key words: Limonium sinense, rDNA ITS, amplification refractory mutation system (ARMS), single nucleotide polymorphism (SNP), molecular identification, genetic relationship.

INTRODUCTION The genus Limonium is considered as the most speciesrich genus of Plumbaginaceae, which is widespread in saline regions or cool alpine areas (Chant, 1993). Many Chinese herbs are made with stems and roots of genus Limonium, such as Limonium sinense (Girard) Kuntze, Limonium bicolor (Bunge) Kuntze and Limonium aureum (Linnaeus) Hill. L. sinense is the folk medicine popularly used as a remedy for bleeding, piles, fever, hepatitis, diarrhea, bronchitis and other disorders (Li, 1978). Recently, L. sinense was demonstrated to possess hepatoprotective action (Chaung et al., 2003) and the major constituents found in the leaves and the roots were flavonoids (Lin and Chou, 2000; Tang and Shen, 2007). It was listed as good restorative drug by Chinese Herbal Medicine (1999). However, L. sinense and L. bicolor are similar in morphological and anatomical characters (Li, 1987); it is difficult to identify them by traditional methods. In recent years, molecular markers have been widely used for species and populations authentication (Qian et al., 2008; Ding et al., 2008b) because of their stability and less effect by external environment (Ding et al., 2008a). Following the first generation markers (restriction fragment length polymorphism, RFLP) and the second generation markers (simple sequence repeats, SSR), single nucleotide polymorphisms (SNPs) have been widely used in crude drugs authentication, genetic diversity and phylogenetic evolution (Sasaki et al., 2004; Xue et al., 2006; Wang et al., 2010; Muleo et al., 2009) due to the advantages such as ease of use, most abundant polymorphisms in genome and ease of

*Corresponding author. E-mail: dingzyc@yahoo.com.cn 86-0515-88233587. Fax: 86-0515-88233991.

Tel:

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Table 1. Plant species in the study and accession numbers of rDNA ITS regions.

Code 1 2 3 4 5 6 7 8 9 10 11 12

Species Limonium aureum Limonium bicolor Limonium sinense Limonium wrightii Limonium rigualii Limonium virgatum Limonium vulgare Limonium narbonense Limonium furfuraceum Limonium delicatulum Limonium interjectum Limonium minutum

Length of ITS1 200 200 200 201 202 203 201 201 202 204 202 202

Length of ITS2 249 249 248 249 248 247 250 250 248 247 247 248

Content of G+ C (%) 45.4 45.7 46 46 49.7 49.8 47.2 47.4 50 48.9 49 49.8

Accession number JQ736797; JQ736798 JQ736799; JQ736800 JQ736801; JQ736802 JQ736803; JQ736804 AJ222854 AJ222855 AJ222839 AJ222838 AJ222856 AJ222851 AJ222843 AJ132332

Table 2. Drug samples tested in this study and authentication results based on the rDNA ITS regions.

Drug sample name Buxue Cao Buxue Cao Buxue Cao

Code B01 B02 B03

Number of drug samples 19 9 2

Place of collection Sheyang, Jiangsu Dafeng, Jiangsu Lanzhou, Gansu

Drug origin L. sinense L. bicolor L. aureum

automation (Ding et al., 2008b; Yoon et al., 2007). The nrDNA ITS region was used for species and population molecular authentication according to fast nucleotide substitution rate (Qian et al., 2008; Ding et al., 2008a; Shen et al., 2005). Amplification refractory mutation system (ARMS), which has also been described as allele-specific PCR, can be used to detect single base mutations (Li et al., 2007). It has been successfully applied to the identification of the species and commercial products (In et al., 2010; Park et al., 2006). In this study, the SNPs of nuclear rDNA ITS region would be studied and diagnostic primers for ARMS could be designed for L. sinense authentication on the basis of SNPs. Moreover, the phylogenetic analysis of Limonium would also be analyzed for providing theoretical basis for genetic relationship between L. sinense and its adulterants.

PCR amplification and sequencing Genomic DNAs were extracted using CTAB method (Doyle and Doyle, 1990) and stored at -20C until use. The two regions of the internal transcribed spacer of nrDNA ITS-1 and ITS-2 were amplified with the primers designed by White et al. (1990). Polymerase chain reaction (PCR) was performed in 30 l reaction volumes including thermophilic buffer (50 mM KCl, 10 mM Tris-HCl pH8.3), 2.0 mM MgCl2, and 10 pmol of each primer, 0.2 mM dNTPs and 1 unit Taq DNA polymerase. The amplification was performed on a GeneAmp PCR System 9700 (ABI) with a temperature profile of 35 cycles at 94C for 50 s, 50C for 40 s and 72C for 90 s. The PCR products were purified using a PCR purification kit, and cycle sequenced on an ABI Prism 3730 automated sequencer.

Data analysis and ARMS analysis All resultant sequences were aligned using Clustal X 1.8 (Thompson et al., 1997) and analyzed using MEGA 4.0 (Tamura et al., 2007). Based on the nucleotide substitutions of L. sinense, one pair of diagnostic primer (LSJB-01, LSJB-02) for authentication was designed. The ARMS amplifications were performed in 30 l of reaction mixture, consisting of 2.0 mM MgCl2, 0.2 mM dNTPs, 10 pmol of each primer and 1 U Taq polymerase. The cycling conditions were: hot start at 94C for 5 min, followed by 30 cycles of 94C for 50 s, 51C for 40 s and 72C for 1.5 min, and final extension at 72C for 7 min. The PCR products were checked by electrophoresis in 1% agarose gel and detected by UVP GDS-8000.

MATERIALS AND METHODS Sample collection A total of 65 fresh samples of genus Limonium were collected from Chinese mainland. Among them, 27 samples were L. sinense and 23 were L. bicolor from Jiangsu province. We added eight samples of L. aureum from Gansu province and seven samples of Limonium wrightii (Hance) Kuntze from Fujian province. Moreover, we used ITS sequences of congeneric species from GenBank (Table 1). Thirty dried herb samples purchased from various markets in China were also analyzed in the study (Table 2).

RESULTS PCR products from all samples were sequenced with

Ding et al.

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Figure 1. Identification of thirty dried herb samples from markets by the diagnostic primer pair (LSJB-01, LSJB-02). M: DL2000 DNA marker.

primers during amplification. The sequences of different fragments have been submitted to GenBank and accession numbers are shown in Table 1. Some other ITS sequences of Limonium from GenBank were also analyzed together. The size of ITS-1 was 200 to 204 bp and ITS-2 was 247 to 250 bp. The ITS sequences of L. sinense and its related species contained 24 variable nucleotide sites. Nine sites were found in ITS1 and 15 in ITS2. The G+C content of ITS1 and ITS2 ranges from 45.4 to 50% (Table 1). In the present study, 24 variable sites existing in ITS regions were SNPs. And all Limonium species could be identified by their SNPs. These sites could be used to distinguish the samples and analyze genetic relationship between these species. Therefore, the variable site 183 was the specific site of L. sinense (Figure 1). Based on the nucleotide substitutions at position 183, one diagnostic primer pair (LSJB-01: 5-ACT GCA CAG TTA AGC CAC-3, LSJB-02: 5-CAA ATA TTT CAA CGA TGC T-3) was designed to authenticate L. sinense from other samples via ARMS. A positive fragment of about 200 bp was successfully amplified in L. sinense but was note in the other species. All sequences were aligned with Clustal X 1.8 and genetic distance was analyzed by using MEGA 4.0. From the genetic distance, Limonium minutum (L.) Chaz. and Limonium rigualii M. B. Crespo and Erben had the smallest difference of 0.005, while L. wrightii and Limonium furfuraceum (Lag.) Kuntze had the largest difference of 0.198. In the NJ tree, L. aureum, L. bicolor, L. sinense and L. wrightii clustered firstly. Then, L. furfuraceum, L. minutum, L. rigualii and Limonium

interjectum Soler and Rossell were gathered, and then assembled with Limonium virgatum (Willd.) Fourr. and Limonium delicatulum (Girard) Kuntze. Finally, Limonium narbonense Mill. and Limonium vulgare Mill. were clustered together with the aforementioned species. Molecular evolutionary evidence showed that L. bicolor and L. aureum had the closest genetic relationship with L. sinense. Thirty dried herb samples were investigated to confirm their origins by using the diagnostic primer pair. The result showed that 19 specimens with a fragment of 200 bp were L. sinense and the others with no segment were adulterants. Subsequently, the rDNA ITS regions of all samples were sequenced to validate the results. It showed that the sequences of the 19 specimens were consistent with L. sinense, and nine adulterant specimens were from L. bicolor and two from L. aureum (Table 2, Figure 1).

DISCUSSION L. sinense has been used as a traditional drug in China for many years (Dong, 2005; Tang and Shen, 2007). The quality of crude drug is important for ensuring consumers safety and efficacy. Sequences of the nrDNA ITS region have been frequently used for the molecular authentication of crude drugs, such as Dendrobium (Ding et al., 2002), Dendrobium loddigesii (Qian et al., 2008), Dendrobium officinale (Ding et al., 2008a), Alisma orientale (Li et al., 2007) and Bupleurum (Yang et al., 2007) by utilizing the universal primers. In the present

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study, ITS sequences of twelve Limonium species were analyzed and the result showed that two closely related species L. sinense and L. bicolor had some specific discrepancies in ITS region. SNPs have been widely used in studies of geoherbalism study (Ding et al., 2008b) and herbs identification (Sasaki et al., 2004; Wang et al., 2010) because of high stability and reliability. In our study, 24 mutation sites were found in sequences of L. sinense and its related species. SNPs were successfully used to identify Limonium by the nucleotide differences at position 183. ARMS is quick and effective during authentication of species and adulterants (Li et al., 2007; In et al., 2010). In the present study, one pair of diagnostic primers was designed to authenticate L. sinense on the basis of SNPs site. All the plants were checked by ARMS, only individuals of L. sinense generated single fragment while the others were all negative. The fragment was about 200 bp and the annealing temperature was 51C. Therefore, this diagnostic primer pair could be used to identify L. sinense quickly and a rapid identification method was established. At the same time, 30 Buxue Cao crude drugs purchased from the market were also authenticated by this primer pair. The result revealed that there were 19 specimens from L. sinense, 9 specimens from L. bicolor and 2 specimens from L. aureum. Conclusion The botanical origin of 30 drug samples was determined; most were from L. sinense and a few were from its adulterants. Compared to other authentication method, ARMS was simple, timesaving and could provide a more rapid and effective means of differentiating genuine plants with good medicinal effect (Ding et al., 2008b; Xu et al., 2006). The present study showed that methods based on SNPs of rDNA ITS regions and ARMS are practical and convenient for authentication of L. sinense and its adulterants. This combined method was also used for crude drugs, and dry samples could be successfully authenticated. So, the combined methods are expected to be useful instructions for quality control of medicinal plants. With the development of DNA molecular markers, identification and genetic diversity analysis of genuine medicinal drugs will become fast and accurate. ACKNOWLEDGEMENTS The authors thank the National Natural Science Foundation of China (No. 31000142), Natural Science Foundation of Jiangsu Province (No. BK2011421), Foundation of Yancheng Institute of Technology (XKR2010003), and Agricultural Science and Technology Project (YKN2011003) for their financial support.

REFERENCES Chant SR (1993). Plumbaginaceae. in Flowering plants of the world, second edition, ed. V. H. Heywood. London: Batsford pp.78-79. Chaung SS, Lin CC, Lin J, Yu KH, Hsu YF, Yen MH (2003). The hepatoprotective effects of Limonium sinense against carbon tetrachloride and beta-D-galactosamine intoxication in rats. Phytother. Res. 17:784-791. Chinese Herbal Medicine (1999). State Administration of Traditional Chinese Medicine, Shanghai: Science and Technology Press. pp.1533. Ding G, Xu GH, Zhang WC, Lu S, Li XX, Gu S, Ding XY (2008a). Preliminary geoherbalism study of Dendrobium officinale Food by DNA molecular markers. Eur. Food Res. Technol. 227:1283-1286 Ding G, Zhang DZ, Feng ZY, Fan WJ, Ding XY, Li XX (2008b). SNP, ARMS and SSH authentication of medicinal Dendrobium officinale Kimura et Migo and application for identification of Fengdou Drugs. Biol. Pharm. Bull. 31:553-557. Ding XY, Xu LS, Wang ZT, Zhou KY, Xu H, Wang YQ (2002). Authentication of stems of Dendrobium officinale by rDNA ITS region sequences. Planta Med. 68:191-192. Dong BH (2005) .Research on the conservation of Limonium sinense in the coast of Jiangsu. Chinese Wild Plant Res. 24:28-30. Doyle JJ, Doyle JS (1990) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19:11-15. In JG, Kim MK, Lee OR, Kim Yjin, Lee BS, Kim SY, Kwon WS, Yang DC (2010). Molecular identification of Korean Mountain Ginseng using an amplification refractory mutation system (ARMS). J. Ginseng Res. 34:41-46. Li HL (1978) Plumbaginaceae, Flora of Taiwan. Editorial Committee of the Flora of Taiwan. Vol. IV, Edited & Published, Taipei pp.90-93. Li SG (1987). Flora of China. Science Press pp.1-166. Li XX, Ding XY, Chu BH, Ding G, Gu S, Qian L, Wang Y, Zhou Q (2007) Molecular authentication of Alisma orientale (Sam.) Juzep - Based on sequences of nrDNA ITS, PCR-RFLP and ARMS. Planta Med. 73:6770. Lin LC, Chou CJ (2000). Flavonoids and phenolics from Limonium sinense. Planta Med. 66: 382-383. Muleo R, Colao MC, Miano D, Cirilli M, Intrieri MC, Baldoni L, Rugini E (2009). Mutation scanning and genotyping by high-resolution DNA melting analysis in olive germplasm. Genome 52:252-260. Park MJ, Kim MK, In JG, Yang DC (2006) Molecular identification of Korean ginseng by amplification refractory mutation system-PCR. Food Res. Int. 39:568-574. Qian L, Ding G, Zhou Q, Feng ZY, Ding XY, Gu S, Wang Y, Li XX, Chu BH (2008). Molecular authentication of Dendrobium loddigesii Rolfe by amplification refractory mutation system (ARMS). Planta Med. 74:470-473. Sasaki Y, Fushimi H, Komatsu K (2004). Application of Single Nucleotide Polymorphisms Analysis of trnK Gene to the Identification of Curcuma Plants. Biol. Pharm. Bull. 27:144-146. Shen J, Ding XY, Zhang WM, Bao SL, Chang J, Tang F (2005). Authentication of Zanthexylum bungeanum Maxin population and adulterants by analysis of rDNA ITS sequences. Acta Pharm. Sin. 40:80-86. Tamura K, Dudley J, Nei M, Kumar S (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24:1596-1599. Tang XH, Ga J, Chen J, Xu LZ, Tang YH, Dou H, Yu W, Zhao XN (2007) Expression of VDAC regulated by extracts of Limonium sinense Ktze root against CCl4-induced liver damage. Int. J. Mol. Sci. 8:204-213. Tang XH, Shen M (2007). Research advancement of the chemical components and pharmacological action for the plants of Limonium Mill. Li Shizhen Med. Mater. Med. Res. 18:1874-1876. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997). The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876-4882. Wang HT, Sun H, Kwon WS, Jin HZ, Yang DC (2010) A PCR-based SNP marker for specific authentication of Korean ginseng (Panax ginseng) cultivar Chunpoong. Mol. Biol. Rep. 37:1053-1057.

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White TJ, Bruns TD, Lee SB, Taylor JW (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications (Innis MA, Golfand DH, Sninsky JJ, White TJ, Eds.), Academic Press, New York pp.315-322. Xue CY, Li DZ, Lu JM, Yang JB, Liu JQ (2006) Molecular authentication of the traditional Tibetan medicinal plant Swertia mussotii. Planta Med. 72:1223-1226. Yang ZY, Chao Z, Huo KK, Xie H, Tian ZP, Pan SL (2007). ITS sequence analysis used for molecular identification of the Bupleurum species from northwestern China. Phytomedicine 14:416-423.

Yoon MS, Song QJ, Choi IY, Specht JE, Hyten DL, Cregan PB (2007). BARCSoySNP23: a panel of 23 selected SNPs for soybean cultivar identification Theor. Appl. Genet. 114:885-899. Xu H, Wang ZT, Ding XY, Zhou KY, Xu LS (2006) Differentiation of Dendrobium species used as "Huangcao Shihu" by rDNA ITS sequence analysis. Planta Med. 72:89-92.

Journal of Medicinal Plants Research Vol. 6(29), pp. 4540-4549,1 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR12.719 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

Ethnomedicinal uses of plant resources in GilgitBaltistan of Pakistan

Arshad Ali Shedayi* and Bibi Gulshan
Departement of Biological Sciences, Karakoram International University, Gilgit, Pakistan.
Accepted 16 July, 2012

A survey was conducted to collect information regarding medicinal and traditional uses of the plant resources from northern areas of Pakistan. Plant species were collected from Gahkuch area, District Ghizer, Gilgit-Baltistan province, Pakistan. A total of 35 plant species belonging to 27 families were found to be commonly used for various medicinal purposes. Among these, 10, 4, 3, 2, 1, 1 and 1 species belonged to families Asteraceae, Cupressaceae, Lamiaceae, Solanaceae, Papilionaceae, Rosaceae and Leguminosae, respectively. Most of the people in the area still depend on herbal medicines for treating different diseases, including asthma, cough, tonic, abdominal pain, jaundice, diarrhea, cancer, headache, diabetes, muscle pain, fever, skin infections, worms, wounds, broken bones, blood pressure, tuberculosis, swellings, anemia, joint pains, inflammation, dyspepsia, arthritis and rheumatism etc. Majority of the medicines prepared by the medicinal plants were taken in direct form, paste form and/or powder form. In some cases, the whole medicinal plants are used while in some other cases, either fruit or leaves/stem or flowers are used as medicine. Besides the use of plants to treat different human diseases, many livestock such as sheep, buffalo, goat and horse were also being treated with herbal medicines. Other than medical use, medicinal plants were also found to be used as fuel, fodder and vegetable/food by local peoples. The people aged 60-80 were found to be the most knowledgeable regarding use of medicinal plants as compared to the younger ones. The major threats to the medicinal palnts of the area were overgrazing, cutting, natural disater and exploitation. Key words: Gahkuch, Ghizer, Gilgit-Baltistan, ethnobotany, ethnomedicine, medicinal plants, traditional uses, diseases. INTRODUCTION Human beings always depend on plants for various purposes like medicines and food etc that has created indigenous knowledge system. Especially, in harsh mountain environment, plants enhance human survival (Mc Corkle, 1992). The elemental/chemical contents of the medicinal plants are very important. To be used as medicines, it is a prerequisite to know organic constituents of the plant material like flavonoids, alkaloids, essential oils, vitamins and glycosides (Desideri et al., 2010). The medicinal plants of Himalaya are habitat specific (Dhar et al., 2000) and their distribution is scattered and restricted to small areas. The northern mountain of Pakistan is well known for its biodiversity; about 3000 species of plants have been reported from the area, out of which at least 124 have medicinal value (Hazarat et al., 2007; UNDP\IUCN, 1999). In Pakistan, the main source of medicinal plants is forest and rangeland. There are 50,000 registered Hakims (traditional/folk experts of medicinal plants) in Pakistan (William and Zahoor, 1999). Rafiq (1997) reported that approximately 400 - 600 medicinal plants are more frequently used in herbal preparation. Moreover, the three mountains ranges of the area viz.: Karakoram, Himalaya and Hindukush collectively contain about 25000 species (about 10 of world plant species), out of which around 10000 are economically or medicinally useful (Pei, 1992). Goodman and Ghafoor (1992) reported 171 species with local ethnobotanical usage

*Corresponding author. E-mail: arshadbio@kiu.edu.pk.

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from Baluchistan province. Khan et al. (2007) also listed 15 plant species from high altitudes of Pakistan, which are being used ethnobotanically. Similar studies have been conducted in different parts of Pakistan and reported different plant species used for curing different diseases such as Hussain et al. (2004), (2006), (2007) and Shah and Hussain (2008). Khal et al. (2011) reported a total of 43 plant species belonging to 28 families from Khunjerab National Park, Gilgit, used to cure 29 different diseases. A total of 117 taxa belonging to 42 families used as medicine were recorded in Ayvacik city Canakkale in Turkey by Uysal et al. (2012). In this study, the ethnobotanical uses of some plants in Gilgit-Baltistan province, Pakistan, were reported.
MATERIALS AND METHODS Gahkuch is the head quarter of District Ghizer in Gilgit-Baltistan province. Its latitude is 36 1700N and longitude is 732650E. The Gahkuch is situated at an elevation of about 5000- 9000 feet. It is one of the vast and serene places to visit round the year due to its beautiful weather and hospitable populace (IUCN, 2006). Several field trips were conducted to collect plant samples and the required information from June to 2010. Plants were pressed, dried and preserved in a herbarium for future use. Plant species were identified with the help of available literature especially flora of Pakistan (Ali and Qaisar, 1995 - 2007). The survey was spread across the seasons so as to obtain maximum data. The information on medicinal uses of the indigenous plants were described after gathering data from the general population, experienced aged and traditional herbal medicine practitioners (Hakeems, etc) and available literature. A total of 100 inhabitants of the village were interviewed. The randomly selected respondents of different ages from 20 to 80 years were interviewed in local language viz.; Shina. A questionnaire was used to elicit information from the local people using standard methods. Information on local name of plants, plant parts used for curing disease, their recipes and mode of administration were recorded.

RESULTS AND DISCUSSION A total 35 species belonging to 27 families were collected. Among these, the families Asteraceae, Cupressaceae, Lamiaceae, Solanaceae, Papilionaceae, Rosaceae, Leguminosae, Elaeagnaceae, Berberidaceae, Capparidaceae, Chenopodiaceae, Polygonaceae, Ephedraceae, Zygophyllaceae, Urticaceae and Plantaginaceae had 10, 4, 3 species, 2, 2, 2, 2, 1, 1, 1, 1, 1, 1, 1, 1, 1 species, respectively. All the species are being used as medicine by the local inhabitants. The results in the Table 1 show the plant species and their uses for treating different diseases in the study area. In an earlier report (Khan et al., 2011), Asteraceae family was found having maximum number of medicinally used species (11.63% spp.) in Khunjerab National Park, Pakistan. Asteraceae family was also found having large number of species used in medicine in Ayvacik of Canakkale area in Turkey (Uysal et al., 2012). It was found that most of the derivatives were being used to

cure diseases in the study area. Earlier, it was reported that 25% all medicinal prescription is based on substances derived from plants or plant derived synthetic analogs (Sara et al., 2009). Hazarat et al. (2010) reported 50 species belonging to 32 families of wild herbs, shrubs and trees of medicinal plants in Usheria valley Dir, Pakistan. Ali and Qaisar (2009) also discovered 83 taxa that were used locally in Chitral, District of Hindu Kush range. In addition, Shinwari et al. (2006) enlisted more than 500 species of flowering plants being used as medicine; Shinwari and Gilanni (2003) listed 45 medicinal plants from Northern Pakistan, while Athar and Siddiqui (2004) reported 95 species as medicinal plants from Pakistan. During present study, it was found that some plant species were used for treatment of only one specific disease, while most of the plants have multiple uses. Likewise, most of the diseases were treated by many different plant species. It was also found that 35 species were to treat 22 different diseases as shown in the Table 1. Only 4 plant species; 11% (spp. number 1, 9, 23 and 28) used to treat one specific disease (3%), 3 plant species; 9% (6, 29 and 31) used to treat 2 different types of diseases (6%), 7 plant species; 20% (4, 12, 22, 26, 27, 33 and 34) used for 3 different types of diseases (9%), 4 plant species; 11% (5, 10, 19 and 32) used to treat 4 different diseases (11%), 4 plant species; 11% (7, 11, 17 and 18) used to treat 5 different diseases (14%), 5 plant species; 14% (13, 14, 15, 20 and 35), used to treat 6 different diseases (17%), 1 plant species; 3% (25) used to treat 7 different diseases (20%), 4 plant species; 11% (1, 16, 21 and 24) used to treat 8 different diseases (23%), 2 plant species; 6% (8 and 30) used to treat 9 different diseases (26%), 1 plant species; 3% (2) used to treat 10 different diseases (29%) and this number is the highest as compared to others. The results were similar to that of Muthu et al. (2006) who reported that a single plant is used for more than one disease, and to treat a single disease, different plants were used in Kancheepuram District of Tamil Nadu, India. Stomachache and gastric diseases were treated by the highest number of medicinal plants (11 (31%)), followed by fever and flu with 10 (29%) plant species, cough 9 (26%), urine/kidney problems (diuretic) and as tonic/stimulant by 8 (23%) different plant species each; for blood related diseases (anemia, hemorrhage, astringent) 7 (20%) different plant species were used; arthritis/rheumatism, skin diseases, heart problems and cancer 5 (14%) different species each, for wounds, cold/catarrh, jaundice and respiratory/lung problems used 4 (11%) different species each, diabetes, perspiration, earache, gonorrhea, ulcer, liver problems, as laxative/ purgative, bites (snake, fish, dog) and as antiseptic 3 (9%) different species used for each, burns, throat diseases, swelling, eye infection, hair fall/dandruff, blood pressure, polyps, warts and worms 2 (6%) different species used for each, while there are 22 diseases treated only by 1 (3%) plant species each; which are as

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Table 1. Plant species and uses.

Spp. no. 1

Plant species and Family Artemisia maritima (Linn) (Asteraceae) Artemisia annum L. (Asteraceae) Artemisia brevifolia Wall. ex DC. (Asteraceae) Artemisia dubia Wall. Ex Bess. (Asteraceae) Artemisia hera-alba Asso (Asteraceae) Artemisia scoparia Waldst. ( Asteraceae) Elaeagnus angustifolia L. (Eleagnaceae) Berberis lyceum Royle. (Berberidaceae)

Parts used Leaves flowers and

Uses The ointment made is used for joint pains. It is used as stomachache remedy, acts as cardiac stimulant, fever and to kill the worms. It is also used as a tonic. A decoction of the whole plant is used for treatment of Malaria. Leaves are used for fever, cough and taken to treat diarrhea. Oil is used in local perfumes due to its pleasant fragrance. Gastronomic herb and fodder for sheep. Leaf powder is used for gastric problem and intestinal worms. Paste of fresh leaves is applied externally for the treatment of wounds and skin infections. Whole plant powder is used for diabetes. A plant decoction is utilized for cooling purposes. Used for muscular pain and fumigation purpose. Used for the treatment of ear pain. The smoke of twigs is considered good for burns. Considered as fodder for goats. The fruits are eaten raw or boiled for sour throat and treatment of cough, flu, cold and various types of fevers. Gum is used for asthma treatment. The roots are used in gonorrhea, chronic diarrhea, piles, and remedy for swollen and sore eyes, broken bones, wounds, ulcers and acute conjunctive. Used as bitter tonic astringent. Leaves are given in jaundice. An ointment made from root powder is mix with oil and applied on broken bones. It is also used for fencing and hedges. The fruits and woods are astringent. Wood is used for carpentry.

Whole plant

Whole plant

Whole plant

Whole plant

Whole plant

Fruits

Roots, fruits, leaves and stem Fruits stem and

Cupressus sempervirens L. (Cupressaceae) Capparis spinosa L. (Capparidaceae) Carthamus tinctorious L. (Asteraceae) Chenopodium album L. (Chenopodiaceae)

10

Fruit, flower

Roots bark is diuretic and expectorant. Used in paralysis, rheumatism and enlarged spleen. Used for arteriosclerosis, kidney disinfectants and tonic. Used for perspiration, hysteria, weight loss and blood imbalance, flowers are laxative.

11

Petals, seeds

12

Whole plant

This plant is said to be laxative and used in hepatic disorder and enlarge spleen.

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Table 1. Contd.

13

Datura stromonium (Linn). (Solanaceae)

Seeds fruits, and leaves Roots, fruits, stem and seed Dried roots and rhizome

The whole plant is poison and narcotic. Leaves are applied to boils, sores and fish bite. Juice of flowers is used in earache. Leaves and seeds are antiseptics. Fruit is sedative. Juice of fruit is used to cure dandruff and falling hairs. Roots and stem are used as anti asthmatic. Stem as cardiac stimulants. The juice of the berries is useful in treating infection of the respiratory passage. Fruits are edible, used for fever and cough. Seeds are used as a cooling medicine. Roots and rhizomes are used in expectorant, diuretic and weak laxative it is used in flavoring tobacco. Liquorices roots used as tooth brush, roots powder for cough and an extract made from the roots after boiling is given in case of cold and flu. It is believed to be good remedy for sore throat. The fruits are used as cough syrups. After decoction of the berries are used for skin and lung problems. Also used for cancer, ulcer, wounds, skin infection, joint pain, hair fall, diabetes, blood pressure, jaundice and heart problems. Locally it is utilized as firewood and forage, particularly for goats. Berries are used for tuberculosis and diabetes. The berries are burnt over coal and smoke is spread in all corners of the house for repelling evils. The paste of berries is applied on painful joints and swelling. Infusion of barriers is diuretic. Berries, wood and oil are used for cancer, polyps, swellings, tumors, and warts. It is also used for curing skin disease, kidney disease.

14

Ephedra jerardiana Wall. (Ephedraceae)

15

Glycerrhiza glabra (Linn). (Leguminosae)

16

Hippophea rhamnoide L. (Asteraceae) Juniperus excelsa Bieb. (Cupressaceae) Juniperus communes L. (Cupressaceae) Juniperus squamata Buch. (Cupressaceae) Medicago sativa L. (Leguminosae) Mentha sylvestris L. (Lamiaceae) Mentha arvensis Linn (Lamiaceae) Melilotus officinalis (L.) (Papilionaceae)

Fruit, stem, and leaves

17

Berries

18

Berries

19

Twigs berries

and

Twigs are burnt as incense and barriers used similarly as that of J. communis and J. excelsa.

20

Whole plant

The plant is tonic. Used in anemia, hemorrhage, earache and jaundice. It is also considered as antibacterial. Leaves and flowers are carminative and stimulant. Used for headache and Stomachache. Its tea is used to cure catarrh, fever, indigestion and profuse mucus discharge, whooping cough, asthma and respiratory inflammation. It is also used for curing digestive problems, acidity and increased fats. The plant is used to treat liver and spleen disorder, asthma and jaundice.

21

Leaves flower

and

22

Whole plant

23

Whole plant

This plant is carminative, aromatic and emollient.

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J. Med. Plants Res.

Table 1. Contd.

24

Pinuss roxburgii Sargent (Pinaceae) Plantago major L. (Plantaginaceae) Rumex hastatus (Don). (Polygonaceae) Rosa indica L. (Rosaceae) Rosa macrophylla Lindl. (Rosaceae) Saussurea lappa (Dcne.) Sch. (Asteraceae) Solanum nigrum L. (Solanaceae) Taraxacum officinale L. (Asteraceae) Thymus serpyllum Linn (Labiateae) Tribulus terrestris L. (Zygophyllaceae) Trigonella foenum graecum L. (Papilionaceae) Urtica dioica (Linn). (Urticaceae)

Resin wood

Resin is stimulant. Internally it is used as stomachic and as remedy for gonorrhea. Wood is stimulant used in burning of body, cough, fainting and ulceration. Wood and oil resin is used in snake bite and scorpion sting. Leaves are astringent and used in inflammation of the skin, malignant ulcer and intermittent fever. Seeds are used in chronic dysentery, diarrhea and constipation, kidney disorder and gonorrhea.

25

Seeds leaves Leaves roots

and

26

and

Used for their sour taste and carminative, stomachic, flavoring and purgative characters.

27

Flower

Oil of the flower is useful in eye disease. Used in fever, stomache- ache and pneumonia.

28

Fruits

The ripen fruits are eaten and contain plenty of vitamin.

29

Roots

The root is used against heart disease of cattle and for toothache. Powdered roots are sprinkled over crops as insecticide. This herb is used for fever skin problems, wounds, diabetes stomach problems and tumors. Rubbing the seeds on the cheeks to remove freckles.

30

Fruit and whole plant leaves root and

31

Used for urinary problems, constipation, dyspepsia and liver problems.

32

Whole plant

Used in stomach trouble and fever. The plant is used as tonic and in whooping cough.

33

Whole plant

It has long been a constituent in tonics also used as an aphrodisiac, diuretic and nerving.

34

Whole plant

Used as appetizer and general tonic for pellagra for treatment of pulmonary disorders. Reduce allergies, stops bleeding, lower blood pressure and heals wounds. Used as a diuretic and to cure rheumatism. Externally it has been used to improve the appearance of the hair and is said to be a remedy for dandruff.

35

Roots leaves

and

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follows; malaria, piles, broken bones, paralysis, hysteria, weight loss, hepatic disorder, boils/sores, sedative, tuberculosis, headache, hysteria/emollient, pneumonia, vitamins deficiency, toothache, antiseptic, freckles, dyspepsia, aphrodisiac, pellagra and allergy. In a similar study conducted by Khan et al. (2011) in Khunjerab National Park, Gilgit, it was found that 9 spp. were used for fever, followed by cough and indigestion 5 spp., while wounds, eye infection, abdominal pain, jaundice, blood pressure and diarrhea had 4 spp. similar to our findings. In a another similar study conducted in Ayvacik city of Canakkale in Turkey, it was found that the taxa used for the treatment of cough were 18.6%, stomachache 13.4%, kidney ailments 11.6%, cold, analgesic, diuretic and hemorrhoid 9.3%, injuries, tonic, abdominal pain, laxative and dyspepsia 6.9% (Uysal et al., 2012). The largest family in the area is Asteraceae which is well known for its aromatic quality. The present study indicates that some plants are invariably used in Swat and other parts of Pakistan for human and animal health care system (Haq and Rehman, 1981; Haq and Gani, 1994; Khan et al., 2003). Most of the species were used for treating arthritis, rheumatism and stomach disorders, jaundice, backache, stomach disorders, ulcer, cold and even cancer. Medicinal plants, since the dawn of civilization, have been used in virtually all cultures as a source of medicine. There is growing interest in medicinal plants as a re-emerging health aid due to the rising costs of prescription drugs in the maintenance of personal health (Hoareau and DaSilva, 1999). Similarly Shinwari et al. (2000a) noted that as overall diversity increases so does medicinal plants species, reporting 345 medicinally used species from the biologically diverse. Plant products are the sources of medicines for 75 - 90% people in the developing countries (Saad et al., 2009). Artemisia is one of the oldest and best known medicinal plants and is still used effectively today by people of all cultures. Artemisia is used in many different ways and one of the most common practices is to insert fresh leaves into the nostrils to clear blocked nasal passages (Van et al., 2005). In Pakistan, more than 60% of the people lives in rural areas where the generation old indigenous knowledge of medicinal plants used against many day to day diseases, still exist and practiced (Shinwari et al., 2006). The leaves, flowers, fruits and seeds are mostly used for treatment because these have more medicinal values, while whole plant and stem are used sometime. Different parts of medicinal plants used as medicine by the local traditional healers among the different plant parts, the leaves were most frequently used for the treatment of diseases followed by the whole plant parts, fruits, stem, root, seed and flowers. The tribal people of western Madhya Par dash of India uses 13 plants for the treatment of jaundice disease (Samvatsar and Diwanji, 2000). Likewise, Khan et al. (2007) discovered fifteen indigenous species used by the locals of Buner, Swat

and Chitral for different diseases. Most of the plant species and the diseases treated are same as that of the present study. The documented medicinal plants were mostly used to cure skin diseases, poison bites, stomachache and nervous disorders in Kancheepuram District of Tamil Nadu, India (Muthu et al., 2006). The present study indicates that some plants are invariably used in Swat and other parts of Pakistan for human and animal health care system (Haq and Rehman, 1981; Haq and Gani, 1994; Sher et al., 2003). Common diseases of the area Although there are many diseases in the area treated by the medicinal plants, the most common diseases of the study area totaled 1; in which both fever and stomachache was 15%, followed by blood pressure 12%, headache ache, headache and respiratory problems 10% each, cancer 9%, asthma 8%, skin diseases 7%, arthritis 6% and then dyspepsia and diabetes 4% each, as shown in the Figure 1. In developing countries, medicinal plants provide a real alternative for primary health care system. According to a report, between 35000 and 70000 plant species are used in folk medicine worldwide (Ali and Qaisar, 2009). The indigenous communities of the Native American (NAS) have learned to use the bio-resources around them for the treatments of diseases; they depend upon the bio-resources for their primary health care because they perceive that the traditional medicines have no or lesser side affect (Kamboj, 2000). The usage of medicinal plants in the area falls in three categories the medicinal plant Solanum nigrum mixed with other herbal medicines has hepatoprotective effect in cirrhotic patients. This protective effect can be attributed to the diuretic, anti-inflammatory, anti-oxidative, immunomodulating properties of the component herb (Fallah et al., 2005). The people living at high altitudes use medicinal plants for different diseases like rheumatism, arthritis, muscular pain, cough, diabetes, fever and migraine (Khan et al., 2007). Forms in which medicinal plants are used Overall, 45% of medicinal plants were used in direct form, followed by paste form (33%) and powdered form (22%) as shown in the Figure 2. According to local inhabitant, most people used medicinal plants as direct form, some use as paste form, and few use as powder form (Fallah et al., 2005). Animal treatment Live stock treated with medicinal plants in the area were sheep 35%, buffalo 30% goat 24% and horse 20%

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Figure 1. Common diseases (%) of the study area (Gahkuch).

Figure 3. Livestock treated by medicinal plants. Figure 2. Forms in which medicinal plants are used.

(Figure 3). The people of the area mostly used Artemisia spp., Hippophae spp. and S. nigrum for the treatment of their livestock. The Artemisia spp. is used for gastric problems and removal of intestinal worms. Various studies have reported on the indigenous use of medicinal plants in the treatment of live stock diseases like oral disease and eye disorder etc. (Tapsoba and Deschamps, 2006). According to Teklehaymanot and Giday (2007), eight species of medicinal plants have veterinary importance. The plant parts used were leaf (62.5%) and root (37.5%). These are used as remedy for seven internal and external illnesses. The number of veterinary important medicinal plants is low compared to those

areas with culture of cattle raring. Giday and Ameni (2003) documented 83 medicinal plants that are used to treat 37 types of livestock aliments. Parts of the plants used The Figure 4 indicates that medicinal plants as a whole were used mostly (28%), followed by fruit (27%), leaves (21%), stem (13%) and flowers (13%). The berries are included in the fruits and twigs in the stem portion. Among the different parts of medicinal plants used as medicine by the local traditional healers, the leaves were most frequently used for the treatment of diseases followed by the whole plant parts, fruits, stem, root, seed and flowers. The tribal people of western Madhya Par

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ailment, followed by leaves and aerial parts; stem and flowers were the least used plant parts (Uniyal et al., 2006). Plant parts were applied as a paste (38%), juice extracted from the fresh plant parts (24%), powder made from fresh or dried plant parts (20%), some fresh plant parts (6%), and decoction (12%) by traditional healers in Kancheepuram District of Tamil Nadu, India (Muthu et al., 2006). Seasons in which plants are collected Figure 5 shows the results of 45% plants collected in summer, 33% in spring and 22% in the autumn by the local inhabitants for healing purpose, while no plants of medicinal importance are collected during winter as no plants are found during this season due to decreased temperature. Most of the flora is available during summer, and these are then collected, used or stored for future use. Medicinal plants used for other purposes The percentage of medicinal plants used for other purposes are as follows; fuel 45%, fodder 44% and as vegetable/food 11% (Figure 6) since the study was conducted in a rural area where more than 80% people depend on agriculture and wild and cultivated plants are used as fuel. The people also domesticate livestock, which again graze on these plants. A plant in one place may be useful as food, feed, fiber and medicine, while in other places, it may be a weed (Shinwari and Malik, 1989). Various medicinal plant species are also used as food along with their medicinal benefits, evaluating their metal content can help to understand the suitability of these plants species (Husain et al., 2009). The 48 woody species of most native plants have uses by the residents of Haramosh and Bagrot valleys, predominantly as medicines, timber, shelter, domestic items and fuel (Wali and Khatoon, 2007). People living in the mountain of Pakistan use plants in many ways, including medicines, timber, wood, firewood, food and fodder (Hussain and Khaliq, 1996). According to Khan et al. (2011), plant species in Khunjerab National Park have been used for food, medicine, shelter, fuel and other purposes since long. Knowledgeable age groups
Figure 6. Other uses of medicinal plants.

Figure 4. Parts of medicinal plants used.

Figure 5. Seasons in which plants are collected.

dash of India use 13 plants for the treatment of jaundice disease (Samvatsar and Diwanji, 2000). In most of the cases (45%), underground parts were used for treating

The people aged 60 to 80 were found to be the most knowledgeable as compared to the younger ones. The percentage of knowledge about the uses of medicinal plants were as follows; 60 - 80 (50%), 40 - 60 (30%), 20 40 (25%) and 1 - 20 (5%) as shown in the Figure 7. The results indicate that with the increase age, the knowledge is also increased that is the age and knowledge of

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Figure 7. The knowledgable age group as found in the study.

passed on from one generation to another after refining and addition with the passage of time wild plants were cleared from their original habitat to replace the desired cultivated crops on large scale. The local communities of different regions of Pakistan have centuries old knowledge about traditional uses of plants occurring in their areas. This indigenous knowledge of plants has been transferred from generation to generation (Bhardwha and Gahkhar, 2005). The older people of Gilgit were noted as being better informants and the vivid reason for this may be their personal experience of using these plants since old times. Respondents under 50 years of age were less aware of the potential of medicinal plants than their older counterparts who have gathered knowledge from the point of view of their traditional health care and their day to day practices. This difference in the perception of the two age classes will likely result in knowledge loss over time (Qureshi et al., 2006). In a study conducted in Rural Niger, it was found that compared to men, women reported more edible plants, different medicinal plants, and less detailed information on construction plants. The interview data indicated that ethnobotanical knowledge increased with age according to Guimbo et al. (2011). Major threats to the medicinal plants The major threats to the medicinal palnts of the area were found to be overgrazing (40%), cutting for fuel and timber (30%), natural disater (25%) and exploitation by the researchers, healers, conservationists and food organizations (5%) (Figure 8). During the last few years, the habitats of medicinal plants all across the Himalaya have been under pressure due to urbanization and exploitation of raw materials by pharmaceutical companies (Tendon, 1996). In Baluchistan, various wild herbs has been collected and sold in the local communities. However very limited scientific knowledge is there. The vulnerability of medicinal plants and species to over exploitation and extinction need to be dealt pragmatically (Khan and Aslam, 2004). The findings were similar to that of Khan et al. (2007) who reported exploitation and overgrazing as the major threats to the Medicinal plants of higher altitudes of Pakistan (Sadia, 2010).
REFERENCES Ali H, Qaisar M (2009). The ethnobotany of Chitral Valley, Pakistan with particular Preference to Medicinal Plants. Pak. J. Bot. 41(4):20092041. Ali SI, Qaisar M (Eds) (1995-1997). Flora of Pakistan in the department of Botany. University of Karachi. Pakistan. 184: 16-25 Athar M, Siddiqui M (2004). Reflection of the taxonomy and distribution of medicinal Flowers of Pakistan, SIDA Contrib. Bot. 21(1):357-368. Dhar U, Rawal RS, Upreti J (2000). Setting priorities for conservation in the Tennessee River village, USA. Q. Res. 25:330-349. Desideri D, Meli MA, Roselli C (2010). Determination of essential and non essential elements in some medicinal plants by polarized x-ray fluorescence spectrometry. Micro Chem. J. 95:174-180.

Figure 8. Major threats to the medical plants.

medicinal plants is directly proportional to each other. The most familiar age group people with the medicinal plants are in-between 50 - 75 than 75 - 100. Older men and women were found to know more about medicinal plants (Quershi et al., 2007) because the older people has more experience to use medicinal plants since in those days hospital resources were not available. The knowledge about the medicinal plants increases with increasing age of the people, but after 75 it starts decreasing because of the memory loss in old aged people. According to Quershi et al. (2010) the knowledge of plants is based on trial and error. Consequently, the authentic knowledge of the uses of medicinal plants

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Fallah HH, Alvian, Heshmat R, Heydari MR, Abbolmali K (2005). The efficacy of Live 52 on liver cirrhotic patients: A randomized, double blind, placebo- controlled first Approach Phytomedicine 12(9):619624. Giday M, Ameni G (2003). An Ethnobotanical survey on plants of veterinary importance in two Woreda of southern Tigray, Northern Ethiopia. Ethiopian J. Sci. 26(2):123-136. Goodman SM, Ghafoor (1992). The ethnobotany of Southern Balochistan, Pakistan with particular reference to Medicinal Plants Filediana: Botany, New series 31(4):1-84. Guimbo ID, Mueller JG, Larwanou M (2011). Ethnobotanical Knowledge of Men, Women and Children in Rural Niger: A mixed-methods approach. Ethnobot. Res. Appl. Vol.9 Haq I, Rehman M (1981). Medicinal Plants of Upper Swat. Hamdard Medicus 30:51-86. Haq I, Gani U (1994). Medicinal Plants of Lower Swat. Pakistan Study Area J. 9:230-236 Hazarat A, Shah J, Ali M, Iqbal I (2007). Medicinal value of Ranunculaceae of Dir Valley. Pak. J. Bot. 39(4):1037-1044. Hazarat A, Shah J, Ahmad S, Nisar M, Jan AK, Sikandar (2010). Medicinal plants of Usheria Valley, Dir NWFP, Pakistan. Pak. J. Bot. 42(1):31-34. Hoareau L, DaSilva EJ (1999). Medicinal plants: a re-emerging health aid. Elec. J. Bot. 2(2):56-70. Hussain F, Khaliq A (1996). Ethno botanical studies on some plants of Dabargia Hills, Swat: 207-215. In Proceedings of first Training Workshop on Ethno botany and its Application to Conservation. NARC, Islamabad. Husain J, Khan AL, Rehman N, Hamayune M, Shah T, Nisar, Bano T, Shinwari ZK, Lee IJ (2009). Proximate and nutrient analysis of selected vegetable species case study of Karak Region pakistan. Afr. J. Biotechnol. 8(12):2725-2729. Hussain F, Sher H, Ibrar M (2004). Ethnobotanical Profile of some plants of district Swat, Pakistan. Pak. J. Pl. Sci. 10(2):85-104. Hussain F, Badshah L, Dastagir G (2006). Folk Medicinal Uses of some Plants of South Waziristan, Pakistan. Pak. J. Pl. Sci. 12(1):27-39 Hussain F, Shah SM, Sher H (2007). Traditional Resource Evaluation of some plants of Mustuj, district Chitral, Pakistan. Pak. J. Bot. 39(2):339-354. IUCN (2006). A survey report on Ghizer district, Gilgit-Baltistan (Unpublished). Kamboj VP (2000). Herbal medicine. Curr. Sci. 95:57-65. Ethno botany Res. applications 122 www. Ethnobotanyjournal.org/vol5/i1547-346505-115.pdf. Khan AM, Aslam M (2004). Medicinal plant of Bolochistan. Project on introduction of medicinal Herbs and species as crops.Ministry of food, Agric. and livestock, Qarshi industry (Pvt) Ltd. pp.3-44. Khan A, Gilani SS, Hussain F, Durrani MJ (2003). Ethnobotany of Gokand Valley, District Bunir, Pakistan. Pak. J. Bio. Sci. 6:363-369. Khan B, Abdukadir A, Qureshi R, Mustafa G (2011). Medicinal Uses of Plants by the inhabitants of Khunjerab National Park, Gilgit, Pakistan. Pak. J. Bot. 43(5):2301-2310. Khan I, Razzaq, Islam M (2007). Ethnobotanical Studies of Some Medicinal and Aromatic Plants at Higher Altitudes of Pakistan. American-Eurasian J. Agric. Environ. Sci. 2(5):470-473. Mc Corkle CM (1992). Plants, animal and people.Agropastrol system st Res. 1 Edition published by Westivew press, inc-Colorado. Muthu C, Ayyanar M, Raja N, Ignacimuthu S (2006). Medicinal plants used by traditional healers in Kancheepuram District of Tamil Nadu, India. J. Ethnobiol. Ethnomed. 2:43. Pei S (1992). Mountain Culture and Forest Resource Management of Himalaya. Pp. 114-120 in Himalayan Ecosystem. Edited by D.W. Tewari. Intel Book Distribution, Dehra Dun, India. Qureshi RA, Ghufran MA, Sultana KN, Ashraf M, Khan AG (2006). Ethnobotanical Studies of Medicinal Plants of Gilgit District and Surrounding Areas. Ethnobot. Res. Appl. 5:115-122.

Rafiq RA (1997). Medicinal plants of Pakistan: conservation, paper presented at the expert consultation on medicinal plant species prioritization for South Asia, IDRC/WWF-India, New Delhi, India. Saad UK, Wazir SM, Subhan M, Zahoor M, Kamal M (2009). Some of the Ethnobotanically Important plants of F. R. Bannu NWFP, Pakistan. Pak. J. Pl. Sci. 15(1):81-87. Sadia R (2010). Scope of herbal based Agro Industrialization in Pakistan. News and Politics Article Forum. Friends Korner. Com. p.1. Sara V, Franca T, Gelsomila F (2009). Traditional uses of medicinal plants in Valvestio (Italy). J. Ethnopharmacol. 121:106-116. Shah M, Hussain F (2008). Ethnobotanical study of some Medicinal Plants of Moun Elum district Bunir, Pakistan. Pak. J. Pl. Sci. 14(2):91-95. Sher H, Midrarullah, Khan AU, Khan ZU, Hussain F, Ahmad S (2003). Medicinal plants of Udigram, District Swat, Pakistan. Pak. J. Forest. 53(1):65-74. Shinwari ZK, Rehman M, Watanab TY, Oshikawa Y (2006). Medicinal and aromatic plants of Pak (a pictorial guide) Kohat Univ. Sci. Technol. Kohat Pakistan p.492. Shinwari ZK, Gilani S (2003). Sustainable harvest of medicinal plants at Bulashber Nullah, Astor (north pak). J. Ethnopharmacol. 84:289-298. Shinwari ZK, Malik S (1989). Plant wealth of Dera Bugti area. Progressive Farm. 9:39-42. Shinwari ZK, Gilani SS, Kohjoma M, Nakaike T (2000a). Status of Medicinal Plants in Pakistani Hindu-Kush Himalayas. in Proceedings of Nepal-Japan Joint Symposium on Conservation of Natural Medicinal Utilization. Resources and their Uses. pp. 235-242 Samvatsar S, Diwanji VB (2000). Plant sources for the treatment of jaundice in the tribals of Westren Madhya Perdash of India J. Ethopharmacol. 73:313-316. Tapsoba H, Deschamps JP (2006). Use of medicinal plants for the treatment of oral disease in Burkina Faro. J. Ethnopharmacol. 104:68-78. Teklehaymanot T, Giday M (2007). Ethnobotanical study of medicinal plants used by People in Zegie Peninsula, Northwestern Ethiopia. J. Ethnobiol. Ethnomedicine 3:12. Tendon V (1996). CAMP workshopplants under threat new list forged. Med. Plants Conserv. Newslett. 2:12-13. UNDP/IUCN (1999). Report on Biodiversity Of Northern Areas, Pakistan. (Unpublished). Uniyal SK, Singh KN, Jamwa P, Lal B (2006). Traditional use of medicinal plants among the tribal communities of Chhota Bhangal, Western Himalaya. J. Ethnobiol. Ethnomed. 2:14. Uysal I, Gucel S, Tutenocakli T, Ozturk M (2012). Studies on the Medicinal Plants of Ayvacik-Canakkale in Turkey. Pak. J. Bot. 44:239-244, Special Issue, Van WBE, Van Oudtshoorn B, Gericke N (2005). Medicinal Plants of South Africa.Briza Publications, Pretoria, p.304 . Wali SK, Khatoon S (2007). Ethnobotanical studies on useful trees and shrubs of Haramosh and Bagrot valley. In Gilgit and Northern Areas Pakistan. Pak. J. Bot. 39(3):699-710. William, Zahoor A (1999). Priorities for Medicinal Plants Research and Development in Pakistan. MPPA, New Delhi, India.

Journal of Medicinal Plants Research Vol. 6(29), pp. 4550-4552, 1 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR12.142 ISSN 1996-0875 2012 Academic Journals

Short Communication

Efficacy of chloroform, ethanol and water extracts of medicinal plants, Malva sylvestris and Malva neglecta on some bacterial and fungal contaminants of wound infections
Payman Zare1*, Razzagh Mahmoudi2, Sina Shadfar3, Ali Ehsani4, Yasaman Afrazeh1, Anoosha Saeedan1, Farzad Niyazpour5, and Babak Seyed Pourmand6
2

Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran. Department of Food Hygiene and Aquatics, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran. 3 Young Researchers Club, Tabriz branch, Islamic Azad University, Tabriz, Iran. 4 Department of Food Hygiene, Faculty of Veterinary Medicine, University of Urmia, Urmia, Iran. 5 Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran. 6 DVM, Large Animal Clinic Private Practitioner, Tabriz, Iran.
Accepted 7 May, 2012

In this study, the efficacy of chloroform, ethanol and water extracts of Malva neglecta and Malva sylvestris on some bacterial and fungal contaminants of wound infections were investigated. The results showed an acceptable efficacy of chloroform, water and ethanol extracts of these medicinal plants (M. neglecta and M. sylvestris) on some bacterial and fungal contaminants of wound infections. Among all obtained extracts, ethanol extracts of both species especially that of M. sylvestris, showed the best activity against bacteria, followed by aqueous extracts. The best of antibacterial minimum inhibitory concentration (MIC) values was 0.4 mg/ml obtained with Streptococcus pyogenes cultures. All extracts were active against Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus vulgaris. Aqueous and chloroform extracts had better antifungal activity. The best of antifungal MIC values was 0.6 mg/ml for M. sylvestris aqueous extract against Aspergillus niger cultures. All extracts had activity against A. niger, Aspergillus fumigatus and Candida albicans. Our results showed that all extracts are active against S. aureus, P. aeruginosa, and P. vulgaris which have been reported to be troublesome bacteria in wound infections, especially in the aspect of antibiotic resistance as multiresistant microorganisms. The ethanol extracts had the highest antibacterial activity than all other solvents. Anthocyanin of M. neglecta has approved bacteriostatic activity. This water soluble pigment can be responsible for acceptable antibacterial effects of aqueous extracts of this plant. Our results add more reasons to the clinical application of these extracts in the prophylaxis and treatment of wound infections. The wide range of the efficacy of these extracts: A. niger, A. fumigatus and C. albicans on different bacterial and fungal agents, and the healing acceleration obtained by these extracts makes them acceptable candidates for the promotion of healing in wound infections. Key words: Malva sylvestris, Malva neglecta, extracts, contamination, antimicrobial agent.

INTRODUCTION So many efforts have been made to discover new antimicrobial compounds from various kinds of sources such as micro-organisms, animals, and plants. One of such resources is folk medicines (Burt, 2004). Systematic screening of them may result in the discovery of novel effective compounds. The increasing prevalence of multidrug resistant strains of bacteria and the recent

*Corresponding author. E-mail: pzare@tabrizu.ac.ir. +984113392374. Fax: +984113357834.

Tel:

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Table 1. Minimal inhibitory concentration (MIC) of M. silvestris extracts.

Extract Ethanol Chloroform Aqueous

S. aureus 0.60 1.00 0.85

P. aeruginosa 0.70 0.95 0.90

S. pyogenes 0.40 0.80 0.60

MIC (mg/ml) P. vulgaris 0.80 1.00 0.65

A. niger 0.80 0.70 0.60

A. fumigatus 1.00 0.90 0.85

C. albicans 0.90 0.70 0.70

Table 2. Minimal inhibitory concentration (MIC) of M. neglecta extracts.

Extract Ethanol Chloroform Aqueous

MIC (mg/ml) S. aureus 0.70 0.95 0.90 P. aeruginosa 0.60 1.00 0.90 S. pyogenes 0.60 0.95 0.90 P. vulgaris 0.75 1.20 0.90 A. niger 0.80 0.80 0.70 A. fumigatus 1.00 1.00 0.95 C. albicans 0.95 0.95 0.90

appearance of strains with reduced susceptibility to antibiotics raises the specter of untreatable bacterial infections and adds urgency to the search for new infection-fighting strategies. Contrary to the synthetic drugs, antimicrobials of plant origin are not associated with many side effects and have an enormous therapeutic potential to heal many infectious diseases (Ferreira et al., 2006; Neves et al., 2009). Malva sylvestris L. (Malvaceae), usually known as common mallow, is native to Europe, North Africa and Asia, and its traditional use has been documented since a long-time ago, although little clinical evidence is available. The Greeks and Romans claimed for its emollient and laxative properties and several ethnobotanical surveys conducted in Europe, highlight the potential of such neglected local resource, which is use today at the brink of disappearance. Roots, shoots, leaves, flowers, fruits, and seeds are applied in infusions, decoctions, poultices, liniments, lotions, baths, and gargles (Barros et al., 2010). Valuable gains have been documented on the bactericidal, bacteriostatic, antifungal and immune-modulatory properties of these M. sylvestris and Malva neglecta extracts (Grierson and Afolayan, 1999). The present study investigates the efficacy of chloroform, ethanol and water extracts of these medicinal plants on some bacterial and fungal contaminants of wound infections.
MATERIALS AND METHODS Sources of plant materials Total aerial parts of wild M. sylvestris and M. neglecta were collected freshly from free pastures in mountain areas of East Azerbaijan, Iran. Preparation of plant extracts Dried grounded powder from total aerial parts of the plants and

were Soxhlet-extracted sequentially with 250 ml of ethanol, and chloromethane. All of the extracts were filtered (through 0.45 mm filters under sterile conditions), dried using a rotary evaporator at 45 C, and were re-suspended in dimethyl sulfoxide (DMS O) to achieve the stock concentration 2 mg/ml (Barros et al., 2010). Individual herb was boiled with 200 ml of distilled water in a conical flask, till the water reduced to 100 ml. The extract was then filtered through filter paper in a sterilized bottle. This made a 50% extract of the herb (Azmi et al., 2010). Antimicrobial activity Different concentrations of these extracts were added to fresh cultures of the test microorganisms in MHB broth media, as previously described by National Committee on Clinical Laboratory Standards (NCCLS)/Clinical and Laboratory Standards. Institute (CLSI) Documents (NCCLS, 2002, 2003). Test microorganisms included Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, Proteus vulgaris, Aspergillus niger, Aspergillus fumigatus and Candida albicans. Broth dilution assay was performed to evaluate antimicrobial activity of the extracts according to NCCLS/NCLI guidelines (NCCLS, 2002, 2003). After 24 and 48 h incubation at 35C on shaker, the test cultures as well as control media were evaluated spectrophotometrically at 570 nm (NCCLS, 2003; Magro et al., 2006), and MIC and minimal bactericidal concentration (MBC) values of extracts were recorded (NCCLS, 2002).

RESULTS AND DISCUSSION The MIC of M. silvestris and M. neglecta extracts are shown in Tables 1 and 2. Among all obtained extracts, ethanol extracts of both species especially that of M. sylvestris, showed the best activity against bacteria, followed by aqueous extracts (Tables 1 and 2). The best of antibacterial MIC values was 0.4 mg/ml obtained with S. pyogenes cultures (Table 1). All extracts were active against S. aureus, P. aeruginosa, and P. vulgaris. Aqueous and chloroform extracts had better antifungal activity. The best of antifungal MIC values was 0.6 mg/ml for M. sylvestris aqueous extract against A. niger cultures

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(Table 1). All extracts had activity against A. niger, A. fumigatus and C. albicans. The prevention and treatment of wound infections especially after contaminated burn and surgical events is a critical issue. The proper interpretation of cultures taken from wounds is uncertain. Multiple organisms are invariably isolated from non-sterile wounds including indigenous aerobic and anaerobic flora, aerobic gramnegative rods, and fungi. Antibiotic trials with routine antimicrobial agents are losing efficacy due to continual development of antibiotic resistances in the causative microorganisms. The antibacterial spectrum of an agent used for burn and surgical wound treatment and prophylaxis should include coverage for a wide range of microorganisms including pathogenic agents and normal flora, especially strains of antibiotic multi-resistant bacteria (S. aureus and P. aeruginosa) with different and varying sensitivity patterns. However, the effect of plant extracts and essential oils on cell wall structure has been supported by Burt (2004). The hydrophobic activity of essential oils leads to their penetration into cell membrane lipids and increase of their permeability and this causes dysfunctions in all vital activities associated with cell membrane, removal of ions and vital compounds, and finally cell death (Palmer et al., 2001; Burt, 2004). Presence of fungal agents including C. albicans and A. niger adds to this coverage problem. The inflammation and healing process of wounds is an additional issue. An ideal treatment may also accelerate the healing process. Natural medicinal plant extracts have an ancient background in this aspect and modern medicine gains valuable benefits with them. The present study shows the acceptable efficacy of chloroform, water and ethanol extracts of medicinal plants of M. sylvestris and M. neglecta on some bacterial and fungal contaminants of wound infections. Previous studies have got valuable gains on bactericidal (Cheng and Wang, 2006), antifungal (Magro et al., 2006), and immunomodulatory (Ghaoui et al., 2008) properties of these plant extracts. Our results show that all extracts are active against S. aureus, P. aeruginosa, and P. vulgaris which have been reported to be troublesome bacteria in wound infections, especially in the aspect of antibiotic resistance as multi-resistant microorganisms. Ethanol extracts had the highest antibacterial activity than all other solvents. The results suggest that essential oils as non-polar organic compounds could be the main active compounds in these plants. Anthocyanin of M. sylvestris has approved bacteriostatic activity (Cheng and Wang, 2006). This water soluble pigment can be responsible for acceptable antibacterial effects of aqueous extracts of both plants. Aqueous extracts of M. sylvestris have shown great antifungal activity against stored products fungi including A. niger (Magro et al., 2006). A. niger is also one of the most important fungal contaminants of wound infections. The efficacy of all extracts especially aqueous extracts against A. niger, A. fumigatus and

C. albicans of the present study can be promising. The polysaccharide ingredients of aqueous extract of M. sylvestris has approved immunomodulatory effects in vitro studies; it has shown to switch off IL-4 transcription and to activate macrophages and T helper-1 lymphocytes (Ghaoui et al., 2008). Ongoing studies of the authors of the present study on the in vivo application of a combination of extracts especially aqueous and ethanol extracts on infectious and non infectious wounds will be complementary to these results. Conclusion Our results add more reasons to the clinical application of these extracts in the prophylaxis and treatment of wound infections. The wide range of the efficacy of these extracts: A. niger, A. fumigatus and C. albicans on different bacterial and fungal agents, and the healing acceleration obtained by these extracts makes them acceptable candidates for the promotion of healing in wound infections.
REFERENCES Azmi AA, Jamali S, Muras R, Zaidi AH (2010). Antibacterial activity joshanda: a polyherbal therapeutic agent used in common cold. Pak. J. Pharmacol. 27:25-28. Barros L, Carvalho AM, Ferreira CFR (2010). Leaves, flowers, immature fruits and leafy flowered stems of Malva sylvestris: A comparative study of the nutraceutical potential and composition. Food Chem. Toxicol. 48:1466-1472. Burt S (2004). Essential oils: their antibacterial propertied application in foods-a review. Int. J. Food Microbiol. 94(3):223-253. Cheng C, Wang Z (2006). Bacteriostatic activity of anthocyanin of Malva sylvestris. J. Forest. Res. 17(1):83-85. Ferreira A, Proena C, Serralheiro MLM, Ara jo MEM (2006). The in vitro screening for acetylcholinesterase inhibition and antioxidant activity of medicinal plants from Portugal. J. Ethnopharmacol. 108:3137. Ghaoui WB, Ghanem EB, Chedid LA, Abdelnoor AM (2008). The effects of Alcea rosea L., Malva sylvestris L. and Salvia libanotica L. water extracts on the production of Anti-egg albumin antibodies, interleukin4, gamma interferon and interleukin-12 in BALB/c mice. Phytother. Res. 22(12):1599-1604. Grierson D, Afolayan AJ (1999). Antibacterial activity of some indigenous plants used for the treatment of wounds in the Eastern Cape, South Africa. J. Ethnopharmacol. 66(1):103-106. Magro A, Carolino M, Bastos M, Mexia L (2006). Efficacy of plant extracts against stored products fungi. Rev. Iberoam Micol. 23(3):176-178. NCCLS (2002). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard M38-A. NCCLS, Wayne, PA. NCCLS (2003). Methods for dilution antimicrobial susceptibility testing of bacteria that grow aerobically; approved standard sixth edition. Document M7-A6. NCCLS. Wayne PA. Neves JM, Matosa C, Moutinho C, Queiroz G, Gomes LR (2009). Ethnopharmacological notes about ancient uses of medicinal plants in Trsos- Montes (northern of Portugal). J. Ethnopharmacol. 124:270-283. Palmer AS, Steward J, Fyfe L (2001). The potential application of plant essential oils as natural preservatives in soft cheese. Food Microbiol. 18:463-470.

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