Muhammad Asaduzzaman et al.

/ Journal of Pharmacy Research 2011,4(11),4296-4298

Available online through Research Article www.jpronline.info ISSN: 0974-6943 Antidiarrheal,analgesic and Cytotoxic activities of crude extract of Oroxylum indicum ( L.) stem bark
Muhammad Asaduzzaman *1, Nishat Nasrin 2, Abdul Muhit1, Sheikh Zahir Raihan 1, Apurba Sarker Apu 2, and Ahasanul Akbar 1 1 Department of Clinical Pharmacy and Pharmacology, University of Dhaka, Dhaka-1000, Bangladesh 2 Department of Pharmacy, East West University, Mohakhali, Dhaka-1212, Bangladesh

Received on: 19-05-2011; Revised on: 08-06-2011; Accepted on:01-07-2011 ABSTRACT

This research work describes the biological investigations of Oroxylum indicum , a plant belonging to the family of Bignoniaceae. Although some investigations have previously been reported with this plant, the species growing in Bangladesh has not been studied yet to date for analgesic, antidiarrhoeal and cytotoxic activities. The stem bark of O. indicum was extracted with three organic solvents namely methanol, ethyl acetate and chloroform by hot extraction method using the Soxhlets apparatus. Then these extracts were evaluated for antidiarrhoeal (using the castor oil-induced diarrhea), analgesic (by acetic acid induced writhing test) and cytotoxic activities (brine shrimp lethality bio-assay). Swiss albino mice were used as the test animals for screening antidiarrhoeal and analgesic activities, whereas cytotoxicity was monitored using brine shrimps. At a dose of 400 mg/kg body weight, the methanol extract significantly (p<0.05) reduced the number of stool (52.17%) and prolonged the onset of diarrheal episode (61.07 3.67 min) in the test groups. Only methanol extract (500mg/kg) of Oroxylum indicum produced 21.36% inhibition of writhing and showed considerable analgesic activity (p<0.05) as compared to the control. In brine shrimp lethality bioassay, the ethyl acetate extract showed promising cytotoxic activity (LC50 = 1.58 g/ml) as compared with vincristine sulfate (LC50 = 0.419 g/ml).

Key words: Oroxylum indicum , Bignoniaceae, Soxhlets apparatus, antidiarrhoeal, writhing test, cytotoxic
INTRODUCTION Epidemiologically Bangladeshi people are prone to several infectious diseases including diarrhea because of unhygienic livelihood condition. According to WHO, diarrhea is the second leading cause of death of children under five years of age. [1] Traditionally urban community intends to use medicinal plants for the treatment of all sorts of disease including diarrhea.[2] Several studies have been performed to establish the role of medicinal plants in diarrheal treatment.[3,4] Medicinal plants play a vital role of discovering the novel drugs those possesses different pharmacological activities. The plant Oroxylum indicum belonging to the family Bignoniaceae is a medium sized deciduous evergreen tree up to 12 m high that is distributed throughout the hilly areas of Bangladesh, Assam, Malacca, Srilanka, Malay Islands and China. The plant is known as Sona, Sonpatti, Kanak in native language. Traditionally the plant is used as diaphoretic, astringent, carminative, diuretic, stomachic, aphrodisiac and has high potential for stimulating digestion and prevent respiratory disorders. [5] Root bark is tonic, anodyne, sudorific, and potentially useful in diarrhea, dysentery etc. Numerous Ayurvedic formulations contain this plant which is used as anti-inflammatory, anti-helminthic, antibronchitic, anti-rheumatic, anti-anorexic and for treatment of leprosy and tuberculosis. [6] Previous phytochemical studies on Oroxylum indicum has reported several compounds such as ellagic acid, [7] chrysin, oroxylin-A, scutellarin,[8] baicalein,[7] pratensol,[9] oroxindin [10] etc. Bark contains large amount of flavonoids, alkaloids, glycosides, sitosterol, p-coumaric acid, and naphthalene compounds. [5] The plant is reported to possess an anti-inflammatory, anti-ulcer, antioxidant, hepatoprotective and immunomodulatory activity. [11,12,13] This biological investigation was aimed to the scientific validation of the traditional uses of Oroxylum idicum stem bark. Present study reports antidiarrheal activity of Oroxylum indicum by castor oil induced challenge,[14] analgesic activity by using acetic acid induced writhing method [15] and cytotoxic activity by brine shrimp lethality bioassay. [16] MATERIALS AND METHODS Collection of plant materials The stem bark of plant Oroxylum indicum was collected from University of Jahangirnagar, Savar, Bangladesh and was taxonomically identified by Md. A. Rahim, Herbarium Assistant, Department of Botany, Jahangirnagar University. A voucher had been deposited to the herbarium for future references (voucher specimen no. DOB/JU/OI-28). Extraction procedure The pulverized coarse powdered of the stem bark (250gm) of the plant Oroxylum indicum was subjected to hot extraction by Soxhlets apparatus with methanol, ethyl acetate and chloroform (1L) by a method described elsewhere.[17] The powder of the plant was placed inside the cotton bag which was loaded into the main chamber of the Soxhlets extractor which was placed onto a flask containing the extraction solvent. The Soxhlet was then equipped with a condenser. The solvent was heated to reflux. After extraction, the solvent was removed, typically by means of a rotary evaporator, yielding the extracted compound. The non-soluble portion of the extracted solid remained in the flask, and was discarded. Test animals Healthy Swiss albino mice (weighed 16-23 gm) of either sex obtained from the animal house of International Centre for Diarrhoeal Diseases and Research, Bangladesh (ICDDR, B) were used as the experimental model for investigating the antidiarrheal and analgesic property. The animals were given standard ICDDR, B formulated food pellets and water and kept in the laboratory environment (12h dark/12h light cycle) for 3 days for acclimatization. The cleaning of the cages was done daily. Artemia salina Leach (brine shrimp eggs) was collected and hatched to get mature nauplii for brine shrimp lethality bioassay. [18] Study of antidiarrhoeal activity by castor oil challenge Four groups of Swiss albino mice, each consisting of five animals, were taken for the study. Group I was kept as control providing only saline while group II and III were considered as test groups giving the methanol and chloroform extract of the plant, respectively and at dose of 400 mg/kg body weight. Group IV was given the standard antidiarrheal drug loperamide orally at a dose of 66.67 g/kg body weight. Extract pre-treatment was made orally 1 hour before the mice were administered with the standard dose of 1.0 ml of castor oil by oral route. The animals were caged individually and examined for the occurrence of diarrhea hourly for 4 hours after the castor oil challenge. [19] Diarrhea was defined as the presence of fluid material in the stool, which stained the absorbent paper placed beneath the cage. The number of diarrheal episodes in terms of drop count was measured and the cumulative number of diarrheal episode up to 4th hour of study was calculated. Study of analgesic activity by acetic acid induced writhing method The peripheral analgesic activity of the methanolic and ethyl acetate extracts of bark Oroxylum indicum was determined by the acetic acid induced writhing method. [15] The inhibition of writhing in Swiss albino mice by the plant extract was compared against inhibition of writhing by the standard analgesic agent, aminopyrine (Sigma-Aldrich), given orally at a dose of 50 mg/kg body weight. Cytotoxic activity by brine shrimp lethality bioassay The cytotoxicity assay was performed on brine shrimp nauplii using the method described by Mayer et. al.,. [18] Simple zoological organism (Artemia salina leach) was used as convenient monitor for the screening. DMSO (Dimethyl sulfoxide) solutions of the fractions were applied to Artemia salina in a one-day in vivo assay. For the experiment, 4 mg of methanol and ethyl acetate extracts were dissolved in DMSO and solutions of varying concentrat ions (400, 200, 100, 50,

*Corresponding author.
Muhammad Asaduzzaman, Lecturer, Department of Clinical Pharmacy and Pharmacology, University of Dhaka, Dhaka 1000, Bangladesh. Tel: +88-02-9661920-73 (Ext-8156), Fax: +88-02-8615583, E-mail: azamanalo@yahoo.com

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25, 12.5, 6.25, 3.123, 1.563, 0.781 g/ml) were obtained by serial dilution technique. The solutions were then added to the pre-marked vials containing ten live brine shrimp nauplii in 5 ml simulated seawater. After 24 hours, the v ials were inspected using a magnifying glass and the number of survived nauplii in each vial was counted. From this data, the percent of lethality of the brine shrimp nauplii was calculated for each concentration. The median lethal concentration (LC50) of the test samples were obtained by plotting percentage of the shrimp killed against the logarithm of the sample concentration and compared with the positive control using vincristine sulphate. Statistical analysis The experimental results are represented as mean SEM (standard error of mean). Statistical analysis of antidiarrheal and analgesic activity was performed by Students t-test. Regression analysis was carried out for analyzing the data obtained from brine shrimp lethality bioassay to study the relationship between different samples and vincristine. RESULTS AND DISCUSSION Antidiarrheal activity of Oroxylum indicum stem bark was investigated by castor oil challenge (1 ml/kg body weight) in the laboratory mice at a dose of 400 mg/kg body weight. Result of the experiment (Table 1) showed that the methanol extract reduced the number of stool more (52.17%) than that observed with the standard drug loperamide (40.58%). On the other hand, the chloroform extract moderately inhibited the number of stool formation (31.88%). The appearance of fluid material in the feces was considered as diarrheal episode. The onset of diarrheal episode was rapid in control group (10.7 1.11 min) and in the group treated with chloroform extract at a dose of 400 mg/kg body weight (14.70 1.69 min). However, the onset of diarrheal episode was found to be delayed (61.07 3.67 min) in the test groups giving the methanol extract at 400 mg/kg body weight. The standard drug loperamide showed the onset of diarrheal episode (60.0 1.58 min), which was less than but comparable to that observed with the methanol extract. Table 1: Castor oil-induced Anti-diarrheal effect of Oroxylum indicum Stem Bark
Test Group N.C (Water) P.C. (Loperamide) Chloroform Extract Methanol Extract Onset of Diarrhea (min) Mean SEM 10.7 60.0 14.4 61.0 1.11 1.58* 1.69 3.67* No. of Defecation Mean SEM 13.8 8.2 9.4 6.6 0.58 0.81 1.47 0.75 % Inhibition 40.58 31.88 52.17

cates that it can be selected for further cell line assay, since many scientists have shown a correlation between cytotoxicity and activity against the brine shrimp nauplii using the extract or isolated compound from plant.[18] The plant possesses several flavonoids which might be the cause of showing cytotoxicity to the cellular level. Table 3: Effect of Methanol and Ethyl acetate extract on brine shrimp nauplii
Conc. Log C (mg/ml) 400 200 100 50 25 12.5 6.25 3.125 1.563 0.781 2.602 2.301 2 1.699 1.398 1.097 0.796 0.495 0.194 -0.107 % Mortality Methanol Ethyl acetate 100 100 100 75 70 70 50 50 41.66 41.66 100 100 91.66 75 75 66.66 58.33 54.54 53.33 54.54 LC 50 ( g/ml) Methanol Ethyl acetate 3.58 1.58 Vincristine Sulphate Conc. Log C % LC 50 (mg/ml) Mortality ( g/ml) 40 20 10 5 2.5 1.25 0.625 0.3125 0.15625 0.078125 1.602 1.301 1 0.698 0.397 0.096 -0.204 -0.505 -0.806 -1.107 100 100 90 90 80 70 60 50 30 20 0.419

Table 4: LC 50 values of the test samples of Oroxylum indicum

Sample Vincristine Sulphate Methanol Ethyl acetate LC 50 ( g/ml) 0.419 3.54 1.58 Regression equation y = 30.403x + 61.484 y = 25.339x + 38.222 y = 19.728x + 48.296 R2 0.9476 0.9353 0.927

Extracts were administered P.O. 1 hr before castor oil administration. Values are expressed as Mean SEM. *p<0.05 when compared with saline-treated group. After screening the analgesic activity, it was found that the percentage inhibition for the methanol, ethyl acetate and chloroform extracts were 21.36, 2.56 and 13.26, respectively (Table 2). The Methanol extract (500mg/kg) of Oroxylum indicum shows significant analgesic activity ( p<0.05) as compared to the control. On the other hand ethyl acetate and chloroform extract of O. indicum shows insignificant analgesic activity. Table 2: Effects of Methanol, Ethyl acetate and Chloroform extract of Oroxylum indicum (bark) and their respective % inhibition of writhing
Treatment Control (vehicle, 10 ml/ kg) Crude Methanol Extract Crude Ethyl Acetate extract Crude Chloroform Extract Aminopyrine
a

100 90 80 70 60 50 40 30 20 10 0 -0.5

y = 19.728x + 48.296 R = 0.927 y = 25.339x + 38.222 R = 0.9353 Linear (% Mortality Methanol) Linear (% Mortality Ethyl acetate)
2 2

%Mortality

0.5

1 Log C

1.5

2.5

Figure 1: Graphical presentation of log concentration of ethyl acetate, methanol and vincristine sulphate versus percent shrimp mortality after 24 h of exposure CONCLUSION Screening of biological activities of the stem bark of Oroxylum indicum reveals that the plant has a very good potential to be used for the development of lead compounds having different types of biological activities. Further extensive phytopharmacological studies are required for the assessment of antidiarrhoeal, analgesic and cytotoxic activities exerted by the plant. REFERENCES
1. 2. 3. 4. 5. 6. WHO, 2009. Diarrheal Disease: Fact Sheet. Available at: http://www.who.int/ mediacentre/factsheets/fs330/en/index.html Chitme HR., Chandra R, Kaushik S, Studies on antidiarrheal activity of Calotropis gigantean in experimental animals, J Pharm Pharmaceuti Sci, 7(1), 2004, 70-75. Atta AH and Mouneir SM, Evaluation of some medicinal plant extracts for antidiarrhoeal activity, Phytotherapy Research, 19, 2005, 481485. Almeida CE, Karnikowski MG, Foleto R, Baldisserotto B, Analysis of antidiarrhoeic effect of plants used in popular medicine, Rev Saude Publica, 29, 1995, 428433. Ghani A, Medicinal plants of Bangladesh with chemical constituents and uses, 2nd, Asiatic society of Bangladesh, Dhaka, 2003, 329-330. Zaveria M and Jain S, Phytopharmacognostical studies on root bark of Oroxylum indicum, vent, International Journal of Pharmaceutical Sciences Review and Research, 4(1), 2010, 132-135. Maitreyi Z, Khandhar A, Jain S, Quantification of Baicalein, Chrysin, Biochanin-A and Ellagic Acid in Root Bark of Oroxylum indicum by RP-HPLC with UV Detection, Eur J Anal Chem, 3, 2008, 245-57. Sankara S and Nair AGR, Flavonoids from the leaves of Oroxylum indicum and

Dose (mg/kg, p.o.)a 500 500 500 50

Writhingsb (Average SEM) 39.00 5.96 30.67 3.34 38.83 1.30 33.83 1.74 20.67 3.17

% Inhibition 21.36c 2.56 13.26 47.0 c

All three extracts and the standard was administered orally. b Writhings are measured as the average SEM. c p<0.05 In cytotoxic test activity, % mortality increased gradually with the increase in concentration of the test samples. In comparison to positive control i.e. vincristine sulphate (LC50 0.419 g/ml), the cytotoxicity exhibited by ethyl acetate soluble fraction (LC50 1.58 g/ml) was found highly significant and that of methanol extract was promising (LC50 3.58 g/ml). On the other hand chloroform soluble fraction demonstrated moderate cytotoxic activity (Table 3, 4 and Figure 1). The degree of lethality was directly proportional to the concentration of the extracts from the lowest concentration (0.781g/ml) to the highest concentration (400g/ml). Moderate cytotoxicity of the plant extract indi-

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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 11.November 2011

4296-4298