1

Chapter 1

The Problem and its Background

Introduction

Health is one of the most important element in a person’s daily

living. A healthy body allows an individual to live an active life and ward

off diseases. It elevates mood and promotes mental awareness as well as

reduced chronic disease risks. Adequate nutrition is essential in early

stage of childhood to ensure cognitive and neurological development,

strong immune system, proper organ development and function, and

health growth. People must be required to have a well-nourished body to

be able to perform new skills, develop ideas and help their communities.

Child malnutrition has a great impact on the society; it pictures poverty

and unhealthy lifestyle. Based on World Health Organization, malnutrition

is not only about being underweight or overweight but it is about deficiency

or lack in healthy food diet. In the statistics of malnutrition in the

Philippines, over 2.7 million people experiences hunger but do not eat

while 1.5 million have gone through the day without eating. Decreased

appetite and weight loss, especially unintentional, may lead to muscle

 2

weakness, infections, and chronic disorders (Glanz, K., and Rimer, B.

K.1995).

Appetite is a term widely used to refer to the desire to eat food. There

are a lot of things that may contribute to decrease in appetite including

medical conditions, environmental conditions and even poverty. Anorexia

is one of the most common diseases that may contribute to low appetite.

Such characteristics of this eating disorder includes low weight, fear of

gaining weight and longing to be thin thus, resulting in food restriction.

Several factors to be considered what causing anorexia nervosa includes;

Obsessive-compulsive disorder, peer pressure, and also genetics and

hormones (Legg, 2016).

Poor appetite can have numerous causes, but may be a result of

physical or psychological factors. Weight loss and loss of appetite is an

effect of some diseases, and a side effect of some drugs. By increasing our

healthy habits, we can decrease our illnesses that are caused by obesity

and malnutrition. There are a several factors that may cause decreased in

appetite. Such factors may differ with age and underlying health

conditions. If left untreated, loss of appetite can result in under nutrition

or malnutrition (Schacter, D. T. et. al, 2011).

 3

Appetite enhancers are formulated to help the body promote

appetite and weight gain with unintentional weight loss or poor food

intake. One of the specific concerns in elderly adults is low appetite. There

may be an underlying disease or condition that they may be experiencing.

Appetite in older adults can affect positively with proper management of

dental care, bowel regularity, and basic hygiene (Suzanne Falck, 2018).

Most of the Filipinos are unaware of healthy living. In order to

prevent malnutrition and other disorder such as anorexia, healthy lifestyle

should be considered. Moderation of alcohol intake, proper diet, exercise,

wise use of medications and avoiding vices are some of the causes

(Godwin, 2018).

From the beginning up to now, nature has a lot to offer. Nature has

been a source of medicinal agents for years and huge numbers of modern

drugs have been derived from natural sources. Plant samples are widely

used when it comes to illnesses and diseases. Plants based medicinal

constituents can be derived from any part of plant like bark, leaves,

flowers, roots, fruits, and seeds. Medicinal plants play a major role in

human health care. Most of the world population relies on the use of

traditional medicine, which is predominantly based on plants, for the

reasons of efficacy of medicinal plants, safety and their cost effectiveness.

 4

Blessed thistle, wormwood, gentian and bitter boost are some of the herbal

plants used for appetite enhancing and even indigestion. In addition, some

plants are considered as important source of nutrition. The Acorus

calamus L. (sweet flag) Family: Acoraceae is a small aromatic plant that

grows like grass, its underground stem or rhizome is succulent and yellow

on the inside, the leaves are grass-like, about 20 to 60 cms long and green

in color. It is commonly seen in rivers and muddy areas. Its distribution:

Along streams in mountains, creeks other moist places with running

water, on boulders, etc., at low and medium altitude in Luzon especially

Laguna. This plant is also found in the province of Benguet and Bontoc

mostly in swamps. Acorus calamus leaves are well known in its alkaloidal

property which gives the efficacy of its appetite enhancer property

(Ashwani Kumar, 2009).

With the current studies present about the appetite enhancer

activity of its roots alone and with the present studies about the chemical

constituents of the leaves of the Sweet flag, the researchers have decided

to make use of the leaves of the Sweet flag, scientifically known as Acorus

calamus from the family Acoraceae, as the source of Methanolic extract for

the determination of appetite enhancer activity (Selvaraj, et.al 2013).

 5

Background of the Study

Sweet flag is a small aromatic plant which possesses grass-like and

is most commonly found in some muddy areas and along river side.The

Sweet flag is scientifically known as Acorus calamus L. is a member of the

Acoraceae family formerly Arum family – Araceae. The said plant is one of

the most common and most frequently used herbal plant among Chinese

and Indians. In other countries, Sweet flag was often added to wines. It

has been medicinally used for various ailments and conditions. The

rhizome can be extracted with fragrant oils used in perfume industry

(Bown and Deni, 2004).

A. calamus L. (Sweet flag) has a long history of use and has

numerous traditional and ethno medicinal applications. Sweet flag is a

useful medicinal plant which gave benefit in different fields of

medicines. This herb is generally used from the Ancient and Vedic periods

due to its wonderful power of rejuvenation of brain, nervous system and

normalizing the appetite. Significant weight loss can occur and long-term

nutrition may be affected, leading to weakness and fatigue that can not

only inhibit daily activities but can also lead to malnutrition, consequently

increasing the risk of morbidity and mortality. Appetite enhancers are

 6

formulated to help the body promote appetite and weight gain with

unintentional weight loss or poor food intake (Motley,1994).

Many therapeutic uses have been attributed to the leaves of Sweet

flag in the system of traditional medicine and as well as in folkloric

practices. A calamus L. has been studied mostly for pharmacological

studies such as anti-oxidant and anti-inflammatory activities brought

about by the phytoconstituents of its rhizome as well as its leaves. For this

reason, the student researchers were able to study further the appetite

enhancer property of sweet flag that is said to be one of its underlying

peculiarities (Singh, et. al 2011).

Conceptual Framework

The researchers want to focus in the therapeutic value of Acorus

calamus leaves for appetite enhancer property. Scientific study was made

in order to examine the therapeutic effectiveness of its constituents.

Researchers believe that sweet flag leaves have abundant active

constituents responsible for its therapeutic activity.

Major chemical constituents identified in sweet flag are the

presence of glycosides, flavonoids, saponins, tannins, polyphenolic

compound and volatile oils. But most of the biological actions of sweet flag
 7

have been attributed to presence flavonoids. The researchers targeted to

conduct physical and phytochemical screening of sweet flag and came up

with design that will satisfy all the parameters set to measure the appetite

enhancer property (Chandra, et. al 2017).

Traditionally, Acorus calamus leaves are western herbal

medicine. It is commonly found in the Philippines which are said to be

used for witchcraft. Further, in some folkloric they are used to stimulate

appetite. The researchers deepen its underlying effect which was being

stated (Balakumbahan, et.al 2010).

Statement of the Problem

The study aimed to determine the Efficacy testing of the Appetite

Enhancer property of the crude extract of Acorus calamus leaves on

Sprague Dawley rats.

Specifically, the study sought to answer the following problems:

1. What percentage yield was identified in the extraction of Acorus

calamus leaves with different solvent system?

2. What are the physicochemical characteristics of Acorus calamus

leaves extract?

3. How safe is Acorus calamus extract when taken orally?

 8

4. What is the efficacy of Acorus calamus leaves extract as appetite

enhacer?

5. What is the appropriate dosage form for the Acorus calamus leaves

extract intended for children?

Hypothesis of the Study

The study would like to prove that the crude extract from Acorus

calamus leaves exhibit the appetite enhancer property. The appetite

enhancer activity from the leaves of Acorus calamus is not significantly

different from that of the commercially available product particularly

Heraclene with the generic name Dibencozide.

Significance of the Study

Nutrition is vital for our body and all of its systems to function

well. Having a good nutrition can help reduce the risk of some illnesses

and diseases. Nowadays, a condition that can change the level of appetite

further in children the loss of appetite is very common, they tend to focus

themselves more of playing with toys, gadgets, playing with their friends

outside that they almost forget about eating nutritious food 3 times a day,

however they spend their eating time buying junk foods. According to

Leann Birch, the individual differences in styles of intake controls appetite,

 9

such chronic dieting, and out-of-control eating. Limited evidence suggests

that the course of development of shared control differs for intermeal

interval, food selection, and meal size (Birch,1995).

The chronic usage of herbs in treating variety of ailments and

diseases is universal, and exists in many different countries. The discovery

of the study can be a big help in pharmaceutical industry. It can be an

alternative treatment for high cost medications. As a result of this high

cost medications, a lot of people are forced to used herbal medications for

an effective yet cheap alternative (Engebretson, 2002).

Appetite enhancers are very important especially to those people

who lack appetite. The overwhelming increase in the prevalence of obesity

and malnourishment in recent years represents one of the greatest threats

to the health and a cause of mortality and morbidity not only in our

country but also in the whole world. Regular exercise, regulating the food

we eat and appetite enhancers are some best solutions to address this

problem (Beth Sissons, 2018).

The researchers aimed to inspire future researchers to conduct

continuous researches on alternative medicines using indigenous plants

which can contribute a lot in pharmaceutical industry. Therefore, the

 10

researchers are looking forward to the success of the study which will be

a great help in the Philippines.

Scope and Delimitation

The study is limited to the determination of the appetite enhancer

property of the crude extract from the leaves of sweet flag from the family

Acoraceae on Sprague Dawley Rats.

The scope comprised of the serial extraction of the crude extract

using n-hexane, methanol, ethyl acetate and 50% methanol for

determining the solvent to be used following computation of percentage

yield. Each of the extract undergone physical and chemical evaluation

specifically the extract that gave most number of positive result in

flavoniods test. The Acorus calamus extract was also subjected to toxicity

testing in mice. Further the formulation of the appetite enhancer dosage

form was done yet it was not able to have its efficacy testing and quality

controls.

Definition of Terms

For better understanding of the study, the following terms have been

identified:
 11

Anorexia. A person with anorexia nervosa struggles to maintain a healthy

weight. The illness occurs in both sexes but mainly in women. Mostly, it

develops in childhood or adolescence. (Treasure, 2019)

Appetite. A psychological phenomenon, an aspect of the mental processes

organizing an individual's behavior (Encyclopedia of food sciences and

nutrition, 2003)

Appetite enhancer. It is a drug, hormone, or compound that

increases appetite and may induce hyperphagia.

Diet. Is the sum of food consumed by a person or other organism. Often

implies the use of specific intake of nutrition for health or weight-

management reasons.

Sprague Dawley rats. It has long and narrow head, longer tail which may

be equal or longer than the body length.

Swiss mice. Common laboratory mice which are smaller than the

sprague dawley rats and was used in the toxicity testing.

 12

Chapter 2

Review of Related Literature and Studies

This chapter includes the review of related literature and studies

relevant in the appetite enhancer property of Acorus calamus L. (Family

Araceae), commonly known as Acorus calamus.

Malnutrition and Anorexia

Nowadays, the most common health problem in the Philippines is

associated with malnutrition. There are various reasons behind

malnutrition such as poverty, lack of food source, wastage, and over

population resulting to unhealthy diet, mental changes, anemia and

growth failure. (Alexandra, 2012).

Inadequately understood alteration happens in the physiological

regulation of appetite; which is then exhibited as less hunger and earlier

satiety. Thus, poor appetite that leads to malnutrition is due to insufficient

caloric intake. It is showed that severely malnourished children often lack

interest to play activities, have a low sense of well-being and are less able

to cope with disease-related treatments. Both physiological and

psychological factors, determines one’s hunger, the desire to eat and fed.

However, for a child up to six years of age, it is quite normal to experience

 13

loss of appetite especially when the child is not feeling well for any reason.

(Khandke, 2011)

Anorexia is one of the common disease that may result to low of

appetite. It is associated with some health complications includes

psychologic disturbances like intense fear of gaining weight and

disturbance of body image and physiologic disturbances such as

hypotension, bradycardia and hypothermia. The researchers show that

anorexia nervosa has the highest mortality rate of psychiatric disorders,

because of its medical complications. (Mehler and Brown, 2015).

It is associated with a high risk of inadequate nutrition that can lead

to muscle weakness, decrease in physical activity, and even in growth and

development. Several medications are presented to enhance the appetite

of an anorexic patient but most of them failed to sustain the condition of

the patient. Anabolic steroids have been used showing some positive

results at the same time promoting serious adverse effects. (Francesco

Landi, et. al)

Many Filipinos are not aware about Anorexia nervosa which is

characterized by setting one's perception to pursuit thinness. It is the most

succeeding eating disorder, with basically unidentified causes and/or

 14

factors. Anorexic persons have a bad habit of consciously losing weight

which lead to dying and follows strict diet especially avoiding foods with

high calorie content. The top candidate for this kind of eating disorder are

female in preadolescence age and males. Patients who are anorexic

commonly observed with sexual abused, low self-esteem and/ or being

bullied. (Wozniak, et. al 2012).

Appetite

There are numerous herbs and herbal formulations which are widely

used to stimulate the appetite in children with non-pathological anorexia.

A study conducted in India where daily consumption of appetizer syrup

was used to enhance appetite, good quality of food intake and make the

children feel the desire to eat. More of these, the study found to be safe

and do not possess any adverse events. Results were obtained from the

baseline prior to appetite stimulating effects of the formulation like

changes in body weight. (Sastry, et. al. 2017).

Dramatic and poorly understood alterations occur in the

physiological regulation of appetite; which is then exhibited as less hunger

and earlier satiety. Thus, inadequate caloric intake consequent to poor

appetite can lead to malnutrition. The authors demonstrated that severely

 15

malnourished children often lack interest in play activities, have a

decreased sense of well-being and are less able to cope with disease-related

treatments. A variety of factors, both physiological and psychological,

determines one’s hunger, desire to eat and satiety. It is quite normal for a

child up to six years of age to experience loss of appetite especially when

the child is not feeling well for any reason. Hence, it is difficult to

differentiate a “normal” loss of appetite from that which requires

intervention. It is only when there is a weight loss or failure to gain weight

that an intervention is called for. External factors such as climate,

temperature, prior meals, or activity may also influence appetite (R. K. S

Tiwari, K. Das 2012).

A decreased appetite occurs when you have a reduced desire to eat.

It may also be known as a poor appetite or loss of appetite. The medical

term for this is anorexia. A wide variety of conditions can cause your

appetite to decrease. These range from mental conditions to physical

illnesses.

Appetite Enhancer

Appetite enhancers are formulated to help the body promote

appetite and weight gain with unintentional weight loss or poor food
 16

intake. One of the specific concerns in elderly adults is low appetite.

Often there may be an underlying disease or condition that they may be

experiencing. Although many agents have been evaluated for their use as

appetite stimulants, the majority of the studies have focused specifically

on cancer associated cachexia/anorexia. Because of the increasing

evidence suggesting that specific physiological processes are responsible

for this syndrome, some of the aforementioned therapies may not be

effective for use in patients outside of the specific cancer population. As

previously noted, larger, more generalized studies would be extremely

useful to confirm the effectiveness of specific agents. The lack of pediatric-

specific dosing continues to make the use of these agents challenging at

best (Kathleen Gura, 2010).

In the US there are available appetite stimulant agents, examples

are megestrol, oxandrolone, and dronabinol. They are indicated for the

treatment for anorexia and to promote weight gain for some patients. The

relative evidences assess the appetite stimulant in patient. These drugs

show an effective treatment option for anorexia but use is limited by

adverse effects. The common adverse effects include. Gastrointestinal

upset, trouble in sleeping, mood changes and testicular failure. Therefore,

 17

the appetite stimulants may be used for the treatment for anorexia.

(Archer, 2014)

Acorus calamus

Sweet flag has a sword-shaped, long slender leaves which produces

a sweet, tangerine like scent when cut. There are a lot of therapeutic claims

attributed to its leaves and rhizomes which includes appetite stimulant

property, increase bowel movement, anti-oxidant and anti-spasmodic

(Mukherjee, et. al 2007).In most experimental studies, the parts used are

the leaves, rhizomes and stem of the plant. Phytochemical studies have

shown positive result in glycosides, saponins, polyphenolic compounds,

flavonoids, tannins, mucilage and volatile oil. (Imam,et.al 2013)

There were few studies done on both hydrophobic and hydrophilic

extracts of A. calamus. The findings of other studies show that the major

chemical constituents of Acorus calamus extract were alpha and beta

asarone. Its bioactive compounds were also found that it is responsible to

its biological activities such antioxidant and antibacterial property. Other

than beta and alpha asarone, hydrocarbon compounds are also present in

acoruscalamus such phenols and flavonoids because of the presence of

hydroxyl group. (Li and Wah, 2017)

 18

Pharmacological activities of Acorus calamus such as antioxidant

activity, antimicrobial activity, and insecticide activity that can be found

in the leaves and rhizomes of a plant sample had been already proven.

Moreover the researcher concluded that even it has different

pharmacological studies or activities, there are still a lot of clinical

experiments that can be conducted which has more activities that shows

a strong result or evidence of its different properties, as well as to conserve

Acorus calamus plant and prevent its extinction. (Balakumbahan and

Rajamani, 2010).Since ancient times, Acoruscalamus has been widely

used in various systems of medicines including Ayurveda, Unani, Siddha,

Chinese medicine, etc. for the treatment of different conditions such as

nervous disorders, appetite loss, bronchitis, chest pain, diarrhea, GIT

disorders, flatulence and vascular disorders. With variety of constituents,

alpha and beta-asarone are the predominant bioactive components. At

high doses, genotoxicity and mutagenecity of such components is

reported. In the previous review, an attempt has been made to discover

and explore traditional uses and pharmacological properties of such plant.

(Rajput and Tonge, 2014.)

Acorus calamus is commonly found in river bottoms and spongy

grounds. It is a semi-aquatic plant throughout the temperate and

 19

subtemperate parts of the region. Hence, it also contains essential oils

which are a unique source of oxygenated sesquiterpenes. Due to its

increase in demand in the public market, sweet flag was reported as

endangered.Such useful medicinal plant, Acorus calamus, gave asset in

various fields of medicine. Acorus calamus is used through wide area in

the pharmaceutical and also in bioethical scale for its great potency and

defense system. This review states the different pharmacological activity of

such plant with distinctive solvent or extract. This herb is widely used from

the Ancient and Vedic periods for the reason of its power of rejuvenation

of nervous system, brain and normalizing the appetite (Tiwari and

Chandra, 2017).

Plant home remedies have been an increase in popularity for the

recent years. In many different countries, herbal plants have been

adapting to these alternative medicines. These medicines include

Ayurvedic, Homeopathic, Unani and other traditional medicinal practices.

Plants are being used to cure various diseases. Active constituents have

been isolated and studies from these plants to yield treatment. Most of the

drugs that are available in the market today were originally made from

plants or have been originated from their constituents. Phyto-chemical

studies help to study different applications of medicinal plants.

 20

Acoruscalamus was also known to many early American settlers and used

for a number of folk remedies. (Avadhani and Selveraj, 2013).

A.calamus combined with Polygala root is used to maintain

intellectual and mental health for the elderly (Hou and Jin, 2008). It is also

used for depressed psychosis and dementia. Other indications include

confusion of the mind, forgetfulness, loss of consciousness, anorexia and

epilepsy (Howes and Houghton, 2003); (Amit, 2013).

Significant weight loss can occur and long-term nutrition may be

affected, leading to weakness and fatigue that can not only inhibit daily

activities but can also lead to malnutrition, consequently increasing the

risk of morbidity and mortality. Children with intestinal failure, those who

have undergone a transplant, and those who have cancer or cystic fibrosis

are often at risk

Acorus calamus is known in the Philippines from the past years up

to the present. According on some literatures there are many plants that

can be considered as appetite enhancers. It is a perennial aromatic herb,

with creeping, branching and rhizomatous rootstock. Rhizomes are

compactly arranged annular rings with numerous rootlets. Leaves arising

from rhizome are linear, distichous, 25 to 50 centimeters long, 1 to 1.5

 21

centimeters wide, with waved margins and stout midribs, base of leaf

sheathed, clasping to each other. Peduncle is compressed. Spathe is green,

elongated, and similar in shape to the leaves. Spadix is 3 to 5 centimeters

long, and 1 centimeter or less in diameter, and bears many flowers.

Flowers are small, concave in shape. Sepals are 6, stamens 6, rarely

flowering in the Philippines. Fruits are berries, turbinate, prismatic, with

pyramidal tops, about 0.2 centimeter diameter.

This plant can be seen along streams in mountains, creeks other

moist places with running water, on boulders, etc., at low and medium

altitude in Luzon (Laguna). It is also found in Bontoc and Benguet

provinces in swamps, at an altitude of about 1,300 meters, as a

naturalized element and occurs in the temperate to subtemperate regions

of Eurasia and the Americas.

The parts used in most of the experimental studies are the leaves,

roots (rhizomes) and stem of the plant. In Traditional medicine rhizomes

are mostly used. Decoction of dried roots and leaves to a pint of boiling

water were used for dyspepsia, gastritis, indigestion, stomach pains,

diarrhea, and asthma.Leaves are used as insect repellant. Rhizomes are

use as masticatory for toothache, stimulant and tonic as well as nervine

sedative; in large doses, nauseant and emetic. Calamus oil is used as

 22

carminative and also used as digestive and to increase the appetite,

furthermore it can be used for scenting snuff and for flavoring alcoholic

beverages (Godofredo U. Stuart Jr., M.D, 2015)

Sweet flag (Acorus calamus), member of the family Araceae, is a

semi-aquatic component of aquatic habitats throughout the temperate to

sub-temperate regions of Eurasia and the Americas. The plant has a rich

ethnobotanical history dating back possibly to the time of Moses in the Old

Testament of the Bible and in early Greek and Roman medicine. Sweet

flag, known to be indigenous to India that has been valued for its rhizome

and fragrant oils which have been used medicinally, in alcoholic beverages,

as a fragrant essence in perfumes and oils, and for insecticidal properties.

Current study investigates sweet flag’s value as an insecticidal,

antibacterial and antifungal agent. (Motley, 1994).

The rhizome is source of an essential oil, which is a oxygenated

sesquiterpenes of great structural variety and responsible for significant

antibacterial, antifungal, and insecticidal properties. Apart from the

terpenes, a few commonly occurring steroids and xanthones had also been

reported. It was also used as a constituent of polyherbal formulation,

namely herbal multiaction skin gel, which was found to be effective against

a variety of specific and nonspecific dermatitis and maggot wounds. The

 23

ethanolic extract of the rhizome is used as antiulcer agent that inhibits

gastric secretion and protects gastroduodenal mucosa against the injuries

caused by pyloric ligation in rats . β-asarone isolated from the volatile

fraction of A. calamus was found to exhibit anticarcinogenic action on

ED50 of SGC cells at a dose level of 25 mcg/ml. Extractives of A. calamus

rhizome for calamus oil are widely used now in pharmaceuticals and

traditional systems of medicines for a number of ailments and in perfumes.

Thus, it is a common item in the international drug market and the raw

material is originated from the natural wild populations. Due to the

indiscriminate collection of the plant materials from forest, its wild

population levels are rapidly decreasing.

Alternative and complementary medicines have gained an increase

in popularity in the recent years. In other countries, a large number of

individuals are engaging with these medicines to complement conventional

allopathic medicines. These complementary medicines include Ayurvedic,

Homeopathic, Unani and other traditional medicinal practices. An ethno-

pharmacological based method has provided new potentials of identifying

new drugs from plants related to many disorders. A large number of drugs

currently available in allopathic medicine were originally isolated from

plants, or have originated from compounds isolated from plants. Phyto-

 24

chemical studies help to understand the applications of medicinal plants.

An attempt has been made in this review to understand the applications

of an Acorus calamus. The rhizome, roots and leaves of the plant have

been used for insecticidal, antifungal, antibacterial and allellopathic

properties (MythiliAvadhani, Immanuel Selvaraj, 2013).

Other study reports phenolic content and antioxidant activities of

methanol extracts of Acorus calamus leaves and rhizomes. The activity of

A. calamus leaf and rhizome extracts were comparable to the standard

antioxidants used. Finally, the study revealed correlation between

phenolics contents and antioxidant activities of A. calamus leaf and

rhizome extracts (Asha Devi 2011).

Acorus calamus (Sweet flag) has a history of use and has many

traditional and ethnomedicinal applications. In ancient times, it has been

used in various systems of medicines such as Ayurveda, Unani, Siddha,

Chinese medicine, etc. for the treatment of various aliments like nervous

disorders, appetite loss, bronchitis, chest pain, colic, cramps, diarrhea,

digestive disorders, flatulence, gas, indigestion, rheumatism, sedative,

cough, fever, bronchitis, inflammation, depression, tumors, hemorrhoids,

skin diseases, numbness, general debility and vascular disorders. Various

therapeutic potentials of this plant have been attributed to its rhizome. A

 25

number of active constituents from leaves, rhizomes and essential oils of

A. calamus have been isolated and characterized constituents, alpha and

beta-asarone are the predominant bioactive components. Though A.

calamus has been used since ancient times, many of its uses are yet to be

scientifically validated. In the present review an attempt has been made to

explore traditional uses and pharmacological properties of A. calamus.

(Sandeep Rajput and Madan Tonge, 2014.)

While rhizomes of Acorus calamus, having high ADH and low LDH

activities, accumulate mainly ethanol, tubers of Solanum tuberosum tend

towards lactic acid fermentation. (Mirjam Sieber, 1991).

The rhizomes of A. calamus are used in the treatment of a wide

variety of human diseases. Administration of the 50% ethanolic extract

(100 and 200 mg/kg) as well as saponins (10 mg/kg) isolated from the

extract demonstrated significant hypolipidemic activity. On the contrary,

the aqueous extract showed hypolipidemic activity only at a dose of 200

mg/kg (Reshma Parab 2002).

Acorus calamus Linn. (family Araceae) commonly known as “sweet

flag” or Waan-Nam, is a well-known medicinal plant. The rhizomes were

utilized extensively by the Chinese, Indians and American Indians as well

as by other cultures, and many of these uses continue to this day (Motley,
 26

1994) including in Thai traditional medicine (Anonymous, 2000). The

rhizomes are considered to possess anti-spasmodic, carminative and

anthelmintic properties and also used for treatment of epilepsy, mental

ailments, chronic diarrhea, dysentery, bronchial catarrh, intermittent

fevers and tumors. It is listed as an insecticide, an antifungal agent, an

antibacterial agent and a fish toxin (Anonymous, 1975). As part of a search

for antimicrobial compounds from plants at the Natural Products Research

Unit, Prince of Songkla University, Thailand, we found that one of

partiallypurified fraction obtained from the crude methanol extract of A.

calamus rhizomes showed antimicrobial activity.

Acorus calamus is a useful medicinal plant which gave benefit in

different fields of medicines. This review deals with the different

pharmacological activity of plant with different extract or solvents. This

herb stimulates the power of intelligence and self-expression in humans.

This herb is generally used from the Ancient and Vedic periods due to its

wonderful power of rejuvenation of brain, nervous system and normalizing

the appetite. Acorus calamus is widely used in the pharmaceutical as well

as on biotechnological scale because it has great potency and defense

system. Acorus calamus has many wide varieties which were used in

different studies possesses anti-inflammatory, anti-spasmodic, anti-

 27

protective and anti-hepatotoxic activities. Article also deals with various

marketed product of Acorus calamus (Rupali Singh, 2011).

Acorus calamus otherwise known as Sweet flag, belongs to the family

Acoraceae. Sweet flag is probably a native of China and India. It is use as

a medicinal plant dates back to Egypt, Greek and Roman times. Sweet flag

was distributed from its native range by rhizomes through trade and

commerce, and arrived in Europe in the 16th Century. In the Malesian

region, it is considered as naturalized and not truly wild. It is found in

many parts of Indonesia, Malaysia, Papua New Guinea, Philippines and

outside Malesia in Indo-China and Thailand. The most active constituents

of Acorus calamus plant are alpha and beta-asarone. The plant has also

been reported for the presence of glycoside, flavonoids, saponins, tannins,

polyphenolic compounds, asarone, sesquiterpenes, volatile oil and bitter

principle. (Nguyen Van Dzu 2016.)

Asarones, stimulates salivary and gastric secretions, relieve

intestinal cramping. In vitro experiments have shown improvement of

alcohol-induced impairment of learning and memory abilities. The 2

stereoisomers α-asarone (trans-isoasarone) and β-asarone(cis-isoasarone)

are reported to have psychoactive effects. This has been attributed to the

structure, which is similar to that of amphetamines and of the

 28

hallucinogenic compound mescaline. Asarone has a relaxing effect on

smooth muscle tissue, and the oil has been found to induce spasmolytic

activity in rabbit intestines, aortae and uteri. Experiments with guinea-pig

ilia have demonstrated that the cortex of the rhizome acts as an

antispasmodic agent. In tests on laboratory animals it also showed

antihistamine, anticonvulsant and antipyretic activity. It has also been

found to act as neuroleptic enhancer, central nervous system depressant,

carcinogen, hypothermic, hypotensive, analgesic, anti-inflammatory,

bronchodilator, respiration inhibitor, hepatotoxin and antifibrillatory.

The rhizomes of sweet flag have been used extensively in traditional

medicine by Chinese, Indians, American Indians and others, and are still

used in many regions. In Roman and Arabic civilizations aphrodisiac

properties were attributed to the rhizome and it was used in North America

and Europe as a panacea; in India, sweet flag has been an important

medicinal aid for stomach complaints and colic in children. Since ancient

times it has been reputed for its stimulant digestive virtues. In India, the

rhizomes are traditionally used in an infusion to treat diarrhea, dysentery,

atonic dyspepsia and asthma, and for their carminative, expectorant,

nauseant, antispasmodic, stomachic, vermifuge, sedative and emetic

properties. In Vietnam, sweet flag is used to treat respiratory disorders

 29

(asthma, inflammation), rheumatism, remittent fevers, snake bites and as

sedative. In Indonesia and Malaysia, the rhizomes are usually used

externally to treat inflammation, rheumatism, lumbago and skin diseases,

and internally after childbirth. In Java, sweet flag is an ingredient of

certain "jamus", and in the Philippines it is used as a masticatory against

toothache and as a stimulant, carminative and anti-rheumatic. In Papua

New Guinea, the leaves of sweet flag are taken as a tonic, and chewed to

relieve toothache. The crushed rhizome is rubbed into the hair to kill lice.

In Brunei, sweet flag is used to treat gastritis and diarrhea and also as a

poison antidote. In Thailand, the rhizomes are used as carminative,

analgesic, anthelmintic, and to treat diarrhea and dysentery. In Japan,

Acorus oil is used as bathing agent, considered to be effective against skin

diseases and to improve blood circulation. In the Unani (Greco-Arab)

system of medicine, sweet flag is used to treat cardiovascular diseases. In

Vietnam, a dose of 2-5 g/day is administered in decoction. In modern

phytotherapy, sweet flag rhizomes ("calami rhizoma") can, on the basis of

their constituents, be called a bitter aromatic. This is principally used as

a stomachic and carminative (internally) and externally as a rubefacient

and in the treatment of seborrhoea (as a bath).

 30

Sweet flag is an amazingly versatile addition to the herbal medicine

cabinet. Historically, it has been used all over the world for many different

purposes. It was listed in the US National Formulary for medicinal use on

humans until 1950. In traditional Chinese medicine it is used to treat

deafness, dizziness and epilepsy. In Ayurvedic medicine it is valued as a

rejuvenator for the brain and nervous system, and as a remedy for

digestive disorders. The Dakotas used it to treat diabetes.

Traditional western herbal medicine uses Sweet Flag as a digestive

bitter and carminative. It perks up the appetite. It stimulates digestion by

promoting the production of bile, stimulating peristalsis of the intestines,

and getting the stomach churning and ready for food. This is what it does

in proper doses. Traditionally, the root was chewed, though a suitable

infusion can be made by soaking it at the top of a closed jar of cold water

overnight. Hot water infusions and alcoholic tinctures are dreadful, will

usually settle heartburn, nausea and gastritis prepared this way.

Sweet flag was included in many of the early herbals and has a rich

history in the Chinese and Indian cultures. Few plants have gained such

various used in diverse cultures. Recent years, ethno-botanical and

traditional uses of natural compounds, especially of plant origin received

much attention as they are well tested for their efficacy and are generally
 31

believed to be safe for human use. It is an effective approach in the search

of new molecules for management of various diseases. Thorough screening

of literature available on sweet flag has been found to be a popular remedy

among many cultures including Unani, Ayurvedic and Chinese

practitioners for treatment of various ailments. In the phytochemical and

biological activities of sweet flag provides detailed evidence for the use of

this plant in different medicines. Regarding the rich background of

biological activities of sweet flag, it seems that there are still a large

number of unaccomplished investigations, particularly clinical trials of

sweet flag and its bioactive compounds.

Major chemical constituents identified in sweet flag are alpha and

beta-asarones along with other constituents such as caryophyllene,

isoasarone, methyl isoeugenol and safrol are also responsible for medicinal

activity but most of the biological actions of sweet flag have been attributed

to presence alpha and beta-asarones. In a recent finding beta-asarone was

shown to possess ameliorative potential in cognitive impairment thereby

suppressing the neuronal apoptosis. Moreover, alpha-asarone is also

noted reduce the excitatory action by stimulation of glutamate uptake and

inhibition of excitatory neurotransmitter transporter mediated current.

(Imam, 2013).
 32

As an appetite stimulant it is the ideal remedy for anorexia, because

of its antianxiety effect the bitter/carminative effects get the digestive

system going, and that makes the person feel hungry. Acorus calamus is

also known for its calming and centering effect that is why it can be both

stimulant and sedative, it puts your energy into balance and gets you

energetically resonating as a whole. For this reason, it is almost without

equal as a treatment for panic and anxiety attacks. Have the person chew

on Sweet flag and breathe deeply, fully and slowly and the anxiety and

panic will fade. Incidentally, its antianxiety effects are the reason why it

was used for quitting smoking, not because it causes a 'distaste for

Tobacco'.

Research findings suggest that sweet flag may have applications for

several ailments for which it has a historical record of use. It may still have

beneficial applications in modern medicine. Its use in perfumes, foods and

beverages is limited because of the carcinogenic phenylpropane derivative

β-asarone present in the extract, but the presence of diploid populations

which seem to be free from this component offers new opportunities for

more extensive use in the future after selection. Sweet flag might have good

prospects for commercial exploitation as a pesticide of plant

origin.(Nguyen Van Dzu, 2016).

 33

For ages, Acorus calamus rhizome has been used for the treatment

of various ailments in traditional and folklore medicine. The plant exhibits

polyploidy, and the composition of the essential oil obtained from the plant

rhizome depends on the karyotype. Of the phytoconstituents reported

from Acorus. rhizomes, α.-asarone and β.-asarone are the predominant

bioactive constituents. Various pharmacological activities of Acorus

calamus rhizome such as sedative, CNS depressant, behavior modifying,

anticonvulsant, antispasmodic, cardiovascular, hypolipidemic,

immunosuppressive, anti-inflammatory, cytoprotective, antioxidant,

antidiarrheal, antimicrobial, anthelmintic, insecticidal, and diuretic have

been reported by different workers. Together with this, some untoward

effects of Acorus calamus rhizome and its constituents α.-asarone and β.-

asarone such as genotoxicity and mutagenicity have also been reported,

which limits its therapeutic usage. Thus, this rhizome is well-known as a

CNS active herb from Ayurvedic tradition and requires further research to

establish the molecular mechanisms of evaluate its activity. (Mukherjee,

et.al., 2008)

It is necessary to have a good health every single day, by doing a lot

of works and losing up energy, people must not forget to have proper diet

and build up supplementation, thus it is very important to consider fuels

 34

for the body most especially to those who struggled in such loss of appetite

and weak food intake. This study parallel with the literatures stated, the

researchers will easily find its way to develop the underlying component of

Acorus calamus leaves for its appetite enhancing property.

Chapter 3

Methods and Procedure

This chapter discusses the procedures, methods, and preparation of

plant sample used to determine the appetite enhancer property from the

extract of sweet flags leaves (Acorus calamus L.) on Sprague Dawley rats.

Setting of the Study

The leaves of sweet flag scientifically known as Acorus calamus were

selectively collected from San Antonio, Nueva Ecija, Philippines, during the

month of July, 2018. The samples were validated at Bureau of Plant

Industry in San Andres St, Malate, Manila for validation and identified as

Acorus calamus with a reference code of PLT-ID-CRPSD-1377-18.

The preparation and experimental works in this research were

conducted at the science laboratory of Centro Escolar University, Malolos,

Bulacan.

Methods of Research
 35

The researchers used experimental method to determine the

appetite enhancer property of the methanolic extract of sweet flags (Acorus

calamus).

Collection and Preparation

The leaves were collected and washed multiple times with water and

air dried, cut into small pieces and powdered using mechanical blender.

About 50 grams were macerated exhaustively and successively with n-

hexane, methanol, ethyl acetate and 50% methanol for 24 hours. Then

filtered and evaporated into dryness over rotavap. The collected residue

was used for the determination of constituents and phytochemical

evaluation.
 36

Collection and preparation of leaves of Acorus calamus

Serial Extraction
n-Hexane Ethyl acetate Methanol 50% Methanol

Rotatory evaporation and Percentage Yield Determination

Physical evaluation and Phytochemical evaluation

Organoleptic Glycosides

Flavonoids

Saponins

Turpenoids

Tannins

Alkaloids

Only that extract that gave the most number of positive results in Flavonoids test
Biological testing

Efficacy
 37

Dosage formulation Toxicity

Organoleptic Evaluation

Figure 1

Flow Chart of the Method

A small amount of the crude extract of sweet flag was observed for the

color, odor, taste and appearance of the sample plant.

Phytochemical Screening

Phytochemical screening is to identify the medicinally active

substances in a given plant. This is done to specifically determine what

component is responsible for the appetite enhancing propert of A.calamus.

Test for Flavonoids

Shinoda Test or Magnesium hydrochloride reduction test:

To the test solution, add small number of pieces of magnesium

ribbon and concentrated hydrochloric acid dropwise. After few minutes,

pink scarlet color appears indicating the presence of flavonoids.

Ferric chloride test:

 38

To the test solution, add few drops of ferric chloride solution. To

show the presence of flavonoids, intense green color was formed.

Bate-Smith and Metcalf test for leucoanthocyanins:

To the test sample, 0.5mL of concentrated HCl was added and

subjected in a water bath for 15 minutes. Observe for any color changes.

The presence of Leucoanthocyanins is indicated by strong red or violet

color.

Alkaline test for flavonoids:

To the test solution, 5 drops of NaOH was added followed by 2mL of

10% HCl. The positive result is indicated by a yellow solution to colorless.

Lead acetate for flavonoids:

A few drops of lead acetate were added to the test solution. The

presence of Flavonoids is indicated by a yellow precipitate formation.

Test for Terpenoids

Libermann-Burchard test:

Crude extract treated with several drops of acetic anhydride, boil

and cool. With the test tube tilted, concentrated sulfuric acid is added the
 39

by the side of the test tube, shows brown ring at the junction of two layer

and the upper layer turns green which shows the presence of sterols and

formation of deep red color indicate the triterpenoids.

Salkowski’s test

With the crude extract treated chloroform and with several drops of

concentrated sulfuric acid and mixed, shaken well and allow to stand for

some time. The indication that there is presence of sterol will appear red

color in the lower layer and formation of yellow colored lower layer indicate

the presence of triterpenoid.

Test for Carbohydrate

Molisch test:

In a test tube treat the 2ml solution with several drops of alcoholic

a*napthol solution. By the side of the test tube, 1 ml of concentrated

sulphuric acid was carefully added. The indication of presence of

carbohydrates was resulted with the formation of violet ring at the

junction.

Fehling’s test:
 40

With the same volume of Fehling A and Fehling B solution are

mixed together and added several drops of sample and boiled, indication

of presence of reducing sugar will result to brick red precipitate.

Test for Tannins

Ferric chloride test:

To this solution, few amount of extract was dissolved in distilled

water ad 2ml of 5% ferric chloride solution was added.

Lead acetate test:

With few amounts of extract, several drops of lead acetate

solution were added. Presence of tannins will result to the formation of

precipitate.

Test for Saponins

Foam test:

The extract was diluted with distilled water and for 15 minutes

was shaken in graduated cylinder. Indication of presence of saponins will

result to the formation of layer of foam.

 41

Test for Glycoside

Borntrager’s test:

To the 3ml test solution, diluted sulphuric acid was added and

for 5 minutes was boiled and then filtered. After, equal volume of benzene

was added to the cold filtrate and mixed well. The organic layer was

separated and ammonia was added to it. The presence of anthraquinone

glycoside was resulted to the formation of pink to red color in ammoniacal

layer.

Keller- Killiani test:

To the 2ml of test solution in a test tube, 3ml of glacial acetic

acid and a drop of 5% ferric chloride were added. With the test tube tilted,

carefully add 0.5 ml of concentrated sulphuric acid by the side of the test

tube. The presence of Cardiac glycosides was resulted to the formation of

blue color in the acetic acid layer.

Test for Alkaloids

Mayer’s reagent

Crude extract was dissolved with 2N HCl and then filtered.

Then the filtrate solution will be treated with several drops of mayer’s
 42

reagent. An appearance of cream precipitate will indicate the presence of

alkaloids.

Dragendorff’s reagent

Crude extract was dissolved with 2N HCl and then filtered.

Then the filtrate solution will be t reated with several drops of dragendorff’s

reagent. An appearance of orange brown precipitate will indicate the

presence of alkaloids.

Wagner’s reagent

Crude extract was dissolved with 2N HCl and then filtered.

Then the filtrate solution will be treated with several drops of wagner’s

reagent. An appearance of reddish brown precipitate will indicate the

presence of alkaloids (Vaghasiya; 2011).

Toxicity Testing

The test substance is administered in a single dose by gavage.

Animals should be fasted prior to dosing (e.g. with the rat, food but not

water should be withheld over-night; with the mouse, food but not water
 43

should be withheld for 3-4 hours). Following the period of fasting, the

animals should be weighed and the test substance administered. After the

substance has been administered, food may be withheld for a further 3-4

hours in rats or 1-2 hours in mice. Where a dose is administered in

fractions over a period of time, it may be necessary to provide the animals

with food and water depending on the length of the period.

Individual weights of animals should be determined shortly before

the test substance is administered and at least weekly thereafter. Weight

changes should be calculated and recorded. At the end of the test surviving

animals are weighed and then humanely killed.

Efficacy Testing

The component of A. calamus after indetifying will be subjected to

efficacy testing, this is to determine whether the plant sample is capable

of giving the target appetite enhancing property to Spague Dawley rats.

Preparation of the test animals

About 25 Sprague Dawley rats were used in the study. Fasting of

rats from food and water for 24 hours before starting the experiment.

Preparation of the plant extract

 44

About 1g crude extract were dissolved and titrated with 1 ml of

distilled watrer, and is administered to rats by oral route.

Efficacy Test for appetite enhancer activity

The rats are randomly divided and selected into 5 groups: group 1

will be treated with Dibencozide 3mg capsule (Positive control), group 2

untreated- distilled water, group 3 treated with extract containing 50

mg/kg test sample group 4 treated with extract containing 100 mg/kg test

sample and group 5 treated with extract containing 150 mg/kg test sample

Dibencozide with a brand name Heraclene was administered

(3mg/kg) in Group I as positive control. The three concentrations

(50mg/kg, 100mg/kg, 150mg/kg) were administered to the remaining

groups (Group 3,4,5) for 7 consecutive days.

The rats in the vehicle group received distilled water and 200g food

every day and those in the untreated group received no intervention. The

food consumption in grams by each cage of 5 rats was measured in 24

hours. The weight comparison will be based on the initial testing up to the

last day of observation (Avicenna, et. al 2013).

 45

Formulation of Dosage Form

Simple syrup was used as a vehicle of AcL methanolic extract. The

components of syrup are sucrose, distilled water, and the active

ingredient.

Physical Evaluation of the Prepared syrup

pH measurement, viscosity testing and physical evaluation were

conducted and results obtained from physical evaluation includes no

crystallization formation, odor, color, and palatability.

Statistical Treatment

The statistical method used in the research was the bartlett’s test

for equal variances. All the raw data used in the research was presented

for the analysis of significant differences within the group of rats during

the process of the treatment.

 46

Chapter 4

Presentation, Analysis and Interpretation of Data

This chapter dealt with the results obtained from the preliminary

screening, physical and chemical evaluation and biological testing of

flavonoids. This also includes the interpretation and analysis of data

collected.

Physicochemical Evaluation

Organoleptic Test

All the extracts of Acorus calamus have the same physical properties

as brown color, viscous, and semi-solid with caramel like odor.

Table 1

Organoleptic Test

Solvent Color Odor Total appearance

n-Hexane Brown Caramel-like odor Viscous, semi-solid

Ethyl acetate Brown Caramel-like odor Viscous, semi-solid

50% methanol Brown Caramel-like odor Viscous, semi-solid

 47

Methanol Brown Caramel-like odor Viscous, semi-solid

Table 2

Computation of percentage yield

Solvent n-Hexane Ethyl Acetate 50% Methanol

Methanol

Trial 1 1.32% 0.90% 2.13% 2.88%

Trial 2 1.18% 0.78% 0.97% 0.97%

Trial 3 0.58% 0.22% 2.36% 5.36%

Average 1.03% 0.63% 1.82% 3.07%

Methanol showed the highest percentage yield average of

3.07% among the chemicals used in the chemical evaluation of the

flavonoidal content of Acorus calamus extract.

Preliminary Screening

In order to identify the flavonoidal content of Acorus calamus extract,

it undergone different chemical tests such as Shinoda test with the

expected result of pink scarlet odor, Ferric chloride test with the expected

result of intense green color, Bate-Smith and Metcalf test for

 48

Leucoanthocyanins with the expected result of strong red or violet color,

Alkaline test with the expected result of yellow solution to colorless and

Lead Acetate test with the expected result of yellow precipitate formation.

All extracts undergone with such tests gave positive results.

50% methanol was used as the solvent for the flavonoidal extraction

of lubigan leaves because it yielded most number of positive result during

phytochemical screening.

The dried Lubigan leaves were extracted using 50% methanol as the

solvent. The percentage yield was 2.88%.

Limit Test

Limit test was conducted in 5 Swiss mice using 2 ml Lubigan leaves

methanolic extract. All the mice survived the limit test. Data obtained from

this test is an important factor in drawing important considerations’

throughout the experiment

Efficacy Testing

Biological Test for Appetite Enhancer Property of Acorus calamus leaves

The appetite enhancer activity was determined by getting the

baseline of each rats and controlling the diet. The study used Sprague
 49

dawley rats as test animals with either male or female sex. The food

were weighed before it was given and collected the remaining food after

8 hours then weighed again. The rats were given the corresponding

dose of drugs and extracts that was computed. Each rats were

monitored after 7 days administration.

The data obtained from getting the baseline and controlling the

diet for testing the appetite enhancer effect of the Flavonoid extract can

be seen in the appendices.

WEIGHT INCREASE IN
PERCENTAGE
150mg/kg 9.91
100mg/kg 9.3
50mg/kg 5.67
NEGATIVE
0.87
POSITIVE 5.13

Figure 2

Weight Increase in Percentage

The graph represents the increase in weight of rats in percentage

and the concentration exhibited the most effective appetite enhancer

property is 150 mg/kg.

 50

Statistical Treatment

Based on statistical analysis performed there was no significant

difference between different concentrations which is 50 mg/kg, 100 mg/kg

and 150mg/kg compared to the positive control (Heraclene Forte 2 mg) but

then there was a significant difference between the concentrations

compared to the negative control (untreated distilled water). 150 mg/kg

concentration showed the highest number of increase in weight of each

rats therefore the researchers conclude that the said concentration was

the most effective.

Dosage Formulation

Preparation of the methanolic extract of Acorus calamus

The dried leaves ware macerated in 50% methanol for 48 hours and

were subjected to rotatory evaporation. Prior to that, the researchers

prepared simple syrup through agitation with the aid of heat with the

following ingredients such as sucrose (80 g) and distilled water (qs.ad). The

distilled water was boiled in the temperature of 100-112 ℃, and was

removed from the heat, the sucrose was added gradually with occasional
 51

stirring until dissolved and viscous. The methanolic crude extract of

Acorus calamus was mixed with the simple syrup until dissolved.

Chapter 5

Summary, Conclusion and Recommendation

This chapter summarizes the results in the investigation of

Appetite Enhancer Activity of theFlavonoids from the leaves of lubigan

scientifically known as Acorus calamus.

The conclusions and recommendations are also presented in this

chapter.

Summary of Findings

Based on the following results of the study, the summary of the findings

are hereby presented:

1. In extracting the plant constituent of lubigan leaves, the

researchers used serial extraction using different solvents such as

n-hexane, methanol, 50% methanol and ethyl acetate and the

percentage yields are as follows, Trial 1- Ethyl acetate: 0.90%, n-

Hexane: 1.32%, methanol 2.88% and 50% methanol: 2.13%, trial 2-

Ethyl acetate: 0.78%, n-Hexane:1.18%, methanol:0.97% and 50%

 52

methanol: 0.97%, trial 3- Ethyl acetate:0.22%, n-Hexane:0.58%,

methanol: 5.36% and 50% methanol: 2.36%.

2. Lubigan has a long-slender leaves which can be found at muddy

places or river side. After several times of maceration and was

subjected to incipient dryness, lubigan extract was brown in color,

has a pungent odor and liquid in its physical appearance. The

solvent which has the highest percentage yield was methanol but

then the most number of positive result in determining the

flavonoidal constituent was 50% methanol.

3. The methanolic extract of the lubigan leaves was prepared again

for toxicity testing. The rats were weighed and their toxic dose was

computed and administered after their 12 hours fasting. upon the

period of observation and trial, 100% survival was obtained,

therefore the lubigan leaves do not have a high range of toxicity.

4. The researchers used the baseline to gather the rats’ minimum

food consumption for a week, the biological testing was done the

week after given the same food consumption on the baseline

conducted. The rats were observed for 2 consecutive weeks and was

weight again. The most effective dose for the appetite enhancing

property of the lubigan leaves is 150mg/kg, because it showed

 53

higher advancement although the 2 remaining concentration also

improved.

5. The use of liquid formulation is the most common due to its ease

of administration, further the absorption of such is more rapid and

efficient than a capsule or a tablet. Therefore the researchers

prepared syrup dosage form. It was done by agitation with the use

of heat and incorporation of the active ingredient, the final

preparation undergone physical tests which includes palatability,

taste (sweet), color (brown), odor (sweet smell) crystallization

formation that does not appear during the process and shelf life. The

pH was also tested by a ph paper and pH meter which showed that

the syrup has a pH of 7.

Conclusions

All doses administered to the rats are effective, yet the most effective

appetite enhancer property of the methanolic extract from lubigan leaves

(Acorus calamus) on Sprague dawley rats is 150 mg/kg.

Recommendations

Based on the study conducted the following recommendations for

further studies are hereby presented:

 54

1. Conduct further pharmacological studies by the use of other parts of

the lubigan plants for the determination of other therapeutic activity that

may be present within the plant.

2. Enhancement of the product formulated to the most convenient dosage

form and unexpensive drug.

 55

BIBLIOGRAPHY

Alexandra, K., (2014), Malnutrition in the Philippines - perhaps a Double Burden. Journal
für Ernährungsmedizin, 11(1), 24.

Alok, R., Paras, J., Binod, S., Pallavi, S., Shama, H.P., (2016) Acorus Calamus L.: An
Insight Review of Botany, Chemistry, Medicinal Uses and Cultural Practice.
Journal of Chemical, Biological and Physical Sciences, 6(3), 1027-1045.

Balakumbahan, R., Rajamani, K., & Kumanan, K., (2010), Acorus calamus: An overview.
Historical Research Station, Tamilnadu Agricultural University, 4(25), 2740-2745.

Bown, D., (2004), Encyclopedia of Herbs & their Uses. Wonderful book on names, culture
(little on Zone info) and harvest, but generalities only for precise use information.
1(2), 45-50.

Devendra, A.K., (2011), Efficacy of a Polyherbal Appetite Stimulant in the Treatment of

Anorexia in Children. Indian Medical Gazette, 2011.

E. Engebretson., (2002), Chapter 1Herbal Medicine. An Introduction to Its History, Usage,

Regulation, Current Trends, and Research Needs, 3(4), 24-28.

Fawzi Mahomoodally, M., (2013). Traditional Medicines in Africa: An Appraisal of Ten

Potent African Medicinal Plants. Evidence-Based Complementary and Alternative
Medicine, 2013, 14.
 56

Francesco, L., Riccardo, C., Matteo, T., Anna Maria, M., Elena, O., Giulia, S., Alex, S., &
Emanuele, M., (2016), Anorexia of Aging: Risk Factors, Consequences, and
Potential Treatments. Nutrients,8(6), 1-10.

Glanz, K., & Rimer, B. K., (1995), Theory at a Glance: A Guide for Health Promotion
Practice. NIH Publication, 5(1), 95–3896.

Greta, W., Maria, R., Zoe, R., (2012), Contribution of social and family factors in anorexia
nervosa. Health Science Journal, 6(2), 257-269.

Immanuel S., (2013), Research Journal of Pharmaceutical, Biological and Chemical

Sciences 4(4), 598-610.

Janet, T., & June, A., (2013), Anorexia Nervosa. A Recovery Guide for Sufferers, Families
and Friends, Second edition.

Katherine L. Godwin, MD., (2018), Eating Disorders Can Mask Malnutrition. 1(3), 37-41.

Kho, S.L., Chan, S.W., (2016), Antioxidant and antibacterial activity of Acorus calamus.
L leaf and rhizome extract. Jurnal Gizi Klinik Indonesia. 13(4), 144-158.

Kumar, A.V., (2011), Medicinal Properties of Acorus calamus, Rajendra Institute of

Medical Sciences, Journal of Drug Delivery & Therapeutics, 3(3), 143-144.

Leann, L.B., & Jennifer, A.F., (1995), Appetite and Eating Behavior in Children. Pediatric
Clinics of North America, 42(4), 931-953.
 57

Lisa R. B., & Zachary A.K., (2016), A Spotlight on Appetite. Neuron Spotlight, 97(1), 729-
741.

Melissa, A., Carin, S., Bryan, L., & Gary, O., (2014), Agents Used as Appetite Stimulants
Drug Class Review, University of Utah College of Pharmacy, 2014

Mohsen, N., Maryam, K., & Michael, P., (2013), The effect of hydroalcoholic extract of
Coriandrum sativum on rat appetite. Avicenna Journal of Phytomedicine, 3(1), 91-
97.

Mythili, A., Immanuel, S., Rajasekharan, P.E., & Tharachand, C., (2013), The Sweetness
and Bitterness of Sweet Flag Acorus calamus L., Research Journal of
Pharmaceutical, Biological and Chemical Sciences. 4(2), 598.

Pulok, K.M., Venkatesan, K., Mainak, M., & Peter, J., (2007), Acorus calamus: Scientific
Validation of Ayurvedic Tradition from Natural Resources, Pharmaceutical
Biology Journal, 45(8), 651-666.

Raj Kumar, S.T., & Krishna, C., (2017), Intercropping of sweet flag (Acorus calamus L.)
with early and late maturing cultivars of rice (Oryza sativa L.). African journal
of agricultural research, 12(20), 1713-1726.

R. Balakumbahan, K. Rajamani, & K. Kumanan, (2010). Horticultural Research Station,

TamilNadu Agricultural University, Pechiparai, 629161. Tamilnadu, India. 2(5),
16-25.
 58

Rupali, S., Pramod, K.S., & Rishabha, M., (2011), Pharmacological Properties and
Ayurvedic Value of Indian Buch Plant (Acorus calamus): A Short Review.
Advances in Biological Research, 5(3), 145-154.

Sastry, J.L.N., Tathed, P.S., Rajiv, K., & Sasibhushan, V., (2016), Clinical evaluation of
efficacy and safety of appetizer syrup as appetite stimulant in children with non-
pathogenic anorexia, Indian Journal of Traditional Knowledge. 16(4), 700-705.

Schacter, D. T., Gilbert, D. T. Wegner, D. M., (2011), Psychology, New York, NY: Worth
Publishers. Appetite, 2(1), 97-102.

Suzanne Falck, MD., (2018), Supplements, Medications, and Lifestyle Changes to Help
Stimulate Appetite. 5(11), 56-59.

Verma, A., & Ahirwar, A.K., (2015), Phytochemical Investigations, Extraction and Thin
Layer Chromatography of Acorus Calamus Linn. International Journal of
Research Studies in Biosciences, 3(4), 18-22.
 59

APPENDIX D

The whole plant of Acorus calamus

 60

APPENDIX E

Results

Dose Compuations

POSITIVE CONTROL

BODY WEIGHT DOSE

RAT 1 266 g = 0.27 kg 0.15 mg x 0.27 kg = 10 ml x 0.0405 mg =

0.0405 mg 0.135 mL
1kg 3 mg

RAT 2 244 g = 0.24 kg 0.15 mg x 0.24kg = 10 mL x 0.036 mg

0.036 mg =0.12ml
1kg 3 mg

RAT 3 228.5 g = 0.23 0.15 mg x 0.23kg = 10 mL x 0.0345 mg

0.0345 mg =0.115 ml
kg 1kg 3 mg

RAT 4 220.5 g = 0.22 0.15 mg x 0.24 kg = 10 mL x 0.033 mg =0.11

kg 0.033 mg ml
1 kg 3 mg

RAT 5 232 g = 0.23 kg 0.15 mg x 0.23 kg = 10 mL x 0.0345 mg

0.0345 mg
1 kg =0.115 ml

3 mg
 61

50 mg/kg :

100 mg/kg :
BODY WEIGHT DOSE

RAT 1 276 g 50mg : 1000 g :: x : 276 g 107mg : 1 mL :: 13.8 mg :

x
= 13.8 mg = 0.1289 mL

RAT 2 275.5 g 107 mg : 1 mL :: 13.775 50 mg : 1000 g :: x : 276 g

mg : x
= 13.775 mg = 0.1287 mL

RAT 3 273.5 g 107 mg : 1 mL :: 13.675mg 50 mg : 1000 g :: x : 273.5

:x g
= 13.675 mg = 0.1278 mL

RAT 4 286.5 g 107 mg : 1 mL :: 14.325 50 mg : 1000 g :: x : 286.5

mg : x g
= 14.325 mg = 0.1338 mL

RAT 5 253.5 g 107 mg : 1 mL :: 12.670 50 mg : 1000 g :: x : 253.5

mg : x g
= 12.670 mg = 0.1184 mL

BODY WEIGHT DOSE

RAT 1 273.5 g 100 mg : 1000 g :: x : 27.35 mg x 1 mL = 0.26

273.5 g mL
= 27.35 mg 107 mg
 62

RAT 2 227 g 100 mg : 1000 g :: x : 227 22.7 mg x 1 mL = 0.21

g mL
= 22.7 mg 107 mg

RAT 3 232 g 100 mg : 1000 g :: x : 23.2mg x 1 mL = 0.22

232.5 g mL
= 23.2 mg 107 mg

RAT 4 266 g 100 mg : 1000 g :: x : 266 26.6 mg x 1 mL = 0.25

g mL
= 26.6 mg 107 mg

RAT 5 275.5 g 100 mg : 1000 g :: x : 27.55 mg x 1 mL = 0.26

275.5 g mL
107 mg
= 27.55 mg

150 mg/kg :

BODY WEIGHT DOSE

RAT 1 321 g 150 mg : 1000 g :: x : 321 48.15 mg x 1 mL =

g 0.45 mL
107 mg
= 48.15 mg

RAT 2 289 g 150 mg : 1000 g :: x : 289 43.35 mg x 1 mL =

g 0.41 mL
107 mg
= 43.35 mg
RAT 3 289.5 g 150 mg : 1000 g :: x : 43.43 mg x 1 mL =
289.5 g 0.41 mL
107 mg
= 43.43 mg
 63

RAT 4 242 g 150 mg : 1000 g :: x : 242 36.3 mg x 1 mL =

g 0.34 mL
= 36.3 mg 107 mg

RAT 5 257.5 g 150 mg : 1000 g :: x : 38.63 mg x 1 mL =

257.5 g 0.36 mL
= 38.63 mg 107 mg

Baseline weight before the oral administration

Day 1 Day2 Day3 Day4 Day5 Day6 Day7 Average

12/04/18 12/05/18 12/06/18 12/07/18 12/08/18 12/09/18 12/10/18 Weight

Negative 86g 83.5g 80.5g 45g 66g 14.5g 46.5g 60.29g

control

Positive 53g 90.5g 72.5g 37.5g 71.5g 45g 38g 58.29g

control

50mg/kg 111g 65g 95.5g 21.5g 43.5g 67.5g 78g 54.57g

100mg/kg 72g 52.5g 81g 66g 16g 78.5g 59.5g 60.79g

150mg/kg 76g 96.5g 113g 104.5g 111g 107g 91.5g 99.93g

=66.77g
 64

One week baseline weight comparison

Initial weight for baseline Dec End of baseline, day of

04,2018 administration

Dec 11,2018

POSTIVE POSTIVE

1). 263.5g 1). 266g

2). 239g 2). 244g

3). 224g 3). 228.5g

4). 217.5g 4). 220.5g

5). 229g 5). 232g

1.

NEGATIVE NEGATIVE

1). 264.5g 1). 266g

2). 272.5g 2). 276g

3). 257g 3). 261.5g

4). 245g 4). 248g

5). 270.5g 5). 275g

50mg/kg 50mg/kg

1). 273g 1). 276g

2). 270.5g 2). 275.5g

 65

3). 267.5g 3). 273g

4). 280.5g 4). 286.5g

5). 251g 5). 253.5

100mg/kg 100mg/kg

1). 2.69g 1). 273.5g

2). 220g 2). 227g

3). 228.5g 3). 232g

4). 261.5g 4). 266g

5). 269.5g 5). 275g

150mg/kg 150mg/kg

1). 318.5g 1). 321g

2). 256g 2). 259g

3). 2.85g 3). 289.5g

4). 238.5g 4). 242g

5). 254g 5). 257g

Food intake in grams during the biological testing

Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Average

12/11/18 12/12/18 12/13/18 12/14/18 12/15/18 12/16/18 12/17/18 Weight
 66

Negative 116g 112.5g 22g 76.5g 29.5g 53.5g 47.5g 65.36g

control

Positive 75.5g 66g 70.5g 62g 39g 50g 68.5g 61.64g

Control

50mg/kg 100.5g 72.5g 48.5g 40.5g 54.5g 61.5g 60g 62.57g

100mg/kg 123.5g 84g 71g 90.5g 72g 28.5g 64.5g 76.29g

150mg/kg 88.5g 78g 98g 101.5g 93.5g 72g 90.5g 88.86g

=70.94

Food intake in grams after administration (week 1)

Day 15 Day 16 Day 17 Day 18 Day 19 Day 20 Day 21 Average

12/18/18 12/19/18 12/20/18 12/21/18 12/22/18 12/23/18 12/24/18 Weight

Negative 118g 105g 98.5g 85.5g 101g 94g 79g 97.29g

Positive 127.5g 61.5g 56.5g 71.5g 60.5g 69g 50.5g 71g

50mg/kg 71g 47.5g 34g 95.5g 42.5g 102g 88.5g 68.71g

100mg/kg 80g 39.5g 52.2g 25g 32.5g 83.5g 79.5g 56.03g

150mg/kg 114g 102g 121.5g 117.5g 106.5g 59.5g 110.5g 104.5g

=79.50
 67

Food administration after administration (Week 2)

Day 22 Day 23 Day 24 Day 25 Day 26 Day 27 Day 28 Average

12/25/18 12/26/18 12/27/18 12/28/18 12/29/18 12/30/18 12/31/18 weight

Negative 85.5g 126.5g 146.5g 81g 114g 101.5g 120.5g 110.79g

Control

Positive 78.5g 91.5g 40.5g 158g 88.5g 76.5g 32.5g 80.86g

Control

50mg/kg 99g 46g 44.5g 113g 131g 61.5g 47.5g 77.5g

100mg/kg 80g 122g 78g 87g 58.5g 39.5g 83g 78.29g

150mg/kg 64.5g 50g 53g 88g 68.5g 62g 51.5g 62.5g

=82.00g

The Weight comparison of the rats from the preliminary test

up to the last day of observation prior to the biological testing.

Preliminary Weight End of observation Dec 31,2018

Dec 11,2018

POSTIVE POSTIVE

1). 266g 1). 300.5g

2). 244g 2). 246g

3). 228.5g 3). 232g

 68

4). 220.5g 4). 239.5g

5). 232g 5). 236g

NEGATIVE NEGATIVE

1). 266g 1). 270g

2).276g 2).276g

3).261.5g 3).263g

4).248g 4).250g

5).275.g 5).279g

50mg/kg 50mg/kg

1). 276g 1). 282g

2). 275.5g 2). 280gg

3). 273.5g 3). 279g

4). 286.5g 4). 293g

5). 253.5 5). 261g

100mg/kg 100mg/kg

1). 273.5g 1). 288.5g

2). 227g 2). 240g

3). 232g 3). 286g

4). 266g 4). 282g

 69

5). 275g 5). 291.5g

150mg/kg 150mg/kg

1). 321g 1). 356g

2). 259g 2). 281g

3). 289.5g 3). 301.5g

4). 242g 4). 279.5g

5). 257g 5). 284g

 70

Beatrice Seann Abuel

#11 Palmera St. Cofradia Malolos City, Bulacan
Beaseann.abuel@gmail.com
+639970870726
PERSONAL INFORMATION
Gender: Female
Birthdate: December 11, 1997
Birthplace: Malolos City, Bulacan
Age: 22 years old
Civil Status: Single
Nationality: Filipino
Religion: Roman Catholic
Language: English and Tagalog
EDUCATIONAL ATTAINMENT
Tertiary Education: Bachelor of Science in Pharmacy
Centro Escolar University – Malolos
Km. 44, Macarthur Highway, City of Malolos,
Bulacan
2015 – 2019
Secondary Education: Holy Infant School
Sta. Isabel Malolos, Bulacan
2012 - 2015
Elementary Education: Holy Infant School
Sta. Isabel Malolos, Bulacan
2008 - 2012
INTERNSHIP TRAINING
Community Internship: 2016 - South Star Drug
(Robinsons Malolos City, Bulacan)
Hospital Internship: 2017 - Internship at Allied Care Experts Medical
Center
(Baliuag, Bulacan)
Manufacturing Internship: 2018 – Internship at Compact Pharmaceutical
Incorporation
(Malinta, Valenzuela)
Major Internship: 2018 – Romel Cruz Hospital
(Matimbo, Malolos Bulacan)
 71

Ricci Mae T. Basco

Blk 7, Lot 119, Phase II, St. Agatha Homes, Tikay Malolos Bulacan
bascoricci@yahoo.com.ph
+639455603909

PERSONAL INFORMATION
Gender:Female
Birthdate: June 22, 1998
Birthplace: Nueva ecija
Age: 20 years old
Civil Status: Single
Nationality: Filipino
Religion: Roman Catholic
Language: English and Tagalog
EDUCATIONAL ATTAINMENT
Tertiary Education:
Secondary Education:
Elementary Education:
INTERNSHIP TRAINING
Community Internship:
Hospital Internship:
Manufacturing Internship:
Major Internship:
Bachelor of Science in Pharmacy
Centro Escolar University – Malolos
Km. 44, Macarthur Highway, City of Malolos,
Bulacan
2015 – 2019
Bethel Christian Academy
Plaridel, Bulacan
2011 – 2015
Lord’s Shepherd Academy
Tabang Guiguinto, Bulacan
2016 - Mercury Drug Corporation
(Puregold Guiguinto Branch)
2017- Allied Care Experts Medical Center
(Baliuag, Bulacan)
2018 – MDB Pharma Trading
(Paombong, Bulacan)
2018 – La Consolacion University General Hospital
(Malolos City, Bulacan)
 72

Jonna B. Esguerra
#0322 Pungo, Calumpit, Bulacan
esguerra.jonna17@gmail.com
+639056963220

PERSONAL INFORMATION
Gender: Female
Birthdate: June 17, 1999
Birthplace: Calumpit, Bulacan
Age: 20 years old
Civil Status: Single
Nationality: Filipino
Religion: Roman Catholic
Language: English and Tagalog
EDUCATIONAL ATTAINMENT
Tertiary Education:
Secondary Education:
Elementary Education:
INTERNSHIP TRAINING
Community Internship:
Hospital Internship:
Manufacturing Internship:
Major Internship:
Bachelor of Science in Pharmacy
Centro Escolar University – Malolos
Km. 44, Macarthur Highway, City of Malolos,
Bulacan
2015 – 2019
Mary Chiles College of Arts and Sciences
Pulilan, Bulacan
2011 - 2015
Pungo Elementary School
Calumpit Bulacan
2008 - 2011
2016 - Mercury Drug Corporation
(Calumpit, Bulacan)
2017 - Sacred Heart Hospital of Malolos Inc.
(Malolos City, Bulacan)
2018 – Primecare Lifesciences Laboratories Corp.
(Hagonoy, Bulacan)
2018 - Sacred Heart Hospital of Malolos Inc.
(Malolos City, Bulacan)
 73

Gabrielle Marie H. Aurelio

#83 Rizal St. Poblacion, Bocaue, Bulacan
aurelio.gabrielle@gmail.com
+639065472298

PERSONAL INFORMATION
Gender: Female
Birthdate: December 22, 1998
Birthplace: Bocaue, Bulacan
Age: 20 years old
Civil Status: Single
Nationality: Filipino
Religion: Roman Catholic
Language: English and Tagalog
EDUCATIONAL ATTAINMENT
Tertiary Education: Bachelor of Science in Pharmacy
Centro Escolar University – Malolos
Km. 44, Macarthur Highway, City of Malolos,
Bulacan
2015 – 2019
Secondary Education: St. Paul College of Bocaue
Turo, Bocaue Bulacan
2011 - 2015
Elementary Education: St. Paul College of Bocaue
Igulot, Bocaue Bulacan
2008 - 2011
INTERNSHIP TRAINING
Community Internship: 2016 - Mercury Drug Corporation
(Bocaue, Bulacan)
Hospital Internship: 2017- Our Lady of Lourdes Hospital
(Sta. Mesa, Manila)
Manufacturing Internship: 2018 - Morishita- Seggs Pharmaceuticals Inc.
(Sta. Maria, Bulacan)
Major Internship: 2018 – Dr. Yanga Hospital Inc.
(Bocaue, Bulacan)
 74

Pamela P. Baluyut
#118 Sta. Rita Macabebe, Pampanga
pamela.baluyut00@gmail.com
+639268017690

PERSONAL INFORMATION
Gender: Female
Birthdate: September 15, 1999
Birthplace: Apalit, Pampanga
Age: 19 years old
Civil Status: Single
Nationality: Filipino
Religion: Roman Catholic
Language: English, Tagalog, Kapampangan

EDUCATIONAL ATTAINMENT
Tertiary Education: Bachelor of Science in Pharmacy
Centro Escolar University – Malolos
Km. 44, Macarthur Highway, City of Malolos,
Bulacan
2015 – 2019
Secondary Education: Colegio De San Lorenzo
Macabebe, Pampanga
2011 - 2015
Elementary Education: Jesus Mary Joseph Montessori School
Macabebe, Pampanga
2008 – 2011
INTERNSHIP TRAINING
Community Internship: 2016 - Mercury Drug Corporation
(Macabebe, Pampanga)
Hospital Internship: 2017- Pampanga Premier Medical Center Inc.
(Sampaloc, Apalit Pampanga)
Manufacturing Internship: 2018 –MDB Pharma Trading
(Paombong, Bulacan)
Major Internship: 2018 - Mercury Drug Corporation
(Macabebe, Pampanga)
 75

Christian A. Domingo
Esguerra St. Poblacion, Pulilan Bulacan
christian.domingo29@gmail.com
+639174533608

PERSONAL INFORMATION
Gender: Male
Birthdate: February 22, 1997
Birthplace: Pulilan, Bulacan
Age: 22 years old
Civil Status: Single
Nationality: Filipino
Religion: Born again Christian
Language: English and Tagalog
EDUCATIONAL ATTAINMENT
Tertiary Education: Bachelor of Science in Pharmacy
Centro Escolar University – Malolos
Km. 44, Macarthur Highway, City of Malolos,
Bulacan
2014 – 2019
Secondary Education: Virgilio V. Dionisio Memoria High School
Pulilan, Bulacan
2010 - 2014
Elementary Education: Balatong Elementary School
Brgy. Balatong Pulilan, Bulacan
2004 - 2010
INTERNSHIP TRAINING
Community Internship: 2015 - South Star Drug
(Robinsons Pulilan, Bulacan)
Hospital Internship: 2016- Our Lady of Mercy General Hospital
(Longos Pulilan, Bulacan)
Manufacturing Internship: 2018 –RoyMed Pharmaceuticals
(Kalayaan village, San fernando Pampanga)
Major Internship: 2019 – Polymedic Hospital
(Guiguinto Bulacan)
76