Ethnopharmacological investigation of the spice Commelina appendiculata C.b. Clarke (Commelinaceae)

Scientific Study, 2012

75 Pages, Grade: A




1.1 General Introduction
1.2 Medicinal plants
1.3 Introduction to commelinaceae family
1.4 Aim of the present study
1.5 Present study protocol

2.1 Plant material
2.2 Biological investigations of Commelina appendiculata
2.2.1 Analgesic activity test Acetic acid induced writhing method Hot plate method Tail immersion method
2.2.2 Neuropharmacological activity test Thiopental sodium induced sleeping time method Hole cross method Open field method
2.2.3 Anti-diarrhoeal activity test Castor oil induced diarrhoea method Magnesium Sulphate-Induced Diarrhea
2.2.4 Cytotoxic activity test Brine shrimp lethality bioassay
2.3 Phytochemical investigations of Commelina appendiculata
2.3.1 Preparation of reagents used for different chemical group tests
2.3.2 Test procedure for identifying different chemical groups

3.1 Biological investigations
3.1.1 Analgesic activity Acetic acid induced writhing method Hot plate method Tail immersion method Discussion
3.1.2 Neuropharmacological activity Thiopental sodium induced sleeping time method Hole cross method Open field method Discussion
3.1.3 Anti-diarrhoeal activity Castor oil induced diarrhoea method Magnesium Sulphate-Induced Diarrhea Discussion
3.1.4 Cytotoxic activity Brine shrimp lethality bioassay Discussion
3.2 Phytochemical screening Discussion
3.3 Conclusion



The present study was intended to assess the plausible pharmacologic properties of the ethanol extract of Commelina appendiculata (EECA) (Family: Commelinaceae). We designed our experiments to find out analgesic, central nervous system (CNS) depressant, antidiarrheal and cytotoxic activities of the aforementioned extract. In the prospect to determine the analgesic activity, acetic acid-induced writhing, hot plate and tail immersion methods in swiss albino mice were conducted at the doses of 100 and 200 mg/kg body weight, p.o. In retrospective, both doses of the extract produced a noteworthy (p < 0.05 and p <0.001) pain-relieving action in a dose dependent manner. In acetic acid induced writhing method, EECA (200 mg/kg) resulted in the most significant analgesic activity with writhing inhibition of 76.27%; on the contrary, the standard drug Diclofenac-Na (25mg/kg) and Aspirin (100 mg/kg) showed 80.72% and 61.94% inhibition respectively. In regard to hot plate and tail immersion tests, similarly, inhibitions of nociceptive thermal stimulus by EECA (200 mg/kg) were maximum of 52.56% and 76.58% correspondingly. Morphine (5mg/kg, i.p.) was used as standard in the precedent study. CNS depressant activity of the extract (100 and 200 mg/kg, p.o.) was assessed by employing thiopental sodium-induced sleeping time model to evaluate sedative potential, hole cross to evaluate motor activity and open field test to estimate exploratory behavior. Consequently, the extract resulted in rapid onset and maximized duration of sleeping time when concomitantly administered with thiopental sodium. In addition, a reduction in motor action and exploratory conduct of mice in hole cross and open field test was found distinctly when treated with the extract (p < 0.05-0.001). In antidiarrheal activity study, the dose (200 mg/kg) likewise associated with most extreme inhibition of defecation (90.64% and 80.95%) in both models independently. Phytochemical screening of ethanol extract of C. appendiculata confirmed the presence of carbohydrates, flavonoids, tannins, glycosides and alkaloids. Notably, substantial level of lethality was found with EECA, involving LC50 value of 26.3 μg/ml, whereas the LC50 of vincristine sulphate was 0.52 μg/ml. The above results from the present study reveal the potential analgesic, central nervous system (CNS) depressant, antidiarrheal and cytotoxic properties of the ethanol extract of C. appendiculata. As a consequence, the plant may be further examined to refine its pharmacologically active natural ingredients.

Key words: Commelina appendiculata, analgesic activity, central nervous system (CNS) depressant activity, antidiarrhoeal activity, cytotoxic activity, phytochemical screening.


1.1 General introduction

At the very beginning, prior to the footprint of modern civilization, health care system for the mankind was solely dependent on the plant diversity. Curing of diseases and caring of health, natural plants played a vital role all over the universe. Through the history of mankind, naturally occurring plant-derived substances with minimal or no industrial processing have been used to treat illness within local or regional healing practices. This traditional practice is now concerning the promising factor for the development of better health care system and getting significant attention in global health debates.

In the last decade, there has been a global upsurge in the use of traditional medicine (TM) and complementary and alternative medicine (CAM) in both developed and developing countries. Today, therefore, certain forms of traditional, complementary and alternative medicines play an increasingly important role in health care reform globally.

The development of traditional medicines has been influenced by the different cultural and historic conditions in which they were first developed. Their common basic is a holistic approach to life, equilibrium between the mind, body and environment, and an emphasis on health rather than on disease. Generally, the treatment focuses on the overall condition of the individual patient, rather than on the ailment or disease. This more complex approach makes evaluation highly difficult, since so many factors must be taken into account.

Geographical point of view, Bangladesh is enriched with diversified natural plants. Natural plants actually are God gifted asset and with proper utilization mankind can have the chance to develop its whole life through the appropriate collaboration of nature and life.


A medicinal plant is any plant which, in one or more of its organ, contains substance that can be used for therapeutic purpose or which is a precursor for synthesis of useful drugs . This definition of Medicinal Plant has been formulated by WHO (World Health Organization) (Sofowora, 1982).

Actually, the plants that possess therapeutic properties or exert beneficial pharmacological effects on the animal body are generally designated as “Medicinal Plants”. Although there are no apparent morphological characteristics in the medicinal plants growing with them, yet they possess some special qualities or virtues that make them medicinally important. It has now been established that the plants which naturally synthesis and accumulate some secondary metabolites, like alkaloids, glycosides, tannins, volatiles oils and contain minerals and vitamins, possess medicinal properties (Ghani, 2005).

Medicinal plants constitute an important natural wealth of a country. They play a significant role in providing primary health care services to rural people. They serve as therapeutic agent as well as important raw materials for the manufacture of traditional and modern medicine. Substantial amount of foreign exchange can be possible to earn by exporting medicinal plants to other countries (Ghani, 2003).

History of medicinal plants

Since medicinal properties of plants were known even to prehistoric men, the discovery of medicinal properties of plants was not certainly based on any scientific data or on the knowledge of chemical constituents of plants. On the contrary, the exploration of the medicinal properties of plants throughout the ages was accomplished principally through careful observation, trial and error, and accidental discovery. And in this process, the human race, over the centuries, has created a vast heritage of knowledge and experience on medicinal plants in different cultures and civilizations. Most of such indigenous knowledge was handed down, through the ages, by oral tradition. The major portion of the present-day knowledge of the medicinal properties of plants is thus the sum total of these observations and experiences. Some of the wonder drugs of modern medicine have their roots in this early knowledge of medicinal plants.

The practice of medicine using medicinal plants flourished most during the Greek civilization when historical personalities like Hippocrates ( born in 460 BC) and Theophrastus (born in 370 BC) practiced herbal medicine. The materia medica of the great Greek physician Hippocrates (460-370 BC) consists of some 300 to 400 medicinal plants which included opium, mint, rosemary, sage and verbena. The far-ranging scientific work of Aristotle (384-322 BC), a Greek philosopher, included an effort to catalogue the properties of the various medicinal herbs known at that time. The encyclopedic work of Dioscorides (1st century AD), De Materia Medica (published in 78 AD), was the forerunner of all modern pharmacopoeias and on authoritative text on botanical medicine. This work features about 600 medicinal plants. Galen, the great Greek pharmacist-physician, is the first person who describe, for the first time in history, procedures and methods of preparing therapeutic recipes including ingredients of both plant and animal origins.

The Arabian Muslim physicians, like Al-Razi and Ibn Sina (9th to 12th century AD), brought about a revolution in the history of medicine by bringing new drugs of plant and mineral origin into general use. The famous medical book, Al-Kanun of Ibn-Sina was the prescribed book of medicine in the schools of Western medicine for several centuries (Ghani, 2003).

The use of medicinal plants in Europe in the, 13th and 14th centuries was based on the Doctrine of Signatures developed by Paracelsus (1490-1541AD), a Swiss alchemist and physician. According to this Doctrine, all plants possess sign, given by the creator, which indicated the use for which illness or diseased organ they were intended .The South American countries have provided world with useful medicinal plants, grown naturally in their forests and planted in the medicinal plant gardens. The current list of the medicinal plants growing around the world includes more than a thousand items (Sofowar, 1982; Ghani, 2003).

Development of modern medicine

Genesis of modern medicine is actually the continuous innovative process of traditionally utilized plant medicine through its improvement and utilization. Traditional medicine is the synthesis of therapeutic experience of generations of practicing physicians of indigenous system of medicine. Throughout the history of mankind, many infectious diseases have been treated with herbals. The traditional medicine is increasingly solicited through tradipractitioners.

Medicinal plants: A natural laboratory

Medicinal plants are rich sources of bioactive compounds and thus serve as important drug material for drug production. It has now been established that the plants synthesize and accumulate some secondary metabolites like alkaloids, glycosides, tannins, volatile oils etc. that may possess a great potential for biological activity and can be a curative agent in therapeutic purposes. As therapeutic uses of plants continued with the progress of civilization and development of human knowledge, scientists endeavored to isolate different chemical constituents from plants, put them to biological and pharmacological tests and thus able to identify and isolate therapeutically active compounds. The 19th century saw the scientific revolution in medicine. The first isolation and crystallization of an active drug from a natural source was the accomplishment of a pharmacist’s assistant, Sertuner (1783-1841), who obtained pure morphine from natural opium in 1803. Pure quinine was isolated from cinchona bark in 1820. Isolation of other important plant derived drugs of modern medicine rapidly followed and many useful drugs have since been discovered and introduced into modern medicine. Drugs like strychnine from Strychonos Nux vomica (1817), emetics from Cephaelis ipecacuanha (1817), caffeine from Thea sinensis (1819), quinine from Chcinchona spp. (1820) and colchicines from Colchicum autumnale (1820) constitutes some examples of such early drugs (Ghani, 1998).

Facilitated by the rapid development of technology of isolation and characterization process that is chromatographic and spectroscopic methods, a large number of therapeutically active plant constitutes have been isolated during last two decades (Ghani, 1998). In the later part of the 19th century the German dye industry led the way toward the deliberate synthesis of new drugs and the molecular modification of existing ones. Still now the medicinal chemists have a great interest in the molecular modification of therapeutic agent isolated from plant sources. For example, taxotere that enjoys an extensive use in present cancer chemotherapy is the synthetic analog of paclitaxel, developed by the scientists of Rhone Poulenc Ror. The current list of medicinal plants growing around the world includes more than thousand items (Ghani, 1998).

Table 1.1: Examples of the large number of therapeutic agents isolated from medicinal plants (Ghani, 1998)

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Status of medicinal plants in Bangladesh

Medicinal plants are an accessible, affordable and culturally appropriate source of primary health care system in Bangladesh. Marginalized, rural and indigenous people, who cannot afford or access formal health care systems, are especially dependent on these culturally familiar, technically simple, financially affordable and generally effective traditional medicines. As such, there is widespread interest in promoting traditional health systems to meet primary health care needs. This is especially true in this country, as prices of modern medicines spiral and governments find it increasingly difficult to meet the cost of pharmaceutical-based health care. Throughout the region, there is strong and sustained public support for the protection and promotion of the cultural and spiritual values of traditional medicines. The total number of plants with medicinal properties in the subcontinent at present stands at about 2000. About 450 to 500 of such medicinal plants have so far been enlisted as growing or available in Bangladesh.

Table 1.2: Examples of the large number of therapeutic agents isolated from medicinal plants (Ghani, 1998).

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1.3 Introduction to commelinaceae family

Herbs annual or perennial, sometimes woody at base. Stems with prominent nodes and internodes. Leaves alternate, distichous or spirally arranged, sessile or petiolate; leaf sheath prominent, open or closed; leaf blade simple, entire. Inflorescence usually of cin-cinni in panicles or solitary, sometimes shortened into heads, sometimes sessile with flowers fascicled, sometimes axillary and penetrating enveloping leaf sheath, rarely flowers solitary and terminal or axillary. Flowers bisexual, rarely unisexual, actinomorphic or zygomorphic. Sepals 3, free or connate only at base, often boat-shaped or craniate, sometimes galeate at apex. Petals (2 or) 3, free, sometimes connate and tubular at middle and free at 2 ends (Cyanotis), sometimes clawed. Stamens 6, free, all or only 2 or 3 fertile; filaments glabrous or torulose villous; anthers parallel or slightly divergent, longitudinally dehiscent, rarely dehiscent by apical pores; staminodes 1–3; antherodes 4-lobed and butterfly like, 3-sect, 2-lobed and dumbbell-shaped, or entire. Ovary 3-loculed, or reduced to 2-loculed; ovules 1 to several per locule, orthotropous. Fruit a loculicidal, 2- or 3-valved capsule, rarely baccate and indehiscent. Seeds few, large; endosperm copious; hilum orbicular or linear. About 40 genera and 650 species: mainly in tropical regions, fewer species in subtropical and temperate regions (Deyuan, 1997).

Scientific classification of Commelina appendiculata

Kingdom: Plantae

Subphylum: Euphyllophytina

Infraphylum: Radiatopses

Subclass: Magnoliidae

Order: Commelinales

Family: Commelinaceae

Genus : Commelina

Species: C. appendiculata


An annual herb, stem creeping, diffusely branched, 40 cm or more long, internodes slightly reddish, roots fibrous at the lower nodes. Leaves 2.5-3.5 × 1.0-1.5 cm, lanceolate to oblong, acute to acuminate, glabrous, sheath surrounding the stem prominent, 1 cm long and ciliate at the open edge. Flowers in leaf opposed cymes emerging from a spathaceous bract, spathes 1-2 cm long, axillary, lanceolate, acuminate, base deeply cordate. Sepals c 4 mm long lanceolate. Petals c 5 × 3 mm, blue or white clawed. Fertile stamens 3 (rarely 2), filaments c 2 mm long, naked, anthers oblong, staminods 2-4, bilobed. Ovary 3-celled, each cell with 1 ovule. Fruit a capsule, mature capsule 2-valved, 5-7 mm long, oblique or oblong with a pointed apex. Seeds 4 mm in ventral cell, oblong, smooth, grey, sometimes flattened appendiculate at both ends, seeds at the dorsal cell shorter or absent.

Flowering and fruiting: June-December. (Siddiqui, 2007).


The plant is distributed in Eastern India and Sri Lanka. In Bangladesh, it is found in Tangail, Mymensingh and Sylhet district (Siddiqui, 2007).

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Figure 1.1. Plant of Commelina appendiculata

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Figure 1.2. Leaf of Commelina appendiculata

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Figure 1.3. Flower of Commelina appendiculata

1.4 Aim of the present study

Nature has been a source of medicinal agent for thousands of years and an impressive number of modern drugs have been isolated from natural sources (Thoundam et al., 2011). Here the studied of Commelina appendiculata (Kandaluya in Bengali) belonging to the family Commelinaceae are widely growing plant throughout Asia including Bangladesh. On the literature available it can be said that this plant yet not tested for its valuable medicinal properties and there are no experimental studies on antinociceptive, neuropharmacological, antidiarrhoeal and cytotoxic activities of this plant. The objective of this study is therefore to investigate the antinociceptive, neuropharmacological, antidiarrhoeal and cytotoxic activities of ethanolic extract of Commelina appendiculata.

1.5 Present study protocol

More specifically, the present study has been designed to observe the following bioactivities using both in vitro and in vivo experimental models as well as to identify the compounds that are present in the ethanolic extract of Commelina appendiculata.

1. Evaluation of analgesic activity of the extract using acetic acid-induced writhing, hot plate and tail immersion tests.
2. Observation of neuropharmacological activity of the extract using hole cross, open field and thiopental induced sleeping time tests.
3. Observation of anti-diarrhoeal activity of the extract using castor oil induced diarrhoea and Magnesium Sulphate-Induced Diarrhea in mice.
4. Evaluation of possible cytotoxic property of the crude extracts using brine shrimp lethality bioassay.
5. Phytochemical screening of the extract of Commelina appendiculata.


2.1 Plant material

Commelina appendiculata was selected for investigation. The whole plant was used for research purpose.

Collection of the plant

The plant of Commelina appendiculata was collected from the local area of Sylhet Sadar, Sylhet during June 2012. Dust, dirt and the undesirable materials were then separated manually.


The collected plant was then identified by the taxonomist of Bangladesh National Herbarium, Mirpur, Dhaka and a voucher specimen has been deposited (DSCB No. 39,321) for further reference.

Preparation of the plant sample

The collected and identified plant was cut into small pieces separately and then dried in the sun and finally dried in a hot air oven at 35-40° C for 24 hours. After complete drying, the whole plant was reduced to coarse powder separately with the help of a mechanical grinder and the powder was stored in a suitable container for extraction. The dried grinded powder weighed by rough balance.

Extraction of the plant material

The plant was extracted by hot extraction method. The powder (100g) of Commelina appendiculata was extracted with 300 ml of ethanol by a Soxhlet apparatus for 2 days at 50°C. The extract was then filtered using a Buchner funnel and a sterilized cotton filter. The solvent was completely removed by rotary evaporator and 5.9 g extract (yield 5.9%) was obtained. This ethanolic extract of Commelina appendiculata (EECA) was subjected to phytochemical and pharmacological activity test.

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Figure 2.1. Hot extraction of Commelina appendiculata

2.2 Biological investigations of Commelina appendiculata

Study of biological activity of the extract of Commelina appendiculata has been adopted in Laboratory of Pharmacognosy and Pharmacology, Department of Pharmacy, Stamford University Bangladesh on mice model.


For the experiment Swiss albino mice of either sex, 4-5 weeks of age, weighing between 25-35 gm, were collected from the animal research branch of the International Center for Diarrhoeal Disease and Research, Bangladesh (ICDDR, B). Animals were maintained under standard environmental conditions (temperature: (24.0±1.0º), relative humidity: 55-65% and 12hrs light/12 hrs dark cycle) and had free access to feed and water ad libitum. The animals were acclimatized to laboratory condition for one week prior to experimentation (Chatterjee, 1993). All protocols for animal experiment were approved by the institutional animal research ethical committee.

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Figure 2.2. Swiss albino mice

Animal feed used

The mice were given special type of chocolate food which was supplied by the ICDDR`B, Animal House, Mohakhali, Dhaka, Bangladesh.

Table 2.1. Type of food used for the mice.

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Materials used for animal house

The materials were used for the mice house are listed in Table 2.2.

Table 2.2. Materials used for animal house.

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Ethnopharmacological investigation of the spice Commelina appendiculata C.b. Clarke (Commelinaceae)
Jahangirnagar University  (Department of Pharmacy)
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ethnopharmacological, commelina, clarke, commelinaceae, pharmacological, investigation
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Pritesh Ranjan Dash (Author)Nabila Morshed (Author), 2012, Ethnopharmacological investigation of the spice Commelina appendiculata C.b. Clarke (Commelinaceae), Munich, GRIN Verlag,


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