Preliminary Phytochemical and Pharmacological Studies on the Root of Pavetta indica Linn

INTRODUCTION

Modern medical technology that developed very rapidly decreased the importance of medicinal plants and the pharmacologically active products derived from them as compared to the synthetic chemical products of the multinational pharmaceutical industry which promoted the concept of modern health care all over the world. The revival of interest in natural drugs, especially those derived from plants, started in the last decades mainly because of the wide spread belief that “Green medicines” are healthier and safer than the synthetic ones. Ever since the WHO in its 29th and 30th assembly gave formal recognition to the traditional medicine, the herbal medicine of ancient times practised during the vedic period [5000 - 3000 B.C] and which was temporarily subdued under the impact of modern medicine have staged a comeback and a “Herbal Renaissance” is blooming across the world. WHO has estimated that 80% of the people in the world rely on traditional medicines for primary health care needs.

In some of the developed countries like Australia, Sweden, Switzerland and Canada the use of Herbal medicine and herbal treatment of diseases are on the increase, especially among the younger generations.

Herbal Churna tablets, Herbal tonics, Herbal soaps, Herbal Shampoos, Herbal talcum powder, Herbal tooth paste and Herbal cosmetics have become popular consumer items. The word “Herbal” has become the symbol of safety for those products, in contrast to the synthetic ones which have become highly unsafe for human consumption once science revealed their adverse effects on human health and environment.

Herbs are staging a comeback and a “Herbal Renaissance” is blooming across the world. They have been prized for their medicinal, flavouring and aromatic qualities for centuries, yet for a while, they were overshadowed by the synthetic products of modern civilization. But once having realized their serious adverse effects people are going back to nature with hopes of safety and security. Today herbs are finding diverse uses in the society from medicines to manure, insecticides, pesticides and many articles of daily use.

Plants, animals and the human beings have intimate ecological relationships since remote past and have evolved along parallel lines cooperating and depending upon each other for existence. Men and animals depended more upon the plants for their very basic needs air, food, fibre, fuel and shelter. The primitive men during the course of their struggle for existence in the forest must have encountered the miseries of pains and sickness, sustained injures and lived under the circumstances of abject poverty, disease and hunger. To liberate themselves from these sufferings they should have looked towards their natural fellow friends - the plants. And this inevitably led to the experimentation through trial and error and discovery of the healing properties of plants. It must have begun with the “miraculous healing effects” of leaves of some plants with which they covered their wounds to avoid flies and dust and with the soothing and stimulating effect of the leaves, root and stems of some plants which they chewed to satisfy their hunger. Once having been realized the significance of the “Wonder Herbs” they started communicating about them to their fellow friends through signs and symbols.

Undoubtedly nature has all along with the disease has created their cure and for every disease that arise on this planet, plant has a cure. Nature keeps in her green bag, the secret of healthy life on this mother earth, perhaps in the luxuriant green cover, the bio-diversity of the flora and fauna.

The herbal revolution has uncovered the unbelievable miraculous muscle relaxants from South America, Arrow poison, Cortisone and contraceptives from “Sapogenin” plants used by aborigines of Central America, India and Africa.

Development of traditional herbal medicine into a modern drug of great therapeutic importance is exemplified by the wonder herb Rauwolfia serpentina, the root of which has been used for insanity, epilepsy and high blood pressure. Some volatile oils are more or less powerful external or internal antiseptics, others posses an analgesic haemolytic or antizymatic action, still others act as sedative, stimulant and stomachics. The knowledge of phytochemical screening has revealed the existence of close relationship between chemical constituents of plants and their taxonomical status.

There are number of other herbs which have come to light in recent years with miraculous therapeutic effects. The rosy periwinkle (Vinca rosea) from the Madagascar tropical forest of the aborigines have provided the million dollar weapon “Vinblastine” to fight childhood leukemia a kind of blood cancer in infants. The pacific Yew (Taxus buccata) preserved and used by the indigenous ethnic communities of the pacific island gave the million dollar worth anticancer drug “Taxa’. The National Cancer Institute of U.S.A. every year examines hundreds of plants used by the ethnic community of the world in search of anticancerous drugs.

Today we stand at the cross roads of ancient, traditional and modern advancement with regard to medicine. The modern medicine has brought with it a host of drugs, none of which is nontoxic and 100% safe for human use and some of them even cause irreversible damage to our physiological systems. It also has no answer to some of the modern diseases ailing the humanity. On the contrary, the herbal medicine which teaches the principles of healthy living has much to offer this progressive world in the treatment of such kind of diseases like cancer, cardiac problems, psychic problems, hepatitis, diabetes, AIDS etc.

There is difference in the mode of action of herbal medicines and modern synthetic medicine. The synthetic medicine attacks the disease directly and helps in destruction of the disease. Also modern medicine attacks the targets blindly, thus affecting the several related metabolic systems of the body including the immune system. In some cases the body becomes drug dependent and there is severe withdrawal symptoms whereas the herbal medicine approaches directly over the head of the disease and destroys it by a process of reinforcement or bolstering up of the patient’s body resistance and immune system. Herbal medicines work selectively and gently without disturbing the other systems of the body. The herbal medicine in the process of curing strengthens the body’s immune system to fight the disease in future also, the synthetic medicine weakens it in the long run and makes the body more susceptible for further attacks.

The herbal medicine also suits the social and cultural needs of the people and influences the patient’s physical, mental and emotional states as well. The herbal drugs prepared with traditional methods through slow grinding and mixing processes conserves all the natural substances within it in the “naturally balanced form” without losing any essential component and maintains the activity and purity of the drug, the very basis which accounts for the minimal side effects of herbal drugs.

Now people are getting conscious of the rare side effects of the modern synthetic medicines and herbs are once again staging a dramatic comeback. Costly modern medicines and complicated prescriptions, the grave side effects caused by them, sometimes even fatal, has gripped a sense of fear in the minds of the people who is now in great dilemma and are anxiously looking back to nature and herbal medicines. People suffering from chronic diseases after losing all hopes from modern synthetic medicines are turning their eye towards herbal medicines.¹

The present day research on indigenous drugs and medicinal plant has made rapid stride depending on vast amount of literatures based on compilation/ cataloging of claims from the folk lore, ancient text of Indian system of medicine Ayurveda, Siddha and Unani, Anthropological, chemical correlation of various species and field surveys. Another issue is convincing septics in the medical world that herbal medicine is not just a poor substitute for conventional medicine but a valuable form of treatment in its own right. The story in China in early 1990’s on the effect of certain Chinese herbs on patients with eczema offers an evidence of skill and art involved in herbal practice. In tailoring the remedy to suit the individual needs of the patient in treating the underlying cause, major improvements were made. This kind of approach in a far cry from the standard medical view of using a single drug to treat a single disease.²

The prime reason for the future of herbal medicine is whether medicinal plants, and the traditional knowledge that informs their use, will be valued for what they are an immense resource of safe, economical, ecologically balanced medicines - or whether they will be yet another life to be exploited for short term profit.

India is now beginning to search her roots in the past and revive her lost glory of the traditional system of medicine which flourished here for several centuries and contributed much to the development of the medical science. To alleviate the sufferings of her large evergrowing populace, she has to revive the traditional folklore medicine and bring it into the mainstream of national health care programme.³ In the light of the above points and considering the importance of natural drugs, the present work and investigation of a plant drug Pavetta indica Linn. was carried out.

MATERIALS AND METHODS

Materials:

The plant Pavetta indica Linn. is widely found throughout India. They found along the road sides, forests and hill areas upto a height of 1500 meters. In Tamil Nadu it is found in Tirunelvelly and nearby districts. For our work the plant Pavetta indica was collected from Tirunelvelly which is about 500 km away from Chennai.

Collection:

The plants were identified by a botanist who authenticated the plant with the available literature. The plants were uprooted by digging and the roots were separated by cutting the aerial parts from the entire plant.

Treatment:

The collected roots were washed with water to remove any dirt, mud and other plant debris. They were made into small pieces and shade dried. The dried roots were powdered by means of wood grinder and the powder was passed through the sieve no.60 for powder analysis and the coarse powder was used for phytochemical and pharmacological work.

Preparation of extracts:

Petroleum ether extract of Pavetta indica: The coarsely powdered shade dried roots of Pavetta indica (100g) was macerated with petroleum ether (60-80°C) for 7 days at room temperature. After completion of maceration it is filtered. The solvent was removed under reduced pressure and concentrated.

Chloroform extract of Pavetta indica: The mare left after petroleum ether extraction was dried and macerated with purified chloroform for 7 days at room temperature. After completion of extraction, the extract was concentrated under reduced pressure.

Ethyl acetate extract of Pavetta indica: The man left after chloroform extraction was dried and macerated with purified ethyl acetate for 7 days at room temperature. After the completion of extraction, the extract was concentrated under reduced pressure.

Acetone extract of Pavetta indica: The marc left after Ethyl acetate extraction was dried and macerated with purified acetone for 7 days at room temperature. After the completion of extraction, the extract was concentrated under reduced pressure.

Methanolic extract of Pavetta indica: The mart left after acetone extraction was dried and macerated with purified methanol for 7 days at room temperature. After the completion of extraction, the extract was concentrated under reduced pressure.

Aqueous extract of Pavetta indica: The mare left after methanolic extraction was dried and finally extracted with distilled water. After completion of extraction the extract was for concentrated under reduced pressure. The above extracts were used for phytochemical studies.

Alcoholic extract: The coarsely powdered shade dried roots of Pavetta indica (500g) was defatted with petroleum ether and then extracted with 2 liters of alcohol (90%) by maceration process for 7 days. The solvent was removed under reduced pressure and concentrated.

Aqueous extract: The coarsely powdered shade dried roots of Pavetta indica (500g) was defatted with petroleum ether and then extracted with 2 litres of distilled water by decoction process for 10 minutes. The solvent was removed by evaporation by the application of heat. The resulting extracts were dried in a desiccator and used for pharmacological studies.

Gross behavioural and toxicity studies:^4,5

This was studied as suggested by Turner (1965). Adult swiss albino mice of either sex, weighing between 20-25g were divided into eight groups of 6 mice each. Group I to VI received the various doses viz., 400, 800, 1000, 1600, 1800 and 2000mg/ kg body weight of ethanolic extract of the roots of Pavetta indica as a fine suspension in 0.5% w/v carboxyl methyl cellulose (CMC) and administered orally.

The Group VII received the solvent control (0.5%) carboxyl methyl cellulose) in a similar manner to the extract and Group VIII was kept to observe normal behavioural pattern.

The similar procedure was followed for the aqueous extract of the root of Pavetta indica.

After the administration of test compounds, the animals were observed continuously for the first 4 hours for behavioural and toxic symptoms and at the end of 24 hours for mortality if any, and was recorded. The animals were observed till the end of 72 hours and the results were recorded.

Screening for anti-inflammatory activity:

Carrageenan-induced rat hind paw edema method:^5,6 This method is based on the technique of Withalin and Domengoj modified by Seth et al. (1972) edema volume was measured by plethysmograph where mercury displacement was used as an index. In this experiment albino rats (male) weighing between 180 - 200g and six in each group were used. Animals were deprived of food and water for 18 hours before the experiment. On the day of the experiment, they were assigned into six groups of 6 animals each. They were marked and numbered for their identification. Group I and group II, received the 1/5th of the maximum tolerated dose (1/5 of 2000 = 400mg) of alcoholic and aqueous extracts (400mg/kg) and group III & IV received the 1/10th of the maximum tolerated dose (1/10th of 2000 = 200mg) of alcoholic and aqueous extracts (200mg/kg) were suspended in 0.5% w/v sodium carboxy methyl cellulose.

Group V and VI were used as a positive control and solvent control by administering Indomethacin (10mg/kg) and 0.5% w/v Carboxyl methyl cellulose (1ml/kg) respectively. All the test compounds and the drug were administered orally.

After 30 minutes of compound administration 0.1m1 of 1% w/v of carrageenan in normal saline was injected into the lateral malleolus of the subplantar region of the left hind paw. To the right hind paw a same dose of normal saline was injected and served as control.

The volume of displacement of mercury was measured in both the paws (immersed upto the predetermined mark) before the administration of carrageenin and immediately after the administration of carrageenan at 0 mts, 30 mts, 60 mts, 120mts, 180mts, 240mts and 8 hours by using plethysmography. The readings were recorded.

Thus the edema volume in solvent control and groups treated with test compound were measured. The % of inhibition of edema (or % protection) was calculated using the following formula:

% inhibition = 100 - Treated/(Solvent Control) x 100

Analgesic Activity:

Eddy’s hot plate method:^5,6 The analgesic activity of various extracts of the root of Pavetta indica were studied by Eddy’s Hot plate method. Analgesics are substances which decrease the pain sensation (pain - killers) by increasing the threshold to painful stimuli. Painful reaction in experimental animals can be produced by applying noxious (unpleasant) stimuli such as thermal (radiant heat as a source of pain), chemical (irritants such as acetic acid and bradykinin) and physical pressure (tail compression). The Eddy’s Hot plate method involves thermal stimulus and heat is applied using a hot plate maintained at 55°C. The procedure is as follows.

The animals are weighed and numbered. Swiss albino mice of either sex weighing between 20-25g were used. Animals were deprived of food and water for 18 hours before the experiment. On the day of the experiment, they were assigned into 4 groups of 6 animals each. They were marked for their identification. Groups I to II received the ethanolic and aqueous extracts of Pavetta indica at a dose level of 200mg/kg respectively. All the extracts were given as a fine suspension in 0.5% carboxy methyl cellulose. Group III and IV served as a positive control and solvent control by administering pentazocine (30mg/kg) and 0.5% w/v carboxyl methyl cellulose (1ml/kg) respectively.

The basal reaction time to radiant heat was taken by placing the mice on Eddy’s hot plate maintained at 55°C. When the animal licks the fore or hind paws or jumps, it is taken as the end point. Normally a mouse jumps or licks the paws within 3.5 seconds. A cut of period of 15 seconds is observed to prevent damage to the paws. Any animal failing to lick the paws or jump in 5 second is rejected from the study.

All the test compounds, standard and solvent control were administered orally. After drug administration note the reaction time at 30 min, 60 min, 90min, 2h, 3h & 4h. As the reaction time reaches 15 seconds, it is considered maximum analgesia and the animal is removed from the source of heat to avoid tissue damage. The readings were recorded. The percentage protection was calculated using the formula:

% protection = 100 - Treated/(Solvent Control) x 100

Antibacterial studies:^7,8

The various root extracts of the plant Pavetta indica were subjected to antibacterial studies against various strains of bacteria. The concentrations of the working stock culture was 106 to 108 c.f.u./ml. The organisms used were Staphylococcus aureus (Gram positive), Escherichia coli (Gram Negative), Pseudomonas aeruginosa (Gram negative), Proteus vulgaris (Gram negative) and Klebsiella pneumoniae (Gram negative).

After confirmation of the identity of the organisms they were used for anti-bacterial studies.

The organisms were maintained on nutrient agar slants. These were tested using nutrient broth. One loopful of the respective cultures in each Slant, which were maintained below 4°C was taken and inoculated in the broth and incubated at 37°C for 24 hrs and were observed for the growth of the organisms with the naked eye for their turbid nature and compared with sterile broth. The presence of turbidity indicates the growth and suitability of the culture for further work.

Preparation of stock culture: From the cultures which were maintained on nutrient agar slants, one loopful of the respective organisms were taken and aseptically transferred to 100m1 of sterile nutrient broth in an Erlenmeyer flask which was plugged with sterile cotton wool. This was shaken thoroughly and incubated at 37°C for 24 hours. This is called stock culture. The stock culture is standardized using the following procedure.

Standardization of stock culture: From this stock culture 1ml was transferred to 99ml of fresh sterile nutrient broth and shaken thoroughly. This is referred to as seeded broth. Seeded broths of the various organisms to be tested were prepared.

lml of this seeded broth was then diluted with 9m1 of sterile water in a culture tube with 0.05% of Tween 80 (containing 8 drops of tween 80 in every 1000m1 of water). This was shaken thoroughly and about l ml of this suspension was transferred to a second culture tube which in addition contains 9 ml of sterile water. This was shaken thoroughly and this was further diluted 10 times with sterile water till 1010 dilution was obtained (up to 10 culture tubes).

Standardization of the seeded broth was done by taking 0.2 ml of the solution from the first culture tube and plating in triplicate on the Mueller Hinton (MH) plate. Plating in triplicate on the MH plate was carried out for the various dilutions. (for all the 10 culture tubes). These plates were then incubated at 37°C for 48 hours and the number of well formed colonies on the plates was counted. The final resulting suspension that contained 106 to 108 c.f.u./ml were taken for in-vitro studies.

Preparation of drug solution: The drug solution was prepared by dissolving the dry extracts of the roots of Pavetta indica in Dimethylformamide (DMF). The DMF was removed from the refrigerator one hour prior to its use and allowed to warm up to the room temperature. The solution of the test drug (extract) at the concentration of 10mg/ml in DMF was prepared. The solubility of the drug was facilitated by warming the drug solution to 60°C for 15 minutes while continuously shaking it. A drug solution of the standard drug ciprofloxacin (1mg/ m1) was also prepared in DMF. Solvent control DMF was maintained throughout the experiment.

Media used for the growth of bacteria was nutrient agar (NA) and nutrient broth (NB).

Nutrient agar media: The nutrient agar was weighed and dissolved in 1000ml of distilled water and adjusted to pH 7.4 to 7.6, autoclaved at 121°C at 15 lb pressure for 15 minutes and used for culturing bacteria.

Nutrient broth media: The nutrient broth was weighed and dissolved in 1000m1 of distilled water and adjusted the pH to 7.4 to 7.6, autoclaved at 121°C at 15 lb pressure for 15 minutes and used for culturing bacteria. In-vitro studies - two fold serial dilution technique: The seeded broth of respective organisms were prepared by transferring l ml of the sub-culture of the respective organism to 99ml of fresh sterile nutrient broth. This inoculated broth was subsequently used for the serial dilution technique.

Series of sterile tubes were placed and l ml of freshly inoculated broth was transferred aseptically. To the first tube another 0.8 ml of the same broth was added so as to make 1.8m1. previously in the first tube alone 0.2 ml of the test drug dissolved in DMSO at a concentration of 10mg/m1 was transferred. Now the first tube alone consists of 2 ml of suspension in it. This was properly mixed and about l ml was transferred to a second tube. The contents of the second tube was mixed thoroughly and l ml from this was transferred to the third and so on up to the fifth tube, so as to provide 1000μg/ml. 500μg/ ml, 250μg/ml, 125μg/ml and 62.5μg/m1 concentrations from the first tube to fifth tube respectively. The addition of the test drugs (all the seed extracts) and all ether manipulations were done under strict aseptic conditions. The control tubes with the solvent alone (solvent control) and also without the solvent, but with the inoculums (Growth control) were maintained throughout the experiment. Ciprofloxacin was used as the positive control. The whole study was carried out in triplicate.

The racks of the tubes were then incubated at 37°C for 24 hours. The observations were recorded at the end of 24 hours. The growth of the organisms were observed in the growth control tubes (only seeded broth) and the other tubes were observed for the inhibition of the growth of the organism was taken as the minimum inhibitory concentration (MIC) of the drug. This was further confirmed by plating also.

RESULTS AND DISCUSSION

Phytochemical studies: The shade dried roots of Pavetta indica were extracted with petroleum ether, chloroform, ethyl acetate, acetone, methanol and water by maceration (cold extraction).

The extracts obtained were subjected to chemical tests to find out the active constituents which showed the presence of Carbohydrates, glycosides, saponins, gums and mucilage and flavonoids in the aqueous extract.

• Carbohydrates, glycosides, saponins, gums and mucilage and flavonoids in the aqueous extract.

• Carbohydrates, glycosides, flavonoids and steroids in the methanolic extract.

• Flavonoids and glycosides in the acetone extract

• Triterpenoids in the petroleum ether extract and steroids in the petroleum ether and chloroform extracts.

Pharmacological studies:

Behavioural and toxicity studies: The ethanolic and aqueous extracts of the roots of Pavetta indica were studied for its acute toxicity and behavioural changes in mice. The extracts were given in various dose levels ranging from 400mg/ kg to 2000mg/kg of body weight (400, 800, 1200, 1600, 1800 and 2000 mg/kg).

All the extracts of Pavetta indica upto a level of 2000 mg/kg showed analgesic activity at the end of 1 hour and all these extracts showed central nervous system depression at the end of 2nd and 4th hour characterised by ptosis, sedation, loss of traction, loss of muscle tone, catatonia, ataxia and analgesia. There was no mortality at the end of 24 hours.

From the above studies, the therapeutic dose was fixed as 1/10th of the maximum tolerated dose as the safe dose for the further pharmacological studies. So it was decided to use 200mg/kg of each extracts of Pavetta indica.

Anti-inflammatory activity: The anti-inflammatory activity of the various extracts of the root of Pavetta indica were carried out by carrageenaninduced paw edema method. The extracts were administered at two dose level 400mg/kg and 200mg/kg body weight.

The results were interpreted based on the difference in the volume of displacement of mercury in plethysmograph after half an hour of post treatment of test extracts and immediately after administering carrageenan. This was compared and statistically calculated for significance with the solvent control which received 0.5% w/v CMC.

All the test extracts were administered orally half an hour before the injection of carrageenan. The difference in the volume of mercury displaced in the plethysmograph was calculated at the end of 1/2 an hour, 1, 2, 3 and 4 hours after injecting carrageenan. The percentage protection at the end of 2nd, 3rd and 4th hour was highly significant with alcoholic extract of the root of Pavetta indica at 400 and 200mg/kg. The aqueous extract was capable of producing significant antiinflammatory activity at 400mg/kg, but in 200mg/ kg 3rd and 4th hour it produced a less significant effect than the higher dose. So all the extracts at a higher dose range are equipotent with that of the positive control indomethacin 10mg/kg.

Analgesic activity: The analgesic activity of the ethanolic and aqueous extracts of Pavetta indica was carried out by Eddy’s hot plate method. The dose was fixed at 200mg/kg. The results were interpreted based on the difference in reaction time for the mice to lick its hind or fore paws or jump when placed on the hot plate between the pre-treatment and posttreatment. This was compared with the solvent control which received 0.5% w/v CMC.

The results showed that all the extracts possessed analgesic activity by prolonging the basal reaction time from 3.5 to 8.8 seconds at 200mg/kg dose level. At the end of 1 hour, the analgesic activity of the alcoholic extracts was comparable and equipotent with that of pentazocine 30mg/kg. The aqueous extract was considered as less potent than the alcoholic extract. After 2 hours the analgesic activity was found to be decreased and may require the next dose for maintaining the analgesic activity.

Anti-bacterial activity:

The ethanolic and aqueous extracts of the root of Pavetta indica were subjected to anti-bacterial studies. The gram positive organism Staphylococcus aureus was found to show intermediate susceptibility to the alcoholic extract of Pavetta indica. The MIC of the ethanolic and aqueous extracts of Pavetta indica was 500μg. But this activity was not significant when compared to the standard drug ciprofloxacin (MIC 7.8μg).

The gram negative organism Proteus vulgaris was found to be susceptible to the alcoholic extract of Pavetta indica. The MIC of alcoholic and the aqueous extracts of Pavetta indica was 500pg. (Ciprofloxacin MIC 7.8pg).

Escherichia coli and Klebsiella pneumoniae were found to be intermediately susceptible to the alcoholic extract of Pavetta indica. The MIC of alcoholic and the aqueous extracts of Pavetta indica was 1000μg. This activity was not significant when compared to the standard ciprofloxacin whose MIC is 15.6μg to E. coli and 7.8μg to K. pneumoniae.

All the five organisms were found to be resistant to the aqueous extract of Pavetta indica and Pseudomonas aeruginosa (gram -ve) was also found to be resistant towards the alcoholic extract of Pavetta indica.

From the above studies it is evident that the organism Proteus vulgaris (gram -ve) is found to be susceptible, the other organisms Staphylococcus aureus (gram +ve), Escherichia coli (gram -ve) and Klebsiella pneumoniae (gram -ve) were found to be intermediately susceptible to the alcoholic extract of Pavetta indica.

CONCLUSION

The pharmacological studies of the different extracts showed that all extracts possessed significant anti-inflammatory activity and analgesic activity to a varying extent. It shows that the steroids, saponins and flavonoids present in these extracts may be possibly responsible for the pharmacological action.

The antibacterial activity of the alcoholic extract was found to be better than the aqueous extract of the plant. This may be probably due to the dissolution of the active constituents in the organic solvent.

Further studies to confirm the anti-inflammatory activity with varying dose levels and with varying acute models will suggest the mode of action. Also the analgesic activity can be carried out with the chemical induced (acetic acid included) model which will differentiate whether it has any peripheral analgesic activity.

Isolation of the active constituents with further techniques and studying their pharmacological, microbiological and toxicity studies will provide a further area for the researchers in future.