INTRODUCTION:

Nature is the best combinatorial chemist and possibly has answers to all diseases of mankind. Failure of some synthetic drugs and its side effects have prompted many researches to go back to ancient healing methods which use herbal medicines to give relief. Till now, natural product compounds discovered from medicinal plants have provided numerous clinically useful drugs. Four billion people or about 80% of the world’s population uses herbal medicine as a part of health care¹. Human brain disorders range which includes Alzheimer’s disease, Parkinson’s disease, depression, epilepsy, schizophrenia, anxiety, Huntington’s disease etc. Psychotherapeutics does not meet properly for therapeutics possibilities for majority of patients with mental health problems but herbal remedies are ultimate therapeutic hope for such patients². Many synthetic drugs because of many unwanted but unavoidable side effects have poor patient compliance. Therefore herbal treatment is being preferred over conventional treatments.

Much attention and so scope is drawn towards herbal remedy of many brain disorders³. Anxiety affects one eighth of the total population of the world and has become a very important area of research interest in psychopharmacology during this decade. Folk medicines have particular values and plants have long been used to treat central nervous system disorders⁴. Leptadenia reticulata is a twining shrub with yellowish colour and green colour flower. Its leaves and roots are useful in cooling, nutritive, stimulant, diuretic, galactogogue, cures eye diseases etc⁵. Mimusops elengi is an ever green tree with creamy flowers and thick brown coloured bark. They are useful in astringent action, dental ailments, cooling, brain tonic, constipation etc⁶. Evolvulus alsinoides is a shrub with more branched leaves useful in nervous exhaustion, memory loss, nootropic agent, improves brain function, concentration etc⁷.

MATERIALS AND METHODS:

Plant Material

Coarsely powdered materials of the plants Leptadenia reticulata (rhizomes), Evolvulus alsinoides (roots), were collected from SKM Siddha and Ayurvedha Company (India) Limited, Erode and Mimusops elengi (bark) was procured from Chakrapani Ayurveda Clinic and Research Center, Jaipur.

Preparation of plant extract

Equal amount of the weighed coarse powders were used for the extraction by successive solvent extraction by Soxhlet apparatus using various solvents. The powder was defatted with petroleum ether and successively extracted with acetone and ethanol. Extract was filtered, concentrated under reduced pressure, dried and the percentage yield was calculated. Phytochemical test was performed to identify the phytoconstituents present in the extract⁸.

Animals

Swiss albino mice (male: 20-25gm) and rats (200-220gm) were used in the present study. The animals were procured from Venkateshwara Enterprises, Bangalore, India. They were provided normal diet and tap water ad libitum and were exposed to 12-h light and 12-h dark cycle. The animals were acclimatized to the laboratory conditions before experiments. Experimental protocol was approved by Institutional Animal Ethical Committee. Care of the animals was taken as per guidelines of the Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA). Experiment was approved by Institutional Animal Ethics Committee.

Toxicity study

Toxicity testing is of paramount importance while screening drugs. Toxicity studies are conducted with the assumption that man will behave in the same manner as the animals. The procedure was followed by using OECD guidelines (Organization of Economic Corporation and Development) 423 (Acute toxic Class method). The method used defined doses (2000 mg/kg, p.o.) and results allow a substance to be ranked and classified according to the Globally Harmonized System (GHS) for classification of chemical which cause acute toxicity. Three male mice 18-25 gm were used for the study, since the herbal extracts are relatively non toxic, the starting dose level of extracts were 5, 50, 300, 2000 mg/kg/bw/p.o. The drug was administered orally to mice which were fasted 3- 4 hrs with water ad libitum before administration of the drug. Body weights of the mice before and after treatment were noted. Any changes in skin and eyes and mucous membrane and also respiratory, circulatory, autonomic, CNS, motor activity, behavioural pattern were observed. And also sign of tremors, convulsion, salivation, diarrhoea, lethargy, sleep and coma were noted⁹.

Pharmacological Screening^{10,
11, 12}

Elevated plus maze

The plus maze was in the shape of a cross or plus with two closed arms each with roof open measuring 30×5×20cm, extending from a central region (5×5) running along a north-south axis and two open arms each measuring 30×5 cm running east-west. The wooden apparatus was elevated to a height of 50cm from the floor in a dimly illuminated room. Mice were placed individually in the central area of the maze with open access to any arm. The amount of time spent on and number of entries in both open and closed arms whereas number of stretch attend postures and head dips in closed arms were measured manually during the 5min test period. An arm entry was defined as all four feet in the arm. Stretch attend posture was defined as mice stretching forward and then retracting to original position from closed (protected) or open (unprotected) arms. Head dipping is defined as mice protruding the head over the edge of closed or open arms down towards the floor. The apparatus was cleaned after each mouse was tested to remove any residue or odour. For the purpose of analysis, open arm stay was quantified as the amount of time that the mouse spent in open and closed arm (open/total×100) and open arm entries were quantified as the number of entries in open and closed arm(closed×total×100).

Rats were treated with the extracts (200 mg/kg, p.o.) and vehicle for 7 days once daily p.o. and the last dose was given on the 7^th day, 60 min before starting the experiment. The standard drug Diazepam was given at a dose of 2 mg/kg, p.o. 60 min before starting the experiment. After proper treatment each rat was placed at the walled region of the maze with its head facing the open arm. During the 5 min experiment, the behavior of the rat was recorded as time spent in open and closed arm and total number of entries in open and closed arm. An arm entry was defined as the entry of all four paws into the arm. After each trial, the elevated plus maze apparatus was wiped clean with ethanol (10%) solution. Group-I: Animals received distilled water (10ml/kg, p.o.), Group-II: Animals received Diazepam (2mg/kg, p.o.), Group-III: Animals received acetone extract, (200mg/kg, p.o), Group-1V: Animals received acetone extract (200mg/kg p.o.).

Stair case method

The staircase is composed of five identical steps 2.5 cm high, 10 cm wide and 7.5 cm deep. The internal height of the walls is constant along the whole length of the staircase. Each animal is used only once. At the end of experimental period mice were placed individually on the floor of the box with its back to the staircase. Total number of steps climbed and total number of rearings were recorded over a period of 3 min. A step is considered to be climbed only if the mouse has placed all four paws on the step.

Mice were treated with the extracts (200 mg/kg, p.o.) and vehicle for 7 days once daily p.o. and the last dose was given on the 7^th day, 60 min before starting the experiment. The standard drug Diazepam was given at a dose of 2 mg/kg p.o. 60 min before starting the experiment. During the 5 min experiment, the behavior of the mice was recorded as time spent in each step and total number of climbing’s in all steps. A climb entry was defined as the entry of all four paws in the stair case. After each trial, the stair case-maze apparatus was wiped clean with ethanol (10%) solution. Group-I: Animals received distilled water (10 ml/kg, p.o.), Group-II: Animals received Diazepam (2mg/kg, p.o.), Group-III: Animals received acetone extract, (200mg/kg, p.o), Group-1V: Animals received acetone extract (200mg/kg p.o.).

Mirror chamber method

The apparatus consists of a mirrored cube open on one side that is placed inside a square wooden box. The mirrored cube measuring 30cm on a side is constructed of five pieces of mirrored glass with one mirrored side and an opposite side painted dark brown. The three mirrored side panes, a top pane and the floor pane faced the interior of the cube. The container box is 40×40×30.5cm.The mirrored cube is placed in the centre of the wooden container to form a 5cm corridor that completely surrounded the mirror chamber. A mirror is also placed on the container wall so that it faced the single open side of the mirrored chamber. The other three walls of the container are painted dark brown. Stop watch is use for recording the activity.

Mice were treated with the extracts (200 mg/kg p.o.) and vehicle for 7 days once daily p.o. and the last dose was given on the 7^th day, 60 min before starting the experiment. The standard drug Diazepam was given at a dose of 2 mg/kg i.p. 60 min before starting the experiment. After proper treatment each mice were placed at the walled region of the apparatus with its head facing the chamber. During the 5 min experiment, the behavior of the mice was recorded as time spent in and out and total number of entries into the chamber. An entry was defined as the entry of all four paws into the chamber. After each trial, the mirror chamber apparatus was wiped clean with ethanol (10%) solution. Animals were placed individually in the chamber of mirrors at a fixed corner and start the stop watch and the following parameters were noted for five minutes: 1. Latency to enter the mirror chamber, i.e., the time in seconds for the first entry into the chamber of mirrors, 2. Number of entries in mirror chamber during five minutes, 3. Total time in seconds spent in the mirror chamber during the 5minute test period.

Neurotransmitter estimation¹³

Rats were sacrificed quickly by using decapitor. The brains were rapidly removed and Tissue was stored in –80° C till the homogenization. Extraction of 5-HT, dopamine and noradrenaline was carried. All estimations were done by using spectrofluorphotometer (Model no: RF- 1501, SHIMADZU). For the estimation of 5-HT, the extracted sample was acidified with 3N Hcl and their native fluorescence was measured at 295/535 nm with respect to blank.Noradrenaline was estimated by adding 0.5 ml 2M pH 6.8 acetate buffer; 0.1 ml I₂ solution; 0.15 ml 0.1 Na₂S ₂O₃.5H ₂O₂; and 0.2 ml alkaline ascorbic acid/ ethylenediamine solution in a sequential order to 0.5 ml of extracted brain sample. The last reagent alkaline ascorbic acid/ethylenediamine solution is very viscous and must be thoroughly mixed. The final sample volume was 1.45 ml. Maximal fluorescence was developed within about 35 min and fluorescence was read at 400/510 nm with respect to blank. For estimating dopamine, 0.5 ml 2M acetate buffer, pH 6.8 (the final pH was about 6.5); 0.1 ml 0.1 N I₂ solution; 0.2 ml alkaline sodium sulphate/ EDTA solution; and 0.25 ml 1:1 glacial acetic acid/concentrated HCl reagents were added in sequential order to 0.5 ml of extracted sample. The tubes were placed in boiling water bath for 45 min, after which they were allowed to cool to room temperature before reading. Fluorescence was read at 335/380 nm with respect to blank.

Statistical analysis

Results were represented as mean+SEM. Data was analysed using a statistical package(Graph pad prism version 3.00 to Windows, Graph pad software, San Diego, California, (USA).Comparison between groups were made using one-way analysis of variance (ANOVA) a post-hoc comparisons were performed using Tukey-multiple comparison test.

RESULTS:

Elevated plus maze

Administration of diazepam 2mg/kg significantly increased the amount the time spent in the open arms and the percentage of open arm entries (p<0.001) compared to the control group was mentioned in Table – 1. Acetone and ethanolic extract of polyherbs at the dose of 200mg/kg significantly increased the time spent in the open arms.

Table – 1. Anxiolytic activity by elevated plus maze method

S. No	Treatment	Time spent (s)		Entries in open arm
S. No	Treatment	Open	Closed	Entries in open arm
1	Control	103.33±2.629	30.00± 2.066	26.00± 1.932
2	Diazepam (2mg/kg, p.o)	119.167±2.857*	17.833± 1.682*	54.00± 1.549*
3	Acetone extract (200mg/kg, p.o.)	103.5± 2.062*	18.667± 0.211*	51.333± 1.333*
4	Ethanolic extract (200mg/kg, p.o.)	93.00± 1.983	24.667± 0.615	33.833± 0.833

n=6, Values are mean ± SEM. Differences were assessed statistically using one-way ANOVA followed by Tukey’s test. *(p<0.001)

Stair case test

Stair case maze was another test for anxiety related behaviour that had been validated using different anxiolytics. In this method mouse explore a rectangular maze that has six steps. The number of steps climbed and the rearing of the mice were recorded as measurement of anxiolytics related behaviour. Acetone and ethanolic extract treated mice showed more step ups and rearing events compared with the standard drug diazepam. The number of steps climbed and rearing was the primary index of anxiolysis in this test was found to be significant (p<0.001). The results were shown in Table-2.

Table – 2.Anxiolytic activity by stair case method

S.No	Treatment	Climbing	Rearings
1	Control	10.33±0.498	10.16±0.354
2	Diazepam (2mg/kg, p.o)	5.5±0.490*	2.16±0.179*
3	Acetone extract (200mg/kg, p.o.)	8.00±0.621*	4.16±0.231*
4	Ethanolic extract (200mg/kg, p.o.)	6.83±0.378*	3.16±0.231*

n=6, Values are mean ± SEM. Differences were assessed statistically using one-way ANOVA followed by Tukey’s test. *(p<0.001)

Table – 3. Anxiolytic activity by mirrored chamber apparatus

S.No	Treatment	Latency of the first entry into the chamber(s)	Total time spent inside the chamber(s)	No of entries into the chamber
1	Control	14.297±0.505	12.772±0.804	5.167±0.703
2	Diazepam (2mg/kg, p.o)	0.195±0.016*	21.113±0.425*	2.667±0.333*
3	Acetone extract (200mg/kg, p.o.)	0.222±0.009*	35.773±1.092	4.833±0.307*
4	Ethanolic extract (200 mg/kg, p.o.)	0.462±0.052*	40.285±0.713	5.667±0.667*

n=6, Values are mean ± SEM. Differences were assessed statistically using one-way ANOVA followed by Tukey’s test. *(p<0.001)

Table – 4. Estimation of biogenic amines in axiolytic state

S.No	Treatment	Norepinephrine(ng/gm)	Dopamine(ng/mg)	5-HT(ng/gm)
1	Control	2.042±0.071	0.912±0.090	1.482±0.082
2	Diazepam (2mg/kg, p.o)	1.172±0.042*	0.528±0.071*	2.225±0.093*
3	Acetone extract (200mg/kg, p.o.)	1.763±0.083*	0.642±0.92*	1.861±0.091*
4	Ethanolic extract (200mg/kg, p.o.)	1.321±0.051	0.638±0.89	1.970±0.051

n=6, Values are mean ± SEM. Differences were assessed statistically using one-way ANOVA followed by Tukey’s test. *(p<0.001)

Mirror chamber method

In this method treatment with the extracts significantly (p<0.001) shortened the latency to enter mirror per entry and increased average time spent per entry in mirror chamber as compared to standard. Increased time spent and number of entries was consistent with anxiolytic activity. The decreased in aversion is the result of an anxiolytic effect expressed by the increased time spent and entries. The primary index was spatiotemporal in nature; it was reduced by anxiolytic drugs and increased by anxiogenic compounds. The decrease in time spent was another indication of reduced “decision making” behaviour. Both parameters were accepted as reliable indicators of anxiety and fearfulness. The results were shown in Table-3.

DISCUSSION:

The extractive value indicates the yield of the extract obtained from the air dried plants powder by successive solvent extraction by Soxhlet extraction. Their percentage yield shows the solubility of the active principles in the organic solvents used based upon the polarity nature. They are then identified and confirmed by the preliminary phytochemical evaluation. Percentage yield of acetone extract was 25.2%W/W and the ethanolic extract was 20.4%W/W. The extract which shows the presence of many phytoconstituents were analysed instrumentally. The presence of the phytoconstituents like alkaloids, flavonoids, carbohydrates, phytosterols, proteins&amino acids, gums&mucilages and resins were responsible for the typical pharmacological effects. The purpose of acute toxicity studies was to determine the LD₅₀ values which helps in determining the safe dose range at which the drug can be used such that there is no harmful or lethal effect on the animal. The LD₅₀ determination was done in mice by OECD guideline 423 and LD₅₀ of extracts were determined (infinity). In this study there was no toxicity/death were observed at the dose of 2000mg/kg body weight in animals. The acute toxicity study showed that at 200mg/kg dose the extracts are safe for consumption and for medicinal uses. The therapeutic dose of the drug was considered as 1/10^th of the LD₅₀ value. Hence, the therapeutic dose used for recording biological response was 200mg/kg, p.o for the extracts.

Benzodiazepines have been extensively used for the last 40 years to treat several forms of anxiety but due to their unwanted side effects, alternative treatment strategies with favourable side effect profiles, credible benefits and moderate costs of interests, especially in primary care settings¹⁴. Medicinal plants are the good sources to find new remedies for these disorders. In the search for an alternative more specific and perhaps cost free therapy, research has been conducted to investigate rational anxiolytic drugs as well as new antidepressant principles. The effects of the acetone and ethanolic extract at 200mg/kg on EPM, stair case and mirror chamber tests were almost equivalent to that of diazepam 2mg/kg. These observations clearly indicate that polyherbs extract exerts anxiolytic activity¹⁵.

To enhance the synergistic action of the herb treatment, the selected suitable herbs were mixed in proper ratio and extracted. The results revealed the synergistic action of the polyherbs, as they produced significant anxiolytic action¹⁶. The phytoconstituents may act through the anxiolytic potential of benzodiazepine/GABA_A receptor related agents while these chemical constituents have been reported to be responsible for anxiolytic and sedative effects observed in different plat extracts. The flavonoid apigenin to the central benzodiazepine receptor possesses important anxiolytic action¹⁷. Despite the fact that the substance with binding abilities was still unknown, leaving undefined whether there was an antagonist or agonist relation upon the receptors, the information was important because cholinergic neurons located in the septum participate in mood states such as: waking up, the motivation and vegetative function through nervous pathway that receive innervations from mescencephalic structures and the brain stem.

Anxiolytics are known to exert their pharmacological action causing an increase in GABA content in brain cerebral hemisphere. It was found that the extracts significantly enhanced the brain GABA concentration which again was synergistic of an anxiolytic action of the herbs. The extract changes the behavioural action correlated to increase in GABA concentration in brain and related in part to the highly sensitive benzodiazepine site of the GABA benzodiazepine receptor chloride ionophore complex¹⁸. Table – 4 represents the neurotransmitters level in rat brain after administration of the acetone and ethanolic extracts. Norepinephrine level was significantly (p<0.05) decreased in brain. A significant (p<0.05) elevation of dopamine concentration was observed with the extract treated animals. Serotonin concentration in brain of rats was significantly decreased compared to control rats. Increased 5-HT activity has been linked to anxiety^{19, 20}.

In summary, the acetone and ethanolic extract of Leptadenia reticulata roots (Asclepiadaceae), Mimusops elengi bark (Sapotaceae) and Evolvulus alsinoides Whole plant (Convolvulaceae) showed significant and potent anxiolytic activity. Further pharmacological and chemical investigations are required to elucidate the exact mechanism of action of these extracts to isolate the active principles responsible for such effects.

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Received on 05.08.2014 Modified on 19.08.2014

Res. J. Pharmacognosy & Phytochem. 7(1): Jan.-Mar. 2015; Page 01-05

DOI: 10.5958/0975-4385.2015.00001.1