INTRODUCTION:

Depression is a common mental disorder that presents with depressed mood, loss of interest or pleasure, feelings of guilt or low self-worth, disturbed sleep or appetite, low energy and poor concentration. These problems can become chronic or recurrent and lead to substantial impairments in an individual's ability to take care of his or her everyday responsibilities. At its worst, depression can lead to suicide, a tragic fatality associated with the loss of about 850 000 lives every year ^[1].

A growing number of herbal medicines are being introduced into psychiatric practice, many of which have comparable efficacy to prescription medications with lower side effects. This makes herbal therapy as desirable alternative treatment for severe depression and anxiety disorders. People from different regions of the world have used herbal medicines to alleviate disorders for many years. Medicinal plants exudates, gums, resins etc were used for the treatment of different diseases. An increasing number of herbal products have been introduced into psychiatric practice, as alternative or complementary medicines (Woode et al., 2010). Similarly, the therapeutic potential of a proving number of herbal medicines has been assessed in several animal models (Zhang, 2004). In fact, these models have contributed to the screening of new psychopharmacological tools and to the understanding of their biological activity (Buller and Legrand, 2001).

Cycas circinalis (Madana Kama raja) was a palm like short sago, dark brown, unbranching plant found in southern parts of India. It was used to treat mental disorders traditionally and found to be effective against Parkinsonism dementia ^[2], treatment of stress^[3] and bacterial infections ^[4]. Artemisia absinthium (Macipattiri) was an herbaceous, perennial plat with hard, woody rhizome and used in earlier days to treat many disorders. Earlier report suggests this herb possess antioxidant ^[5], Antiparasitic ^[6], hepatoprotective ^[7], neuroprotective ^[8], memory enhancer ^[9] in the preclinical research. Nardostachys jatamansi (Jatamashi) flowering plant of the valerian family with many CNS uses like anticonvulsant^[10], catalepsy ^[11], enhancing memory ^[12] and antidepressant ^[13]. 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 ^[14]. Toxicity studies are of the acute toxicity, subacute toxicity and chronic toxicity studies. The aim of the present work is to evaluate the toxicity of the polyherbal Cycas circinalis (Madana Kama raja), Artemisia absinthium (Macipattiri) and Nardostachys jatamansi (Jatamashi) ethanolic extract and to find out the therapeutic dose by acute and sub acute toxicity method and to screen antidepressant activity in another research and to report on its pharmacological mechanism. The purpose of the combined polyherbal treatment is to improve the synergistic action of the herbal medicines for the treatment of depression. Synergistic action of the drug to cures the disease at the faster rate and now a day all drugs are become resistant as the practitioners move to the combination therapy for the ailments. Thus, to prove the synergistic effect of the above selected plants the present study is carried out.

MATERIALS AND METHODS:

Plant collection –

Coarsely powdered materials of the plants Cycas circinalis (flower), Nardostachys jatamansi (roots), Artemisia absinthium (whole plant) were collected from SKM Siddha and Ayurvedha Company (India) Limited, Erode, Tamil Nadu, India.

Extraction –

Equal amount (250gm) of the weighed coarse powder of each plant part was mixed primarily and used for the extraction by successive solvent extraction by Soxhlet apparatus using various solvents (Petroleum ether, Chloroform, Acetone, Ethanol and Water -Cold maceration). From the weight of each extractive residue, the extractive values were calculated in percentage. All the above extracts were used for identification of constituents by preliminary phytochemical tests ^[15],
[16].

TOXICITY STUDY

Animals

The experimental protocol was approved by Committee for the Purpose of Control and Supervision of Experiments on Animals and Institutional Animal Ethics Committee (IAEC) Registration number (1012/c/06/CPCSEA) of RVS College of Pharmaceutical Sciences, Sulur, Coimbatore - 641402. Swiss albino male mice weighing 20-25gm and Albino wistar rats of either sex weighing 160 -180 gm each were housed at 24±2°C with 12:12 hour light and dark cycle. They had free access to food and water ad libitum. The animals were acclimatized for a period of 7 days before the study. All the experiments were carried out between 10.00 to 16.00 hour at ambient temperature. The animals were drawn at random for test and control groups.

Acute toxicity study

Acute toxicity study is generally carried out for the determination of LD₅₀value in experimental animals. The LD₅₀determination was done in mice by OECD guideline 423 acute oral Class method. The aim of performing acute toxicity study is for establishing the therapeutic index of a particular dug and to ensure the safety. Adult albino mice (25-30gm) were chosen for the study. They were maintained as per the standard laboratory conditions and provided with normal chow diet and water ad libitum. At the day of the experiment the animals were fasted for 12 hours and the extract was premixed with 1% gum acacia at the doses of Group I 5mg/kg, Group II 50mg/kg, Group III 300mg/kg and Group IV 200mg/kg were administered per orally (3 animals per group). 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 noted ^{[17], [18]}. The abnormal signs of the animals were comparable with the control group animals Group VI.

GROSS BEHAVIOURAL STUDIES

Central actions of the extracts were analysed by the method described by Irwin. Briefly, animals were observed for 30min after oral administration of the extracts. The procedure involved an initial phase of undisturbed observation and a later manipulative phase during which animals were subjected to the least provocating stimuli. In the initial phase, the animal was observed for body position, locomotion, rearing, respiration, salivation, urination, skin colour, tremors, staggering and gait. In the later phase, the effect on grip strength, limb tone, abdominal tone, passivity, writhing reflex, pinnal reflex, corneal reflex, ipsilateral flexor reflex (IPR), pupil size, pain response and lacrimation were also observed.

Scoring

The effects of the test substance on the animal were scored with the use of nine degrees, with a scale ranging from 0 to 8. Scoring was performed at the time of peak. The basal score for normal signs or effects was 4 for supranormal. The basal score for abnormal signs were 0 and the maximal score was 8.

The profile was divided into three parts behavioural, neurological and autonomic.

Behavioural profile

· Awareness - The alertness or stupor was recorded.

· Visual placing, stereotypy, passivity was scored.

The mouse was grasped with thumb and index finger in order to hold the dorsal skin. In this situation, the mouse was held in a walking position. The scores were as follows:

Score 0 An unaffected mouse moves its head and limbs and tries to escape.

Score 2 : If the mouse, still grasped in the same manner, held in vertical position, it struggles.

Score 4 : When the unaffected mouse is placed in the supine position on the back of the observer’s hand in first so that the thumb can support the mouse’s head, it tries to escape.

Score 6 : The unaffected mouse tries to escape when held vertically by one fore paw.

Score 8 : The unaffected mouse tries to escape when held vertically by one fore paw or by one hind paw.

Mood

The unaffected mouse grooms itself frequently. Its grooming was scored 4. Vocalization (basal score 0) may point to a noxious stimulus. Restlessness (basal score 0) was absent in unaffected mouse. Irritability (basal score 0) was an extension of restlessness. Fearfulness (basal score 0) was also recognised.

Motor Activity

This includes spontaneous activity (basal score 4) of the mice when placed in a bell jar. If the animal sleeps score was 0, if there was little activity the score is 1. Lesser activities were scored as 2 and 3. Excessive inquisitive activity was scored 6, constant walking (score 6), walking with some running (score 7), agitated spurts (score 8).

A similar test was performed with the same scoring, where the animals were removed and placed on a table. This was called reactivity (basal score 4).The touch response (basal score 4) when the animal was touched with a pencil or forceps at various parts. The pain response (basal score 4) was graded when a small artery clamp was attached to the base of the tail.

Neurological Profile

Central Excitation

The startle response (basal score 0) of the animal to a loud noise was recorded. The degree of elevation of tail was recorded as straub response (basal score 0).The degree of tremor (basal score 0) and the convulsions (basal score 0) were recorded.

Motor Inco-ordination

The body position (basal score 4) and the limb position (basal score 4) were noted. A staggering gait (basal score 0) may indicate ataxia, abnormal gait (basal score0) may indicate muscular relaxation or may be related to ataxia.

The somersault test (basal score 0) was also performed. The mouse was picked up by tail and tossed in the air so that a somersault of 2 and 3 turns made before landing upon a pad of sponge rubber. This procedure was repeated until 5 trials have been made, the scoring was as follows:

Standing on four feet in all trials: 5/5, score 0

Lying on one side: If 1/5 or 2/5, score is1, If 3/5 or 4/5, score is 2, If 5/5 score is 3

Lying on back: If 1/5 or 2/5, score is 4, If 3/5 or 4/5, score is 5, If 5/5 the score is 6

Slowly regaining from a supine or side position, the score is 7.

Remaining on the back score is 8.

Muscle Tone

The limb tone (basal score 4) was estimated by grasping a forepaw of the mouse and noting the resistance to extension of the paw. The grip strength (basal score 4) measured by allowing the animal to grasp a pencil in the horizontal position and noting the ease with which the animal drops to the table. The body tone (basal score 4) and the abdominal tone (basal score 4) were estimated by noting the muscle tension in comparison with control animals.

Reflexes

The pinna (basal score 0) reflex was tested by touching the center of the pinna with a hair or other fine instrument. For eliciting corneal (basal score 4) reflex, a stiff hair touched the cornea which causes the animals to withdraw. In the ipsilateral flexor (basal score 4) reflex, a toe-pad was compressed with a forceps which causes the animal to flex its leg in a retiring movement/position.

Autonomic Profile

Ocular Signs

The pupil size (basal score 4) was compared before and after administration of drug. A wide palpebral opening (basal score 4) indicates sympathomimetic activity and a narrow one sedative activity. Exopthalmos (basal score 4) indicates sympathetic stimulation.

Secretary signs

The urination (basal score 0) of the animal was noted. Salivation (basal score 0) was also observed.

General signs

Writhing (basal score 0) was checked in animals. Hypothermia, skin colour (basal score 4), respiratory rate (basal score 4) were also recorded.

Sub acute toxicity study

The cumulative toxicity of a substance on target organs or physiological and metabolic effects at low dose on prolonged exposure can be studied by sub acute toxicity. The results from sub acute toxicity studies can provide valuable information, which helps in selecting dose levels ^[19]. The long term safety level of a compound can be predicted from acute or shorter than subacute studies. Hence, 1/20^th (100mg/kg), 1/10^th (200mg/kg) and 1/5^th (400mg/kg) of doses were selected for the sub acute study. Swiss adult albino rats (180 – 210gm) were used. At the day of the experiment the animals were fasted for 12 hours and the extract was premixed with 1% gum acacia at the doses of Group I Control, Group II 100mg/kg, Group III 200mg/kg and Group IV 400mg/kg was administered per orally. Each group contains 6 animals. At the 28^th day end of the study animals were sacrificed blood was collected for haematological and biochemical studies. Vital organs like liver, kidney, heart and brain was isolated and weighed. Histological examination was carried out for liver and kidney ^{[20], [21]}.

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 was made using one-way analysis of variance (ANOVA) post-hoc comparisons were performed using Tukey-multiple comparison test.

RESULTS:

The extractive value indicates the yield of the extract obtained from the air dried plant 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 showed the presence of alkaloids, flavonoids, phytosterols, terpenoids, carbohydrates, and phenolic compounds. The purpose of acute toxicity studies is 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 animals did not show any specified abnormal signs in the parameters assessed which were given in the Table – 03. All the scores were normal and the extract showed non toxic signs in the behavioural profile of the animals and the results were shown in Table – 04. No noticeable weight change or weight variation was seen in the animal’s body weight and also there was no change in the weight of vital organs of the animals (Table – 05 AND 06). The haematological and the biochemical parameters of the animals were found to normal and there were no significant changes (Table – 07AND 08). No remarkable changes in the kidney and liver cells, even at the high dose of 400mg/kg, which clearly shows that the smaller doses of the extract were safe and non-toxic.

Table – 1: Extraction of Air dried plant materials of Artemisia Absinthium, Cycas Circinalis and Nardostachys Jatamansi

Plant	Part used	Method of Extraction	Solvents	Average value of extractive (%W/V)
Artemisia absinthium	Whole plant	Continuous Hot percolation by Soxhlet apparatus and Aqueous extraction by Cold maceration	Petroleum ether (60-80°C)	10.11
			Chloroform	3.2
Cycas circinalis	Flower		Acetone	1.2
Cycas circinalis	Flower		Ethanol (95%)	21.4
Nardostachys jatamansi	Root		Water	22.3

Table – 2: LD₅₀value of the ethanolic extract

Animal used – Swiss albino mice Weight of animals – 20-25gm

No. of animals – 03; Route of administration – per oral

No. of animals	Dose	No. of death of animals
3	5mg/kg	0
3	50mg/kg	0
3	300mg/kg	0
3	2000mg/kg	1

LD₅₀– 2000mg/kg; ED₅₀– 200mg/kg

Table – 3: Data showing the effect of ethanolic extract on the specified parameters on mice

Muscle tone						Reflexes
Treatment	Dose	Limb tone	Grip strength	Body tone	Abdominal tone	Pinna	Corneal	IPR	Writhing	Pupil size
Normal score	_	4	4	4	4	4	4	4	0	4
Ethanolic extract	100
	200
	400

Table-3…Cont…

Autonomic profiles
Palpebral opening	Exopthalmos	Urination	Salivation	Skin Colour
4	0	0	0	4

Dose in mg/kg, p.o. The scores of normal responses are not indicated.

Table – 4: Data showing the effect of ethanolic extract on behavioural profile of mice

Awareness
Treatment	Dose	Alertness	Visual placing	Passivity	Stereotypy
Normal score	mg/kg	4	4	0	0
Ethanolic extract	100
	200
	400

Table – 4:… Cont….

Mood					Motor activity
Grooming	Vocalization	Restlessness	Aggression	Fearfulness	Reactivity	Spontaneous activity	Touch response	Pain response
4	0	0	0	0	4	4	4	4

Table – 4:… Cont….

CNS excitation					Posture		Motor incordination
Startle response	Straub tail	Tremors	Twitches	Convulsions	Body posture	Limb position	Staggering gait	Abnormal gait	Righting reflex
0	0	0	0	0	4	4	0	0	0

Dose in mg/kg, p.o. The scores of normal responses are not indicated.

Table – 5: Effects of Extracts on Weight (g/100g) of Vital organs of Rat

Extract	Dose (mg/kg)	Liver	Kidney	Heart	Brain	Body weight (gm)
Extract	Dose (mg/kg)	Liver	Kidney	Heart	Brain	Initial	Final
Control	-	4.50±0.40	1.40±0.24	0.64±0.10	1.82±0.28	180	179
Ethanolic extract	100	4.45±0.42	1.41±0.21	0.66±0.11	1.80±0.25	180	179
	200	4.56±0.41	1.42±0.25	0.64±0.14	1.84±0.26	181	179
	400	4.53±0.40	1.52±0.20	0.67±0.12	1.82±0.29	210	212

n=6 animals in each group, P<0.001

Table – 6: Effects of extracts on Haematological Parameters in Rat

Extracts	Dose (mg/kg)	RBC (10⁶/cumm)	Total WBC (10³/cumm)	ESR (mm/1^st hr)	Clotting time (sec)	PCV (%)	Hb (g %)
Control	-	4.6±0.57	7.3±0.22	3.5±0.4	111±10.2	41.1±2.82	13.76±0.66
Ethanolic extract	100	4.61±0.45	7.48±0.24	3.33±0.8	106±9.9	45.41±4.71	13.56±0.65
	200	4.67±0.50	7.67±0.21	3.32±1.00	109±10.2	46.31±2.84	13.86±0.67
	400	4.69±0.51	7.54±0.20	3.4±1.0	110±10.3	47.1±2.85	13.76±0.56

n=6 animals in each group, P<0.001

DISCUSSION:

The purpose of acute toxicity studies is 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 2000 mg/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 200 mg/kg, p.o for the extracts. The phytochemical tests showed the presence active phytoconstituents in the crude extracts. The presence of phytoconstituents like alkaloids, flavonoids, carbohydrates, tannins, phytosterols, proteins and amino acids, gums and mucilage and resins are responsible for the typical pharmacological effects.

The cumulative toxicity of a substance on target organs or physiological and metabolic effects at low dose on prolonged exposure can be studied by sub acute toxicity. The results from sub acute toxicity studies can provide valuable information, which helps in selecting dose levels. The long term safety level of a compound can be predicted from acute or shorter than subacute studies. Sub acute toxicity studies are generally carried out for a period ranging from a few days to three months. Rats treated with extracts were observed daily for a period of 14 days for signs of toxicity and/or mortality. None of the animals exhibited any abnormal behaviour in any dose range tested. Also, there was no change in fur coating, eyes and respiratory functions. There was no significant difference in food and water consumption between the treated and the control groups. Extract treated animals produced moderate increase in bodyweight of the animals over a period of 28 days. In addition, changes in weight of organs such as liver, kidney, heart and brain were observed. Haematological and biochemical parameters were also analysed. Histopathological changes produced if any on vital organs were studied.

These observations presented in below

Parameter Results

Bodyweight - Moderately increased

Weight of vital organs- No change

Haematological studies- No toxicity

Biochemical studies- No toxicity

Histopathological studies- No change

Control

Ethanolic extract treated 400mg/kg

Fig – 01: T.S. of Rat liver showing normal structures treated with ethanolic extract

Control

Ethanolic extract treated 400mg/kg

Fig – 02: T.S. of Rat kidney showing normal structures treated with ethanolic extract

The study revealed that extracts did not show any adverse effect on the growth of the animals tested. There was no change in the weight of the vital organs. All these confirm that the extracts were free from adverse effects and there was no mortality when administered at a very high dose, thus proving the safety of the plant extracts.

Liver is the principal and most important organ for drug metabolism and kidney is the major organ for drug excretion. Hence, liver and kidney were subjected to histopathological examination. These studies show that, the liver of animals treated with the extracts showed normal histopathological features at low and high doses as reflected by absence of congestion of hepatocyte and focal steatosis. There was absence of congestion of central vein and inflammation of portal tract when compared with control. They show normal epithelial cells, no inflammation, no congestion, no necrosis and no cell damage when compared to the controls. Gross examination of liver and kidney did not reveal any abnormalities.

After 28 days there were also no treatment related changes in haematological parameters between control and treated groups indicating the extracts were non toxic to the circulating cells and not interfered with their production. In addition most of the biochemical parameters were also unchanged by the doses given. The relevance of this result may be associated with the biological value of the plants extracts. The present investigation demonstrates that at doses consumed in the extracts may be considered as relatively safe, as it did not cause either any lethality or changes in the general behaviour in both acute and subacute toxicity studies in rats. Studies of this type were needed before a phytotherapeutic agent can be generally recommended for use. Now days, all drugs were become resistant due to various reasons and the practitioners move to the combination therapy for the ailments. Synergistic action of the polyherbal drugs cure the disease at the faster rate and the purpose of the combined polyherbal treatment were to evaluate the synergistic action of the herbal medicines for the treatment of the various diseased conditions.

CONCLUSION:

A World Health Organization survey indicated that about 70–80% of the world’s population rely on non-conventional medicine, mainly of herbal source in their primary health care ^{[22], [23]}. Although medicinal plants may produce several biological activities in humans, generally very little is known about their toxicity and the same applies for this research. Because safety should be the overriding criterion in the selection of medicinal plants for their use in healthcare system. To determine the safety of drugs and plant products for human use, toxicological evaluation is carried out in various experimental animals to predict toxicity and to provide guidelines for selecting a safe therapeutic dose in humans. The present investigation demonstrates that at doses consumed in the traditional medicine, the ethanolic extract of Cycas circinalis (flower), Nardostachys jatamansi (roots), Artemisia absinthium (whole plant) may be considered as relatively safe, as it did not cause either any lethality or changes in the general behaviour in both acute and sub acute toxicity studies in experimental animals. Studies of this type are needed before a phytotherapeutic agent can be generally recommended for use.

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Received on 02.02.2014 Modified on 18.03.2014

Res. J. Pharmacology & P’dynamics. 6(2): April- June 2014; Page 94-100