INTRODUCTION:

Stress is a common phenomenon that is experienced by all individual. When stress becomes extreme, it is harmful for the body and hence needs to be treated. Stress plays an major role in the pathogenesis of a variety of diseases including hypertension, peptic ulcer, immunodepression, reproductive dysfunction and behavior disorder¹. Evidence related that stress impairs learning and memory and encounter several disorders including anxiety and depression². Stress mapping or screening of biologically active constituents of natural origin, mainly from plant kingdom³. Drugs having Antistress properties induce a state of non-specific resistance against stressful conditions.

Empirical use of medicinal herbs has been widely disseminated since ancient times to treat a wide range of diseases. In current days, the interest in alternative therapies has raised markedly across the globe⁴.

Drugs derived from plant source are emerging as alternative therapies in the treatment of many diseases including psychiatric disorders⁵. Medicinal plants playing significant role in the search of novel pharmacotherapy to treat psychiatric illnesses⁶. Plants have always been an exemplary source of drugs and many of the available drugs have been derived directly or indirectly from them⁷.

Drugs like benzodiazepines, certain CNS stimulants such as amphetamines and caffeine as well as some anabolic steroids are routinely used by people to combat stress. The incidence of toxicity and dependence has limited the therapeutic usefulness of these drugs⁸. Alternative are clearly needed because of the inability of the current therapies to manage condition of disease⁹. The first drugs used to treat pathologic condition of the CNS were based on natural resources¹⁰. People from different area of world using herbal medicine to alleviate affective disorders¹¹. The herbal formulations claimed to enhance physical endurance, mental functions and non-specific resistance of the body have been termed as adaptogen⁸.

Various plants are being used in complementary and alternative medicines for management of stress¹². The potential utility of safer and cheaper herbal medicines as Antistress agents have been reported as they can withstand stress without altering the physiological functions of the body.

Ficus benghalensis (FB) commonly known as Alada mara in Kannada is a rich source of medicinal value having multidimensional curative properties. Different parts of the tree have been found to possess medicinal properties; leaves are used for treating ulcers, aerial roots for gonorrhea whereas seeds and fruits are cooling and tonic. In India, milky juice (latex) of stem bark of F. benghalensis is used for the treatment of rheumatism and other inflammatory diseases¹³.

Photochemical investigation of Ficusbenghalensis explored wide variety of constituents which are responsible for its wide range of pharmacological activities. They include ketones, flavonoids, flavonols, sterols, pentacyclic triterpenes and triterpenoids, furocoumarin, tiglic acid ester and some other esters¹⁴. Flavanoids have been acknowledged for their interesting medicinal properties¹⁵. Different parts of Ficus benghalensis reported to possess anthelmentic, analgesic, immunomodulator, hypolipidemic, antidiabetic and antiallergic activities¹⁶. Flavonoids isolated from leaves of Ficus benghalensis has been reported for Antistress activity¹⁷. However, Antistress (adaptogenic) activity of fruits of title plant has not been scientifically validated till date. Hence the present study was undertaken to evaluate antistress activity.

MATERIALS AND METHODS:

Plant material:

The fruits of Ficus benghalensis was identified and authenticated by Dr. Paramanna D (Needagi) Professor & HOD, Dept. of Botany S.B Arts & KCP Science College, Vijayapur, Karnataka. Then sufficient amount of fruits of Ficus benghalensis were collected from in and around the garden of Vijayapur city, Karnataka and the sample has been preserved in the herbarium of the college.

Preparation of extract:

The fruits were shade dried at room temperature and ground to coarse powder and then extracted with methanol by Soxhlet’s extraction method. Thereafter, the extract was concentrated using rotary flash evaporator. The yield of the extract obtained was 7.4 %. The obtained crude extract was stored in airtight container in refrigerator below 10 ⁰C for further studies.

Preliminary phytochemical screening:

Preliminary phytochemical screening was carried out on test extract for the detection of phytoconstituents by following literature reported methods^18,19.

Experimental animals:

The Wistar albino rats of 150 - 200 gm and Swiss albino mice 20 - 30 gm of either sex was used in the experimentation. After randomization into various groups, animals were acclimatized for period of 10 days under standard husbandry condition as follows.

· Room temperature: 27 ± 3^◦

· Relative humidity: 65 ± 10%,

· 12 hr light/dark cycle

All the animals were fed with rodent pellet diet (VRK Nutritional Solutions, Pune, India) and water ad libitiumunder strict hygienic condition. Study protocol was approved from Institutional Animals Ethics Committee (IAEC) before initiation of the experiment.

Acute toxicity study (LD₅₀):

An acute toxicity of methanolic extract of Ficus benghalensis fruits was performed on female albino mice (20-30 gm). The animals were fasted overnight prior to the experiment. Fixed dose (OECD Guideline No. 423) method was adapted for toxicity studies²⁰. 1/40^th, 1/20^th and 1/10^th LD₅₀ cut off value of the extract were selected as screening doses for the anti- stress activity.

Evaluation models for anti-stressactivity of methanolic extract of Ficus benghalensis fruit extract

Anoxia Stress Tolerance Test

Albino mice of either sex weighing 20-30 gm were selected and divided into five groups of six each.

Group I - Control, received distilled water

Group II - Std. (Withania somnifera, 100mg/kg,p.o.)

Group III - MEFBF (125mg/kg, p.o.)

Group IV - MEFBF (250mg/kg, p.o.)

Group V -MEFBF (500mg/kg, p.o.)

Animals were treated as shown above for the three weeks. At the end of 1^st, 2^nd and 3^rd week i.e. on 7^th, 14^th and 21^st day 1 hr. after the treatment. Stress was induced in all the groups of animals by placing each mouse individually in the air tight bottle of 250 ml capacity to record anoxia time. The moment when the animal showed the first convulsions removed immediately from the bottle and resuscitated if needed. The time duration of animal entry into the air tight bottle and the appearance of the first convulsion were recorded as anoxia time. Appearance of convulsion was very sharp end point, as delay by minute of removal of the animal from the vessel may lead to death of the same.

Swimming Endurance test in mice:

Albino mice of either sex weighing 20-30gm divided into five groups of six animals each for the test as below

Group I -Control, received distilled water

Group II - Std. (Withania somnifera, 100mg/kg, p.o.)

Group III - MEFBF (125 mg/kg, p.o.)

Group IV – MEFBF (250 mg/kg, p.o.)

Group V – MEFBF (500 mg/kg, p.o.)

Treatment was given to mice for 7 days. On seventh day 1 hr. after treatment, all the mice were subjected to swimming endurance test. The mice were allowed to swim individually in swimming tank 30cm height with 20cm diameter containing water of 25 cm height maintained at 26+ 1⁰ C temperature. The end point was taken when the animals remained at the bottom of swimming tank for 10 sec. The mean swimming time for each group was calculated.

Immobilization Stress in rats²¹

In the present study, adult albino rats of either sex weighing 150 – 200gm were divided into six groups of six animals each.

Group I - Normal control

Group II - Stress control

Group III - Standard (Withaniasomnifera, 100mg/kg, p.o.)

Group IV - MEFBF (125 mg/kg, p.o.)

Group V - MEFBF (250 mg/kg, p.o.)

Group VI - MEFBF (500 mg/kg, p.o.)

The treatment was made as stated above for 10 days 1hr. before the exposure of stress. Stress was induced by immobilizing rats with head down, supine position by fixing the forelimbs and hind limbs to a wooden board inclined at an angle of 60⁰, daily 2hrs. for a period of ten days.

Biochemical estimations:

At the end of 10^th day one hour after drug treatment the blood was collected from retro orbital plexus in sodium citrated tubes under mild ether anesthesia using disposable syringe and needle for estimation of biochemical parameters, such as serum glucose (GOD-POD method), cholesterol (CHOD-PAP method), triglycerides (GPO-Trinder method), BUN (Blood Urea Nitrogen, GLDH-UREASE method) using Erba Chem Semi-auto analyzer and ready reagent kits.

The rats then scarified and their organs such as liver, spleen and adrenal glands were removed. The weight of organs such as liver, spleen and adrenal glands after washing with alcohol was recorded per 100g body weight of animal.

RESULTS:

Preliminary phytochemical screening:

Preliminary phytochemical investigation on methanolic extract of Ficus benghalensis fruit indicated the presence of flavonoids, alkaloids and tannins.

Acute toxicity study:

MEFBF was studied for acute toxicity at dose of 2000 mg/kg i.p. in female albino mice. The extract did not cause any mortality (0/3 mice died) of the animals at dose of 2000mg/kg, even at repeated dosing using 3 new mice. Hence, 5000mg/kg was taken as LD₅₀ cutoff value as per fixed dose method of OECD guideline number 423.

Anoxia stress tolerance time in mice:

In the anoxic tolerance test, the time taken for the mice to exhibit clonic convulsions was taken as the end point. The graded doses (125, 250, 500mg/kg) of the test extract demonstrated dose and duration dependent significant delay in clonic convulsions on 7^th, 14^th and 21^st day compared to control group. Antistress effect of the higher dose (500 mg/kg) of the test extract was found closer to that of the standard drug. The results are presented in Table-1.

Table 03. Effect of MEFBF on serum biochemical changes in immobilization stress in rats

Groups	Treatment	Dose mg/kg	Biochemical estimations
Groups	Treatment	Dose mg/kg	Glucose mg/dl	Total Cholesterol mg/dl	Triglyceride mg/dl	BUN mg/dl
I	Normal Control	--	99.5±4.2	53.3±3.5	58.9±2.2	23.1±1.0
II	Stress Control	Vehicle	149.4±6.1^@	139.8±2.9^@	90.3±2.5^@	38.4±0.9^@
III	Standard (W S)	100	109.1±4.3^***	59.6±3.0^***	56.6±1.8^***	26.9±0.8^***
IV	MEFBF	125	129.0±2.4^*	126.4±2.5^*	80.9±2.1^ns	33.6±1.5^*
V	MEFBF	250	119.1±3.2^***	99.3±3.1^***	75.3±2.3^***	30.5±0.9^***
VI	MEFBF	500	113.0±2.6^***	66.4±3.1^***	70.4±2.5^***	28.9±0.8^***

The values are expressed as Mean ± SEM, (n=6),

Where^@p< 0.001, * p< 0.05, ** p< 0.01, *** p< 0.001 as compared to stress control.

DISCUSSION:

In the current study, anti-stress activity of MEFBF was demonstrated at different dose levels (125, 250 and 500 mg/kg) against anoxia stress tolerance test, swimming endurance time and immobilization stress models in experimental animal like mice and rat.

Anoxia is a more severe stress. All the body functions including cellular respiration depends on oxygen supply to them. Any lack of this vital element plays major role on all body mechanisms. Increase in adaptation during anoxic stress by any drug could be considered as its major anti-stress effect^22-24. The results of the anoxic tolerance test showed that MEFBF significantly delayed the latency of post anoxic convulsions in experimental animals, thereby confirm its anti-stress activity.

Increase in swimming endurance time has reported in mice when pre-treated with anti-stress agents²⁵ and the test has been utilized to investigate the adaptogenic activity of different agents, based on the fact that swim endurance reflects physical endurance²⁶. In the present investigation the results indicate clearly that the extract of Ficus benghalensis have the properties whereby they increased the physical endurance as well as the overall performance in mice.

Experimental animals exposed to an Immobilisation stress resulted in hyperglycemia, this is because during stressful condition adrenal cortex secrets excess cortisol²⁶. Excessive secretion of cortisol maintains the internal homeostasis through the process of gluconeogenesis and lipogenesis^27. The results of the current study revealed that the extract of the Ficus benghalensis exhibited promising effect in controlling hyperglycemia indicating the ability to prevent the alterations on adrenal cortex and helping in maintaining the homeostasis.

The mechanism by which stress rises serum cholesterol is likely to be related to the enhanced activity of hypothalamo-hypophyseal axis (HPA) resulting in liberation of catecholamines and corticosteroids. This could lead to increase in blood cholesterol level, since epinephrine is known to mobilize lipids from adipose tissues. The effect of stress on serum triglycerides has been shown to be variable. The increase in release of catacholamines leads to elevated levels of glucose and BUN^28-29. In Immobilisation stress model, the test extract reduced the elevated levels of serum biochemical parameters in dose dependent manner.

Stress induces adreno-medullary response in man. Adrenaline in turn stimulates Beta 2 receptors on the pituitary glands causing greater release of ACTH, which can stimulate the adrenal medulla as well as cortex. So adrenal gland weight increases. Cortisol increases mRNA levels in liver cells, this lead to increase in weight of liver. Spleen constricts to release more red blood cells (RBC) during stress, so its weight decreases during stress^30-32. The rats pretreated with Withenia Somnifera and MEFBF significantly reversed altered organs weight of adrenal glands, liver and spleen thus supports the anti-stress effect of MEFBF against immobilization stress model.

The literature reports indicated that extracts of medicinal plants containing flavonoids and tannins known to possess significant anti-stress activity^33.In our study also flavonoid and tannin contents of crude extract of the title plant (evidenced by phytochemical analysis) may be responsible for observed anti- stress (adaptogenic) activity.

CONCLUSION:

In conclusion, the methanolic extract of Ficus Benghalensis fruit exhibited dose dependent significant antistress activity by increasing the capacity to tolerate stress in experimental animal as well as restoring the altered biochemical parameters and organs weight. Thus the fruit extract of the title plant acts as an adaptogenic agent in management of stress and stress related diseases.

ACKNOWLEDGEMENT:

The authors are thankful to Principal, BLDEA’s SSM College of Pharmacy & Research Centre, Vijayapur for providing necessary facility to carry out the research work.

CONFLICT OF INTEREST:

The authors do not have any conflict of interest.

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Received on 17.06.2019 Modified on 21.07.2019

Research J. Pharm. and Tech. 2020; 13(1): 191-196.

DOI: 10.5958/0974-360X.2020.00039.6

Groups	Treatment	Dose (mg/kg)	Duration of anoxia stress tolerance time (min)
Groups	Treatment	Dose (mg/kg)	7^th Day	14^th Day	21^st Day
I	Control	--	30.2 ± 0.87	36.5 ± 1.12	39.9 ± 1.58
II	Std. (Withania somnifera)	100	56.5± 2.4***	59.0 ± 1.45***	64.5 ± 2.0***
III	MEFBF	125	36.4 ± 1.5***	41.5 ± 1.5***	44.3 ± 1.7***
IV	MEFBF	250	39.4 ± 1.2***	43.3± 1.6***	47.5± 1.3***
V	MEFBF	500	46.0 ± 1.1***	48.2± 1.9 ***	51.1± 1.4***

Groups	Dose mg/kg	Organs weight (gm/100 gm b.w.)
Groups	Dose mg/kg	Liver	Adrenal glands	Spleen
Normal control		3.58 ± 0.20	0.013 ± 0.001	0.380 ± 0.005
Stress control		5.63±0.17^@	0.098±0.031^@	0.231±0.004^@
Standard (W S)	100	3.53±0.13^***	0.017 ± 0.001^***	0.370±0.009^***
MEFBF	125	4.81±0.12^**	0.024±0.001^**	0.275 ±0.005^**
MEFBF	250	4.62±0.11^***	0.021±0.001^**	0.310±0.004^***
MEFBF	500	4.13±0.15^***	0.018±0.001^**	0.348±0.006^***

Preliminary phytochemical screening:

Experimental animals:

Acute toxicity study (LD50):

Acute toxicity study (LD₅₀):