INTRODUCTION:

Stress is the disturbed homeostatic condition of the organism, and it is represented by nonspecific response of the body to any demand imposed on it ¹. Stress is the part of life, which co-exist and is integrated with organism. Even in most of the circumstances stress has to be suffered by the organism at the cost of potential damage.

Stress brings various changes in physiological condition of the organism, but various mechanism of the body will counteract to maintain homeostasis. In some situations, body’s adaptation to stress is like reward, which is responsible for improvement of body’s performance beyond its capacity. However, if organism suffers strong acute or chronic stress, body is unable to maintain homeostasis. Under this condition, various types of diseases and disorders will develop, and even it may lead to death, if it is not managed at proper time. Hazardous stressful situation can be managed by using antistress agents. They act by improving resistance and tolerance of the body to any stress situation². Various medicinal plants have shown antistress activity such as Ashwagandha, Ginseng, and Tulsi. But still the need of effective antistress agents to combat noxious stressful situations of life is high.

The present study was therefore undertaken to explore antistress activity of Vitis vinifera and Chicorium intybus. Because, both the plants have been used traditionally for various diseases and disorders. Vitis vinifera belongs to vitaceae family commonly known as wine grape or common grape, which is widely grown throughout the world³.Complex matrix of grape seeds contains complex phenols, tannins, sugars, protein, oil, fiber, mineral and salts etc⁴. Proanthocyanidins are group of polyphenolic bioflavonoids present in Vitis vinifera seed, which are responsible for the potential pharmacological activities⁵.Vitis vinifera has been reported for its pharmacological actions like antibacterial and antioxidant^6-8, cardioprotective action^9-10, antidiabetic¹¹, antilisterial¹² and antithrombotic action¹³.

Chicorium intybus belonging to Asteraceae family, is an erect perennial plant. Its phytochemical constituents are inulin, phytosterols, lactones, flavonoids, triterpenoids, sesquiterpene, coumarins (including cichorin), caffeic acid derivatives, tannins, vitamins, pectins and fats^14-15. Chicorium intybus is reported for its pharmacological activities like antidiabetic¹⁶ antimalarial¹⁷, hepatoprotective¹⁸, tumor inhibitory activity¹⁹, antibacterial²⁰, antioxidant activity²¹, gastroprotective²², anticonvulsant²³, immuno modulatory activity²⁴and antilithiatic activity²⁵.

While reviewing the reported pharmacological actions and uses of Vitis vinifera and Chicorium intybus, it was found useful for several diseases that are postulated to be induced by stress. The present work is planned to study the antistress activity of Vitis vinifera and Chicorium intybus in a stress induced animal model.

MATERIALS AND METHODS:

Plant material:

The selected plants namely, fruits of Vitis vinifera (common grapes) and roots of Cichorium intybus (chicory) were procured from Bangalore, Karnataka. The collected material were identified, confirmed and authenticated by Dr. Athaulla khan, Botanist. The voucher specimen (MMU/RMGM/VV-CI/2008-09) was maintained.

Extraction of Vitis vinifera:

The collected fruits of Vitis vinifera (grapes) were pressed to isolate seeds. The cleaned and dried seeds were powdered using mechanical grinder. Powdered material was packed in the thimble and placed in a Soxhlet extractor. Petroleum ether was taken in the round bottomed flask and heated at 60 ⁰C for 6 h to remove fatty material from seed powder. Powdered material was re-extracted with 95% ethanol at 65 ⁰C. Extract was filtered and the filtrate was evaporated using rotary evaporator. Finally the viscous liquid was evaporated on water bath to a dry residue. Vitis vinifera seed extract was found to be brown in color, non sticky solid, with percentage yield of 12% w/w.

Extraction of Chicorium intybus:

Cichorium intybus roots were isolated, chopped into small pieces and dried under shade for 2 weeks. The dried slices of roots were grounded to powder and charged in the thimble of the Soxhlet΄s assembly. About 1.5 liter of 95% ethanol as a solvent was taken in the flask and heated at 70 ⁰C. The extract was evaporated using rotary evaporator and dried. Cichorium intybus root extract was found to be yellowish brown in color and as a Sticky mass, with percentage yield of 14% w/w.

Preliminary phytochemical screening:

Vitis vinifera seeds and Cichorium intybus root extracts were subjected to systematic chemical test analysis. The presence of different phytoconstituents was investigated, as described by the well established methods^26-27.

Experimental animals:

Experimental studies were carried out using normal adult Swiss Albino mice (24±2 g) and Albino rats (200±20 g) of either sex of Wistar strain. Animals were housed in clean and sanitized polypropylene cages under standard environmental conditions of relative humidity (50±5%), room temperature (25±2^oC) and photocycle (12:12h of light/dark period with lights on 0700h). Feed of animals was dietary pellets (pellets of Amruth Lab. Bangalore). Drinking water was maintained ad libitum. All animals were acclimatized and habituated to laboratory conditions for seven days prior to experiment, to minimize nonspecific stress conditions. Ethical clearance was obtained for procuring the animals and for evaluating antistress activity of Vitis vinifera and Cichorium intybus. (Approval No. MMUCP/ IAEC/03/ 2008-09).

Acute toxicity study:

Six adult albino mice in the age group of 90±10 days were used for the study. All the six female mice were nulliparous and nonpregnant. Mice were fasted for four hours prior to the administration of the extract. But mice were allowed free access to water as and when required. To avoid coprophagy mice were fasted in wire bottomed cages. Dose of Vitis vinifera seeds and Cichorium intybus roots extracts were fixed at 5000mg per kg body weight of mice (Limit test dose as per OECD guideline no.425 received from CPCSEA). Intra gastric administration of extract into the mice was done in a single dose. Individual mice were observed continuously for the first 30 m after the administration of extracts. Then mice were observed after every 30 m during the first 4 h. later on observation continued for 48 h and daily thereafter for a period of 14 days for delayed toxicity. Parameters of observation were mortality, moribund status and gross behavior. Gross behavior observations were behavior, neurological and autonomic responses.

Dose selection:

The dose of plant extracts selected in this investigation was 250 and 500 mg/kg body weight of animals. Diazepam was used as standard drug, and its dose was 5 mg/kg body weight of animal, based on previous reported studies²⁸.

Treatment protocol:

The rats were randomly assigned into nine groups, and each group consisted of six animals as follows:

Group 1: Albino rats of this control group received vehicle.

Group 2: Animals of this stress control group were administered with vehicle, and then subjected for cold water swimming stress.

Group 3: In this standard group, animals were administered with diazepam at the dose of 5 mg/kg body weight, and then subjected for cold water swimming stress.

Group 4: Albino rats of this group were administered with Vitis vinifera seeds extract at the dose of 250 mg/kg body weight, and then subjected for cold water swimming stress.

Group 5: Albino rats of this group were administered with Vitis vinifera seeds extract at the dose of 500 mg/kg body weight, and then subjected for cold water swimming stress.

Group 6: This group of animals were administered with Cichorium intybus root extract at the dose of 250 mg/kg body weight, and then subjected for cold water swimming stress.

Group 7: Albino rats of this group were administered with Cichorium intybus root extract at the dose of 500 mg/kg body weight, and then subjected for cold water swimming stress.

Group 8: This group of animals was administered with Vitis vinifera seeds extract at the dose of 500 mg/kg body weight. Animals were not subjected for cold water swimming stress.

Group 9: Albino rats of this group were administered with Cichorium intybus root extract at the dose of 500 mg/kg body weight. Animals were not subjected for cold water swimming stress.

Rats of all groups were treated with vehicle, extracts and diazepam by oral route in the above order. The treatment was given daily for a period of 10 consecutive days. Drugs were administered to rats by oral route using oral gavages. Food was withheld for a further 1 h after the administration of the drugs. On tenth day, animals of group 2 to group 7 were subjected to cold water swimming stress for an acute period, after one hour of drug administration. Cold water swimming stress was accomplished as described by previous investigators^29-30. In this model rats were allowed to swim in the cold water of transparent tub (60cm depth × 40cm width) containing water to the depth of 50 cm maintained at 15±5^oC. Water level was maintained up to the neck region. Rats suffered stress as they were not allowed to stand on bottom or grasping the side of the tub. Rats were removed from the water after 15 minutes. To avoid further stress, rats were anaesthetized with anaesthetic ether to stage of surgical anesthesia. Blood samples were withdrawn from retro orbital sinus of the eye (inner canthus of the eye) using microhaematocrit capillary tube. Unclotted blood for separation of plasma was collected in ice cold tubes containing heparin (100 unit heparin/ ml of blood). Blood was centrifuged at 3000 rpm for 10 minute and supernatant was separated as plasma and stored in cold condition (±4^oC) until further processing for estimation of plasma corticosterone. Rats were euthanized by cervical dislocation method. Adrenal glands were dissected out, washed with alcohol and their weights were recorded with respect to their body weights (expressed as weight of adrenal gland / 100 g of body weight of rats).

Estimation of plasma corticosterone formed during activation of adrenocortical activity in albino rats:

It was carried by the well-established method of Mattingly, as follows³¹.

Materials: Fluorescence reagent: it was prepared by mixing concentrated sulphuric acid and absolute alcohol in the ratio of 7:3. Then it was cooled .The reagent was colorless and stable at room temperature.

· Standard stock solution of corticosterone: 50 mg of corticosterone was dissolved in 50 ml of absolute alcohol. 1 ml of above solution was diluted up to 100 ml with distilled water to produce 10µg/ml stock solution. Both these solutions were kept at cold condition to remain stable for long period. Working standard solution was prepared by diluting above stock solution to produce 1µg/ml.

· Sample: It was obtained by extraction of corticosterone from plasma. Two ml of above separated plasma was taken into a 250 ml of conical flask and 15 ml of methylene chloride (organic solvent) was added into it, to extract the corticosterone. The conical flask was shaken well using mechanical shaker for 10 minute. Two layers were formed. The upper layer was aspirated and discarded. 10 ml of lower layer of methylene chloride extract was collected into a 20 ml of glass stoppered extraction tube.

· Reagent blank: Two ml of distilled water was taken into a conical flask and remaining procedure was carried as per that of sample treatment.

· Standard: Two ml of corticosterone solution of known strength (1µg/ml) was taken into a conical flask and remaining procedure was carried as per that of sample treatment.

Fluorimetric analysis method:

Either 10 ml of plasma extract or reagent blank or standard was taken into a 20 ml glass stoppered extraction tube. 5ml of fluorescence reagent was added into it. The extraction tube was shaken vigorously for 20 second. Again supernatant methylene chloride layer was sucked off. The lower acid extract layer was taken into a glass cell and reading was recorded using Systronic photofluorimeter. Excitation was done at 470 nm and readings were taken at peak transmission at 530nm. The concentration of plasma corticosterone was determined as follows:

Concentration of plasma corticosterone=

Reading of unknown X 100

Reading of known

The concentration of plasma corticosterone was expressed as µg/100 ml plasma. The obtained results were expressed as mean µg/100 ml plasma for each group with standard error of mean. The results of this investigation were compiled in the table no 1 and graphically represented in figure no.1 and 2.

Statistical analysis:

The data of this investigation was subjected to one way analysis of variance, followed by Dunnet΄s test for comparison between the groups of treatment. Difference with p<0.05 was considered as significant.

RESULTS:

Qualitative studies have shown the presence of carbohydrates, proteins and phenolic compounds in Vitis vinifera seed extract. Even tests for flavonoids were found to be positive for extract, indicating its presence in the Vitis vinifera seed. Test for fixed oils was found to be positive in seeds extract of Vitis vinifera.

Qualitative studies on Cichorium intybus root extract exhibited presence of carbohydrates, proteins and phenolic compounds. Tests for phytosterols and triterpenoids were found to be positive revealing the presence of steroids and triterpenes.

During acute toxicity studies of both plant extracts, mice showed normal behavioral, neurological and autonomic response. Animal showed normal body weight gain during 14 days. There was no significant change in body weight as compared to control group. There was no noticeable toxicity and no deaths till the dose tested (up to the dose of 5000 mg per kg body weight of animal).

As shown in table 1, cold water swimming stress of albino rats for acute period produced extremely significant (p<0.0001) elevation in plasma corticosterone and adrenal gland weight, when compared to control. Compared to the stress control group, Vitis vinifera at the dose of 250 mg per kg body weight of animal, exhibited very significant (p<0.01) decrease in plasma corticosterone concentration and adrenal gland weight. Group of rats pretreated with Vitis vinifera at the dose of 500 mg per kg body weight of animal, exhibited extremely significant (p<0.0001) decrease in plasma corticosterone concentration and adrenal gland weight, when compared to stress control group. Compared to the stress control group, Cichorium intybus at the dose of 250 mg per kg b. w. of animal, exhibited significant (p<0.02) decrease in plasma corticosterone concentration and extremely significant (p<0.0001) decrease in adrenal gland weight. Group of rats pretreated with Cichorium intybus at the dose of 500 mg per kg b. w. of animal, exhibited very significant (p<0.01) decrease in plasma corticosterone concentration and extremely significant (p<0.0001) decrease in adrenal gland weight, when compared to stress control group.

TABLE NO.1: Effect of extracts of Vitis vinifera and Cichorium intybus on adrenocortical activity in stress induced rats.

Sl. No.	Group	Plasma corticosterone (µg/100ml)	Adrenal gland (mg/100g of b. w.)
1	Control	21.84±0.98	6.35±0.07
2	Stress control	43.51±2.04^a	8.27±0.14^a
3	Standard + stress	24.20±1.82^b	6.43±0.10^b
4	Vitis vinifera (250 mg/kg b. w.) + stress	34.41±1.89^ce	7.00±0.05^bh
5	Vitis vinifera (500 mg/kg b. w.) + stress	25.03±1.7^bf	6.51±0.11^bf
6	Cichorium intybus (250 mg/kg b. w.) + stress	34.97±1.96^de	7.14±0.07^bh
7	Cichorium intybus (500 mg/kg b. w.) + stress	30.15±2.27^cf	6.55±0.08^bf
8	Vitis vinifera (500 mg/kg b. w.)	22.76±1.30^g	6.46±0.06^g
9	Cichorium intybus (500 mg/kg b. w.)	24.71±1.4^g	6.46±0.04^g

Values shown for each group as the mean ± S.E.M. obtained from six observations.

· Where ^ap<0.0001 and ^bp<0.0001 represent extremely significant, compared to control group and stress control group respectively.

· Where ^cp<0.01 and ^dp<0.02 represent very significant and significant respectively, compared to stress control group.

· Where ^ep<0.01, ^fp>0.05 and ^hp<0.001 represent very significant, non-significant and highly significant respectively, compared to standard group (Diazepam).

· Where ^gp>0.05 represent nonsignificant, compared to control.

DISCUSSION:

Any type of stress, regardless of the nature (whether physical or psychological), will stimulate Hypothalamic-Pituitary-Adrenal (HPA) axis of the organism³². In this pathway, stressors are able to activate nerve cells and hypothalamus, so that CRH is secreted from the paraventricular nucleus. Then CRH reaches the pituitary gland through hypothalamicohypophysial portal system. Anterior pituitary gland releases adrenocorticotropic hormone (ACTH), under the regulation of CRH². ACTH is carried by the blood to the adrenal gland; there it stimulates adrenal cortex to secrete corticoid hormones such as Cortisol or corticosterone into the blood³². Several studies have reported that stress increases plasma corticosterone level and altered adrenal gland weight³³. So estimation of plasma corticosterone with assessment of adrenal gland weight is the index of hypothalamo- hypophyseal- adrenocortical axis activation, and important markers to evaluate stress³⁴.

In this investigation cold water swimming stress model was used to induce effect on adrenocortical activity in rats, as described by previous investigators^{29-30, 35}.Cold swimming stress has been used frequently to induce acute stress. When the organism is exposed to cold condition of environment, it leads to stress resulting in damage to the body³⁶.Even it is well documented that cold swimming stress elicits an increase in the plasma corticosterone level³⁰. In this model, rodents are subjected to swim in a tank of cold water, at a temperature of 10 ⁰C till exhaustion³⁰. Due to cold swimming stress, temperature regulatory center in the hypothalamus is activated, and this results in activation of HPA axis, which is responsible for stressful response²⁹.

In this study the results reveal that cold water swimming stress of rats exhibited elevation in the plasma corticosterone and adrenal gland weight, which is in accordance with the previous studies of other antistress agent^{30, 35}. Pretreatment with diazepam prevented elevation of plasma corticosterone and adrenal gland weight in albino rats due to stress. This is in agreement with previous reported studies³⁷. Pretreatment with the extracts of Vitis vinifera seeds and Cichorium intybus roots extracts have shown significant protective affect against cold water swimming stress, by preventing rise in plasma corticosterone and adrenal gland weight. Thus, Vitis vinifera and Cichorium intybus extracts have exerted antistress effect by regulating homeostasis mechanism through hypothalamic pituitary adrenal axis of the animal.

CONCLUSION:

The protective effect of Vitis vinifera and Cichorium intybus extracts on plasma corticosterone level and weight of adrenal gland during stress, justifies their use as antistress agents. The observed antistress effect of Vitis vinifera and Cichorium intybus extracts was found to be through hypothalamic pituitary adrenal axis of the animal.

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