Evaluation of Anti-stress (adaptogenic) Activity of Luffa cylindrica Leaves Extract

INTRODUCTION

Stress basically is a reaction of mind and body against change in the homeostasis. The productive stress is called Eustress while harmful stress is called Distress. If the stress is extreme, the homeostatic mechanisms of the organism become deficit and the survival of the organisms threatened. Under these conditions, stress triggers a wide range of body changes called General Adaptation Syndrome (GAS). The stimuli, which produce GAS, are called as Stressors. The stressors are ranging from physical to psychological factors including cold, heat, infection, toxins, major personal disappointment etc.1 In the stress-filled environment we live in, successful adaptation to stress is a prerequisite for survival. In the indigenous system of medicine, there are many herbal drugs and formulations recommended to enable one to withstand stress without altering the physiological functions of the body. This, drug induced state of resistance against aversive stimuli is termed as adaptogenic activity and the drugs, named adaptogens.2 The medicinal substance causing non-specifically increased resistance (SNIR) was variously named as adaptogen or athenkropic.3 The plant adaptogen is defined as “Smooth pro-stressors which reduce reactivity of host defense systems and decrease damaging effects of various stress due to increased basal level of mediators involved in the stress response.”4 A number of plants possess adaptogenic activity due to diverse classes of chemical compounds.

India has a rich history of using plants for medicinal purposes. We all are aware that In-dia is one of the richest sources of medicinal plants. Nature has provided a complete storehouse of remedies to cure all ailments of mankind. The history of herbal medicine is as old as human civilization. Plants have been used in the treatment of various diseases from the time of imme-morial and the use of plant as a source of medicine lies deep in the history of mankind. Thou-sands of plant species growing throughout the world have medicinal uses containing active con-stituents that have direct action in the body. Hence herbal drugs are valuable as well as precious gift from nature to mankind.5,6

Luffa cylindrica (Cucurbitaceae) is also known as Torai. This is grown and harvested before maturity and eaten as a vegetable popular in Asia and Africa. The ripe dried fruit is also the source of the loofah or plant sponge.5 The major chemical components of Luffa cylindrica are triterpenoid saponins, as well asflavonoids.6-8 The plant is reported to be having anti-inflammatory,9 anti-fungal,10 analgesic and sedative,11 antimyocardial ischemia,12 anti-hypertriglyceride,13 immunostimulation,14 anti-allergic,15 antiasthmatic, anti-tussive andexpec-torant effects.16 Different parts of this plant are traditionally claimed to be used for the treatment of broadspectrum of ailments including snake bites, convulsions, cramps, tetanus, emetic, cathartic,dropsy, nephritis, chronic bronchitis, asthma, sinusitis and fever to be list a few.17

The antistress property of the title plant has been also described in ancient Indian Medicinal Plants Texts.18,19 However, the scientific data on anti – stress property of the leaves of the title plant has not been documented in the literature so far. In view of this, the present study was undertaken.

MATERIALS AND METHOD

Plant material

The plant sample was identified and authenticated by Dr. M. B. Mulimani, Professor of Botany, S.B Arts and Science College Bijapur, Karnataka. Then fresh mature leaves of Luffa cylindrica were collected from the surrounding fields of kenchammanahalli, Huvinahadagali Taluk, Bellary Dist, Karnataka and the sample was preserved in the herbarium of the college.

Preparation of extract

Freshly collected leaves were cleaned, shade dried at room temperature, coarse powdered and then extracted with 70% hydroalcohol by Soxhlet’s extraction method. Thereafter, the extract was concentrated using rotary flash evaporator. The yield of the extract obtained was 19.16 %. The obtained crude extract was stored in airtight container in refrigerator below 100 C for further studies.

The ethanolic extract of Luffa cylindricaleaves was dissolved in distilled water and subjected to the following studies.

1. Preliminary phytochemical screening.

2. Acute toxicity study.

3. Antistress activity.

   3.1. Anoxia Stress Tolerance Test in Mice.

   3.2. Swimming Endurance Test in Mice.

   3.3. Cold Restraint Stress in Rats.

Preliminary phytochemical screening

Preliminary phytochemical screening was carried out on test extract for the detection of phytoconstituents by following literature reported methods.20,21

Experimental animals

he albino rats of Wistar strain 150 -200 g and albino mice 20 - 30 g of either sex were used in the experimentation. The animals were procured from Sri Venkateshwara Enterprises, 4304, 13thmain 2nd cross, Subramanyanagar, Bangalore-21 (237/ CPCSEA). After randomization into various groups, animals were acclimatized for period of 10 days under standard husbandry condition as follows.

          Room temperature: 27 ± 3⁰

          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 libitium under strict hygienic condition. Study protocol was approved from Institu-tional Animals Ethics Committee (IAEC) before initiation of the experiment.

Acute toxicity study (LD50)

An acute toxicity of Luffa cylindrica leaves extractwas performed on female albino mice (20-30 g). The animals were fasted overnight prior to the experiment. Fixed dose (OECD Guide-line No. 423) method of CPCSEA was adapted for toxicity studies.22 1/20th, 1/10th and 1/5th LD50 cut off value of the extract were selected as screening doses for the antistress activity.

Evaluation of antistress activity

Anoxia stress tolerance time in mice

Principle

All the body functions, including cellular respiration depends on the oxygen supply. Any lack of vital element will play havoc on all body mechanisms and increase in adaptation during stress by any drug could be considered as its major antistress effect. During stress, adaptogens are capable of increasing succinate dehydrogenase (SDH) in the brain. The enzyme SDH is re-sponsible for utilization and conservation of energy in the cellular system of the organism, which helps adaptive processes during stress.

Method23

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

Group I - Control, received 0.5 ml of distilled water

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

Group III - EELCL(50 mg/kg, p.o.)

Group IV - EELCL (100 mg/kg, p.o.)

Group V- EELCL (200 mg/kg, p.o.)

Animals were treated as shown above for the three weeks. At the end of 1st, 2nd and 3rd week i.e. on 7th, 14th and 21st day 1 hr. after the treatment. Stress was induced in all the groups of animals by placing each mouse individually in the hermetic vessel of 300 ml capacity to record anoxia time. The moment when the animal showed the first convulsions removed immediately from the vessel and resuscitated if needed. The time duration of animal entry into the hermetic vessel 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 ves-sel may lead to death of the same.

Swimming endurance test in mice

Principle

Mice when forced to swim in a restricted space from which they cannot escape, become immobile after an initial period of vigorous activity. It has been suggested that the observed immobility signifies behavioural “despair” resembling a state of mental depression and has been used to screen anti-depressants. It is now recognised that this behavioral depression is fairly a common consequence of stress. It is also evident that the animals ability to cope with the stress largely influenced by the neurochemical consequence of stress. Thus exposure of rats to inescapable and severe stress leads to depletion of central nor adrenaline and serotonin, postulated to be the cause of endogenous depression.

Method23 Albino mice of either sex weighing 20 -30 g divided into five groups of five animals each for the test as below

Group I - Control, received 0.5 ml of distilled water

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

Group III - EELCL (50 mg/kg, p.o.)

Group IV - EELCL (100 mg/kg, p.o.)

Group V - EELCL (200 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 (30 cm height with 20 cm 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 timefor each group was calculated.

Cold Restraint Stress in rats23

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

Group I - Normal control

GroupII - Cold stress control received 1 ml of distilled water

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

Group IV - EELCL (50 mg/kg, p.o.) Group V - EELCL (100 mg/kg, p.o.)

Group VI - EELCL (200 mg/kg, p.o.)

Grouping and treatment of animals was done as shown above. The hind and limbs of rats were tied and subjected to cold stress by exposing them to cold environment 4 ± 1 ºC for 2 hr. daily in refrigerator. The drug treatment was given daily for 10 days 1hr. before the exposure of stress challenge. Animals were scarified at the end of specific period and blood was collected by carotid bleeding under mildether anesthesia using disposable syringe and needle for estimation of biochemical parameters, such as serum glucose (GOD-POD method), cholesterol (CHOD-PAP method), triglycerides (GPO-Triender method), BUN (Blood Urea Nitrogen, GLDH-UREASE method) using Erba Chem Semi-auto analyzer and ready reagent kits.

The weight of organs such as liver, spleen, testes and adrenal gland after washing with alcohol was recorded per 100 g body weight of animal.

Statistical analysis

The results generated from the present investigation were subjected to statistical analysis using ANOVA followed by Turkey Kramer Multiple Comparison Test. Results are expressed as Mean ± SEM.

RESULTS

Preliminary phytochemical screening

Preliminary phytochemical investigation on ethanolic extract of Luffa cylindrica leaves indicated the presence of saponin, glycosides, flavonoids, alkaloids and tannins.

Acute toxicity study

EELCL was studied for acute toxicity at dose of 2000 mg/kg i.p. in female albino mice and extract was found to be lethal (2/3 animals died). However, the extract dose of 300 mg/kg was found to be safe which was evident by observing no mortality of the animals. Hence, 1000 mg/kg was considered as LD50 cut off value as per fixed dose method, OECD, guideline No. 423 of CPCSEA. The screening doses selected for the antistress activity were:

1. 50 mg/kg (1/20th of 1000 mg/kg).

2. 100 mg/kg (1/10th of 1000 mg/kg).

3. 200 mg/kg (1/5th of 1000 mg/kg).

Antistress activity

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 (50, 100, 200 mg/kg) of the test extract demonstrated dose and duration dependent significant delay in clonic convulsions on 7th,14th and 21st day compared to control group received vehicle alone. The lower dose of the test extract ( 50 mg/kg) did prolong the clonic convulsions at the end of 1st and 2nd week, but the results found statisti-cally not significant. Antistress effect of the higher dose (200 mg/kg ) of the test extract was found closer to that of the standard drug. The results are presented in Table -1.

Values are expressed as Mean ± SEM, (n=5),*p< 0.05, **p>0.01 and ***p>0.001 as compared to control.

Swimming endurance test in mice

There was dose dependent significant increase in swimming performance time observed in mice seven days pretreated with graded doses (50, 100 and 200 mg/kg) of the EELCL. The percentage increase in swimming performance time was found to be 55 to 89. However, the ef-fect of test extract on swimming performance time was found to be less potent than the refer-ence standard drug, Withania sominifera The results are tabulated in Table - 2.

Values are expressed as Mean ± SEM, (n=5), *p<0.05,**p< 0.01 and ***p<0.001 as compared to control.

Cold Restraint Stress

Effect on biochemical parameters

Cold restraint stress adversely affected the serum concentration of various biochemical parameters. The induction of cold restraint stress significantly elevated the serum cholesterol, triglycerides, BUN and glucose levels in stress control rats compared to normal control group. Animals pretreated for ten days with test extract at different dose levels (50, 100 and 200mg/kg) showed significant and dose dependent fall in all the biochemical parameters, as compared to the stress control animals. The results are displayed in Table -3.  

Values are expressed as Mean ± SEM (n=5), *p< 0.05, **p< 0.01 and ***p< 0.001 compared to the stress control, @p< 0.001 compared to normal control

Effect on weight of organs

Cold stress significantly increased the weight of liver, adrenal glands and decreased the testes and spleen weight. Ten days pretreatment with graded doses of EELCL significantly and dose dependently ameliorated the cold stress induced altered organs weight. The results are represented in Table – 4.

Values are expressed as Mean ± SEM, (n=5), *p< 0.05, **p< 0.01, ***p< 0.001 as compared to stress control and @p< 0.001 compared to normal control.

DISCUSSION

Since the introduction of adptogens, several plants that had once been used as tonics in the Ayurvedic medicine, have been investigated for their antistress property due to their adapto-genic and rejuvenating properties.24

In the present study, antistress activity of EELCL was investigated at different dose le-vels (50, 100 and 200 mg/kg) against anoxic tolerance test, swimming endurance test and cold restraint stress animal models.

The swimming endurance test is most widely used physical stress model for the assess-ment of adaptogenic activity of a novel drug. This paradigm is based on the observation that an-imals when forced to swim in water eventually assumed a characteristic immobile posture, de-void of any activity. The appearance of immobility therefore, reflects a state of reduced stamina, fatigue and tiredness with the end point being the movement when the animal could not swim further and started drowning. The results of the swimming test demonstrated the marked increase in swimming time in mice, pretreated for seven days with test extract with enhanced physical performance and thus confirming its antistress property.

Anoxia is a very severe stressor. All the body functions including cellular respiration de-pends on oxygen supply to them. Any lack of this vital element plays havoc on all body mechanisms. Increase in adaptation during this stress by any drug could be considered as its major antistress effect. The results of the anoxic tolerance test showed that EELCL significantly delayed the latency of convulsions in experimental animals, which therefore confirm its antistress activity.

The mechanism by which stress rises serum cholesterol is likely to be related to the enhanced activity of hypothalamohypophyseal axis (HPA) resulting in liberation of catecholamines and corticosteroids. This could lead to increase in blood cholesterol level, since epinephrine is known to mobilise 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.25-27 In cold restraint stress model, the test extract reduced the elevated levels of serum biochemical parameters in dose dependant 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. Soits weight decreases during stress.28-30 This stress induced changes of organs weight were significantly reversed by the test extract in dose dependent man-ner and thus supports the antistress effect of EELCL against cold restraint stress model.

The literature reports indicated that extracts of medicinal plants containing flavonoids and tannins known to possess significant antistress activity.31In our study also flavonoid and tannin contents of crude extract of the title plant may be responsible for observed antistress/ adaptogenic activity.

CONCLUSION

In conclusion, the findings from the present study suggest that 70% hydro alcoholic leaf extract of Luffa cylindrica demonstrated increased resistance against different aversive stimuli in a nonspecific manner thus test extract could have adaptogenic effect. However, the present study did not include the tests for establishing the exact mechanism of action.