EVALUATION OF ANTI-STRESS (ADAPTOGENIC) ACTIVITY OF LUFFA CYLINDRCIA FRUIT EXTRACT

About Authors:
Sachin Kaushik*, Dr. shivakumar Hugar
P.G. Department of Pharmacology,
B.L.D.E.A.’s college of Pharmacy,
Bijapur- 586103,
Karnataka,India
*sachin_2004_kaushik@yahoo.com

ABSTRACT:
In the present study antistress activity was assessed against anoxia tolerance, swimming endurance and cold restraint stress models. In anoxia tolerance test, the mean time of appearing first convulsion in mice was taken as end point to determine the time of anoxia.  In swimming endurance test the mean time of swimming performance was recorded.The end point taken was when the animals remained at the bottom of swimming tank for 10 sec. Biochemical estimation like glucose, cholesterol, triglycerides and BUN and weight of organs were measured in cold restraint stress model.

Reference Id: PHARMATUTOR-ART-1341

INTRODUCTION:
According to Oxford dictionary, stress means pressure or tension. Stress can be defined as an injury or threat to our physical and mental well being, loss or a perception of loss, or challenge that we fear which is partly or totally beyond our control. Stress is basically a reaction of mind and body against change in homeostasis. It is also a major factor that constitutes individuals daily life.
Stress is body’s physical, mental or chemical reaction when we get excited or confused or we otherwise feel unsafe or threatened. If daily demands are easy and well balanced an individual is fine. It is when one decides that pressure is unreasonable or the situation is upsetting, that the potential for damage occurs. That’s when one feels stressed. Stress reactions begin in our minds. As long as humans have been around there has been stress.
Some of the more common symptoms of stress include problems with sleep, depression, anxiety, irritability, fatigue and lethargy. A large proportion of all illness is believed to occur because of stress. High-stress modern living is probably the main factor causing chronic disease.

A unique class of herbal products called "adaptogens". Adaptogens have the most broad-spectrum healing properties of any herbal medicines, but their unique value is that they specifically relieve stress. Perhaps the single most important property of an adaptogenic plant is the proven ability to combat stress in all forms. Herbal drugs have gained importance in recent years because of their efficacy and cost effectiveness. Adaptogens are such herbal agents, which help the body to overcome excess stress even in chronic cases. Since the introduction of adaptogens, several plants have been investigated, which were once used as tonics due to their adaptogenic and rejuvenating properties in traditional medicine. The drugs of plant origin are gaining increasing popularity and are being investigated for remedies of a number of disorders including antistress (adaptogenic) activity.

Luffa cylindrica (family: Cucurbitaceae) isone such plant cultivated for different purposes such as vegetable and spice.The leaves and fruits of this plant are used as a highly nutritive vegetable in many countries, particularly in India and China. The major chemical components of Luffa cylindrica are triterpenoid saponins, as well as flavonoids. Different parts of the title plant are used in the traditional system of medicine for the treatment of liver diseases, ulcer, cough, diarrohea and stress disorders. The plant is said to be having diverse pharmacological activites viz. abortifacient, anti HIV, anti-inflammatory and antidiabetic activites. However, no reports on anti stress activity of the fruit of the title plant available in the literature so far. In view of this, the present investigation was under taken.

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METHODOLOGY:
Plant material
The plant was identified and authenticated by Dr. M. B. Mulimani, Professor of Botany, S.B Arts and Science College Bijapur, Karnataka. Then fresh mature fruits of Luffa cylindrica were collected from the garden of Bijapur and the sample preserved in the herbarium of the college.
Preparation of extract
Freshly collected fruits were cleaned, shade dried at room temperature, coarse powdered and then extracted with 70% hydroalcohol by Soxhlet extraction method. Thereafter, the extract was concentrated using rotary flash evaporator. The yield of the extract obtained was 18.0 %. The obtained crude extract was stored in airtight container in refrigerator below 10 ⁰C for further studies.
Experimental animals
The albino rats of Wistar strain 150 -200 g and albino mice 20 - 30 g of either sex was used in the experimentation. The animals were procured from Sri Venkateshwara Enterprises, 4304, 13^th main 2^nd 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 ± 3^?

           Relative humidity: 65 ± 10%

               12 hr light/dark cycle

All the animals were fed with rodent pellet diet (VRK Nutritionals Industries, Pune, India) and water ad libitium under 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 Luffa cylindrica fruits extractwas performed on female albino mice (20-30 g). The animals were fasted over night prior to the experiment. Fixed dose (OECD Guideline No. 423) method of CPCSEA was adapted for toxicity studies. 1/20^th, 1/10^th and 1/5^th 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 responsible for utilization and conservation of energy in the cellular system of the organism, which helps adaptive processes during stress.

Method
Albino mice of either sex weighing 20 -30 g were selected and divided into five groups of five each.
Group I - Control, received distilled water
Group II - Std. (Withania somnifera, 100 mg/kg, p.o.)
Group III - EELCF(50 mg/kg, p.o.)
Group IV - EELCF (100 mg/kg, p.o.)
Group V - EELCF (200 mg/kg, p.o.)

Animals were treated as shown above for the three weeks. At the end of 1^st and 2^nd week i.e. on 7^th and14^th 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 vessel 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 behavioural 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.

Method
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 distilled water
Group II      -     Std. (Withania somnifera 100 mg/kg, p.o.)
Group III     -     EELCF (50 mg/kg, p.o.)
Group IV     -     EELCF (100 mg/kg, p.o.)
Group V       -     EELCF (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 ±1⁰C 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 rats
Method
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         - Control
Group II         - Standard ( Withania Somnifera, 100 mg/kg, p.o.)
Group III       - EELCF (50 mg/kg, p.o.)
Group IV       - EELCF (100 mg/kg, p.o.)
Group V         - EELCF (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).
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.

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RESULTS:
Acute toxicity study
EELCF 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 (1/20^th of 1000 mg/kg).

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

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

Antistress activity

Anoxia stress tolerance time in mice
The test extract could able to exhibit duration dependent significant antistress activity at 200 mg/kg dose only. This was evident by observing subsequent significant increase in anoxia stress tolerance time on 7^th and 14^th day when compare to control group. Though, there was increase in anoxia stress tolerance time demonstrated by test extract at 50 and 100 mg/kg doses also, but the results seen statistically not significant. Antistress effect of 200 mg/kg dose of the extract was found closer to that of standard drug, Withania somnifera. The results are tabulated in Table – 1.

Swimming endurance test in mice
There was dose related significant increase in swimming performance time seen in mice pretreated with graded doses (50, 100 and 200 mg/kg) of the test extract. The antistress/ adaptogenic effect was found to be statistically significant only at 100 and 200 mg/kg doses. The increase in swimming performance time was found to be 10.40 to 28.86 %. However, the effect of test extract on swimming performance time was found to be less potent than the reference standard drug. The results are tabulated in Table – 2.

Cold Restraint Stress
a)   Effect on biochemical parameters
There was marked elevation of biochemical parameters seen in positive control (stressed group) compared to normal control (unstressed group). The EELCF has significantly reduced the elevated levels of serum glucose, cholesterol, triglycerides and BUN at doses 50, 100 and 200 mg/kg in dose dependent fashion, when compared with positive control. The results are given in table – 3.

b)  Effect on organs weight
Cold stress significantly increased the weight of liver, adrenal glands and decreased the testes and spleen weight. The dose dependant significant protection on weight of organs was observed in animals pretreated with test extract at higher doses (100 and 200 mg/kg) only when compared to stressed group. The extract dosed at 50 mg/kg also showed reasonably good protection on altered weight of the organs, but the results were found to be statically not significant.  However, the antistress effect of EELCF at dose of 200 mg/kg was found almost similar to that of standard. (Table-4)

Table- 1
Effect of EELCF on anoxia time in mice

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

Table – 2
Effect of EEPPL on swimming endurance time in mice

Values are Mean ±SEM, (n=5)
*p<0.01 and **p< 0.001 vs. control

Table – 3
Effect of EELCF on biochemical parameters in cold restraint stress induced rats

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

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Table – 4
Effect of EELCF on organs weight in cold restraint stress induced rats

Values are Mean ±SEM, (n=5)
*p< 0.05, **p< 0.01, ***p? 0.001 and ns - not significant vs control.

DISCUSSION:
There are various models used for the screening of anti-stress activity of drug which include food deprivation stress, sound stress, cold restraint stress, water immersion restraint stress, activity wheel stress, anoxia stress tolerance and foot shock stress models. In the present  investigation, anti-stress property of graded doses (50, 100 and 200 mg/kg)  EELCF was assessed using anoxia tolerance and swimming endurance tests (acute stress models) and cold restraint stress (chronic stress) technique.
The anoxia tolerance test and swimming endurance test are well known physical stress models for the evaluation of antistress property of the drug. In the swimming endurance test, the mice are forced to swim in a restricted space from which they cannot escape. This induces the characteristic behavior of immobility. It has been well demonstrated that drugs with antistress activity increase swimming endurance time and latency of post anoxic convulsions. In the present study,  results of the swimming endurance test and anoxia tolerance test indicate clearly that the EELCF have the properties, whereby, they increase the physical endurance as well as the overall performance in mice.

During cold restraint stress, serum glucose, triglycerides, cholesterol and BUN levels significantly increases which was found to be significantly reversed in animals pretreated with Luffa cylindrica fruit extract. Lowering of stress induced hyperglycemia and reduction in elevated levels of biochemicals indicates the antistress property of the test extract.

The literature review reveals that plant extracts rich in flavonoids and tannins known to possess significant antistress activity. In the current investigation also flavonoid and tannin contents of crude extract of the title plant could be the reason for observed antistress/ adaptogenic activity.

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