EVALUATION OF ADAPTOGENIC ACTIVITY OF THE FRUIT EXTRACT OF Phyllanthus emblica AND ROOT EXTRACT OF Withania

somnifera BY USING MICROORGANISMS

BY SHAIK SHAHEENA CH.DINESH BABU

UNDER THE SUPERVISION OF N.KANAKADURGA DEVI M.PHARM.,Ph.D

KOMMAREDDY VENKATA SADASIVA RAO SIDDHARTHA COLLEGE OF PHARMACEUTICAL SCIENCES 2014

Any individual who can adapt easily to changing situation(physical, mental, microbial, chemical) can stay

healthy

ADAPTOGENS was first coined by Dr.NIKOLAI LAZAREV a Russian scientist only 50 years ago but Indian Physicians practicing before 5000years-RASAYANAS

It is sold in the market as Modern Nutraceuticals with different labels

An ADAPTOGEN is a substance that demonstrates a NONSPECIFIC

enhancement of the body’s ability to resist a stressor

NON SPECIFIC – NOT CAUSED BY SPECIFIC AGENT

RESISTANCE – THE POWER OF RESISTING

PROPERTIES OF ADAPTOGENS:

•The substance is relatively Non – Toxic to the recipient•It has Non specific activity and increases resistance of organism to broad spectrum of adverse biological, chemical and physical factors•These substances tend to help, regulate or normalize organ and system function within the organism

HOW DO THEY WORK ……….?

One theory suggested that Adaptogens function primarily due to anti-oxidant and free radical scavenging activity

Most of the adpatogens are Anti-oxidants but substances having Anti-oxidant properties ( Green Tea, Rosemary, Cranberry) are not sufficient to make it an adaptogen.

Amphoteric is a substance that normalizes function of an organ or a system within the body

All the herbs are Amphoterics but none of them qualify as adaptogens.

So Adaptogens act as broad spectrum amphoterics to living organisms.

Later research postulated that adaptogens work primarily by affecting Hypothalamic/Pituitary/ Adrenal (HPA) axis and Sympathoadrenal System (SAS)

Thus, adaptogens modulate our response to stress and help regulate the interconnected endocrine, immune and nervous system

This re-regulation of a highly disordered stress system is achieved by metabolic regulators such as cytokines, catecholamines, glucocorticoids, cortisol, serotonin, nitric oxide, sex hormones, corticotrophin releasing factor (CRF)

This broad array of biochemical activators explain why adaptogens have Anti- Inflammatory, Anti-Oxidant, Anxiolytic, Anti-Depressant or nervine effects as well

Adaptogens can help people handle stress by providing :

•Anti- Oxidant activity•Liver protection and Anti toxin activity•Improved blood sugar metabolism•Less craving for alcohol and sugar•Improved immune resistance•Increased stamina and energy•Improved muscle tone•Increased strength•Faster recovery•Better focus and concentration•Less anxiety•Better sleep•Better motivation and productivity•A feeling of well being•Better moods

Stress induced health problems:

•Angina•Asthma•Auto immune Diseases•Cancer•Cardiovascular disease syndrome•Common cold•Depression•Diabetes( Adult onset type II )•Headaches•Hypertension•Immune suppression•Irritable Bowel Disease•Menstrual irregularities•Pre menstrual tension•Rheumatoid arthritis•Ulcerative colitis•Ulcers

The plants with Adaptogenic activity is

1. Red ginseng2. American ginseng3. Siberian ginseng4. Ashwagandha5. Astragalus6. Liquorice7. Tulsi8. Satavari9. Amla10.Ginger

Ashwagandha Root ( Withania somnifera)

This herb is one of the most prominent Rasayana ( rejuvenative ) remedies of Ayurveda

It is one of the few calming Adaptogens and has traditionally been used for Anxiety, Bad Dreams, Insomnia and Nervous Exhaustion

It acts as Anti spasmodic and Anti Inflammatory, in treating Fibromyalgia, restless leg syndrome, mild Tourette’s Syndrome and Osteo arthritis

It is an immune amphoteric useful for hypo and hyper immune conditions

It enhances male fertility (sperm count and sperm motility)Due to its Iron content, it benefits iron deficient anemia

Ashwagandha also stimulates thyroid function

Amla : Shining through the Three Stages of Stress

It is one of the most used herbs in the world as it is a part of many formulas such as Triphala, consumed by millions daily with their meals as delicious and digestive condiment

The general aim of the Adaptogen therapy lies in their ability to reduce stressful agitation reactions during the ALARAM PHASE of the stress response

Support the physiological rallying during the RESISTANCE PHASE of stress response

And prevent or at least delay the state of fatigue during the FINAL STAGE of the stress response

Doing so they provide a certain level of protection against long-term stress and eventual exhaustion and systemic decay

Amla supports all the three stages of stress reactions and is shown in tests to be an Anti- Stress Immunomodulating Adaptogen that supports us in emotional and cognitive wellness

Amla provides a healthy balance between the aggressive factors of digestion ( the acids and enzymes) and the protective factors of digestion ( the mucous, structural cells and alkalinity)

AIM OF THE WORK:TO EVALUATE ANTI-STRESS ACTIVITY OF CRUDE EXTRACT OF THE FRUIT OF Phyllanthus emblica AND ROOT EXTRACT OF Withania somnifera USING MICROORGANISMS LIKE Escherichia coli, Staphylococcus aureus AND Lactobacillus

PLAN OF THE WORK:1.By subjecting the microorganisms

to stress conditions like exposure to UV light in the presence of an extract

2.Testing of survival of microorganisms even after subjecting to stress by COLONY COUNT METHOD

The fruits are washed thoroughly to remove impurities and pulped

Then passed through 1/8” sieve followed by 1/16” sieve to obtain fine particles

The product is pasteurized at 80-850

C and sterilized for storage

PREPARATION OF CRUDE EXTRACT OF AMLA FRUIT:

10 grams of root powder is dissolved in 1000 grams of distilled water

Boil at 1000C for 6 hours

Store at room temperature and it is the crude extract of roots of ashwagandha

PREPARATION OF CRUDE EXTRACT OF ASHWAGANDHA ROOT:

Prepare nutrient agar medium

Then prepare fresh subcultures of the required strains of microorganism

Prepare agar plates by

using pour plate

method

Allow the plate to dry for

approximately 5 minutes

Using flamed forceps gently

press each disc to agar to ensure disc is attached to

agar

Allow the discs to soak

in fruit extract of Amla/ root

extract of Ashwagandha

Let the plates be in refrigerator for 10

minutes and incubate the plates at 370C for

24 hours

After incubation expose the

plates to UV light for

15minutes and then Incubate again for 24

hours at 370C

E.coli S.aureus

Lactobacillus

FOR FRUIT EXTRACT OF AMLA

E.coli S.aureus

Lactobacillus

FOR ROOT EXTRACT OF ASHWAGANDHA

EVALUATION OF ANTI-STRESS ACTIVITY:

Following aseptic conditions from each agar plate transfer two loop full of sample around the disk into 10 ml of normal saline solution

From that serial dilution was performed for all three strains of Amla fruit extract and Ashwagandha root extract

ASHWAGANDHA

E.coli

S.aurues

Lactobacillus

AMLA

E.coli

S.aurues

Lactobacillus

COLONIES ARE COUNTED USING QUBEC COLONY COUNTER

EXPERIMENTAL RESULTS:No of colonies at 106 dilutionS.NO TYPE OF EXTRACT E.COLI S.AUREUS LACTOBACILLUS

1 AMLA 128 112 72

2 ASHWAGANDHA 144 172 168

NUMBER OF CELLS PER MILLI METER OF ORIGINAL SAMPLE = NUMBER OF COLONIES × DILUTION

S.NOTYPE OF EXTRACT

E.COLI S.AUREUS LACTOBACILLUS

1 AMLA 1.28X108 1.12X108 0.72X108

2 ASHWAGANDHA 1.44X108 1.72X108 1.68X108

NUMBER OF CELLS PRESENT AT 106 DILUTION:

A more elaborated results can be seen when number of colonies are converted into number of cells In the presence of E.coli, Ashwagandha shows an increase in the activity by 11% to the activity of AmlaWhile there is a significant increase of 35% in the anti-stress activity of Ashwagandha to Amla in the presence of S.aureusIn the presence of Lactobacillus there is a sharp rise in the activity of Ashwagandha to Amla by an increase in 57%

E.coli S.aureus Lactobacillus0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

1.28

1.12

0.720000000000001

1.44

1.721.68000000000001

COMPARISON OF CELLS PRESENT AFTER EXPOSURE TO STRESS IN THE PRESENCE OF EXTRACTS OF AMLA AND ASHWAGANDHA

amlaashwagandha

CONCLUSIONS AND SUMMARY:Ashwagandha and Amla both are said to have Adaptogenic activity according to Ayurvedic medicines

The results show that even after exposure to stress conditions (UV light) for a considerable period of 15minutes the microorganism managed to survive

This was seen when a diluted sample of the organism in the presence of both the extracts after exposure to stress produced a good number of colonies

Though E.coli is said to be an organism that can resist heat, high temperatures and other forms of stress like exposure to UV light other organisms like S.aureus and Lactobacillus also showed excellent results

NOVELTY OF THE WORK:Normally we see that evaluation of adaptogenic activity of an herbal formulation or an extract is carried out by determining its effects on restrain stress (by immobilization of rats). The use of albino mice or albino wistar rat is commonly seen.

The novelty of the work is the use of microorganisms to evaluate the activity of adaptogens in the herbal extract.

Microorganisms of different strains are used to evaluate the anti-microbial activity of the Flavonoids present in the herbal formulations but a procedure to evaluate anti-stress activity of microorganisms in the presence of an adaptogen has never been performed

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[10] Rege NN, Thatte UM, Dahanukar SA. Adaptogenic properties of six rasayana herbs used in ayurvedic medicine. Phytotherapy Res 1999; 13:275–91. [11] Muruganandam, A. V.; Kumar, Vikas; Bhattacharya, S. K.; Effect of poly herbal formulation, EuMil, on chronic stress-induced homeostatic perturbations in rats; Indian Journal of Experimental Biology 40 (10) : 1151-1160 October, 2002 [12] Biosourcing.Com Private Limited, Bhubaneswar [13] http://www.anniesremedy.com/herb_detail205.php [14] Studies on Effect of root extracts of Withania somnifera on some clinically isolated bacterial pathogens by Puja S.A and Gandhi M.B[15] Lapage S., Shelton J. and Mitchell T., 1970, Methods in Microbiology', Norris J. and Ribbons D., (Eds.), Vol. 3A, Academic Press, London[16] http://aminj.myweb.uga.edu/KIRBY-BAUER.html[17] Brock Biology of Microorganisms 11/e

THANK YOU AND STAY STRESS FREE