“UTILIZATION OF ASHWAGANDHA (WITHANIA SOMNIFERA)

ROOT POWDER IN FORMULATION OF HEALTH FOODS”

THESIS SUBMITTED TO

ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY

IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF

MASTER OF SCIENCE

(FOOD SCIENCE & TECHNOLOGY)

By

MRIDU NARAYAN

B.A.Sc. (H) (FOOD TECHNOLOGY)

POST GRADUATE & RESEARCH CENTRE

ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY

RAJENDRA NAGAR, HYDERABAD- 500 030.

OCTOBER 2007

CERTIFICATE

Ms. MRIDU NARAYAN has satisfactorily prosecuted the course of

research and that the thesis entitled “UTILIZATION OF

ASHWAGANDHA ROOT POWDER IN FORMULATION OF

HEALTH FOODS” submitted, is the result of original research work and is

of sufficiently high standard to warrant its presentation to the examination. I

also certify that the thesis or part thereof has not been previously submitted

by her for the degree of any university.

DATE: Dr. N. LAKSHMI DEVI

PLACE: Major Advisor

ACKNOWLEDGEMENTS

First and foremost, I offer my obeisance to the Almighty for

having bestowed his grace.

I would like to express my earnest gratitude and heartful

thanks to Dr. N. Lakshmi Devi Professor, Department of Foods and

Nutrition, Postgraduate research centre, Rajendra nagar, Hyderabad

for reposing confidence in me and accepting me as her student. I

further extend my sincere gratitude for her meticulous guidance,

diligence, writing interest, constant encouragement and suggestions

throughout the course of work.

I place with deep sense of gratitude and respect, my heartful

thanks to Dr. Kamini Devi, Professor, Department of Foods and

Nutrition and Dr. K. Malla Reddy Professor and Head, Department

of Horticulture, members in my advisory committee for their valuable

suggestions, able guidance and encouragement to complete my

research work.

I am profoundly indebted to Dr. S. Sumathi, Professor and

Head, Department of Foods and Nutrition, Rajendra nagar for

providing me a milieu in the form of necessary facilities conducive

enough to my research work completion. I also feel equally important

to thank Dr.M. Usha Rani, Dr. Uma Maheshwari,

Dr. T.V Hymavati, Mr. B. Srinivasan and all other faculty members

for their kind cooperation during my study.

I take it as a privilege to receive the blessings of my father

Shri. Kapilesh Narayan, who was the main source of inspiration for

taking up this course and the study. I also take it as equally

important to owe my sincere regards and gratitude to my mother

Smt. Renu and brother Pranjal who wished success in all my ventures

and provided constant encouragement.

I take pleasure in expressing sincere thanks to my colleagues

who supported me in all my efforts and extended their timely

assistance. My grateful thanks to my friends Prerna, Dolly, Silk,

Ragini ,Priya, Noor and Sanjeev for their cooperation and

encouragement.

My special thanks to V. Kumari madam, Shakuntala madam,

Venkataiah, Yedukondalu, and Shobha for their help and

coordination.

Finally I find it necessary to extend my heartful thanks to

those behind the curtain, whose unfailing belief in me has shown me

this day.

(Mridu Narayan)

DECLARATION

I, Ms. MRIDU NARAYAN hereby declare that the thesis entitled

“Utilization of Ashwagandha root powder in formulation of health

foods” submitted to Acharya N.G. Ranga Agricultural University for the

degree of MASTER OF SCIENCE is a result of original research work

done by me. It is further declared that the thesis or any part thereof has not

been published earlier in any manner.

(MRIDU NARAYAN)

LIST OF CONTENTS

CHAPTER NO. TITLE PAGE No.

I Introduction 1-4

II Review of Literature 5-32

III Materials and Methods 33-42

IV Results and Discussion 43-61

V Summary and Conclusion 62-66

Literature cited 67-77

Appendices 78-99

LIST OF TABLES

TABLE

No. TITLE

PAGE

No.

1

Proximate and Withaferine content of Ashwagandha

root powder

44

2

Effect of storage condition and packaging material on

chemical properties of Ashwagandha powder

46

3

Microbiological count of Ashwagandha powder

stored in different packaging materials

48

4 Mean sensory scores of deep fried products

52

5 Mean sensory scores of Missi roti and Chutney

powder 53

6 Chemical composition and cost of developed products 60

LIST OF FIGURES

FIGURE

No. TITLE

PAGE

No.

1

Schematic experimental design of storage study of

Ashwagandha root powder in different packaging

materials

35

2

Schematic experimental design of product

development

36

3 Mean scores for the sensory evaluation of Pappu Chakalu

54

4 Mean scores for the sensory evaluation of Namakpara

55

5 Mean scores for the sensory evaluation of Muruku

56

6

Mean scores for the sensory evaluation of Chutney powder

58

7 Mean scores for the sensory evaluation of Missi Roti

59

LIST OF APPENDICES

APPENDIX

No. TITLE

PAGE

No.

I

Method of preparation of products 78-82

II Score card for sensory evaluation of products 83

IIIA Estimation of moisture 84

IIIB Estimation of crude fiber 85

IIIC Estimation of reducing and total sugars 87

IIID Estimation of fats 90

IIIE Estimation of proteins 91

IIIF Estimation of total Ash 94

IV Quantitative estimation of Withaferine A 96

V Microbiological analysis 99

LIST OF ABBREVIATIONS

°C : Degree centigrade

g : Gram

LDPE : Low density polyethylene

PP : Polypropylene

PET : Polyethylene terepthalate

SD : Standard deviation

mg : Milligram

IS : International Standard

WS : Withania somnifera

RT : Room temperature

% : Percentage

LIST OF PLATES

PLATE NO.

TITLE PAGE NO.

1 Ashwagandha roots and root powder

38

2 Ashwagandha root powder incorporated Muruku

38

3 Ashwagandha root powder incorporated Pappu Chakalu

39

4 Ashwagandha root powder incorporated Namakpara

39

5 Ashwagandha root powder incorporated Missi roti

40

6 Ashwagandha root powder incorporated Chutney powder

40

Author : MRIDU NARAYAN

I.D. No. : FST/2005-04

Title of thesis : UTILIZATION OF ASHWAGANDHA

(Withania somnifera) ROOT POWDER IN

FORMULATION OF HEALTH FOODS

Degree : MASTER OF SCIENCE

Faculty : INTERFACULTY P.G. PROGRAMME

Major Field of study : FOOD SCIENCE & TECHNOLOGY

Major advisor : DR. (Mrs.) N. LAKSHMI DEVI

University : ACHARYA N. G. RANGA

AGRICULTURAL UNIVERSITY

Year of submission : October 2007

ABSTRACT

Clinical and experimental evidence indicates that phytochemicals

identified from traditional medicinal plants are presenting an exciting

opportunity for the development of new type of therapeutics. Such foods in

addition to their basic nutritive value and natural being help us to function

better and more effectively. The idea of health filled foods is to produce

packaged food which retained its nutrition and appeared and tasted as close

to nature’s intentions as possible.

Ashwagandha is a valued herb in Ayurveda medicine and as such was

used and cultivated for centuries in India. It possesses therapeutic value

against a large number of ailments such as mental diseases, asthma,

inflammation, arthritis, rheumatism, tuberculosis, infections, fever, male

sexual disorders and a variety of other diseases including cancer.

The present study was thus undertaken with the objective to exploit the

potential benefits of the plant in formulation of various health foods.

The nutrient analysis revealed that Ashwagandha roots are a good

source of dietary fiber (28.8%) and minerals (10.1%) apart from having

Withaferine A content of 0.16% which accounts for its multiple medicinal

applications.

The shelf life of Ashwagandha powder was studied by storing in three

different packaging materials viz., Low density polyethylene (LDPE),

Polypropylene (PP) and Polyethylene terepthalate (PET) for three months

and was analyzed for physical, chemical and microbiological parameters

before and after storage period.

No major differences were found on physical characteristics as well as

on proteins, fat, total ash and total sugar content of the powder on storage in

different packaging materials. Though a progressive increase in moisture

content from 5.2% to 5.7% in LDPE, 6.3% in PP and 8.7% in PET was

observed at room temperature, the gain of moisture was more at ambient

temperature as compared to low temperature (refrigeration) storage.

The microbial load of the Ashwagandha root powder with 450 cfu/ml

colonies initially increased on storage for three months both at ambient and

refrigeration conditions in all the three packaging materials. The percent

increase in the microbial count of the powder in LDPE was least both at

room temperature (1000 cfu/ml) and refrigeration (850 cfu/ml) as compared

to PP (8000 cfu/ml, 1000 cfu/ml) and PET (10,000 cfu/ml, 1000 cfu/ml).

No yeast and mold growth was detected initially in the sample. The

colony forming units were nil even after 90 days of storage, except for the

powder stored in PET films (1000cfu/ml).Among all the three packaging

materials, LDPE showed the best results both at refrigeration and room

temperature and could be suggested for storage of root powder than others.

Ashwagandha root powder was incorporated directly as powder after

sieving or as boiled extract in a number of recipes like fruit-whey beverages,

bakery items, savoury and snack items, at 5, 7.5, 10 and 13 percent levels.

Among all the three categories of food products, savoury and snack products

like Namakpara, Muruku, Pappu chakalu, Chutney powder and Missi roti

(chapathi) were successfully prepared and standardized. Sensory results

revealed that the Ashwagandha incorporated recipes were almost

comparable to standard recipes with respect to taste, flavour and overall

acceptability.

Thus it was concluded that the Ashwagandha root powder is safe to

store in LDPE pouches at ambient temperatures without any significant

changes in its physical, chemical and microbiological properties.

It was observed that spices such as asafoetida, cumin, black pepper,

chillies etc effectively mask the typical odour and bitter taste of

Ashwagandha. The results of the study also suggest that the Ashwagandha

root powder should preferably be used in salty products as it does not go

well with sugar based products, specially the bakery items and beverages.

Recent years have witnessed a phenomenal growth in the

market demand for traditional snack foods. These are unique in their

character and composition, cheap in price and are accessible to the poor.

According to one estimate the consumption of traditional foods in India is

more than 30 times the quantity of processed style foods like bread, biscuits,

jams, ice-creams, soft drinks and confectionary items. Hence Ashwagandha

incorporated traditional recipes like Namakpara, Muruku, Pappu chakalu,

Chutney powder and Missi roti can be explored for consumption as a step in

this direction will go a long way in improving the health and well being of

people of all economic groups.

CHAPTER I

INTRODUCTION

Medicinal plants are the local heritage with global importance. World

is endowed with a rich wealth of medicinal plants. The variety and sheer

number of plants with therapeutic properties is quite astonishing. It is

estimated that around 70,000 plant species, from lichens to towering trees,

have been used at one time or another for medicinal purposes.

The Indian Pharmacopia (1966) recognizes eighty five drug plants

whose ingredients are used in various pharmaceutical preparations.

(Bhattacharjee, 1998).

Over time, the practice of herbal medicine has grown more complex.

Science has enabled us to process natural substances into pills, tinctures and

powders.

Today medicinal plants in the form of ‘Herbal Neutraceuticals ‘are

assuming a middle ground between food and drug. There has been a

resurgence in the consumption and demand for these plants due to a growing

body of evidence that supports their role in maintaining health and

contributing to the treatment of diseases. (Tyler, 1996).

Herbs or botanicals may have health benefits that are not derived from

the plant’s nutrient composition. The curative properties of medicinal plants

are due to the presence of various complex chemical substances of different

composition which are found as secondary plant metabolites. These

Scientific name: - Withania somniferaCommon name:- Indian Winter Cherry (English),

Indian ginseng, ashwagandha, punir (hindi),

Distribution:- India, North AfricaPart of plant used:- Roots, Leaves

metabolites according to their composition are grouped as alkaloids,

glycosides, corticosteroids, essential oils etc. (Narayan Das Prajapati and

Purohit SS 2003)

With an emphasis on quality and standardization, food manufacturers

may find herbals to be a new source of functional ingredients. Herbal

additives have begun to appear in conventional foods ranging from teas and

juices to snack chips and energy bars.

Adding botanicals to foods to create functional products raises many

issues including appropriate regulation, safety, stability and efficacy.

Individual food herbal dynamics will create even more challenges including

ways to mask the unpleasant flavours of some herbs. However as our

knowledge base grows and confines the suspected actions of many herbal

compounds, our future food supply could be dramatically different than it is

today. (Goldberg, 1994)

Withania Somnifera, popular as Ashwagandha is also explored as a

medicinal plant. It is a native to Mediterranean region and found growing

naturally in forests particularly in drier regions of India. It is an important

cultivated medicinal crop of India, with the annual production of about

12,000 tons both from wild as well as cultivated sources.

Several types of alkaloids are found in this plant, Withanolides

(Withaferine A, Withanone, Withanolide I, II, III etc.) are the most

important bioactive constituents of roots that are believed to account for the

multiple medicinal applications of Ashwagandha (Sharma et al 1985).

Ashwagandha possesses therapeutic value against a large number of

ailments such as mental diseases, asthma, inflammation, arthritis,

rheumatism, tuberculosis, infections, fever, male sexual disorders and a

variety of other diseases including cancer. (Asthana et al, 1989, Singh et al,

1998).

The root is used as an aphrodisiac, tonic, and diuretic. It is useful in

sexual & general weakness. It promotes urination, acts as a narcotic and

removes functional obstruction of body.

The root powder is applied locally on ulcers and inflammation and on

tubercular glands while paste of its roots is used for curing skin diseases,

bronchitis and ulcers. (Sharma and Ravindra, 2004).The root is also said to

have been used to treat snake bites. In some areas warm leaves are used for

providing comfort during eye diseases. (Bhattacharjee, 1998)

In India, the Ayurvedic systems of medicine have been in use for over

three thousand years. But the age-old Indian systems of medicine have been

neglected mainly because of the allopathic system of medical treatment. This

is despite the fact that our country has a long history of local health

traditions.

However the market for herbal products and medicines in the

developed countries is growing at a faster rate. The WHO estimated that 80

percent of the population of developing countries still relies on traditional

plant drugs for their primary health care needs.

Plants contain many physiologically and /or pharmacologically

active compounds; their consumption may reduce the risk of some common

degenerative diseases in the majority of people. Food fortification with these

plants or their active principles is a fertile field for research and

development.

A wide range of food products like drinks, confectionary items, chips,

tea and traditional foods like chapatti, dosa, dal, idli etc. can be developed

using them.

Though a number of herbal products containing Ashwagandha in the

form of tablets, capsules, powders, rasayans, tonics etc. are available on

shelf, but the bitter taste of Ashwagandha root powder is considered as a

limitation to its use in food products. Hence the present study is an attempt

to utilize the powder of Ashwagandha (Withania somnifera) roots in

formulation of various health foods with the following objectives:-

GENERAL OBJECTIVE:

Utilization of Ashwagandha root powder in formulation of health

foods.

Specific Objectives:

1. To study the shelf life of Ashwagandha root powder using different

packaging materials.

2. To carry out storage stability of the powder by means of physico-

chemical & microbiological analysis

3. To standardize products using the root powder at different levels & test

the acceptability by sensory evaluation

4. To analyze the physico-chemical properties of the developed products.

CHAPTER II

REVIEW OF LITERATURE

Medicinal plants have served as a common link between the

traditional and modern medical science as they are the main source of

medicaments involved through the centuries.

They have been widely used for treatment of various human ailments in all

cultures throughout history and today extensive scientific documentation

now exists concerning their use in number of health conditions.

The word ‘drug’ comes from the old Dutch word “drogge’ meaning

‘to dry’ as pharmacists physicians and ancient healers often dried plants for

use as medicines. Some of these drugs are made from plant extracts while

others are synthesized to mimic a natural plant compound.

Even today about 80% of the world’s population particularly that living in

the rural area is dependent on traditional medicines for their health security.

Twenty five percent of all such prescriptions contained plant extracts or

active principles obtained from higher plants.

Of the estimated 250,000- 500000 plant species of the world more

than 2/3rd occur in the tropical forests of developing countries but only a

small percentage of these plants have been investigated phytochemically

active and only a fraction of that has been subjected to biological or

pharmacological screening.

Since each plant may contain hundreds or even thousands of

metabolites, each with differing biological activity there is currently a

resurgence of interest in the plant kingdom as a possible source of new lead

compounds for introduction into therapeutically screening programs.

(Akerele, 1992).

Clinical and experimental evidence indicates that phytochemicals

identified from traditional medicinal plants are presenting an exciting

opportunity for the development of new type of therapeutics. This has

accelerated the global effort to harness and harvest medicinal plants that bear

substantial amount of potential phytochemicals showing multiple beneficial

effects in combating various public health problems. (Tyler, 1996)

Functional foods in addition to their basic nutritive value and natural

being help us to function better and more effectively by helping us directly

in prevention and treatment of illness and disease. The idea of health filled

foods is to produce packaged food which retains its nutritional and appears

and tastes as close to nature’s intentions as possible.

(Goldberg, 1994)

Ashwagandha is a valued herb in Ayurveda medicine and as such was

used and cultivated for centuries in India. Annual production of drug is

12,000 tons both from wild as well as cultivated sources. While the annual

requirement of the drug in Indian market is about 3000 tons. The present

market rate of dried root is about Rs. 80 per kg and the wholesale rate is

about Rs. 5000 per quintal. (www.primaryinfo.com). Ashwagandha root has

great demand in crude drug market and the drug is mainly used in Ayurvedic

& Unani preparations. (Sharma, 2004).

It possess therapeutic value against a large number of ailments such as

mental diseases asthma, inflammation, arthritis , rheumatism, tuberculosis,

infections , fever, male sexual disorders and a variety of other diseases

including cancer.(Asthana et al., 1989, Singh et al., 1998)

The present study was thus undertaken with the objective to exploit

the potential benefits of the plant in formulation of various health foods. The

literature furnished below provides an overview of the following aspects:-

2.1 Description of plant

2.2 Traditional Ayurvedic uses of Ashwagandha

2.3 Composition of Ashwagandha

2.4 Therapeutic properties

2.5 Safety and dosage

2.6 Products on shelf

2.7 Packaging materials

2.1 DESCRIPTION OF PLANT

Withania somnifera, also known as the Indian Ginseng is a perennial

plant that belongs to the family solanaceae. It is commonly known as

“Ashwagandha” or “Asgandh” (smells like a horse) and is an ancient

Ayurvedic drug. The crop is widely distributed in India, Pakistan, Srilanka,

Mediterranean regions, Canaries, South Africa, Syria and Turkey (Nasir et

al., 1972; Jaffari et al., 1966)

In India it is cultivated in an area of about 4000 Ha, mainly in the

drier parts of Manasa, Neemuch and Jawad tehsils of the Mandsaur district

of MP, in Punjab, Sind, Rajasthan and South India (Sharma, 2004)

Ashwagandha is an evergreen medium sized undershrub 30-150 cm

long with ascending branches and stellate- tomentosic shoots. (Chadha,

1976)

The leaves are ovate and hairy while stems and branches are also

covered with minute star shaped hairs. It has one or more fairly long

tuberous roots and short stem. The flowers are greenish yellow borne in

auxiliary clusters while the fruits are smooth red when ripe, globose, and

enclosed in an inflated and membranous calyx.

The roots of the plant are 20-30 cm long and 6-12 mm in diameter

with few (2-3) lateral roots of slightly smaller size & straight unbranched.

The outer surface is buff to grayish – yellow with longitudinal wrinkles and

contains soft solid mass with scattered pores in the center. (Bhattacharjee,

1998)

The roots have a strong odour and a mucilaginous, bitter and acrid

taste. The young tuberous and older roots have distinctive macroscopic and

microscopic characters.

Soil and climate

It grows well in well drained sandy loam or light red soil of 7-8 pH with

good organic matter & drainage. The plant prefers a subtropical dry climate

and is grown in late rainy season (kharif crop). An annual rainfall of 660-

750mm is best suited for its growth. The root development improves if one

or two winter rains are received. (Sharma, 2004)

Cultivation

Before cultivation the land should be ploughed twice to make the filth fine

and weed free. After leveling it is divided into beds of suitable size having

proper irrigation channels.

Ashwagandha is a rainfed crop and require little irrigation after the crop get

established. However additional irrigation helps in better root growth.

There is no experimental evidence on the effect of fertilization on root yield

of this crop (Bhattacharjee, 1998). The crop is harvested for roots by digging

in January to March i.e. 150-180 days after sowing.

The roots are then separated, dried and graded according to its length and

diameter. They should be solid, brittle and pure white from inside. The crop

yields 3-4 quintals of dry roots and half quintals of seed per hectare.

Grading

The roots after cultivation are carefully hand-sorted into four grades, based

on the thickness and uniformity of the pieces. (Sharma, 2004)

A – Grade- root pieces up to 7 cm in length, solid, with 1-1.5 cm diameter,

brittle & pure white from inside.

B- Grade- root pieces up to 5 cm in length, solid, with a diameter of less

than 1 cm, brittle & white from inside

C- Grade- root pieces up to 3-4 cm in length, side branches solid, with a

diameter of 1 cm or less.

D- Small root pieces, semi- solid, very thin and yellowish on the inside.

2.2 TRADITIONAL AYURVEDIC USES

Ashwagandha has been mentioned in Ayurvedic classics as a rasayana

and in Ayurveda it is stated that Rasayana is the best profitable means of

obtaining body tissues like- Rosa Rakhta Mamsa etc of very high quality.

The plant finds extensive uses in the indigenous medicinal system (Charka,

1941).

The leaves of the plant are used for tumor and tubercular glands in

Ayurveda and Unani systems (Chopra, 1958). Leaves are bitter in taste and

used as an anti-helmanthic. They are also given as infusion in fevers.

A fomentation of the leaf is used to cure sore eyes. It cures boils and

swellings of the hands and feet causing the boils to ripen and burst. The

leaves are also used as a hypnotic in alcoholism and the fresh leaf- juice is

applied on anthrax pustules.

The roots are used in constipation, rheumatism, in cases of nervous

exhaustation, loss of memory, loss of muscular energy and spermatohoea. It

infuses fresh energy and vigor and is good for the treatment of syphilis

rheumatic fever etc. The roots of the plant are also used as nutrient and

health restorative agent in pregnant women and old people. The decoction of

the root boiled with milk and” vegetable oil’ is recommended for curing

sterility in women (Kirtikar and Basu, 1999).

The tuber has a bitter acrid taste. It is aphrodisiac , tonic, anthelmintic

thus useful in “vata” and “ kapha” inflammations, Psoriasis, bronchitis,

asthama , constipation, ulcers, marasmus of children, senile debility (Thattey

and Dhanukar, 1989)

Rajputs regard the root as useful in rheumatism and dyspepsia. In

Punjab it is used for lumbar pains. The ground root and bruised leaves are

employed as a local application to carbuncles, ulcers and painful swellings.

The seeds are employed to coagulate milk. They are diuretic and hypnotic.

The sutas use a decoction of the root for colds and chills while

Transvaal sutos administer it to tone up the uterus in women who habitually

miscarry. The sutos also take an infusion of the bark for asthma and apply an

ointment of the leaf to bed sores.

Europeans used to take a decoction of the root for chest complaints

and decoction of the leaf externally and internally in the treatment of

haemmorrhoids. (Kirtikar and Basu 1999)

The root in combination with other drugs is prescribed for snake bite

and scorpion sting. (Charka, 1941)

The decoction of the root mixed with pepper (piper longum), butter

and honey (25-50 g) or the powdered root with milk or clarified butter is

used as an aphrodisiac and in-seminal debility.

The decoction along with milk and clarified butter is considered as a

cure for female sterility, if taken for a few days after the menstrual period.

An enema of the roots (with bark removed) is given to feverish infants.

In Ayurvedic literature it has been stated that Ashwagandha

administered with milk to toddlers and growing children for 15 days

nourishes them as rain does to tender crops. (Sarma, 1956)

When taken with milk, ghee, oil or water (15 days) it imparts strength

for the emaciated body. It gives luster virya and bala (strength). (Thayyil

Kumara Krishnan, 1960).

2.3 COMPOSITION

Phytochemical studies on this plant has showed the presence of

various chemical constituents which have been isolated and identified by

chemical and spectroscopic means and tested for their biologically active

constituents.

The fruits of this plant contain a high proportion of free amino acids

as evidenced by a strongly positive Ninhydrin Colour reaction. The presence

of these amino acids may be explained by the fact that a proteolytic enzyme

chymase is present in the berries of this plant (Atal and Shwarting 1960)

It contains a large number of alkaloids and withniols (0.13- 0.68%).

Withaniol group of alkaloids (withanine and pseudowithanine) are

therapeutically important. Root parts contain higher percentage of alkaloid

and are collected from cultivated plants also. (Pullaiah, 2006).The total

alkaloid content in the roots of Indian types has been reported to vary

between 0.13% and 0.31%. (Sharma, 2004)

In all 13 components have been obtained chromatographically. This

includes Choline, tropanol, pseudotropanol, Cuscokygrens, 3-

tigloyloxytropana, Iso pelletierine, Anaferine, anahygrine, Withasommnine

and several other lactones.

Withanolides (Withaferin A, Withanone, Withanolide I, II, III etc.) are

the most important bioactive constituents of roots that are believed to

account for the multiple medicinal applications of Ashwagandha (Sharma et

al 1985). These are compounds specific for the solanaceae family and in

particular for the genus withania, and are used as marker compounds.

(Bruneton, 1999)

The analysis of W. somnifera root, stem and leaf confirmed the

presence of Withferine A and Withanolide D in all parts of the plant, but

with significant differences in ratio. Withaferine D was dominant in the

roots (0.193%) while minor in leaves whereas a rather a high amount of

withaferine A (0.236%) was found in leaves.

Stem contained lowest percentage of total A and D withanolides (0.055%)

(Ganzera et al., 2003).

The roots are also reported to contain starch, reducing sugars,

hentriacontane, glycosides, dulcitol, withanicil (0.08%) (Sharma, 2004)

Ashwagandha roots contain a large reserve of iron. (Kupppurajan et

al., 1980). It is worthwhile to note that Ashwagandha contains free amino

acids like valine tyrosine, proline, alanine and glycine in abundance. (Atal

and Shwarting 1960))

2.4 THERAPEUTIC PROPERTIES

Ashwagandha, i.e. Withania Somnifera (solanaceae) is an Ayurvedic

medicinal plant having many common properties as ginseng. (Patwardhan et

al 1988) thus is also sometimes called as the Indian ginseng. This plant has

been investigated for chemical, pharmacological and therapeutic efficacy.

(Sharma and Dandiya 1991).

The compounds known as withanolides are believed to account for the

multiple medicinal applications of Ashwagandha. These molecules are

steroidal and bear a resemblance, both in their action and appearance, to the

active constituents of Asian ginseng (Panax ginseng) known as

ginsenosides. Indeed, Ashwagandha has been called “Indian ginseng” also.

Ashwagandha is in use for a very long time for all age groups and both sexes

and even during pregnancy without any toxic effects. (Sharma et al 1985)

Adaptogenic Properties

Withania somnifera energizes our system to help us fight with stress

in easier and better way. It resets the immune system, nerves and hormones

to a healthier state so that a truly great adaptogenic effect is experienced.

Adaptogenic activity of Withania somnifera root extract was

evaluated in Chronic Stress rat models against adaptogens like Panax

ginseng. CS induced significant hyperglycaemia, glucose intolerance,

increase in plasma corticosterone levels, gastric ulcerations, male sexual

dysfunction, cognitive deficits, immunosuppression and mental depression

in the rats. The results indicated significant anti-stress activity in WS

extracts comparable to Panax ginseng. (Bhattacharya, 2003)

Shukla et al (2001) reported the protective effect of Withania

somnifera in Swiss albino rats in stress induced neuronal degeneration.

Study suggests that probably the herbal drug has cytoprotective properties.

In another study the levels of corticosterone were estimated by the

HPLC method in the adrenal glands of stressed (5 h constant swimming)

male albino mice treated with Trichopus zeylanicus, Withania somnifera and

Panax ginseng preparations were compared with non-treated stressed and

normal controls. The treatments increased the corticosterone levels in all the

groups indicating the anti- stress properties of all the three. (Singh et al.,

2000)

Anti- anxiety and Anti- depressant Herb

Ashwagandha is found to be of great help in anxiety, depression and

other psychiatric disorders. Its traditional benefits in Ayurvedic Medicine

have been validated by modern herbal research. Anxiety, depression, panic

attacks, phobia are very well supported by this herb & the effectiveness is

comparable to modern drugs.

Bhattacharya SK et al (2000) found that WS extract induced

anxiolytic & antidepressant effects comparable to that produced by

lorazepam, and imipramine, both standard drugs. The investigations thus

support the use of WS as a mood stabilizer in clinical conditions of anxiety

and depression in Ayurveda.

Antioxidant Properties

Ashwagandha or Withania somnifera is one of the first liner herbal

remedy with antioxidant, free radical scavenging properties.

This herb was tested on rats and found that it increases three natural

antioxidants in rat brain - catalase, superoxide dismutase and glutathione

peroxidase. This study validated its therapeutic effects popular in Ayurvedic

herbal medicine. This antioxidant effect partly explains its other effects like

anti-aging, anti-stress, anti-inflammatory and cognition facilitation.

(Esposito and Iuvone, 2003)

The WS extracts showed a dose-dependent free radical scavenging

capacity and a protective effect on DNA cleavage. These results were

confirmed by a significant protective effect on H2O2-induced cytoxicity and

DNA damage in human non-immortalized fibroblasts. (Russo et al., 2001)

The effects of Ashwagandha on copper-induced lipid peroxidation and

antioxidant enzymes in aging spinal cord of Wistar rats were investigated.

The activity of glutathione peroxidase (GPx) decreased significantly in the

spinal cord from adult to aged mice. Treatment with Ashwagandha

successfully attenuated GPx activity and inhibited lipid peroxidation as well

as protein oxidative modification induced by copper in a dose dependent

manner. The results indicate the therapeutic potential of Ashwagandha in

aging and copper-induced patho-physiological conditions. (Gupta et al.,

2003).

A double blind clinical trial to study the effect of WS on prevention of

process of ageing in 101 male healthy adults in age group of 50-59 yrs

indicate that there is increase in haemoglobin, RBC, hair melanin and seated

stature in the treated group which is statistically significant than the

placebo.( Kuppurajan et al, 1980)

Brain Positivity & Ashwagandha

Ashwagandha benefits the brain and some of the neurochemicals

positively.It is used in India to treat mental deficits in geriatric patients,

including amnesia.

It also benefits by helping to de-addict from morphine. Additionally it

possesses nootropic like effect on brain and nerves.

A study at Department of Pharmacology, University of Texas, found

GABA like activity in Withania somnifera. The results suggested that the W.

somnifera extract contains an ingredient which has a GABA-mimetic

activity. This activity was correlated with Anti-anxiolytic effect (Mehta,

1991)

Researchers in Pakistan isolated six withanolides from Ashwagandha.

Out of six, four withanolides displayed inhibitory potential against

butyrylcholinesterase, while three were active against acetylcholinesterase.

This activity helps to retain Acetylcholine for longer time by slowing down

its breakdown. (Choudhary et al., 2005)

Studies showed that treatment with Ashwagandha slowed the

development of tolerance to analgesic effect of morphine.WS (100 mg/kg)

also suppressed morphine-withdrawal jumps, a sign of the development of

dependence to opiate. (Kulkarni et el., 1997)

Ashwagandha (50, 100 and 200 mg/kg; orally) also reversed the

scopolamine (0.3 mg/kg)-induced disruption of acquisition and retention and

attenuated the amnesia produced by acute treatment with electroconvulsive

shock (ECS), immediately after training. Chronic treatment with ECS, for 6

successive days at 24 h intervals, disrupted memory consolidation on day 7.

(Dhuley,1998)

Neuritic regeneration and synaptic reconstruction is induced by

withanolide. (Kuboyama et al., 2002)

Ashwagandha: potential in cancer

Ashwagandha or Withania is one such herb whose role as anticancer

herb is widely studied. It doesn't possess properties like chemotherapy but

showed anti- proliferate and preventive properties against many chemicals

that possess carcinogenic effect.

In Ayurveda, the extracts from W. somnifera are distinctively

employed for the treatment of arthritis and menstrual disorders. Because

these conditions involve angiogenic processes it was hypothesized that the

W. somnifera extracts might contain angiogenesis inhibitors.

The applicability of this drug as a radio sensitizer in cancer therapy needs to

be explored. (Devi et al., 1996).

Twelve Withanolides from the leaves of withania somnifera were

tested for anti-proliferative activity in lungs, colon, breast, central nervous

system human tumor cell lines. Withaferine A showed inhibitory activity.

Thus it was concluded that Adding WS as dietary supplement might prevent

the growth of tumor in human. (Jayaprakasam et al., 2003)

In another study, the leaf extract of WS was investigated against the

mutagenic effects of MNNG (N-methyl-N'-nitro-N-nitrosoguanidine), a

highly toxic chemical. This study reported significant protection against the

mutagenic effect of MNNG by Withania somnifera. (Kaur, et al, 2004)

Field raised and Laboratory raised leaves of Withania somnifera was

tested for anti-proliferative and anti-oxidative properties. A significant anti-

proliferative activity in human tumor producing cells was found in field

raised plants .It was not found to protect against oxidative damage by high

glucose and hydrogen peroxide to human tumor cells. The study suggested

its potential role as anti-tumor. (Christina, 2004)

A similar study from All India Institute of Medical Sciences, New

Delhi, India, found Withania somnifera extract to prevent (DMBA) (2,4-

Dimethylbenzoic Acid )-induced squamous cell carcinoma of skin in Swiss

albino mice. This study indicated that Withania somnifera hydro-alcoholic

root extract (WRSE) possesses potential chemopreventive activity.

In yet another study a significant increase in the life span and a

decrease in the cancer cell number and tumour weight were noted in the

tumour-induced mice after treatment with Ashwagandha .(Christina et al.,

2004 )

Gupta et al (2003) reported the reversal of paclitaxel induced

neutropenia by Withania somnifera in mice. The findings of the study

suggest the potential of W. somnifera as an adjuvant during cancer

chemotherapy for the prevention of bone marrow depression associated with

anticancer drugs.

Thus incorporation of withanolides in the diet may prevent or

decrease the growth of tumors in human. (Jayaprakasam et al., 2003)

Cardiovascular & Diabetic protection with Withania somnifera

Ashwagandha or Withania somnifera has in its arsenal the weapons

for cardiac and diabetic disorders as well. This botanical is truly versatile in

the beneficial effects for us. The cardiac protective action is exerted by

helping in ischemic and reperfusion injury. It supports focal ischemia as

well. (Gupta, 2004)

A study showed Cardio-protection against Ischemic & reperfusion

Injury by Ashwagandha (Manikandan, 2004).In yet another study

effectiveness of WS was reported in focal ischemia. Pretreatment with its

extract for 30 days prevented motor impairment and significantly decreased

the raised levels of MDA (Malondialdehyde), the marker of oxidative

stress). (Chaudhary et al., 2003)

Six mild NIDDM and hypercholesterolemic persons were treated with

Withania somnifera for one month. At the end of one month, the blood

glucose level was lower comparable with oral hypoglycemic drug. There

was significant increase in urine volume, urine Na+ while serum cholesterol,

triglycerides, LDL & VLDL cholesterol were reduced. This reflected

hypoglycemic, diuretic and anti-hypercholesterolemic effects of

Ashwagandha (Andallu, 2000)

Ashwagandha: Immunity Boosting Herbal Remedy

Withania somnifera enhances body’s immunity and safeguards against

various diseases and infections. Strengthening of immunity is done in

various ways i.e. by promoting the bodily defense mechanisms

like increasing the White blood cells count.

Methanolic extract of Withania somnifera root was investigated for its

immunostimulant effect on Nitric oxide (NO) production in J774

macrophages. Ashwagandha caused dose dependent rise in nitric oxide

production. Induction of nitric oxide production in macrophages explains its

immune boosting effects. (Esposito and Iuvone, 2003).

Another study suggested that administration of an extract from the

powdered root of Ashwagandha stimulates immunological activity in mice.

( www.holistic-herbalist.com)

Aggarwal et al studied immunomodulatory activity of Ashwagandha

extract in experimental immune inflammation. A protective effect in

cyclophosphamide-induced myelosuppression was observed in animals

treated with its extract, revealing a significant increase in white blood cell

counts and platelet counts. (Agarwal, 1999)

Ashwagandha & Alzeimer’s disease

Ashwagandha is found to support Parkinson's disease and Alzheimer's

disease. Both of these affect the senior citizens most and are incapacitating

in one way or the other. With Parkinson's disease, the patient is left to great

mental trauma because of the tremors and with Alzheimer's disease, the

person experiences dementia.

It’s usefulness in Parkinson's disease is because of its L-dopa content

and several other inherent actions. Alzheimer's disease is relieved by

positive effects of this botanical on Cholinergic receptors. (Arai H, 2000)

Besides above, there are other properties in traditional Ayurvedic

medicine that supports its efficacy in these disorders

Ashwagandha in Miscellaneous & Versatile Role

Ashwagandha apart from being a valuable adaptogenic and

rejuvenative herb is additionally useful in several other ways too.

WS exerts anti-inflammatory, antiviral, antibacterial, anti ulcer

activity. It is found promising in curing side effects of fungicide. It relieves

CCl4 induced hepatotoxicity. Intestinal absorption is restored.

Five plants (Myrtus communis, Apium graveolens, Matricaria

chamomilla, Withania somnifera and Achillea santolina) grown in Iraq

were assessed for their anti-inflammatory activity on intact rats by

measuring the suppression of carrageenan-induced paw

edema. Acetylsalicylic acid was used as the standard drug.

Results showed that the plants possessed varying degrees of anti-

inflammatory activity and were classified in the following descending order

of activity: W. somnifera > A. graveolens > A. santolina > M. chamomilla >

M. communis.( Al-Hindawi, 1992)

Antibacterial Activity of Ashwagandha:

Researchers evaluated alcoholic and aqueous extract of Ashwagandha

against experimental murine salmonellosis. It was found to possess strong

anti-bacterial activity against a range of bacteria including Salmonella

typhimurium. WS didn't induce the lysis of erythrocytes like

Chloramphenicol. Treated animals revealed better survival rate and lesser

bacterial load in vital organs. (Owais et al., 2005)

Another study also found potent antibacterial activity against S.

typhimurium and E. coli. In Ashwagandha. Additionally it exerted

synergistic effect with Isoniazid and Rifampicin. ( Okunade , 2004 )

The methanolic and ethanolic extracts of the aerial parts of the plant

showed significant antimicrobial activity against gram positive bacteria.

However none of these extract were effective against gram negative bacteria.

(Jaffer et al 1988)

Withanolides from WS could also be used in curing the side effects of

anti-fungal drug carbendazim. The drug affects renal tubules and

hepatocytes advesely. Treatment of carbendazim-treated rats with the

powder of tuberous root of Withania somnifera (Ashwagandha) for 48 days

resulted in complete cure of these organs. The results indicate that

Ashwagandha would be an effective curative for carbendazim-induced

histopathological changes in the liver and kidney. (Akbarsha , 2007).

Anti inflammatory properties

The anti inflammatory activity and protective effect against ccl4

induced hepato- toxicity of the alcoholic extract of leaves of W. somnifera

has also been assessed.

The leaves were found to possess marked effects in sub acute

inflammation and hepato toxicity. A comparison of the anti – inflammatory

properties revealed the extract (at 1 g/kg dose) to be as active as 50 mg / kg

of phenylbutzoneand 10 mg/kg of hydrocortisone (Sudhir et al., 1986).

Ashwagandha and Arthiritis

Ashwagandha is a good natural remedy for Arthritis. Most of the

joints pain in arthritis is a result of degenerative disease in body due to

wrong posture, wear and tear of tissues and aging.

In clinical studies Ashwagandha root powder has been found to be

useful in cases of acute rheumatoid arthiritis especially of recent onset & to

some extent in cases of acute exacerbations of chronic rheumatoid arthiritis.

(Bector, 1968).

2.5 SAFETY AND DOSAGE

Ashwagandha side effects are not present in any significant way and

its use can safely be done on regular basis. Its safety profile is based on

research.

In India, Ayurvedic practitioners are using this herb in all age groups

in male and female with good results. There is no problem of using it in

children. Though no thorough clinical research is backing this use.

Under normal therapeutic dosage Ashwagandha side effects are not

present if it is used for more than 180 days. However it is a good practice to

give a break of few days, say one week, after six weeks of its use.

(www.holistic-herbalist.com)

There are no dietary restrictions with its use. In Ayurvedic medicine,

there is general recommendation about using moderate quantities of spices,

chillies and sour items with it. Usually people feel some sort of refreshing

energy & some warmth after using this herb. It is never hot or stimulating in

action. Its benefits build up over a period of few weeks.

Ashwagandha Side Effects:

Ashwagandha is well tolerated and without any significant side

effects. No significant drug interactions have been found.

Though some persons have complained of slight drowsiness after

using it while with majority of persons there had been no trouble at all. The

persons who might have felt some drowsiness can try it after meals but even

then if the complaint persists, it can be taken in the evening.

The crude powder of WS is slightly hard to digest and it can cause

some heaviness in abdomen and flatulence especially in those who have

weak digestion. (www.holistic-herbalist.com)

Evaluation of Subacute Toxicity:

In a research subacute toxicity of Ginseng and Ashwagandha was

assessed in rats. The study went on for 90 days and there were no toxicity in

these herbs individually. There was significant increase in body weight, food

consumption and liver weight, and improved hematopoiesis was observed.

Brain, heart, lung, liver, spleen, kidneys, stomach, testis and ovaries were

normal on gross examination and histopathologically. Subacute toxicity

studies in rats did not reveal any toxicity. (Aphale, 1998)

Another study, mentioned that Ashwagandha possesses no toxicity up

to a dose of (100 mg/kg; orally for 180 days) and does not cause significant

changes in biochemical parameters in the blood serum of rats. (Dhuley,

2000)

Ashwagandha is not found addictive in any way. So it is safe even

when one is using it for long times over a period of months.

Drug Interactions with Sedatives & Anxiolytics:

It should not be taken with sedatives and anti-anxiety drugs on

personal basis. However it is a safe practice to take it under the qualified

supervision. There might be some need of adjustment of dosage after some

time.

WS is found to potentiate the effects of Barbiturates so it is better not

to take it with them without mentioning it without the advice of your doctor.

These interactions are based more on inference and not on clinical research

study.

Pregnancy:

Large dosage of WS is abortifacient (Ability to cause abortion). The

normal dosage is generally safe and we use it in India along with other herbs

even during pregnancy. However it is better to be taken under the

supervision of qualified herbalist.

Breastfeeding or Nursing Mothers:

In Indian scenario of Ayurvedic Medicine, there appears to be no

restriction of its use in nursing mothers and the Ayurvedic practitioners use

this herb. However its use in nursing mothers is not founded on clinical

studies.

Ashwagandha is found to be completely safe up to 100 mg per kg of

body weight in a single dosage. This comes out to be 21 gm per day in an

average adult man. And the therapeutic dosage in most cases is under 10 gm

per day.

The effects of larger dosage are majorly on gastro-intestinal tract. The

crude powder is a bit hard to digest and hence most of the Ayurvedic

formulations are polyherbal thus this already balances this slight adverse

effect. Larger dosage can cause heaviness in abdomen, nausea, vomiting and

diarrhea.

In an experiment WS was given to mice as 25% of diet (exceeding

large dosage). There had been microscopic lesions on organs like liver,

lungs, vascular congestion and inflammation and tubular congestion of

kidneys. Still it is fully safe in human and animals even on fairly large

dosage. (www.holistic-herbalist.com).

2.6 COMMERCIALLY AVAILABLE PRODUCTS

There are many compositions prepared using Ashwagandha herb which are

useful for a wide range of body disorders. (www.ayurveda-herbs.com)

Ashwaganbdhadi Lepa: A paste form of Ashwagandha and some other

herbs like Licorice, Katuki, Manijistha etc.

Ashwagandha Rasayana: A simple but very much effective formulation of

Ashwagandha, that works for rejuvenation for the body.

Aswagandharishta: Alcoholic preparation (Arista) of Ashwagandha. The

other composition includes Safed musali, manjistha, Harda, Turmeric,

Licorice, Bark of Arjuna, Sandal wood, Elaichi, Cinnamon, Dry ginger,

Pepper, Honey and many more.

Chyavanprasa Leham: The thick sauce (Leha) like formulation. It is on of

the most common formulation that is widely used for all ages of life. Amla is

one of the chief ingredient and the other composition includes more than 40

ingredients, where Ashwagandha is one.

Godhumadyam ghritam: A ghee (Ghrita) based preparation that is named

after the presence of Wheat (also called Godhuba). The composition

includes herbs like Ashwagandha, Asparagus, Black gram, Licorice, Elaichi,

coriander and more along with ghee and milk etc.

Ashwagandha Taila: An oil (Taila) formulation of Ashwagandha, that is

for increase of mass, when applied locally. The composition includes

Ashwagandha, Asparagus, Kusta, Jatamanshi, kantakari.

List of other products available on shelf: (www.Primaryinfo.com)

Ashwaganda Extract Products

Jarrow Ashwagandha-Sensoril

Pravek Chyawanprash

Herbal Tonic

Ashwaganda Capsuless

Ashwaganda root Products

Natural Herbal Rejuvenative

Optimized Ashwagandha Extract (Stimulant Free)

Morpheme Ashwaganda Supplements

Ashwagandha root tincture

2.7 PACKAGING MATERIALS

Urbanization and the time lag between harvest, production and

consumption of food has given rise to urgent need of packaging food

material (Paranjpe, 2001). Packaging must protect against a variety of

assaults including physical damage, chemical attack, and contamination

from biological vectors including microorganisms, insects and rodents.

Environmental factors such as oxygen and water vapor will spoil food if

they are allowed to enter the food freely.

Certain packaging materials particularly those that are included in the

category of plastics have a tendency to slowly migrate into the food which is

termed as global migration. This can sometimes prove to be hazardous for

human health. (Sushma, 2005). Thus it is very important to ensure that the

packaging material used is of food grade quality i.e. migration is less than 60

ppm as per the Indian standards (IS 9845). Also the packaging material must

be compatible with the food.

The selection of packaging material for a particular use depends on a

number of factors. Apart from the technical properties of the different

materials their actual use will depend mostly on the cost and availability in a

particular area. Furthermore, there may be particular marketing reasons for

choosing a certain type of package. Among the important plastic materials

available as films and semi rigid containers for food packaging are

polyamide (Nylon), Polyesters (PET, Mylar), Polyethylene, polypropylene,

polystyrene, polyvinyl chloride (saran) and polyvinyl chloride. Three plastic

films viz., low density polyethylene (LDPE), polypropylene (PP) and

polyethylene terepthalate (PET) were selected for storing the Ashwagandha

root powder.

Polyethylene

Low-density polyethylene is heat sealable, inert, odour free and shrinks

when heated. It is a good moisture barrier but has relatively high gas

permeability, sensitivity to oils and poor odour resistance. It is less

expensive than most films and is therefore widely used.

Polypropylene

Polypropylene is a clear glossy film with a high strength and is puncture

resistance. It has moderate permeability to moisture, gases and odours,

which is not affected by changes in humidity. It stretches, although less than

polyethylene.

Polyethylene terepthalate

PET (Polyethylene terephthalate) is a strong but lightweight form of clear

polyester. It exists both as an amorphous (transparent) and a semi-crystalline

(opaque and white) thermoplastic, and can be made into either a resin or a

film. It is a hard, stiff, strong, dimensionally stable material that absorbs

very little water. It has good gas barrier properties and good chemical

resistance except to alkalis.

CHAPTER III

MATERIALS AND METHODS

The present study is an attempt to develop health foods by

incorporating Ashwagandha root powder and also test its storage stability in

different packaging materials. This chapter includes detailed description of

the methodology followed in the study under the following heads:-

3.1 Location of the study

3.2 Procurement & processing of raw material

3.3 Storage studies of root powder

3.3.1 Proximate principles (moisture, protein, ash, fat, crude fibre,

total Sugars) and Withaferine A (active constituent) estimation

3.3.2 Microbiological evaluation of Ashwagandha root powder

3.4 Standardization and organoleptic evaluation of Ashwagandha

incorporated recipes

3.5 Nutrient analysis of developed products

3.6 Statistical analysis of data

3.1 LOCATION OF THE STUDY

The entire study was planned at the Post Graduate and Research

centre, Acharya N.G. Ranga Agricultural University, Rajendra Nagar,

Hyderabad. The microbiological analysis was carried out at Department of

Agricultural Microbiology and Bioenergy, College of Agriculture, Acharya

N.G. Ranga Agriculture University (ANGRAU).

The analysis of Withaferine (active constituent) in root powder and

developed products was done at Natural Products laboratory, Department of

Chemistry, Osmania University, Hyderabad.

3.2 PROCUREMENT AND PREPARATION OF RAW MATERIAL

For the present investigation, fresh Ashwagandha (Withania

somnifera) roots of A- grade quality were procured in a single lot from

herbal garden, ANGRAU and stored under refrigeration conditions. The

roots were then properly washed under running water to remove adhering

foreign particles, mud, dust etc., dried at low temperature (50°-60° C) and

ground under hygienic conditions to powder form using traditional stone

pestle & mortar as well as electric grinder. The powder so obtained was

sieved twice to remove the coarse particles and stored in air tight containers

till further analysis and product development. The yield of the powder was

found to be about 65 %.

The raw material i.e. branded sugar, flour, hydrogenated fat, salt,

spices, etc as required for product development were procured from local

super market. The packaging materials viz., Low density polyethylene

(LDPE), Polypropylene (PP) and Polyethylene terepthalate (PET) were

purchased from STB Polypack private limited, Kattedhan, Hyderabad.

METHODOLOGY

I. Storage of powder under different packaging & temperature

conditions

Figure 1: Schematic experimental design of storage study of Ashwagandha root powder in different packaging materials.

ASHWAGANDHA ROOT POWDER

POLY ETHYLENE TERPTHALATE

(PET)

POLY PROPYLENE (PP)

ROOM TEMPERATURE

REFRIGERATION

LOW DENSITY POLYETHYLENE

(LDPE)

PHYSICO - CHEMICAL & MICROBIOLOGICAL ANALYSIS

II. Product development & Analysis

Figure 2: Schematic experimental design of product development

3.3 STORAGE STUDIES OF ROOT POWDER

Thirty grams each of root powder was kept in three different

packaging materials i.e. LDPE, PP, & PET, selected on the basis of their

properties. The packed powder was kept for a period of three months under

refrigeration and at room temperature.

DEVELOPMENT OF PRODUCTS

INCORPORATION OF ASHWAGANDHA ROOT POWDER AT DIFFERENT LEVELS

(5%, 7.5%, 10%, 13%)

ACCEPTABILITY STUDIES BY SENSORY EVALUATION

CHEMICAL ANALYSIS

DATA COMPILATION AND STATISTICAL ANALYSIS

3.3.1 PHYSICO – CHEMICAL ANALYSIS

The analysis of the powder was done on the basis of both physical and

chemical parameters initially and after 90th day of storage. Physical changes

were estimated by noticing changes in the following i.e. colour, appearance,

odour, flowability while chemical analysis included estimation of moisture ,

protein, total ash, fiber, reducing and non reducing sugar contents.(AOAC,

1990) and Withaferine A ( HPLC Method).

3.3.2 MICROBIOLOGICAL ANALYSIS

The powder was assessed for microbiological quality with respect to

total plate counts of bacterial, yeast and mold colonies over a period of three

months. Total plate count and yeast and mold count was done for all the

samples initially and after 90th day of storage, as per the procedures outlined

by Cruick et al, 1975.

3.4 STANDARDIZATION AND ORGANOLEPTIC EVALUATION OF

ASHWAGANDHA INCORPORATED RECIPES

To test the palatability and acceptability of the root powder in foods,

many recipes were tried. Common traditional snack items like Namakpara,

Muruku, Pappu chakalu, Missi roti, and Chutney powder were successfully

prepared & standardized using the root powder after conducting a number of

trials for their acceptability.

Plate 1: Ashwagandha roots and root powder

Plate 2: Ashwagandha root powder incorporated Muruku

Plate 3: Ashwagandha root powder incorporated Pappu Chakalu

Plate 4: Ashwagandha root powder incorporated Namakpara

Plate 5: Ashwagandha root powder incorporated Missi roti

Plate 6: Ashwagandha root powder incorporated Chutney powder

Ashwagandha root powder was incorporated at 5%, 7.5%, 10% levels

in standard basic recipes. Detailed methods of preparation & recipes of all

the five products are mentioned in appendix I

Other products like fruit based whey drink, biscuits and cakes were

also tried but were not very much accepted.

SENSORY EVALUATION

Sensory evaluation of the control and experimental products was

conducted to assess the acceptability by a panel of 10 judges selected from

the staff of the department of Foods and Nutrition, PGRC, ANGRAU,

Hyderabad.

A 5-point Hedonic scale was used for the evaluation and the score

card (Appendix II) was prepared keeping in view the quality characteristics

of the developed products (Peryam and Pilgrim, 1957).

3.5 NUTRIENT ANALYSIS

All six samples viz. Ashwagandha root powder and five products of

most accepted combination were analyzed in duplicate for the following

parameters viz moisture, fat, protein, reducing & total sugar, crude fiber &

total ash by using standard method of AOAC (1990). Methodology for the

estimation of above parameters is given in Appendix III. The estimation of

Withaferine A (active constituent) was done using HPLC method (Appendix

IV).

3.6 STATISTICAL ANALYSIS

The data was compiled, tabulated and subjected to statistical analysis

at the end of the study as per procedures laid by Snedecor and Cochran

(1989).The mean values of sensory scores, standard error and critical

difference for all parameters were statistically calculated using Analysis of

variance (ANOVA).

CHAPTER - IV

RESULTS AND DISCUSSION

In this chapter results and discussion are presented under the following

headings:-

4.1 Proximates and Withaferine A (active constituent)

content

4.2 Storage studies of Ashwagandha root powder

4.2.1 Physico-Chemical analysis

4.2.2 Microbiological analysis

4.3 Development of products

4.4 Organoleptic evaluation of products

4.5 Chemical analysis and cost of developed products

4.1 PROXIMATES AND WITHAFERINE A

(ACTIVE CONSTITUENT) CONTENT

Withania somnifera belonging to family Solanaceace is an important

medicinal plant used in traditional Indian system of medicine through the

restoration of a healthy balance of life forces. (Kirtikar K.R and Basu, 1991)

Its wide range of clinically proven biological activities has been

attributed to the presence of group of compounds referred as Withanolide.

Majorly the roots are used as therapeutic drugs. Though the process of root

growth and development has been studied in Ashwagandha, the information

on the physico- chemical constituents is lacking. So far no published data

could be obtained with regard to the biochemical screening of this plant.

Hence, one of the objectives of this study was to evaluate the biochemical

composition of Ashwagandha roots.

The proximate principles and Withaferine A content of fresh Withania

somnifera root that was processed into powder is given in table I

Table 1: Proximate and Withaferine A content of Ashwagandha root powder

CONSTITUENT Percentage ( g/ 100g)

Moisture 5.1

Total sugars 10.0

Reducing sugars 2.4

Total ash 10.1

Proteins 5.8

Fat 6.0

Crude fiber 28.8

Withaferine A (%) 0.16

The present study carried out on the plant root powder revealed the

presence of proximate constituents as proteins (5.8 %), fat (6 %), reducing

(2.4 %) and non reducing sugars (7.6 %). The values indicate that

Ashwagandha root is not a good source of these nutrients.

The Ashwagandha root powder was found to be significantly rich in

crude fiber (28.8%) and mineral content (10.1%). Hence apart from the

medicinal uses of Ashwagandha it can also be used for development of fiber

rich products and in dietary supplements.

The percentage of Withaferine A (0.16%) that is the major active constituent

present in the root of the plant was also estimated (Table 1).

4.2 STORAGE STUDIES OF ROOT POWDER

4.2.1 PHYSICO-CHEMICAL ANALYSIS

The Ashwagandha root powder was assessed for various physical

parameters before and after 90 days of storage. No major differences were

found in physical characteristics of the powder on storage in different

packaging materials.

Appearance Fine powder

Colour Brown to brownish green

Odour Horse like smell

Taste Mild bitter

CHEMICAL ANALYSIS

The Ashwagandha root powder kept under different packaging materials at

room temperature and refrigeration was analyzed on the basis of physico-

chemical and microbiological tests. The following table illustrates the results

obtained.

Table 2: Effect of storage condition and packaging material on chemical

properties of Ashwagandha root powder

* Initial values

As can be seen from the above table no significant change was

observed in total sugars, total ash, proteins and fat content of samples over

storage period in all the packaging materials.

Particulars Moisture

(g/100g)

Total sugars

(g/100g)

Total ash

(g/100g)

Proteins

(g/100g)

Fat

(g/100g)

Crude fiber

(g/100g)

ROOM

TEMPERATURE Initial Final Initial Final Initial Final Initial Final Initial Final Initial final

LDPE 5.2* 5.7 10.0*

10.0 10.1* 10.1 5.8* 5.8 6.0* 6.0 28.8* 28.8

PP 6.3 9.9 10.2 5.8 6.0 28.5

PET 8.6 10.1 10.1 5.8 6.0 28.7

REFRIGERATON

LDPE 5.4 10.1 10.1 5.8 6.0 28.7

PP 5.4 10.0 10.0 5.7 6.0 28.8

PET 5.5 10.0 10.2 5.8 6.0 28.7

It was observed that the moisture content (5.2%) progressively

increased in all samples during storage. At ambient temperatures, a highest

gain in moisture was found in PET (8.7%) followed by PP (6.3%) while

LDPE showed the least increase (5.7%). At refrigeration temperature, the

moisture content of Ashwagandha root powder packed in PET was 5.5%

while it was 5.4% in both PP and LDPE. Lower water vapour transmission

rate (WVTR) of LDPE than PET might be the reason for its better moisture

barrier properties.

The gain in moisture was more at ambient temperatures as compared

to low temperature (refrigeration) storage. It may be due to more absorption

of moisture from the atmosphere by the powder being hygroscopic in nature.

Similar trend was also described by Helkel et al (1972) in dried mango

sheets. However the root powder retained its free flowing character both at

ambient and refrigeration temperatures.

With regard to packaging material, the samples stored in LDPE

pouches gained least moisture in both conditions of storage as compared to

PP and PET. It can be attributed to the low water vapour transmission rate

(WVTR) of LDPE than PET that makes it a good moisture barrier.

4.2.2 MICROBIOLOGICAL ANALYSIS

Both total plate counts and yeast and mold counts of the samples kept

at RT and refrigeration were enumerated by standard method initially and

after 3 months of storage. The results so obtained are compiled in table 3

given below:-

Table 3: Microbiological count of Ashwagandha powder stored in different packaging materials

* Initial values

Microbial load of the Ashwagandha root powder packed in different

packaging materials increased when stored for three months at ambient and

STORAGE TYPE

AND CONDITION

TOTAL PLATE COUNT YEAST & MOLD COUNT

ROOM

TEMPERATURE Initial

After 90 days

(cfu/ ml) Initial

After 90 days

(cfu/ ml)

LDPE * 450 cfu /ml 1000 * NIL

Nil

PP 8000 Nil

PET 10,000 1000

REFRIGERATION

LDPE 850 Nil

PP 1000 Nil

PET 1000 Nil

refrigeration conditions. The increase in microbial growth of the powder

packed in LDPE was less at room temperature (2.2 times) than in PP

(18 times) and PET (22 times). Same trend was observed at refrigeration

conditions also where the growth was observed to be 1.9 times in LDPE and

2.2 times in both PP and PET, than the initial values.

The percent increase in the moisture content of Ashwagandha root

powder packed in PP and PET was more than that in LDPE (Table 2) and

this might be the reason for a higher microbial load of the root powder

packed in them.

In the present study, initially no yeast and mold growth was detected

in all the samples packaged in the three films. The colony forming units

were nil even after 90 days of storage, except for the Ashwagandha root

powder packed in PET films in which 1000 yeast and mold colonies were

observed.

The storage study of Ashwagandha root powder carried out on the

basis of physico-chemical and microbiological analysis in different

packaging materials for a period of three months revealed that out of the

three films viz., LDPE, PP and PET, LDPE was most suitable.

Since percent fat (6%) in Ashwagandha root powder remained

constant over 90 days of storage irrespective of the packaging material used,

it can be interpreted that Ashwagandha is not prone to oxidative changes and

thus does not require an expensive packaging material that has good oxygen

barrier properties like PET. The critical factor for the shelf life of

Ashwagandha root powder happens to be moisture content which can be

easily controlled by using an inexpensive packaging material like LDPE.

4.3 DEVELOPMENT OF PRODUCTS

To test the palatability and acceptability of Ashwagandha root powder

a number of recipes from beverages, bakery to savoury and snack items were

tried. The root powder was incorporated directly after sieving or as an

extract (in case of beverages) at 5, 7.5, 10 and 13 percent levels for weight

of raw ingredients.

The fruit and whey based beverages as well as bakery items (cakes

and biscuits) prepared after incorporating Ashwagandha root powder at

different levels (5, 7.5, 10 percent) were not acceptable when evaluated by

the panelists in terms of color, texture, taste and appearance. Ashwagandha

probably does not go as well with sweet products as with the savouries.

Fruit –whey beverage

Fruit based whey beverage was prepared using whey, as obtained

from milk & juice from different fruits like pineapple, mango, lemon and

their combinations etc. Ashwagandha root extract was made by boiling the

root powder for 2 minutes in 20ml water and then filtering it. The extract

was incorporated in the recipe at different percentages. The product so

obtained when compared with control was quite unacceptable in terms of its

colour that was dull & brownish (muddy), though the taste of the product

was acceptable but a typical Ashwagandha smell dominated its flavour.

Bakery products

Sweet biscuits and fruit cake were tried by incorporating the powder

into the flour before sieving.

The dough development time for Ashwagandha biscuits increased by

1.5 minutes with increased levels of incorporation. A higher amount of

liquid (water/ milk) was required for dough development as compared to

control. The high fiber content of Ashwagandha root powder could be a

reason for this behaviour. Chen et al (1988) reported an increase in dough

development time with increase in level of apple pomace that was used as a

source of fiber in wheat flour and concluded that the increase could be due

to the slower rates of hydration.

As compared to the control biscuit, biscuits with Ashwagandha were darker

in colour and became overcooked in a short period of time only. Unlike

control they had cracked surfaces and became hard on keeping.

On the other hand Ashwagandha incorporated cake recipes did not

bake properly. Even after 80-85 minutes of baking, an uncooked core was

found at the center. The size of the core also increased with increase in level

of incorporation. It can be attributed to the fibre’s functional quality of

imbibing water which thereby gives a highly thick batter unlike that of

control. Similar observations were found by Rao et al (1985) and Deshpande

(1982) also who reported an increase in peak viscosity with increase in level

of fibre as the fibre itself imbibed a lot of water.

4.4 ORGANOLEPTIC EVALUATION OF STANDARDIZED RECIPES

Among all the three categories of food products, savory and snack

products like Namkpara, Muruku, Pappu chakalu, Chutney powder and

Missi roti (chapatti) were successfully prepared and standardized. The

flavour of Ashwagandha root powder that was unacceptable in sweet based

products (as discussed in 4.3) was effectively masked by the spices added in

the savories. The developed products were subjected to sensory evaluation at

5 point hedonic scale by a panel of selected judges along with the control

samples. The mean scores of sensory evaluation of developed products are

presented in the table:-

Table 4: Mean sensory scores of Deep fried products SENSORY

ATTRIBUTES CONTROL 5% 7.50% F VALUE

STANDARD ERROR

CRITICAL DIFFERENCE

NAMAKPARA

COLOR 4.6 ± 0.73 4.3± 0.70 4.0± 0.53 1.12 0.26937 0.80038

APPEARANCE 4.7± 0.45 4.6± 0.49 4.3± 0.45 1.3 0.19047 0.56595

TEXTURE 3.7± 0.45 4.7± 0.45 4.3± 0.70 5.0± 0.22335 0.66364

TASTE 4.4±0.49 5.0± 0 4.1± .12 2.3 0.28965 0.86064 OVERALL ACCEPTABILITY 4.1± 0.64 4.7± 0.45 4.1±0.83 1.5 0.26937 0.80038

MURUKKU COLOR 4.7± 0.45 4.4±0.49 4.1±0.35 2.57 0.17017 0.5294

APPEARANCE 4.8± 0.35 4.3± 0.70 4.0± 0.53 3.82 0.22335 0.66364

TEXTURE 4.0± 0.53 4.1± 0.64 3.7±0.70 0.72 0.25643 0.76193 TASTE 4.6±0.73 4.1± .64 3.7±0.70 2.31 0.28171 0.83705 OVERALL ACCEPTABILITY 4.4±0.73 4±0.53 3.9±0.64 1.3 0.26082 0.77496

PAPPU CHAKALU COLOR 4.8±0.37 4.1±0.69 4.0±0.58 4.56 0.250924 0.7562 APPEARANCE 4.6±0.47 4±0.82 3.6±1.11 1.84 0.37515 1.31059 TEXTURE 4.0±0.58 4.5±0.76 3.8±0.90 1.05 0.33884 1.02116 TASTE 4.1±1.07 4.5±0.50 3.5±0.50 2.37 0.33054 0.99615 OVERALL ACCEPTABILITY 4.1±0.69 4.5±0.50 3.5±0.50 4 0.36004 0.76724

** Values of mean ± standard deviation significant at 5% level

Table 5: Mean sensory scores of Missi roti and Chutney powder

** Values of mean ± standard deviation significant at 5% level

PAPPU CHAKALU

The results indicate that though scores for colour & appearance of

control sample were higher than the experimental samples, the product with

5% incorporation displayed greater acceptability with respect to texture,

taste & overall acceptability.

A significant decrease in scores of colour and appearance of products

with increasing level of incorporation can be attributed to the darkening

effect of the root powder. Lesser scores for taste & overall acceptability of

Pappu chakalu with 7.5% root powder were due to slight bitter notes of

Ashwagandha that were probably masked at 5% level. Moreover, the

experimental recipes were crispier than control, which was more acceptable

at 5% level.

SENSORY ATTRIBUTES Control 5% 7.50% 10% 13% F

VALUE STANDARD

ERROR CRITICAL

DIFFERENCE MISSI ROTI

COLOR 4.1±0.33 4.0±0.87 3.8±0.66 4.0±0.50 - 0.45 0.23504 0.68075 APPEARANCE 4.1±0.60 4.0±0.87 3.9±0.78 4.0±0.50 - 0.15 0.26516 0.768

TEXTURE 3.6±0.86 4.0±0.71 3.8±0.66 3.9±0.78 - 0.32 0.28543 0.8267 TASTE 4.1±0.60 3.9±0.60 4.0±0.50 4.3±0.43 - 0.63 0.20321 0.58856

OVERALL ACCEPTABILITY 4.1±0.60 4.1±0.78 4.0±0.71 4.1±0.60 - 0.06 0.25551 0.74006

CHUTNEY POWDER COLOR 4.7±0.47 - 4.5±0.50 4.5±0.50 4.7±0.47 0.2 0.2173 0.64106

APPEARANCE 4.5±0.50 - 4.5±0.50 4.3±0.47 4.5±0.50 0.14 0.22047 0.65042 TEXTURE 4.0±0.82 - 4.2±0.69 4.1±0.69 4.2±0.69 0.07 0.32274 0.95212

TASTE 4.7±0.47 - 4.2±0.69 4.1±0.69 3.7±0.75 1.94 0.29344 0.86568 OVERALL

ACCEPTABILITY 4.8±.37 - 4.3±0.47 4.3±0.75 3.8±0.90 1.94 0.29344 0.86568

The higher crispiness of Ashwagandha incorporated recipes can be

attributed to its high dietary fiber content. Fiber has higher capacity of

imbibing water, thus leaves the product comparatively less hydrated than

control. Similar observations were made by Ahluwalia and Amarjeet Kaur,

2001 in Chapathi incorporated with Isabgol as a source of dietary fiber. The

lower moisture content of the prepared dough gives higher crispiness to the

final product.

0

2

4

6

MEA

N S

CO

RES

COLOUR APPEARANCE TEXTURE TASTE OVERALL ACCEPTABILITY

CONTROL 5% 7.50%

Figure 3: Mean scores for the sensory evaluation of Pappu chakalu

NAMAKPARA

The mean sensory scores for control and experimental samples of

Namakpara show only a slight variation in terms of colour, appearance, taste

and overall acceptability that was found to be insignificant on statistical

analysis. The texture of Ashwagandha incorporated recipes was more

acceptable than control with 5% powder incorporated Namakpara scoring

highest. It can be attributed to the higher crispiness of the experimental

products that gave a better mouthfeel as compared to control.

0

2

4

6

MEA

N SC

OR

ES

COLOUR APPEARANCE TEXTURE TASTE OVERALL ACCEPTABILITY

CONTROL 5% 7.50%

Figure 4: Mean scores for the sensory evaluation of Namakpara

MURUKU

The results as interpreted from the mean scores revealed that the

control sample received highest scores for all the parameters except texture.

It was also observed that variations in scores were significant only in

appearance of the products. The comparatively low acceptability of

Ashwagandha incorporated recipes in terms of colour and appearance may

be due to the dark brown colour given by the root powder that gave an

unappealing over-cooked appearance to the products.

0

2

4

6

MEA

N SC

OR

ES

COLOUR APPEARANCE TEXTURE TASTE OVERALL ACCEPTABILITY

CONTROL 5% 7.50%

Figure 5: Mean scores for the sensory evaluation of Muruku

CHUTNEY POWDER

The mean sensory scores for Chutney powder showed only slight

variations among all the samples that were statistically insignificant. Only

slight differences were found in control and experimental samples with

respect to the product’s colour, appearance and texture. Among the

experimental samples, only minor differences in mean scores for all

parameters were found between 5 and 10 percent Ashwagandha powder

incorporated products.

The Chutney powder with 13 % level of incorporation (Fig. 5)

received highest scores for colour and appearance. The brown colour as

given by Ashwagandha to the Chutney powder could be a reason for its

higher eye appeal. However, comparatively lesser scores for taste and

overall acceptability were given to the product which might be due to slight

bitterness in the product on account of higher levels of Ashwagandha

powder incorporation.

0

2

4

6

ME

AN S

COR

ES

COLOUR APPEARANCE TEXTURE TASTE OVERALL ACCEPTABILITY

CONTROL 8% 10.00% 13%

Figure 5: Mean scores for the sensory evaluation of Chutney powder

MISSI ROTI

The differences found in mean sensory scores of Missi roti for all parameters

viz. colour, appearance, taste, texture & overall acceptability were not found

to be statistically significant. Among the experimental samples Missi roti

with 10% root powder received the highest scores for all parameters. The

mean scores for taste of experimental products indicated that the flavour of

Missi roti was enhanced with addition of Ashwagandha. This was probably

because the other ingredients present in Missi roti dominated its flavour

thereby masking the unpleasant notes of Ashwagandha and thus imparting a

better flavour to the final product.

1

3

5

MEA

N SC

ORE

S

COLOUR APPEARANCE TEXTURE TASTE OVERALL ACCEPTABILITY

CONTROL 5% 7.50% 10%

Figure 7: Mean scores for the sensory evaluation of Missi roti

4.5 CHEMICAL ANALYSIS AND COST OF DEVELOPED PRODUCTS

The developed products were analyzed for their chemical composition as

given in the following table:-

Table 6: Chemical composition and cost of developed products

The Withaferine A content of the developed products at 7.5%

Ashwagandha powder incorporation was found to be 0.11 g per Kg of the

products ( i.e. 0.011g / 7.5 g root powder) which is comparable to the initial

Withaferine A content of the roots (0.16 g % , 0.012g / 7.5g root powder) as

given in Table 1. This clearly indicates that high temperature treatment

during processing has no effect on the active constituent present in

Ashwagandha roots that impart various medicinal properties to it and hence

it can be easily used in developing heat processed products like Muruku,

Pappu chakalu, Namakpara, Missi roti.

PRODUCTS

(with 7.5%

incorporation)

MOISTURE

(g %)

CRUDE

FIBER

(g %)

FAT

(g %)

PROTEIN

(g %)

TOTAL

ASH

(g %)

TOTAL

SUGARS

(g %)

WITHAFERINE

A ( g / Kg of

Product)

COST

(Rs/ kg.)

NAMAKPARA 3.1 0.6 29.0 11.1 0.6 14.3 0. 113 50.00

MURUKU 3.4 3.4 30.5 10.9 1.3 15.4 0.113 60.00

PAPPU

CHAKALU 2.5 3.5 26.1 10.5 1.3 15.6 0.113 60.00

CHUTNEY

POWDER 7.9 2.1 14.4 22.3 2.5 12.4 0.116 45.00

MISSI ROTI 10.3 3.0 17.5 13.0 2.9 11.8 0.115 40.00

The present market rate of Ashwagandha roots is about Rs. 80 per kg

and the cost of processed root powder comes out to be approximately

Rs. 130/Kg. On incorporating the Ashwagandha root powder at the rate of

7.5% in the products, the cost of products increased by Rs. 10/Kg. Keeping

in mind the prospective medicinal properties of the powder the increase is

marginal.

CHAPTER V

SUMMARY AND CONCLUSION

Keeping in line with the growing awareness on health and fitness

worldwide, there has been considerable debate on healthy lifestyle and

eating habits.

Increasingly in India too, owing to the alarming rise in high-risk

diseases, consumers are becoming more cautious and are switching to

healthier food habits. Thus there is a need to introduce new range of foods

with therapeutic benefits that can restore unwillingly created health

imbalance in people. Hence, the present study was designed to bring out the

enormous potential of Ashwagandha, one of the important herbs in

Ayurveda, the principle constituents of whose root compounds

(Withanolides) are believed to account for its multiple medicinal

applications.

The fresh roots of Ashwagandha were dried, ground to powder form

and its proximate principles and Withaferine A content were analyzed. The

study revealed that while as protein, reducing & non reducing sugars, and

fats were present in minor amounts, the root is rich in crude fiber & mineral

content. The percentage of Withaferine A, i.e. the active principle present in

the root of the plant was also estimated and found to be 0.16 g%.

For testing the palatability and acceptability of Ashwagandha root

powder in foods a number of recipes from savouries, snacks to bakery items

were tried. The root powder was incorporated at 5%, 7.5%, 10% and 13%

levels for weights of raw ingredients. Among the developed products,

savories were more accepted than sweet based bakery items like cakes and

biscuits.

The recipes of successfully developed products viz Namakpara,

Muruku, Pappu chakalu, Missi roti, and Chutney powder were standardized.

Sensory evaluation of the developed products by selected panel of

judges revealed that 5% level of incorporation of Ashwagandha powder in

Namakpara, Muruku, Pappu chakalu, while upto 10% in Missi roti and

Chutney powder was very well accepted.

The accepted products with their controls were analyzed for moisture,

fat, protein, total ash, reducing and total sugars, crude fiber and Withaferine

A (active principle) by standard analytical procedures.

For storage study of the powder in different packaging materials,

thirty grams each of root powder was kept in LDPE, PP, & PET for a period

of three months under refrigeration and at room temperature. The analysis of

the powder was done on the basis of physical, chemical and microbiological

parameters initially and after 90 days of storage.

No major differences were found in physical characteristics of the

powder over storage in different packaging materials.

Though a continuous picking up of moisture by the product in all the

three samples was observed, but the powder however retained its free

flowing character in all the packaging materials.

Microbial load of the Ashwagandha root powder packed in different

packaging materials increased when stored for three months at ambient and

refrigeration conditions. The percent increase in microbial load of the

powder in LDPE was least both at RT and refrigeration temperature.

No yeast and mold growth was detected initially in the sample. The

colony forming units were nil even after 90 days of storage, except for the

powder stored in PET film.

LDPE showed the best results both at refrigeration and room

temperature and is thus suggested for storage of root powder over other

types of packaging materials.

RECOMMENDATIONS AND FUTURE RESEARCH

A number of herbal products containing Ashwagandha in the form of

tablets, capsules, powders, rasayans, tonics etc are available on shelf. But the

bitter taste of Ashwagandha root powder is a limitation to its use in food

products. Hence an attempt was made to evolve recipes by incorporating it

in commonly consumed foods. It was observed that spices like asafoetida,

cumin, black pepper, chillies effectively masked the typical odour and bitter

notes of Ashwagandha. The results revealed that the incorporated recipes

were almost comparable to standard recipes with respect to taste, flavour and

overall acceptability.

The results of the study also revealed that the Ashwagandha root

powder should preferably be used in salty products as it does not go well

with sugar based products, specially the bakery items. Further research thus

is recommended in this direction.

Unconventional products are not readily accepted, especially in the

case of food items, a wide publicity is therefore needed regarding the use of

such unconventional sources as nutrient and therapeutic supplements.

New innovations such as promoting the use of unconventional medicinal

plant sources like usage of Ashwagandha in day to day food items can slow

down the progression of degenerative diseases.

Moreover a number of other products like- chips with added St. John's

Wort (herb that fights depression), ice cream with added ginseng (an

energizer) and beverages with various sundry herbs such as Echinacea

(improve immune function) and yohimbe (improve virility) are available on

shelf in the west. Ashwagandha being one of the major herb with extensive

medicinal properties has a lot of potential that needs to be tapped in

development of more such therapeutics.

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APPENDIX I PREPARATION OF RECIPES MURUKU

Ingredients Control (g) 5%(g) 7.5%(g)

Rice flour 75 70 65

Black gram flour 25 25 25

Ashwagandha root powder - 5 7.5

Vanaspati 15 15 15

Asafoetida 1 1 1

Cumin 3 3 3

salt 1.5 1.5 1.5

METHOD:- 1) Mix rice and black gram/ Ashwagandha flour.

2) Add all the dry ingredients.

3) Heat vanaspati and add to the flour.

4) Make it into soft dough adding sufficient water.

5) Press the dough through a mould and deep fry.

NAMAKPARA

Ingredients Control (g) 5%(g) 7.5%(g) Wheat flour (Maida) 100 95 92.5 Ashwagandha root powder - 5 7.5 Salt 3.5 3.5 3.5 Vanaspati 7 7 7 Cumin powder 0.6 0.6 0.6 Bishop’s weed (Ajowan) 1 1 1 Chilli powder 0.6 0.6 0.6 Black pepper 0.8 0.8 0.8 Refined oil As reqd As reqd As reqd

METHOD:- 1) Mix all the dry ingredients.

2) Add vanaspati and mix well.

3) Make stiff dough adding enough water.

4) Knead the dough well and roll into thin roties.

5) Cut into diamond shaped pieces and deep fry.

PAPPU CHAKALU

Ingredients Control (g) 5%(g) 7.5%(g)

Rice flour 75 70 65

Black gram flour 25 25 25

Ashwagandha root powder - 5 7.5 Bengal gram dhal (soaked) 10 10 10 Vanaspati 15 15 15

Chilli powder 3 3 3

Cumin 3 3 3

salt 1.5 1.5 1.5

METHOD:-

1) Mix rice and black gram/ Ashwagandha flour

2) Mix all the dry ingredients along with soaked bengal gram dhal.

3) Add vanaspati and mix well.

4) Make into soft dough using enough water.

5) Knead the dough well.

6) Flatten into thin round form and deep fry.

MISSI ROTI (CHAPATHI)

Ingredients Control (g) 5%(g) 7.5% (g) 10%(g) Jowar 30 29 28.5 28 Wheat flour (Atta) 40 39 38.5 38

Bengal gram flour 30 29 28.5 28

Ashwagandha root powder

- 5 7.5 10

Spinach 15 15 15 15 Salt To taste To taste To taste To taste

Chilli powder 1.2 1.2 1.2 1.2 Cumin powder 4 4 4 4 Oil to fry As reqd. As reqd. As reqd. As reqd. METHOD:-

1. Mix jowar, wheat flour and bengal gram flour / Ashwagandha root

powder

2. Add all the dry ingredients and spinach

3. Add sufficient amount of water to make it into soft dough.

4. Divide dough into small balls and role them in the form of rotis.

5. Shallow fry on tawa ( heated iron plate) with oil

CHUTNEY POWDER

Ingredients Control(g) 7.5% (g) 10% (g) 13% (g)

Roasted Chana 25.2 25.2 25.2 25.2 Ashwagandha Root Powder

- 1.05 1.4 1.82

Roasted Groundnut 24.8 24.8 24.8 24.8

Dhania Powder 2.4 2.4 2.4 2.4 Cumin Power 6 6 6 6 Chilli Powder 2.8 2.8 2.8 2.8 Garlic 2.5 2.5 2.5 2.5 Salt 3.2 3.2 3.2 3.2 Dried/ fried Curry leaves

As per taste As per taste

As per taste As per taste

METHOD:

1. Ground roasted Chana and groundnut

2. Add all dry ingredients to it.

3. Mix well and serve.

APPENDIX II

SCORE CARD FOR SENSORY EVALUATION OF PRODUCTS

NAME OF EVALUATOR: DATE: PRODUCT: INSTRUCTIONS:

Please evaluate the following samples according to the attributes given below Rinse your mouth between each sample and follow the quality description

provided.

SENSORY ATTRIBUTES SAMPLES A B C D

COLOUR Highly appealing 5 Appealing 4 Moderately appealing 3 Slightly appealing 2 Not appealing 1 APPEARANCE Highly appealing 5 Appealing 4 Moderately appealing 3 Slightly appealing 2 Not appealing 1 TEXTURE Excellent 5 Very good 4 Good 3 Fine 2 Poor 1 TASTE Highly acceptable 5 Acceptable 4 Moderately acceptable 3 Slightly acceptable 2 Not acceptable 1 OVERALL ACCEPTABILITY Highly acceptable 5 Acceptable 4 Moderately acceptable 3 Slightly acceptable 2 Not acceptable 1 Highly appealing 5 SUGGESTIONS / REMARKS:

SIGNATURE APPENDIX III

METHODS OF CHEMICAL ANALYSIS

IIIA ESTIMATION OF MOISTURE

The moisture content of the samples was determined by following the

method of AOAC (1990)

Procedure:

1. A petridish was weighed with lid

2. Ten grams of homogenous sample was weighed into the petridish and

spread evenly for uniform drying

3. The petridish was left in oven at 100-105 *C with the lid open for

about 15-17 hours.

4. The petridish was cooled in a dessicator with the lid closed for about

1-2 hours

5. The petridish with dried sample was weighed

6. The procedure of heating and cooling was repeated till a constant

weight was achieved.

Formula used:

% Moisture of the sample = (W2-W1) - (W3- W1) X 100

_________________

(W2-W1)

Where:

W1= initial weight of the empty petridish (with lid) (gm)

W2= weight of the empty petridish with sample before drying (gm)

W3= weight of the empty petridish with sample after drying (gm)

IIIB ESTIMATION OF CRUDE FIBER

The crude fiber content of the samples was determined using the procedure

of AOAC (1990).

Principle:

The sample is allowed to boil with sulphuric acid (0.255 N) and dilute

sodium hydroxide (1.25 % W/V). The remaining residue after these

digestions is taken as crude fiber.

Reagents:

1. Dilute sulphuric acid (0.255N)

2. Sodium hydroxide (1.25% W/V)

3. Alcohol

4. Ether

Procedure:

1. 2-5 gm of moisture and fat free sample was weighed into a 500 ml

beaker

2. 200 ml of boiled sulphuric acid (0.255 N0 was added and the mixture

was boiled for 30 min. keeping the volume constant by the addition of

distilled water at frequent intervals.

3. After boiling, the mixture was filtered through a muslin cloth and the

residue was washed with hot water till free from acid.

4. Then the residue was transferred into the same beaker and 200 ml of

boiled sodium hydroxide was added and boiled for 30 minutes,

filtered followed by washing with hot water.

5. Later the mixture was washed with few ml of alcohol and ether.

6. The residue was transferred to a crucible and dried over night at 80-

100 ° C.

7. Weight of the crucible was noted down.

8. Then the crucible was transferred to a muffle furnace at 600 ° C for 2-

3 hrs.

9. The crucible was removed from furnace and kept in a dessicator for

cooling and the weight of crucible was taken after cooling.

Calculations:

% Crude Fiber in the sample = (100- (moisture + fat) X A

W1

Where:

W1 = weight of the sample

W2 = weight of the crucible & sample before heating at 600° C

W2-W3 = A = Weight of crude fiber

IIIC ESTIMATION OF REDUCING SUGARS AND TOTAL SUGARS

Estimation of reducing sugars was done by AOAC (1990) procedure.

Reagents required

1. 45% lead acetate- weigh 45g of lead acetate, dissolve in distilled

water and make up the volume to 100 ml

2. 0.1% phenolphthalein- weigh 10mg of phenolphthalein and dissolve

in 10ml of ethanol.

3. Standard glucose solution - weigh 50mg of glucose, dissolve and

make upto 100ml in distilled water

4. Potassium oxalate

5. Concentrated HCl and concentrated sulphuric acid

6. Copper reagent A: weigh 0.25g anhydrous sodium carbonate, 2.5 g

sodium potassium tartarate, 2g sodium bicarbonate and 20g anhydrous

sodium sulphate, dissolve and make upto 100ml in distilled water.

7. Copper reagent B: weigh 1.5 g copper sulphate dissolve in distilled

water and add one or two drops of concentrated sulphuric acid and

make upto 10 ml with distilled water.

8. Alkaline copper reagent: mix 25 ml reagent A and 1ml reagent B just

before use.

9. Arsenomolybdate reagent: weigh 2.5g of ammonium molybedate into

45ml of distilled water add 2.1 ml of conc. sulphuric acid and mix the

solution well. Dissolve 0.3 g of sodium arsenate in 2.5ml distilled

water and add to the above solution. Mix the solution, store in a

brown bottle and incubate at 37ºC for 24 hours before use.

Procedure:

Extraction of reducing sugar

1. Take 1g food sample or /1ml of fruit or honey/ 1g jam etc.

2. Add 25ml water and simmer for 20 min

3. Cool, and add 5ml of 45% of lead acetate keep for 30 min

4. Add 3 spatulas of potassium oxalate and make up the volume to 50ml

and Filter (reducing sugar extract). For total sugars

1. take 10ml of above extract , add 1ml of conc sulphuric acid and boil for 5 min

2. cool, add few drops if phenolphthalein indicator 3. neutralize with 1N NaoH till a faint pink color appears 4. make up the volume to 25ml (total sugars

(Nelson – Somoyogi Method)

1. Take 0.1 -0.5 ml glucose solution in five different test tubes (tube no.2

to 6)

2. Take 0.1ml reducing sugar extract into separate test tubes (tube no. 7

and 8)

3. Run a blank simultaneously without any sugar solution (tube no.1)

4. Add 1ml copper reagent in all the tubes and keep in a boiling water

bath for 20min

5. Cool the tubes and add 1.0ml arsenomolybdate solution and mix well.

6. Make up the solution in each test tube to 25 ml with distilled water in

a volumetric flask.

7. Keep about 5ml of this solution and discard the rest of the solution.

8. Read the absorbance of the solution in a spectrophotometer at 520nm

9. Plot the standard graph and calculate the sugar content of the

unknown samples based on the values obtained from the standard

graph.

Observation:

Standard/sample Conc. of

glucose (µg)

Alkaline

copper

reagent(ml)

Arsenomolybdate

(ml)

O.D

values

Blank

Standard

0.1 50 1.0 1.0

0.2 100 1.0 1.0

0.3 150 1.0 1.0

0.4 200 1.0 1.0

0.5 250 1.0 1.0

Sample

Red. Sugar 1.0 1.0

Calculations:

------- O.D. of reducing sugar extract contains = µg glucose

i.e. 0.1 ml reducing sugar extract contains = µg glucose

1.0 ml reducing sugar extract contains = µg glucose

50 ml reducing sugar extract contains = mg glucose

i.e. 1.0 gm sample contains = mg glucose

100 gm sample contains = x 100 = gm glucose

IIID ESTIMATION OF FAT

The fat content of the sample was determined using the method of AOAC

(1990).

Principle:

Fat is estimated as the crude extract of moisture free sample.

Reagent:

Petroleum ether (60-80 ° C b. pt.)

Procedure:

1. 5-10 g moisture free sample was weighed accurately into a thimble

with Whatman no. 1. Filter paper.

2. The thimble was placed in the soxhlet apparatus and extracted with

petroleum ether for 16 hours

3. The ether extract was filtered into a weighed beaker.

4. The flask was rinsed four to five times with small quantities of

petroleum ether.

5. All the rinsing were pooled to the beaker.

6. Petroleum ether was evaporated and the flask with the residue was

dried in a hot air oven at 80- 100 ° C for 1-2 hours.

7. The flask was later cooled in a dessicator and weighed.

8. The flask was later cooled in a dessicator and weighed.

Calculation:

% fat content of the sample= Weight of ether extract (gm) X 100

Weight of the sample (gm)

IIIE ESTIMATION OF PROTEINS

The crude protein content of the sample was estimated by Microkjeldahl

method as per the procedure of AOAC (1990) and calculated as the product

of percent Nitrogen and a multiplication factor.

Principle:

The nitrogenous compounds of the material to be tested are converted into

ammonium sulphate by boiling with concentrated Sulphuric acid (H2SO4).

It is subsequently decomposed by addition of excess of alkali and the

liberated ammonia absorbed into boric acid solution containing mixed

indicator by steam distillation. Ammonia forms a loose compound,

ammonium borate, with boric acid, which is titrated directly against

standardized HCl.

Chemical reagents:

1. Digestion mixture (98 g of potassium sulfate and 2 gm of copper

sulfate)

2. Concentrated H2SO4

3. Sodium hydroxide (NaOH)- 40%

4. Boric acid – 4%

5. Mixed indicator : a) 0.2% Bromocresol green ( in alcohol)

b) 0.2% methyl red (in alcohol), a; b = 1:3

6. Standardized HCl (0.5N)

Procedure:

1. The digestion unit was switched on and the temperature was set to

375 ° C

2. 500 mg of the sample was weighed into the digestion tube.

3. 1 g of digestion mixture added to the tubes and 10 ml of concentrated

H2SO4 was added carefully along the sides of the tubes.

4. The tubes were kept in the digestion unit and digestion was done for 2

hours or till the contents of the tube turned sea green or sea blue in

colour.

5. The tubes were removed and cooled for 10 min and 50 ml of distilled

water was added carefully from the sides while the tubes were slightly

hot.

6. A 250 ml conical flask was taken and 40 ml of 4% boric acid and few

drops of mixed indicator was added to it.

7. 50 ml of NaOH solution was slowly added to each of the digestion

tubes.

8. The digestion tube and conical flask was flask was kept on their

respective places in the Gerhardt’s distillation unit and distillation was

done for 3 min.

9. The contents of the conical flask turned green after distillation. This

was titrated with 0.5 N standard HCl to a permanent pink coloured

end point.

10. A blank was also run simultaneously without any sample in the

digestion tube.

Calculations

1ml of 0.5 N HCl= 14.007 g of nitrogen.

1g of Nitrogen= 100/16= 6.25 g of protein.

% protein content of the sample= T.V x 14.007 x N x factor x 100

W

Where; T.V. = Titre value (ml)

N= Normality of HCl (N)

W= Weight of the sample (mg)

III F ESTIMATION OF TOTAL ASH

The ash content of the sample was determined by AOAC (1990) method.

Principle:

The sample is ignited at 600 ° C to burn off all the organic material. The

inorganic material does not volatilize at that temperature is called ash.

Equipment:

Muffle furnace, dessicator

Procedure

1. Clean a porcelain crucible , heat at 200*C for about 1 hour, cool in a

dessicator and take the weight of the crucible (W1)

2. Add 2-5 g of dry sample (grain), 10g jelly, jam etc., 25g of juice or

fresh fruit and take the weight of the crucible (W2)

3. Heat the crucible continuously with a low flame until the material

begins to char and continue till charring is complete.

4. Then transfer the crucible to muffle furnace which is already heated to

500-600º. Continue ashing until a grey or white ash is obtained.

5. It may require 4-6 hours

6. Cover the crucibles and transfer carefully while it is still hot in a

dessicator

7. Weigh the crucible along with ash (W3)

8. Repeat the process of heating the crucible in muffle furnace for half

an hour, cool and take the weight (W4) till two consecutive readings

are same.

Calculation:

Weight of the sample taken = W2-W1 g.

Weight of the ash obtained = W3- W1 g.

% Ash in the sample= weight of the ash x 100 or (W3- W1) x 100

Weight of the sample (W2- W1)

APPENDIX IV

QUANTITATIVE ESTIMATION OF WITHAFERINE A IN

ASHWAGANDHA

Chemicals and reagents:

Methanol, toluene, ethyl acetate, formic acid, ammonia solution

Reagent preparation:

5% methanolic sulphuric acid: mix 5 ml concentrated sulphuric acid with

methanol carefully, cool and make up the volume to 100ml with methanol.

Standard preparation:

Weigh 5mg of Withaferine A standard . dissolve it in 10 ml of chloroform(

0.5 μg/L)

Sample preparation:

1. Weigh 500 mg of sample in 100 ml round bottom flask.

2. Add 30 ml of 2 N HCl.

3. Shake well, then add10 ml of methanol.

4. shake the mixture thoroughly

5. reflux the mixture for about 1 hour at 50 ° C

6. cool the solution and filter through Whattmann paper number 1

7. extract the filtrate with chloroform and collect the chloroform layer

8. evaporate it just to dryness

9. reconstitute it with 1ml of chloroform( 500μg/μL)- TEST SOLUTION

Stationary phase

HPTLC Al sheets silica gel 60F254 precoated platecut to 20X 10 cm

prewashed with methanol and then heated in oven for 10 minutes at 110°C.

Mobile phase: Toluene: Ethyl acetate: formic acid (5:5:1)

(Volume= 15ml)

Sample/ standard application

Apply with the help of camag ATS-4 or linomat 5, 10 μL of test solution and

2 μL, 4 μL,6 μL,8 μL,10 μL of standard on precoated layer, 8mm from

bottom edge, band length 8 mm distance between track 11.3 mm distance

from the side 15mm.

Development chamber

Camag Twin trough chamber of 20X 10 cm with S.S. lid.

Tank saturation: - 10 minutes with filter paper

Plate equilibrium: - none

Development distance: - 80mm

Visulalization- observation in U.V cabinet at 254 nm and 366 nm.

Post chromatographic derivatization:-

Dip the plate in 5% methanolic sulphuric acid and heat at 120 °C for 5-7

minutes.

Photodocumentation:-

In 254 nm (before derivatization) and in visible light (After derivatization)

Measurement mode: - UV absorbance/ reflectance

For Quantification:-

Using camag scanner 3 with CATS software slit micro, 6X 0.45 mm

Scan 214 nm, 366nm (before derivatiation) and 580nm (after derivatization)

For identification

Take spectra at 190 nm- 400nm using CAMAG scanner 3 with win CATS

software, slitmicro 6X 0.45 mm.

APPENDIX V

MICROBIOLOGICAL ANALYSIS

Total bacterial counts: (Cruik et al., 1975)

For estimating bacterial population in different samples, dilution plate

method was followed (Cruik et al., 1975)

One gram of sample was thoroughly mixed in 9 ml of sterile saline water.

From this, 1 ml of sample was transferred through a sterile pipette to a

screw- cap tube containing 9 ml sterile saline water, giving a dilution of 102.

Similarly, serial dilutions were made.

One ml of the sterile serially diluted sample was placed in a sterile

petridish to which cooled Plate count agar medium was added and mixed

thoroughly with the suspension and then allowed to set and incubated at 28

±2°C for 48 hrs. Individual colonies were counted and multiplied.

Yeasts and molds

Dilution plate method was followed for yeasts and mold content. (Cruik et

al., 1975). Potato dextrose agar medium was used for estimating the fungal

population. Samples were diluted in the sterile saline before plating.