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Shimoga Shimoga Shimoga Shimoga –––– 577 201577 201577 201577 201

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This is to certify that the dissertation entitled Standardization Of Sitopaladi Standardization Of Sitopaladi Standardization Of Sitopaladi Standardization Of Sitopaladi

Churna Churna Churna Churna is a bonafide research work carried out by Mr.Mr.Mr.Mr.Amith Kumar BAmith Kumar BAmith Kumar BAmith Kumar B.... Submitted in

partial fulfillment for the award of the degree of “Master of Pharmacy” “Master of Pharmacy” “Master of Pharmacy” “Master of Pharmacy” in PharmacognosyPharmacognosyPharmacognosyPharmacognosy

by the Rajiv Gandhi University of HRajiv Gandhi University of HRajiv Gandhi University of HRajiv Gandhi University of Health Sciences, Karnataka, Bangaealth Sciences, Karnataka, Bangaealth Sciences, Karnataka, Bangaealth Sciences, Karnataka, Bangalorelorelorelore

Date : Dr.Kiran Kumar Hullatti, M.Pharm, Ph D.

Place : Shimoga Lecturer, Dept. of Pharmacognosy, National College of Pharmacy, Shimoga. - 577 201.

“STANDARDIZATION OF SITOPALADI CHURNA”

By

AMITH KUMAR B

Reg. No: 09PG606

Dissertation Submitted to Rajiv Gandhi University Of Health

Sciences, Karnataka, Bangalore.

In Partial Fulfillment Of The Requirements For The Degree Of

MASTER OF PHARMACY

IN

PHARMACOGNOSY

Under The Guidance Of

Dr. Kiran Kumar Hullatti, M.Pharm, Ph D, DPPH

Department Of Pharmacognosy.

National College Of Pharmacy.

Shimoga – 577 201

Karnataka, India.

February-2011

Rajiv Gandhi University of Health Sciences,

Karnataka, Bangalore

DECLARATION BY THE CANDIDATE

I here by declare that the matter embodied in the dissertation entitled

Standardization Of Sitopaladi Churna is a bonafide and genuine research work carried

out by me under the guidance of Dr. Kiran Kumar Hullatti assistant professor,

Department of Pharmacognosy, National College of Pharmacy, Shimoga. The work

embodied in this thesis is original and has not been submitted the basis for the award

of degree, diploma, associateship (or) fellowship of any other university (or)

institution.

Date :

Place : Shimoga Amith kumar B

ENDORSEMENT BY THE HOD, PRINCIPAL/HEAD OF THE INSTITUTION

This is to certify that the dissertation entitled Standardization Of Sitopaladi Churna is a

bonafide research work carried out by Mr.Amith Kumar B. submitted in partial fulfillment

for the award of the degree of “Master of Pharmacy” in Pharmacognosy by the Rajiv Gandhi

University of Health Sciences, Bangalore, Karnataka. He carried out this work in the

library and laboratories of National College Of Pharmacy, Shimoga, under the guidance of

Dr. Kiran Kumar Hullatti , assistant professor, Dept. of Pharmacognosy, National College

Of Pharmacy, Shimoga.

Mr. S M Prasanna Dr. B. K. Sridhar

Head Of The Department, Principal,

Dept. of Pharmacognosy, National college of Pharmacy,

National College Of Pharmacy Shimoga: 577 201.

Shimoga: 577 201

Date : Date :

Place : Shimoga Place : Shimoga

COPYRIGHT

Declaration by the Candidate

I hereby declare that the Rajiv Gandhi University of Health Sciences, Karnataka

shall have the rights to preserve, use and disseminate this dissertation/ thesis in print or

electronic format for academic/ research purpose.

Date : Signature of candidate

Place : Shimoga Amith kumar B.

© Rajiv Gandhi University of Health Sciences, Karnataka.

ACKNOWLEDGEMENT

I believe that it is through God’s grace every achievement is possible.

Parents, Guru and God are the wonderful avatars of god.

Today, at the acme of my dissertation, with heartiness, I gratefully remember

my devine god Elu Koti Mailaralingeshwara, Huligemma, my parents,

teachers, friends, relatives and well wishers; as one flower cannot make a

garland. This presentation would not have taken shape without their

wholehearted encouragement and live involvement.

From deepest depth of my heart, I express my love and gratitude to

my beloved parents Mrs. Suman Bondade & Mr. Laxman Rao Bondade, my

wife Supriya, sister Sapana, Yash Navale, my niece Diya Yash Navale my

grandfather and grandmother, my father in-law Dr. Khamithkar N Suresh, my

mother in-law Mrs. Savatri and Patige, Navale,families all my relatives and

friends for their love, support and constant encouragement throughout my

career.

With a deep sense of gratitude, I express my indebtedness to my

guide Dr.Kiran Kumar Hullatti, my Lecturers Dr. G Narayana Murthy, & Mr. S

M Prasanna, Head of the department, Dept of pharmacognosy, National

College of Pharmacy, Shimoga. for his valuable guidance, boundless

enthusiasm and constant inspiration throughout the entire course of the

work. I shall for ever remain indebted to him for having inculcated in me a

quest for excellence, a spirit of diligence and perseverance.

I express my sincere thanks to our respected Principal,

Dr. B. K. Sridhar, National College of Pharmacy, Shimoga, for his cooperation,

encouragement and for providing all the facilities, which enabled me to

complete this dissertation work successfully.

My special thanks to Mr.M.L.Vijaykumar, Mr.K.C.Prashanth,

Mr.M.C.Ravi, Dept. of Pharmacognosy & Mr. Manzoor Ahmed, Dr.A.

Satishkumar Shetty, Head of the department, Dept of pharmaceutical

analysis, National College of Pharmacy, Shimoga, for their everwilling help

and moral support during the dissertation work.

I am also expressing my special thanks to Mr.Ganesh.H.G for his

encouragement and support throughout the course.

I also express my special thanks to, Mr.Manjunath Rao, Mr.Shailendra,

Mr.Santhosh, Mr.Omprakash, Mr.Vishwendra, Mr.Parashuram, Mr.Ramesh, for

their encouragement and support during the course of my study.

I am also thankful to Ganesh, Venkoba, Basavaraj, Venkatesh, Raju,

Nanjundappa, Sangappa, Chandru, Nalini, Mahalakshmi, Gururaj,

Ravishankar, Arun, Kashinath, Kousalyamma, and Philomena for their

support throughout the course.

I am also thankful to the teaching and non teaching staff of SCS

college of pharmacy, Harapanahalli.

I am also thankful to library staff and all other teaching and non-

teaching staff of National College of Pharmacy, Shimoga, for their co-

operation during the course of my study.

I wish to express my special thanks to my colleagues, batchmates and

friends Neelanjan, Ravi Sheeri, Naresh, Akshta, Nataraja, Rakesh, Manjunath,

Siddalinga Swamy, Arpan, for their inspirational support during my course. I

wish to thank all my juniors, Indudhar, Veeresh, Prem, Prithvi for their

cheerful support during my dissertation work.

I am also expressing my special thanks to Mr.Ganesh.H.G for his

encouragement and support throughout the course.

Last but not the least I thank my computer teacher Ms.Sudha Rani De,

and my well-wishers Apurva, Mr.Subhramanya, Chaitra, Kala,

Mrs.Vasundhara, Lakshmikanth, Jaymin, Amit, Deepa, Naveen, Balaji,

Srinivas, Santosh, Sanjay, Niranjan and thanks to all those whoever related to

me and my work…

Thank you one & all…

Amith Kumar Bondade

Dedicated to my parents,

my wife, and people

who care for me…

LIST OF ABBREVIATIONS USED

Lt : Liter

mg : Milligram

gm : Grams

hrs : Hour

μg/ml : Microgram per milliliter.

TLC : Thin layer chromatography.

HPTLC : High performance thin layer chromatography

W.H.O. : World Health Organization

kg : Kilogram

SDM : Sri Dharmasthala Manjunatha ayurveda

cm : Centimeter

h/r : height/radius

g/cc : grams/cubic centimeter

nm : nanometer

Rf : Kilogram

ppt : Precipitate

ABSTRACT

Department of Pharmacognosy,NCP,Shimoga

Abstract

Standardisation of herbal formulation is essential in order to assess the quality of

drugs, based on the concentration of their active principles. The present paper reports on

standardisation of Sitopaladi churna, a poly herbal ayurvedic medicine used as remedy

for cold, cough, congestion, bronchitis, TB, asthma, emphesema, flu, sinus congestion,

inflammation, sore throat, laryngitis, pharyngitis, bronchitis, trachyitis, sinus headache,

respiratory allergy, seasonal sneezing, wheezing, excess mucous. Sitopaladi churna was

prepared as per Ayurvedic Formulary of India. In-house preparation and three marketed

have been standardised on the basis of organoleptic characters, physical characteristics,

physico-chemical properties, TLC and HPTLC for the estimation of marker compounds,

piperine and cinnamaldehyde. The set parameters were found to be sufficient to evaluate

the churna and can be used as reference standards for the quality control/quality

assurance laboratory of a Pharmaceutical house.

Keywords: Sitopaladi churna, physicochemical parameters, polyherbal formulation,

standardisation.

Tables of contents

Sl. No. Topics Page No.

1 INTRODUCTION 1-3

2 OBJECTIVE 4-5

3 REVIEW OF LITERATURE 6-19

4 MATERIALS AND METHODS 20-36

5 RESULTS AND DISCUSSION 37-63

6 SUMMARY AND CONCLUSION 64-65

7 BIBLIOGRAPHY 66-68

List of Tables

Sl No. Name of Tables Page

No.

1 Relationship between angle of repose and type of flow 32

2 Relationship between Hausner’s ratio and type of flow 32

3 Relationship between compressibility index and type of flow 33

4 Ash values of ingradients used to prepare Sitopaladi Churna 38

5 Ash values of in-house and marketed products of Sitopaladi Churna 38

6 Extractive values of churnas 40

7 Angle of repose of Sitopaladi Churna 40

8 Bulk Density of Sitopaladi Churna 42

9 True Density Sitopaladi Churna 42

10 Carr’s Index of Sitopaladi Churna 44

11 Hausner’s Ratio of Sitopaladi Churna 44

12 Preliminary phytochemical screening of alcoholic extracts of churnas 45

13 Fluorescent test for In-house formulation of Sitopaladi churna 48

14 Fluorescent test for SDM formulation of Sitopaladi churna 48

15 Fluorescent test for Baidyanath formulation of Sitopaladi churna 49

16 Fluorescent test for dabur formulation of sitopaladi churna 49

17 Organoleptic Properties Of Different Sitopaladi Churna 50

18 Measurement of starch grains present in sitopaladi churna 52

19 Determination of Rf values of piperine in methanolic extract of

sitopalaci churna by TLC 54

20 Rf values and peak areas of methanolic extract for piperine by HPTLC 54

21 Rf values and peak areas of methanolic extract for cinnamaldehyde by

HPTLC 58

List of Figures

Sl No. Name of Figures Page

No.

1 Candy sugar 13

2 Vamsalochana 14

3 Piper Longum 14

4 Cardamamum 15

5 Cinnamon Zeylanicum 15

6 Photos of Powder microscopy 52

7 TLC of methanolic extract of sitopaladi churna for piperine 53

8 3-D spectra of all tracks at 350nm (piperine) 55

9 3-D spectra of all tracks at 350nm (cinnamaldehyde) 58

INTRODUCTION

Department of Pharmacognosy,NCP,shimoga 1

INTRODUCTION:

In the long struggle to overcome the powerful forces of nature, the

human beings have always turned towards plants for food, shelter, clothing, and

healing. Even today herbal medicine plays an important role in the management of

diseases. Though we are in 21st century where modern technology and scientific

discoveries are ushering remarkable changes in our lives, nevertheless, the story of

plants as herbal medicines definitely continues to unfold, however, quietly and

independently1.

The contributions of Indians in the field of medicine are not known completely

to the world even today. It is needless to emphasize the rich herbal resources of the

vast subcontinent and its varying climatic zones with variety of vegetation ranging

from the alpine to the tropical regions. Their judicious use in the interest of the

suffering humanity is the need of the hour. The Indian science of herbs possesses the

capacity to lead the world. The only need is encouragement of investigation.

In recent years we have seen a rash of prescription drugs removed from the

market due to some extremely dangerous, even deadly side effects. Herbal medicines

are now in great demand in the developing world for primary health care not because

they are inexpensive but also for better cultural acceptability, better compatibility with

the human body and minimal side effects. It is for this exact reason that many people

are now turning to alternative medicine, such as herbal medicines, to treat everything

from acne to cancer, because herbal medicines has less side compared to anyother

medicines.

The Indian ancient systems of medicine, ayurveda commands respect from a

very large section of the population in our country and also arouse cautions welcome

from some part of the world. Herbal medicines are in huge demand even in the

INTRODUCTION


modern era. On study of ayurvedic literature, one comes across several references of

permitting the use of a substitute drug when the classical drug is not available. This is

based on its therapeutic use and clinical efficacy. There were certain classical drugs in

ayurveda whose supply was limited and seasonal. By the time efforts were made to

identify these drugs, their supply has declined and commercial substitution started.

It was during this period that commercial manufacture of ayurvedic medicines

started. Taking advantage of the commercialization of ayurvedic drugs, some

unethical persons and pharmacies exploited the market by manufacturing adulterated

spurious products. To prevent this adulteration, some sort of uniformity in the

manufacture of ayurvedic medicines was needed.

Standardization is a system to ensure that every packet of medicine that is

being sold has the correct substances in the correct amount and will induce its

therapeutic effect2. It is an essential factor for ayurvedic formulation in order to assess

the quality of drugs based on the concentration of their active principle. It is very

important to establish a system of standardization for every plant medicine in the

market, since the scope of variation in different batches of medicine is enormous.

Plant material when used in bulk quantity may vary in its chemical content

and therefore, in its therapeutic effect according to different batches of collection for

e.g. collection in different season and/or collection from sites with different

environmental surrounding or geographical location. The increasing demand of the

population and chronic shortage of authentic raw materials have made it incumbent,

so there should be some sort of uniformity in the manufacturing of Ayurvedic

formulations so as to ensure quality of the product. The World Health Organisation

(WHO) has appreciated the importance of medicinal plants for public health care in

developing nations and has evolved guidelines to support the member states in their

INTRODUCTION


efforts to formulate national policies on traditional medicine and to study their

potential usefulness including evaluation, safety and efficacy3.

“Sitopaladi churna” is a Polyherbal Ayurvedic medicine which contains; Sitopala

(candy sugar), Vamsalochana (silicious substance from Bambusa Arundinaceae),

Pippaliphala(fruits of Piper Longum), Ela (fruits of Elettaria Cardamamum), and

Twak (bark of Cinnamomum Zeylanicum)4

and used as remedy for cold, cough,

congestion, bronchitis, TB, asthma, emphysema, flu, sinus congestion, inflammation,

sore throat, laryngitis, pharyngitis, bronchitis, trachyitis, sinus headache, respiratory

allergy, seasonal sneezing, wheezing, excess mucous5. Standardization of Sitopaladi

churna is based on its organoleptic, physical, physico-chemical and chemical

properties.

OBJECTIVES

Department of Pharmacognosy,NCP,Shimoga 4

OBJECTIVES AND PLAN OF WORK

The present study deals with the standardization of sitopaladi churna

1. Collection of the ingredients for sitopaladi churna

2. Preparation of sitopaladi churna

3. Powder microscopy

4. Physico-chemical evaluation

5. Physical characteristics of powder formulations

6. Phytochemical investigations

7. Standardization based on TLC and HPTLC profiles

8. Compilation of data

2.1 Plan of work

1. Preparation of sitopaladi churna: Churna will be prepared as per the procedure

given in ocean of pharmaceutics.

2. Powdered microscopy: It will be done for the examination of fibers, starch,

lignin, etc.

3. Physico-chemical evaluation: The parameters such as extractive values, ash

values will be studied.

4. Physical characteristics of powder formulation: The parameters such as tap

density, bulk density, angle of repose, Hausner ratio, Carr’s index will be

studied.

5. Phytochemical investigations: To confirm the various secondary metabolites

preliminary phytochemical investigation will be performed.

OBJECTIVES


6. Standardization based on TLC and HPTLC profiles: all the preparations will

be subjected for chromatographic fingerprint analysis and HPTLC.

7. Compilation of data collected by in-house preparation and marketed products

will be carried out in order to establish the standards for sitopaladi churna.

REVIEW OF LITERATURE


A.2 Review of literature:

PIPER LONGUM:

PIPERINE

Family : piperaceae

Synonyms

English name – long pepper

Hindi name – pippal

Sanskrit name- pippali

Gujarati name – pippal

Habitat : It is found throughout India especially in the warmer places. It is also found

in Malaysia, Indonesia, Singapore, Sri Lanka and south Asian regions.

Botanical classification

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnolipsida

Order : Piperales

Family : Piperaceae

Genus : Piper

Species : longum

http://www.ayushveda.com/herbs/piper-longum.htm



Morphology : It is a creeper that spreads on the ground or may take support of other

trees. Leaves are 2 to 3 inch long. The older leaves are dentate, dark in color and heart

shaped. The younger leaf is ovate in shape and contains 5 veins on them. Flowers are

monoceous and male and female flowers are borne on different plants. Male flower

stalk is about 1 to 3 inch long and female flower stalk is ½ to 1 inch long. Fruit is

long. When it ripes it attains red color and when it dries it attains black color. It is one

inch in diameter. The plant flowers in rains and fruits in early winters.

According to ayurveda it has 4 varieties

1. Pippali

2. Gaja pippali

3. Saheli

4. Vana pippli

Chemical Constituents

It contains aromatic oil that is about 0.7 %, piperine 4 to 5 % and an alkaloid and

pipalartine. Besides this it contains sesamin and piplasterol. The root contains

pipperin 0.15 to 0.18 %, pippalartin (0.13 to 0.20 %), pipperleguminin, sterols and

glycosides

Indication

Inflammation, Pain, Nervine weakness, Tastelessness, Indigestion, Loss of appetite,

Constipation, Pain abdomen, Piles, Liver related problems, Anemia, Heart related

problems, Blood disorders, Cough, Asthma, Hiccups, General body weakness,





Tuberculosis, Decreased sperm count, Menstrual disturbances, Skin related disorders,

Worms

Oil and paste- it is used in applying on wounds and skin related ailments. It

suppresses pain and also reduces inflammation6.

CINNAMON ZEYLANICUM:

Kingdom : Plantae

Phylum : Magnoliophyta

Class : Magnoliopsida

Order : Laurales

Family : Lauraceae

Genus : Cinnamomum

Species : Cinnamomum zeylanicum

Domain : Eukarya7

Common Names : cinnamon bark, Dalchini, Ceylon cinnamon.

Habitat : It is a species that is found in various states of central and southern parts of

the country. It originated in India and living in warm climates, semi, dry and mild. It

grows in houses and cultivated farmland and is associated with the tropical deciduous

forest, subcaducifolia, subperennifolia, evergreen; mountain cloud forests of oak and

pine.

Morphology : Tree of up to 20 m tall, thick bark and pale color. The leaves are bright

and elongated. The flowers are grouped and fruits originate in purple.



Indications

Inappetence, dyspepsia, gastrointestinal spasms, colitis, gastritis, bronchitis,

emphysema, bronchiectasis, asthma, amenorrhea, dysmenorrhea.

ActivePrinciples

Essential oil: Cinnamaldehyde (50-75%), eugenol (4-10%), traces of carbides

terpénicos (pinene, cineol, phellandrene, linalol), and metilamilcetona; carbohydrates,

mucilages, tannins, traces of coumarin.

Uses : Stimulating appetite, eupéptico, Carmine, antiseptic, spasmolytic, emenagogo,

antidismenorréico and is commonly used for infertility8.

ELETTARIA CARDOMOMUM:

Kingdom : Plantae


Class : Liliopsida

Order : Zingiberales

Family : Zingiberaceae

Common Names: Bastard cardamom, Cardamom seeds, Malabar

Habitat : Southern India; cultivated in other tropical areas.

Medicinal Parts : Seed

Uses : the medicinal actions, which include appetite stimulation, carminative,

stimulant and stomachic actions. Flatulent dyspepsia is avoided, foods are better

digested and assimilated, the blood is thinned, the heart and digestive tract mildly

stimulated and so forth.

http://www.appliedhealth.com/index.php?option=com_content&view=article&id=108252&Itemid=206



http://www.appliedhealth.com/index.php?option=com_content&view=article&id=100006&Itemid=206#ItemId108254










Morphology : It is a tall, herbaceous perennial herb with branching subterranean

rootstock, from which arise a number of upright leafy shoots, bearing alternate,

elliptical or lanceolate sheathing leaves. The flowers are borne in panicles, arising

from the base of vegetative shoots. The flowers are bisexual but self-sterile, and open

in succession from the base towards the tip. Fruits are trilocular capsules, fusifrom to

oviod, pale green to yellow in colour, containing 15-20 hard, brownish black, angled

and rugose seeds, covered by a thin mucilaginous membrane10

.

Bambusa arundinacea (Vansalochan)

Kingdom : Plantae


class : Liliopsida

Order : Poales

Family : Bambusaceae

Genus : Bambusa

Species : Arundinacea

Other Names

English : Thorny bamboo

Hindi : Bams, Kantabams

Kannada : Bedru

Malayalam : Mula Llli

Sanskrit : Vamsah, Venuh



Tamil : Mulmunkil

Telugu : Vedurubiyyam, Veduruppu

Habitat: Throughout India, in areas up to 2100 m elevation

Morphology: A tall thorny bamboo upto 30 m in height with many sterns, tufted on

stout root stock, nodes prominent, the lowest rooting, internodes upto 45 cm long,

stem sheath coriaceous, orange-yellow, streaked, glabrous or puberulous beneath,

base rounded, ciliate, tip stiff, midrib narrow, leaf sheaths with a short bristly auricle,

ligule short; spikelets glabrous, yellow or yellowish green, in very long panicles, often

occupying the whole stem, floral glumes, 3-7 in number, the uppermost 1-3 male or

neuter, lodicules 3, hyaline, 1-3 nerved, ciliate; fruits oblong grains, beaked by the

style base, grooved on one side.

Chemical Constituents: Analysis of tender shoots gave protein, carbohydrates,

calcium, phosphorus, iron, magnesium, sodium, copper, chlorine, thiamine, vitamin-C

etc.

Plant part used: Roots, leaves, axillary bud, fruit and vansalochan

Indication

Skin related ailments, Beauty enhancer, Inflammation, Heart related problems,

Menstrual disturbances, Blood impurity, Fever, Cough and asthma, General body

weakness, Dysurea, Poisoning

http://www.ayushveda.com/herbs/bambusa-arundinacea.htm



Uses

The roots are sweet, astringent, cooling, laxative, depurative, diuretic and tonic. They

are useful in vitiated conditions of kapha and pitta, leprosy, skin diseases, burning

sensation, discolorations, strangury, ringworm, leucorrhea, arthralgia and general

debility. The leaves are sweet, astringent, cooling, emmenagogue, ophthalmic,

vulnerary, constipating and febrifuge. They are useful in vitiated conditions ofpitta,

ophthalmopathy, lumbago, hemorrhoids, diarrhea, gonorrhea, amenorrhea,

dysmenorrhoea, wounds, skin diseases and fever. The sprouts are acrid, bitter,

laxative, thermogenic, anti-inflammatory, digestive, carminative, antihelmintic and

diuretic. They arc useful in inflammations, ulcers and wounds, dyspepsia, nausea,

intestinal worms, flatulence and strangury. The grains are acrid, sweet, thermogenic,

anthelmintic, aphrodisiac, alexeteric and tonic. They are useful in vitiated conditions

of kapha, urorrhea, intestinal worms11

.

Candy sugar:

Kingdom: Plantae

Division: Magnoliophyta

Class: Liliopsida

Order: Cyperales

Family: Poaceae (formerly Graminae).

Genus: Saccharum 12

Sugarcane is indigenous to tropical South Asia and Southeast Asia.[2]

Different

species likely originated in different locations, with S. barberi originating in India and

S. edule and S. officinarum coming from New Guinea.[2]

Crystallized sugar was

http://open-encyclopedia.com/Plant

http://open-encyclopedia.com/Flowering_plant

http://open-encyclopedia.com/Monocotyledon

http://open-encyclopedia.com/Cyperales

http://open-encyclopedia.com/Poaceae

http://en.wikipedia.org/wiki/South_Asia

http://en.wikipedia.org/wiki/Southeast_Asia

http://en.wikipedia.org/wiki/Sugarcane#cite_note-Sharpe-1

http://en.wikipedia.org/wiki/India

http://en.wikipedia.org/wiki/New_Guinea

http://en.wikipedia.org/wiki/Sugarcane#cite_note-Sharpe-1



reported 5,000 years ago in the Indus Valley Civilisation, located in modern-day

Pakistan and northwest India.

Sugarcane cultivation requires a tropical or temperate climate, with a minimum of

60 centimetres (24 in) of annual moisture. It is one of the most efficient

photosynthesizers in the plant kingdom. It is a C4 plant, able to convert up to 1 percent

of incident solar energy into biomass.[4]

In prime growing regions, such as India,

Pakistan, Peru, Brazil, Bolivia, Colombia, Australia, Ecuador, Cuba, the Philippines,

El Salvador and Hawaii, sugarcane can produce 20 lb (9 kg) for each square meter

exposed to the sun13

.

Fig.no.1 Candy sugar (16gms)

http://en.wikipedia.org/wiki/Indus_Valley_Civilisation

http://en.wikipedia.org/wiki/Pakistan

http://en.wikipedia.org/wiki/Tropical

http://en.wikipedia.org/wiki/Temperate

http://en.wikipedia.org/wiki/Photosynthetic_efficiency

http://en.wikipedia.org/wiki/Photosynthesis

http://en.wikipedia.org/wiki/Plant_kingdom

http://en.wikipedia.org/wiki/C4_plant

http://en.wikipedia.org/wiki/Sugarcane#cite_note-3

http://en.wikipedia.org/wiki/India

http://en.wikipedia.org/wiki/Pakistan

http://en.wikipedia.org/wiki/Peru

http://en.wikipedia.org/wiki/Brazil

http://en.wikipedia.org/wiki/Bolivia

http://en.wikipedia.org/wiki/Colombia

http://en.wikipedia.org/wiki/Australia

http://en.wikipedia.org/wiki/Ecuador

http://en.wikipedia.org/wiki/Cuba

http://en.wikipedia.org/wiki/Philippines

http://en.wikipedia.org/wiki/El_Salvador

http://en.wikipedia.org/wiki/Hawaii



Fig.no.2 Vamsalochana (8gms)

Fig.no.3 Piper Longum (4gms)



Fig.no.4 Cardamamum (2gms)

Fig.no.5 Cinnamon Zeylanicum(1gm)



Karunakar shukla et al, (2007) has reported standardization of lavan bhaskar

churna was carried out as per the guidelines of World Health Organization such as,

extractive value, ash value, phyto-constituents, micromeritic parameters and

subjected for spectroscopic studies with references to spectrum and overlain

spectrum. The results were found in close proximity of three batches. This study on

lavan bhaskar churna was reproducible precise and may be considered as a method

for its quality control14

.

Jeganathan NS et al, (2008) has reported standardization of a siddha formulation,

amukkara curanam by HPTLC was investigated for the estimation of marker

compounds withaferine A and piperine contents in a prepared standard and

commercial formulation by using HPTLC method of analysis. The two

formulations were subjected to methanol, ethyl acetate and chloroform extractions

by using soxhlet apparatus. The chromatogram was developed using

chloroform:methanol (8.5:1.5 v\v) and toluene:ethyl acetate (7:3 v\v) as mobile

phases for the estimation of withaferine A and piperine and was checked at 220nm

and 254nm respectively. The proposed HPTLC method was found to be rapid,

simple and accurate for quantitative estimation of withaferine A and piperine in

different formulation extracts15

.

Gopala simha KR et al, (2007) has reported standardization of ayurvedic polyherbal

formulation, nyagrodhadi churna by specific parameters both for the raw materials

and finished products. The obtained values of physical and chemical parameters

can be adopted to laydown new pharmacopoeial standards for the traditional

preparation of nyagrodhadi churna with batch to batch consistency. The phyto



chemical constituents found to be present in raw materials used for the preparation

of nyagrodhadi churna possibaly facilitate the desirable therapeutic efficacy and

also to know the pharmacological action16

.

Aswatha Ram HN et al, (2009) has reported standardization of avipattikar churna a

polyherbal formulation was prepared as per ayurvedic formulary of India in-house

prepretion and two marketed products have been standardised on the basis of

organoleptic characters, physical characteristics and physico-chemical properties.

The set parameters were found to be sufficient to evaluate the churna and can be

used as a reference standards for the quality control / quality assurance laboratory

of a pharmaceutical house 17

.

Mohapatra P et al, (2008) has reported standardization of a polyherbal formulation-

Hingwastak churna. In-house preparation and three marketed samples have been

standardizad on the basis of organoleptic characters, physical characteristics and

physico-chemical properties. The set parameters were found to be sufficient to

evaluate the churna and can be used as reference standard for the quality control /

quality assurance laboratory of a pharmaceutical house18

.

Jain Sanjay et al (2009)an attempt to standardize Dashamularishta, a traditional

formulation, used in the normalization of physiological processes after child birth.

Four marketed preparations and three in-house preparations were used for the

study. The various parameters performed included organoleptic characteristics,

physicochemical and toxicological parameters. HPTLC was carried out for

quantitative analysis of piperine in all the formulations. The results obtained were



considered as tools for assistance to the regulatory authorities, scientific

organizations and manufacturers for developing standards19

.

M Rajani et al, (2008) has reported commercialization of the manufacture of these

medicines to meet this increasing demand has resulted in a decline in their quality,

primarily due to a lack of adequate regulations pertaining to this sector of

medicine. The need of the hour is to evolve a systematic approach and to develop

well-designed methodologies for the standardization of herbal raw materials and

herbal formulations. In this chapter, various methods of phytochemical

standardization, such as preliminary phytochemical screening, fingerprint profiling,

and quantification of marker compound(s) with reference to herbal raw materials

and polyherbal formulations, are discussed in detail and suitable examples are

given20

.

Sanjay jain et al (2010) attempt to standardize “Chopchiniyadi Churna” an

ayurvedic polyherbal formulation used in the treatment of rheumatism, epilepsy,

anthelmintic, malignant ulcers and some diseases of the central nervous system.

Three marketed and one in-house formulations were used for the study. All the

formulations were standardized on the basis of organoleptic characters, physical

characteristics and physico-chemical properties. The set parameters were found to

be sufficient to evaluate the churna and can be used as reference standards for the

quality control/quality assurance purposes21

.

K R Gopala simha et al(2006) reported standardization of the ayurvedic medicine,

yogaraja guggulu was achieved by following modern scientific quality control

procedures both for the raw material and the finished product. The obtained



values/ranges of physical and chemical parameters can be adopted to lay down

new pharmacopoeial standards to be followed for traditional prepration of yogaraja

guggulu with batch to batch consistency. The phytochemical constituents found to

be present in the raw material used for the preparation of yogaraja guggulu

possibly facilitate the desirable therapeutic efficacy of the medicinal formulation as

a whole in knowing the mechanisms of pharmacological action22

.

Neeraj K Sriwastava et al, (2010) reported standardization of Ajmodadi churna, a

polyherbal ayurvedic medicine used as a carminative and an antispasmodic, and is

a strong wormifuge, and helps in all painful conditions like sciatica and stiffness in

back and also restores normal digestive functions. Ajmodadi churna was prepared

as per Ayurvedic Formulary of India. In-house preparation and the marketed drug

have been standardized on the basis of organoleptic characters, physical

characteristics, and physico-chemical properties. The set parameters were found to

be sufficient to evaluate the churna and can be used as reference standards for the

quality control/quality assurance laboratory of a Pharmaceutical house23

.

MATERIALS AND METHOD

Department of Pharmacognos,NCP,Shimoga 20

4.1 Collection and identification of plant materials

Plant material - Sitopaladi churna consists of five ingredients viz., Sugar candy,

Bambusa arundinacea, Piper longum, Eletarria cardamom and Cinnamonum

zeylanicumm. All these ingredients were procured from the local market of Shimoga,

and Belgaum, Karnataka, India.

4.2 Determination of physico-chemical constants for individual ingredients of

Sitopaladi churna and for formulations

4.2.1. Foreign organic matter- 250 g or the quantity specified in the individual

monograph, of the original sample was weighed accurately and spread out in a thin

layer. The samples were inspected with the unaided eye or with the use of a

magnifying lens (6X or 10X) and the foreign organic matter were separated manually

as completely as possible and weighed. The percentage of foreign organic matter was

weighed and determined with reference to the weight of the drug taken.

4.2.2. Loss on drying (LOD) - About 2-5 g of the prepared air dried individual

materials were accurately weighed in a previously dried and tared flat weighing

bottle. The samples were distributed evenly and were placed in the drying chamber

(Oven). Drying was carried out by heating to 100-105°C, the bottle was removed

from the oven and the bottle was closed promptly and allowed to cool to room

temperature and then weighed. The experiment was repeated till two consecutive

weighing did not differ by more than 5 mg, unless otherwise stated in the test

procedure. The loss in weight on drying was then calculated.

4.2.3. Ash value- Ash content of the crude drug is generally taken to be the residue

remaining after incineration. It represents the inorganic salts naturally occurring in the



drug and adhering to it, but may also include inorganic matter added for the purpose

of adulteration.

Total ash is the residue remaining after incineration. Acid insoluble ash is the part of

the total ash, which is insoluble in dilute hydrochloric acid. Water-soluble ash is the

part of total ash, which is soluble in hot water.

4.2.3.1. Total ash- About 2g of the individual powdered ingredients of Sitopaladi

churna were accurately weighed in a tared silica crucible. The powdered drug was

spread as a fine layer at the bottom of the crucible. The crucible was incinerated at a

temperature not exceeding 450°C until free from carbon. The crucible was cooled and

weighed. The procedure was repeated till a constant weight was observed. The

percentage of the total ash was calculated in triplicate with reference to the air dried

drug.

4.2.3.2. Acid insoluble ash- The ash obtained as described in the determination of

total ash was boiled with 25 ml of hydrochloric acid for 5 min. The insoluble ash was

collected on an ashless filter paper by filtration and it was washed with hot water. The

insoluble ash was transferred into a tared silica crucible, ignited, cooled and weighed.

The procedure was repeated till a constant weight was observed. The percentage of

acid insoluble ash was calculated with reference to the air-dried drug.

4.2.3.3. Water-soluble ash

To the crucible containing the total ash, add 25 ml of water and boil for 5 minutes.

Collect the insoluble matter in a sintered-glass crucible or on an ashless filter-paper.

Wash with hot water and ignite in a crucible for 15 minutes at a temperature not



exceeding 450°C. Subtract the weight of this residue in mg from the weight of total

ash. Calculate the content of water-soluble ash in mg per g of air-dried material.

4.3. Extractive values- Extractive value is a measure of the content of the drug

extracted by solvents. Extractive value can be water soluble, ethanol soluble and ether

soluble extractives. Extractive value unless and otherwise prescribed, is carried out by

maceration.

4.3.1 Water soluble extractive- 4 g of previously weighed air-dried powdered

individual ingredients of Sitopaladi churna were taken in a glass stoppered flask and

macerated with 100 ml of chloroform water (1:99). It was shaken frequently for 6 h

and then allowed to stand for 18 h. It was filtered rapidly taking precautions against

loss of the solvent. 25 ml of filtrate was evaporated to dryness in a tared flat-bottomed

petri dish, dried at 105°C, cooled in a desiccator and weighed. The percentage of

water-soluble extractive was calculated with reference to air-dried drug24

.

Note – Ether and alcohol soluble extractive, follow the same procedure of water

soluble extractive excepting the solvent (water) which is replaced by ether and

alcohol.

4.4. Fluorescence analysis - The powdered samples were exposed to Ultraviolet light

at wavelength of 254 nm and 366 nm. Fluorescence analysis was carried out in

accordance with the procedure reported by Kokoshi et al. One mg of powdered drug

was placed on a micro slide and observed under UV 366, UV 254 and in day light to

observe the fluorescent characteristics of powder, if any. One mg powdered drug was

placed on a micro slide and treated with one ml 1N HCl and observed under UV 366,

UV 254 and in day light while wet. One mg powdered drug was placed on a micro



slide and treated with one ml 1N NaOH and observed after a few minute in day light,

under UV 366, UV 254. One mg powdered drug was placed on a micro slide and

treated with one ml 1N NaOH in one ml methanol and observed under UV 366, UV

254 and in day light while still wet. One mg powdered drug was placed on a micro

slide and treated with one ml 50% KOH and observed under UV 366, UV 254 and in

day light while still wet. One mg powdered drug was placed on a micro slide and

treated with one ml of 50% sulphuric acid and observed under UV 366, UV 254 and

in day light while still wet. One mg powdered drug was placed on a micro slide and

treated with one ml of conc. sulphuric acid and observed under UV 366, UV 254 and

in day light while still wet. One mg powdered drug was placed on a micro slide and

treated with one ml of 50% HNO3 and observed under UV 366, UV 254 and in day

light while still wet. One mg powdered drug was placed on a micro slide and treated

with one ml of Conc. HNO3 and observed under UV 366, UV 254 and in day light

while still wet. One mg powdered drug was placed on a micro slide and treated with

one ml of acetic and observed under UV 366, UV 254 and in day light while still wet.

One mg powdered drug was placed on a micro slide and treated with one ml of iodine

and observed under UV 366, UV 254 and in day light while still wet25

.

4.5. Powder microscopy- The powder of Sitopaladi churna was examined for its

microscopic characters. The powder was passed through sieve no. 60 and was boiled

with chloral hydrate to remove colouring matter and viewed under microscope for

fibers, starch and other characters. The clarified powder was later stained with

phloroglucinol in the presence of hydrochloric acid for the lignified structures like

stone cells were viewed under microscope.26



4.6. Preparation of Sitopaladi churna: The In-house churna (I) was prepared as per

the procedure given in Ayurvedic Formulary of India. All the ingredients were

powdered separately, passed through 80 # sieve and then mixed together in specified

proportions to get uniformly blended churna.

Formula of In-house churna27

:-

S.NO. INGREDIENTS QUANTITY (parts)

1 Sitopala (Sugar Candy) 16

2 Bambusa arundinacea (Vamsa) (S.C.) 8

3 Piper longum (Pippali) (Fr.) 4

4 Elattaria cardamom (Ela) (Sd.) 2

5 Cinnamomum zeylanicumm (Tvak) (St. Bk) 1

4.7.Measurement of starch grains of sitopaladi churna:

Equipment

Use a microscope with an ocular micrometer to measure the size of small objects. The

scales should be calibrated using a stage micrometer, consisting of a glass slide of

usual size, upon which a scale is engraved or photographed, usually 1 or 2 mm long,

in 0.1 and 0.01 mm divisions. The ocular micrometer consists of a small disc of glass,

across the diameter of which a 100-line scale is engraved or photographed. The disc is

placed into the eyepiece.



4.8.Calibration of the ocular micrometer

Place the ocular micrometer scale in the eyepiece of the microscope. Put the stage

micrometer on the microscope stage and focus on the scale divisions. Both scales

should be sharply defined in the field of view. Turn the eyepiece to place the scales in

a parallel position and, if necessary, move the stage micrometer until the starting lines

of both scales coincide. Now find another point, as far along the scale as possible,

where two other division lines are exactly superimposed. Count the number of

divisions on the ocular micrometer and the corresponding length on the stage

micrometer scale, in order to determine the length that is equivalent to one division on

the ocular micrometer scale; for example, if 100 divisions on the ocular micrometer

scale are equal to 30 divisions on the stage micrometer scale, since the divisions on

the stage micrometer scale are 0.01 mm apart, 100 ocular micrometer divisions are

equivalent to 0.30 mm, therefore each division on the ocular micro-meter scale

represents 3.0 μm. Since the calibrations apply only for a particular lens combination,

it is advisable to determine and record the ocular micrometer values for the most

frequently used combinations.

Method

Place the specimen on the microscope stage and focus on the object to be measured.

Superimpose the ocular micrometer scale and read off the dimensions of the object.

Multiply the number of scale divisions by the micrometer value to give the actual

dimension in micrometers. By this method, using a 40x objective and a 6x eyepiece,

measurements are correct to the nearest 2 μm, i.e. a dimension of 20 μm is liable to an

error of 2 μm, or 10%, or ±5%; a dimension of 100 μm is liable to an error of 2% or

±1%. For curved and elongated objects, the measurement of lengths may be made



using a microscope equipped with a drawing apparatus or a camera lucida. The

instrument should be set up so that the image of the object, the drawing paper and the

pencil are in focus simultaneously. With the stage micrometer in place, trace the line

of the stage micrometer scale upon the paper fastened on a drawing board. Tilt the

drawing board, if necessary, until the divisions drawn upon the paper are equally

spaced. The magnification is determined by measuring the distance between selected

lines on the drawing paper and dividing by the distance between the corresponding

lines on the stage micrometer. Place the specimen on the microscope stage and trace

the image of the object on the paper. Superimpose a colored thread along the length of

the object drawn on the paper and after straightening it, measure the length of the

thread by means of a ruler graduated in millimeters; divide the measured length by the

magnification to give the actual length of the object24

.

4.9. Preparation of extracts

Alcoholic extract - The powdered churna was macerated with methanol for 2 days.

After the completion of maceration residue was removed by filtration followed by the

evaporation of solvent and extract was concentrated.

4.10. Phytochemical analysis of extract: Following chemical tests were carried out

for different extracts of Sitopaladi churna to identify the presence of various

phytochemical constituents.

4.5.2 Detection of alkaloids

The small portions of fractions are stirred separately with a few drops of dilute

HCl and filtered and then subjected to test for alkaloids.



4.10.1. Test for alkaloids

Mayer’s test:- Few ml of extract was treated with Mayer’s reagent. Formation

of cream or pale yellow ppt indicates the presence of alkaloids.

Wagner’s test:- Few ml of extract was treated with Wagner’s test. Formation

of reddish brown ppt indicates the presence of alkaloids.

Dragendroff’s test:- Few ml of extract was treated with Dragendroff’s

reagent. Formation of reddish brown ppt indicates the presence of alkaloids.

Hager’s test:- Few ml of extract was treated with Hager’s test reagent.

Formation of yellow ppt indicates the presence of alkaloids.

Tannic acid test:- Few ml of extract was treated with Tannic acid. Formation

of buff ppt indicates the presence of alkaloids.

4.10.2. Test for triterpenes

Liberman-Burchard’s test:- Extract was treated with few drops of acetic

anhydride, boiled and cooled. Few drops of sulphuric acid were added through sides

of test tube. Formation of reddish colour ring at the interface indicates the presence of

steroids and triterpenes.

Salkowski’s Test:- Extract was treated with chloroform and filtered. The

filtrates were treated with few drops of Conc. Sulphuric acid, shaken and allowed to

stand. Appearance of red or violet colour at the interface indicates the presence of

triterpenes.



Tschugajew’s test:- Extract of substance in CHCL3 was mixed with Acetyl

chloride in excess. Add a little quantity of Zncl2 and boil. Appearance of eosine red

colour with yellow fluorescence indicates the presence of triterpenes.

Hirschorn test:- Substance treated with trichloro acetic acid and warm.

Formation of red to yellow colour indicates the presence of triterpenes.

4.10.3. Test for steroids

Liberman-Burchard’s test:- Extract was treated with few drops of acetic

anhydride, boiled and cooled. Few drops of sulphuric acid were added through sides

of test tube. Formation of reddish colour ring at the interface indicates the presence of

steroids and triterpenes.

Sulphur test:- To the chloroform solution of the sample, powder sulphur was

sprinkled on the surface, if sulphur shrinks down, it indicates the presence of

cholesterol.

Salkowski’s Test:- Extract was treated with chloroform and filtered. The

filtrates were treated with few drops of conc. sulphuric acid, shaken and allowed to

stand. Appearance of red or violet colour at the interface indicates the presence of

triterpenes.

4.10.4. Test for carbohydrates

Molisch’s test:- Filtrate was treated with 2 drops of alcoholic α-naphthol

solution in a test tube and 2 ml of conc. sulphuric acid was added carefully along the

sides of the test tube. Formation of violet ring at the junction indicates the presence of

Carbohydrates.



Barford’s test:- Filtrate was treated with Barfoed’s reagent and heated on a

water bath. Orange red precipitate indicated presence of mono and disaccharides.

Benedict’s test:- Filtrate was treated with Benedict’s reagent and heated on

water bath. Formation of orange red precipitate indicates the presence of reducing

sugars.

Fehling’s test:- Filtrate was hydrolyzed with dil.HCl, neutralized with alkali

and heated with Fehlings-A & Fehlings-B solutions. Formation of red precipitate

indicates the presence of reducing sugars.

4.10.5. Test for glycosides

Chrysarobin test:- 0.1gm of powder was dissolved in H2SO4 ,deep red

solution is produced indicates the presence of glycosides

Anthraquinone glycoside test:- The drug was powdered and extracted with

ether or any water immiscible solvents. The filtered ethereal extract is made alkaline

either with caustic soda or NH3. the aq. Layer shows pink colour, turns red to violet

colour indicates the presence of Anthraquinone glycoside.

4.10.6. Test for flavonoids

FeCl3 test:- Extract was treated with FeCl3 solution. Formation of green to

black color indicates presence of flavonoids.

Shinoda test:- Extract was treated with the mixture containing piece of

magnesium ribbon and Hcl. Formation of red colour indicates presence of flavonoids.



NaOH test:- To the extract add 10% NaOH was added. Formation of yellow

colour indicates presence of flavonoids.

Lead acetate test:- To the extract add 10% Lead acetate solution was added.

Formation of yellow colour ppt. indicates presence of flavonoids.

Mineral acid test:- To the drug add conc. H2SO4 was added. Formation of

orange colour indicates presence of flavonoids.

Zn/Hcl test:- Pinch of zinc dust was added to extract and conc. Hcl was

added. Formation of red colour indicates presence of flavonoids29

.

Standardization - The marketed sample of Sitopaladi churna belonging to SDM,

Baidynath, Dabur company and the in-house preparation were standardized based on

their organoleptic characters, physical characteristics and physico-chemical

properties.

4.11. DETERMINATION OF PHYSICO-CHEMICAL PARAMETERS OF

SITOPALADI CHURNA

4.11.1. Organoleptic evaluation - Organoleptic evaluation refers to evaluation of

formulation by color, odour, taste, texture etc. The organoleptic characters of the

samples were carried out based on the official method.

4.11.2. Ash values and extractive values were determined as described earlier.

4.11.3. Determination of physical characteristics of formulations - Physical

characteristics like bulk density, tap density, angle of repose, Haussner ratio and

Carr’s index were determined for different formulations. The term bulk density refers



to method used to indicate a packing of particles or granules. The equation for

determining bulk density (Db) is Db=M/Vb where M is the mass of particles and Vb is

the total volume of packing. The volume of packing can be determined in an

apparatus consisting of graduated cylinder mounted on mechanical tapping device that

has a specially cut rotating can. 100gm of weighed formulation powder was taken and

carefully added to cylinder with the aid of a funnel. The initial volume was noted and

sample was then tapped until no further reduction in volume was noted. The initial

volume gave the bulk density value and after tapping the volume reduced, giving the

value of tapped density. Angle of repose has been used as an indirect method

quantifying powder flowability, because of its relationship with interparticle cohesion.

The fixed funnel and the free standing cone method employs a method that is secured

with its tip at a given height (H), above the glass paper that is placed on a flat

horizontal surface. Powder or granules were carefully poured through the funnel until

the apex of the conical pile just touched the tip of funnel. Thus, with R being the

radius of the conical pile. Tan α = H/R or α = arc tan H/R, where α is the angle of

repose.



Table no.1 Relationship between angle of repose and type of flow

Flow property Angle of repose

(degree)

Excellent 25-30

Good 31-35

Fair 36-40

Passable 41-45

Poor 46-55

Very Poor 56-65

Very-very Poor >66

Hausner’s ratio is related to interparticle friction and as such can be used to predict

the powder flow properties. The equation for measuring the Hausner’s ratio is Df/Do.

Where, Df = Tapped density and D0 = Bulk density. Relationship between Hausner’s

ratio and type of flow

Table no. 2 Relationship between Hausner’s ratio and type of flow

Flow property Hausner’s ratio

Excellent 1-1.11

Good 1.12-1.18

Fair 1.19-1.25

Passable 1.26-1.34

Poor 1.35-1.45

Very Poor 1.46-1.59

Very-very Poor >1.6



Carr’s compressibility index is another indirect method of measuring the powder

flow from bulk density. The equation for measuring Carr’s index is %

Compressibility = Df – Do/Df x 100. Where Df = tapped density, D0 = Bulk density.

Table no. 3 Relationship between compressibility index and type of flow29

Flow property Compressibility index

(%)

Excellent ≤10

Good 11-15

Fair 16-20

Passable 21-25

Poor 26-31

Very Poor 32-37

Very-very Poor >38

4.12. Thin layer chromatography

Thin layer chromatography is mainly used for qualitative screening of plant

extract which serve as a very important tool in the overall phytochemical research

studies.

Procedure:

Slurry of silica gel G was prepared in distilled water and poured over a glass

plate to form a thin film. The prepared plates were allowed for setting (air-drying).

After setting, the plates were kept in an oven at 100 to 1200 C (30 min) for activation.

The extracts were applied to the activated plates (1 cm above from the bottom). It was



then kept in previously saturated developing chamber containing mobile phase, and

allowed to run 3/4th of the height of the plate. The developed plate was removed, air

dried and observed under ultraviolet light and florescent compound identified and

sprayed with vanillin sulphuric acid then compared with standard drug spot and

calculate the Rf value using following formula.

Rf = Distance traveled by solute

Distance traveled by the solvent

Standard used – piperine

Mobile phase - toluene : ethyl acetate (7:3)

Spraying reagent – vanillin sulphuric acid, UV at longer and shorter wavelength.

4.13. High-performance thin-layer chromatography (HPTLC)- Quantitative

densitometric HPTLC analysis was performed to develop the characteristic fingerprint

profile for the methanolic extract of Sitopaladi Churna.

4.13.1. Estimation of Piperine in Sitopaladi Churna:-

Preparation of piperine standard solution- A stock solution of standard Piperine (1

mg/ml) was prepared by transferring 5 mg of Piperine, accurately weighed, into a 5

ml volumetric flask, dissolving in 2 ml methanol. It was then sonicated for 10 min and

the final volume of the solutions was made up to 5 ml with methanol to get stock

solutions containing 1 mg/ml.

Preparation of sample solution- Accurately weighed 100 mg of dried methanolic

extract of Sitopaladi Churna was transferred to a 10 ml volumetric flask dissolving in

5 ml of methanol. It was then sonicated for 10 min and the contents of the flask were



filtered through Whatman No. 1 paper (Merck, Mumbai, India). The final volume of

the solution was made up to 5 ml with methanol to get stock solution of 20 mg/ml.

Mobile phase- benzene: ethyl acetate (2:1)

Instrumentation And Chromatographic Conditions- HPTLC was performed on 20

cm × 10 cm aluminum backed plates coated with silica gel 60F254 (Merck, Mumbai,

India). Standard solution of Piperine and sample solution were applied to the plates as

bands on the same chromatographic plate by use of a Camag (Muttenz, Switzerland)

Linomat V sample applicator equipped with a 100 μl Hamilton (USA) syringe.

Ascending development was performed at room temperature (28 ± 2°C) using mobile

phase in a Camag glass twin-trough chamber previously saturated with mobile phase

vapour for 20 min. After development, the plates were dried and then scanned at 254

nm with a Camag TLC Scanner- 3.

4.13.2. Estimation of Cinnamaldehyde in Sitopaladi Churna:-

Preparation of Cinnamaldehyde standard solution- A stock solution of standard

Cinnamaldehyde (1 mg/ml) was prepared by transferring 5 mg of Cinnamaldehyde,

accurately weighed, into a 5 ml volumetric flask, dissolving in 2 ml methanol. It was

then sonicated for 10 min and the final volume of the solutions was made up to 5 ml

with methanol to get stock solutions containing 1 mg/ml.

Preparation of sample solution- Accurately weighed 100 mg of dried methanolic

extract of Sitopaladi Churna was transferred to a 10 ml volumetric flask dissolving in

5 ml of methanol. It was then sonicated for 10 min and the contents of the flask were

filtered through Whatman No. 1 paper (Merck, Mumbai, India). The final volume of

the solution was made up to 5 ml with methanol to get stock solution of 20 mg/ml.



Mobile phase- Petroleum ether: Dichloromethane: Formic acid (4:2:0.1).

Instrumentation And Chromatographic Conditions- HPTLC was performed on 20

cm × 10 cm aluminum backed plates coated with silica gel 60F254 (Merck, Mumbai,

India). Standard solution of Cinnamaldehyde and sample solution were applied to the

plates as bands on the same chromatographic plate by use of a Camag (Muttenz,

Switzerland) Linomat V sample applicator equipped with a 100 μl Hamilton (USA)

syringe. Ascending development was performed at room temperature (28 ± 2°C), with

the mobile phase in a Camag glass twin-trough chamber previously saturated with

mobile phase vapour for 20 min. After development, the plates were dried and then

scanned at 254 nm with a Camag TLC Scanner- 3.

RESULTS AND DISCUSSION


5. Results:

The present work, standardization of sitopaladi churna is based on the

comparative studies (quality control parameters) of in-house formulation prepared as

per Ayurvedic Formulary of India, and three marketed products of Sri Dharmasthala

Manjunatha ayurveda, Baidyanath, and Dabur.

5.1 Collection:

Sitopaladi churna consists of five ingredients viz., Sugar candy, Bambusa

arundinacea, Piper longum, Eletarria cardamom and Cinnamonum zeylanicum. All

these ingredients were procured from the local market of Shimoga, and Belgaum,

Karnataka, India.

5.2 Visual examination of ingredients of Sitopaladi churna:

Visual inspection provides the simplest and quickest means by which to establish

identity, purity and, possibly, quality in terms of colour, consistency, odour or taste,

from the specifications.

5.3 Determination of quality control parameters for ingredients and

formulations:

Various physiochemical parameters were evaluated for ingredients and

formulations as per guidelines described by W.H.O, results are given below:



Table no. 4 Ash values of ingredients used to prepare Sitopaladi Churna:

Table no. 5 Ash values of in-house and marketed products of Sitopaladi Churna:

Sl.no. Name of the

ingradient

Total ash Acid insoluble

ash

Water soluble

ash

01. Cinnamon

zeylanicum

2.85% 2.80% 1.95%

02. Piper longum 3.4% 1.37% 2.6%

03. Cardamum 6.6% 3.6% 3.3%

04. vamsalochana 86% 83.45% 3.9%

Sl.no. Name of the product Total ash Acid insoluble

ash

Water soluble

ash

01. In-house 23.7% 22.15% 1.7%

02. Sri Dharmasthala

Manjunatha Ayurved

(SDM)

22.45% 21.55% 0.75%

03. Baidyanath 25.55% 24.15% 1.1%

04. Dabur 25.6% 27.0% 1.2%



Ash values of ingredients used to prepare Sitopaladi Churna:

All the ingredients of sitopaladi churna were subjected for ash values and the

results are given in the table no.4. where total ash of Cinnamon zeylanicum is reported

to be 2.85%, piper longum showed 3.4%, cardamom showed 6.6% and vansalochana

showed the highest value of 86%. The acid insoluble ash of Cinnamon zeylanicum is

2.80% piper longum showed lowest 1.37%, cardamom showed 3.6% and

vansalochana showed the highest value of 83.45%.

Ash values of in-house and marketed products of Sitopaladi Churna:

Sitopaladi churna of different brands and in house were subjected for ash

values and the results are given in the table no.5. Total ash, of in house is reported to

be 23.7%, SDM showed 22.45%, Baidyanath showed 25.55% and Dabur showed the

highest value of 25.6%. The acid insoluble ash of in house is reported to be 22.15%,

SDM showed lowest of 21.55%, Baidyanath showed 24.15% and Dabur showed the

highest value of 27.0%. The water soluble ash of in house is reported to be highest of

1.7%, SDM showed 0.75%, Baidyanath showed 1.1% and Dabur showed the value of

1.2%.



Table no.6 Extractive values of churnas:

Flow properties of churna:

Table no. 7 Angle of repose of Sitopaladi Churna:

Sl.no Name of product Water extract Alcohol extract Ether extract

01 In house 2.824% 1.832% 0.096%

02 SDM 3.264% 1.456% 0.056%

03 Baidyanath 2.984% 1.856% 0.016%

04 Dabur 2.752% 1.72% 0.056%

Sl.no Brand name of

the product

Height of

the pile (cm)

Radius of the

circle(cm)

Ø=tan-1

(h/r)

Flow

01. In house 2.8 4.7 30.75 Passable

02. SDM 3.0 3.87 37.73 Passable

03. Baidyanath 3.6 4.87 36.27 Passable

04. Dabur 3.0 4.07 36.35 Passable



Extractive values of churnas:

Water extract, alcoholic extract, and ether extract of the formulations are

reported in table no.6. Water extracts of in house formulation was found to be

2.824%, SDM has highest % of water extract of 3.264% which means comparatively

it contains more polar constituents ,Baidyanath showed 2.984% and Dabur showed

2.752%. Alcohol extract of in house formulation was found to be 1.832%, SDM has

showed 1.456%,Baidyanath showed highest of 1.856% and Dabur showed 1.72%.

Ether extract of in house formulation was found to be 0.096% which means

comparatively it contains more non polar constituents, SDM has showed 0.056,

Baidyanath showed 0.016% and Dabur showed 0.056%.

Angle of repose of Sitopaladi Churna:

The angle of repose is the angle between the surface of a pile and the ground

under it. This test method is used for the determination of the angle of repose of free-

flowing powders. The report of angle of repose of all the churnas are discussed in

table no.7 and it states that flow property of all the formulations are passable. The

ratio of height of the pile and radius of in house is determined to be 30.75 that has

excellent flow property, where as SDM, Baidyanath, Dabur showed 37.73, 36.27,

36.35 respectively which have fair flow property.



Table no. 8 Bulk Density of Sitopaladi Churna:

Sl.no Brand name of the

product

Mass of

powder(gms)

Bulk

volume(ml)

Bulk

density(g/cc)

01. In house 20 36 0.555

02. SDM 20 35.5 0.563

03. Baidyanath 20 36 0.555

04. Dabur 20 51 0.392

Table no.9 True Density Sitopaladi Churna:


product

Weight of

powder

True volume

of powder

True density

01. In house 20 26 0.769

02. SDM 20 25 0.8

03. Baidyanath 20 25.5 0.784

04. Dabur 20 36 0.555



Bulk Density of Sitopaladi Churna:

Bulk density of sitopaladi churna of different brands and in house formulation

was determined and reported in the table no.8 among which Dabur showed lowest

Bulk Density of 0.392 g\cc and SDM showed highest lowest Bulk Density of 0.563

g/cc, Baidyanath and in house showed 0.555 g/cc. This parameter helps in selecting

containers for packing the formulations.

5.4.3 True Density Sitopaladi Churna:

True density of sitopaladi churna of different brands and in house formulation

was determined and reported in the table no.9 among which Dabur showed lowest

True Density of 0.555 and in house showed 0.769, SDM showed the highest value of

0.8, baidyanath showed 0.784. This parameter helps in selecting containers for

packing the formulations.



Table no.10 Carr’s Index of Sitopaladi Churna:


product

Tapped

density

Fluff

density

Carr’s

index

Flow

01. In house 0.642 0.555 13.5 Excellent

02. SDM 0.666 0.563 15.4 Good

03. Baidyanath 0.645 0.555 13.9 Excellent

04. Dabur 0.444 0.392 11.7 Excellent

Table no. 11 Hausner’s Ratio of Sitopaladi Churna:


product

Tapped

density

Bulk

density(g/cc)

Hausner’s

ratio:

01. In house 0.642 0.555 1.15

02. SDM 0.666 0.563 1.18

03. Baidyanath 0.645 0.555 1.16

04. Dabur 0.444 0.392 1.13



Table no.12. Preliminary phytochemical screening of alcoholic extracts of

churnas:

Sl

no.

Test In house SDM Baidyanath Dabur

01 Alkaloids

Mayer’s test +ve +ve +ve +ve

Wagner’s test -ve -ve -ve -ve

Dragendroff’s test +ve +ve +ve +ve

Hager’s test +ve +ve +ve +ve

Tannic acid test -ve -ve -ve -ve

Pyridine alkaloids +ve +ve +ve +ve

02 Triterpenes

Salkowaski test +ve +ve +ve +ve

Liberman-storch +ve +ve +ve +ve

Morawaski test +ve +ve +ve +ve

Tschugajew’s test +ve +ve +ve +ve

Hirschorn test +ve +ve +ve +ve

03 Steroids

Liberman buchard

test

+ve +ve +ve +ve

Sulfur test +ve +ve +ve +ve

Salkowski test +ve +ve +ve +ve

04 Carobohtdrates

Molisch test -ve -ve -ve -ve



Fehling’s test -ve -ve -ve -ve

Starch test +ve +ve +ve +ve

05 Glycosides

0.1gm powd.+H2so4 +ve +ve +ve +ve

Anthraquinone +ve +ve +ve +ve

06 Flavonoids

Extract+Fecl3 +ve +ve +ve +ve

Shinoda test +ve +ve +ve +ve

Flavonoids+ NaOH +ve +ve +ve +ve

Lead acetate test +ve +ve +ve +ve

Mineral acid test +ve +ve +ve +ve

Zn/Hcl test +ve +ve +ve +ve

07 Proteins

Biurette test -ve -ve -ve -ve

Xanthoprotic test +ve +ve +ve +ve

Millions test -ve -ve -ve -ve



Carr’s Index of Sitopaladi Churna:

Carr’s Index of sitopaladi churna of different brands and in house formulation

was determined and reported in the table no.10. among which in house baidynath and

Dabur showed excellent flow property (13.5, 13.9, 11.7 respectively).

Hausner’s Ratio of Sitopaladi Churna:

Hausner’s Ratio of sitopaladi churna of different brands and in house

formulation was determined and reported in the table no.11, among which Dabur

showed lowest Hausner’s Ratio (1.13) and SDM showed highest Hausner’s Ratio

(1.18) where in house showed 1.15 and Baidyanath showed 1.16

Preliminary phytochemical screening of alcoholic extracts of churnas:

Alcoholic extracts of sitopaladi churna of different brands and in house

formulation were subjected for Preliminary phytochemical screening which is

reported in table no.12. according to this report the churnas showed the presence of

Alkaloids, Triterpenes, Steroids, Carobohtdrates, Glycosides, Flavonoids, and

Proteins



Table no. 13 Fluorescent test for In-house formulation of Sitopaladi churna:

Sl.no Reagent used Day light At 254nm At 366nm

01 1N NaoH in water LB B LG

02 1N Hcl PB LB LG

03 1N NaoH in methanol GB DB G

04 50% KOH B DB LG

05 50% HNO3 LG B G

06 Conc. H2SO4 DB DB G

07 Iodine water IB B DG

08 50% H2SO4 B B LG

09 Conc HNO3 LB B G

10 CH3COOH B DB G

LB: light brown, B: brown, LG: light green, PB: pale brown, DB: dark brown, IB:

intense brown

Table no.14 Fluorescent test for SDM formulation of Sitopaladi churna:



02 1N Hcl CB or LB LB LG

03 1N NaoH in methanol YB B LG

04 50% KOH LB PB LG

05 50% HNO3 YG LB LG

06 Conc. H2SO4 DR DB DG



07 Iodine water YB DB DG

08 50% H2SO4 LB B LG

09 Conc HNO3 GB B G

10 CH3COOH PB B LG


intense brown, CB: cloudy brown,

Table no.15 Fluorescent test for Baidyanath formulation of Sitopaladi churna:



02 1N Hcl CB DB LG

03 1N NaoH in methanol PB B G

04 50% KOH B PB LG

05 50% HNO3 GB B LG


07 Iodine water GB DB DG

08 50% H2SO4 LG LB LG

09 Conc HNO3 GB B G

10 CH3COOH PB B LG


intense brown, DR: dark red

Table no.16 Fluorescent test for dabur formulation of sitopaladi churna:


01 1N NaoH in water YB B LG

02 1N Hcl CG PB LG



03 1N NaoH in methanol CB LB PG

04 50% KOH GB LB LG

05 50% HNO3 GB B G


07 Iodine water GB DB DG

08 50% H2SO4 B LB LG

09 Conc HNO3 B LB LG

10 CH3COOH YG PB LG


intense brown, DR: dark red, GB: greenish brown

Table no.17 Organoleptic Properties Of Different Sitopaladi Churna

Sl.no Name of product Appearance Colour Taste Odour

01 In house Powder Pale brown Sweet

pungent

Characteristic

02 SDM Powder Pale brown Sweet

pungent

Characteristic

03 Baidyanath Powder Light ash Sweet Characteristic

04 Dabur Powder Light ash Sweet

pungent

Characteristic



Fluorescent analysis for formulations of Sitopaladi churna:

All the churnas were subjected for Fluorescent analysis with different reagents

and viewed under UV at 254 and 366nm which are reported in table nos.13,14,15,16

and this analysis is considered as on of the quality control parameter.

Organoleptic Properties Of Different Sitopaladi Churna

Organoleptic properties of sitopaladi churna were reported in table no.17. The

color of in house and SDM formulations were light brown and Baidyanath and Dabur

formulations were found to be light ash. The taste of In house, SDM, Dabur

formulations were found to be sweet pungent and that of Baidyanath was found to be

sweet.



Fig.no.6 Photos of Powder microscopy:

a. Fibers of In house b. Fibers of Dabur

c. Fibers of Baidyanath d. Fibers of SDM

Table no.18 Measurement of starch grains present in sitopaladi churna:

Sl no. Name of product Length of starch grains

in microns (µ)

1 In house 0.093

2 SDM 0.0837

3 Baidyanath 0.0901

4 Dabur 0.0952



Powder microscopy of Sitopaladi churna:

Figure no. 6 shows the fibers present in the churna.

Measurement of starch grains present in sitopaladi churna:

Strach grains of Sitopaladi churna was calculated with the calibration factor of 0.007.

The results were reported in the table no.18. , where in house showed the starch grains

of size 0.093µ, SDM showed about 0.0837µ, Baidyanath showed 0.0901µ, Dabur

showed 0.0952µ.

Fig.no.7 TLC of methanolic extract of sitopaladi churna for piperine

1 2 3 4 5 1 2 3 4 5

7a. TLC at 254nm 7b. TLC at 365nm



Table no.19. Determination of Rf values of piperine in methanolic extract of

sitopalaci churna by TLC

Sl no. Methanolic extract of Rf values

1 Standard 0.31

2 In house 0.31

3 SDM 0.30

4 Baidyanath 0.32

5 Dabur 0.30

High Performance Thin Layer Chromatography

Table No.20 Rf values and peak areas of methanolic extract for piperine by

HPTLC

Track Peak Start

Rf Start Height

Max Rf

Max Height

Max %

End Rf End Height

Area Area %

1 1 0.51 2.5 0.58 369.3 75.36 0.63 33.9 12515.5 77.22

1 2 0.64 34 0.68 120.8 24.64 0.73 1.8 3691.7 22.78

2 1 0.46 2 0.55 542.3 73.88 0.6 50.5 18689.4 77.11

2 2 0.6 51.3 0.64 191.7 26.12 0.71 0.9 5546.4 22.89

3 1 0.5 1.3 0.55 23.4 44.18 0.59 2.7 668.4 44.83

3 2 0.59 3.3 0.64 29.5 55.82 0.68 4.4 822.5 55.17

4 1 0.48 0.2 0.55 90 55.03 0.59 8.3 2429 54.59

4 2 0.6 8.5 0.64 73.6 44.97 0.69 3.2 2020.6 45.41

5 1 0.5 1.8 0.56 82.5 51.17 0.6 7.7 2275 50.67

5 2 0.6 8.1 0.65 78.8 48.83 0.7 5.4 2214.8 49.33

6 1 0.52 1.5 0.57 69.4 53.5 0.62 6.6 1964.4 52.81

6 2 0.62 6.7 0.67 60.3 46.5 0.73 2 1755.3 47.19



Fig.no.8 3-D spectra of all tracks at 350nm (piperine)

HPTLC of piperine

Mobile phase- benzene: ethyl acetate (2:1)

8a. Standard piperine 1mg/ml (volume applied 2ul)



8b. Dabur 10mg/ml (volume applied 2ul)

8c. Baidyanath 10mg/ml (volume applied 2ul)



8d. SDM 10mg/ml (volume applied 2ul)

8e. In house 10mg/ml (volume applied 2ul)



8f. HPTLC at 254nm 8g. HPTLC at 365nm

Table No.21 Rf values and peak areas of methanolic extract for

cinnamaldehyde by HPTLC

Track Peak Start

Rf Start Height

Max Rf

Max Height

Max %

End Rf

End Height

Area Area %

1 1 0.48 0.9 0.53 13.8 1.81 0.57 2.3 422.7 0.85

1 2 0.65 11.8 0.8 697.1 91.38 0.87 47.6 48221.7 96.58

1 3 0.87 47.8 0.89 52 6.81 0.96 2.3 1286.3 2.58

2 1 0.47 3.1 0.53 25.2 3.27 0.57 10.7 936.2 1.38

2 2 0.61 11.8 0.8 743.8 96.73 0.96 3.2 66670.3 98.62

3 1 0.2 1.2 0.24 145.4 23.76 0.27 45.4 3072.2 14.93

3 2 0.27 45.6 0.29 54.8 8.95 0.34 1 1269.3 6.17

3 3 0.67 6.1 0.76 411.9 67.29 0.84 8.6 16240.2 78.91

4 1 0.2 1 0.24 29.3 32.04 0.26 16.3 593 25.21

4 2 0.26 16.4 0.28 24.5 26.87 0.33 2.7 678.6 28.85

4 3 0.71 1.7 0.75 37.5 41.1 0.8 5.6 1080.3 45.93

5 1 0.2 1.7 0.23 113.5 53.55 0.26 47.2 2278.1 48.12

5 2 0.26 47.7 0.28 81.2 38.29 0.33 1.6 1965 41.5

5 3 0.71 1 0.75 17.3 8.16 0.79 7.1 491.2 10.38

6 1 0.19 1.1 0.23 92.7 57.56 0.25 35.9 1929.1 52.35

6 2 0.26 36.2 0.28 55.8 34.63 0.33 2.4 1433 38.88

6 3 0.71 1.8 0.76 12.6 7.81 0.78 5.9 323.1 8.77



Fig.no.9 3-D spectra of all tracks at 350nm (cinnamaldehyde)

HPTLC of cinnamaldehyde

Mobile phase- toluene: ethyl acetate (9:1.5)

9a. Standard cinnamaldehyde volume applied- 2ul



9b. Standard cinnamaldehyde: volume applied- 4ul

9c. Dabur : volume applied- 2ul



9d. Baidyanath : volume applied- 2ul

9e. SDM : volume applied- 2ul



9f. In house : volume applied- 2ul

9g. HPTLC at 254nm 9h. HPTLC at 365nm



TLC of methanolic extract of sitopalaci churna for piperine The alcoholic extract of all the churnas were subjected for TLC with the standard

reference piperine. The Rf values are given in the table no.19

High Performance Thin Layer Chromatography

It provides the rapid and positive analysis of plant drugs leading to semi

quantitative/ quantitative information on chief constituents of the formulations. It can

provide fingerprinting for monitoring the identity and purity of drug and detection of

adulteration/ substitution.

HPTLC fingerprinting and quantification of piperine with standard shows the

present of piperine in all the formulations with the Rf value, 0.55 of Dabur and

Baidyanath, 0.56 of SDM, 0.57 of in house with their relative % of, 44.83, 54.59,

50.67, 52.81 of Dabur, Baidyanath, SDM, In house respectively.

HPTLC fingerprinting and quantification of cinnamaldehyde with standard

shows the present of cinnamaldehyde in all the formulations, 0.29 of Dabur, 0.28 of

Baidyanath, 0.28 of SDM, 0.28 of in house with their relative % of, 6.17, 28.85, 41.5,

38.88 of Dabur, Baidyanath, SDM, In house respectively.

SUMMARY AND CONCLUSION


SUMMARY AND CONCLUSION:

The present study deals with, standardization of Sitopaladi churna which is

based on the comparative studies (quality control parameters) of in-house formulation

prepared as per Ayurvedic Formulary of India, and three marketed products of Sri

Dharmasthala Manjunatha ayurved, Baidyanath, and Dabur.

First the ingredients of the formulation were subjected to determine foreign

organic matter manually and subjected for powder microscopy, determination of ash

values like total ash, acid insoluble ash, and water soluble ash.

The churna was prepared as per the procedure given in Ayurvedic Formulary

of India there after it was subjected to, powder microscopy and compared with that of

the marketed products. Physical parameters like Angle of repose, Hausner’s ratio,

Bulk density, tap density, True density, Carr’s compressibility index were determined

and reported.

All the churna were subjected for determination of physicochemical

parameters like Fluorescent analysis, determination of ash values like total ash, acid

insoluble ash, and water soluble ash, preliminary phytochemical screening,

Organoleptic evaluation, Thin layer chromatography (TLC), and High-performance

thin-layer chromatography (HPTLC). HPTLC was done for quantification of piperine

and cinnamaldehyde.

The results of physical, physico chemical parameters like ash value, extractive

value, preliminary phytochemical screening, physical properties like bulk density, tap

density, true density, carr’s index, Hausner’s ration and analytical parameters like

TLC and HPTLC of all the marketed formulations of sitopaladi churna contains the

SUMMARY AND CONCLUSION


intended ingredients with their standard values, there is significant difference in water

soluble ash of SDM when compared to others. Difference found in the values of bulk

density and carr’s index of SDM and dabur can be considered significant, rest of the

values determined are have very insignificant difference.

BIBILOGRAPHY


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