Researth Artitle

Development of quality control methods forpolyherbal formulation, Chyawanprash

RaIlul P Kasar1, K S Laddha1*,Jayesh Chaudhary2 and Anil Shulda2IMedicinal Natural Products Research Laboratory, Pharmaceutical Division

University Institute of Chemical Technology, N M Parikh Marg, Matunga (E), Mumbai- 400 019, Maharashtra, India

2VedicLife Sciences, 118 Morya House, OffNew Link Road, Andheri (W), Mumbai-400 053

*Correspondent author, E-mail: ksladdha@udct.org; ks14@udct.org

Received 22 March 2005; Accepted 12 September 2005

Abstract

Chyawanprash is a traditional polyherbal formulation, which is widely used as tonic,

rejuvenator, anabolic, immunomodulator and memory enhancer. Chyawanprash contains the pulp

of Emblka officinalis Gaertn. as the prime ingredient, along with powder and extract of

several other herbs. It is observed that the consistency and taste varies from one manufacturer to

another. Even these variations are observed in the same pharmaceutical company in different

manufacturing batches. Hence, it is the need of the hour to standardize the raw materials to obtain

product consistency. With the advent of new analytical tools and sophisticated instrmpentaltechnology, the quality assurance profile for a crude drug or its bioactive constituents can be made

possible. 'I\vo in-house batch of Chyawanprash was prepared according to the procedure described

in text Charaksamhita and one Chyawanprash sample was procured from market. These

batches were evaluated by physicochemical methods and its bio-efficacywas determined byvarious

biological methods like antioxidant study and microbial contamination study and finally stability

study was performed on the formulation according to the ICH guidelines.

The results of two in-house batches were found in close proximation with the marketed

batch. The method used for determination of quality control of Chyawanprash was found to be

precise and reproducible and can be used for quality control of other marketed formulations.

Keywords: Chyawanprash, Phytoconstituents, Physicochemical Parameter, Standardization,

Stabilitystudy.

IPe code; Int. d.7 - A61K 35/78

Introduction

Chyawanprash is very commonly

used health supplement and medicinesince centuries. It is a jam preparation,

which contains 35-40 natural ingredients.

In general, the formulation has a sweet

and tangy taste with a fine aroma. Since

the day of its known inception dating back

1500 BC to the age of technology itremained in the hearts of Indians

irrespective of political, cultural and

scientific upheavals.

The word Chyawanprashcomposed of two words, 'Chyawana'and 'Prasha'. The former stands for the

name of a sage. The word also denotes"degenerative change". The later word

denotes a drug or diet, which is fit for

ingestion. Chyawanprash is otherwiseknown as Balya, which increases the

anabolism I. Apart from this, it is helpful

in kshaya (emaciation). It is also

reputed as an anxiolyticand nervine tonic

in Ayurveda.It is geriatric tonic and is an

adaptogenic progeny and hence prevents

the harmful effectof stress2• In the modern

time it cures cough, asthma and act as an

immunomodulator and memory

enhancer3,4. It promotes growth in

chUdren, increases the sexual power and

digestive fire. The entire Indian

Chyawanprash market stands around 50­60 tonnes5, even though only very few

quality control methods for its evaluation

have been attempted6-8• Due to lack ofsuitable quality control standards of

Ayurvedic drugs it is difficult to ensure

uniformity of their composition and

consequently the efficacy of finalproducts9• Even the reported official

methods for quality assurance of

Chyawanprash do not include completeanalysis parameters10•

Chyawanprash falls, by virtue of

its consistency and dosage form, under

the category ofAwaleha-paka group of

Ayurvedic formulationsll. Generally

Chyawanprash includes four class of

herbal drugs: Dashmula Class (tenroots); Chaturjata Class (four aromatic

plant); substitution of Ashtavarga(Ashtavarga, orchids herbal drug whichare not commercially available in modern

era) and general class (other than former

classes). The main plants used in the

preparation of Chyawanprash are giveninTable 1.

Vol 5(1) January-February 2006 ---'m

Researth Arl;tle

In the present work,

Chyawanprash was prepared strictly

according to the method quoted in the

ancient Aymvedictext 'Charaksamhita'under the supervision of an Ayurvedic

expert. This paper reports the

characteristic features and phytochemical

evaluation of the finished products

samples prepared by Medicinal Natural

Product Research Laboratory, VICT,Mumbai and coded as CU-l and CU-2in

collaboration with Potdar Ayurvedic

Medical College, Mumbai, respectivelyand marketed formulation coded as M-1.

Materials and Methods

Materials

Herbs

Theplants used in Chyawanprashwere collected from local market of

Mumbai and identified by Botany

Department. of Zandu' Pharmaceutical

Limited, Mumbai and voucher specimen

were deposited in MNPR Lab, VICT,

Mesua ferrea

Piper languID

Mumbai. 1\vobatches of the product were

prepared as per the procedure described

in Ayurvedic text 'Charaksamhita'.

They were named as CU-l and CU-2,

respectively and one marketed

formulation of Chyawanprash (Ml) was

procured for the study.

Awala fruits

Solvents and chemicals

Hexane, toluene, ethanol, ethylacetate, acetic acid, formic acid, methanol

of HPLCand as AR grade were used.

Piperine, epicatechin, catechin and

gallic acid, DPPH (Sigma)chemicals were used in

experimental part.

Instruments

Camag HPTLC system

employing Linomate IV,1\vintrough

chamber and Camag TLC scanner III

equipped with CATS,V.4.06 softwareused.

UVspectrum was recorded on Mis Jasco,

.. Kjeldhals apparatus.

Methods

Morphological, physical and

chemical evaluation including qualitative

test and quantitative analysis of

phytoconstituents were done. Biological

evaluation for free radical scavenging

(anti-oxidant) activityand microbiological

test (e.g. Bioburden level) along with

stability study were carried.

Morphological characteristics

Colour, odour, taste, consistency

and texture of the product vary from onemanufacturer to another. Hence, these

parameters are also important for quality

control. The samples prepared during this

study are brownish black, semisolid,

sweet, astringent, granulated, rough in

texture and possess sweet aroma.

Physical and chemical evaluation

For physical and chemical

evaluationl2.14 all three samples (lOg each)

were extracted separately with n-hexane,

methanol and distilled water by cold

Adhatoda vasica

m--- ~Natural Product Radiance

Researlh Artille

Table 1: Plants used in Chyawanprash3, 5, 28

Pueraria tuberosa DC.

Dioscorea bulbifera Linn.

-

Coronion name

Aduso ki patti, Vasa

Agarkashta, Agar, KalaagarVanshalochan

Punainava, Gadhapuran

Kachur, Sathi kchora

Mustak, Nagarmotha, Motha:Amla,Awala

Pushkarmool, pohkarrrlul

Jeevanti

Kakakshi, Kaknasa, KauathodiNeelkamal

Van-mug, Mataki, Mudgaparni

Bhueawala, Bhumyamalaki ,Pippali

Kakad singi, Sringi, Karkatshringi

Safed chandan, Chandansaar

Bala, Bariyara

Mashparni, Van-uqadh, Mashvan

Harad, Haritaki, Abhaya.

Guduchi, AmritaDraksha

Bel, Bilva

Shalparni, Sarivan

Gambhari, Kashmarya

Shyonaka, Sonapatha, Aralu

Arani, Aagnimantha

Brihat Kantkari, Vanbhantha

Laghu kantakari, Kashtakari

Patha, Padhal, Patal

Gokharu, Gokshura

Pithwan, Prishniparni, Devala

Tejpatta, Patra, TamalpatraDalchini

Elaichi

Nagakeshar

Shatawari

(Substitution for Meda, Mahameda)

Barahikand, Varahikand

(Substitution for Rddhi, Vridhi)

Vidarikand ..

(Substitution for Jivak, Risabhaka)

Ashwagandha

(Substitution for Kakoli, Kshirkakoli)

Dashmula Class

General Class

&Wendl.

~oJanicai name'

Withania somnifera Dunal

Chaturjata ClassCinnamomum tamala Nees & Eberm.

Cinnamomum zeylanicum BlumeElettaria cardamomum Maton

Mesuaferrea Linn.

Substitution of Ashtavarga

Adhatoda vasica Nees

Aquilaria agallocha Roxb.

Bambusa arundinacea (Retz.) Roxb:

Boerhaavia dijfusa Linn.Curcuma zedoaria Rose.

Cyperus rotundus Linn.

Ef1Jblica officinalis Gaertn.

(Averagewt # 21g)lnula racemosa Hook f.

Leptadenia reticulata Wight & Am.

Martynia diandra Glox.

Nymphaea stellata Willd.Phaseolus trilobus Ait.

Phyllanthus amarus Schum.

Piper longum Linn.

Pistacia integerrima Stew. ex BrandisSantalum album Linn.

Sida cordifolia Linn.

Teramnus labialis Spreng.Terminalia chebula Retz.

Tinospora cordifolia Meirs.

Vitis vinifera Linn.

Asparagus racemosus Willd.

. Cj-:: .. ' ~. '

'-Aegle '~armelos Corr. ex Roxb.

Desmodium gangeticum DC.

Gmelina arborea Roxb.

Oroxylum indicum Vent.

Premna integrifolia Linn.Solanum indicum Linn.

Solanum xanthocarpum Schrad.

Stereospermum suaveolens DC.

Tribulus terrestris Linn.

Uraria picta Desv.

SNo.

1

d "2 '3A5678'9,10

11

121314151617

18

19202122232425262728293031

32

333435

36373839

Vol 5(1) January-February 2006 m

Resear,h Art;,'e

maceration. The extractive value, angle of

rotation, ester value and free fatty acid

value were determined by method

described in Indian Pharmacopoeia.

Chyawanprash (5g) from each batch was

extracted separately with 100 ml distilled

water by maceration and their pH,refractive index and total solid content

were determined.

(i) Physicochemical andphytochemical evaluation(Qualitative Test)15

Aqueous and methanol extract of

Chyawanprash prepared byhot extraction

were used in the present study.

Physicochemical and phytochemical

screening of all formulations were

done by using reported methods 16-20

(Tables 2-4).

(ii) Quantitative determination ofphytoconstituents 21,22

Chromatographic Analysis:

HPTLCanalysiswas performed on a Camag

HPTLCsystem. The plate used was HPTLC

254 silica gel 60 (E. Merck). Camag

HPTLCsystem equipped with a sample

applicator Linomat IV, Twin trough

developing chamber, Integration software

system, CATSV.4.06, TLCscanner III inabsorbance/reflectance mode and selected

common wavelength 254nm for piperine,

epicatechin, catechin and gallic acid. The

solvent system comprising of toluene:

ethyl acetate: formic acid: ethanol [6: 4:

0.3: 0.4] gave proper resolution for

piperine, epicatechin, catechin and gallic

acid [Table 4, Fig. 1].

Spectrophotometric studies:

Chyawanprash (10g) from all three

batches was extracted separately withdistilled water till exhaustion. The

solution was illtered and diluted to 100ml

with distilled water. All the three batches

were studied spectrophotometrically for

spectrum measurement, colourimetric

analysis like total tannin by folin-denis

method, carbohydrates by anthrone

reagent and total alkaloids by

Dragendorff's reagent. Samplepreparationfor determination of alkaloid was done

by extraction with 10% acetic acid in

methanol till exhaustion, filtered anddiluted to 25 ml with 10% acetic acid in

methanol.

Biological evaluations

DPPH radical scavengingactivity23,24: This assay is based on the

measurement of the scavenging ability of

+

+

+

+

+

+

+

+

Marketed sample

MI

+

____________________________________ -'Natural Product Radiance

Table 2: Phytochemical evaluation Chyawanprash sample

S.No.

Analytical parameter In-bouse preparation

CutAlkaloids

+I+

(Dragendorff's reagent) 2

IAmino acids i+ !+,(Ninhydrin reagent) 3

!Carbohydrates I+ !+

(Anthrone reagent) 4IFixed oils and fats I+ I+

(Gravimetric method) 5

IFlavonoids !+ I+

(Shinoda test) 6

IGlycoside

!+ I+

(Anisaldehyde reagent) 7 IPhenolic (Folin-Denis reagent)

I+

I+

8Phytosterol ++

(Liebermann Burchard reagent) 9

ISaponinsI+ I+

(Hemolytic method) _im

Researlh Arlj,le

Table 3: Physicochemical evaluation of Chyawanprash14-2O

~"

"" _ ,i

S.No.Analytical parameter m-house preparationMarketed sample

cUt

CU2Ml

1

+Angle of Rotation

(0.05% solution)

-10-14 -17

2

Conductivity (mv) (5 % solution) 227202 208

3

+Crude Fat (Hexane Fraction) 0.583 %0.96 %I0.87%

4

+Extractivevalues:Water soluble

68.15 %62.38%64.90%Methanol soluble

80.78%72.26 %69.60%

5

+Ester value hexane fraction 3.2254.3011.722

6+Free fatty acid hexane fraction 0.28050.3740.328

7

+Lignanecontents 4.213%6.421%6.75 %w/w

8

+L.O.DE\.

8.98 %11.22 %15.5 %

9

I pH (5% solution) 3.0~3.503.38

10

I Refractive Index at 29°C, 68%

1.3338

. 1.3391.339

humidity (5% solution)11

+Saponification value 3.5062I4.675 I2.050(Hexane fraction)

12

+Silicacontents 0.133 %0.198 %0.508 %

13

Spectrum measurement 272,731 nm276,731 nm272, 731 nm

14

+TotalAcidity 0.030 %0.028%0.050%

(% in term of anhydrous citric acid)15 , +TotalAsh

12.19 %17.88 %16.18%

16

+rotal Calories (kcallg) 1.9722.6751.002

17

I +TotalCarbohydrate 50.66%68.438%63.63%

18

I +Reduced sugar-(Anthrone reagent) 85.41%86.109%92.68%

19 1 +Totalfibres

2.154 %3.27%3.377%w/w

20

• Total Nitrogen (N glkg) 0.7730.43650.088

(Kjeldhals method) 21

+TotalProtein (glkg) 4.832.728

I0.55

22

I Total Solid (Brix) (5% solution) 4%3.5%4.2%

23

I +rotal Tannin (Folin-Denis reagent)1.804 %1.465 %

I2.89 %

24I Wt/ml (1 % solution)

0.99690.99941.005

+Valuesare mean of three experiments Vol 5(1) January-February 2006

ED

Research Article

Table 4: Quantitative analysis of phytoconstituents from Chyawanprash by HPTLCmethod"

Quantitative analysis of phytoconstituents (%w/w)Chyawanprash Coded

Gallic acidPiperineEpicatechinCatechin

""-"~T

-1---j- -

CUI0.9320.102I

0.225

I0.025

I

I

CU20.7080.053

!0.1998 0.019I IM1

1.5740.073I

0.331 0.022I ."Values are mean of three experiments

Table 5: Test of DPPH radical scavenging activity of Chyawanprash"

Contents r*EC50 pglml

f

~~ v, '",:_!CatechinI0.9875I3.2

II

Tannic Acid I0.9869I

7.48

Awala ethanol extract

I0.9924

I

71.6

Dashmula ethanol extractI

0.9888513.3

Chyawanprish ethanol extract

0.9937,114.7

700

HXXl

Piperine

Gallic acid

antioxidants towards the stable radical 2, "

2-diphenyl-1-picryl-hydrazyl[DPPH].Thefree radical DPPH is reduced to the

corresponding hydrazine when it reacts

with hydrogen donors. DPPH scavenging

activity was measured by

spectrophotometeric method. To an

ethanol solution of DPPH (lOOllM,

2.5ml), 0.5ml oftest compound dissolved r* Correlation coefficient

in ethanol was added at different 'Values are mean of three experiments,

concentration (500-800011Wml). Equalamount of ethanol was added to the

control. Absorbance was recorded at 517

nm at regular intervals of 30 sec up to 5minutes (Table 5).

The purpose of stability testing

--.-------,.-----,--.--'-+'--I~--------;0.47 0.67 0.87

is to provide,evidence on how the qu3)ityof drug substance or drug product varies

with time under the influence of varietyof environmental factors such as

temperature, humidity and light and

enables to recommend storage condition

and to predict the shelf life. Stabilitystudyfor Chyawanprash was performed ataccelerated condition Le. 40°C± 2°CJ.75%

Fig. 1: Chromatogram of catechin, piperine,epicatechin and gallic acid by HPTLC method

-~ --~ .. _ .. --------_.~~-~-, ,0.07 0.27

0-'-

200

100 ,,---

500

400

S~ability study

liquefied casein-soybean digest agar.

The samples were incubated at

30-35°C for 5 days and the numbers ofcolonies formed were counted after

5 days.

Bioburden leve[25:It is based

on determination of microbial

contamination limits in medicinal plantmaterials. Different limits are set

according to the use of the material. A

large number of bacteria and fungi forms

the naturallyoccurring microflora ofherbsand aerobic spore-forming bacteria.

Bioburden level may indicate the qualityof an herbal formulation. The total viable

aerobic count of the material being

examined is determined by using plate

count method. Sample after treatment

with sodium chloride-peptone buffersolution (pH 7.0) was inoculated on

Microbiological test

m~ ~Natural Product Radiance

Researlh Artille

After Aftllr After After After10 30 60 180 240

days dllY. days days days

DaY'

Accelerated tilting for Chyawanpraeh

70.00%

f :~:~~:4MO%

30.00%

2MO%

110.00%""' 0.00%

4) At 366 nrn (After Spraying _reagent)

CB Chyawanprash -B Chloroform fraction

CC Chyawanprash -C Chloroform fractionDASH Dashmula churna methanol fraction

HA Chyawanprash -A Hexane fraction

2) At 254 nrn

Aftor AftorMlirAfterAfter10

3060180240day,

dllysday.dllYIdllyl

DOYI

Allcolllratlld tIlltlng forChYllwllfIpralh

Fig.2: Stabilitystudyfor Chyawanprashon storage conditionsin climaticZonesIII and N (Acceleratedtest-40°C ±2°C/ 75%RH± 5%RH)

Fig. 3: Fingerprint of Chyawanprash with different

mobile phase by HPTLC

.•.•.,..•,,,

-

3) At Visible (Spraying reagent- ;

Anisaldehyde reagent)

B) Mobile Phase-Ethyl acetate: Acetic acid: Methanol (8:2:3)

1) At 366 nrn

A) Mobile phase - Toluene: Ethyl acetate (7:1)

CHA Chaturjata class methanol fraction

HB Chyawanprash -B Hexane fraction

HC Chyawanprash -C Hexane fraction

CA Chyawanprash -A Chloroform fraction

RH ± 5% RH (recommended for climatic

zone III and IV)(Fig. 2).

Results and Discussion

Chyawanprash samples (CUI,

CU2) were prepared in the laboratoryaccording to the standard text.

Comparative evaluation was done usingvarious parameters, viz. extractive value,

spectrum measurement, colourimetric,

titremetric analysis and fingerprinting by

HPTLC.The data analysis revealed that

physico-chemical parameters were in

close proximity for both in-house batches(CUI and CU2) and marketed

Chyawanprash (M!). Thus all these

parameters listed in Table 2 and Table 4

together can be used successfully for

analysis of Chyawanprash and will serve

as a basis for fixing standardization

parameters. For determining purity and

potency of the formulations, proceduresmentioned here could be followed in QC/

QAlaboratory. Stabilitystudies indicated

the best use of Chyawanprash should be

within one year after manufacturing(Fig. 1).

Chyawanprash contains many

phytoconstituents like phenols (gallic

acid, catechin, epicatechin) and alkaloids

(piperine). Out of which phenolics are

considered to be responsible for its

antioxidant activity(Table 5) and piperine

is responsible for bioavailabilityenhancer.

Thus considering the importance of theseingredients attempt has been made todetermine the content of these constituents

in the Chyawanprash (Table 4 and Fig. 1).

The fingerprint of Chyawanprash was

developed with different mobile phases

(Fig. 3).

Vol :j(1) Januqry-February 2006 ~m

Re.ear,h Art;,'e

Table 6: Detennination of total viable aerobic count in

Chyawanprash fonnulation

Bioburden level indicates the

qualityof Chyawanprashformulation. Theformulation shows lower microbial

contamination limits than WHOguidelines

(Table6). DPPHradical scavengingeffects

of Chyawanprash ethanol extracts areshown in Table 5. All the studies showed

appreciable free radical scavenging

activity.However, Chyawanprash showed

the comparable activityas that of Awalaethanol extract. Thus; the formulation will

be a rational combination in the

preparation of Chyawanprashto blend the

Ayurvediccriteria, which is being used for

centuries along with the above mentioned

parameters to get a noble product.

Conclusion

. MicrobesUsed

Number of

Bacterial colonies

per gram of sample

(Aerobic bacteria)

Number of Fungal

colonies per gram of

sample

Microbialcontamination limits

for herbal

fonnulation, as

per WHOguidelines

Aerobic bacteria,

maximum I05/g

Mould Propagules,

maximum I03/g

Microbialcontaminationin

Chyawanprash'

7500 colonies/g

850 colonieslg

Observation

as per WHOguidelines

Lower than

Microbial

contamination

limits for

internal herb

uses

Lower than

Microbial

contamination

limits for

internal herb uses

"Marketed Chyawanprash are used for Bioburden study.

Note - Aerobic bacteria used: Enterobacteria (G-), Staphylococcus aureus(G+), Streptococcus (G+)

and fungal mould propagules used: Trichoderma viride

Katiyar CK, Brindavanam MB and Tiwari P,

Immunomodulatory Products from

Ayurveda, SN Upadhyay (Ed), Narosa

Publishing House, New Delhi, India, 1997,

pp.163-187.

3. Sharma RK (Translator), Charaka

Samhita, Bhagwandas Chowkhamba

Sanskrit Series Office,Varanasi, 1988,

Chapl:l, para/verse no.62-74, 20.

2. Verma M and Singh RH, Physiological,

Endocrine and Metabolic studies on the

Chyawanprash, J Res Indian Med, 1973,

8(2), 1-10.

1. Ojha JK, Bajpai HS, Sharma PV, Khanna MN,

Shukla PK and Sharma TS, Chyawanprash as

an anabolic agent, J Res Indian Med, 1973,

8(2),11-14.

References

Authors are thankful to Dr. J M

Pathak and Dr. V R Naik, Zandu

Pharmaceutical Limited, Mumbai for

the identification of plants and helpfuldiscussion. Authors also thank to

Dr. K R Kohli (Former Dean of Poddar 4.

AyurvedicMedical College, Mumbai) for

helping in the preparation of

Chyawanprash formulation.

Acknowledgement

small step towards the development of

quality control methods for a polyherbal

preparation. This will also help to

produce uniform standard products,which will restore faith in Ayurvedic

system.

The present work was carried out

for the preparation and standardization

of Chyawanprash. The results obtainedwere equally comparable with that of

marketed formulations analysed by

gravimetric, titremetric and HPTLC-UVmethod. The results indicate some

qualitative and quantitative difference indifferent batches studied. These methods

were also employedto analyzecommercial

samples to illustrate their application in

qualitative (fingerprint) and quantitative

determination, demonstrating their

feasibility in the quality control of

Chyawanprash. Hence, these parameters

and the developed methods may beconsidered as a tool for assistance to the

regulatory authorities, scientific

organizations and manufacturers in

developing standards.So far since no attempt has been

made to evaluatea polyherbal formulation

by calculating cumulative indices of

their ingredients, the present work is a

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