development of quality control methods for polyherbal
TRANSCRIPT
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: [email protected]; [email protected]
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
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Chyawanprash. Hence, these parameters
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regulatory authorities, scientific
organizations and manufacturers in
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made to evaluatea polyherbal formulation
by calculating cumulative indices of
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