Standardization of Ayurvedic
polyherbal formulation, Kutajastaka
Pravahi Kvatha
Ashok Kumar Tiwari*, Neelesh Dwivedi, Manoj Kumar Tripathi, Aakanksha Tiwari,
Pawan Kumar Ahirwar, Sharda Prasad Tripathi
Ayurveda Sadan, Arogyadham, JRD Tata Foundation for Research in Ayurveda
and Yoga Sciences, Deendayal Research Institute, Chitrakoot, Satna, Madhya
Pradesh, India-485334
*Corresponding Author E-mail: ashokckt77@yahoo.com,
gangagargi@gmail.com
ABSTRACT:
Ayurvedic medicine Kutajastaka
Pravahi Kvatha, known to be
effective in Daha (burning sensation); Raktatisara (Diarrhoea with
blood); Sula (pain); Amadosa;
(products of impaired digestion and metabolism) and Sarvatisara
(all types of diarrhoea), has been standardized by
following modern scientific quality control procedures for the finished
product. The results of physico-chemical parameters
viz. specific gravity (1.01),
total solids (4.45%) and pH
(4.50) were found. Microbiological
limit test and heavy metals Pb, Cd, As, Hg were
also found within the limits set by Ayurvedic Pharmacopiea of India (API). The obtained values can
be adapted to lay down new pharmacopoeial standards
with batch to batch consistency. The phytochemical
constituents found in the raw material used for the preparation of Kutajastaka Pravahi Kvatha facilitate the desirable therapeutic efficacy of the
medicinal formulations a whole in elements and also could help in understanding
the underlying mechanism of pharmacological action.
KEYWORDS: Standardization,
Ayurvedic formulation, Kutajastaka
Pravahi Kvatha, Pharmacopoeial standards.
INTRODUCTION:
Kutajastaka Pravahi Kvatha (KPK),
an Ayurvedic polyhearbal formulation,
consists of Kutaja (Holarrhena
antidyssentrica Wall.) and others 8 ingredients
in Kvatha Curna
form. It supposedly has multi-faceted action in Daha
(burning sensation); Raktatisara (Diarrhoea
with blood); Sula (pain); Amadosa;
(products of impaired digestion and metabolism) and Sarvatisara
(all types of diarrhoea) (Anonymous, 2000).
Preparation of KPC is based on traditional methods in accordance with the
procedures given (Anonymous, 2011; Sastri, 2007).
Due to lack of modern pharmacopoeial
standards laid down and followed for processing of KPC using traditional
methods, the medicine prepared may not have the desire quality and batch to
batch consistency. Hence, there is a need for standardization of KPK following
scientific parameters including organoleptic
characters, physico-chemical analysis,
chromatographic pattern, heavy metals and microbial screening. The work was
undertaken to standardize and validate Ayurvedic
medicine, Kutajastaka
Pravahi Kvatha used in
treatment of varies diseases. Standardization of KPK was carried out following
Good Manufacturing Practices (GMP) for preparation of Ayurvedic
medicine. Standardization guidelines for herbal product provided by Word Health
Organization (Anonymous, 1998) and European Agency for the Evaluation of
Medicinal Products (EMEA) have also been followed.
Kutajastaka Pravahi Kvatha (KPK)
an Ayurvedic compound formulation, formulated
with 9 ingredients viz. Kutaja (Holarrhena antidyssenterica
Wall.-stem barck), Ativisa
(Aconitum heterophyllum Wall. - root), Musta (Cyperus rotundus Linn. - rhizome), Balaka
(Coleus vettiveroides K.C. Jacos.
- root), Lodhra (Symplocos
racemosa Roxb. - stem
bark), Raktacandana (Pterocarpus
santalinus Linn. f. - heart wood), Dhataki (Woodfordia fruticosa Kurz. - flower), Dadima (Punica granatum Linn. - dried fruit) and Patha
(Cissampelos pareira
Linn. - root) prepared as per AFI (Table 1).
Table 1: Ingredients of Kutajastaka
Pravahi Kvatha
|
Sanskrit /Hindi name |
Botanical name |
Parts used |
Portion |
|
Kutaja |
Holarrhena antidyssentrica
Wall. |
Stem bark |
1 part |
|
Ativisã |
Aconitum heterophyllum Wall. |
Root |
1 part |
|
Musta (Mustã ) |
Cyperus rotundus
Linn. |
Rhizome |
1 part |
|
Bãlaka (hrîvera) |
Coleus vettiveroides K.C. Jacos. |
Root |
1 part |
|
Lodhra |
Symplocos racemosa Roxb. |
Stem bark |
1 part |
|
Candana (Raktacandana) |
Pterocarpus santalinus
Linn. f. |
Heart Wood |
1 part |
|
Dhãtakî |
Woodfordia fruticosa Kurz. |
Flower |
1 part |
|
Dãdima |
Punica granatum
Linn. |
Dried Fruit |
1 part |
|
Pãthã |
Cissampelos pareira
Linn. |
Root |
1 part |
MATERIALS AND METHODS:
Collection and authentication
of raw materials
Kutaja (Holarrhena
antidyssenterica Wall.-stem barck),
Ativisa (Aconitum heterophyllum
Wall. - root), Musta (Cyperus
rotundus Linn. - rhizome), Balaka
(Coleus vettiveroides K.C. Jacos.
- root), Lodhra (Symplocos
racemosa Roxb. - stem
bark), Raktacandana (Pterocarpus
santalinus Linn. f. - heart wood), Dhataki (Woodfordia fruticosa Kurz. - flower), Dadima (Punica granatum Linn. - dried fruit) and Patha
(Cissampelos pareira
Linn. - root), were collected during year 2012 from Chitrakoot
forest range. Whereas Kirãta (Swertia chirata Buch Ham.-whole plant was collected from herbal
garden, Chitrakoot, Arogyadham
in 2012. All the materials were
authenticated with the help of taxonomist Dr. RLS Sikarwar
at Deendayal Research Institute, Chitrakoot,
Satna (MP).
Preparation of the Kutajastaka Pravahi
Kvatha
All the ingredients used were of pharmacopoeial quality (Anonymous, 1998; Anonymous, 1990).
Cleaned, washed, dried and grind
the Kutaja, Ativisa, Musta, Balaka, Lodhra, Raktacandana, Dhataki, Dadima and Patha individually, crushed all the ingredients
into pieces of 1-3 cm. Weighed separately and mixed them in equal proportions
(1:1:1:1:1:1:1:1:1) to ensure a homogenous mixture. Boiled the crush of all the
ingredients together in 16 times water and reduce to 1/8th, filtered
through muslin cloth and heated further to concentrate it to1/4th,
added approved preservatives, these were stored in an airtight containers to
protect from light and moisture. Two samples were prepared at research
laboratory Ayurveda Sadan, Chitrakoot Batch-A and Batch-B where Batch-C was prepared
by Chitrakoot Rasshala
Pharmacy, Chitrakoot.
Physico-chemical tests
Organoleptic characters, specific gravity and physico-chemical
analysis of all the samples were carried out. Quantitative analysis for total
solids, pH of filtrate of 10% w/v aqueous solution were checked in
triplicate according to the prescribed Standard methods in Indian Pharmacopoeia
(Lohar and Singh, 2008; Anonymous, 2008 and
Anonymous, 1996).
TLC profile
For HPTLC, 1 ml Pravahi
Kvatha with 10 ml methanol, filtered each of the extracts
and combined, add 5 gm of anhydrous sodium sulphate,
filtrate and concentrated. TLC of extracts of all the samples was carried out
on Silica Gel 60 F254 precoated plates
(0.2 mm thickness; from Merck India Limited Mumbai). A TLC applicator from Camag Linomat-5 (Camag
Switzerland 140443) was used for band application and photo documentation unit
(Camag Reprostar-3: 140604) was used for
documentation of chromatographic fingerprints. The mobile phase used was
Toluene: Ethyl acetate: Methanol: Water (5: 3.5: 1: 0.5). The plate was
developed over a distance of 9 cm in a saturated development chamber (Twin
trough chamber (10 x 10 cm with SS lid, and visualized under visible light,
254nm and 366nm. After spraying with Anisaldehyde-sulphuric
acid followed by heating at 110 0C for 5-10 min (Wagner and Bladt, 2004 and Wall, 2005).
Test for microbial limits
The microbiological screening was done for the
finished product using the procedure after Pelczar et
al., 1993 and Anonymous, 2010.
Heavy metal
Heavy metal analysis (lead, cadmium,
arsenic and mercury) were carried out using Atomic absorption spectrophotometry (Shimadzu-Model-AA-7000). All samples are
digested with concentrated HNO3: HClO4 (4:1). Standards
solutions are made for different dilution to get linear calibration (Merck). Pb and Cd were performed using
graphite oven method, while As (Arsenic) were determined as hydride method and
Hg were determined using cold absorption method (Anonymous, 2010 and Anonymous,
2010a).
RESULTS AND DISCUSSION:
The three batches of the homogenized samples were
observed as Pravahi Kvatha,
light brown in colour; with the presence of an odour and tasting slightly bitter. Physicochemical data was
obtained after subjecting the samples to various analytical parameters. The average
specific gravity was 1.01. The total solid was 5.63%, while the pH value
recorded was 5.50. All of the physicochemical properties recorded during this
study are as shown in Table 2.
Table 2: Physico-chemical
parameter of Kutajastaka Pravahi
Kvatha
|
Name of Pravahi
Kvatha |
Specific gravity |
Total solids (%) w/w |
pH |
|
Kutajastaka Pravahi Kvatha (Batch A) |
1.02 |
4.49 |
5.57 |
|
Kutajastaka Pravahi Kvatha (Batch B) |
1.01 |
4.43 |
5.63 |
|
Kutajastaka Pravahi Kvatha (Batch C) |
1.02 |
4.45 |
5.71 |
|
Average value |
1.01 |
4.45 |
5.50 |
HPTLC fingerprint profile of the formulations are
depicted in (Figs. 1-4) indicates the presence of all the ingredients in
proportional quantity in the formulations. This confirms the batch- to- batch
consistency of the finished products. Development of fingerprint profile would
serve as a reference standard of the formulation. The TLC plate was examined
under 254nm, 366nm, after derivatization 366nm and
visible light. The Rf values and colours of the bands obtained were recorded (Table 3-6).
|
T1 T2 T3 |
T1 T2 T3 |
|
Fig.1.TLC profile of Kutajastaka
Pravahi Kvatha under 254 nm (before derivatization)
|
Fig.2.TLC profile of Kutajastaka Pravahi Kvatha under 366
nm (before derivatization) |
Track 1: Batch A, Track 2: Batch B, Track 3: Batch
C.
Track 1: Batch A, Track 2: Batch B, Track 3: Batch
C.
Table 3: Rf
values in test solution of Kutajastaka Pravahi Kvatha at 254 nm (before derivatization)
|
Rf values |
Kutajastaka Pravahi Kvatha |
||
|
Batch A |
Batch B |
Batch C |
|
|
Rf 1 (black) |
0.09 |
0.09 |
0.09 |
|
Rf 2(black) |
0.12 |
0.12 |
0.12 |
|
Rf 3(blak) |
0.16 |
0.16 |
0.16 |
|
Rf 4 (black) |
0.21 |
0.21 |
0.21 |
|
Rf 5 (black) |
0.31 |
0.31 |
0.31 |
|
Rf 6 (black) |
0.34 |
0.34 |
0.34 |
|
Rf 7 (black) |
0.43 |
0.43 |
0.43 |
|
Rf 8 (black) |
0.53 |
0.53 |
0.53 |
|
Rf 9 (black) |
0.62 |
0.62 |
0.62 |
Table 4: Rf
values in test solution of Kutajastaka Pravahi Kvatha at 366 nm (before derivatization)
|
Rf values |
Kutajastaka Pravahi Kvatha |
||
|
Batch A |
Batch B |
Batch C |
|
|
Rf 1 (brown |
0.08 |
0.08 |
0.08 |
|
Rf 2(brown) |
0.15 |
0.15 |
0.15 |
|
Rf 3(brown |
0.23 |
0.23 |
0.23 |
|
Rf 4 (blue |
0.31 |
0.31 |
0.31 |
|
Rf 5 (blue) |
0.52 |
0.52 |
0.52 |
|
Rf 6 (blue) |
0.58 |
0.58 |
0.58 |
|
Rf 7 (blue) |
0.68 |
0.68 |
0.68 |
|
Rf 8 (blue) |
0.75 |
0.75 |
0.75 |
|
Rf 9 (blue) |
0.81 |
0.81 |
0.81 |
|
Rf 10(blue) |
0.91 |
0.91 |
0.91 |
|
Rf 11(blue) |
0.95 |
0.95 |
0.95 |
Table 5: Rf
values in test solution of Kutajastaka Pravahi Kvatha at 366 nm (after derivatization)
|
Rf values |
Kutajastaka Pravahi Kvatha |
||
|
Batch A |
Batch B |
Batch C |
|
|
R 1(sky blue) |
0.08 |
0.08 |
0.08 |
|
Rf 2(sky blue) |
0.16 |
0.16 |
0.16 |
|
Rf 3 (sky blue) |
0.22 |
0.22 |
0.22 |
|
Rf 4 (red ) |
0.25 |
0.25 |
0.25 |
|
Rf 5 (purple) |
0.30 |
0.30 |
0.30 |
|
Rf 6 (light) |
0.35 |
0.35 |
0.35 |
|
Rf 7 (yellow) |
0.39 |
0.39 |
0.39 |
|
Rf 8 (light brown ) |
0.47 |
0.47 |
0.47 |
|
Rf 9(pink ) |
0.52 |
0.52 |
0.52 |
|
Rf 10(brown) |
0.72 |
0.72 |
0.72 |
|
Rf 11(sky blue) |
0.77 |
0.77 |
0.77 |
|
Rf 12(light brown) |
0.82 |
0.82 |
0.82 |
|
Rf 13(light orange) |
0.92 |
0.92 |
0.92 |
Table 6: Rf
values in test solution of Kutajastaka Pravahi Kvatha at visible light
(after derivatization)
|
Rf values |
Kutajastaka Pravahi Kvatha |
||
|
Batch A |
Batch B |
Batch C |
|
|
Rf 1 (brown) |
0.25 |
0.25 |
0.25 |
|
Rf 2 (light brown) |
0.36 |
0.36 |
0.36 |
|
Rf 3 (blue) |
0.53 |
0.53 |
0.53 |
|
Rf 4 (light brown) |
0.72 |
0.72 |
0.72 |
|
Rf 5 (light black) |
0.83 |
0.83 |
0.83 |
|
Rf 6 (blue) |
0.90 |
0.90 |
0.90 |
The microbial profile of the Kutajastaka
Pravahi Kvatha was found
satisfactory. Total bacterial count (18-20 cfu/gm),
Yeast and Moulds counts (25-30 cfu/gm) were reported
less than the limit set by API (API limit total bacterial count -105
cfu/gm and yeast and mould -103 cfu/gm). Pathogenic bacteria, i.e., Salmonella, Pesudomonas, Staphyllococcus
and E.coli were not detected in samples
(Figure 5a-f).
Figure 5. Microbiological limit test in compound
formulation of Kutajastaka Pravahi
Kvatha
Heavy metals if present in the drug may
carcinogenic and toxic. In the present study the level of heavy metals viz. Pb, Cd, As, Hg are within limit
set by WHO (Table 7). These results thus the revealed that the formulation was
safe for consumption.
Table1 7: Determination of heavy metals in Kutajastaka Pravahi Kvatha
|
Parameter |
Kutajastaka Pravahi Kvatha |
Actual Conc. unit |
API Limites |
||
|
Batch
A |
Batch
B |
Batch
C |
|||
|
Lead
(Pb) |
0.2111 |
0.2354 |
0.2342 |
ppm |
10 ppm |
|
Cadmium
(Cd) |
ND |
ND |
ND |
ppm |
0.3 ppm |
|
Arsenic
(As) |
0.0204 |
0.0202 |
0.0204 |
ppm |
03 ppm |
|
Mercury
(Hg) |
0.0172 |
0.0176 |
0.0178 |
ppm |
01 ppm |
Note: ND-Not detected
CONCLUSION:
Ayurvedic medicine, Kutajastaka Pravahi
Kvatha has been standardized intervention with modern
scientific quality control measures in the traditional preparation described in
Yogaratnakara. The results obtained could be
used to lay down a set of new pharmacopoeial
standards for the preparation of Kutajastaka Pravahi Kvatha, to obtain optimal
efficacy of the medicine.
ACKNOWLEDGEMENT:
Authors are grateful to Sri Abhay
Mahajan, Organizing Secretary, Deendayal
Research Institute, Chitrakoot for providing the
research facilities. Funding authors are also thankful to Department of AYUSH,
Ministry of Health and Family Welfare, Government of India, for financial
support under the scheme “Centre of Excellence”.
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Received on 26.04.2016 Modified on 30.05.2016
Accepted on 28.06.2016
©A&V Publications All right reserved
Res. J. Pharmacognosy and Phytochem.
2016; 8(3): 103-108.
DOI: 10.5958/0975-4385.2016.00019.4