STANDARDIZATION OF AN AYURVEDIC FORMULATION - TRIKATU CHURNA USING BIOANALYTICAL TOOLS

Abstract

Trikatu Churna (TC) is an ancient traditional Ayurvedic preparation prescribed for a wide range of disorders. Though TC is an age old formulation, there are very few references on its quality control and standardization. In this work, an attempt has been made to standardize TC by qualitatively evaluating the preliminary phytochemicals. Piperine content of TC was determined using HPTLC. Evaluation of safety potential of TC samples and stability evaluation by comparative study of the in house TC formulation with marketed TC formulations with respect to their piperine content is a value addition to the current work.

Full text

Shailajan Sunita et al / IJRAP 2011, 2 (6) 1676-1678
International Journal of Research in Ayurveda & Pharmacy
ISSN 2229-3566
Research Article www.ijrap.net
STANDARDIZATION OF AN AYURVEDIC FORMULATION: TRIKATU CHURNA USING
BIOANALYTICAL TOOLS
Shailajan Sunita*, Sayed Neelam, Joshi Harshvardhan, Tiwari Bhavesh
F-13, Herbal Research Lab, Ramnarain Ruia College, Matunga (E), Mumabai – 400 019 India
Received on: 01/10/11 Revised on: 06/11/11 Accepted on: 15/11/11
*Corresponding author
Dr. Sunita Shailajan, Associate Professor in Botany, Ramnarain Ruia College, Matunga (E), Mumbai – 400 019 India
Email: sunitashailajan@gmail.com
ABSTRACT
Trikatu Churna (TC) is an ancient traditional Ayurvedic preparation prescribed for a wide range of disorders. Though TC is an age old formulation, there are very few
references on its quality control and standardization. In this work, an attempt has been made to standardize TC by qualitatively evaluating the preliminary phytochemicals.
Piperine content of TC was determined using HPTLC. Evaluation of safety potential of TC samples and stability evaluation by comparative study of the in house TC
formulation with marketed TC formulations with respect to their piperine content is a value addition to the current work.
KEY WORDS: Trikatu Churna, standardization, piperine, HPTLC evaluation, safety evaluation
INTRODUCTION
Trikatu Churna (TC) is an Ayurvedic polyherbal formulation of
Maricha (Piper nigrum L., fruit), Pippali (Piper longum L., fruit)
and Sunthi (Zingiber officinale Rosc, rhizome)1. Since ancient times,
it is being prescribed for Agnimandya (digestive impairment), Gala
roga (throat diseases), Svasa (asthma), Kushtha (skin diseases),
Pinasa (sinusitis), Kasa (cough) and Slipada (filariasis).
Modern bioanalytical techniques have been routinely used for
assessing the quality of the raw materials and traditional
formulations and there are reports on the standardization of some of
the herbal preparations widely used in Ayurveda2-5. Though TC is an
age old formulation, there exist meager references on its quality
control and standardization. Hence, there is a need to standardize TC
using the same.
TC (in house) was prepared in the Herbal Research Laboratory,
Mumbai using standardized raw materials as per the classical
reference1. Traditional formula composition of TC is given in Table
1. Preliminary phytochemicals of TC were evaluated as per standard
methods6.
Piperine (Figure 1), a major alkaloid of Maricha and Pippali
(ingredients of TC) is reported as a bioavailability enhancer, anti-
inflammatory, anticonvulsant and antiulcer agent7-9. There are
reports on extraction of piperine using various extraction techniques
and its estimation using analytical tools from single herbs and
polyherbal formulations2,5,10,11. But, there are no methods reported
for its estimation from the complex matrix of TC. Hence, the
piperine content of TC and its ingredients was estimated using
HPTLC.
HPTLC method was applied to evaluate the impact of storage
periods on the piperine content of stability samples and also to
comparatively evaluate marketed samples of TC with the in house
TC formulation. Acute toxicity studies were conducted to check the
safety potential of TC in mice.
MATERIALS AND METHODS
Materials
Raw materials used for the preparation of TC and three different
marketed brands of TC (M-01, M-02, M-03) were procured from
Bharat Aushadhi Bhandar, Pydhonie, Mumbai and authenticated by
Herbal Research Lab, Ramnarain Ruia College. They were dried in
the incubator at 45º C for a week, powdered and sieved through 85-
mesh (BSS) sieve followed by their storage in air tight containers at
room temperature.
Standard and reagents
The organic solvents and chemicals of analytical grade were
procured from Merck Specialties Private Limited, Mumbai. Standard
piperine (≥ 99 % purity) was procured from Sigma Aldrich
Chemical Company, Germany.
Proximate analysis of raw materials
The quality of raw materials used in the preparation of TC was
assessed by determining the proximate parameters like foreign
matter, ash value (total ash, acid insoluble ash and water soluble ash)
and loss on drying using the standard pharmacopoeial methods12.
In house preparation of TC
TC was prepared by mixing the fine powders of Maricha, Pippali
and Sunthi in equal proportions as per the classical reference1 and
stored in air tight container at room temperature.
Preliminary phytochemical evaluation
The qualitative phytochemical tests were carried out to evaluate the
presence of major phytoconstituents in TC6.
Preparation of standard solution of piperine
Stock solution of piperine (1000.0 μg/mL) was prepared by
dissolving 10.0 mg of accurately weighed standard in small amount
of methanol and the volume was made up to 10.0 mL in standard
volumetric flask. Aliquots of 20.0-80.0 μg/mL were prepared from
this stock solution for calibration curve. Quality control samples
[LQC: MQC: HQC (25.0, 40.0 and 65.0 μg/mL)] were prepared for
precision, accuracy and ruggedness studies.
Extraction of piperine from TC, its ingredients and marketed
samples
Extraction of marker components from TC was a daunting task
because of its complex polyherbal matrix. Hence, the extraction
conditions were optimized to achieve good fingerprinting and to
resolve piperine efficiently. Different solvents and varied solvent to
sample ratios were tested and finally TC and its ingredients (each
1.0 g) were extracted with 10.0 mL of methanol, vortexed for 1-2
min and kept standing overnight at room temperature. The mixture
was filtered through Whatmann filter paper No. 41 (E. Merck,
Mumbai, India) and the filtrate was used for further analysis. Similar
extraction procedure was followed for marketed samples of TC.
HPTLC conditions
Chromatographic separation was achieved on HPTLC plates
precoated with silica gel 60 F254 (E. Merck) of 0.2 mm thickness
with aluminum sheet support. Samples were spotted using CAMAG
Linomat IV Automatic Sample Spotter (Camag Muttenz,
Switzerland) equipped with syringe (Hamilton, 100 μL). Plates were

Shailajan Sunita et al / IJRAP 2011, 2 (6) 1676-1678
International Journal of Research in Ayurveda & Pharmacy
developed in a glass twin trough chamber (CAMAG) pre-saturated
with mobile phase. Scanning device used was CAMAG TLC
Scanner II equipped with CATS 3 software. The experimental
condition was maintained at 20 ± 2oC. Detection of piperine was
possible after derivatizing the plates with anisaldehyde sulphuric
acid reagent and photo documentation with CAMAG Reprostar 3 at
550 nm. ICH guidelines were followed for the validation of the
developed HPTLC method13.
Solvent system
Solvent system consisting of toluene-ethyl acetate-glacial acetic acid
(8: 2: 0.1, v/v/v) was used to resolve and quantify piperine from the
matrix of TC, its ingredients and marketed samples.
Estimation of piperine from TC
Samples [10.0 μL, filtrates (obtained as per section “extraction of
piperine from TC, its ingredients and marketed samples)] were
applied in triplicate to a pre-coated silica gel 60 F254 HPTLC plate
(E. Merck) with the Camag Linomat IV sample spotter. The plate
was developed and analysed as per the optimized chromatographic
condition (as per section “HPTLC conditions”).
Method application
The developed HPTLC method was applied to study the effect of
storage on the stability of TC samples stored at different time
periods (at room temperature) in terms of piperine content. Three
different marketed samples of TC (M-01, M-02 and M-03) were
individually compared with the in-house formulation with respect to
their piperine content.
Safety evaluation
As a safety parameter, acute oral toxicity of TC was conducted on
Albino Swiss female mice weighing 18-22 g. Animals were
purchased from Haffkine Institute, Mumbai and after seven days of
acclimatization, animals were divided into two groups containing
three mice per group for the evaluation of toxicity. First group (test)
received aqueous slurry of TC orally at the dose of 2.0 g/kg body
weight of the animal while the second group (control) received 2.0
mL of distilled water. Study was conducted as per the methodology
laid down in the OECD guideline 425 viz., fixed dose procedure
(evident toxicity). Toxicity was evaluated in terms of mortality,
daily food, water intake, body weight and general behavioral
changes14.
RESULTS AND DISCUSSION
Standardization is an essential factor for ASU preparations in order
to assess their quality based on the concentration of chemical or
bioactive marker3. Modern bioanalytical techniques like HPTLC and
HPLC are being used to achieve the aforesaid objectives. In the
current work, an attempt has been made to standardize TC using
HPTLC.
The results of proximate analysis TC and its ingredients for the
parameters like ash values (total ash, acid insoluble ash and water
soluble ash), loss on drying and foreign matter are represented in
(Table 2). It was observed that the values obtained were in
compliance with the limits documented in the Pharmacopoeia15-17. In
house preparation of TC was carried out as per the classical
reference (Table 1).
The preliminary phytochemical evaluation of TC revealed the
presence of alkaloids, tannins and essential oils, flavonoids,
glycosides and resins as major phytochemicals. Piperine content of
TC and its ingredients (Maricha and Pippali) was determined using
HPTLC technique.
The HPTLC method for estimation of piperine was validated in
terms of specificity, precision, sensitivity, recovery and ruggedness
as per ICH guidelines. Response for piperine was found to be linear
in the range of 20-80 µg/mL (r2 = 0.991) which resulted as a
regression equation y = 11.838 x -98.064. This equation was used to
determine piperine content of TC, its ingredients and marketed
samples. Developed HPTLC method was found to be precise with %
RSD < 2 % for intra-day and inter-day precision. LOD and LOQ
value for piperine was found to be 2.0 and 6.0 μg/mL respectively.
Average recovery at three different levels of piperine for formulation
was found to be 99.16 %. Method was found rugged for the
parameters like change in analyst, change in mobile phase
composition and change in spotting volume etc.
Among the various solvent systems tested, the mixture containing
toluene-ethyl acetate-glacial acetic acid (8: 2: 0.1, v/v/v) gave the
best resolution for piperine (Rf = 0.43) from the formulation matrix
which enabled its quantification as well as phytochemical fingerprint
(Figure 2). The identity of the band of piperine in TC was confirmed
by comparing its UV absorption spectra with that of the standard.
TC samples subjected to storage up to 4 months, showed variation in
piperine content. It was observed that the content of piperine
decreased on prolonged storage (Table 4, Figure 3) when compared
to freshly prepared formulation. The results of stability studies are
supported by the frequent references in classical Ayurvedic texts
regarding use of Churna18. Thus, it can be recommended that this
formulation should be consumed when fresh. The comparative study
of in-house TC formulation with other marketed formulations
revealed that the piperine content was more in the freshly prepared
in-house formulation (Table 5, Figure 4).
In acute toxicity studies, no significant change in body weight, food
intake and water intake of the animals was observed compared to
animals of control group and also no mortality was recorded. Thus,
at the dose empirically used in traditional medicine, the formulation
in the form of an aqueous slurry (2.0 g/kg body weight of animals),
can be considered with a wide margin of safety for oral use.
Such reproducible modern techniques can make the traditional
Ayurvedic medicines more acceptable in the local and global
market. Thus, rationally designed, carefully standardized, synergistic
traditional formulations and botanical drug products with robust
scientific evidence can be used as an alternative to modern medicine.
CONCLUSION
Results of the present study can be used to characterize the samples
in industry to check their uniformity. The obtained values of
physical, chemical and biological parameters for TC can be adopted
to lay down new pharmacopoeial standards to be followed in its
preparation with batch to batch consistency.
ACKNOWLEDGMENT
Authors are thankful to Ms. Gauri Swar for technical assistance.
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Table 1: Traditional for mula composition of TC
Ingredients
Quantity
Sanskrit name
Botanical identity
Maricha
Fruit of Piper nigrum L.
1 part
Pippali
Fruit of Piper longum L.
1 part
Sunthi
Rhizome of Zingiber officinale Rosc.
1 part
Table 2: Proximate analysis of raw materials and TC
Samples
Foreign matter
Total ash
Acid insoluble ash
Water soluble ash
Loss on drying
% Mean (n = 3) ± S. D.
Maricha
0.463 ± 0.036
4.276 ± 0.080
0.176 ± 0.023
2.360 ± 0.051
14.856 ± 0.427
Pippali
1.557 ± 0.030
3.987 ± 0.060
0.108 ± 0.036
2.093 ± 0.034
12.076 ± 0.308
Sunthi
0.350 ± 0.037
5.176 ± 0.036
0.684 ± 0.053
3.433 ± 0.043
9.596 ± 0.355
TC
Not applicable
5.765 ± 0.200
0.337 ± 0.012
3.216 ± 0.037
9.273 ± 0.012
Table 3: Piperine content in TC and its ingredients using HPTLC
Sample
Piperine content (mg/g)
[Mean ± S. D., n=3]
Maricha
3.53 ± 0.12
Pippali
1.65 ± 0.24
Sunthi
---
TC
3.14 ± 0.55
Table 4: Stability study of TC in terms of piperine content using HPTLC
Storage period
(month)
Piperine content (mg/g)
[Mean ± S. D., n=3]
0
3.14 ± 0.55
2
2.48 ± 0.06
4
0.50 ± 0.10
Table 5: Comparative study of in-house TC and marketed formulations in terms
of piperine content using HP TLC
Name of formulation
Piperine content (mg/g)
[Mean ± S. D., n=3]
In-house TC
3.14 ± 0.55
M-01
2.24 ± 0.11
M-02
2.67 ± 0.04
M-03
2.88 ± 0.17
Figure 1: Structure of piperine
Figure 2: HPTLC detection of piperine from TC and its ingredients at 550 nm, Track
details - 1: Maricha, 2: Pippali, 3: Piperine, 4: Sunthi, 5: TC
Figure 3: Stability samples of TC on HPTLC at 550 nm, Track details - 1: 0 month
sample, 2: 2 month sample, 3: piperine, 4: 4 month sample
Figure 4: In house and marketed samples of TC on HPTLC at 550 nm, Track details -
1: TC, 2: M-01, 3: piperine, 4: M-02, 5: M-03
Source of support: Nil, Conflict of interest: None Declared