INTRODUCTION:
Herbal
formulations show the number of problems when quality aspect is considered.
This is because of nature of the herbal ingredients and different secondary
metabolites present therein. Mainly, variation in the chemical profile of the
herbal due to intrinsic and extrinsic factors (growing, harvesting, storage and
drying processes) [1-3].
Ayurveda
is practiced widely in India, Srilanka and other countries[4] and Ayurvedic
preparations are either of herbal origin, mineral origin, animal origin or
combination of them. The safety and efficacy of these formulations is closely correlated
with the quality and the source of raw materials used in their production[5].
It
is generally possible to estimate a phytochemical marker for the ingredient
plant raw material using various analytical techniques like TLC, HPLC and
HPTLC. It is possible to detect the presence of the marker phytochemical
compound and also quantify it to ascertain the limits in the final
formulation[4,6,7]
Chromatographic
fingerprint have been suggested to check for authenticity or provide quality
control of herbal medicine[8]. Chromatography has the advantage of separating a
complicated System into relatively simple sub-systems and then presenting the
chemical patterns of herbal medicine in the form of a chromatogram. The World
Health Organization (WHO) accepts fingerprint chromatography as an
identification and quality evaluation technique for medicinal herbs since
1991[9]. Fingerprints can be a unique identification utility for herbs and
their different species)[10,11], and can be used for modeling pharmaceutical activities[12].
Now, chromatographic fingerprint technique plays an important role in
controlling the quality of TCM for the systemic characterization of
compositions of samples and focusing on the identification and assessment of
the stability of the components[13]. The Patent proprietary Ayurvedic medicines
are sold over the counter in pharmacies; these products appear to represent a
major share of branded traditional medicine in India. Nevertheless systems like
Ayurveda still need to gain an empirical support of modern medical sciences to
make them credible and acceptable for all. An innovative research effort to
define the advantage of traditional system of medicine with respect to their
safety and efficacy could result in a better utilization of these complementary
systems of medicine[14].
Marichyadi
Vati is an important ayurvedic formulation which is official in Ayurvedic
Formulary of India is combination of ingredients (Piper nigrum, Piper longum, Hordeum vulgare, Punica granatum, Saccharum
officinarum).The formulation is dispensed for the disorder of respiratory
tract. It eliminates the aggravated kapha in the respiratory tract and in the
digestive channels. It also regulates the path for vatta and helps minimize gas
formation in the abdomen, being hot in nature[15]. Therapeutically it is also
used for the treatment of kasa
(cough) and svasa (asthma) [16].
Piperine is one of the major constituent of Marichyadi Vati. Molecular formula
C17H19NO3 density 1.193 g/cm3, melting point 130°C, in animal studies, piperine
also inhibited other enzymes important in drug metabolisms [17].
MATERIAL AND
METHODS:
Instrumentation and Chromatographic
Conditions
The method developed involved
the use of Yongline acme 9000 pump, Shimadzu system controller and Shimadzu
UV-VIS 1700 detector. The column used was LiChrosorb NH2 (10 cm) and
AUTOCHROM 3000 software was used as integrator. A flow rate of 1.5 ml/min was
maintained. The optimized mobile phase was found to be Methanol: Water (69:31
v/v) was passed through a 0.22µm membrane filters and degassed by
ultrasonication under vacuum before use. The injection volume was 20µL and the
effluent was monitored for UV absorption at 343 nm was used for quantitative
estimation. All separations were performed at ambient temperatures. The
optimized method was then validated for limits of detection, linearity, range,
precision and accuracy and specificity.
Procurement of Crude Drugs and Marketed
Formulations.
The
raw materials were purchased from the local market of Ujjain for the formulation
of Marichyadi Vati, and marketed formulation was obtained as a gift sample
(i.e. Marichydi Vati manufactured by Shree Baidynath Ayurved Bhavan Pvt. Ltd. Bamhani, Gopalgang, Dist. Seoni
M.P. Batch no. 08), from Mahakal Ayurveda Sansthan, Ujjain.
Authentication of Plant Material
The plants were identified by Dr. S. K.
Billore (Professor and head of department), botany department of Madhav Science
College, Ujjain (MP) and voucher specimen (MIPS/P/001/2009) of Piper longum Linn., (MIPS/P/002/2009) Piper nigrum Linn., (MIPS/S/001/2009) Saccharum
officinarum Linn., (MIPS/P/003) Punica granatum Linn, (MIPS/H/001/2009) Hordeum
vulgare Linn. were deposited in
the of department of Pharmacognosy, Mahakal Institute of Pharmaceutical
Studies, Ujjain (MP).
Preparation of
Formulations15
A fine powder of Pippali, Marica,
Yavakshar, Dadima (phala, tvak) was prepared by passing the required quantity
through the sieve no. 80.
Table 1: Ingredients used in the formulation of Marichyadi Vati (300
vati)
|
S.No.
|
Botanical name
|
Local name
|
Plant part used
|
Quantity (in
gms)
|
|
1
|
Piper nigrum
|
Marica
|
Fruit
|
12
|
|
2
|
Piper longum
|
Pippali
|
Fruit
|
12
|
|
3
|
Hordeum vulgare
|
Yavakshar
|
Salt
|
06
|
|
4
|
Punica granatum
|
Dadima (Phalatvak)
|
Bark
|
24
|
|
5
|
Saccharum
officinarum
|
Guda
|
Dried concentrated form of juice
|
96
|
Preparation of standard
solution of Piperine
Standard solution was prepared
by dissolving accurately weighed (100 mg) piperine in 100 ml methanol into a
100 ml volumetric flask (Stoke A) containing 1000 micro gram of peperine per ml
of the solution and further dilutions were made from stoke A [18, 19].
Standard
plot of Piperine
Serial
dilutions containing 2, 4, 6, 8 and 10 μg/ml piperine in methanol were
prepared from the stock solution of piperine (100 mg/100ml). Each dilution was
chromatographed on HPLC and area under the peak of piperine recorded (Table I).
Retention time of piperine was observed to be 7.7 min. A standard curve of
piperine was prepared by plotting the actual amount of piperine present in
different dilutions against the area under the peaks of piperine observed by
injecting above serial dilutions. The intercept and the slope of the standard
plot was observed to be 0.00 and 70.52 respectively, with coefficient of
correlation as 0.999 (r2).
Sample Preparation:
Accurately weighed 4 gm of Marichyadi Vati was refluxed for 1 hr
with 100 ml of methanol. The extract was filtered and concentrated till the
semisolid mass was obtained and from that 100 mg was dissolved and diluted up
to 100 ml with methanol in volumetric flask. Same procedure was used for the
preparation of solution for estimation of piperine for marketed formulations.
The
same procedure was performed separately for, the powdered crude drug of Piper longums (Pippali) and piperine
extract. Each of the solutions was subjected to HPLC analysis and the area
under the peak of piperine was recorded.
Figure: Calibration graph of standard
peperine.
Figure: HPLC
Chromatogram of isolated piperine from crude drug.
Figure: HPLC
Chromatogram of extract of laboratory preparation.
Figure: HPLC
Chromatogram of extract of marketed formulation.
Figure: Overlay chromatogram of crude drug,
sample and standard piperine.
Figure: HPLC
chromatogram of piperine (Standard solutions) showing the precision.
Validation Parameters
Selectivity and peak purity
Selectivity was checked by
using prepared solutions of MV and available standards optimizing separations
and detection. The purity of the peaks was checked by multivariate analysis.
Three spectra corresponding to upslope, apex and down slope of each peak were
computer normalized and superimposed. Peaks were considered pure when there was
a coincidence between the three spectra (match factor was =98%).
Linearity, limits of detection
and quantification
The linearity of the detector
response for the prepared standards was assessed by means of linear regression
regarding the amounts of each standard, measured in mg,
and the area of the corresponding peak on the chromatogram. Linearity was also
confirmed for MV prepared sample solutions. After chromatographic separation,
the peak areas obtained were plotted against concentrations by linear
regression. Limits of detection and quantification were determined by
calculation of the signal-to-noise ratio. Signal-to-noise ratios of
approximately 3:1 and 10:1 were used for estimating the detection limit and
quantification limit, respectively of the methods.
Precision
The repeatability of the
injection integration was determined for both standard piperine and the content
of piperine in MV. A standard solution containing reference compounds and
prepared sample solutions was injected. MV samples were also prepared 3 times
to evaluate the repeatability of the process. The mean amount and S.D. values
were calculated. The precision was calculated at two different concentrations
high and low tested in the concentration range. For standardization, sample was
injected at six different concentrations and linearity was noted.
Accuracy
The accuracy of the method was
determined by analyzing the percentage of recovery of the piperine in MV. The
samples were spiked with two different amounts (50,100 mg)
of standard compounds before sample preparation. The spiked samples were
extracted by triplicate and analyzed under the previously established optimal
conditions. The obtained average contents of the target compounds were used as
the “real values” to calculate the spikes recoveries.
Robustness
For the determinations of the
method’s robustness a number of chromatographic parameters, such as column
package and size, mobile phase composition and gradient ratios, flow rate and
detection wavelength, were varied to determine their influence on the
quantitative analysis. Inter day and intraday variability was studied for the
sample, by injecting the same concentration of the sample on three different
days and the standard error mean was calculated.
Statistics
When applicable one-way or
two-way analyses of variance was used to assess the observed differences in the
piperine content. Differences were considered to be statistically significant
when the P-value was <0.05.
Table: Analytical
parameters of HPLC procedure for the piperine estimation.
|
S.No.
|
Parameters
|
Value
|
|
1.
|
Absorption
maxima
|
343
nm
|
|
2.
|
Beer’s
law limit
|
2-10 mg/ml
|
|
3.
|
Regression
equation(y=mx + c)
|
y =
70.89x+2.843
|
|
4.
|
Intercept
(a)
|
2.843
|
|
5.
|
Slope
(b)
|
70.89
|
|
6.
|
Correlation
coefficient (r2 )
|
0.999
|
|
7.
|
LOD
|
0.6
|
|
8.
|
LOQ
|
2.0
|
|
9.
|
Precision(n=3,%
RSD)
|
0.35
|
|
10.
|
Accuracy
(%)
|
99.3
|
Table: Piperine content.
|
S.No.
|
Name
|
Piperine
content ((mg/ml)
|
%
RSD
|
|
1.
|
Crude
|
0.99±0.10
|
10.18
|
|
2.
|
MKT
|
13.52±0.60
|
4.47
|
|
3.
|
Lab
|
1.32±0.10
|
1.29
|
Mean ± SD of three
determinations.
Table: Data of recovery
|
S.No.
|
Amount of piperine (mg/ml)
|
Recovery %
|
|
In sample
|
Added
|
Estimated
|
|
1
|
10
|
8
|
7.5
|
98.75
|
|
2
|
10
|
10
|
7.9
|
98.80
|
RESULTS AND DISCUSSION:
The formulation follows Lambert-Beer’s law
limit in the concentration range of 2- 10 µg/ml with λmax at
343 nm having piperine content as 13.52±0.60 and 1.32±0.10 %w/w in MKT and
Laboratory formulations respectively.
The HPLC chromatogram of formulation has
retention time as 7.7 minutes. The HPLC method was validated by defining the
linearity, peak purity, limit of quantification and detection, precision,
accuracy, specificity and robustness. For the qualitative purpose the method
was evaluated by taking into account the precision in the retention time, peak
purity, and selectivity of piperine elutes. A high repeatability in the
retention time was obtained with (% R.S.D.) value lower than 1.5% for both
standard and samples even at higher concentration. The peak purity was studied
in the major peaks. Linearity, limit of detection (LOD), limit of
quantification (LOQ), accuracy and precision were evaluated for quantification
purposes. Thus LOD and LOQ found to be 0.6 and 2 mg/ml respectively which
suggest full capacity for quantification of piperine content in different
laboratory batches of formulation. R2 value for the regression
equation of the piperine is: 0.9978 this confirms the linearity of the method.
The recovery was performed at two levels by adding known amounts with
pre-analyzed sample of formulation found to be close to 98.75, 99.45% and a
higher repeatability indicate a satisfactory accuracy in the proposed methods
.Finally the robustness of the method was also assessed. Minor modification of
the initial mobile phase gradient had no effect on the peak resolution of the
compound. This confirms that the formulation is properly standardized as per
WHO guidelines and all parameters are found within the limits.
CONCLUSION:
Most
of the Ayurvedic formulation is lacked in their defined quality control parameters
and methods of its evaluation. WHO has emphasized the need to ensure the
quality of medicinal plant products by using modern controlled technique and
applying suitable standareds.
The
developed method of estimation can be considered as the protocol for the
evaluation of Marichyadi vati which will assist the regulatory authorities,
scientific organizations and manufacturers in developing standards.The method
used for evaluation is found to be precise and reproducible and help to produce
uniform standard products, which will restore faith in Ayurvedic system.The
developed method can also be applied to various polyherbal formulations for the
quantitation of piperine and can be used as a routine quality control method in
pharmaceutical industries.
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