Quantitative Estimation of Rosmarinic
Acid in Ocimum sanctum Extracts and Polyherbal Formulation by HPTLC
Bincy Raj1*,
Salma Khanam2, Soosamma
John3 and Madhavi T3 Ayesha
Siddique1
Dayananda Sagar
College of Pharmacy, Kumaraswamy Layout, Bangalore-
560 078
ABSTRACT:
Rosmarinic acid is an important
bioactive content having analgesic and inflammatory activity. The content of Rosmarinic acid in Ocimum sanctum and
three polyherbal formulations was estimated by HPTLC.
KEYWORDS:
INTRODUCTION:
Ocimum sactum is ranked
among few wonder herbs for having enormous curative effect. Due to this the
plant is considered as highly sacred, worth worshipping and hence was given the
name of sacred, Tulsi or holy basil in India1.
Different part of the plant has been reported to exhibit several medicinal
properties2-4 like analgesic5, anti-oxident6,7, antibacterial and anthelmentic
activity.
The
chemical constituents of Ocimum sanctum indicate volatile oil and triterpine. Rosmarinic acid have
been isolated and characterized from the leaves of Ocimum sanctum. This compound can be used as marker compound to evaluate
and standardize extracts and formulations containing Tulsi.
Literature survey reveals that very few validated HPTLC8-10 methods
for estimation of Rosmarinic acid in extract and
formulation.
MATERIAL AND METHODS:
Whole
plant of Ocimum sanctum was collected from Natural
Remedies and authenticated by RRI Bangalore. Formulation F-1 (marketed), each
10ml contains 100mg of Ocimum sanctum, formulation F-2 (marketed),
each 5ml contains 100mg of Ocimum sanctum and
formulation F-3 (marketed), each capsule contain 250mg of Ocimum sanctum.
The
powdered drug (100g) was refluxed with 70% methanol, filtered and then
concentrated under reduced pressure. 1g of extract was dissolved in 100ml
methanol and filtered through whatman No 1 filter
paper. The same procedure was applied for the extract as well as for the polyherbal formulations.
The
apparatus used was: Camag HPTLC system, consisting of
Linomat V spotting device and Scanner III with winCat 4 software; TLC aluminium
sheets silica gel F254 precoated layer (10X10 cm).
Toluene, ethyl acetate and formic acid in the ratio of 9:1:0.2 was used as
mobile phase. The chromatogram was developed up to 80mm and scanned at 254 and
366. Spectrum analysis was done in the range of 200 – 700nm using D2
lamp and tungsten lamp. The spectrum showing λ max is given in the fig 1.
The λ max for rosmarinic acid was found to be
320nm with Rf
value 0.62.
Methanolic solution
of Rosmarinic acid (50ng/µl) was used for the
application on TLC plate. The five standard levels (2-6µl) of standard Rosmarinic acid were applied in triplicate on TLC plate.
The
chromatogram was scanned at 320nm and average peak areas of three samples were
calculated. The HPTLC chromatogram of extract along with standard Rosmarinic acid is shown in the fig 3. The HPTLC method was
validated according to ICH guidelines. The recovery of analyte
from the sample was confirmed by analysis of sample of known content. The
solutions were spiked with three different known concentrations and analysis of
each sample was repeated six times. The %RSD and % recovery was calculated. The
repeatability was studied by analysing each sample
for six times under the same analytical procedure and intermediate precision
was studied by analysing each sample for six times on
different days. The %RSD / CV was obtained.
RESULTS AND DISCUSSION:
The Rf value of standard Rosmarinic acid was found to be 0.62 and the peak area of
the drug was reproducible as indicated by low coefficient of variation 1.65-1.81.
A good linear relationship(r=0.996) was observed between the range 100 -400ng.
Linearity curve of Rosmarinic acid is shown in the
fig 2. The recovery of the method by this method was found to be more than
99.6-101%, which indicate high accuracy of proposed HPTLC method. The low
percentage RSD (2.1-2.3) indicates that the proposed HPTLC method is highly
precise. More over the limit of quantification was found to be 100ng. The
content of Rosmarinic acid in dried extract and
formulations F-1(marketed), F-2(marketed) and F-3(marketed) was found to be
1.44, 0.1, 0.05 and 0.15%w/w respectively. The highest amount of Rosmarinic acid was found to be in the dried plant extract.
The result of all parameter was within limit. The method was found to be
simple, sensitive and accurate. The good recovery of Rosmarinic
acid showed the reliability and suitability of the method.
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Received on 08.11.2009
Accepted on 31.12.2009
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Research Journal of Pharmacognosy and Phytochemistry. 2(1): Jan.-Feb. 2010, 46-48