Estimation of Andrographolide
Content in Aqueous Extract of Siddha Formulations
by HPTLC
A Rajasekaran*,
R Arivukkarasu, M Linda
KMCH College of Pharmacy, Coimbatore, Tamilnadu, India
*Corresponding Author E-mail: rsekaran2001in@yahoo.co.in
ABSTRACT:
Indigenous
system of Siddha medicine is playing a vital role in the
prevention of viral infections such as dengue fever, chikungunya
and swine flu. The main aim of the study was to estimate the andrographolide content in aqueous extracts of Siddha formulations containing Andrographis paniculata supplied
in Tamilnadu for prevention of dengue fever, chikungunya and swine flu. HPTLC method was adopted for
determining the content of and rographolide in Siddha formulations using and rographolide as marker compound. The HPTLC method was
performed using HPTLC aluminium sheets precoated with silica gel 60 GF254 as stationary
phase and ethyl acetate: n-hexane (8.5:1.5 v/v) as the mobile phase. The
developed chromatogram was scanned at 233 nm using Camag
scanner III. The Rf
value of standard and rographolide and andrographolide in the samples were found to be in the
range of 0.31 to 0.33.Siddha formulation
collected from Siddha VaidhyaNilayam
(Dharmapuri Siddha Traditional
Formulation) showed more content of andrographolide
compared to other Sidhha formulations.
KEYWORDS: Andrographolide, Siddha formulations,
Andrographispaniculata, HPTLC, King of bitters.
INTRODUCTION:
In the recent years the Siddha formulations is widely used in a most
effective way to prevent the epidemic diseases such as dengue fever, chikungunya and swine flu without causing any side effects.
Nilavembu Kudineer and Nilavembu Kudineer Choornamare the Siddha polyherbal formulations in aqueous decoction form is being
distributed by Govt. of Tamilnadu and by many Siddha manufacturers, which mainly contains Andrographispaniculataalong
with 8 other herbs for the treatment of dengue, chikungunya
and swine flu1-3. Andrographis paniculata (Burm. F) Wall ex Nees,
commonly known as the “king of bitters,” is an herbaceous plant belonging to
the family Acanthaceae4. Andrographolide is the major active constituent of this plant which is a bicyclic diterpenoid lactone (Fig 1) having several pharmacological activities5.
Fig 1. Chemical structure of andrographolide
Andrographolide has beneficial roles in the management of diseases
like common cold6, inflammation7, microbial diseases8,
gastrointestinal problems9, cardiovascular diseases10,
hepatotoxicity11, respiratory diseases12, cancer13,
HIV14 and diabetis15. The present study was undertaken to
determine the percentage of active ingredient andrographolide
present in various siddha formulations for treating viral
infections.
MATERIALS AND
METHODS:
Equipment:
A Camag
HPTLC system comprising of Camag Linomat
5 Sample applicator, Camag microliter
syringes, Camag Twin trough chambers, Camag UV detection chamber and Camag
TLC scanner 3 was used the HPTLC studies.
Chemicals and solvents:
Andrographolide was procured from Sigma Aldrich chemical company. Ethylacetate and n-hexane were purchased from Himedia. Merck HPTLC silica gel 60GF254 precoated aluminium
plates was used to perform the HPTLC.
Collection of Siddha formulations:
Five
Siddha formulations containing the Andrographis paniculata was obtained from different local shops and
the details are given in Table 1.
Table 1. Details of the Siddha formulation collected
|
Formulation ID |
Formulation |
Date of procurement |
|
DSTF |
Dharmapuri Siddha Traditional Formulation |
03-01-15 |
|
MTT |
Maha Thikthakam Kashayam Tablets |
08-01-15 |
|
NKC |
Nilavembu Kudineer
Choornam |
26-01-15 |
|
NK |
Nilavembu Kudineer |
24-06-15 |
|
NKP |
Nilavembu Kashaya
Podi |
26-06-15 |
Preparation of aqueous extracts of Siddha formulations:
Two
g of the Siddha formulations was accurately weighed and
extracted by sonication in 20 ml distilled water for 30 min. The aqueous
extract was filtered through Whatman filter paper No.1 and then concentrated
and the residue obtained (0.1 g) was utilized for HPTLC studies.
Preparation of standard andrographolide solution:
Ten
mg of standard andrographolide was accurately weighed
and transferred into ten ml volumetric flask, and then the solution was made up
to ten ml with methanol. From this stock solution of andrographolide
further dilutions were made for estimation.
Determination of λmax
of andrographolide:
The
sensitivity of HPTLC method depends upon the proper selection of wavelength by
UV detector. Shimadzu 1700 Pharmaspec UV-Vis
spectrophotometer was used to determine the absorption maxima of standard andrographolide solution (1 mg/ml) in methanol.
Estimation of andrographolide
content in aqueous extracts of Siddha formulations
by HPTLC:
The samples and standard
were spotted on precoated HPTLC aluminium
sheets silica gel 60 GF254 (10 x 10 cm, 0.2 mm thickness) as 6 mm
wide band positioned 10 mm from the bottom of the plate and 15mm from side of
the plate by using a automatic TLC applicator Camag Linomat V with nitrogen flow providing a delivery speed of
150 nl/s by Camag microliter syringe. These conditions were kept constant
throughout the analysis of samples. The linear ascending development was
performed in a Camag twin trough glass chamber 10 x
10 cm which is previously saturated with the mobile phase for 10 min. The
mobile phase used was ethyl acetate: n-hexane (8.5:1.5v/v). The plates were
developed to 8 mm from the bottom of plate and after development the plates
were dried in air. The quantification of andrographolide
was done by using Camag TLC Scanner Model III
equipped with Wincats software. The applied scan
conditions were 6 mm x 0.45 mm slit width, wavelength 233 nm and absorption
mode. The plates were photographed at 254 nm.
RESULTS AND
DISCUSSION:
The
absorbance maximum (λmax) of standard
andrographolide was obtained at 233 nm and hence all
the detections of andrographolide were carried out at
233 nm. The solvent system of ethyl acetate: n-hexane (8.5:1.5 v/v) gave a good
resolution for standard andrographolide and Rf value was
found to be 0.33.
Estimation
of andrographolide content in aqueous
extract of Siddha formulations:
The main constituent andrographolide in Siddha formulations
was well resolved from other constituents and appeared in the chromatogram at Rf value in
the range of 0.31 to 0.33. The percentage
of andrographolide determined in each Siddha formulation was provided in Table 2.
Table
2.Estimation of andrographolide content in aqueous
extract of Siddha formulations
|
S. N |
Formulation ID |
Formulation |
Amount of andrographolide present % w/w |
|
1 |
DSTF |
Dharmapuri Siddha Traditional
Formulation |
1.1612 |
|
2 |
MTT |
MahathikthakamKahsayamTablet |
0.4175 |
|
3 |
NKC |
NilavembuKudineerChoornam |
1.1017 |
|
4 |
NK |
NilavembuKudineer |
0.1180 |
|
5 |
NKP |
NilavembuKashayaPodi |
0.4470 |
Fig 2. Chromatogram of Siddha formulation collected from
Dharmapuri siddha traditional
formulation (DSTF)
Fig 3. HPTLC profile of marker andrographolide with aqueous extracts of all Siddha formulations
CONCLUSION:
Among
the five Siddha formulations estimated for andrographolide content, Dharmapuri
Siddha Traditional Formulation (DSTF) showed
more content of andrographolide compared to other
formulations.
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Received on 09.12.2015 Accepted on 27.12.2015
© Asian Pharma
Press All Right Reserved
Asian J. Pharm.
Ana. 5(4): October- December, 2015; Page 206-208
DOI: 10.5958/2231-5675.2015.00032.0