Estimation of Andrographolide Content in Aqueous and Methanolic
Extracts of Marketed Nilavembu Kudineer Siddha formulations by HPTLC Technique
Marimuthu Murugan, Mohanraj Shankar, Manojkumar
Duraisamy, Melina D. Cruz, Rajasekaran Aiyalu, Arivukkarasu Ramasamy*
KMCH College of Pharmacy, Coimbatore, Tamilnadu, India.
*Corresponding Author E-mail: phytoarivu@gmail.com
ABSTRACT:
Andrographis paniculata is one of the important ingredient used
in various Siddha formulations. The present study is mainly aimed to compare
various marketed nilavembukudineer churna on the basis of andrographolide
content. HPTLC method was used for this determination using marker compound
andrographolide. 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 233nm using Camag scanner III. The reference standard
andrographolide Rf value was found to be 0.55.Fromthe five market formulations
screened, the andrographolide content in methanolic and aqueous extracts
showed the discrepancy in content amount representing by order MFM-1
MFA-1MFM-2MFA-2
-3.The other market formulation extracts
both methanol and aqueous extracts does not confirms the presence of
andrographolide.
KEYWORDS: Andrographis
paniculata, HPTLC,
Nilavembu, Andrograpolide.
INTRODUCTION:
Andrographis paniculata (Burm.f.) Wall. ex Nees., (Family: Acanthaceae)
(English name-King of Bitters, Tamil name-Nilavembu) is an annual herbaceous
plant and is extensively cultivated in Southern Asia, China and some parts of
Europe. Andrographis paniculata is widely used to get rid of body heat, dispel
toxins from the body; prevent common cold1,
upper respiratory tract infections including sinusitis and feverand as an
antidote against poisons of snakes and insects. The plant has been reported to
posses antimalarial2,
anti-inflammatory3, antioxidant4, antihepatitis5,
antihyperglycemic6,
anthelmintic7, antibacterial8,
antipyretic9 and anticancer activity10. Andrographolide is bicyclic diterpenoid
which is the constituent of this plant and one of the ingredients used in
formulation of Nilavembukudineer siddha formulation. It is also supplied
to the people by government of tamilnadu as it has potent antiviral activity
against viruses causing dengue and chikungunya11.
The present study was
undertaken to find the sample with maximum andrographolide content among the
formulations collected around coimbatore.
MATERIAL AND METHODS:
Equipment:
A Camag HPTLC system comprising of Camag
Linomat5 Sample applicator, Hamilton microliter syringe, Camag twin trough
chambers, and Camag TLC scanner 3 was used the HPTLC studies.
Table 1. Formulation in Methanol and Aqueous Identity
|
Formulation Name
|
Formulation in Methanol Identity
|
Market Formulation in Aqueous Identity
|
|
Market Formulation-1
|
Market-Formulation in Methanol in MFM-1
|
Market Formulation in Aqueous MFA-1
|
|
Market Formulation-2
|
Market-Formulation in Methanol in MFM-2
|
Market Formulation in Aqueous MFA-2
|
|
Market Formulation-3
|
Market-Formulation in Methanol in MFM-3
|
Market Formulation in Aqueous MFA-3
|
|
Market Formulation-4
|
Market-Formulation in Methanol in MFM-4
|
Market Formulation in Aqueous MFA-4
|
|
Market Formulation-5
|
Market-Formulation in Methanol in MFM-5
|
Market Formulation in Aqueous MFA-5
|
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 Nilavembu Kudineer Siddha formulations
containing the Andrographis paniculata was obtained from different local
shops and the details are given in Table 1.
Preparation of methanolic extracts of Siddha
formulations:
1g of the Siddha formulations was
accurately weighed and extracted by sonication in 10ml methanol for 30 min. The
Marketed formulations methanolic extract (MFM) was filtered through Whatman
filter paper No.1 and then concentrated. The residue obtained (0.1g) was
utilized for HPTLC studies.
Preparation of aqueous extracts of Siddha
formulations:
1g of the Siddha formulations was
accurately weighed and extracted by sonication in 10ml distilled water (MFA) for
30 min. The aqueous extract was filtered through Whatman filter paper No.1 and
then concentrated. The residue obtained (0.1g) 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 UV-Vis
spectrophotometer was used to determine the absorption maxima of standard
andrographolide solution (1mg/ml) in methanol.
Estimation of andrographolide content in
methanol and aqueous extracts of Siddha formulations by HPTLC:
The samples and standard were spotted on
precoated HPTLC aluminium sheets silica gel 60 GF254 (10x 10cm, 0.2mm
thickness) as 6 mm wide band positioned 10mm from the bottom of the plate and
15mm from side of the plate by using a automatic TLC applicator CamagLinomat V
with nitrogen flow providing a delivery speed of 150nl/s by Camag microliter
syringe. These conditions were kept constant throughout the analysis ofsamples.
The linear ascending development was performed in a Camag twin trough glass
chamber 10 x10cm which is previously saturated with the mobile phase for 10min.
The mobile phase used was ethylacetate: n-hexane (8.5:1.5v/v)12. 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 0.45mm slit width, wavelength 233nm and absorption
mode. The plates were photographed at 254nm.
Fig 1: Chromatogram of Andrographolide In
Nilavembu Kudineer Churnam Marketed Formulation in Methanol (MFM ) Marketed
Formulation in Aqueous (MFA)
Market-Formulation in Methanol in MFM-1, Market-Formulation
in Methanol in MFM-2Market-Formulation in Methanol in MFM-3, STD
Andrographolide, Market Formulation in Aqueous MFA-1, Market Formulation in
Aqueous MFA-2, Market Formulation in Aqueous MFA-3, Market-Formulation in
Methanol in MFM-4, Market-Formulation in Methanol in MFM-5, STD
Andrographolide, Market Formulation in Aqueous MFA-4, Market Formulation in
Aqueous MFA-5
RESULTS AND DISCUSSION:
The absorbance maximum (λmax) of
standard andrographolide was obtained at 233nm and hence allthe detections of
andrographolide were carried out at 233nm. 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 0.55. The chromatogram of the plate was exhibited in
Fig 2 and
overlay of the spectrum was given in Fig 3 and Densitogram was given in Fig 4. From
the five market formulations screened, the andrographolide content in
methanolic and aqueous extracts were calculated exhibited in Table 2. The
discrepancy in content amount by representing increasing order MFM-1> MFA-1>
MFM-2> MFA-2 > MFM-3.
Fig 2: Overlay of samples Market-Formulation
in Methanol in MFM-1, Market-Formulation in Methanol in MFM-2Market-Formulation
in Methanol in MFM-3, STD Andrographolide, Market Formulation in Aqueous MFA-1,
Market Formulation in Aqueous MFA-2, Market Formulation in Aqueous MFA-3.
Fig 3: Overlay of samples Market-Formulation
in Methanol in MFM-4, Market-Formulation in Methanol in MFM-5, STD
Andrographolide, Market Formulation in Aqueous MFA-4, Market Formulation in
Aqueous MFA-5.
Fig. 4 Densitomatogram of
MFM-Market-Formulation in Methanol and MFA-Market Formulation in Aqueous
Table:2:Amount of Andrographolide estimatd in Nilavembu
Kudineer Choornam
|
Name of the formulation
|
Rf value
|
Area of peak
|
Amount of Andrographolide in µg
|
% of Andrographolide
|
|
MFM-1
|
0.08, 0.20, 0.37, 0.47, 0.55, 0.66, 0.78, 0.90
|
25226.8
|
7.49µg
|
0.74%
|
|
MFM-2
|
0.16, 0.41, 0.46, 0.55, 0.71, 0.78, 0.88
|
9068.4
|
2.69 µg
|
0.26%
|
|
MFM-3
|
0.45, 0.54, 0.74, 0.77
|
1484.4
|
0.44 µg
|
0.044%
|
|
MFM-4
|
0.07, 0.16, 0.32, 0.58, 0.73, 0.79, 0.85, 0.92
|
Nil
|
Nil
|
Nil
|
|
MFM-5
|
0.05, 0.09, 0.28, 0.59, 0.70, 0.80, 0.86
|
Nil
|
Nil
|
Nil
|
|
Standard
|
0.55
|
33637.1
|
10 µg
|
|
|
MFA-1
|
0.12, 0.44, 0.55, 0.66, 0.72, 0.77
|
16989.6
|
5.05µg
|
0.50%
|
|
MFA-2
|
0.45, 0.57, 0.73 0.79, 0.89
|
3698.6
|
1.09 µg
|
0.10%
|
|
MFA-3
|
0.02, 0.09, 0.33, 0.41, 0.60, 0.70
|
Nil
|
Nil
|
Nil
|
|
MFA-4
|
0.03, 0.14, 0.30, 0.58, 0.70, 0.81
|
Nil
|
Nil
|
Nil
|
|
MFA-5
|
0.36, 0.60, 0.70, 0.81, 0.85
|
Nil
|
Nil
|
Nil
|
Among five formulations estimated, methanolic
extract of MFM-1, MFA-1, MFM-2, MFA-2
MFM-3 showed andrographolide
content of 0.74%, 0.50%, 0.26 %, 0.10% and 0.04% respectively. The other
marketed formulation extracts both methanol and aqueous extracts does not
confirms the presence of andrographolide. The percentage of andrographolide determined
in each Siddha formulation was provided in Table 3.
Table 3. Estimation of andrographolide
content in methanol and aqueous extract of Siddha formulations
|
Formulation Code
|
Amount of Andrographolide
|
|
*MFM-1
|
7.49 µg
|
|
MFM-2
|
2.69 µg
|
|
MFM-3
|
0.44 µg
|
|
MFM-4
|
Nil
|
|
MFM-5
|
nil
|
|
* MFA-1
|
5.05 µg
|
|
MFA-2
|
1.09 µg
|
|
MFA-3
|
Nil
|
|
MFA-4
|
Nil
|
|
MFA-5
|
Nil
|
*MFM-Market-Formulation in
Methanol*MFA-Market Formulation in Aqueous.
CONCLUSION:
Among five formulations estimated, methanolic
extract of MFM-1, MFA-1, MFM-2, MFA-2
MFM-3 showed andrographolide
content other marketed formulation extracts both methanol and aqueous extracts
does not confirms the presence of andrographolide.
REFERENCES:
1.
Cįceres DD et al.
Prevention of common colds with Andrographis paniculata dried extract. A Pilot
double blind trial. Phytomedicine. 1997; 4(2):101-104.
2.
Mishra K, Dash AP, Swain
BK, Dey N. Anti-malarial activities of Andrographis paniculata and
Hedyotiscorymbosa extracts and their combination with curcumin. [available at
http://www.malariajournal. com/content/8/1/26] Malar J 2009; 8:26.
3.
Xia YF et al.
Andrographolide attenuates inflammation byinhibition of NF-kappa B activation
through covalentmodification of reduced cysteine 62 of p50. JImmunol. 2004; 173
(6):4207-4217.
4.
Nees K, Sheeja PK,
Shihab GK. Antioxidant and anti-inflammatory activities of the plant
Andrographis paniculata. Immunopharmacol Immunotoxicol. 2006; 28:129-40.
5.
Singh RP, Banerjee S,
Rao AR. Modulatory influence of Andrographis paniculata on mouse hepatic and
extrahepatic carcinogen metabolizing enzymes and antioxidant status. Phytother
Res. 2001; 15:382-90.
6.
Zhang XF, Tan BK.
Antihyperglycaemic and anti-oxidant properties of Andrographis paniculata in
normal and diabetic rats. Clin Exp Pharmacol Physiol. 2000; 27: 358-63.
7.
Singh S, Mehta A, John
J, Mehta P. Anthelmintic potential of Andrographis paniculata, cajanuscajan and
silybummarianum. Pharmacog J. 2009; 1(4):71-73.
8.
Burm F, Kumar OA, Naidu
LM, Rao KG. In vitro antibacterial activity in the extracts of Andrographis
paniculata. International Journal of Pharm Tech Research. 2010; 2:1383-5.
9.
Chandra R, Kumarappan
CT, Kumar J, Mandal SC. Antipyretic activity of JURU-01 - a polyherbal
formulation. Global J. Pharmacol. 2010; 4(1):45-47.
10. Kumar RA, Sridevi K, Kumar NV, Nanduri S,
Rajagopal S. Anticancer and immunostimulatory compounds from Andrographis
paniculata. J Ethnopharmacol. 2004; 92:291-5.
11. Kalaiarasi R et al. A combination of
nilavembukudineer and adathodaimanapagu in the management of dengue fever.
International Journal of Current Research. 2013; 5(04): 978-981.
12. Rajasekaran A, Arivukkarasu R, Linda M.Estimation of
Andrographolide Content in Aqueous Extract of Siddha Formulations by
HPTLC. Asian J. Pharm. Ana. 2015; 5(4);
206-208.
DOI: 10.52711/2231-5675.2023.00039