Phytochemicals are often used to describe the large number of secondary metabolic
compounds found in plants and animals with known protective and human consumers
functions (Harborne, 1982). Acanthus ilicifolius (Family:
Acanthaceae) is commonly known as holy mangrove and used as a traditional medicine
to treat pain, inflammation and ulcer in Parangipettai coastal village, Southeast
coast of India. Furthermore, analgesic, anti-inflammatory, hepatoprotective
and antimicrobial activities were validated (Babu et
al., 2001). Rapid and systematic measurement of specific plant metabolites
are a serious challenge for analytical chemists, phytochemists and biochemists
because of their inherent structural diversity and dietary impact. In this context,
in 2000, the US Food and Drug Administration (FDA) issued a draft of Guidance
for Industry Botanical Drug Products (FDA, 2000). Recently,
many separation techniques have been proposed to separate, identify and quantify
the phytoconstituents in plants. Among that, high performance liquid chromatography
is a key technique to quantify the secondary metabolites (Satyavani,
2013). There was insufficiency of scientific data in the phytochemicals
of A. ilicifolius. Therefore, the present study aimed to evaluate phytochemicals,
qualitative and quantitative methods using HPTLC.
MATERIALS AND METHODS
Collection of plant material: Fresh leaves of A. ilicifolius were
collected from Parangipettai coast, Tamil Nadu, India during January 2008. The
vouchered specimen (AUCASMB 01/2008) was deposited in the herbarium of C.A.S.
in Marine Biology, Annamalai University, Parangipettai, India.
Extraction: About 3 kg leaves of A. ilicifolius were dried over
polythene cover in shade drying method at 21°C and pulverized using a mixer
grinder. The coarse powder was used only for the preparation of extract. One
kilogram powdered material of A. ilicifolius was cold macerated with
3000 mL of benzene, chloroform, acetone, ethyl acetate, methanol and ethanol
for three days. After that, the suspension was filtered and the residues were
removed. The filtrate of each extract was evaporated and dried at 40°C under
reduced pressure in a rota-evaporator to separate the benzene, chloroform, acetone,
ethyl acetate, methanol and ethanol extract. The final residual extract was
used for further experiments.
Qualitative analysis of phytochemicals: Different extracts viz., benzene,
chloroform, acetone, methanol and ethanol in A. ilicifolius were used
to determine the preliminary phytochemicals followed by the method of Evans
Preparation of standard solution
Gallic acid: The 10 mg of gallic acid was dissolved in methanol and making
upto 10 mL with methanol. Then 1 mL was pipetted out from stock solution and
made upto 10 mL with methanol to get the final concentration of 100 μg
Quercetin: The 10 mg of quercetin was dissolved in methanol and making
upto 10 mL with methanol to get final concentration of 1000 μg mL-1.
Lupeol: The 10 mg of lupeol was dissolved in chloroform and making upto
10 mL with chloroform. Then 1 mL was pipette out from the stock solution and
made upto the volume 10 mL with chloroform to get the final concentration of
100 μg mL-1.
Estimation of gallic acid and quercetin: The 100 mg of ethanolic extract
was dissolved in ethanol and making upto 10 mL to get the concentration of 10
mg mL-1 (Test GA and Test Q). The solution was filtered and used
for chromatographic analysis.
Estimation of lupeol: The 100 mg of the ethanolic extract was dissolved
in chloroform and making upto 10 mL to get the concentration of 10 mg mL-1
(Test L). The solution was filtered and used for chromatographic analysis.
HPTLC analysis: The samples were spotted in the form of bands with CAMAG
microlitre syringe on a precoated silica gel GF254 plates (20x20 cm with 0.2
mm thickness, E. Merck) using camag linomat V. Automatic sample spotter of band
width 7 mm. The plates were developed in a solvent system in CAMAG glass twin
trough chamber previously saturated with the solvent for 30 min. The distance
was 8 cm subsequent to the scanning, TLC plates were air dried and scanning
was performed on a CAMAG TLC scanner in absorbance at 254, 280 and 538 nm operated
by Wincats software 4.03 (Table 1).
|| Quantification of gallic acid, quercetin and lupeol in A.
|| Phytochemical screening of A. ilicifolius extracts
|P: Positive, N: Negative
RESULTS AND DISCUSSION
Phytochemical studies: Qualitative results of A. ilicifolius extracts
indicated the presence of alkaloids, flavonoids, phenols, carbohydrate, tannins,
terpenoids, glycosides and proteins significantly high amount in ethanolic extract
of A. ilicifolius. The chloroform and acetone extracts having alkaloids,
flavonoids, phenols, carbohydrate, tannins in moderate amount. Ethanolic extract
of A. ilicifolius showed the presence of higher levels of terpenoids
and phenolics as compared to other extracts (Table 2). Hence,
the quantification was carried out in the ethanolic extract.
HPTLC: High performance liquid chromatography method is a very desirable
method for phenolic constituents estimation from A. ilicifolius.
Toluene:Ethyl acetate:Formic acid (6:4:0.8 v/v/v) as mobile phase gave the best
resolution of gallic acid and quercetin (Rf-0.22, 0.36), respectively. Toluene:Ethyl
acetate (7:3 v/v) as mobile phase for lupeol with Rf values 0.70 of the other
components of the methanolic extract of A. ilicifolius. The identity
of band of gallic acid, quercetin and lupeol in A. ilicifolius extract
was confirmed by overlay in UV absorption spectra with those of the standards
gallic acid and quercetin, while identity of bands of lupeol in A. ilicifolius
extract was confirmed by overlay in visible spectra with those of the standard
lupeol using CAMAG TLC scanner 3. The peaks of gallic acid, quercetin and lupeol
in A. ilicifolius extract were confirmed by overlaying the absorption
spectra (Fig. 1). The amount of gallic acid, quercetin and
lupeol was 12.26, 58.45, 71.89 μg mL-1, respectively.
HPTLC chromatogram of ethanolic extract
of Acanthus ilicifolius
, (a) Standard gallic acid, (b) Gallic acid
of AIEEt, (c) Standard lupeol, (d) Lupeol of AIEEt, (e) Standard quercetin
and (f) Quercetin of AIEEt
In conclusion this the first report of gallic acid, quercetin and lupeol was
identified and quantified from A. ilicifolius. Quantification of gallic
acid, quercetin and lupeol showed good resolution and separation. Further, it
will be used to herbal formulation for therapeutic applications.
The authors are grateful to the authorities of Annamalai University and BioMed
Research Management Services, Portonovo, Tamil Nadu, India for providing the