OPTIMIZATION OF MICROWAVE ASSISTED EXTRACTION ... - IJPRD

IJPRD, 2011; Vol 3(5): July 2011 (128 – 134) International Standard Serial Number 0974 – 9446 

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OPTIMIZATION OF MICROWAVE ASSISTED EXTRACTION OF LUTEOLIN FROM LEAVES OF Vitex negundo LINN AND ITS 

COMPARISON WITH CONVENTIONAL EXTRACTION METHOD 

ABSTRACT 

Available online on www.ijprd.com 

Sunita Shailajan* 1 , 

Manasi Yeragi 1 

1 Herbal Research Lab, Ramnarain Ruia College, Matunga, Mumbai. 

The edifice of green chemistry is built on the premise of the reduction 

in waste, materials, hazards, energy and cost involved in the design of 

chemical process. The use of microwave has come to the force in view 

of the unique benefits like energy efficiency, enhanced reaction rates 

and increased yields. In the present work Microwave assisted 

extraction (MAE) and conventional extraction technique has been 

investigated to determine the content of luteolin from leaves of Vitex 

negundo Linn. Luteolin, a flavonoid, is reported to occur widely in 

many medicinal plants. It has been shown to have important biological 

activities. The effect of single factors such as microwave power, 

microwave irradiation time are evaluated and standardized. 

Microwave assisted extraction (MAE) and Liquid Liquid extraction 

(LLE) technique has been comparatively evaluated for their efficiency 

to extract the content of luteolin from leaves of Vitex negundo. 

Luteolin content from the plant matrix using MAE extraction was 

found to be 0.5260±0.0869 (mg/0.5g±SD) and by LLE extraction was 

found to be 0.3316 ± 0.0978 (mg/0.5g±SD).The luteolin content by 

microwave assisted extraction was found to be more than the luteolin 

content by Liquid Liquid extraction technique. The HPTLC method for 

the quantification of luteolin was found to be simple, precise, specific, 

sensitive and accurate and can be used for quality control of raw 

materials. 

. 

Correspondence to Author 

Sunita Shailajan 

Herbal Research Lab, Ramnarain 

Ruia College, Matunga, Mumbai. 

Email 

sunitashailajan@gmail.com 

Key Words 

Vitex negundo, Microwave 

assisted extraction, 

Conventional extraction 

technique, Luteolin, High 

Performance Thin Layer 

Chromatography 

128

International Journal of Pharmaceutical Research & Development ISSN: 0974 – 9446 

INTRODUCTION 

Vitex negundo L. (Verbenaceae) is a hardy plant 

distributed in East Asia, south west China, throughout 

India and cultivated in Pakistan. All parts of the plant 

possess a multitude of phytochemical secondary 

metabolites which impart an unprecedented variety of 

medicinal uses to the plant. It is used for treatment of 

a wide spectrum of health disorders in traditional and 

folk medicine; some of which have been 

experimentally validated. The plant is a component of 

a number of commercially available Herbal, Ayurvedic 

and Unani formulations [1] . Vitex negundo leaves are 

reported to have various therapeutic activities like 

analgesic, anti-asthamatic, anti-rheumatic, astringent, 

antifungal, febrifuge, sedative, vermifuge and brain 

tonic. Vitex negundo leaves contain glucononitol, phydroxybenzoic 

acid,5-hydroxyisophthalic acid, 3,4 

dihydroxybenzoic acid, vitamin C, carotene, casticin , 

orientin , isoorientin , luteolin, luteolin-7-0-glucoside, 

corymbosin, gardenins A and B, iridoid- agnuside, 

aucubin, nishindaside and negundoside etc [2] . Luteolin 

is one of the constituent of Vitex negundo leaves and 

possesses antioxidant 


[3] , cardiovascular [4,5] , anti- 

carcinogenic, anti-mutagenic, anti-angiogenic [6-9], 

estrogenic [10] , anti-obesity [11] , anti-viral [12] , 

antibacterial [13] and gastroprotective properties [14] . 

The conventional methods operate through cell 

permeation followed by solubilising the active 

constituents by the extracting solvent. These 

conventional methods are time and solvent 

consuming, thermally unsafe. Liquid-liquid extraction, 

also known as solvent extraction and partitioning, is a 

method for extraction of a substance from one liquid 

phase into another liquid phase. The Microwave 

Assisted Extraction Process is a high-speed method 

used to selectively extract target compounds from 

various raw materials. Microwave assisted extraction 

uses energy of microwave radiation to heat solvents 

quickly and efficiently. With the increasing demand 

for more environmental friendly methods, 

Microwave assisted extraction is used for fast 

extraction of constituents from plant matrix [15, 16] . 

OBJECTIVE 

Present study describes the development and 

optimization of rapid, reliable and sensitive method 

of extraction of luteolin from Vitex negundo leaves 

using MAE and its comparison with conventional 

method (LLE) of extraction for amount of luteolin 

using HPTLC technique. This is the first report for 

quantitation of luteolin from Vitex negundo Linn. 

leaves using different extraction techniques. MAE 

thus, appears to be the most efficient and 

reproducible method of extraction and provides an 

alternative for extraction of pharmacologically 

important phytoconstituent like luteolin. 

EXPERIMENTAL 

Plant Material 

Leaves of Vitex negundo were collected from Malvan 

region of Maharashtra, India and were authenticated 

by Agharkar Institute (Auth08-72) and voucher 

specimens were deposited in Herbal Research Lab of 

Ramnarain Ruia College. The samples were powdered 

and sieved through 85 mesh size and stored in airtight 

containers for further use. 

Chemicals 

Analytical grade solvents Toluene, Ethyl acetate, 

Formic acid, Distilled Water were procured from 

Qualigens Fine Chemicals, Mumbai. Standard luteolin 

(≥ 98% purity) was procured from Sigma Aldrich, 

Germany. (Figure 1) 

Figure1. Structure of luteolin 

Microwave- assisted extraction 

129


Microwave assisted extraction was performed on a 

modified domestic microwave oven (Model 

GMC25E09MRGX, Godrej, India) equipped with a 

magnetron of 2450 MHz with nominal maximum 

power of 700 W operated at 3 power levels and the 

time controller. Methanolic extract was selected for 

analysis since it gave maximum extraction in 

comparison with other solvents. Linearity was 

performed by varying the amount of drug and 

concentration of solvent. 500 mg dissolved in 10ml 

Methanol gave good resolution of bands using HPTLC 

technique which was optimised for further analysis. 

The plant powder was placed into the round bottom 

flask fitted with a water condenser in addition with 

solvent subjected to different time of irradiation and 

irradiation power. Before extraction the crude powder 

was soaked in 1 ml of distilled water for 2 minutes. 

After the extraction time had elapsed the vessels were 

allowed to cool to room temperature and the samples 

were centrifuged at 4000rpm. 

Optimisation of microwave power 

The extraction procedure was employed at 10%, 20%, 

30%, 40% and 50 % for microwave power optimisation. 

The extracts were further analysed by HPTLC for the 

concentration of Luteolin to optimize the power. 

Optimisation of microwave time 

The extraction procedure was employed at 1min, 3 

min, 5 min, and 10 min for microwave time 

optimisation. The extracts were further analysed by 

HPTLC for the concentration of Luteolin to optimize 

the power. 

Conventional Extraction technique 

Liquid-liquid Extraction (LLE) was performed in a clean 

stopper tube with different extraction solvents; the 

exhaustive extraction was performed on optimised 

amount of plant material with solvent. The extraction 

was performed for 12 hours on rotary shaker at 12-15 

rpm. 

HPTLC conditions 

Chromatographic separation was achieved on HPTLC 

plates (20 X 20 cm) precoated with silica gel 60 F254 (E. 

Merck) of 0.2 mm thickness with aluminium sheet 

support. Samples were spotted using CAMAG Linomat 

Table1. Linearity parameters 


IV Automatic Sample Spotter (Camag Muttenz, 

Switzerland) equipped with syringe (Hamilton, 100 µL). 

Plates were developed in a glass twin trough chamber 

(CAMAG, 20 X 10 cm) presaturated with mobile phase. 

Scanning device used was CAMAG HPTLC Scanner 2 

equipped with CATS3 software. The experimental 

condition was maintained at 25 ± 2 0 C. 

HPTLC FINGERPRINTING PROFILE 

Standard Stock and sample solution 

The stock solutions were transferred to volumetric 

flask in order to obtain aliquots of luteolin (20- 150 

µg mL -1 ) and volume was made upto 10 ml with 

methanol. Accurately weighed (500 mg) of plant 

powder was placed in a stoppered tube and 10 mL of 

methanol was added to it. The sample solutions 

extracted by MAE and by LLE techniques were further 

analyzed using HPTLC technique. 

Further three quality control samples (LQC: MQC: 

HQC) of Luteolin (25, 60, 120 µg mL-1) were prepared 

for precision, accuracy and ruggedness studies. 

Solvent system 

Solvent system consisted of Toluene: Ethyl acetate: 

Formic acid (3: 3: 0.8 v/v/v) has been used in this 

method to resolve and to quantitate luteolin from 

Vitex negundo leaves. 

Calibration Curve for luteolin 

For calibration curve the plate was scanned and 

absorption spectra were recorded at start, middle 

and end position of the band to check the purity of 

the band. The plates were scanned at 254 nm using 

CAMAG TLC Scanner 2 and CATS software. The peak 

areas were recorded. Calibration curve of luteolin 

was obtained by plotting peak areas vs. 

concentration of luteolin applied. 

METHOD VALIDATION 

ICH guidelines were followed for the validation of the 

developed analytical method (CPMP/ICH/281/95 and 

CPMP/ICH/381/95). (Table 1) 

130


y = mx + c, y is peak area, m is the slope, x is the concentration and c is the intercept. 

Instrumental Precision 

Instrumental precision was checked by repeated 

scanning (n = 7) of the same spot of luteolin (100 µg 

mL -1 ) and further expressed as %CV. 

Repeatability 

The repeatability of the method was affirmed by 

analysing 60 µg mL -1 of luteolin on a TLC plate (n = 5) 

and expressed as %CV. 

Inter-Day and Intra-Day Precision 

Variability of the method was studied by analysing QC 

samples (25, 60, 120 µg mL -1 ) of luteolin on the same 

day (intra-day precision) and on different days 

(interday precision) and the results were expressed as 

%CV. 

Limit of Detection and Limit of Quantitation 

For the evaluation of limit of detection (LOD) and limit 

of quantitation (LOQ) different concentrations of the 

standard solutions of luteolin were applied along with 

methanol as blank and determined on the basis of 

signal-to-noise (S/N) ratio. LOD was determined at an 

S/N of 3: 1 and LOQ at an S/N of 10: 1. 

Recovery 

The accuracy of the method was assessed by 

performing recovery study at two different levels (25 

and 50 % spiking of luteolin in plant matrix). The 

percent recoveries and the average percent recoveries 


Parameters Result 

Linearity range 20 to 150 µg mL -1 

Slope (m) 15.672 

Intercept(c) 226.63 

Correlation coefficient (R) 0.997 

LOD 10 µg mL -1 

LOQ 20 µg mL -1 

System Suitability (n =5 % CV) 0.09 

Instrument Precision (n=6 % CV) 0.11 

Intraday (precision) (n=3 % CV) 1.62 

Interday (precision) (n=3 % CV) 1.12 

were calculated. It was carried out for sample 

extracted by MAE and LLE. 

Specificity 

Specificity was ascertained by analyzing standard 

compounds and samples. The band for luteolin from 

sample solution was confirmed by comparing the Rf 

and spectra of the band to that of the standard. The 

peak purity of the compound was analysed by 

comparing the spectra at three different levels, i.e. 

start, middle, and end positions of the bands. 

Ruggedness 

Ruggedness of the method was assessed by 

deliberately incorporating the small variations in the 

optimized chromatographic condition. Effect of 

change in analyst, change in mobile phase 

composition [Toluene: Ethyl acetate: Formic acid 

(3.1: 3: 0.8 v/v/v) and Toluene: Ethyl acetate: Formic 

acid (3: 3: 0.9 v/v/v)] and change in spotting volume 

(9 µL and 11 µL) on the response and Rf of quality 

control samples was observed. 

RESULTS 

For the extraction procedure employed for 

optimisation of power and time 30% power and 3 

min time was optimised for the extraction procedure 

according to the luteolin content.Of the various 

solvent systems tried, mixture containing Toluene: 

Ethyl acetate: Formic acid (3: 3: 0.8 v/v/v) gave the 

131


best resolution of luteolin (Rf = 0.49) from the other 

components of the methanolic extract of Vitex 

negundo leaves. The identity of bands of luteolin in 

plant matrix was confirmed by overlay in UV 

absorption spectra with that of the standard luteolin 

using Camag TLC scanner 2. The purity of band of 

luteolin in the plant extract was confirmed by 

overlaying the absorption spectra at the start, middle 

and end position of the bands (Figure 4). 

Figure 3. Linearity of luteolin 

CONC 

3000 

2500 

2000 

1500 

1000 

500 

0 

LINEARITY OF LUTEOLIN 

y = 15.672x + 226.63 

R 2 = 0.9967 

0 50 100 150 200 

AREA 

Figure 4. Spectrum of MAE extracted and LLE 

extracted sample with luteolin standard 

The pharmacologically active phytoconstituent 

luteolin was quantified from Vitex negundo leaves by 

HPTLC densitometric method using MAE and LLE 

extraction technique. The developed method was 

validated in terms of precision, repeatability and 

accuracy (Table 1). The linearity range for luteolin 

was found to be 20 to 150 µg mL -1 with correlation 

coefficients (r values) 0.997, (Table 1).The TLC 

densitometric method was found to be precise with 

average % RSD for intra-day precision 1.62 and for 

inter-day precision 1.12 for different concentrations 


of luteolin (Table 1). This indicates that the method is 

precise. The LOD for luteolin was found to be 10 µg 

mL -1 and LOQ value was found to be 20 µg mL -1 

(Table 1) respectively. The average recovery at three 

different levels of luteolin was found to be 98.71 % 

(Table 2). Luteolin content from the plant matrix 

using MAE extraction was found to be 0.5260±0.0869 

(mg/0.5g±SD) and by LLE extraction was found to be 

0.3316 ± 0.0978 (mg/0.5g±SD). Ruggedness of the 

method by change in analyst and change in mobile 

phase composition showed variations within 

acceptable limits. Change in spotting volume at 9 and 

11 µL did not affect the Rf of examined 

phytoconstituent but change in response was 

observed which was within acceptable limits. Hence 

by using 0.5g of plant drug with 10 ml methanol with 

30% power gave the maximum yield of Luteolin in 5 

mins by MAE extraction than with LLE extraction for 

which the same amount of drug and solvent was 

used. This is the first method reported for 

quantitation of free luteolin content in leaves of Vitex 

negundo extracted by MAE and LLE technique (Fig. 

2). 

Figure 2. Chromatographic plate of LLE and MAE 

extract of Vitex negundo leaves with luteolin 

A B C 

Track A: LLE extract 

Track B: Luteolin Standard 

Track C: MAE extract 

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Table 2. Recovery of luteolin from Vitex negundo leaves 

DISCUSSION 

Microwave extraction can be performed at higher 

temperatures and extraction time can be reduced 

drastically. It is an innovative solvent-extraction 

technology, offers a superior alternative to conventional 

techniques owing to its efficient volumetric heat 

production. Considering the biological importance and 

wide occurrence of luteolin in plant materials this 

method has great advantages over the conventional 

extraction techniques. This method can be applied for 

the extraction of high-value compounds from natural 

sources including medicinally important constituents, 

phytonutrients, neutraceutical, functional food 

ingredients and pharma actives from biomass. 

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