INTRODUCTION:

Cynodon dactylon (L.) Pers. is a rapid growing perennial grass belonging to the family Poaceae¹. Also known as Durva (meaning, which is cut or eaten by animals), Bermuda grass, Dog’s Tooth grass, Devil’s grass, Couch grass, Scutch grass, Indian Doab, Dhub, Doob and Durba in different parts of world², C. dactylon is found abundantly as weed and can readily take possession of any uncultivated area. The whole herb as well as its root stalk is used for medicinal purpose³. It is used as a folk remedy for various ailments like calculus, convulsions, cough, cramps, cystitis, diarrhea, dropsy, dysentery, epilepsy, hemorrhoids, leucoderma, headache, hemorrhage, hypertension, bronchitis, asthma, measles, snakebite, sores, urogenital disorders, warts, wounds, eye disorders etc. It is also useful against pains, inflammations, toothache and grippe in children.

The expressed juice of plant act as astringent and is applied to bleeding cuts and wounds to stop bleeding⁴. Various fractions of this plant possess pharmacological actions like antioxidant⁵, antidiarrheal⁶, immunomodulatory⁷, antiulcer⁸, antiarrhythmic⁹, CNS depressant¹⁰, cardioprotective¹¹, hepatoprotective¹² and high antidiabetic potential along with significant hypoglycemic and hypolipidemic effect^13-14. C. dactylon contains β-sitosterol, β-carotene, vitamin C, palmitic acid, triterpenoids, alkaloids like ergonovine and ergonovinine, phenolic phytotoxins like ferulic acid (FA), syringic acid, vanillin acid and p-coumaric acid¹⁵. The plant also contains crude proteins, carbohydrates and mineral constituents like oxides of magnesium, phosphorous, calcium, sodium and potassium¹⁶. Various phenolics compounds have attracted the attention of food and medical scientists because of their fragrant aroma, antioxidant and anti-inflammatory properties¹⁷. Ferulic acid is also known to possess pharmacological actions^18-19.

Traditional method for extraction of ferulic acid is by refluxing for 4–5 h in 70% ethanol. However, ferulic acid is heat sensitive and its structure would be destroyed during long time heating in ethanol²⁰. Therefore, there is always a need for better extraction techniques which are more efficient (in terms of time, consumption of solvent and energy input), conserve the integrity of the target molecule and increases the extraction yield. One such technique is microwave-assisted extraction (MAE), in which microwave energy is used to heat solvent in contact with the sample in order to partition phytoconstituents from the sample matrix into the solvent^21-22. Extraction of ferulic acid by microwave-assisted extraction (MAE) from Radix Angelicae sinensis has been reported²⁰.

Various chromatographic analytical techniques have been utilized for phytochemical analysis of C. dactylon like TLC²³, HPTLC using β-sitosterol as a marker for quantitation^24-25, HPLC for fingerprint development¹¹ and separation of carotenoids²⁶ and GC-MS for determination of bioactive components²⁷. The HPTLC analysis of Lycopodium clavatum (stem) using ferulic acid for the purpose of quality control has been reported¹⁹.

Though ferulic acid is known to be one of the phytoconstituents of C. dactylon, but work related to its MAE, identification and quantitation from C. dactylon was still unexplored. The present work therefore describes the process of microwave assisted extraction (MAE) of ferulic acid from the whole plant of C. dactylon in conjunction with gradient solvent polarity technique followed by the development and validation of a simple and precise HPTLC method for quantitative determination of ferulic acid in the extracts of C. dactylon whole plant.

MATERIAL AND METHODS:

Plant Material and Chemicals:

Fresh whole plant of Cynodon dactylon (L.) was collected during December 2010 from garden of CCRAS, New Delhi, India and send to National Institute of Sciences Communication and Information Resources (NISCAIR), New Delhi for identification and the specimen voucher (NISCAIR/RHMD/Consult/2010-2011/1632/230) has been procured. After authentication, the whole plant was cleaned, dried under a gentle stream of air in the laboratory for 4-5 days till no loss in weight (temperature 25±2⁰C) and powdered in an electric grinder. Solvents and chemicals were used of analytical grade (E. Merck, Germany). The standard ferulic acid (≥ 98%) was procured from Sisco Research Laboratory, Mumbai.

MAE method:

Powdered whole plant of C. dactylon (4 g each) was extracted with variety of solvents ranging from non-polar to polar i.e. n-hexane, dichloromethane, ethyl acetate, acetone and ethanol in a domestic microwave (900W, frequency 2450MHz) separately. Before microwave irradiation, a pre-leaching time of 5 min was given to each suspension. The various experimental conditions for optimization of extraction parameters are given in Table 1.

Standard preparation:

Standard stock solution containing 1mg mL^-1 of ferulic acid was prepared by dissolving 10 mg ferulic acid in 10 mL methanol. The stock solution was further diluted to attain final concentration of 25 μg mL^-1 for HPTLC analysis.

Sample preparation:

Each of the concentrated extract was re-dissolved in methanol and filtered through 0.45μm filter. The concentration of individual sample extracts used for HPTLC analysis is given in Table 2.

HPTLC Instrumentation:

Camag Switzerland HPTLC system equipped with an automatic (TLC sample applicator) Linomat 5 fitted with 100 µL syringe (Hamilton, Switzerland), TLC scanner device 4 (for multi wavelength scanning), TLC visualizer, winCATS planar chromatography manager software version1.4.5 and twin trough glass tank (20 x10 cm) was used for the analysis of FA.

Calibration curve of Ferulic acid:

2 μL, 4 μL, 6 μL, 8 μL, 10 μL and 12 μL of ferulic acid solution (25 μg mL^-1), corresponding to different concentrations (50, 100, 150, 200, 250 ng and 300 ng spot^-1, respectively) were applied to the TLC plate for preparing six points linear calibration curve.

Chromatography:

The sample solution and standard solution were applied on the stationary phase i.e. TLC plate consisting of aluminum sheets pre-coated with silica gel 60F₂₅₄ using Linomat 5 applicator. The plates were developed in twin trough glass tank using a mixture of Toluene: Ethyl acetate: Formic acid (8.5: 1.5: 0.1, v/v/v) as mobile phase at room temperature (28±2⁰C). The composition of the mobile solvent was optimized to achieve good separation. Wavelength for detection of ferulic acid was selected after evaluation of complete UV spectrum of ferulic acid. Quantitative analysis of the chromatogram was performed in the remission /absorbance mode at λ 270 nm for ferulic acid. The slit dimension was 6.00 mm X 0.40 mm, macro scanning speed 100 nms^-1 and data resolution 1 nm step^-1 and developed plate was visualized at different wavelengths (Figure 1A-1B).

Fig.1A. HPTLC fingerprint of MAE extracts and ferulic acid at 254 nm

MAE Extracts (1 and 8 = n-Hexane; 2 =Dichloromethane; 3= Ethyl acetate; 4= Acetone; 6 and 7= Ethanol), FA= Ferulic acid and 5= Cylindrin.

Fig. 1B. HPTLC fingerprint of MAE extracts and ferulic acid at 366 nm

MAE Extracts (1 and 8 = n-Hexane; 2 =Dichloromethane; 3= Ethyl acetate; 4= Acetone; 6 and 7= Ethanol), FA= Ferulic acid and 5= Cylindrin.

Method Validation:

In order to be a useful method for qualitative and quantitative estimation, the method was examined on the parameters of specificity, linearity, precision, accuracy and recovery²⁸.

Specificity:

The specificity of the method was determined by analyzing the sample along with the standard ferulic acid. The band for ferulic acid in the ethanol extract sample was confirmed by comparing the R_f and UV spectra of the band to that of the standard, Figure 2-3.

Linearity and range :

For linearity, six different concentrations 50, 100, 150, 200, 250 and 300 ng spot^-1of ferulic acid (25 µg mL^-1) were applied to the HPTLC plate. A six point calibration curve was obtained by plotting the concentration of standard ferulic acid versus peak area as evident in Table 5. Regression analysis data for ferulic acid was obtained, Table 5.

Repeatability (intraday) :

Six replicates of 4 µL of ferulic acid (25µg mL^-1) were analyzed by the proposed method to determine variation due to the chromatographic conditions (system precision). The % RSD of R_f and peak area were calculated and given in Table 5.

Limit of detection and Limit of quantitation (LOD and LOQ):

The ferulic acid in sample extract was identified on the basis of R_f and UV-Vis spectral overlaying with the standard. Standard was diluted and applied on HPTLC plate to plot the calibration curve. LOD was determined based on the lowest concentration detected by the instrument from the standards while the LOQ was determined based on the lowest concentration quantified in the sample accurately. The results are shown in Table 5.

Recovery:

For percentage recovery, three known concentrations i.e. 50, 75, 100 ng spot^-1 of standard ferulic acid solution (25 µg mL^-1) were spiked on band preloaded with 2 µL of extract (ethanol). The bands were applied in triplicates and analyzed using the developed method. The results obtained are presented in Table 5.

RESULTS AND DISCUSSION:

MAE Mechanism:

Microwave-assisted extraction consists of heating the solvent in contact with the sample by means of microwave energy. The process involves disruption of hydrogen bonds as a result of microwave-induced dipole rotation of molecules and migration of ions, which enhances penetration of the solvents into the matrix, allowing dissolution of the components to be extracted^[29-30]_.Usually higher the dielectric constant, higher is the degree of microwave absorption. Therefore, it is best to choose a solvent that has a high dielectric constant as well as a high dissipation factor as evident in Table 3.

Tab. 3. Physical factors for commonly used solvents in MAE

Solvent	Boiling Temp. (⁰C)	Dielectric Constant^a (ε)	Dissipation Factor (tan δ)
n-Hexane	68.7	1.89	0.10 x 10^-4
Dichloromethane	39.8	8.93	4117 x 10^-4
Ethyl acetate	71.1	6.02	5316 x 10^-4
Acetone	56.2	20.7	5555 x 10^-4
Ethanol	78.5	24.3	2500 x 10^-4

^a= determined at 20⁰C

Effect of solvent nature and volume on extraction yield:

4 g of crushed whole plant was extracted with 20 mL of different solvents (mentioned in Table 1) for 120 s. The microwave power was 900 W and the temperature was 50⁰C. The result of extraction yield is shown in Table 1. The yield of FA reached the maximum when the ethanol was used solvent. Different solvent volumes (10, 15, 20 mL and 25 mL) of ethanol were taken for the extraction of FA, while other conditions (microwave power, irradiation time and temperature) were same as above. The yield of FA reached maximum when the solvent volume was 20 mL, as evident in Table 2 and Table 4. Therefore, 20 ml of ethanol was chosen for optimum extraction of FA from whole plant of C. dactylon.

Tab. 4. Optimization of MAE conditions for FA extraction

Parameters	Conditions
Ethanol Volume (mL)	10	15	20	25
Microwave Power (W)	700	800	900	1000
Irradiation time (s)	30	60	120	180
FA Yield (µg/100 g dry plant, w/w)	231.22	250.54	261.49	255.60

Effect of microwave power:

Table 4, indicates that there was signiﬁcant improvement in extraction yield with the increase in microwave power between 800 W and 900 W. However, a sharp decrease in extraction yield was obtained between 900 W microwave power and 1000 W microwave power with the increase in extraction time. No signiﬁcant increase in extraction yield was observed by using higher power level with increasing extraction time. Based on the above observation 900 W microwave power was considered to be optimum.

Effect of irradiation time:

Effect of microwave irradiation time (30, 60, 120 s and 180 s) at 900 W microwave power on the extraction yield of FA were calculated, Table 4. Three intervals were studied during the process of microwave extraction. Initially, a short rise in extraction yield was seen between 30 s and 60 s which indicated the ﬁrst quantities extracted located at the surface of root particles, representing approximately 250.54 µg of the FA. Between 60 s and 120 s irradiation time, extraction yield of FA was highest (261.49 µg/ 100 g plant) due to internal warming of the natural moisture located in the plant cells. Since no signiﬁcant difference in extraction yield was obtained between 120 s and 180 s extraction time, hence irradiation time of 120 s was considered optimum for maximum extraction of FA.

Chromatography:

Composition of the mobile phase was varied based on polarity to achieve good separation and resolution. The desired resolution with symmetrical and reproducible peaks were achieved by using a mixture of Toluene: Ethyl acetate: Formic acid (8.5: 1.5: 0.1, v/v/v) as mobile phase, Figure 1A- 1B. Identification of ferulic acid in the sample extracts was done by overlaying of UV spectra and matching of R_fvalue with the standard ferulic acid as evident in Figure 2 and Figure 3, scanned at 270 nm.

Fig. 2. HPTLC chromatograph of standard ferulic acid at 270 nm

Fig. 3. HPTLC chromatograph of MAE extract (ethanol) of C. dactylon whole plant at 270 nm

Method validation:

The method was validated for its linearity, precision, accuracy, LOD and LOQ. The calibration equation shown in Table 5 indicates the response is linear function of concentration versus peak area in the range of 50 to 300 ng spot^-1 of ferulic acid. The slope (m), intercept (c), correlation coefficient (r) and r- square (r²) were 14.51, - 448.5, 0.998 and 0.997 respectively. The method showed acceptable precision with %RSD values less than 4% for the peak areas and R_fasevident inTable 5. Thus, the method was found suitable for the purpose of analysis. The limit of detection (LOD) and quantitation (LOQ) were found 16.314 ng and 49.437 ng respectively, which indicate adequate sensitivity of the method. The recovery analysis at three different level of ferulic acid was done and the % recovery was found to be in the range 97.36 % to 103.61 % as shown in Table 5, indicating adequate sensitivity and accuracy of the method.

Tab. 5. HPTLC method validation data of FA

Parameters	Results
Linearity Range (ng spot^-1) Linear equation Slope (m) Intercept (C) Correlation coefficient (r) r-square (r²) Standard deviation (sd)	50 – 300 y = 14.51x - 448.5 14.51 - 448.5 0.9987 0.997 71.91
Precision R_f of FA = 0.12 Intraday ( n=6), (%RSD) Repeatability of peak area Repeatability of R_f	2.30 3.84
Limit of Detection(LOD)	16.314 ng
Limit of Quantitation (LOQ)	49.437 ng
Specificity	Specific
Recovery (%) Level 1- 50% Level 2- 75% Level 3- 100%	97.366 102.481 103.611

Quantitative determination of ferulic acid:

Ferulic acid was quantitatively determined in extracts of C. dactylon whole plant using the proposed and validated HPTLC method. The amount of ferulic acid in the MAE extracts (ethyl acetate, acetone and ethanol) was found to be in the range of 75.89 – 261.79 µg per 100 g dry whole plant of C. dactylon as evident in Table 2. Among all the MAE extracts, the concentration of ferulic acid was found to be the highest in ethanol extract followed by remaining MAE extract in the order ethyl acetate > acetone. Ferulic acid was not detected in n-hexane extract and dichloromethane extract due to less polar nature of the extracting solvent. Further, identification and quantitation of the other secondary metabolites present in whole plant of C. dactylon by the developed method or other new method is currently under progress.

CONCLUSION:

Solvent polarity based MAE of ferulic acid in the whole plant of C. dactylon Linn. and its quantitation by HPTLC is reported. MAE extract obtained from ethanol showed highest yield of extract as well as highest amount of ferulic acid. In the proposed study 20 mL of ethanol, microwave irradiation time of 120 s and microwave energy input of 900 W were found to be most favorable conditions for the maximum extraction of ferulic acid from the whole plant of C. dactylon. The proposed extraction technique was found to be rapid, simple, eco-friendly and economical because of its various attributes like minimization of solvents consumed, energy required and time utilized for extraction. The developed analytical method was found to be simple, sensitive and accurate and can be employed for fingerprint development and quantitative analysis of FA in C. dactylon and its preparations for quality control purpose.

ACKNOWLEDGEMENT:

The authors are grateful to Prof. M. P. Singh (Principle), M.M.H. College, Ghaziabad and Director General, CCRAS, New Delhi for providing necessary facilities. The authors are grateful to Dr. Roopak Kumar, Vice President, Arbro Analytical Division, New Delhi for necessary help related to study. The authors also appreciate the kind help extended by Dr. D. K. Aggarwal, R.O. (Botany), CCRAS and Dr. H. B. Singh, Scientist, NISCAIR, New Delhi for plant material identification.

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Received on 10.08.2011 Modified on 19.08.2011

Asian J. Research Chem. 4(9): Sept, 2011; Page 1460 -1465

Solvent used for MAE	Yield of MAE crude extract (mg)	Sample conc. (mg mL^-1)	Yield of FA (µg) / 100 g dry whole plant
n- Hexane	16.9	11.26	Not detected
Dichloromethane	17.7	11.80	Not detected
Ethyl acetate	28.0	18.66	86.23
Acetone	25.5	17	75.89
Ethanol	40.5	27	261.49

Powdered whole plant (g)	Solvent used for extraction	Solvent volume (mL)	Irradiation time (s)	Microwave power input (W)	% extractive yield
4	n- Hexane	20	120	900 W	0.421
4	Dichloromethane	20	120	900 W	0.1067
4	Ethyl acetate	20	120	900 W	0.699
4	Acetone	20	120	900 W	0.6374
4	Ethanol	20	120	900 W	1.0120