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HPTLC methods for the Rapid Determination of Adhatoda vasica L. Glycyrrhiza glabbra L., Phyllanthus embelica L. and Camellia sinensis L. in a polyherbal formulation (INSTY).

Byline: Zeeshan Ahmed Sheikh, Aqib Zahoor, Kiran Shafiq, Saleha Suleman Khan, Ajmal Khan and Khan Usmanghani

Summary:

Introduction:

An Insty Granule is a polyherbal formulation, and it is widely used to treat upper respiratory tract infections. It contains expectorant, anti-inflammatory, mucolytic and anti- pyretic properties. The major active constituents are vasicine, gallic acid, caffeine and glycyrrhizin. Insty a poly herbal formulation of eight herbs were investigated for its phytochemical evaluation. Biomarkers of about four herbs both qualitatively and quantitatively were investigated. Methods: The solvent systems used were ethyl acetate: chloroform: ethanol: ammonia (6: 3: 1: 1) for vasicin, ethyl acetate: chloroform: formic acid (12: 15: 3) for gallic acid, ethyl acetate: methanol: water (100: 13.5: 10) for caffeine, and methanol: water: acetic acid (70: 30: 0.5) for glycyrrhizin. Results: The methods showed a good linear relationship (r2 = 0.999) in the concentration range 251500 ng per spot. It was found to be linear, accurate, precise, specific, robust and can be applied for quality control and standardization.

Conclusion: In present study rapid and inexpensive qualification methods for the quality control of Adhatoda vasica L. Glycyrrhiza glabbra L., Phyllanthus embelica L. and Camellia sinensis L. on thin layer chromatography (TLC) were developed and validated on silica gel. HPTLC is most suitable technique because of quantification of number of samples at low operating cost, easy sample preparation, short analysis time and analytical assurance.

Keywords: Insty Granule; Biomarkers; glycyrrhizin; HPTLC.

Introduction

Conventional phytomedicine are naturally occurring, plant-based medicines which have been used to promote health, treat and cure illnesses and enhance immunity throughout the world. Traditional phytomedicines are getting considerable interest in global health debate [1]. But, the major hindrance in the approval of herbal remedies is the lack of standard quality control profiles. The standardization of herbal products i.e. the fingerprinting of the markers/biomarkers in the final product has insinuation in effectiveness and safety. Due to the complex nature and natural inconsistency of the chemical compounds in poly-herbal drugs, it is not easy to set quality control parameters. It is suggested that the modern analytical techniques are probably supportive to resolve these problems [2,3]. HPTLC is most suitable technique because of quantification of number of samples at low operating cost, easy sample preparation, short analysis time and analytical assurance [4,5].

Insty Granule is a polyherbal formulation, and it is widely used to treat upper respiratory tract infections. It contains expectorant, anti-inflammatory, mucolytic and anti-pyretic properties. The major active constituents are vasicine, gallic acid, caffeine and glycyrrhizin. From such polyherbal formulations identification, separation and quantification of chemically active compounds is very difficult [6].

According to literature survey, above mentioned chemical constituents has various medicinal properties. The major active constituent of Adhatoda vasica (Nees) commonly known as Bansa is vasicine [7]. Vasicine, is a major bioactive pyrroquinazoline alkaloid, has been reported as bronchodilator [8,9], a respiratory stimulant and hypotensive in action. It is also reported to have antihistaminic effect against histamine-induced bronchospasm [10]. The fruits of Phyllanthus embelica consumed as fruit or in the form of food products as a rich source of vitamin C [11]. Gallic acid is the active component of Embelica officinalis fruit and has been reported as antioxidant, hepatoprotective and cardio- protective [12,13]. It is hydrolysable tannin and has been found to show cytotoxicity against cancer cells without damaging healthy cells and used as an astringent in cases of internal haemorrhage [14].

Caffeine is the major alkaloid in dry leaves of Camellia sinensis spp. present in the concentration of about 2-5% [ 15]. After metabolism in the liver, due to anti-inflammatory and broncho-protective effect, caffeine is converted by the cytochrome P450 oxidase enzyme system into metabolic dimethylxanthines, viz. theophylline which relaxes smooth muscles of the bronchi, and is used to treat asthma [16]. The main compound in the roots and stolons of Glycyrrhiza glabra is sweet flavored glycyrrhizin which is a oleanane-type triterpene saponin [17]. Traditionally, it is used as a laxative, emmenagogue, contraceptive, galactagogue, anti- asthmatic drug and antiviral agent [18]. The liquorice extract is very beneficial for the treatment of sore throat, cough and bronchial catarrh. It has anti-tussive and expectorant effects that help to expel clogging in the upper respiratory tract as it enhances tracheal mucus secretion. The demulcent action is also related to glycyrrhizin [19].

The development in chromatographic techniques made it feasible to quantify the chemical constituents in polyherbal formulations with comparatively little extraction technique using high performance thin layer chromatography (HPTLC) [20]. Present study deals with development and validation of methods for quantification of some of the important marker compounds viz. vasicine, gallic acid, caffeine and glycyrrhizin in insty granules with some modifications to suit the sample.

Experimental

Chemicals and Reagents

The formulation was developed in indigenous herbal pharmaceutical company Herbion Pakistan (Pvt) Ltd. Karachi. All the chemicals and solvents used in the experiments were of analytical grade. Vasicine standard was in house isolated (purity 85%). Gallic acid standard (purity 99%) was purchased from Across Organics, Belgium. Caffeine standard (purity 99.3%) was purchased from Merck, Darmstadt, Germany. Glycyrrhizin standard (purity 98.6%) was purchased from Sigma-Aldrich.

Instrumentation

Spotting device: Linomat V Automatic sample applicator linked to Wincats software;

Syringe: 100 L; TLC development chamber: Glass twin trough chamber (20 x 20 cm);

Densitometer: TLC Scanner linked to WinCats software; all were purchased from CAMAG (Muttenz, Switzerland).

HPTLC plates: 20 x 10 cm, 0.2 mm thickness precoated with silica gel 60 F254 and silica gel 60 RP 18 F254S TLC plate (0.16-0.2 mm thickness) was purchased from Merck, Darmstadt, Germany.

Standard and Sample Solutions Preparation vasicine, gallic acid, caffeine and glycyrrhizin

A stock solution of vasicine, gallic acid, caffeine and glycyrrhizin was prepared by dissolving 6.5 mg,2.5,6.0mg,5.0mg were dissolve in methanol and making up the volume of each component to 25 ml respectively. From this stock solution standard solutions vascicine of 13 g/ml to 156 g/ml were prepared by transferring aliquots (0.5, 1 to 6 ml); From stock gallic acid solution standard solutions of 10 g/ml to 60 g/ml were prepared by transferring aliquots (1 to 6 ml) ; of stock solution to 10 ml volumetric flasks and adjusting the volume with methanol; From this stock solution caffeine standard solutions of 24 g/ml to 140 g/ml; were prepared by transferring aliquots (1 to 6 ml) and from glycyrrhizin stock solution standard solutions of 20 g/ml to 120 g/ml were transferred to 10 ml of each component volumetric flasks and adjusting the volume with methanol.

Sample Preparation for Vasicine, Caffeine, Gallic acid and Glycyrrhizin

About 3.0 g of formulation powder was dissolved in 20 ml water and 3 ml hydrous ammonia and was extracted with chloroform (30 ml x 5). Chloroform fraction was collected in same flask and evaporated to dryness under vacuum. The dry residue was dissolved in 10 ml methanol. The obtained solution was filtered through a filter with pore size 0.45 m and used for chromatography.Gallic acid sample 5 gm of formulation powder was dissolved in 25 ml methanol and heated at water bath for 10 min. it was cooled and filtered through Whatmann filter paper. Similarly Glycyrrhizin 5 gm of formulation powder was dissolved in 25 ml of 70 % methanol and sonicated for 15 min. the solution was carefully filtered through Whatman filter paper.

Calibration Curve of Vasicine, Gallic Acid, Caffeine and Glycyrrhizin

Vasicine 10 l of standard solution was applied in triplicate on TLC plates. The plate was developed in a solvent system of ethyl acetate:

Chloroform: ethanol: ammonia (6: 3: 1: 1) up to a distance of 8 cm. After development, the plates were dried in air and scanned densitometrically at 300 nm. The peak areas were recorded. Calibration curve of vasicine was prepared by plotting peak areas vs. concentration (Fig. 1). Gallic acid 10 l of standard solution was applied in triplicate on TLC plates. The plate was developed in a solvent system of ethyl acetate: chloroform: formic acid (12: 15: 3) up to a distance of 8 cm. After development, the plates were dried in air and scanned densitometrically at 273 nm. The peak areas were recorded. Calibration curve of gallic acid was prepared by plotting peak areas vs. concentration (Fig. 2). Caffeine 10 l of standard solution of caffeine was applied in triplicate on TLC plates. The plate was developed in a solvent system of ethyl acetate: methanol: water (100: 13.5: 10) up to a distance of 8 cm. After development, the plates were dried in air and scanned densitometrically at 254 nm.

The peak areas were recorded. Calibration curve of caffeine was prepared by plotting peak areas vs. concentration (Fig. 3). Glycyrrhizin 10 l of standard solution was applied in triplicate on silica gel RP 18 F254S TLC plates. The plate was developed in a solvent system of methanol: water: acetic acid (70: 30: 0.5) up to a distance of 8 cm. After development, the plates were dried in air and scanned densitometrically at 254 nm. The peak areas were recorded. Calibration curve of glycyrrhizin was prepared by plotting peak areas vs. concentration (Fig. 4).

Quantification of Vasicine, Caffeine, Gallic Acid and Glycyrrhizin

Vascicine 10 l of sample solution was applied in triplicate on a pre-coated silica gel 60 F254 TLC plate (0.2 mm thickness) along with 10 l of standard solution in triplicate with the CAMAG Linomat V automatic sample applicator. Plate was developed in the solvent system of ethyl acetate: chloroform: ethanol: ammonia (6: 3: 1: 1) and scanned at 300 nm.. The amount of vasicine in the sample was calculated using the respective calibration curves. Caffeien 10 l of sample solution was applied in triplicate on a pre-coated silica gel 60 F254 TLC plate (0.2 mm thickness) along with 10 l of standard solution in triplicate with the CAMAG Linomat V automatic sample applicator. Plate was developed in the solvent system of ethyl acetate: methanol: water (100: 13.5: 10) and scanned at 254 nm.

Gallic acid 10 l of sample solution was applied in duplicate on a pre-coated silica gel 60 F254 TLC plate (0.2 mm thickness) along with 10 l of standard solution in duplicate with the CAMAG Linomat V automatic sample applicator. Plate was developed in the solvent system of ethyl acetate: chloroform: formic acid (12: 15: 3) and scanned at 273 nm. Glycyrrhizin 10 l of sample solution was applied in triplicate on a pre-coated silica gel 60 RP-18 F254S TLC plate (0.16-0.2 mm thickness) along with 10 l of standard solution in triplicate with the CAMAG Linomat V automatic sample applicator. Plate was developed in the solvent system of methanol: water: acetic acid (70: 30: 0.5) and scanned at 254 nm. The peak areas and absorption spectra of all the four component were recorded. The amount of glycyrrhizin in the sample was calculated using the respective calibration curves.

Validation of the Methods

ICH guidelines were (CPMP/ICH/381/95; CPMP/ICH/281/95) followed for the validation of the analytical procedure. The method was validated for precision, repeatability and accuracy. Instrumental precision was checked by repeated scanning of the same spot of vasicine (130 ng), gallic acid (100 ng), caffeine (240 ng) and glycyrrhizin (200 ng) seven times and was expressed as coefficient of variance (% CV). The repeatability of the method was confirmed by analyzing 130 ng/spot of standard solution of vasicine, 100 ng/spot of standard solution of gallic acid, 240 ng/spot of standard solution of caffeine and 200 ng/spot of standard solution of glycyrrhizin after application on the TLC plate (n = 6) and was expressed as % CV.

Robustness of the method was studied by analyzing aliquots of standard solution of vasicine (260, 520, 780 ng/spot), gallic acid (200, 300, 400 ng/spot), caffeine (480, 720, 960 ng/spot), glycyrrhizin (800, 600, 200 ng/spot), on the same day (intra-day precision) and on different days (inter-day precision) and the results were expressed as % CV. Accuracy of the method was tested by performing recovery studies at three levels (50 %, 100 % and 125 % addition). The percent recovery as well as average percent recovery was calculated. For the determination of limit of detection and limit of quantification different dilutions of the standard solutions of vasicine, gallic acid caffeine and glycyrrhizin were applied along with methanol as the blank and determined on the basis of signal to noise ratio.

Results and Discussion

Insty is a poly-herbal formulation, consisting of 8 ingredients of plant origin (Table-1) and it is widely used to treat upper respiratory tract infections. It contains expectorant, anti-inflammatory, mucolytic and anti-pyretic properties. The major active components in this formulation are vasicine in the aerial parts of Adhatoda vasica and glycyrrhizin in roots of Glycyrrhiza glabra that are responsible for its several activities. Adhatoda vasica and Glycyrrhiza glabra are the major herbs of the formulation and responsible for the activity of the preparation. Caffeine from Thea sinensis and gallic acid from Phyllanthus emblica. TLC densitometric methods were developed using HPTLC for the quantitative estimation of four marker compounds from the poly-herbal formulation Insty. Solvent systems were optimized to achieve best separation of the marker components from the other components of the formulation.

After several trials of solvent systems, the one containing ethyl acetate: chloroform : ethanol: ammonia (6: 3: 1: 1) gave best resolution of vasicine (Rf = 0.33), ethyl acetate: chloroform: formic acid (12: 15: 3) gave best resolution of gallic acid (Rf = 0.58), ethyl acetate: methanol: water (100: 13.5: 10) gave best reolution of caffeine (Rf = 0.46) and methanol: water: acetic acid (70: 30: 0.5) gave best resolution of glycyrrhizin (Rf = 0.51) in the presence of other number of compounds in the sample extract and enabled the quantification of marker compounds.

The presence of compounds in the sample was confirmed by comparing the Rf with vasicine standard (Fig. 5-A), gallic acid standard (Fig. 6-A), caffeine standard (Fig. 7-A) and glycyrrhizin standard (Fig. 8-A) and the HPTLC chromatograms by overlaying their spectra with those of their respective standards; vasicine standard and in sample (Fig. 5-B and 5-C), gallic acid standard and in sample (Fig. 6-B and 6-C), caffeine standard and in sample (Fig. 7-B and 7-C) and glycyrrhizin standard and in sample (Fig. 8-B and 8-C) using CAMAG TLC Scanner 3.

Linearity and Range

The relationship between the concentration of standard solutions and the peak area was linear within the concentration range of 130 - 1560 ng/spot with a correlation coefficient of 0.988 for vasicine, 100 - 600 ng/spot with a correlation coefficient of 0.992 for gallic acid, 240 - 1400 ng/spot with a correlation coefficient of 0.994 for caffeine and 200 - 1200 ng/spot with a correlation coefficient of 0.978 for glycyrrhizin.

The purity of the bands due to vasicine, gallic acid, caffeine and glycyrrhizin in the sample extract was confirmed by overlaying the chromatogram recorded at start, middle and end position of the band in the sample tracks.

Precision

The methods were validated in terms of precision, repeatability and accuracy (Table-2 and 3). The repeatability of sample application and measurement of peak area for active compound were expressed in terms of relative standard deviation (RSD %). Method repeatability was obtained from R.S.D. value by repeating the assay three times in same day for intra-day precision. Intermediate precision was assessed by the three sample sets on different days (inter-day precision). The intra- and inter-day variation was carried out at three different concentration levels.

Accuracy

The pre-analyzed samples were spiked with extra 50, 100, and 150% of the standards. The average percent recovery at three different levels was found and the results are presented in Table-3.

Limits of Detection and Limit of Quantification

In order to estimate the limit of detection (LOD) and limit of quantitiation (LOQ), blank methanol was spotted three times following the same methods. The signal to noise ratio were determined. LOD were considered as 3:1 and LOQ as 10:1. LOD and LOQ were experimentally verified by dilution known concentrations until the average responses were approximately three or ten times the standard deviation of the responses for three replicate determinations.

Specificity

The specificity of the method was ascertained by analyzing standard drug and sample. The spots for analytes were confirmed by compairing the Rf and spectra of the spot with that of standard. The peak purity of analytes were assessed by compairing the spectra at three different levels, i.e. peak start, peak apex and peak end positions of the spot.

Quantification

Vasicine, gallic acid, caffeine and glycyrrhizin content in a poly-herbal composition Insty were quantitatively determined by the proposed methods (Table-4). The methods developed were found to be suitable for the quantification of these marker compounds.

Conclusion

We established TLC densitometric methods for the quantification of four bioactive compounds viz., vasicine, gallic acid, caffein and glycyrrhizin using HPTLC. The methods were found to be simple, precise, specific, sensitive and accurate and can be used for their quantification in the polyherbal formulations and also in routine quality control of the raw materials as well as formulations containing any or all of these compounds.

Acknowledgements

The authors are thankful to honorable CEO Mr. Nadeem Khalid and COO Mr. Abid Mumtaz for their generous funding to support this study. Thanks also due to Herbion Pakistan (Pvt) Limited, Karachi, Pakistan for providing research facilities.

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Title Annotation:High performance liquid chromatography
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Date:Aug 31, 2015
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