Quality Control Of Herbal Materials Research Articles

Antidiabetic Activity of Polyherbal Formulation in Streptozotocin – Nicotinamide Induced Diabetic Wistar Rats

Glycosmis pentaphylla, Tridax procumbens, and Mangifera indica are well-known plants available throughout India and they are commonly used for the treatment of various diseases including diabetes mellitus. The antidiabetic activity of the individual plant parts is well known, but the synergistic or combined effects are unclear. The concept of polyherbalism has been highlighted in Sharangdhar Samhita, an Ayurvedic literature dating back to 1300 AD. Polyherbal formulations enhance the therapeutic action and reduce the concentrations of single herbs, thereby reducing adverse events. The aim of the present study is to formulate a polyherbal formulation and evaluate its antidiabetic potential in animals. The polyherbal formulation was formulated using the ethanol extracts of the stem bark of G. pentaphylla, whole plant of T. procumbens, and leaves of M. indica. The polyherbal formulation contains the ethanol extracts of G. pentaphylla, T. procumbens, and M. indica in the ratio of 2:2:1. The quality of the finished product was evaluated as per the World Health Organization's guidelines for the quality control of herbal materials. The quality testing parameters of the polyherbal formulation were within the limits. Fingerprint analysis of the polyherbal formulation showed effective separation at 366 nm, and it revealed that the active compound present in the polyherbal formulation and the active compounds present in all the three extracts were the same. The acute toxicity studies of the polyherbal formulation did not show any toxic symptoms in doses up to 2000 mg/kg over 14 days. The oral antidiabetic activity of the polyherbal formulation (250 and 500 mg/kg) was screened against streptozotocin (50 mg/kg; i.p.) + nicotinamide (120 mg/kg; i.p.) induced diabetes mellitus in rats. The investigational drug was administered for 21 consecutive days, and the effect of the polyherbal formulation on blood glucose levels was studied at regular intervals. At the end of the study, the blood samples were collected from all the animals for biochemical estimation, and the animals were sacrificed and the liver and pancreatic tissues were collected for histopathologic analysis. Polyherbal formulation showed significant antidiabetic activity at 250 and 500 mg/kg, respectively, and this effect was comparable with that of glibenclamide. The antidiabetic activity of polyherbal formulation is supported by biochemical and histopathologic analysis.

Rapid simultaneous determination of marmelosin, umbelliferone and scopoletin from Aegle marmelos fruit by RP-HPLC.

The surge of interest in naturally occurring phytochemicals with high therapeutic potential has led to the discovery of many molecules, out of which naturally occuring coumarins such as marmelosin, umbelliferone and scopoletin present in Aegle marmelos (Bael) fruit shows good therapeutic potential. The aim of the present work is to develop and validate Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method for simultaneous determination of marmelosin, umbelliferone and scopoletin in A. marmelos fruit extracts. The chromatographic separation was performed with isocratic elution of 55:45 (%, v/v) methanol-water containing 0.1% acetic acid as mobile phase. The method used to analyse the extract of A. marmelos showed good resolution with retention time within 12min. The relative concentrations of above phytoconstituent were determined in A. marmelos fruits. The method was found to give compact peaks for scopoletin, umbelliferone and marmelosin (Rt of 4.6, 6.5 and 11.3min respectively) and were linear over the range 5-30μgml(-1) (R(2) = 0.9655), 2-10μgml(-1) (R(2) = 0.9964) and 2-10μgml(-1) (R(2) = 0.9862) respectively. The mean recoveries for marmelosin, umbelliferone and scopoletin at three concentrations were in the range of 98.8-102.9, 98.8-101.1 and 94.2-98.3% respectively. The relative standard deviation of accuracy, precision and repeatability were within 2%, indicating the method produced highly reproducible results. Therefore this simple, precise and accurate method enables simultaneous separation of this phytoconstituent and hence can be successfully applied in analysis and routine quality control of herbal material and formulation containing A. marmelos.

Development of validated HPTLC method for quantification of stigmasterol from leaf and stem of Bryophyllum pinnatum

This study presents the report of TLC densitometric method, which has been developed and validated for quantification of stigmasterol from petroleum ether extract of leaves and stems of Bryophyllum pinnatum. The separation was performed on TLC aluminum plates precoated with silica gel 60 F254. Good separation was achieved in mobile phase using Chloroform:Ethanol (9.8:0.2v/v). Determination and quantitation were performed by densitometric scanning at 490nm in reflection/absorbance mode. This method gave compact spots at Rf 0.45 corresponding to stigmasterol. The method was validated using ICH guidelines in terms of precision, repeatability and accuracy. Linearity range for stigmasterol was 50–300ng/spot with correlation coefficient R2±SD=0.990±5.05% in the concentration range 50–300ng/spot. The LOD and LOQ were found to be 15 and 45ng, respectively. The contents in crude extract obtained from leaf and stem were found to be 0.1545±0.005% w/w and 0.1383±0.002% w/w respectively. HPTLC method was found to be simple, precise, accurate and convenient method for rapid screening of active constituents present in petroleum ether extracts of plant and can be used for analysis and routine quality control of herbal material and formulations containing B. pinnatum.

TLC-DENSITOMETRIC QUANTIFICATION OF NEGUNDOSIDE, URSOLIC ACID, EUGENOL, LUPEOL, AND β-SITOSTEROL USING HPTLC FROM VITEX NEGUNDO LEAVES

Vitex negundo Linn (Verberaceae) is an important drug in Indian, Chinese, and Malaysian Systems of medicine. The leaves are reported to show anti-inflammatory, anti-ulcer, anti-implantation, and analgesic activities. They contain negundoside, nishindaside, agnuside, luteolin, vitexicarpin, ursolic acid, and β-sitosterol. Lupeol, an important marker compound has not been reported from V. negundo until recently. Our preliminary TLC experiments indicated the presence of lupeol in the leaves of V. negundo. Subsequently, lupeol was isolated by preparative TLC. The identity of lupeol was confirmed by comparing the mass spectra [m/z value 427.8 (M+1) and 425.8 (M−1)]; melting point (215°C, using DSC) and co-TLC with the standard compound. A High Performance Thin Layer Chromatography (HPTLC) method for the quantification of five marker compounds, namely, negundoside, ursolic acid, eugenol, lupeol, and β-sitosterol from the leaves of V. negundo was developed and validated as per ICH guidelines. The amounts of negundoside, ursolic acid, eugenol, lupeol, and β-sitosterol were found to be 1.048, 0.013, 0.115, 0.116, and 0.043% w/w, respectively. The developed methods were found to be accurate, precise, and reproducible. They can be used for routine quality control of herbal material and formulations containing these compounds.

Hyphenated chromatographic analysis of bioactive gallic acid and quercetin in Hygrophila auriculata (K. Schum) Heine growing wildly in marshy places in India by validated HPTLC method

ObjectiveA simple, accurate, and rapid high-performance thin-layer chromatographic (HPTLC) method for simultaneous quantification of the two biologically active flavonoidal compounds, gallic acid and quercetin, in Hygrophila auriculata (K. Schum) Heine (HA) has been established and validated. MethodsChromatography was performed on aluminium foil-backed silica gel 60 F254 HPTLC plates with the binary mobile phase toluene: ethyl acetate: formic acid (5:4:1, v/v/v). Ultraviolet detection was performed densitometrically at the maximum absorbance wavelength, 270nm. The method was validated for precision, recovery, robustness, specificity, and detection and quantification limits, in accordance with ICH guidelines. ResultsThe system was found to give compact spots for gallic acid (GA) and quercetin (QE) (Rf value of 0.31 and 0.50, respectively). The limit of detection (23 and 41 ng band−1) limit of quantification (69 and 123 ng band−1), recovery (99.4–99.9 and 98.7–99.4%), and precision (i.e ≤1.98 and 1.97) were satisfactory for gallic acid and quercetin respectively. Linearity range for GA and QE were 100–1000 (r2= 0.9991) and 150–900 ng band−1 (r2= 0.9956) and the contents estimated as 0.28±0.01% and 0.41±0.01% w/w respectively. ConclusionsThis simple, precise and accurate method gave good resolution from other constituents present in the extract. The method has been successfully applied in the analysis and routine quality control of herbal material and formulations containing Hygrophila auriculata (K. Schum) Heine.

Validation of the method for the simultaneous estimation of bioactive marker gallic acid and quercetin in Abutilon indicum by HPTLC

ObjectiveTo establish and validate an simultaneous estimation of the two biomarker compounds gallic acid (GA) and quercetin (QE) from methanolic extract of Abutilon indicum (AI). MethodsChromatography was performed on aluminium foil-backed silica gel 60 F254 HPTLC plates with the binary mobile phase toluene-ethyl acetate-formic acid (5:4:1, v/v/v). Ultraviolet detection was performed densitometrically at the maximum absorbance wavelength, 270nm. The method was validated for precision, recovery, robustness, specificity, and detection and quantification limits, in accordance with ICH guidelines. ResultsThe system was found to give compact spots for GA and QE (Rf value of 0.31 and 0.50, respectively). The limit of detection (23 and 41 ng band-1) limit of quantification (69 and 123 ng band-1), recovery (99.4–99.9 and 98.7–99.4%), and precision (≤1.98 and 1.97) were satisfactory for gallic acid and quercetin respectively. Linearity range for GA and QE were 100-1000 (r2= 0.9991) and 150–900 ng band-1 (r2 = 0.9956) and the contents estimated as 0.69% ± 0.01% and 0.57% ± 0.01% w/w respectively. ConclusionsThis simple, precise and accurate method gave good resolution from other constituents present in the extract. The method has been successfully applied in the analysis and routine quality control of herbal material and formulations containing AI.

Quantification of Allethrin using HPTLC from Annona Squamosa (Custard Apple)

Annona squamosa (Custard Apple) is delicious fruit and it is traditionally used for curing a variety of ailments such as Ulser, dysentery, Antiovulatory, hair problems, burning sensation, hysteria (fearful state of mind) and fainting spells. There are no reports of quantification of Allthrine from this plant. Hence a HPTLC densitometric method has been developed and validated for quantification of this marker compound. Allethrine was quantified from methanolic extract using the solvent system of Petroleum ether: Diethyl ether (7: 3, v/v). The method was validated using ICH guidelines in terms of precision, repeatability and accuracy. Linearity range for Allethrine was 40–320 ng/spot and its content was 0.01046% w/w in peel and 0.01042% w/w in seed. This simple, precise and accurate method gave good resolution from other constituents of extract. It can be adopted for routine quality control of herbal material and formulations containing Annona squamosa (Custard Apple).

Study on quality control of herbal material of paeony formula granule by near infrared diffuse reflection spectroscopy

To develop a novel method to determine fastly and nondestructively the content of paeoniflorin, albiflorin and moisture in Radix Paeoniae Alba with near infrared diffuse reflection spectroscopy. Multivariate calibration models based on PLS algorithm were developed to correlate the spectra and the corresponding values determined by the reference method. The corelation coefficients (R2) of the calibration models were 0.938, 0.943 and 0.976, and the prediction average relative deviation for paeoniflorin, albiflorin and moisture content were 6.5%, 0.23% and 3.8%, respectively. The NIRS determination method is rapid, accurate, nondestructive and non-pollution. It can dispose the samples without complicated pretreatment. It is qualified to analyze rapidly traditional Chinese medicine whose components are complex. NIRS can be used to control the quality of herbal material of paeony formula granule.

HPTLC densitometric quantification of stigmasterol and lupeol from Ficus religiosa

This study presents the first report of TLC densitometric method, which has been developed and validated for simultaneous quantification of the two marker compounds (stigmasterol and lupeol) from methanolic extract using the solvent system of toluene:methanol (9:1, v/v). The method employed TLC aluminum plates precoated with silica gel 60 F254 as the stationary phase. Densitometric analysis of stigmasterol and lupeol was carried out in the reflectance mode at 525nm. The system was found to give compact spots for stigmasterol and lupeol (Rf value of 0.37 and 0.60, respectively). The method was validated using ICH guidelines in terms of precision, repeatability and accuracy. Linearity range for stigmasterol and lupeol was 80–480ng/spot and 150–900ng/spot and the contents were found to be 0.06±0.005% w/w and 0.12±0.02% w/w, respectively. The limit of detection (LOD) value for stigmasterol and lupeol were found to be 20 and 50ng, and limit of quantification (LOQ) value were 60 and 100ng, respectively. This simple, precise and accurate method gave good resolution from other constituents present in the extract. The method has been successfully applied in the analysis and routine quality control of herbal material and formulations containing Ficus religiosa.

DNA FINGERPRINTING OF TINOSPORA CORDIFOLIA USING RAPD ANALYSIS

Tinospora cordifolia Linn. is one of the important immunomodulating agents in Ayurveda, the traditional Indian system of medicine. In this study, we analyze different samples of Tinospora cordifolia for genetic polymorphism using Random Amplified Polymorphic DNA (RAPD) markers. Analysis of well-characterized marker compounds is now the most popular method for identifying and quality control of herbal materials. However, in many herbal species the chemical composition of the plant changes with environment growing and processing conditions, which lowers the reliability of authentification methods. For the determination of genetic polymorphism, one hundred and twenty decamer oligonucleotide primers were used to screen twenty selected samples. Total DNA extracted from the stem by using ethanol precipitation was used as the template in the PCR reaction. Four primers OPA-16, OPC-7, OPC-13 and OPG-5, showed reproducible distinct polymorphic patterns. The primers possess abilities in detecting variations in twenty collected samples of Tinospora cordiofolia. It can be applied to detect the presence of the drug in formulations. A low genetic diversity among the Tinospora cordifolia samples was revealed by RAPD analysis.

Simultaneous Quantification of Bergenin, (+)-Catechin, Gallicin and Gallic acid; and Quantification of β-Sitosterol using HPTLC from Bergenia ciliata (Haw.) Sternb. Forma ligulata Yeo (Pasanbheda)

In the present paper we report our work on development and validation of TLC densitometric method for simultaneous quanti fi cation of Bergenin, (+)-Catechin, Gallicin and Gallic acid, and quanti fi cation of β -Sitosterol using HPTLC.To the best of our knowledge, this is the fi rst report of simultaneous quanti fi cation of these four bioactive compounds viz. Bergenin, (+)-Catechin, Gallicin and Gallic acid using HPTLC from this plant. Simultaneous quanti fi cation of Bergenin, (+)-Catechin, Gallicin and Gallic acid was carried out from the methanolic and hydrolysed extract (2N HCl) using the Solvent System of Toluene: Ethyl acetate: Formic acid (6: 6: 1, v/v/v). β -Sitosterol was quanti fi ed from petroleum ether extract using the Solvent System of Toluene : Methanol (9: 1, v/v). The methods were validated using ICH guidelines in terms of precision, repeatability and accuracy. The thin layer chromatography densitometric methods were found to be precise with RSD for intra-day in the range of 2.62–4.26, 0.35–2.5, 0.61–1.83, 1.14–1.57 and 0.15–0.52 and for interday in the range of 1.90–4.27, 0.85–3.05, 0.97–1.45, 0.58–1.27 and 0.26–0.61 for different concentrations of Bergenin, (+)-Catechin, Gallicin, Gallic acid and β -Sitosterol respectively. The linearity ranges for Bergenin, (+)-Catechin, Gallicin and Gallic acid were found to be the same (160 –720 ng/spot) with correlation coef fi cients ( r -values) of 0.999, 0.997, 0.998 and 0.996 respectively. The linearity range for β -Sitosterol was 80–480 ng/spot with correlation coef fi cient ( r -values) of 0.995. Instrumental precision was 3.39, 3.36, 3.05, 1.20 and 0.85 (% RSD) for Bergenin, (+)-Catechin, Gallicin, Gallic acid and β -Sitosterol respectively. Accuracy of the method was checked by conducting recovery studies at three different levels for both the compounds and the average percentage recoveries obtained were 100.38, 101.45, 102.38, 99.9 and 99.92% respectively for Bergenin, (+)-Catechin, Gallicin, Gallic acid and -Sitosterol. The amount of Bergenin, (+)-Catechin and Gallic acid was 0.22, 0.063 and 0.25 % w/w respectively whereas Gallicin was not in detectable amount. Developed method permitted simultaneous quanti fi cation of Bergenin, (+)-Catechin, Gallicin and Gallic acid, and showed good resolution and separation from other constituents of extract and was found to be simple, precise, speci fi c, sensitive and accurate. It can be adopted for routine quality control of herbal material and formulations containing Bergenia ciliata .

Quality Control of Herbal Material and Phytopharmaceuticals with MS and NMR Based Metabolic Fingerprinting

Metabolic fingerprinting techniques have received a lot of attention in recent years and the annual amount of publications in this field has increased significantly over the past decade. This increase in publications is due to improvements in the analytical performance, most notably in the field of NMR and MS analysis, and the increased awareness of the different applications of this growing field. Metabolomic fingerprinting or profiling is continuously being applied to new areas of research such as drug discovery from natural resources, quality control of herbal material, and discovering lead compounds. In this review the current state of the art of metabolic fingerprinting, focussing on NMR and MS technologies will be discussed. The application of these two analytical tools in the quality control of herbal material and phytopharmaceuticals forms the major part of this review. Finally we will look at the future developments and perspectives of these two technologies in the quality control of herbal material.

Simultaneous quantification of bergenin, catechin, and gallic acid from Bergenia ciliata and Bergenia ligulata by using thin-layer chromatography

Bergenia ciliata Sternb. and Bergenia ligulata Wall. (Saxifragaceae) are reputed drugs of Ayurveda, commonly known as Paashaanbhed. A simple TLC method has been developed for the simultaneous quantification of bergenin, catechin, and gallic acid from different parts of B. ciliata and B. ligulata using HPTLC plate precoated with silica gel 60 F 254. The method was developed in toluene:ethyl acetate:formic acid (4:6:1, v/v) and validated in terms of precision, repeatability, and accuracy. The linearity range for bergenin, catechin and gallic acid were found to be 160–800, 160–480 and 160–560 ng/spot respectively with correlation coefficients of 0.999, 0.999 and 0.999, respectively, which were indicative of good linear dependence of peak area on concentration. The method permits reliable quantification and showed good resolution and separation from other constituents of extract. Accuracy of the method was checked by conducting recovery studies at three different levels for all the three marker compounds and the average percentage recoveries were found to 99.29%, 98.66%, and 99.23%, respectively. The rhizomes were found to contain higher concentration of bergenin, catechin, and gallic acid than other parts of the plants. The proposed method was found to be simple, precise, specific, sensitive, and accurate. It can be used for routine quality control of herbal material and formulations containing bergenia species.