@article {1377, title = {A Unique Method for Simultaneous HPLC Analysis of Gallic Acid, Glycyrrhizic Acid and E-Guggulsterone in a Polyherbal Formulation}, journal = {Pharmacognosy Research}, volume = {17}, year = {2025}, month = {January 2025}, pages = {295-306}, type = {Original Article}, chapter = {295}, abstract = {

Background: The therapeutic effects of herbal medicines are exerted due to the presence of different phytoconstituents and the effects are further potentiated when compatible herbals are formulated together. High Performance Liquid Chromatography (HPLC) is an important analytical tool towards standardization of polyherbal formulation. Complex nature, co- existence with other constituents trace quantities of active phytoconstituents and their simultaneous estimation are challenges encountered in HPLC analysis of polyherbal formulation. Simultaneous estimation of active biomarkers in polyherbal formulation by HPLC provides a unique method for verifying the quality and efficacy of polyherbal formulation. Materials and Methods: Present research work is about inventing an unique, selective and sensitive HPLC (High Performance Liquid Chromatography) method for simultaneous estimation of Gallic acid, Glycyrrhizic acid and E-Guggulsterone from a polyherbal formulation containing thirteen Ethnobotanical herbs, reported to have Cardioprotective effects, marketed as Collasyn tablets by Dr. Palep Research Labs Ltd. in Indian market. Reverse-phase column (C18, 250 mM X4.6 mM i.d., 5μm) was used to analyze the marker compounds Gallic acid, Glycyrrhizic acid and E-Guggulsterone at λmax 224 nm. The mobile phase was composed of acetonitrile and 0.05\% OPA (Ortho-phosphoric acid) in water (50:50) with a flow rate of 1 mL/min. Results: A unique HPLC method is developed for simultaneous estimation of active markers-Gallic acid, Glycyrrhizic acid and E-Guggulsterone from the polyherbal formulation. Quantitative analysis shows Gallic acid, Glycyrrhizic acid and E-Guggulsterone to be 11.47\%, 1.05\% and 0.63\% respectively in the polyherbal formulation. The developed method is validated in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals guidelines. The method shows good linearity in a relatively wide range of Concentration of 50-150 mcg/mL. The method is also found to be precise, accurate and robust. Conclusion: This unique and validated method for simultaneous estimation of cardioprotective biomarkers Gallic acid, Glycyrrhizic acid and E-Guggulsterone from polyherbal formulation, marketed as Collasyn tablets by Dr. Palep Research Labs Ltd. is important for its quality control. It helps in harnessing the complete plant potential for therapeutics. Thus, this approach to standardizing polyherbal formulations will be instrumental in verifying the quality and efficacy of the polyherbal extract. The simultaneous estimation of constituents from plants presents a significant advancement in phytochemical analysis, offering a comprehensive understanding of plant properties and their potential applications. The methodologies employed demonstrate high efficiency and accuracy, allowing for the effective quantification of multiple compounds in a single analytical run. This approach not only streamlines the process of phytochemical profiling but also enhances the ability to explore the therapeutic potentials of various plant species.

}, keywords = {Cardioprotective, HPLC, Method development, Method validation, Polyherbal formulation}, doi = {10.5530/pres.20252013}, author = {Bharati Gawade and Swati Patil} } @article {1275, title = {HPTLC Method Development and Validation for Simultaneous Quantification of Purpurin and Alizarin in Rubia cordifolia L. Roots and their Marketed Preparations}, journal = {Pharmacognosy Research}, volume = {16}, year = {2024}, month = {June 2024}, pages = {652-661}, type = {Original Article}, chapter = {652}, abstract = {

Background: Rubia cordifolia L. a spiky perennial climber of Rubiaceae family, is a rich reservoir of anthraquinone containing compounds purpurin and alizarin. It emerges as a versatile powerhouse possessing diverse pharmacological properties which includes antibacterial, antitumor, antioxidant, antiplatelet, potent blood purifier, diuretic, anti-stress, neuroprotective, anti-mutagenic, anticancer, anti-inflammatory, antidepressant, antidiabetic and vasodilation. Thereby it offers a holistic approach for disease prevention through its pharmacological properties. Objectives: However due to lack of standardization for R. cordifolia roots and its derived marketed herbal products, its quality control remains a daunting task..The present study aims to develop a simple, novel, reliable and specific HPTLC method for quantifying purpurin and alizarin in R. cordifolia roots as well as in its marketed preparations. Materials and Methods: The separation and quantification of purpurin and alizarin has been carried out on HPTLC plates pre-coated with Silica gel 60 F254 as stationary phase and toluene, ethyl acetate, glacial acetic acid (6:3.5:0.5; v/v/v) as mobile phase. Determination and quantification were performed by densitometric scanning at 254 nm. Results: The developed method gave compact spots at Rf 0.57{\textpm}0.007 for purpurin and 0.73{\textpm}0.008 for alizarin. The method was validated as per International Council for Harmonization Q2(R1) guidelines for specificity, precision, robustness, accuracy and recovery. Linearity range for purpurin and alizarin was 100-2000 ng/spot, which showed good regression coefficient for purpurin and alizarin; R{\texttwosuperior}=0.9967 and R{\texttwosuperior}=0.9941 at 254 nm respectively. The LOD and LOQ for validated marker compounds purpurin and alizarin at 254 nm were found as 23.87{\textpm}0.52, 71.61{\textpm}0.84 ng/spot and 18.36{\textpm}0.72, 55.08{\textpm}0.26 ng/spot respectively. The content of purpurin and alizarin in R. cordifolia plant extract was found as 17.960{\textpm}0.658, 34.645{\textpm}1.153 μg/mg, marketed preparation A, contains 21.741{\textpm}0.746 and 6.824{\textpm}0.485 μg/mg while marketed preparation B, contains 18.289{\textpm}1.014 and 3.031{\textpm}0.234 μg/mg of the sample. Conclusion: The developed method was found to be simple, precise, accurate, economical and convenient for rapid screening of bioactive marker purpurin and alizarin present in methanolic extracts of R. cordifolia roots and its marketed preparations.

}, keywords = {Alizarin, Densitometry, HPTLC, Method validation, Purpurin, Rubia cordifolia L.}, doi = {10.5530/pres.16.3.76}, author = {Rizwan Ahmad and Zeeshan Fatima and Sadath Ali and Suneela Dhaneshwar and Sayeed Ahmad} } @article {654, title = {Isolation, Method Development, and Validation of Diterpenes in Coffee Oil: Identification by Multiple Reaction Monitoring Approach with Atmospheric Pressure Chemical Ionization Mode}, journal = {Pharmacognosy Research}, volume = {13}, year = {2021}, month = {April 2021}, pages = {42-48}, type = {Original Article}, chapter = {42}, abstract = {

Objectives:\ The aim of the present study is to isolation, identification, and quantification of free diterpenes in coffee oil of\ Coffea arabica.\ Materials and Methods:\ For the isolation of free diterpenes, saponification reaction step was employed and followed with a liquid-liquid extraction procedure applied. Cafestol and kahweol were identified by multiple reaction monitoring (MRM) of positive ionization (MRM) mode of atmospheric pressure chemical ionization (APCI) mass spectrometry detection. The quantification of free diterpenes was done by high-performance liquid chromatography with diode array detector. The developed method was validated according to the international conference on harmonization guidelines.\ Results:\ The ion transitions of the precursor to the product ion at (M + H)+m/z\ 317.20{\textrightarrow}146.90, 281.00, 299.00 for cafestol and 315.10{\textrightarrow}144.90, 278.90, 296.90 for kahweol were observed. The proposed method was validated for linearity with excellent correlation coefficient of cafestol and kahweol were found to be 0.9997 and 0.9996, respectively. The intra-day and intermediate precisions and repeatability showed the percentage relative standard deviation was \<1\%. The recovery rate for cafestol and kahweol were observed to be in the acceptable limit of 95.303\%{\textendash}98.539\% and 95.963\%{\textendash}97.174\%, respectively. The limit of detection of cafestol and kahweol were found to be 6.81 and 7.35 ppm, respectively. The limit of quantitation (LOQ) was found to be 22.72 and 24.52 ppm, respectively.\ Conclusion:\ The developed method is simple, rapid, precise, accurate and it is recommended for efficient assays in routine work.

}, keywords = {Atmospheric pressure chemical ionization, Cafestol, Coffea arabica, Kahweol, Method validation}, doi = {10.4103/pr.pr_66_20}, author = {Naveen Puttaswamy and Lingaraju Harakanahalli Basavegowda and Deepak Machettira and Chandrappa Siddappa and Rashmi Ramesh and Shyam Prasad Kodimule} } @article {93, title = {Development and Validation of Veratric Acid in Tabebuia avellanedae using Liquid Chromatography-Electrospray Ionization-Mass Spectrometry/Mass Spectrometry-Multiple Reaction Monitoring-Based Assay Coupled with 1,1-Diphenyl-2-Picrylhydrazyl Method}, journal = {Pharmacognosy Research}, volume = {12}, year = {2020}, month = {May 2020}, pages = {107-111}, type = {Original Article}, chapter = {107}, abstract = {

Objective: The aim of the present study is to develop a new simple, precise, and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of veratric acid in Tabebuia avellanedae Linn. Materials and Methods: The quantification of veratric acid was done using collision-induced dissociation multiple reaction monitoring scan of mass spectrometry technique. The developed method was validated according to the International Conference on Harmonization guidelines. The free radical scavenging activity was performed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. Results: The ion transitions of the precursor to the productions were principally protonated ion [M + H]+ at m/z 182.8 \>139.20, 124.20, and 77.0 for veratric acid. The proposed method was validated for linearity with an excellent correlation coefficient of 0.9978. The intraday and intermediate precisions and repeatability showed the percentage relative standard deviation was \<2\%. The accuracy for the determination of veratric acid was within 82.20\%{\textendash}97.65\%. The limit of detection and limit of quantitation were 0.66 and 2.21 ppm, respectively. The half-maximal inhibitory concentration value for the DPPH radical scavenging activity of ascorbic acid and quality control sample (hydroalcoholic extract of T. avellanedae) was found to be 17.79 and 36.44 μg/ml, respectively. Conclusion: The developed LC-MS/MS method is a simple, rapid, precise, accurate, and it is recommended for efficient assays in routine work. T. avellanedae exhibited strong DPPH radical scavenging activity.

}, keywords = {1, 1-diphenyl-2-picrylhydrazyl, Method validation, Multiple reaction monitoring, Tabebuia avellanedae, Veratric acid}, doi = {10.4103/pr.pr_77_19}, author = {Puttaswamy Naveen and Harakanahalli Basavegowda Lingaraju and Siddappa Chandrappa and Srikant Anitha and Kodimule Shyam Prasad} }