Quantification Of Marker Compounds Research Articles

Development of Simultaneous HPTLC Method and Validation for the Quality Assessment of Ayurvedic Formulation-Ayush Kvatha Churna by Using Marker Compound Rosmarinic Acid, Trans-Cinnamaldehyde and Piperine.

Ayurveda emphasizes the propagation of nature in maintaining health. In the present scenario, we have seen the faith of people in herbal drugs during the Covid 19 outbreak. The raises in the number of peoples have been using herbal drugs to boost immunity against infectious diseases shows the popularity of this ancient system of medicine. The standardization of Ayush Kvatha Churna (AKC), work set out to establish a straightforward, accurate and sensitive HPTLC method for the identification and quantification of marker compounds. The Rosmarinic acid, trans-Cinnamaldehyde and Piperine were used for the estimation of markers in Ayush Kvatha Churna by using HPTLC with a solvent system, consisting of Toluene: Ethyl acetate: Ethyl alcohol: Formic acid (5.6:2.4:2: 0.3v/v/v/v). The Rf value 0.33 for Rosmarinic Acid, 0.69 for Piperine and 0.77 for trans-Cinnamaldehyde was observed and it is exactly complying with the corresponding bands in Ayush Kvatha Churna. The technique has been effectively verified and validated, enabling it to be used for the standardization or quantitative analysis of Rosmarinic acid, trans-Cinnamaldehyde and piperine in Ayush Kvatha Churna.

High Performance Thin Layer Chromatographic Standardization and Quantification of Marker Compounds in an Ayurvedic Polyherbal Formulation Padmakadi Churna

Indian Journal of Natural Products,2022,36,1,24-27.DOI:10.5530/ijnp.2022.1.4Published:February 2023Type:Original ArticleAuthors:Ravichand Meshram, Umakant Sahu, Arun K S Parihar, Prashant K Gupta, and Nagendra Singh Chauhan Author(s) affiliations:Ravichand Meshram1, Umakant Sahu2, Arun K S Parihar2, Prashant K Gupta1,3, Nagendra Singh Chauhan2,* 1Shri Narayan Prasad Awasthi Government Ayurved College, Raipur, Chhattisgarh, INDIA. 2Drugs Testing Laboratory Avam Anusandhan Kendra, G.E. Road, Raipur, Chhattisgarh, INDIA. 3Department of Kaumarabhritya, All India Institute of Ayurveda, New Delhi, INDIA. Abstract:The aim of this study was to find out a simple, accurate and sensitive HPTLC method for the detection and quantification of marker molecule Gallic and Piperine in Padmakadi churna for standardization. Ethanolic extraction was performed for analysis. HPTLC was done using gallic acid and Piperine as a standard. The mobile phase was a mixture of Toulene:ethyl acetate:Ethanol:Formic acid (6:3:1:0.3 v/v/v) and detection at 254 nm. The Rf was detected at - 0.39 for Gallic acid and Rf - 0.55 for Piperine was identified in Padmakadi Churna. The simple and sensitive HPTLC method was successfully developed for determination of Gallic acid and Piperine in Padmakadi Churna. The proposed HPTLC method was found to be simple, fast and inexpensive, and can be used for the routine quality control of raw materials. Keywords:Gallic acid, HPTLC, Padmakadi Churna, Physiochemical, Piperine, Standardization.View:PDF (510.58 KB)

Beneficial role of Boehmeria nivea in health and phytochemical constituents.

The leaf and stem extracts of Boehmeria nivea (BN) collected from three different regions in Korea were screened for their antioxidant, neuroprotective, estrogenic, insulin secretion, and α-glucosidase inhibitory activity. We also examined whether BN extracts regulate cancer cell growth, inflammatory-related gene expression, and lipid accumulation in cellular system. Leaf extracts possessed greater antioxidant, anti-proliferative in cancer cells, neuroprotective, estrogenic activity, and inhibitory effect on pro-inflammatory gene expression than stem extracts. Leaf and stem extracts inhibited lipid accumulation in three T3-L1 adipocytes but did not affect glucose-stimulated insulin secretion in INS-1 cells. We isolated and identified the phytochemical constituents in the n-butanol and ethyl acetate fractions of BN leaves by combining silica gel column chromatography with mass spectrometry and 1 H- and 13 C-NMR analysis. The active compounds (caffeic acid, isoquercitrin, p-coumaric acid, and rutin) exhibited ABTS and DPPH radical scavenging activity, which may contribute to the biological activities of BN leaf extract. An analytical method was developed to quantify marker compounds for the discrimination of BN collected from different regions. Our results support the use of this analysis method for accurate identification and quantification of marker compounds in BN for the development of functional foods. PRACTICAL APPLICATIONS: Boehmeria nivea (BN) has been used as a raw material for the textile industry or traditional herbal medicine. The current study established the biological activities and active components of BN. Our results showed that BN leaf and stem extracts exhibit antioxidant, neuroprotective, and estrogenic activity. BN leaf extract also inhibited cancer cell growth, inflammatory mediators and cytokines production, and lipid accumulation in vitro. Moreover, the bioactive compounds, such as caffeic acid, isoquercitrin, p-coumaric acid, and rutin, exert ABTS and DPPH radical scavenging activities. Therefore, BN could potentially be a promising source of bioactive phytochemicals for the development of functional foods or drugs.

High-performance thin-layer chromatographic standardization and quantification of marker compounds in an Ayurvedic polyherbal formulation: Krishnadi Churna

High-performance thin-layer chromatographic standardization and quantification of marker compounds in an Ayurvedic polyherbal formulation: Krishnadi Churna

Identifikasi Senyawa Marker Dominan Ekstrak Daun Sirih Hitam dan Kuantifikasi Berdasarkan Perbedaan Lokasi Tanam

It has been revealed that the benefits of black betel leaf extract have created an opportunity for the black betel plant to be commercialized. The several benefits of black betel leaf extract are antimicrobial, anti-inflammatory, cytotoxic, and antioxidant. The extraction method uses maceration techniques. Identification of active markers as antimicrobials (Streptococcus mutans, Streptococcus sanguinis, Candida albicans) begins with Bioautography Thin Layer Chromatography (TLC), for isolation from macerated extracts followed by Vacuum Liquid Chromatography Fractionation (KCV), conventional Column Subfraction (KK), Layer Chromatography Thin Preparative (TLC). Quantification of marker compounds using a Thin Layer Chromatography (TLC) Scanner. Samples of black betel leaf (P. betle L. var Nigra) were collected from Samarinda (East Kalimantan), Wonosobo (Central Java), Godean (Yogyakarta), Banyuwangi (East Java) in June 2020. The results of the TLC test for bioautography of 70% ethanol extract and the ethanol fraction of 96% black betel leaf showed that the active spot of ethanol extract 70% for antibacterial activity on Streptococcus mutans Atcc 25175 and Streptococcus sanguinis Atcc 10556 was Rf 0.729 cm, the active spot of the ethanol fraction 96% for the activity of the fungus Candida albicans ATTC 10231; Rf 0.486 cm. Purification using TLC multi eluent with eluent I Hexane: Ethyl Acetate (7: 3) as non polar eluent and eluent II Chloroform: Ethyl Acetate (4: 1) as polar eluent on different TLC plates. The results obtained in the form of a single stain with Rf = 0.4 in eluent I and Rf = 0.73 in eluent II. The results of measuring the levels of the dominant marker compound using a TLC scanner showed that the highest marker compound area was from betel leaf grown in the Godean area (Yogyakarta) with the number 29869.4.

A Review on Phytochemical Characterization of Kwatha–Ayurvedic Polyherbal Formulation

A paradigm shifts from the conventional approach of a single drug-based system to polyherbal formulations is recently observed in the modern pharmaceutical industry. The rising demand has led to a decrease in the quality and efficacy of herbal medicines. In order to ensure the sustained demand and therapeutic efficacy, it is vitally important to devise proper methods of standardization from the raw drugs to finished polyherbal formulations. However, there is no consensus regarding how these herbal medicines should be standardized. The phytochemical standardization by multiple markerbased fingerprint profiling along with preliminary screening and quantification of marker compounds can assure the reproducibility of the activity of the polyherbal formulations to an extent. The clinically pertinent scientific data to support the asserted synergistic therapeutic effects of Ayurvedic poylyherbal formulations is inadequate. In this review, we discuss the phytochemical standardization and pharmacological studies of kwatha, a major form of herbal remedies in Ayurvedic pharmacology, and the underlying concept of synergism. Kwatha (decoction) is the backbone of self-administered herbal preparations in India. Since the majority of the active principles of plants are water-soluble, herbal decoctions that are usually prepared in water formulate the potent and effective Ayurvedic medicines.

Monitoring of Chemical Markers in Extraction of Traditional Medicinal Plants (Piper nigrum, Curcuma longa) Using In Situ ReactIR.

The fingerprinting and quantification of marker compounds from medicinal plants is a domain of the herbal industry for quality/quantity control parameters. The main objective of this study is the application of the in situ ReactIR technique for measuring the concentration of different components during the extraction process of different medicinal plants. In this study we have performed the extraction of two-marker compounds, viz. piperine from Piper nigrum and curcumin from Curcuma longa plants, using various solvents (dichloromethane and methanol). The progress of extraction was monitored using an in situ Fourier transform infrared (FTIR) probe instrument and an automated reactor. In this communication, using the in situ ReactIR technique we developed a method which demonstrates the relative quantification of marker analytes, optimizes extraction time and type of solvents to be used for different analytes during the extraction process. To the best of our knowledge, this is the first report of relative quantification and structural information of marker compounds during the process of extraction using in situ FTIR. The present study highlights the real-time monitoring, in situ quantification, and structural information of marker compounds during the process of extraction of medicinal plants using in situ FTIR.

Applicability of high-resolution NMR in combination with chemometrics for the compositional analysis and quality control of spices and plant-derived condiments.

Over the last years, the consumption of spices and plant-derived condiments has increased considerably, owing to new culinary trends. Unfortunately, the current marketing channels make them highly vulnerable to adulteration and food fraud. High-resolution nuclear magnetic resonance (NMR) is a powerful tool for the compositional study of spices and plant-derived condiments. It allows the chemical characterization of a wide range of polar and non-polar metabolites, and provides unique structural information not available by other techniques. The chemometric-based analysis of NMR 'fingerprints' has been used to discriminate samples according to species and geographical origin and to detect adulterations, among other applications. The comprehensive identification and quantification of marker compounds can be achieved even in complex mixtures, demonstrating a great potential for high-throughtput quality control applications. © 2020 Society of Chemical Industry.

Botanical formulation, TADIOS, alleviates lipopolysaccharide (LPS)-Induced acute lung injury in mice via modulation of the Nrf2-HO-1 signaling pathway

Ethnopharmacological relevanceTADIOS is an herbal formulation prepared from a mixture of Taraxacum officinale (L.) Weber ex F.H.Wigg, Dioscorea batatas Decaisne and Schizonepeta tenuifolia (Benth.) Briquet. These plants have traditionally been used in Asia to treat a variety of respiratory diseases. A bulk of literature on traditional Korean medicine describe their activities and functions for respiratory problems. Therefore, we hypothesized that the combination of these plants might be effective in alleviating respiratory symptoms. Aim of the studyIn this study, we investigated whether TADIOS ameliorates LPS-induced acute lung injury via regulation of the Nrf2-HO-1 signaling pathway. Materials and methodsThe LPS-induced acute lung injury mouse model was used to determine the anti-inflammatory and anti-oxidative stress effects of TADIOS. The amount of marker compounds contained in TADIOS was quantified using high-performance liquid chromatography (HPLC) analysis. The protein level of pro-inflammatory cytokines in culture supernatant was measured by ELISA. Changes in the RNA level of pro-inflammatory cytokines in mice lungs and RAW264.7 cells were measured by quantitative RT-PCR. The relative amounts of reactive oxygen species (ROS) were measured by DCF-DA assay. Western blot analysis was used to evaluate expression of cellular proteins. Effects of TADIOS on antioxidant responsive elements (AREs) were determined by luciferase assay. The severity of acute lung injury was evaluated by Hematoxylin & Eosin (H&E) staining. To test the effects of TADIOS on LPS-induced oxidative stress, myeloperoxidase (MPO) activity and the total antioxidant capacity were measured. ResultsTADIOS was prepared by extraction of a blend of these three plants by ethanol, and quality control was performed through quantification of marker compounds by HPLC and measurement of bioactivities using cell-based bioassays. In the murine macrophage cell line RAW264.7, TADIOS effectively suppressed the production of pro-inflammatory cytokines such as IL-6 and IL-1β, and also ROS induced by LPS. When RAW264.7 cells were transfected with a luciferase reporter plasmid containing nucleotide sequences for AREs, TADIOS treatment increased the level of relative luciferase units in a dose-dependent manner. In the LPS-induced acute lung injury mouse model, orally administered TADIOS alleviated lung damage and neutrophil infiltration induced by LPS. Consistent with the in vitro data, treatment with TADIOS inhibited the LPS-mediated expression of pro-inflammatory cytokines and oxidative stress, and activated the Nrf2-HO-1 axis. ConclusionOur data suggest the potential for TADIOS to be developed as a safe and effective therapeutics for the treatment of acute respiratory distress syndrome.

Fast and easily applicable LC-UV method for analysis of bioactive anthrones from Aloe leaf latex

Aloe leaf latex is a commonly used plant preparation in traditional medicine. However, quality control on the content of medicinally important constituents is often limited. Hence, establishing a reliable quality control method to identify and quantify bioactive markers is important to ensure safety and efficacy.In the present study, a novel liquid chromatographic (LC) method was developed and validated for efficient analysis of bioactive markers to evaluate the quality of aloe leaf latex. Quantification of marker compounds was possible in only 7 min on a monolithic column using gradient elution with 0.1 % formic acid in acetonitrile and water as mobile phases. The major compounds (aloins A and B) could be baseline separated together with related compounds within 10 min. The method showed excellent linearity with determination coefficients (r2) of 0.9999. Detection limits were 0.017 and 0.013 μg/mL, while quantification limits were 0.057 and 0.043 μg/mL for aloin A and aloin B, respectively. Relative standard deviation (RSD) values for intra- and inter-day precision were less than 2% and recoveries for both aloins were close to 100 %. The robustness was evaluated using an experimental design. The method was applied to some aloe leaf latex samples from Ethiopia. Aloin contents varied from 14 to 35 % and two unknown peaks were tentatively identified as aloinoside and microdontin.

Integration of mass spectral fingerprinting analysis with precursor ion (MS1) quantification for the characterisation of botanical extracts: application to extracts of Centella asiatica (L.) Urban.

IntroductionThe phytochemical composition of plant material governs the bioactivity and potential health benefits as well as the outcomes and reproducibility of laboratory studies and clinical trials.ObjectiveThe objective of this work was to develop an efficient method for the in‐depth characterisation of plant extracts and quantification of marker compounds that can be potentially used for subsequent product integrity studies. Centella asiatica (L.) Urb., an Ayurvedic herb with potential applications in enhancing mental health and cognitive function, was used as a case study.MethodsA quadrupole time‐of‐flight analyser in conjunction with an optimised high‐performance liquid chromatography (HPLC) separation was used for in‐depth untargeted fingerprinting and post‐acquisition precursor ion quantification to determine levels of distinct phytochemicals in various C. asiatica extracts.ResultsWe demonstrate the utility of this workflow for the characterisation of extracts of C. asiatica. This integrated workflow allowed the identification or tentative identification of 117 compounds, chemically interconnected based on Tanimoto chemical similarity, and the accurate quantification of 24 phytochemicals commonly found in C. asiatica extracts.ConclusionWe report a phytochemical analysis method combining liquid chromatography, high resolution mass spectral data acquisition, and post‐acquisition interrogation that allows chemical fingerprints of botanicals to be obtained in conjunction with accurate quantification of distinct phytochemicals. The variability in the composition of specialised metabolites across different C. asiatica accessions was substantial, demonstrating that detailed characterisation of plant extracts is a prerequisite for reproducible use in laboratory studies, clinical trials and safe consumption. The methodological approach is generally applicable to other botanical products.

HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY ANALYSIS AND FREE RADICAL SCAVENGING POTENTIAL OF SOUTH INDIAN ORTHODOX BLACK TEA

Objective: The objective of this study was to evaluate the free radical scavenging potential and high-performance thin-layer chromatography (HPTLC) fingerprinting of the ethanolic extract of south Indian orthodox black tea (OBT).Methods: Phytochemical analysis was carried out using standard methods, and free radical scavenging activity of the extract was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), superoxide anion (SOD), and hydroxyl radical scavenging capacities. The ethanolic extract of OBT was loaded in the pre-coated HPTLC plates (silica gel 60 F 254) E-MERCK KGaA. HPTLC was carried out with toluene: ethyl acetate: diethylamine (7:2:1), chloroform: methanol:formic acid (8.5:1.0:0.5), and butanol: isopropyl alcohol (1:1) as mobile phase for alkaloids, flavonoids, and terpenoids, respectively.Results: HPTLC results confirmed that the extract contained several potential active components such as flavonoids, alkaloids, and terpenoids as the slides revealed multicolored bands of varying intensities. Extract of OBT reliably showed the total phenolics 132.27 mg/g, flavonoids 72.52 mg/g, and alkaloids 66.01 mg/g of dry matter. The IC50 value of OBT for DPPH was found to be 372.22 μg/ml, SOD 311.93 μg/ml, NO 362.17 μg/ml, hydroxyl radical 342.14 μg/ml, and reducing power 178.54 μg/ml.Conclusion: The HPTLC fingerprinting profile developed for ethanolic extract will help in proper identification and quantification of marker compounds. The ethanolic extract of OBT was found to possess a wide range of phytochemicals with excellent antioxidant properties. This information may help to choose the best beverage to be consumed in the future.

Histo-Chromatographic Finger Printing Profiles of the Root of Plumbago zeylanica Linn and Quantification of Marker Compound, Plumbagin

Histo-Chromatographic Finger Printing Profiles of the Root of Plumbago zeylanica Linn and Quantification of Marker Compound, Plumbagin

Quantification of marker compounds in Cirsium setidens Nakai by HPLC-DAD

An effective method of high performance liquid chromatography-diode array detector (HPLC-DAD) was developed for the simultaneous quantification of two marker compounds, pectolinarin and pectolinarigenin, in Cirsium setidens Nakai, a Korean wild herb. The two marker compounds of C. setidens were separated on a C18 column with a gradient system. The calibration curves of the two standard compounds exhibited good linearity (R 2 >0.9992). The limits of detection (LOD) values of pectolinarin and pectolinarigenin were 0.52 and 0.41 µg/mL, respectively, while their limits of quantification (LOQ) values were 1.57 and 1.25 µg/mL, respectively. The relative standard deviations (RSDs) of the data of the intra- and inter-day experiments were less than 1.65 and 2.78%, respectively. The results of the recovery test were 98.06-105.81% with an RSD value of 1.87-3.50%. In conclusion, the results of validation showed that the proposed HPLC method was useful for the quantification of the two marker compounds in C. setidens.

HPTLC fingerprinting analysis of Evolvulus alsinoides (L.) L.

Plants and plant-based products are the basis of many of the modern pharmaceuticals we use today for various ailments. High performance thin layer chromatography (HPTLC) is a valuable tool for the investigation of herbal products with respect to different aspects of their quality. To study the HPTLC fingerprinting of ethanolic extract of Evolvulus alsinoides (L.)L. A CAMAG HPTLC system equipped with LINOMAT 5 applicator, a TLC scanner 3, REPROSTAR 3 and WIN CATS-4 software were used. The study revealed the presence of steroids, terpenoids and glycosides in ethanolic extract of E alsinoides . The HPTLC fingerprinting profile developed for ethanolic extract will help in proper identification and quantification of marker compounds. By isolating and identifying marker compounds, new drugs can be formulated to treat various diseases.

HPTLC Fingerprinting of different leaf extracts of Tylophora indica (Burm f.) Merill.

Tylophora indica is very popularly used for the treatment of asthma based on its traditional use for asthma. Tylophora is perennial climbing plant native to the plains, forests, and hills of southern and eastern India. A method has been developed for different extracts of Tylophora indica for HPTLC fingerprinting analysis for identification and quantification of marker compound. For chloroform extract-Chloroform(90): Methanol (5) : Ethyl acetate (5) v/v, Methanol Extract-Toluene(5): Chloroform(90), Ethyl acetate(5) v/v and for Petroleum ether extract-Hexane(40) : Ethyl acetate (60) v/v. The HPTLC fingerprinting profile developed for different extracts of Tylophora indica will help in proper identification and quantification of marker compound.

Development and densitometric standardization of Convolvulus pluricaulis containing herbal medicinal products by quantification of marker compound

Convolvulus pluricaulis (Fam. Convolvulaceae) is a perennial herb with a small woody-branched rootstock and occurs in tropical and subtropical countries. It is available in Asian market as Shankhpushpi. A thin layer chromatographic method with densitometric UV detection at λ=226 nm has been developed for standardization of Convolvulus pluricaulis containing herbal medicinal products by quantification of one isolated marker compound. The isolated marker compound was characterized as 3β, 23, 24-trihydroxyolean-12-en-28-oic acid. TLC analysis was performed on aluminium backed silica gel 60 F254 plates with n-hexane-ethyl acetate, 7:4 (v/v), as a mobile phase. Under these experimental conditions the method was highly sensitive (the limit of detection was 14.2ng) and recovery was satisfactory (from 94.46% to 97.24%). The results obtained during validation of the method were confirmed in standardization of herbal syrups and tablets by quantification of isolated marker, because high precision and accuracy were achieved.