Bioactive Marker Compounds Research Articles

Simultaneous rapid detection of glycyrrhizin and sennoside A in Daiokanzoto samples by lateral flow immunoassay.

Glycyrrhizin (GLY) and sennoside A (SA) are characteristic bioactive marker compounds of the Kampo medicine Daiokanzoto. Their accurate detection in blends of Rhei rhizoma and Glycyrrhizae radix of several species (4:1 or 4:2) is essential for quality control and to ensure therapeutic efficacy. A rapid, efficient assay can significantly facilitate their detection. To establish a rapid qualitative assay for GLY and SA detection, a lateral flow immunoassay (LFA) was developed using specific monoclonal antibody (mAb) nanoparticles. This assay harnesses the competitive binding of mAb nanoparticles to the immobilized analytes on test strips and free analytes in the samples. Two conjugates for detecting GLY and SA, GLY-bovine serum albumin and SA-human serum albumin, were separately immobilized on the test zones of LFA strips. The detection mechanism is reliant on the visual detection of color changes in the test zones. When GLY and SA were present in samples, they contended with the immobilized conjugates on the strip to bind with the mAb nanoparticles and produced distinct color patterns in the test zones. The limits of detection of the assay for GLY and SA were both 3.13 μg/mL. The capability of the LFA was substantiated using plant samples and Daiokanzoto, and its alignment with indirect competitive ELISA results was confirmed. The introduced LFA is a groundbreaking procedure that offers a rapid, straightforward, and sensitive method for simultaneously detecting GLY and SA in Daiokanzoto samples. It is instrumental in ensuring product quality.

Correlation of bioactive marker compounds of an orally applied Morus alba root bark extract with toxicity and efficacy in BALB/c mice.

Introduction: In traditional Chinese medicine, the root bark of Morus alba L. is used to treat respiratory infections. Recently, anti-inflammatory and multiple anti-infective activities (against influenza viruses, corona virus 2, S. aureus, and S. pneumoniae) were shown in vitro for a standardized root bark extract from M. alba (MA60). Sanggenons C and D were identified as major active constituents of MA60. The aim of the present preclinical study was to evaluate, whether these findings are transferable to an in vivo setting. Methods: MA60 was orally administered to female BALB/c mice to determine 1) the maximum tolerated dose (MTD) in an acute toxicity study and 2) its anti-influenza virus and anti-inflammatory effects in an efficacy study. A further aim was to evaluate whether there is a correlation between the obtained results and the amount of sanggenons C and D in serum and tissues. For the quantitation of the marker compounds sanggenons C and D in serum and tissue samples an UPLC-ESI-MS method was developed and validated. Results: In our study setting, the MTD was reached at 100mg/kg. In the efficacy study, the treatment effects were moderate. Dose-dependent quantities of sanggenon C in serum and sanggenon D in liver samples were detected. Only very low concentrations of sanggenons C and D were determined in lung samples and none of these compounds was found in spleen samples. There was no compound accumulation when MA60 was administered repeatedly. Discussion: The herein determined low serum concentration after oral application once daily encourages the use of an alternative application route like intravenous, inhalation or intranasal administration and/or multiple dosing in further trials. The established method for the quantitation of the marker sanggenon compounds in tissue samples serves as a basis to determine pharmacokinetic parameters such as their bioavailability in future studies.

The chemistry and activity-orientedcharacterization of isoflavones difference between roots of Pueraria lobata and P. thomsonii guided by feature-based molecular networking.

Isoflavones are important chemical components in Pueraria species with various biological activities. This study proposed an integrated strategy combining feature-based molecular networking (FBMN), chemometrics and activity evaluation for isoflavone analysis in the roots of P. lobate (PLR) and P. thomsonii (PTR). Based on the strategy, a total of 68 isoflavones were annotated in the two Pueraria species, and 11 significant difference isoflavones between PLR and PTR were identified by chemometric methods. Additionally, the correlation coefficient between the characteristic isoflavones and hypoglycemic activity were calculated, and 7 isoflavones were further confirmed as bioactive marker compounds. This approach provided guidance for the discovery of active markers among different products.

The Efficient Synthesis and Anti-Fatigue Activity Evaluation of Macamides: The Unique Bioactive Compounds in Maca.

Macamides are a class of amide alkaloids that are only found in maca and are widely considered to be its bioactive marker compounds. More than thirty macamide monomers have been identified in recent years; however, it is difficult to obtain a single macamide monomer from the maca plant because of their similar structures and characteristics. We used the carbodiimide condensation method (CCM) to efficiently synthesize five typical macamides, including N-benzyl-hexadecanamide (NBH), N-benzyl-9Z,12Z,15Z-octadecenamide, N-(3-methoxybenzyl)-9Z,12Z-octadecenamide, N-benzyl-9Z,12Z-octadecenamide, and N-(3-methoxybenzyl)-9Z,12Z,15Z-octadecadienamide. All the synthesized macamides were purified by a one-step HPLC with a purity of more than 95%. NBH is the most abundant macamide monomer in natural maca, and it was selected to evaluate the anti-fatigue effects of macamides. The results indicated that NBH could enhance the endurance capacity of mice by increasing liver glycogen levels and decreasing blood urea nitrogen, lactate dehydrogenase, blood ammonia, and blood lactic acid levels. Macamides might be the active substances that give maca its anti-fatigue active function.

Simultaneous densitometric determination of β-sitosterol and lupeol through validated HPTLC method in different plant parts of Uraria picta (Jacq.) Desv. ex DC. – A dashmool species

Abstractβ-sitosterol (BS) and lupeol (LU) exhibit a number of biological activities and are the important bioactive marker compounds in pharmaceutical science. In the present study, a simple, precise, accurate and validated high performance thin layer chromatographic (HPTLC) method was developed for simultaneous quantification of these two compounds in leaves, stem and roots of Uraria picta, a critically endangered medicinal plant and one of the important constituents of ten plants ayurvedic formulation called Dashmoola. Standards and test samples were applied on TLC aluminum plate precoated with 0.2 mm layer of silica gel 60F254. The plate was run in a twin glass chamber comprising toluene: methanol: chloroform (8:1:1, v/v/v) as a mobile phase. The plates were immersed in anisaldehyde-sulfuric reagent and then heated at 105 °C for 5 min in CAMAG TLC plate heater for appearance of bands. Densitometric scanning was performed at λmax = 525 nm using tungsten light source in CAMAG TLC Scanner4 armed with WinCATS software. RF values of BS and LU standards and those of test samples were found to be 0.53 ± 0.01 and 0.63 ± 0.01 respectively. The method was further validated by following the International Conference of Harmonization (ICH) guidelines. For BS and LU, the linear regression data for the calibration plots revealed a satisfactory linear association with r2 = 0.995 and 0.998, respectively. Linear range for both BS and LU was 200–600 ng/band. Accuracy of the method was evaluated by performing recovery study at three different levels by standard addition methods with an average recovery of 99.86% and 101.07%. The results revealed that the leaf samples of U. picta contained highest concentration of BS (0.150 ± 0.02%) while its root samples confined the highest concentration of LU (0.149 ± 0.01%). The developed method can be applied for routine and quality control analysis in different herbal formulations containing U. picta species.

UPLC-QTof-MS chemical profiling and characterization of antiproliferative and anti-inflammatory compounds from seven Hypericum species in China

Many Hypericum species have been used as medicinal plants, herbal teas, and dietary supplements around the world. A previous ethnobotanical survey found that seven Hypericum species in southern China (H. daliense, H. hengshanense, H. himalaicum, H. japonicum, H. petiolulatum subsp. yunnanense, H. seniawinii, and H. wightianum) have been used traditionally to treat inflammation and infectious diseases, but studies on the chemical compositions and bioactivities of these folk medicines are limited. The current study aimed to characterize bioactive marker compounds correlated with two in vitro bioassays for cytotoxicity and anti-inflammation. Methanolic extracts of these species were chemically profiled using UPLC-QTof-MS metabolomics, characterizing the 52 major components from these Hypericum species. To chemically differentiate these seven species, 112 chemical features were characterized as potential chemotaxonomic markers, out of which 89 were tentatively identified. Using a chemometric approach, antiproliferative activity was tracked with chemical markers. H. japonicum showed antiproliferative effects on SMMC-7721 human hepatocarcinoma cell line with an IC50 value of 59.8 μg/mL. Comparison of H. japonicum to six other Hypericum species revealed that betulinic acid, isojacareubin, and taxifolin are potential antiproliferative compounds. In addition, a primary in vitro anti-inflammatory assay revealed that all the seven extracts inhibited NO production in lipopolysaccharide-induced RAW 264.7 murine macrophage cells at 100 μg/mL, indicating that anti-inflammatory compounds may exist in all the seven species. Out of the 52 major components identified from these species, 11 have been previously reported to possess anti-inflammatory properties. These findings provide a scientific rationale for traditional uses of the seven Hypericum species in China and merit further study for the development of new medicinal products.

Identification and estimation of bioactive constituents Negundoside, Berberine chloride, and Marmelosin by HPLC and HPTLC for development of quality control protocols for Ayurvedic medicated oil formulation

BackgroundAnu Taila is an ancient medicated oil Ayurvedic preparation that is commonly used for nasya karma. It contains more than 25 herbs and goat milk as per the Ayurvedic Formulary of India (AFI). It strengthens the neck, shoulder, and chest muscles and improves the capacity of sense organs. It delays the aging process and reduces hair fall. Recent studies showed that it is also useful in COVID-19. In the current study, an attempt to develop quality control protocols and evaluate the standardization parameters like refractive index, iodine value, saponification value, peroxide value, acid value, rancidity, HPTLC fingerprint profile along with major bioactive compound and quantification of Berberine chloride, Negundoside, and Marmelosin by HPLC. Establishing quality protocol and standard parameters like physicochemical parameters and estimation of bioactive compounds of this preparation is significant for quality control.ResultsIn this study, HPTLC identifies bioactive chemical compounds like Berberine chloride, Marmelosin, Negundoside, glycyrrhizin, and para hydroxybenzoic acid (PHBA), Lupeol, Embelin, and Solasodine, which were present in the Anu Taila formulation. HPLC was used to estimate the bioactive marker compounds Negundoside, Berberine chloride, and Marmelosin were present in the Anu Taila formulation. The quantitative evaluation of Berberine chloride (0.0013%), Marmelosin (0.0366%), Negundoside (0.0086%) is present in Anu Taila formulation.ConclusionThe study reveals that sufficient quality control parameters were followed during the preparation of the formulation. Physicochemical analysis was carried out as per the guidelines of Ayurvedic Pharmacopeia of India. HPTLC and HPLC profiles generated in this particular study can be considered as a preliminary tool ascertaining the authenticity of Anu Taila.

The Standardized Wood Extract of Albizia myriophylla: Its Potential as an Active Ingredient in an Anti-inflammatory Herbal Gel Formulation.

Albizia myriophylla has been used in Thai folk medicine for treating inflammation-related diseases. The wood of this medicinal plant is traditionally used as a single herbal drug in the form of an aqueous decoction and as a component in several Thai herbal formulations for the remedy of fever, sore throat, and aphthous ulcers. This study aimed to evaluate in vivo the anti-inflammatory potential and possible mechanism of action of the standardized wood extract of A.myriophylla as well as to investigate the anti-inflammatory activity and physicochemical properties of the developed herbal gel formulation containing standardized wood extract of A.myriophylla. Results of quantitative HPLC analysis demonstrated that the standardized wood extract of A.myriophylla contained 22.95 mg/g of 8-methoxy-7,3',4'-trihydroxyflavone, a bioactive marker compound of A.myriophylla. The standardized wood extract of A.myriophylla (1% w/v) exhibited remarkable inhibition (54.4 - 80.3%) in the croton oil model of topical inflammation at all assessment times, comparable to standard indomethacin (55.3 - 63.6%). Real-time quantitative reverse transcription-polymerase chain reaction was performed to clarify the anti-inflammatory mechanism of standardized wood extract of A.myriophylla, and the result showed that this standardized extract decreased lipopolysaccharide-induced nitric oxide synthase mRNA levels in a dose-dependent manner. The developed herbal gel containing standardized wood extract of A.myriophylla (1% w/w) had good physicochemical characteristics and exhibited potent inhibition (51.4 - 77.8%) of inflammation in a rat ear edema model at all assessment times, comparable to indomethacin gel (33.3 - 40.5%). The notable anti-inflammatory activity of standardized wood extract of A.myriophylla and its developed herbal gel formulation indicates their potential application as natural anti-inflammatory agents.

Effect of extraction procedure on the yield and biological activities of hydroxychavicol from Piper betle L. leaves

Piper betle Linn is one of the most common ethnomedicinal plants with its extract being popularly used in the modern product to enhance functionality. However, extraction methods always lead to differences in biological activities. The objectives of this study were to evaluate the effects of the extraction methods on the yield and biological activities of hydroxychavicol from P. betle L. extracts and to determine the correlation between the hydroxychavicol content and biological activities of P. betle L. extracts such as antioxidant, antimicrobial, and anticancer properties. The purity of the hydroxychavicol and its concentration (quantitative) in the crude extracts were also evaluated using a reverse-phase HPLC while GC–MS was employed to determine other components (qualitative). The results showed that only certain extraction procedures gave high yields of hydroxychavicol as well as remarkable biological activities. The chloroform extract following boiling with water (M2) gave the highest percentage of hydroxychavicol content based on the HPLC analysis. M2 and pure hydroxychavicol actively inhibited all the five cancer cell lines studied except A549. M2 showed more effective inhibition activity against MCF 7 with an IC50 of 1.74 ug/mL. M2 extract also showed strong antibacterial activity against all the bacteria strains as well as a strong antifungal activity against Candida albicans. There was, however, a weak correlation between the hydroxychavicol content and the biological activities of P. betle L. extracts. In conclusion, extraction procedures greatly affect the yield and biological activities of hydroxychavicol from P. betle L. The designation of a single compound such as hydroxychavicol as a bioactive chemical marker compound in the P. betle L. extracts, however, is not enough to determine the biological activities of the extract.

Development and Validation of a Novel, Rapid Gradient HPLC Method for Simultaneous Estimation of Bioactive Marker Compounds in a Mixture of Convolvulus pluricaulis, Withania somnifera and Bacopa monnieri Extracts.

The use of herbs as medicine is an ancient form of healthcare known to mankind. Standardization of herbal medicines is however a challenging task and is the major bottleneck in their acceptance as the primary therapeutic option. The aim of this study was to develop and validate a simple, rapid HPLC method for standardizing the mixture of extracts of three Medhya Rasayanas (neurotonic), Convolvulus pluricaulis, Withania somnifera and Bacopa monnieri. Simultaneous estimation of the respective bioactive markers of these plants viz., scopoletin, withaferin A, bacoside A 3, bacopaside II, jujubogenin and bacosaponin C has been reported for the first time. The method was developed using Waters Hybrid X-Bridge shield with BEH technology 2.5μm, 4.6 × 75mm column and validated according to ICH guidelines. The 20minutes run time makes the method eco-friendly. The method was linear over a range of 12.5-400ng/10μL for scopoletin and 62.5-2,000ng/10μL for withaferin A, bacoside A 3, bacopaside II, jujubogenin and bacosaponin C with detection limits of 8.0, 48.3, 30.4, 40.7, 15.6 and 18.9ng/10μL and quantification limits of 24.5, 146.5, 92.2, 123.4, 47.4 and 57.4ng/10μL, respectively. The correlation coefficient for each analyte was >0.999. The intra-day and inter-day precision was <2%. These results confirmed the precision, accuracy and robustness of the proposed method.

Simultaneous determination of macaenes and macamides in maca using an HPLC method and analysis using a chemometric method (HCA) to distinguish maca origin

Macamides and Macaenes are the bioactive marker compounds in maca (Lepidium meyenii Walp., Brassicaceae) tuber. To simultaneously quantify these two types of compound, HPLC method was studied. To distinguish and group the growing regions of different maca samples, Hierarchical cluster analysis, a chemometric method, was applied to analyze the HPLC data. The calibration curves obtained using the HPLC method showed satisfactory linearity with determination coefficients >0.9998. The precision and repeatability relative standard deviation values were <4%, and the accuracy relative standard deviation value was <5%. The limits of detection was <0.1 μg/ml and the limit of quantification was <0.3 μg/ml. Our HPLC method was successfully used for the separation and determination of macamides and macaenes in Maca within 45 min, i.e., two macaenes (9-oxo-10E,12Z-octadecadienoic acid and 9-oxo-10E,12E-octadecadienoic acid) and five macamides (N-benzyl-9-oxo-10E,12Z-octadecadienamide, N-benzyl-9-oxo-10E,12E-octadecadienamide, N-benzyl-9Z,12Z,15Z-octadecatrienamide, N-benzyl-9Z,12Z-octadecadienamide and N-benzyl-hexadecanamide). The HPLC method was applied to analyze and quantify the seven compounds in thirty maca samples with different colors and origins. The origins of all the maca samples were distinguished and grouped using hierarchical cluster analysis of the HPLC data. Accordingly, the metabolism of macaenes and macamides in maca post-harvest processing has also been proposed. The HPLC method is efficient to simultaneously quantify the macamides and macaenes in maca. Analyzing the HPLC data using hierarchical cluster analysis can distinguish maca growing origins.

HPLC fingerprinting coupled with linear discriminant analysis for the detection of adulteration in Orthosiphon aristatus

Orthosiphon aristatus known as java tea is one of the medicinal plants commonly used in some herbal medicines especially for diuretics and to treat diabetes. Orthosiphon aristatus leaves could be substituted or mixed with Eupatorium riparium and Acalypha siamensis because of the similarity in their morphological leaves. To detect contamination of O. aristatus from the two similar plants, we developed an HPLC fingerprinting in combination with multivariate analysis. By using the developed method, we could also simultaneously determine two bioactive marker compounds in O. aristatus namely rosmarinic acid and sinensetin. Fingerprint chromatogram of the three samples showed a different chromatogram profile, so it could be used to distinguish the three samples. A combination of HPLC fingerprint and linear discriminant analysis was successfully grouped the three samples and adulterated O. aristatus with E. riparium and A. siamensis. The result indicated that the developed method could detect adulterated samples of O. aristatus from the other two plants used in this study and for the quantification of rosmarinic acid and sinensetin in O. aristatus simultaneously.

Simultaneous microwave assisted extraction and HPTLC quantification of mangiferin, amarogentin, and swertiamarin in Swertia species from Western Himalayas

Swertia is one of the most widely traded genus of 150 species, many of which are used in Ayurvedic, Chinese, Tibetan and traditional medicine. Swertia herb is mostly traded and exported in dried or in extract forms. In this present study, a microwave-assisted extraction (MAE) method coupled with simultaneous quantification of swertiamarin, amarogentin, and mangiferin from different species of Swertia using high performance thin layer chromatography (HPTLC) was developed. A three-level factor Box-Behken statistical design was used for optimization, where microwave power, irradiation time, and solvent-to-solid ratio were used as independent variables whereas swertiamarin, amarogentin and mangiferin were used as the dependent variables. Densitometric-HPTLC method was developed for the simultaneous estimation of swertiamarin, amarogentin, and mangiferin using mobile phase of ethyl acetate: acetic acid glacial: formic acid: water (10:1.2:1.2:2.7 v/v/v/v) and stationary phase as silica gel F254 HPTLC plate. In this study, five different Swertia species viz. S. angustifolia, S. cordata, S. chirata, S. paniculata, and S. nervosa collected from Western Himalayan, India were used to screen the elite Swertia species. Optimal extraction conditions to achieve the maximum yield of swertiamarin, amarogentin, and mangiferin were found to be 5.86%, 0.710%, and 4.93% respectively which were very close to the predicted values of 5.916%, 0.711%, and 5.084% respectively by keeping microwave power (530 W), time (4 min), and solvent-to-solid (30 mL). Densitogram patterns obtained from all the test samples and standard compounds revealed that the peaks corresponding to Rf 0.57, 0.64, and 0.74 were meant for swertiamarin, mangiferin, and amarogentin respectively. HPTLC studied showed that S. paniculata and S. angustifolia contained high content of bioactive compounds and could be used as a substitute for S. chirata, whereas S. cordata and S. nervosa could not be recommended as substitutes for S. chirata. Thus, the newly developed optimized MAE and HPTLC-densitometry method for simultaneous quantification of three bioactive marker compounds in five species of Swertia can be effectively implemented for quality control of herbal formulations.

HPTLC method for quantitative determination of hydroxycinnamic acid derivatives in Solanum lycopersicum

The study was aimed to validate and optimize high performance thin layer chromatography (HPTLC) method for the determination of phenolic compound hydroxycinnamic acid derivatives from the fruits of Solanum lycopersicum (tomato). The presence of caffeic acid and coumaric acid in the aqueous extracts of S. lycopersicum was observed in spectrophotometer at 200-400 nm. The phenolic functional group was recorded by FTIR analysis. In the HPTLC analysis, the pre-coated silica gel was used as the stationary phase. The solvent mixture containing toluene:ethyl acetate:formic acid:methanol (3:6:1.6:0.4) was identified as an optimum ratio were used as a mobile phase. The chromatograms of the extract was scanned by densitometer at 327 nm. The Rf values (0.67 and 0.69) and finger print data were recorded by WIN CATS software. The developed HPTLC methods for bioactive marker compounds present in the fruit were found to be simple, accurate, and economical.

An emerging strategy for evaluating the grades of Keemun black tea by combinatory liquid chromatography-Orbitrap mass spectrometry-based untargeted metabolomics and inhibition effects on α-glucosidase and α-amylase

Quantitative analysis and untargeted liquid chromatography mass spectrum (LC–MS) based metabolomics of different grades of Keemun black tea (KBT) were conducted. Quantitative analysis did not show tight correlation between tea grades and contents of polyphenols, but untargeted metabolomics analysis revealed that high-grades KBT were distinguished from the low-grades. S-plot and Variable Importance (VIP) analysis gave 28 marker compounds responsible for the discrimination of different grades of KBT. The inhibitory effects of KBT on α-amylase and α-glucosidase were positively correlated to tea grades, and the correlation coefficient between each marker compound and inhibitory rate were calculated. Thirteen compounds were positively related to the anti-glycemic activity, and theasinensin A, afzelechin gallate and kaempferol-glucoside were confirmed as grade-related bioactive marker compounds by chemical and bioassay in effective fractions. This study suggested that combinatory metabolomics and bioactivities assay provided a new strategy for the classification of tea grades.

Quantitative Analysis and monitoring of Bioactive Marker Compounds from Polygalae Radix by High Performance Liquid Chromatography.

Quantification monitoring of 74 samples of Polygalae Radix collected from Korean and China was conducted following the analysis method from Chinese Pharmacopoeia (CP) 2015 edition. However, this method is considered not accuracy enough because tenuifolin was chosen to be the only marker component for the quality control of Polygalae Radix. The de-sugar products of onjisaponins and oligosaccharide esters including tenuifolin(1), 3,4,5-trimethoxycinnamic acid(2), 4-methoxy cinnamic acid methyl ester(3) were isolated from the alkaloid hydrolysis products of extracts from Polygalae Radix. A novel simultaneous quantification of the three components by HPLC-UV was developed, and quantification mon-itoring for 17 samples collected from China and Korea was conducted. The results from the new analysis method improved complicated preprocessing method and presented the standard as the using of grand contents.

Comparative Phytochemical Analysis of Chinese and Bay Starvine (Schisandra spp.): Potential for Development as a New Dietary Supplement Ingredient

ABSTRACTSchisandra chinensis (Chinese starvine) is a popular dietary supplement with a rich history of use in traditional Chinese medicine. Schisandra glabra (bay starvine) is the only North American representative of the genus, and little is known about its history of traditional use, chemistry, and potential biological activity. In this study, we conducted comparative high-performance liquid chromatography-diode array detector (HPLC-DAD) analysis on S. glabra and S. chinensis fruits. Additional characterization of S. glabra was performed by liquid chromatography-Fourier transform mass spectrometry (LC-FTMS). Quantitative analysis of four bioactive marker compounds revealed that S. glabra does not have statistically higher levels of schisandrin A or schisandrol B than S. chinensis. S. glabra has lower levels of schisandrol A and γ-schisandrin. Total phenolic contents of the two species' fruits were not statistically different. S. glabra had higher total tannin content than S. chinensis. We discuss the relevance of this analytical analysis to the study of S. glabra as a potential dietary supplement ingredient and give specific consideration to the conservation challenges involved in commercially developing a regionally threatened species, even in semicultivated conditions.

Hop (Humulus lupulus L.) Extract and 6-Prenylnaringenin Induce P450 1A1 Catalyzed Estrogen 2-Hydroxylation.

Humulus lupulus L. (hops) is a popular botanical dietary supplement used by women as a sleep aid and for postmenopausal symptom relief. In addition to its efficacy for menopausal symptoms, hops can also modulate the chemical estrogen carcinogenesis pathway and potentially protect women from breast cancer. In the present study, an enriched hop extract and the key bioactive compounds [6-prenylnarigenin (6-PN), 8-prenylnarigenin (8-PN), isoxanthohumol (IX), and xanthohumol (XH)] were tested for their effects on estrogen metabolism in breast cells (MCF-10A and MCF-7). The methoxyestrones (2-/4-MeOE1) were analyzed as biomarkers for the nontoxic P450 1A1 catalyzed 2-hydroxylation and the genotoxic P450 1B1 catalyzed 4-hydroxylation pathways, respectively. The results indicated that the hop extract and 6-PN preferentially induced the 2-hydroxylation pathway in both cell lines. 8-PN only showed slight up-regulation of metabolism in MCF-7 cells, whereas IX and XH did not have significant effects in either cell line. To further explore the influence of hops and its bioactive marker compounds on P450 1A1/1B1, mRNA expression and ethoxyresorufin O-dealkylase (EROD) activity were measured. The results correlated with the metabolism data and showed that hop extract and 6-PN preferentially enhanced P450 1A1 mRNA expression and increased P450 1A1/1B1 activity. The aryl hydrocarbon receptor (AhR) activation by the isolated compounds was tested using xenobiotic response element (XRE) luciferase construct transfected cells. 6-PN was found to be an AhR agonist that significantly induced XRE activation and inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced XRE activity. 6-PN mediated induction of EROD activity was also inhibited by the AhR antagonist CH223191. These data show that the hop extract and 6-PN preferentially enhance the nontoxic estrogen 2-hydroxylation pathway through AhR mediated up-regulation of P450 1A1, which further emphasizes the importance of standardization of botanical extracts to multiple chemical markers for both safety and desired bioactivity.

Validated high performance thin layer chromatography method for the quantification of bioactive marker compounds in Draksharishta, an ayurvedic polyherbal formulation

Draksharishta is an ayurvedic polyherbal formulation with Draksha (Vitis vinifera L., Vitaceae) as chief ingredient prescribed for digestive impairment, respiratory disorders and weakness. These herbal medicines containing biologically active compounds play a significant role. Therefore it is necessary to carry out the chemical standardization of bioactive marker compounds present in the polyherbal ayurvedic formulation like Draksharishta. The aim of the present work was to develop and validate a HPTLC method for determination of gallic acid, catechin and resveratrol in commercially available marketed and in-house prepared formulations of Draksharishta. This is the first report of quantification of bioactive marker compound resveratrol using HPTLC in Draksharishta. The method employed silica gel precoated thin layer chromatography plates with F254 as the stationary phase. The respective mobile phases were used to develop the plates which separated bands according to the marker compound. Camag scanner V was used for densitometric scanning. Further, the method was validated according to International Conference of Harmonization (ICH) guidelines. The Rf values of the three marker compounds were measured. Correlation coefficients were calculated from the standard graph of linearity. Accuracy, precision and recovery were all within the required limits. The developed HPTLC methods for bioactive marker compounds present in in-house and marketed formulations were found to be simple, accurate, precise and robust.

Quality evaluation and pattern recognition analyses of bioactive marker compounds from Farfarae Flos using HPLC/PDA.

The flower bud of Tussilago farfara L., called Farfarae Flos, has traditionally been used in Oriental medicine for the treatment of bronchitis and asthma. To establish a standard for quality control as well as the reliable identification of Farfarae Flos, the contents of five standards, rutin (1), isoquercetin (2), 3,5-dicaffeoylquinic acid (3), tussilagone (4), and tussilagonone (5), were determined by quantitative high-performance liquid chromatography (HPLC)/photodiode array (PDA) analysis. The five standards were separated on a YoungJinBioChrom Aegispak C18-L (250-mm×4.6-mm, 5-µm) column by gradient elution using 0.03% trifluoroacetic acid in water (A), with acetonitrile (B) as the mobile phase. The flow rate was 1.0 mL/min, and the UV detector wavelength was set at 220 nm. The method was successfully used in the analysis of Farfarae Flos from different geographic origins with relatively simple conditions and procedures, and the results demonstrated satisfactory linearity, recovery, precision, accuracy, stability, and robustness. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of 62 Farfarae Flos samples. This result indicated that the established HPLC/PDA method is suitable for quantitation and pattern recognition analyses for the quality evaluation of Farfarae Flos.