Comparative Phytochemical Investigation of the Sources of Ayurvedic Drug Patha: A Chromatographic Fingerprinting Analysis

Standardization of herbal drugs based on their chemical and biological activity profile is an important prerequisite for acquiring the herbal market. The main problem encountered in standardization of Ayurvedic drugs is proper identification of the source plant. The present study was aimed to establish identification characters, quality control parameters, chemical and biological parameters for roots of three plants Cissampelos pareira, Cyclea peltata and Stephania japonica (Fam. Menispermaceae) which are being used as source of Patha, in the market. All the three plant were subjected for evaluation of quality control parameters as per WHO guidelines and root extracts and total alkaloidal fractions were subjected for HPTLC and HPLC fingerprinting analysis using a marker compound Bebeerine isolated from roots of Cissampelos pareira. The parameters studied clearly indicated the significant differences among the three plant materials. The roots of Cissampelos pareira can be distinguished from other two plants by presence of high concentration of alkaloids especially the presence of high concentration of pharmacologically active alkaloid bebeerine, which was found to be present in very low concentration in Stephania japonica and absent in roots of Cyclea peltata. The roots of Cyclea peltata were found to contain high concentration of saponins and comparatively in low concentration in Cissampelos pareira where as it was found to be absent in roots of Stephania japonica.     

Combinative method using HPLC fingerprint and quantitative analyses for quality consistency evaluation of an herbal medicinal preparation produced by different manufacturers

A combinative method using HPLC-DAD fingerprint and quantitative analysis was developed and validated for manufacturer-to-manufacturer quality consistency evaluation of Yiqing preparations. For fingerprint analysis, 22 peaks were selected as the characteristic peaks to evaluate the similarities of different samples collected from different manufacturers. The similarities of 12 Yiqing samples were beyond 0.90, indicating that samples from different manufacturers were, to some extent, consistent. Additionally, simultaneous quantification of nine markers including berberine, aloe-emodin, rhein, emodin, chrysophanol, baicalin, baicalein, wogonoside, and wogonin in Yiqing was performed to interpret the quality consistency. The results from the quantitative data showed that the contents of these nine marker compounds were quite consistent for batches produced within one manufacturer and significantly different from manufacturer-to-manufacturer. This study demonstrated that a combination of the chromatographic fingerprint and quantitative analysis offers an efficient way to quality consistency evaluation of herbal preparation.     

Quality assessment of Rhizoma et Radix Notopterygii by HPTLC and HPLC fingerprinting and HPLC quantitative analysis

This paper describes an improved quality assessment method for Rhizoma et Radix Notopterygii (the rhizome and root of Notopterygium incisum Ting ex H.T. Chang or Notopterygium forbesii Boiss). The method was established by using fingerprinting and quantitation of marker compounds (isoimperatorin, notopterol and bergapten) in this herbal medicine. The authentication of Rhizoma et Radix Notopterygii using high performance thin-layer chromatography (HPTLC) fingerprinting was achieved by comparing the colors and Rf values of the bands in TLC fingerprints with those of the marker compounds. The HPLC fingerprints of 16 batches of herbal samples from different regions of China showed similar chromatographic patterns. Five peaks were selected as characteristic peaks, and three of these were identified by using LC-MS-MS techniques. The relative retention times of these characteristic peaks in the HPLC fingerprint were established as an important parameter for identification of Rhizoma et Radix Notopterygii. Finally, the pharmacologically active marker compounds isoimperatorin, notopterol and bergapten in this herb were quantitatively determined using a validated reverse-phase HPLC method.     

Development of an in-line HPLC fingerprint ion-trap mass spectrometric method for identification and quality control of Radix Scrophulariae

Chromatographic fingerprinting has been widely accepted as a crucial method for qualitative and quantitative analyses of bioactives within traditional Chinese medicine. A fingerprint provides detailed information, specific for any given herb, thus facilitating the quality control measures of a given traditional Chinese medicine. In this article, quality assessment of Radix Scrophulariae was achieved by using high performance liquid chromatography combining diode-array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI/MS). Eight batches of sample obtained from different origins in China were used to establish the fingerprint and quantitative analyses. By comparing the retention times, UV and MS spectral data with reference standards, four characteristic peaks in the chromatograms were confirmed as corresponding to acetoside, angoroside C, cinnamic acid, and harpagoside. In addition, other two characteristic peaks were tentatively identified, following the literature interpretation of HPLC-ESI-MS and LC-MS/MS (affording structural information) to be sibirioside A and scrophuloside B₄, respectively. The results indicated that the newly developed HPLC-DAD-MS fingerprint method would be suitable for quality control of Radix Scrophulariae.     

Cytotoxic Indole Alkaloids against Human Leukemia Cell Lines from the Toxic Plant Peganum harmala

Bioactivity-guided fractionation was used to determine the cytotoxic alkaloids from the toxic plant Peganum harmala. Two novel indole alkaloids, together with ten known ones, were isolated and identified. The novel alkaloids were elucidated to be 2-(indol-3-yl)ethyl-α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside (2) and 3-hydroxy-3-(N-acetyl-2-aminoethyl)-6-methoxyindol-2-one (3). The cytotoxicity against human leukemia cells was assayed for the alkaloids and some of them showed potent activity. Harmalacidine (compound 8, HMC) exhibited the highest cytotoxicity against U-937 cells with IC₅₀ value of 3.1 ± 0.2 μmol/L. The cytotoxic mechanism of HMC was targeting the mitochondrial and protein tyrosine kinase signaling pathways (PTKs-Ras/Raf/ERK). The results strongly demonstrated that the alkaloids from Peganum harmala could be a promising candidate for the therapy of leukemia.     

中药附子的研究进展

目的探讨附子的药效物质基础,为附子的进一步开发和利用提供参考。方法通过查阅国内外相关文献,进行归纳、分析,对附子的化学成分、生物活性及质量控制进行综述。结果附子的主要成分为生物碱类,其中以乌头碱骨架的C-19型二萜生物碱为主,其次为C-20型二萜生物碱,以海替生碱型、维替碱型、纳哌啉型、光翠雀碱型骨架类型为主;除生物碱外,还含有黄酮、皂苷、神经酰胺类等成分。附子的生物活性主要包括强心、抗肿瘤、抗炎镇痛、增强免疫、抗衰老等作用。附子药材质量控制方法包括高效液相色谱(HPLC)指纹图谱研究及主要成分的含量测定研究。结论附子的现代研究取得了一定进展,但在阐明其传统药效作用及机制方面仍存在一些问题,需要进一步深入探讨。     

Quality Assessment of Psoralea fructus by HPLC Fingerprint Coupled with Multi-components Analysis

Psoralea Fructus, the dried and ripe fruit of Psoralea corylifolia L., have been used as traditional medicine. There is substantial evidence that multiple constituents are responsible for the beneficial effects of this medicine. To effectively control the quality of this herbal medicine, HPLC fingerprint analysis was performed on a SinoChrom ODS-BP column with mobile phase of a gradient prepared from H₂O and CH₃CN, which the conditions used for gradient elution were: 0–10 min, 5–45% CH₃CN; 10–45 min, 45–70% CH₃CN; 45–50 min, 70–100% CH₃CN; 50–60 min, 100–100% CH₃CN, and the flow rate was 1.0 ml/min. It was obtained on the basis of the chromatographic data from 28 batches of samples, which contained 26 common peaks and 13 peaks were identified by the electrospray ionization-mass spectrometry as psoralen, isopsoralen, isobavachin, neobavaisoflavone, bavachin, corylin, broussochalcone B, psoralidin, isobavachalcone, bavachinin, corylifol A, bavachalcone and backuchiol. The contents of these 13 compounds were also simultaneously examined. By using principal component analysis, 28 batches of samples collected from 6 producing locations with different collecting time were evaluated and differentiated. In summary, the data as described in this study offer valuable information for quality control and proper use of Psoralea Fructus.     

Development of HPLC fingerprint for species differentiation and quality assessment of Rhizoma Smilacis Glabrae

Rhizoma Smilacis Glabrae (RSG) is a commonly used herbal material in functional food and Traditional Chinese Medicine. A HPLC chromatographic fingerprint was developed for its quality control and species differentiation. Nine peaks were found in the chromatogram of RSG and all these peaks were identified by diode array detection and electrospray ionization–MS/MS: 5-O-caffeoylshikimic acid, taxifolin, engeletin, isoengeletin, trans-resveratrol, astilbin and its three stereoisomers. Six of these constituents were consistently found in 18 batches of samples. The standard fingerprint of RSG was generated by mean simulation of all tested samples. Using the standard fingerprint, RSG could be easily differentiated from Rhizoma Smilacis Chinae and Rhizoma Heterosmilacis, the two species that can be confused with RSG.     

LC/MS fingerprinting of Shenmai injection: a novel approach to quality control of herbal medicines

Chromatographic fingerprinting has been recommended as a potential and reliable strategy for the quality control of herbal medicines. Although varieties of chromatographic techniques, particularly HPLC, have been widely employed, hyphenated chromatographic approach has not been sufficiently exploited in chromatographic fingerprinting. In this work, LC/MS fingerprinting of Shenmai injection was developed. Thirty ginsenosides as well as seven ophioponins were selected to construct the LC/MS fingerprint using selective ion monitoring (SIM) mode, while previous HPLC fingerprint [H.J. Zhang, Y.J. Wu, Y.Y. Cheng, J. Pharm. Biomed. Anal. 31 (2003) 175-183] only represents the ginsenosides. Subsequently, the proposed LC/MS fingerprints were applied to identifying the product manufacturers. All the samples were accurately classified based on their LC/MS fingerprints in conjunction with principal components analysis (PCA). This study would be potentially helpful to improve the quality control ability of fingerprinting-based strategy for complex herbal medicines.     

Development of a chromatographic fingerprint for the chloroform extracts of Ganoderma lucidum by HPLC and LC-MS

A new high-performance liquid chromatographic (HPLC) fingerprinting method was developed for the quality control of Ganoderma lucidum. Twenty-nine batches obtained from three different origins in China were used to establish the fingerprint. The constituents of these samples were separated with a Kromasil C(18) column (250 mm x 4.6 mm, 5 microm) by linear gradient elution using water-acetic acid (100:0.1, v/v) and acetonitrile as mobile phase components at a flow rate of 0.8 ml/min and detector wavelength at 254 nm. Mean chromatograms and correlation coefficients of samples were calculated by the software "Similarity Evaluation System for Chromatographic Fingerprint of TCM". There were 19 common peaks in this fingerprint. Eleven of these common peaks were tentatively identified with reference to literature data based on their liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS) and UV data. This profile was then successfully used to identify and assess the differences among samples from various origins with the aid of similarity analysis. The diverse similarities among different samples indicated that the quality of G. lucidum was not stable and the products from different areas were inconsistent. All results showed that the developed fingerprint assay was specific and could further serve for quality identification and comprehensive evaluation of G. lucidum.     

An approach based on antioxidant fingerprint–efficacy relationship and TLC bioautography assay to quality evaluation of Rubia cordifolia from various sources

In this work, an approach based on antioxidant fingerprint–efficacy relationships and TLC bioautography assay was developed for quality evaluation of traditional Chinese medicine (TCM). First, chemical fingerprints of 20 batches of different sources of Rubia cordifolia were established by HPLC. The antioxidant activities of these samples were evaluated by 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay. Then, five active components from fingerprint peaks were determined by both multiple correlation analysis and TLC bioautography assay. Similarity analysis and hierarchical clustering analysis (HCA) based on these active peaks were chosen to evaluate the quality of R. cordifolia from different sources. The samples with similarities below 0.9 had poorer antioxidant activities and those having stronger activities fell into the same cluster in HCA. Finally, five batches of commercial samples were processed in the same way to verify the feasibility of this method. The results suggested that chemical fingerprinting combined with similarity analysis based on fingerprint–efficacy relationship and TLC bioautography could evaluate the antioxidant activity of R. cordifolia from various sources. The established technique would also provide an easy method of evaluating the quality of other TCMs.     

Botanicals in Ribes nigrum bud-preparations: An analytical fingerprinting to evaluate the bioactive contribution to total phytocomplex

Abstract

Context. Ribes nigrum L. (Grossulariaceae) is among the most commonly used herbal medicines and it is popularized for its alleged tonic effect and curative and restorative properties. The current practice of identifying herbal extracts is by measuring the concentration of the main botanicals. Their concentrations are used to characterize the herbal preparations and fingerprinting is recommended by the main Pharmacopeias as a potential and reliable strategy for the quality control of complex mixtures.

Objective: The aim of this research was to perform an analytical study of R. nigrum bud-preparations, in order to identify and quantify the main bioactive compounds, obtaining a specific chemical fingerprint to evaluate the single class contribution to herbal preparation phytocomplex.

Materials and methods: The same analyses were performed using a high-performance liquid chromatograph-diode array detector both on University lab preparations and on commercial preparations from different Italian locations. Different chromatographic methods were used to analyse the macerated samples, two for polyphenols and one for terpenic compounds.

Results. Ribes nigrum was identified as a rich source of anti-inflammatory and antioxidant compounds. The observed analytical firgerprint demonstrated that these bud-preparations represent a rich source of terpenic and polyphenolic compounds, especially catechins and phenolic acids.

Discussion and conclusion: Analytical fingerprinting could be an important tool to study the assessment of chemical composition and bioactivities of plant-derived products, helping to find new sources of natural health-promoting compounds: this study allowed the development of an effective tool for quality control through botanical fingerprinting of bud preparations.     

Rapid investigation of α-glucosidase inhibitory activity of Phaleria macrocarpa extracts using FTIR-ATR based fingerprinting

Phaleria macrocarpa, known as “Mahkota Dewa”, is a widely used medicinal plant in Malaysia. This study focused on the characterization of α-glucosidase inhibitory activity of P. macrocarpa extracts using Fourier transform infrared spectroscopy (FTIR)-based metabolomics. P. macrocarpa and its extracts contain thousands of compounds having synergistic effect. Generally, their variability exists, and there are many active components in meager amounts. Thus, the conventional measurement methods of a single component for the quality control are time consuming, laborious, expensive, and unreliable. It is of great interest to develop a rapid prediction method for herbal quality control to investigate the α-glucosidase inhibitory activity of P. macrocarpa by multicomponent analyses. In this study, a rapid and simple analytical method was developed using FTIR spectroscopy-based fingerprinting. A total of 36 extracts of different ethanol concentrations were prepared and tested on inhibitory potential and fingerprinted using FTIR spectroscopy, coupled with chemometrics of orthogonal partial least square (OPLS) at the 4000–400 cm⁻¹ frequency region and resolution of 4 cm⁻¹. The OPLS model generated the highest regression coefficient with R²Y = 0.98 and Q²Y = 0.70, lowest root mean square error estimation = 17.17, and root mean square error of cross validation = 57.29. A five-component (1+4+0) predictive model was build up to correlate FTIR spectra with activity, and the responsible functional groups, such as −CH, −NH, −COOH, and −OH, were identified for the bioactivity. A successful multivariate model was constructed using FTIR-attenuated total reflection as a simple and rapid technique to predict the inhibitory activity.     

TLC and HPTLC Fingerprints of Various Secondary Metabolites in the Stem of the Traditional Medicinal Climber,Solena amplexicaulis

Aim of this study was to develop a TLC and a HPTLC fingerprint profiles for various secondary metabolites of methanol extracts of the stem of the traditional medicinal climber, Solena amplexicaulis. These studies were carried out as per the methods of Harborne and Wagner et al. The profiles of various individual secondary metabolites were made and developed for authentication. The methanol extract of the stem showed the presence of 6 alkaloids, 6 flavonoids, 2 glycosides, 9 saponins and 3 terpenoids. Owing to the presence of rich variety of secondary metabolites, the stem extract of S. amplexicaulis is expected to exhibit therapeutic properties.     

叶下珠药材的高效液相色谱指纹图谱研究

目的对叶下珠的乙醇提取物进行指纹图谱研究。方法采用高效液相色谱法,在一定的洗脱条件下,以柯里拉京为对照品,比较不同产地及采收时期的11批叶下珠药材乙醇提取物的指纹图谱。结果在指纹图谱中找到了10个共有特征峰,建立了叶下珠药材的高效液相色谱指纹图谱和标准。结论指纹图谱为叶下珠药材的质量控制提供了依据。     

Design,Development and Rationalization of Sarpagandha Ghanvati

Sarpagandha ghanvati is a classical Ayurvedic formulation widely prescribed for anxiety and insomnia. It contains Sarpagandha (roots of Rauwolfia serpentina L. (Benth.) Ex Kurz; Family: Apocyanaceae), Khurasani ajowan (Hyocyamus niger L.; Family: Solanaceae) seeds, Jatamansi (Nardostachys jatamansi DC. Family: Valerianaceae) roots and Pipplamul (root of Piper longum L.; Family: Piperaceae). The objective of this study was to make a comparative evaluation of Ghanvatis and tablets of this formulation. Two tablet formulations were prepared; one incorporating only powders of all ingredients; the other with ethanol extracts of the first three ingredients and powder of Piper longum root. Similarly, two types of Sarpagandha ghanvati pills were prepared; one as per Ayurvedic Formulary of India; the other with ethanol extracts of the first three ingredients and powder of Piper longum root. Alcohol extracted 0.22% w/w of total alkaloids as against 0.061% w/w extracted by water. Tablets prepared with powders of all the ingredients had friability more than 3.0% where as those prepared with ethanol extract had very low friability. Ghanvatis, prepared as per the Ayurvedic formulary, did not show reserpine although other alkaloids were present. They showed less content uniformity and lower drug release. Ethanol extracted reserpine along with other alkaloids. Ghanvatis made with the alcoholic extracts exhibited better content uniformity and drug release than the traditional formulation. Tablets prepared with powders or extracts of the ingredients exhibited good content uniformity but the release of alkaloids from the tablets of powders was only 80%. Tablets of the extracts had good content uniformity with 90% release of the total alkaloids. Tablets prepared with alcoholic extracts using 1% polyvinylpyrrolidone as binder and 5% dried starch powder as disintegrating agent confirmed to all the requirements. Thus, the study shows tablets made with the extracts are superior to Ghanvatis and powder tablets.     

Toxicity evaluation of some traditional African spices on breast cancer cells and isolated rat hepatic mitochondria

Background

Fagara leprieuri (FL), Fagara xanthoxyloïdes (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) are used in many African countries as food spices or in traditional medicine to treat several maladies. In this work, we (a) investigate whether the crude spice extracts present selective cytotoxicity for breast cancer cell lines and (b) investigate whether the same extracts affect the bioenergetics and calcium susceptibility of isolated liver mitochondrial fractions.

Results

All extracts were cytotoxic to the cell lines studied, with the exception of MW, which was less toxic for a normal cell line. Interestingly, some of the extracts did not depolarize mitochondria in intact breast cancer MCF-7 cells, although this effect was observed in a normal breast cancer cell line (MCF-12A). All extracts increased hepatic mitochondrial state 2/4 respiration and decreased the respiratory control ratio and the transmembrane electric potential. Also, the extracts induced the mitochondrial permeability transition (MPT).

Conclusions

Mitochondrial toxicity may be part of the mechanism by which the spices tested cause inhibition of proliferation and death in the cell lines tested. This study also warrants caution in the excessive use of these spices for human consumption.     

不同产地黄连指纹图谱及质量评价(英文)

通过HPLC指纹图谱和主要生物碱成分的含量测定,对来自27个不同产地的88批黄连药材进行分析。色谱柱为Phenomenex Gemini-NX C₁₈（250 mm×4.6 mm,5μm）,流动相为乙腈-30 mmol/L碳酸氢铵溶液（含0.7%氨水,0.1%三乙胺）;流速1.0 mL/min;检测波长270 nm,柱温30℃。得到分离度、重现性均较好的黄连药材HPLC指纹图谱,标示了12个共有色谱峰,对27个来自不同产地黄连样品的色谱指纹图谱进行了相似度评价;并测定了不同产地黄连样品中6个主要生物碱成分的含量,建立了黄连药材的真伪鉴别方法和其质量优劣的评价方法。     

High performance liquid chromatographic fingerprint evaluation of the quinolizidine alkaloids from commercial Radix Sophorae Flavescentis

Fifteen commercial samples of Radix Sophorae Flavescentis were collected from different parts of China. HPLC analysis showed that the commercial samples all contained quinolizidine alkaloids and a total of nine chromatographic peaks were identified by referring to standard compounds, HPLC/MS analysis and comparison of the physicochemical data of the isolated peak with the literature. The contents of the major alkaloids were determined and the ratio of the major alkaloids contents was shown to be correlated with their source of origin. The commercial samples from China gave a distinct HPLC pattern showing the main alkaloids contents. The sample preparations were researched and the extraction conditions of the alkaloids were optimized. Reproducible HPLC fingerprints can be obtained for the quinolizidine alkaloids under the well-controlled extraction conditions. The HPLC fingerprint analysis method is suitable for the quality control of the Radix Sophorae Flavescentis and the standardization of phytomedicines.     

CYP3A-mediated apoptosis of dauricine in cultured human bronchial epithelial cells and in lungs of CD-1 mice

Dauricine is the major bioactive component isolated from the root of Menispermum dauricum DC and has shown promising pharmacologic activities with a great potential for clinical use. Recently, we found that intraperitoneal exposure of dauricine produced selective pulmonary injury in mice. A quinone methide metabolite of dauricine was identified and is suggested to be associated with the pulmonary toxicity of dauricine. The present study evaluated the apoptotic effect of dauricine in cultured cells and mice, determined the change in cellular glutathione (GSH) contents after exposure to dauricine, investigated the role of GSH depletion in dauricine-induced cytotoxicity and apoptosis, and examined the role of CYP3A in dauricine-induced GSH depletion and apoptosis. Dauricine was found to induce apoptosis in NL-20 cells. Additionally, intraperitoneal administration of dauricine caused GSH depletion and apoptosis in lungs of mice. Treatment with ketoconazole, an inhibitor of CYP3A, reversed cellular GSH depletion in lungs of mice given dauricine and showed protective effect on dauricine-induced apoptosis in lungs of mice. This indicates that metabolic activation is involved in dauricine-induced GSH-depletion, cytotoxicity and apoptosis. The glutathione depletor l-buthionine sulfoximine showed potentiating effect on cytotoxicity and apoptosis induced by dauricine. We propose that dauricine is metabolized to a quinone methide intermediate which depletes cellular GSH, and the depletion of GSH may trigger and/or intensify the cytotoxicity and apoptosis induced by dauricine.