Discrimination of Baphicacanthis Cusiae Rhizoma et radix and its adulterant species and establishment of an assay method for quality control.

BACKGROUND: Baphicacanthis Cusiae Rhizoma et Radix, commonly known as Nan-Ban-Lan-Gen (NBLG), is an essential traditional Chinese medicine that possesses diverse bioactivities, particularly noteworthy for its antiviral properties. Although NBLG has been listed in the Chinese Pharmacopoeia as an independent Chinese medicine, the establishment of a comprehensive quality standard for NBLG remains elusive. The absence of assay for marker compound in its quality standards has led to the lack of corresponding quality control measures for NBLG-containing preparations, and its discrimination from adulterant species in the market which thereby can significantly impact the efficacy and safety of NBLG-containing products. METHODS: Ultra-high performance liquid chromatography (UHPLC) coupled with quadrupole-time-of-flight mass spectrometry (Q-TOF-MS) was employed for comprehensive profiling of the chemical constituents of NBLG, the stem of Baphicacanthus cusia (Nees) Bremek (NBLJ), and the roots of Isatis indigotica Fort. (Bei-Ban-Lan-Gen, BBLG). Additionally, multivariate analysis was conducted to compare the chemical components of NBLG with those of NBLJ and BBLG. Furthermore, we established an optimized and validated HPLC method to obtain the fingerprint of NBLG and quantify the content of 2-benzoxazolinone and acteoside in the samples. RESULTS: A total of 73 compounds belonging to six classes were assigned in NBLG, with alkaloids being the most abundant and diverse species. High compositional similarities with significant differences in content were observed between NBLG and NBLJ. Moreover, the chemical profile of BBLG markedly differed from that of NBLG. An informative high performance liquid chromatography (HPLC) fingerprint of NBLG comprising seven characteristic peaks that can be used for quality assessment was established. Notably, we propose a quality control standard for NBLG, stipulating that the limit of content in dry weight for both 2-benzoxazolinone and acteoside should not be less than 0.010%. CONCLUSION: This study provides the most comprehensive chemical information to date on NBLG, offering valuable insights into its authentication and quality control. Our findings highlight the importance of comprehensive chemical profiling to differentiate potential substitutions and adulterations of herbal medicines, particularly when the original source is scarce or unavailable. These results can aid in the development of quality control measures for NBLG-containing preparations, ensuring their safety and efficacy.

Study on triterpenoic acids distribution in Ganoderma mushrooms by automatic multiple development high performance thin layer chromatographic fingerprint analysis.

Ganoderma--"Lingzhi" in Chinese--is one of the superior Chinese tonic materia medicas in China, Japan, and Korea. Two species, Ganoderma lucidum (Red Lingzhi) and G. sinense (Purple Lingzhi), have been included in the Chinese Pharmacopoeia since its 2000 Edition. However, some other species of Ganoderma are also available in the market. For example, there are five species divided by color called "Penta-colors Lingzhi" that have been advocated as being the most invigorating among the Lingzhi species; but there is no scientific evidence for such a claim. Morphological identification can serve as an effective practice for differentiating the various species, but the inherent quality has to be delineated by chemical analysis. Among the diverse constituents in Lingzhi, triterpenoids are commonly recognized as the major active ingredients. An automatic triple development HPTLC fingerprint analysis was carried out for detecting the distribution consistency of the triterpenoic acids in various Lingzhi samples. The chromatographic conditions were optimized as follows: stationary phase, precoated HPTLC silica gel 60 plate; mobile phase, toluene-ethyl acetate-methanol-formic acid (15 + 15 + 1 + 0.1); and triple-development using automatic multiple development equipment. The chromatograms showed good resolution, and the color images provided more specific HPTLC fingerprints than have been previously published. It was observed that the abundance of triterpenoic acids and consistent fingerprint pattern in Red Lingzhi (fruiting body of G. lucidum) outweighs the other species of Lingzhi.

Chemical pattern-aided classification to simplify the intricacy of morphological taxonomy of Epimedium species using chromatographic fingerprinting.

Epimedium herb (Yinyanghuo), one of the popular Chinese materia medica, is a multiple species colony of Epimedium genus belonging to Berberidaceae. There are five species of Epimedium that have been officially adopted in Chinese Pharmacopoeia under the same crude drug name 'Yinyanghuo' comprising Epimedium brevicornu, E. koreanum, E. sagittatum, E. pubescens, and E. wushanense. In addition, non-official species like E. acuminatum, E. miryanthum and E. leptorrhizum are also mix-used. Frequently, the morphological taxonomical identification is very difficult during on-site inspection for species authentication in the market. Researchers are often bewildered by the multiple species ambiguity when putting this crude drug in use. Referring to the bioactive constituents that are vital for therapeutic efficacy, the key to clarifying the multiple species confusion should rely on analysis of the bioactive composition. It is well known that medicinal Epimedium herbs contain special C-8 prenylated flavonol glycosides which contribute to various bioactivities and the major four, epimedin A (A), epimedin B (B), epimedin C (C) and icariin (I), are unanimously used as bioactive markers for quality control. In this study, HPLC-DAD fingerprinting was performed for investigating the molecular spectrum of various Epimedium species. It was found that the four major flavonoids constitute the middle part of the chromatographic profiles to form a specific region (named as 'ABCI fingerprint region') being dominant in the HPLC profiles of all medicinal Epimedium species, and the five official species express five different 'ABCI' patterns (different peak: peak ratios). Our study found that the convergent tendency of the 'ABCI region' among multiple species of Epimedium could facilitate differentiation of complex commercial samples based on similar bioactive composition should confer similar bioactivities. Merging the different species that possess the same 'ABCI region' pattern into the same group can create a simpler bioactive-fraction-aided classification array by clustering the commercial samples into three bioactive ingredients-based fingerprint patterns - 'E.b. pattern', 'E.k. pattern' and 'extensive E.w. pattern'. This approach offers the feasibility of characterizing and quality-controlling complex samples in the same genus designated under a single herbal drug entity on the premise of possessing the same bioactive ingredients pattern and supported by long-term traditional usage.

Study of the destructive effect to inherent quality of Angelicae dahuricae radix (Baizhi) by sulfur-fumigated process using chromatographic fingerprinting analysis.

The after-harvesting sun-dried process of Angelicae dahuricae radix (Chinese name: Baizhi) was previously the traditional treatment for commodity. Over recent decades the natural drying process for some fleshy roots or rhizomes of Chinese materia medica has been replaced by sulfur-fumigation for curtailing the drying duration and pest control. We used high performance liquid chromatography (HPLC) and high performance thin-layer chromatography (HPTLC) fingerprinting analysis to investigate the potential damaging effect of the sulfur-fumigating process. The experimental conditions were as follows. HPTLC analysis was carried out on pre-coated silica-gel 60 plate, twice development was performed with two solvent systems (mobile phase) A, chloroform-ethyl acetate (10:1) and B, hexane-chloroform-ether (4:1:2); the fluorescent images were observed under UV 365 nm. HPLC was preceeded on Zorbax SB-C(18) column; the linear gradient elution was conducted with mobile phase prepared from methanol-0.5% acetic acid; column temperature was at 25 degrees C; the detection wavelength was 250 nm. We found serious degradation of the majority of coumarins in sulfur-fumigated Baizhi. The destructive effect was manifested by the defaced chromatographic profile and verified by imitating the sulfur dioxide reaction with the constituents in Baizhi in the laboratory. It is suggested that sulfur-fumigation process is an unacceptable approach for processing herbal drugs.

[Study of HPLC-DAD fingerprint on complex traditional Chinese medicine proprietary preparation-Baoji pills].

OBJECTIVE: Based on 'Back-tracking' method, identification and quality evaluation of complex traditional Chinese medicine (TCM) preparation of Baoji pills (BJP) were carried out by HPLC fingerprint analysis. METHOD: HPLC-DAD fingerprint of BJP was conducted with Zorbax SB-C18 column and non-linear elution with the mobile phase consisted of acetonitrile-0.5% glacial acetic acid at column temperature 30 degrees C and detective wavelengths of 250 nm and 283 nm. From the established chromatographic pattern of BJP, track backward to the corresponding crude herbal drugs in the formula, attribution ofmost peaks in the BJP fingerprint can be disclosed. RESULT: The BJP HPLC fingerprint consisted of 44 peaks among which 35 peaks were assigned by parallel comparison with the fingerprint of the 10 corresponding crude drugs in the formula such as pueraria, pummelo peel, and magnolia bark, etc. and 22 peaks we reidentified by comparison with the chemical reference substances. CONCLUSION: The established HPLC fingerprint represents the whole character of BJP, which enhanced the specialty for control and assessment of the product quality. It exemplified much more effective for quality control than selecting any marker for qualitative or quantitative testing target. And the Back-tracking' experimental method extended the study mentality for complex formula TCM products chromatographic fingerprinting analysis.