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.

Characterization of deglycosylated metabolites of platycosides reveals their biotransformation after oral administration.

Platycodon grandiflorus is a well-known edible and medicinal plant that has been developed for dietary supplements or functional foods to relieve pulmonary disorders. Platycosides are the main active constituents of P. grandiflorus with multiple pharmacological activities. However, their metabolic fates after dietary consumption are still unclear. Herein, 25 deglycosylated metabolites of platycosides were identified, most of which were identified in vivo for the first time. Notably, 3-O-ß-d-glucopyranosyl platycosides could be absorbed into the bloodstream, and their structures were unambiguously characterized with the aid of chemically prepared standards, including two new compounds (M3 and M11). These findings reveal that both intestinal bacterial metabolism and hydrolysis of ester linkage at C-28 by carboxylesterases in liver are the possible in vivo deglycosylation metabolism pathway of platycosides. This study greatly facilitated our understanding of the fate of the platycosides after dietary consumption of P. grandiflorus products.

Cough Inhibition Activity of Schisandra chinensis in Guinea Pigs.

Chronic cough is very common in respiratory clinics, and no effective drugs are available. Schisandra chinensis (Turcz.) Baill. (S. chinensis), an important traditional Chinese medicine, has been extensively prescribed for patients with a persistent cough. Preliminary research indicated that 95% ethanol extracts (EE) of S. chinensis showed remarkable antitussive activity in guinea pigs exposed to cigarette smoke (CS). To find out the antitussive ingredients of S. chinensis, EE was divided into four fractions according to the polarity: petroleum ether extract (PEE), ethyl acetate extract (ECE), n-butyl alcohol extract, and residue extract. The antitussive, antioxidant, and anti-inflammatory effects of the four fractions were evaluated in a guinea pig model of cough hypersensitivity induced by CS exposure. Eighteen main constituents of the two effective fractions, PEE and ECE, were identified using ultra-high-pressure liquid chromatography electronic spray ion time-of-flight mass spectrometry. The cough inhibition activities of compound 1, 3, 9, 10, 17 were evaluated on citric acid induced acute cough guinea pigs. The results showed that the antitussive activity of EE was almost all contained in PEE and ECE. The 16 major peaks in PEE were identified as 15 lignans (1-12 and 14-16) and 1 triterpene (compound 13), and 3 major peaks (1, 17, and 18) in ECE were also identified as lignans. Three doses of five compounds brought about a significant decrease in number of cough efforts (P < .01), and the cough inhibition rates were between 40.9% and 85.1%. Therefore, lignans are the antitussive ingredients of S. chinensis.

Chemical profiling and cytotoxicity assay of bufadienolides in toad venom and toad skin.

ETHNOPHARMACOLOGICAL RELEVANCE: Toad venom and toad skin have been widely used for treating various cancers in China. Bufadienolides are regarded as the main anticancer components of toad venom, but the difference on composition and anticancer activities of bufadienolides between toad venom and toad skin remains unclear. METHODS: Fractions enriched with free and conjugated bufadienolides were prepared from toad venom and toad skin. Bufadienolides in each fraction were comprehensively profiled by using a versatile UHPLC-TOF-MS method. Relative contents of major bufadienolides were determined by using three bufogenins and one bufotoxin as marker compounds with validated UHPLC-TOF-MS method. Furthermore, cytotoxicity of the fractions was examined by MTT assay. RESULTS: Two fractions, i.e., bufogenin and bufotoxin fractions (TV-F and TV-C) were isolated from toad venom, and one bufotoxin fraction (TS-C) was isolated from toad skin. Totally 56 bufadienolides in these three fractions were identified, and 29 were quantified or semi-quantified. Bufotoxins were identified in both toad venom and toad skin, whereas bufogenins exist only in toad venom. Bufalin-3-conjugated bufotoxins are major components in toad venom, whereas cinobufotalin and cinobufagin-3-conjugated bufotoxins are main bufotoxins in toad skin. MTT assay revealed potent cytotoxicity of all the fractions in an order of TV-F>TV-C>TS-C. CONCLUSIONS: Our study represents the most comprehensive investigation on the chemical profiles of toad venom and toad skin from both qualitative and quantitative aspects. Eight bufotoxins were identified in toad skin responsible for the cytotoxicity for the first time. Our research provides valuable chemical evidence for the appropriate processing method, quality control and rational exploration of toad skin and toad venom for the development of anticancer medicines.

Characterization of oxygenated metabolites of ginsenoside Rb1 in plasma and urine of rat.

Oxygenated metabolites have been suggested as the major circulating metabolites of ginsenosides. In the current study, 10 oxygenated metabolites of ginsenoside Rb1 in plasma and urine of rat following iv dose were characterized by comparison with chemically synthesized authentic compounds as quinquenoside L16 (M1 and M2), notoginsenoside A (M3), ginsenoside V (M4 and M7), epoxyginsenoside Rb1 (M5 and M9), notoginsenoside K (M6), and notoginsenoside C (M8 and M10), 9 of which were detected as in vivo metabolites for the first time. After oral administration of ginsenoside Rb1, M3, M4, and M7 were observed as major circulating metabolites and presented in the bloodstream of rat for 24 h. Characterization of the exact chemical structures of these circulating metabolites could contribute greatly to our understanding of chemical exposure of ginsenosides after consumption of ginseng products and provide valuable information for explaining multiple bioactivities of ginseng products.

Catechins and procyanidins of Ginkgo biloba show potent activities towards the inhibition of ß-amyloid peptide aggregation and destabilization of preformed fibrils.

Catechins and procyanidins, together with flavonoid glycosides and terpene trilactones, are three important categories of components in the standard extract of Ginkgo biloba leaves (EGb761). In this research, catechins and proanthocyanidins were found to exist in both the extract of Ginkgo leaves and Ginkgo products. By comparing with reference compounds, six of them were identified as (+)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin and procyanidins B1 and B3. The activities of these polyphenols in the inhibition of Aß42 aggregation and the destabilization of preformed fibrils were evaluated using biochemical assays, which showed that all six of the polyphenols, as well as a fraction of the extract of Ginkgo biloba leaves (EGb) containing catechins and procyanidins, exerted potent inhibitory activities towards Aß42 aggregation and could also destabilize the performed fibrils. Catechins and procyanidins can therefore be regarded as the potent active constituents of EGb761 in terms of their inhibition of Aß42 aggregation and destabilization of the fibrils. Although quantitative mass spectroscopic analysis revealed that the catechins and procyanidins are only present in low concentrations in EGb761, these components should be studied in greater detail because of their potent inhibitory effects towards Aß42 aggregation and their ability to destabilize preformed fibrils, especially during the quality control of Ginkgo leaves and the manufacture of Ginkgo products.

Quantitative analysis of the flavonoid glycosides and terpene trilactones in the extract of Ginkgo biloba and evaluation of their inhibitory activity towards fibril formation of ß-amyloid peptide.

The standard extract of Ginkgo biloba leaves (EGb761) is used clinically in Europe for the symptomatic treatment of impaired cerebral function in primary degenerative dementia syndromes, and the results of numerous in vivo and in vitro studies have supported such clinical use. The abnormal production and aggregation of amyloid ß peptide (Aß) and the deposition of fibrils in the brain are regarded as key steps in the onset of Alzheimer's Disease (AD), and the inhibition of Aß aggregation and destabilization of the preformed fibrils represent viable approaches for the prevention and treatment of AD. Flavonoid glycosides and terpene trilactones (TTLs) are the two main components of EGb761 which represent 24 and 6% of the overall content, respectively. In our research, seven abundant flavonoid glycosides 1-7 were isolated from the extract of Ginkgo biloba leaves and characterized by spectroscopic analysis. Furthermore, an ultra-high performance liquid chromatography method was established for the simultaneous quantification of these seven flavonoids. The inhibitory activities of these flavonoids, as well as four TTLs, i.e., ginkgolides A, B, and C and bilobalide (compounds 8-11), were evaluated towards Aß42 fibril formation using a thioflavin T fluorescence assay. It was found that three flavonoids 1, 3 and 4 exhibited moderate inhibitory activities, whereas the other four flavonoids 2, 5, 6 and 7, as well as the four terpene trilactones, showed poor activity. This is the first report of the inhibition of Aß fibril formation of two characteristic acylated flavonoid glycosides 6, 7 in Ginkgo leaves, on the basis of which the structure-activity relationship of these flavonoids 1-7 was discussed.

Transformation of ginsenosides from notoginseng by artificial gastric juice can increase cytotoxicity toward cancer cells.

Multicomponent metabolic profile of notoginseng saponins in artificial gastric juice was qualitatively and quantitatively investigated, showing that ginsenosides were transformed via multiple pathways including deglycosylation, dehydration, hydration, and oxygenation. A total of 83 metabolites was identified by using UPLC-Q-TOF-MS, among which 16 new dammarane glycosides were further characterized by comparing with synthesized authentic compounds. Transformation time-course of notoginseng saponins in artificial gastric juice was quantitatively measured for the first time, showing rapid degradation of primary ginsenosides and concomitant formation of deglycosylation, hydration, and dehydration products. It was further demonstrated that the resultant metabolites exhibited enhanced cytotoxicity toward cancer cells. The extensive metabolism of ginsenosides within a transit time span in stomach, together with the formation of metabolites with diversified chemical structures possessing enhanced biological activities, indicated an important role of transformation in gastric juice in the systematic effects of ginsenosides.

Quantitative comparison of ginsenosides and polyacetylenes in wild and cultivated American ginseng.

Quantitative comparison of seven ginsenosides in wild and cultivated American ginseng revealed that the Rg(1)/Rd ratio presented a significantly large difference between cultivated and type-I (one of the defined chemotypes) wild American ginseng, facilitating this ratio as a characteristic marker for differentiating these two groups. Similarly, the ratio (Rg(1)+Re)/Rd, and the ratio of protopanaxatriol (PPT)-type ginsenosides to protopanaxadiol (PPD)-type ginsenosides showed a large difference between these two groups. On the other hand, type-II wild samples were found to have high Rg(1)/Rb(1) and Rg(1)/Re ratios and low panaxydol/panaxynol ratio, which is entirely different from Type-I American ginseng, but is very similar to that of Asian ginseng. This not only suggests that the chemotype should be taken into consideration properly when using these parameters for differentiating American and Asian ginseng, but also indicates that type-II wild American ginseng may have distinct pharmacological activities and therapeutic effects.