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Consistency in quality of traditional Chinese medicine granules is an important factor to ensure reproducible clinical efficacy. In this study rhubarb dispensing granules were utilized to construct an efficacious near-infrared spectroscopy (eNIRS) assay by combining NIRS and biopotency. A NIR method for assaying rhubarb dispensing particles was established, and information on different batches was collected. The diarrhea-inducing biopotency of rhubarb dispensing granules was determined based on a constipation model induced by diphenoxylate in mice. The animal protocol was approved by the Animal Ethic Committee of 302 Hospital of Chinese PLA People's Liberation Army (ID: IACUC-2019-0010). Ten anthraquinones were determined in rhubarb dispensing granules by UPLC. The correlation between NIR and biopotency was analyzed and five characteristic bands that correlated highly with bioactivity were identified, including 4 011-4 390, 4 859-5 461, 7 012-7 493, 10 992-11 312 and 11 871-12 489 cm-1. There were some differences in the main bands of different chemical constituents. In summary, five active bands based on NIRS were identified and found to be able to achieve rapid on-line detection of rhubarb dispensing granule quality. This research model may also provide reference for quality control of other Chinese medicine dispensing granules.
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A chemical fingerprint is an important mean for quality control of traditional Chinese medicine (TCM); however, there is much redundant information in a conventional fingerprint that can affect its availability and accuracy. In this work, the antibacterial biopotency of Scutellariae Radix (Huangqin, HQ) was determined according to the parallel line method of quantitative response. HPLC was adopted to detect the chemical fingerprint of HQ; Grey relational analysis (GRA) was used to identify the primary effective components. The results showed that the antibacterial biopotency of 15 batches of HQ ranged from 0 to 1 000 U·g-1 and the average potency was 556.29 ± 258.57 U·g-1 (1 U is equivalent to the bacteriostatic activity of 2.25 μg gentamicin). There were 34 characteristic peaks in the fingerprints of the samples and their similarities were 0.255-0.991. Eight components (P33, P30/baicalein, P19/baicalin, P15, P29, P34, P31/wogonin and P28) are positively related to antibacterial biopotency and selected from the top ten components of the grey correlation sequence to define the antibacterially effective components fingerprint of HQ. This fingerprint can clearly distinguish the commodity specification and grade, and can also characterize the morphology, components and the bacteriostatic potency differences of HQ. In summary, we established an antibacterially effective components fingerprint which provides simplified information on the antibacterial activity of Scutellariae Radix and could significantly improve the efficacy, specificity, and discriminative ability of the fingerprint for HQ, and could be a useful reference for the comprehensive quality evaluation of other TCM.
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Objective To evaluate the ability of the antiplatelet aggregation of ten anthraquinone derivatives in Rhei Radix et Rhizoma by using bioassay method, and to screen for the indicators that can be used to control the quality of rhubarb wine processing product. Methods Platelet aggregation instrument was used to determine the platelet aggregation rate induced by ADP in vitro and calculate the antiplatelet aggregation rates of 10 anthraquinone derivatives (aloe-emodin, rhein, emodin, chrysophanol, physcion, aloe-emodin-8-O-β-D-glucoside, rhein-8-O-β-D-glucoside, emodin-8-O-β-D-glucoside, chrysophanol-8-O-β-D-glucoside, and physcion-8-O-β-D-glucoside) at different concentrations. The biopotency was calculated by bioavailability software. In order to verify the accuracy of the bioassay results, the estimated inhibition constant of rhein and chrysophanol-8-O-β-D-glucoside in P2Y12 protein receptor were determined by molecular docking software. Results The bioassay results showed that the antiplatelet potencies of rhein and emodin were significantly higher than those of aloe-emodin, chrysophanol and physcion. Compared with the antiplatelet biopotency of aspirin, the antiplatelet potencies of rhein and emodin were 5.02 and 5.15 times higher than that of aspirin, which indicated that rhein and emodin have strong inhibitory effect on ADP-induced platelet aggregation. However, the antiplatelet potencies of aloe-emodin, chrysophanol and physcion were equivalent of that of aspirin. The antiplatelet potencies of aloe-emodin-8-O-β-D-glucoside, rhein-8-O-β-D-glucoside, emodin-8-O-β-D-glucoside, chrysophanol-8-O-β-D-glucoside, and physcion-8-O-β-D-glucoside were higher 4.13, 4.46, 9.31, 5.46, and 7.80 times than that of aspirin, respectively, which indicated that the five anthraquinone glucosides had a strong ability to antagonize ADP-induced platelet aggregation. The results of molecular docking showed that P2Y12 protein had different selectivity to 10 anthraquinone derivatives, especially for rhein, chrysophanol-8-O-β-D-glucoside, and the estimated Ki value were 5.73 and 2.51 μmol/L, respectively, indicating that rhein, chrysophanol-8-O-β-D-glucoside produced a strong inhibitory effect on P2Y12 protein at a lower concentration level. Moreover, the activity of chrysophanol-8-O-β-D-glucoside was stronger than rhein, which was consistent with their measured intensity of antiplatelet aggregation. Conclusion All results showed that there were some differences among antiplatelet potencies of 10 anthraquinone derivatives, and finally the screening of rhein, emodin can be used as evaluation indicators to control the quality of rhubarb wine processing product.
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The quality control of Chinese materia medica (CMM) has been the focus of CMM modernization, and how to demonstrate the consistency of product quality and clinical efficacy has become an important research aspect of development and innovation of CMM. Due to the heterogeneity of CMM, it is necessary to combine biopotency with other detection methods to guarantee the quality of medicines. This article reviewed the research status of CMM biopotency, analyzed the global local government regulations about biopotency and the difficulties of development in CMM, and discussed development ideas of biopotency based on PK-PD for CMM. Its main aim is to provide reference for the construction of the follow-up quality evaluation system, and to promote recognition from the international drug administration management on CMM quality standard.
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This study attempts to evaluate the quality of Chinese formula granules by combined use of multi-component simultaneous quantitative analysis and bioassay. The rhubarb dispensing granules were used as the model drug for demonstrative study. The ultra-high performance liquid chromatography (UPLC) method was adopted for simultaneously quantitative determination of the 10 anthraquinone derivatives (such as aloe emodin-8-O-β-D-glucoside) in rhubarb dispensing granules; purgative biopotency of different batches of rhubarb dispensing granules was determined based on compound diphenoxylate tablets-induced mouse constipation model; blood activating biopotency of different batches of rhubarb dispensing granules was determined based on in vitro rat antiplatelet aggregation model; SPSS 22.0 statistical software was used for correlation analysis between 10 anthraquinone derivatives and purgative biopotency, blood activating biopotency. The results of multi-components simultaneous quantitative analysisshowed that there was a great difference in chemical characterizationand certain differences inpurgative biopotency and blood activating biopotency among 10 batches of rhubarb dispensing granules. The correlation analysis showed that the intensity of purgative biopotency was significantly correlated with the content of conjugated anthraquinone glycosides (P<0.01), and the intensity of blood activating biopotency was significantly correlated with the content of free anthraquinone (P<0.01). In summary, the combined use of multi-component simultaneous quantitative analysis and bioassay can achieve objective quantification and more comprehensive reflection on overall quality difference among different batches of rhubarb dispensing granules.
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The current quality control (QC) pattern for Chinese materia medica (CMM) lacks suitable methods and indicators to evaluate their safety and efficacy effectively, which impedes the smooth development of CMM. In this review, main problems of the current QC pattern for CMM, principally focused on the content determination of constituents, were summarized and the inspiration from the QC of biological products was introduced. With the aim at introducing a suitable tool to the QC of CMM, biopotency assay and its feasibility in the QC pattern for CMM were analyzed and confirmed by relevant researches with years of practice. From the applications of biopotency assays in the QC of CMM in the last 10 years, we propose that biopotency assays should be an integral part of the QC pattern for CMM, for these assays can make the QC indicators related to the clinical safety and efficacy, supplementing the existed QC system of CMM.
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Ovulation induction in hypothalamic amenorrhea using gonadotropin- releasing hormone(GnRH) pulse therapy is complicated by widely variant patient responses ranging from anovulation to multiple pregnancy. Route of administration(intravenous vs subcutaneous), pulse therapy, GnRH dose, infusion interval, or hormone preparation may contribute. We evaluated the bioactivity of 4 GnRH preparations(Relisorm,Serono; Lutrelef,Ferring; Factrel,Ayerst; GnRH,Sigma) in a rat anterior cell bioassay. Dispersed rat anterior pituitary cells were placed for 48 hrs at 5x105 cells/well, washed and incubated with GnRH. The GnRH was diluted according to the manufacturer's culture medium(10(-12) to 10(-5)M). GnRH stimulated immunoreactive luteinizing hormone(LH) production was assested in culture medium after 4 hrs by radioimmunoassay(RIA). A linear dose-response relationship was exhibited by all preparations from 10(-10) to 10(-7)M. Msximal LH production was 249+/-24 ng/ml/4hrs(mean+/-SEM) and was not different among the preparations tested(ANOVA, p>0.05). The minimal effective dose of GnRH was 10-10M for all preparations(basa1=27+/-4ng/ml/4hrs:mean+/-SEM). No significant differences were noted for MED, or dose-response slope(p<0.05, ANOVA and slope test for parallelism, respectively). In addition, bioactive LH and immuno and bioactive follicular stimulating hormone(FSH) dose responses were confirmed. We concluded that the principal variability of patient response seen with GnRH pulse therapy cannot be attributed to the bioactivity of these commercial GnRH preparations. But rather, most of the variability is due to the inherent individualism in patient response or other factors of the treatment protocol.