Improvement of Quality Standard for Mongolian Medicine Juniperus rigida / 中国药房

OBJECTIVE：To provide scientifi c evidence for improving the quality standard of Mongolian medicine Juniperus rigida. METHODS ：Totally 10 batches of J. rigida from different places were taken as samples to observe their characters and identify them by microscope ；TLC method was adopted to qualitatively identify isoquercitrin ，quercitrin，amentoflavone， podocarpusflavone A and hinokiflavone ；the contents of total ash ，acid-insoluble ash ，ethanol-soluble extract and heavy metals were determined by related method stated in 2020 edition of Chinese Pharmacopeia （part Ⅳ）. The contents of above 5 components in samples were determined by HPLC. RESULTS ：The powder of J. rigida was green or yellowish green ，polygonal tracheids ， closely arranged in longitudinal with unequal stomatal ；epidermal cells were nearly rectangular ；sclerenchyma cells were quasi rectangular and the wall beadedly thickening. Results of TLC showed that the spots of the same color were found in the corresponding positions of chromatogram for test sample and substance control. The contents of total ash ，acid-insoluble ash and ethanol-soluble extract in 10 batches of samples were 7.37％-11.18％，0.75％-2.98％，16.55％-26.42％，respectively；average contents were 8.51％，1.27％，22.35％. The contents of lead ，arsenic，cadmium，mercury and copper were 2.00-5.44，0.65-1.65， 0.044-0.100，0.034-0.160，4.59-6.79 mg/kg，respectively；average conte nts were 3.73，0.97，0.078，0.061，5.23 mg/kg. The linear ranges of isoquercitrin ，quercitrin，amentoflavone，podocarpus- flavone A and hinokiflavone were 4.98-20.02，49.99-199.96， 19.94-99.96，9.99-40.00，20.20-159.98 μg/mL（all r＞0.999 7）； com RSDs of precision ，repeatability and stability （24 h） tests were all less than 3.00％（n＝6）；the average recoveries were 话：0993-2057878。E-mail：Tanghuishz@qq.com 100.62％-102.96％，RSDs were 1.21％-1.88％（n＝6）. Average contents of the above-mentioned 5 compounds in 10 batches of samples were 0.089-0.379，1.379-4.250，1.077-2.026，0.162-0.423， 0.016 9-0.117 0 mg/g，respectively. CONCLUSIONS ：The qualitative and quantitative analysis methods of Mongolian medicine J. rigida are established. It is preliminarily proposed that the total ash content shall not exceed 10.22％，the acid-insoluble ash content shall not exceed 1.53％，ethanol-soluble extract content shall not be less than 17.88％，heavy metal lead should not exceed 5 mg/kg，arsenic should not exceed 2 mg/kg，cadmium should not exceed 0.3 mg/kg，mercury should not exceed 0.2 mg/kg，copper should not exceed 20 mg/kg.

Natural compounds as potential inhibitors of SARS-CoV-2 main protease: An in-silico study

Objective:To explore natural compounds as potential inhibitors against main protease (Mpro) of SARS-CoV-2. Methods:In the current study, systematic molecular docking analysis was conducted using AutoDock 4.2 to determine the binding affinities and interactions between natural compounds and Mpro. Selected natural compounds were further validated using a combination of molecular dynamic (MD) simulations and molecular mechanic Poisson-Boltzmann surface area (MM/PBSA) free energy calculations. Results:Out of twenty natural compounds, four natural metabolites namely, amentoflavone, guggulsterone, puerarin, and piperine were found to have strong interaction with Mpro of SARS-CoV-2 based on docking analysis. During MD simulations, all four natural compounds bound to Mpro at 50 ns and MM/G/P/BSA free energy calculations showed that all four shortlisted ligands had stable and favorable energies with strong binding to Mpro protein. Conclusions:Guggulsterone is a potential inhibitor of COVID-19 main protease Mpro. Further in vitro and pre-clinical studies are needed.

Primary study on scientific connotation "carbonizing retains characteristics" of Platycladi Cacumen / 中草药

Objective: To study the scientific connotation "carbonizing retains characteristics" of Platycladi Cacumen (PC). Methods: Chemical constituents of PC and Platycladi Cacumen Carbonisata (PCC) of different carbonizing degree were compared by HPLC characteristic chromatogram, and the chromatographic peaks were assigned; PC and PCC of different carbonizing degree and the characteristic components changed before and after being carbonized were used to compare the impact to hemostasis in vitro and inhibition of zebrafish cerebral hemorrhage. Results: Chemical compositions of PC were affected by different carbonizing degree, when carbonizing degree is moderate, the content of myricitrin, quercitroside, isoquercitroside, amentoflavone and hinokiflavone original in PC were significantly reduced and the amount of quercetin and kaempferol which were newly produced were higher. In vitro hemostatic experiments showed that compared with the blank group, the APTT and FIB of PC which was carbonized moderately were significantly different (P < 0.01) and TT was significantly different (P < 0.05). It also had obvious inhibition effect on zebrafish cerebral hemorrhage when the concentration of PC carbonized moderately was 50 μg/mL (P < 0.01). The enhanced hemostasis was significantly related to newly produced compositions quercetin and kaempferol. Conclusion: Chemical compositions of PC changed significantly after being carbonized and the hemostatic effect was enhanced,which were related to the processing degree. The scientific connotation and traditional processing requirements of "carbonizing retains characteristics" was preliminarily analyzed when the carbon medicine was processed by this study, which provides a certain idea for the research on traditional processing theory of carbon medicine.

Anticomplementary and anti-oxidant activities of constituents from Juniperus convallium / 中草药

Objective: To investigate the chemical constituents from Juniperus convallium, as well as their anticomplementary and antioxidant activities. Methods: The constituents were isolated and purified by column chromatography over silica gel, Sephadex LH-20, ODS-C18, and preparative HPLC. Their structures were identified by spectra analysis. The cell hemolysis assay was used to evaluate the anticomplementary activities and the targets through classical and alternative pathways. Also the anti-oxidant activities were tested by DPPH, ABTS and FRAP methods. Results: A total of 17 compounds were obtained from the ethyl acetate extract of J. convallium and identified as amentoflavone (1), cupressuflavone (2), cupressuflavone-4″’-O-β-D-glucosides (3), naringenin-7-O- glycoside (4), apigenin (5), tiliroside (6), quercetin 3-O-β-D-glucoside (7), quercetin-3-O-rhamnoside (8), hypolaetin-7-O-β-D- glucopyranoside (9), isomassonianoside B (10), (+)-isolariciresinol 2a-O-β-D-glucoside (11), (+)-isolarisiresinol 3a-O-β-D-glucopyranoside(12), cryptomeridiol (13), 3β-hydroxysandaracopimeric acid (14), (1R,3R,4aR,4bS,7R,10aR)-7-ethenyl-1,2,3,4,4a,4b,5,6,7,9,10,10a- dodecahydro-3-hydroxy-1,4a,7-trimethyl-1-phenanthrene methanol (15), 4-hydroxy-5-methyl-coumarin (16) and β-sitosterol (17). Compounds 1-15 and 17 showed anticomplementary activities in different degrees (CH50: 0.05-3.99 mmol/L, AP50: 0.58 -19.13 mmol/L). The flavonoids, especially the biflavonoids, are the important anticomplementary constituents in J. convallium. Further analysis of structure-activity relationship showed that phenolic hydroxyl and glycosidic groups influenced their anticomplementary activity. Only the flavonoids (1-3, 5-9) and lignans (10-12) showed different degrees of antioxidant activities due to their hydroxyl groups. Conclusion: All the 17 compounds are isolated from J. convallium for the first time. The flavonoids and lignans are the important anticomplementary and antioxidant constituents in J. convallium with a certain structure-acticity relationship. This study provides a good reference for further research on the pharmacological substance and quality control of J. convallium.

Protective effect of amentoflavone on radiation-reduced damage in mice bone marrow / 国际药学研究杂志

Objective To investigate the protective effect and possible mechanism of amentoflavone (AF) on bone marrow cells of mice injured by irradiation. Methods Primary bone marrow cells of male C57BL/6 mice were cultured and randomly divided into 4 groups (normal control, radiation control,AF 2.5μmol/L and 5μmol/L), with 3 samples in each group. After treated with AF for 12 h, the cells were injured by 12 Gy 60Coγirradiation. 6 h and 12 h post-irradiation, apoptosis was evaluated by using Hoechst 33258 stain, cell cycle was determined by flow cytometry while the level of TNF-α was tested by ELISA. Results The cell cycle arrest and apoptosis were not significantly affected by Amentoflavone. Amentoflavone (5 μmol/L)could significantly inhibit the production of TNF-α on cell supernatant of mouse bone marrow cells at 6 h or 12 h after radiation and 2.5 μmol/L Amentoflavone could significantly inhibit the production of TNF-α at 6 h after radiation. Conclusion Taken together, the data suggest that AF may have radioprotection against damage in mice bone marrow by inhibiting the production of TNF-α.

Preliminary Study on the Processing Mechanism of Cacumen Platycladi / 中国药师

To establish an HPLC method for the determination of myricetrin, quercitrin, myricetin, quercetin, kaempferol, amentoflavone and hinokiflavone in Cacumen Platycladi and processed Cacumen Platycladi, and compare the difference before and after the processing. Methods:The method described in Chinese pharmacopoeia was used in the processing. The chromato-graphic column was Welch Ultimate LP-C18 (250 mm × 4. 6 mm,5 μm),the mobile phase was methanol and 0. 2% phosphoric acid with gradient elution, the detection wavelength was 330 nm, and the flow rate was 1 ml·min-1 . The column temperatuhe was 35℃, the injection volume was 20μl. Results:The linear relationship respectively of myricetrin, quercitrin, myricetin, quercetin, kaempfer-ol, amentoflavone and hinokiflavone was good(r≥0. 999 0). The average recovery was within the range of 97. 6%-101. 1% (RSD≤1. 14%, n=6). The content of myricetrin, quercitrin, amentoflavone and hinokiflavone in Cacumen Platycladi was reduced after the processing, and the content of quercetin and kaempferol was increased after the processing. Conclusion: The method is simple,and can be used in the determination of Cacumen Platycladi and its processing product. The results of the content show that the flavones in Cacumen Platycladi can be changed by the processing.

Protective effect of amentoflavone on radiation-reduced damage in mice bone marrow / 国际药学研究杂志

Objective To investigate the protective effect and possible mechanism of amentoflavone (AF) on bone marrow cells of mice injured by irradiation. Methods Primary bone marrow cells of male C57BL/6 mice were cultured and randomly divided into 4 groups (normal control, radiation control,AF 2.5 μmol/L and 5 μmol/L), with 3 samples in each group. After treated with AF for 12 h, the cells were injured by 12 Gy60Co γ irradiation. 6 h and 12 h post-irradiation, apoptosis was evaluated by using Hoechst 33258 stain, cell cycle was determined by flow cytometry while the level of TNF-α was tested by ELISA. Results The cell cycle arrest and apoptosis were not significantly affected by Amentoflavone. Amentoflavone 5 μmol L) could significantly inhibit the production of TNF- α on cell supernatant of mouse bone marrow cells at 6 h or 12 h after radiation and 2.5 μmol/L Amentoflavone could significantly inhibit the production of TNF- α at 6 h after radiation. Conclusion Taken together, the data suggest that AF may have radioprotection against damage in mice bone marrow by inhibiting the production of TNF-α.

In vitro activity of the hydroethanolic extract and biflavonoids isolated from Selaginella sellowii on Leishmania (Leishmania) amazonensis

This study is the first phytochemical investigation of Selaginella sellowii and demonstrates the antileishmanial activity of the hydroethanolic extract from this plant (SSHE), as well as of the biflavonoids amentoflavone and robustaflavone, isolated from this species. The effects of these substances were evaluated on intracellular amastigotes of Leishmania (Leishmania) amazonensis, an aetiological agent of American cutaneous leishmaniasis. SSHE was highly active against intracellular amastigotes [the half maximum inhibitory concentration (IC50) = 20.2 µg/mL]. Fractionation of the extract led to the isolation of the two bioflavonoids with the highest activity: amentoflavone, which was about 200 times more active (IC50 = 0.1 μg/mL) and less cytotoxic than SSHE (IC50 = 2.2 and 3 μg/mL, respectively on NIH/3T3 and J774.A1 cells), with a high selectivity index (SI) (22 and 30), robustaflavone, which was also active against L. amazonensis (IC50 = 2.8 µg/mL), but more cytotoxic, with IC50 = 25.5 µg/mL (SI = 9.1) on NIH/3T3 cells and IC50 = 3.1 µg/mL (SI = 1.1) on J774.A1 cells. The production of nitric oxide (NO) was lower in cells treated with amentoflavone (suggesting that NO does not contribute to the leishmanicidal mechanism in this case), while NO release was higher after treatment with robustaflavone. S. sellowii may be a potential source of biflavonoids that could provide promising compounds for the treatment of cutaneous leishmaniasis.

Chemical constituents of Lobelia chinensis / 中草药

Objective: To investigate the chemical constituents in the dried whole plant of Lobelia chinensis. Methods: Column chromatography, such as silica gel, MCI, ODS, Sephadex LH-20, C18 reverse-phased silica gel columns, and preparative HPLC were used to isolate the compounds. Spectroscopic methods like MS, 1H-NMR, and 13C-NMR were used to elucidate their structures. Results: Fifteen compounds were isolated from 95% ethanol extract of L. chinensis, including twelve flavonoids: quercetin (1), rutin (2), luteolin (3), apigenin (4), hesperidin (5), quercetin-3-O-α-L-rhamnoside (6), quercetin-7-O-α-L-rhamnoside (7), quercetin-3- O-β-D-glucoside (8), amentoflavone (9), naringenin (10), hesperetin (11), and eupafolin (12), and three coumarins: 5, 7- dimethoxy-coumarin (13), isoscopoletin (14), and scoparone (15). Conclusion: Compounds 6-12 are isolated from the plants in genus Lobelia L. for the first time. Compounds 5, 13, and 14 are isolated from L. chinensis for the first time.

Determination of five flavonoids in Cacumen Platycladi Carbonisatum by UPLC / 中草药

Objective: To establish a UPLC method for simultaneous determination of five flavonoids (myricetin, quercitrin, quercetin, kaempferol, and amentoflavone) in Cacumen Platycladi Carbonisatum. Methods: The analysis was performed on a Waters Acquity UPLC system with a Waters BEH C18 column (50 mmx2.1 mm, 1.7 mm). The five flavonoids were separated with gradient mobile phase consisting of 0.05% aqueous formic acid and methanol. The temperature of column was 30°C, and the injection volume was 2 μl/min, detection wavelength was 360 nm. Results: The five flavonoids including myricetin, quercitrin, quercetin, kaempferol, and amentoflavone had good linearity (r≥0.999 5) within the linear ranges. The average recovery rate was 96.85% - 99.01% with RSD ≤4.00%. Conclusion: The developed UPLC method is simple, sensitive and accurate and has the good repeatability in separation, which is available for the quality control of Cacumen Platycladi Carbonisatum.

TLC Qualitative Studies of 11 Species of Selaginella Medicinal Material / 中药新药与临床药理

Objective To establish an effective qualitative discrimination method for Selaginella medicinal materials.Meth- ods Thin layer chromatography(TLC)method was used.Results The TLC method has a good specialization for identifying Selaginella medicinal materials and can distinguish Selaginella rnoellendorfii from other 10 familiar species in northern areas of Guangdong province.Conclusion The method can help to control the quality of Selaginella moellendorfii Tablet.

HPLC Fingerprint Differentiation of Herba Selaginellae moellendorfii and Amentoflavone Determination / 中药新药与临床药理

Objective To establish the HPLC fingerprint of Herba Selaginellae moellendorfii for identification and comparison with other species of the same genus and to determine the content of amentoflavone.Method Separation was performed on ZORBAX SB-C18 chromatographic column,acetonitrile(A)-0.5 %solution of acetic acid in water(B)as mobile phase with gradient elution and the flow rate was 1.0 mL?min-1.The detection wavelength was at 270 nm.The content determination of amentoflavone carried out synchronously.Results The characteristic eighteen peaks in the chromatogram consisted of the common pattern of Herba Selaginellae moellendorfii.The fingerprint coupling with similarity and Principal Component Analysis differentiated and classified the various species of selaginella.Nine species could be divided into 3 classes.The content of amentoflavone in Herba Selaginellae moellendorfii was 0.8～1.0 %.Conclusions The weighted similarity coefficient was calculated from 13 batches of Herba Selaginellae moellendorfii samples as high as more than 0.98.Among 9 species of Selaginella,5 species(S.biformis,S.involvens,S.doederileinii,S.trachyphylla,S.tamariscina)were sorted out by means of Principal Component Analysis in the same class with S.moellendorfii.Which indicated the possibility of bio-equivalence among the herbs of these six species,while S.picta,S.uncinata and S.delicatula are considered as adulterants of S.moellendorfii as their dissimilar fingerprints.