Antioxidative and Cytoprotective Effects of Ganoderma applanatum and Fomitopsis pinicola in PC12 Adrenal Phaeochromocytoma Cells.

Considering the impact of oxidative stress on the development of many diseases, together with the role of natural antioxidants in maintaining physiological balance in humans, medicinal mushrooms are potential sources of bioactive compounds against many diseases. In the present work, in vitro evaluation of the biological activities of the alcoholic extracts of two wild tree mushrooms, namely, Ganoderma applanatum and Fomitopsis pinicola, has been performed. Extraction of G. applanatum (GAE) and F. pinicola (FPE) was conducted with 60% ethanol and 100% ethanol sequentially. UPLC-MS/MS identification was conducted on the two mushrooms extracts. A total of 15 substances were identified in GAE, including 3 spiro meroterpenoids and 12 triterpenoids; a total of 14 chemical constituents were iden¬tified in FPE, including 8 triterpenoids, 4 triterpene glycosides, 1 lanosterol, and 1 lanostanoid. The resulting extracts were examined for their in vitro antioxidative and cytoprotective effects against AAPH-induced oxidative damage. Our results demonstrated that both extracts have potent antioxidative activities, when GAE was 0.2 mg/mL, the clearance rates of DPPH and ABTS have reached 93.34% and 99.93%, respectively. When FPE was 1.4 mg/mL and 0.6 mg/mL, the scavenging rates of DPPH and ABTS have reached 91.76% and 100%, respectively. Both the alcoholic extracts of G. applanatum and F. pinicola were able to protect the AAPH-induced damage and could effectively inhibit cell aging via ß-galactosidase (SA ß-gal) staining activity test and scanning electron microscopy analysis.

Characterization and Comparative Analysis of Chloroplast Genomes in Five Uncaria Species Endemic to China.

Uncaria, a perennial vine from the Rubiaceae family, is a typical Chinese traditional medicine. Currently, uncertainty exists over the Uncaria genus' evolutionary relationships and germplasm identification. The complete chloroplast genomes of four Uncaria species mentioned in the Chinese Pharmacopoeia and Uncaria scandens (an easily confused counterfeit) were sequenced and annotated. The findings demonstrated that the whole chloroplast genome of Uncaria genus is 153,780-155,138 bp in full length, encoding a total of 128-131 genes, containing 83-86 protein-coding genes, eight rRNAs and 37 tRNAs. These regions, which include eleven highly variable loci and 31-49 SSRs, can be used to create significant molecular markers for the Uncaria genus. The phylogenetic tree was constructed according to protein-coding genes and the whole chloroplast genome sequences of five Uncaria species using four methods. The topology of the two phylogenetic trees showed no difference. The sequences of U. rhynchophylla and U. scandens are clustered in one group, while the U. hirsuta and U. macrophylla are clustered in another group. U. sessilifructus is clustered together with the above two small clades. New insights on the relationship were revealed via phylogenetic research in five Uncaria species. This study will provide a theoretical basis for identifying U. rhynchophylla and its counterfeits, as well as the species of the Uncaria genus. This research provides the initial chloroplast genome report of Uncaria, contributes to elucidating the chloroplast genome evolution of Uncaria in China.

[Three-dimensional multi-component quality evaluation of Chinese medicine based on proportion consistency of active components: a study of Salvia miltiorrhiza].

To reveal the law of the proportion consistency of the active components in Chinese medicine and explore a new way to evaluate the quality of Chinese medicine, the present study investigated the content of tanshinone Ⅱ_A, cryptotanshinone, tanshinone Ⅰ, salvianolic acid B, rosmarinic acid, caffeic acid, and lithospermic acid in 895 samples of Salvia miltiorrhiza from 87 literature articles. The samples with salvianolic acid B and tanshinones(total content of tanshinone Ⅱ_A, tanshinone Ⅰ, and cryptotanshinone) meeting the requirements of Chinese Pharmacopoeia were analyzed for proportion consistency of components, and the proportion distribution of components was compared. The results showed that the proportions in and between tanshinones and salvianolic acids were stable. The content ratios of cryptotanshinone to tanshinone Ⅱ_A, rosmarinic acid to salvianolic acid B, and salvianolic acid B to tanshinone Ⅱ_A fluctuated from 0.1 to 1.7, from 0.02 to 0.14, and from 10 to 60, respectively. It indicated that the content proportions of different components were not fixed, but fluctuated in a certain range. The fluctuation range of components in the same group was small, but that in different groups was large. The content proportions of active components were stable for Chinese medicinal materials with fixed varieties or origins. The samples whose content proportions were out of the fluctuation ranges were generally unqualified medicinal materials. We proposed a "three-dimensional multi-component" quality evaluation model of Chinese medicine based on the above findings. Specifically, "three-dimensional" means to accurately evaluate the quality of Chinese medicine from varieties, proportions, and content of active components, and "multi-component" means to highlight the information of multiple components in Chinese medicine, especially the quality markers and equivalent component groups. The proportion of components in Chinese medicine can be used as an important index for the quality evaluation of Chinese medicine. The proportions of active components are stable in Chinese medicinal materials with fixed varieties or places of origin.

The complete chloroplast genome sequence of traditional Chinese medicine Uncaria macrophylla (Rubiaceae).

Uncaria macrophylla (Rubiaceae) is a medicinal vine plant of the Rubiaceae family that was distributed in East Asia and Southeast Asia. The first complete chloroplast genome of Uncaria macrophylla was sequenced and assembled in this study. The genome is 155,138 bp in length and contained 129 encoded genes in total, including 79 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The phylogenomic analysis showed that U. macrophylla was closely related to Uncaria rhynchophylla according to the current sampling extent.

Systematic investigation on the distribution of four hidden toxic Aconitum alkaloids in commonly used Aconitum herbs and their acute toxicity.

Yunaconitine (YAC), crassicauline A (CCA), 8-deacetylyunaconitine (DYA), and 8-deacetylcrassicauline A (DCA), as hidden toxic Aconitum alkaloids, are detected in some products of processed Aconitum carmichaelii lateral root and poisoning cases. The distribution and toxicity of these four components in Aconitum herbs should be further systematically studied for medication safety. This study developed a new UHPLC-QQQ-MS/MS method to determine ten Aconitum alkaloids, including aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconine, YAC, CCA, DYA, and DCA, for Aconitum herbs simultaneously. YAC and CCA were founded in some samples of unprocessed A. carmichaelii lateral root (7.04%), A. carmichaelii root (9.43%), A. brachypodum root (6.00%), and A. ouvrardianum root (100%). Four hidden toxic Aconitum alkaloids were detected in processed A. carmichaelii lateral root (2.56%) and A. vilmorinianum root (100%). Four hidden toxic Aconitum alkaloids played significant roles in the classification of Aconitum herbs by OPLS-DA analysis. The acute toxicity test was performed by up-and-down procedure (UDP). The oral administration of the half lethal dose (LD50) of YAC, CCA, DYA, and DCA to female ICR mice was 2.37 mg/kg, 5.60 mg/kg, 60.0 mg/kg, and 753 mg/kg, respectively. The LD50 by intravenous injection was 0.200 mg/kg, 0.980 mg/kg, 7.60 mg/kg, and 34.0 mg/kg, respectively. The LD50 of unprocessed A. carmichaelii lateral root, A. vilmorinianum root, and A. brachypodum root to mice orally was 1.89 g/kg, 0.950 g/kg, and 0.380 g/kg, respectively. Symptoms of Aconitum alkaloid poisoning in mice were decreased activity, fur erect, palpebral edema, vomiting, polypnea, and convulsions. The main change of organs was flatulence. No poisoning or death occurred in mice at the maximum dosage (27.0 g/kg) of A. ouvrardianum root orally. To better control the quality and safety of Aconitum herbs, this study provides favorable support for improving the existing standards to strengthen the supervision of the four hidden toxic Aconitum alkaloids.

Genus Picrasma: A comprehensive review on its ethnopharmacology, phytochemistry and bioactivities.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Picrasma belongs to the Simaroubaceae family and contains six species which are mainly distributed in tropical and subtropical regions of Asia and America. The barks, roots, stems, branches, or leaves of several Picrasma species have been applied as folk medicines to treat fever, sore throat, dysentery, eczema, nausea, loss of appetite, diabetes mellitus, cancer, and hypertension. AIM OF THE STUDY: A systematic summary on the botanic characterization, ethnopharmacological uses, phytochemistry, bioactivities and toxicity of species belonging to Picrasma was presented to facilitate the exploitation of the therapeutic potential of these plants. MATERIALS AND METHODS: The literatures about Picrasma were retrieved from a series of scientific search engines including Web of Science, SciFinder, PubMed, CNKI, Google Scholar, Elsevier, Wiley, ACS publications, and SpringerLink between 1970 and 2020. Plant names were validated by "The Plant List" (www.theplantlist.org). RESULTS: As ethnopharmacological uses, Picrasma species are valuable folk medicines to treat fever, inflammation, dysentery, eczema, cancer, diabetics, skin infection, and so on. Up to now, a total of 361 compounds including 126 alkaloids, 132 quassinoids, 67 triterpenoids, and 36 miscellaneous compounds were reported from Picrasma species. Quassinoids and alkaloids are the principal constituents in the genus. The extracts and phytochemical constituents of Picrasma species demonstrate a wide range of bioactivities including cytotoxic, anti-inflammatory, antimicrobial, and other activities. CONCLUSIONS: Picrasma species are widely used as traditional medicines, have diverse chemical constituents with obvious biological activities. Nevertheless, further studies are required on the Picrasma species to assert the ethnopharmacological uses, clarify their bioactive constituents, determine pharmacological actions, and toxicity. Therefore, the present review may provide a critical clue for future studies and further exploitations on Picrasma species.

Current advances in environmental stimuli regulating the glycyrrhizic acid biosynthesis pathway.

Glycyrrhizic acid, the main active ingredient of licorice, has good antibacterial, anti-tumor, anti-viral, anti-inflammatory, and immunostimulatory activities. However, the content of glycyrrhizic acid fluctuates greatly in different licorice cultivars, and production depends on plant sources, which greatly limits its development and applications. Therefore, increasing glycyrrhizic acid content has become a research priority. In recent years, regulation of the glycyrrhizic acid biosynthesis pathway has been analyzed, the downstream synthesis pathway in licorice has been fully investigated, some key genes have been cloned, polymorphisms have been studied, and the content of glycyrrhizic acid was shown to be regulated by environmental stimuli. This work has provided a basis for studying the regulation mechanism of the glycyrrhizic acid synthesis pathway. This review summarizes and discusses relevant research to provide a current understanding of the glycyrrhizic acid synthesis pathway and its regulation in licorice.

[Effects of different carbon sources on growth and active component contents in Salvia miltiorrhiza and S. castanea f. tomentosa hairy roots].

Salvia miltiorrhiza(Sm) and Salvia castanea f. tomentosa(Sc) hairy roots were used as experimental materials to study the effects of six different carbon sources, galactose, fructose, lactose, glucose, arabinose and sucrose(control), on fresh weight, dry weight, contents and yields of salvianolic acids and tanshinones. The results showed that galactose was most beneficial to the growth of two kinds of hairy roots, while lactose and arabinose were not conducive to their growth. As for Sm hairy roots, fructose significantly promoted the accumulation of salvianolic acid B, and the content increased by 5.801 times and 10.151 times compared with the control group, respectively. Glucose significantly promoted the accumulation of salvianolic acids. The content and yield of rosmarinic acid were 7.674 times and 9.260 times of that of the control group, and the content and yield of salvianolic acid B were 5.532 times and 6.675 times of the control group. For the hairy roots of Sc, galactose significantly increased the content and yield of rosmarinic acid, reaching 7.820 times and 9.944 times of the control group, respectively. Fructose promoted the increase of the content and yield of cryptotanshinone, reaching 9.242 times and 6.609 times of the control group, respectively. The study confirmed the optimal carbon source for the hairy root culture of Sm and Sc, and provided theoretical guidance for large-scale production of Sm drug-derived components and the utilization of Sc.

[Promoting tanshinone synthesis of Salvia miltiorrhiza root by a seed endophytic fungus, Phoma herbarum D603].

The interaction of endophytes and host plant is an effective mean to regulate the growth and secondary metabolism of medicinal plants. Here we want to elucidate the effects and mechanism of Phoma herbarum D603 on the root development and tanshinone synthesis in root of Salvia miltiorrhiza by endophyte-plant coculture system. The mycelium of P. herbarum D603 was colonized in the root tissue space, and formed a stable symbiotic relationship with host plant. The in vitro activities analysis showed that the concentration of IAA produced by D603 can reach(6.45±0.23) µg·mL~(-1), and this strain had some abilities of phosphorus solubilization and siderophore production activities. The coculture experiment showed that strain D603 can significantly promote the synthesis and accumulation of tanshinones in the root of S. miltiorrhiza, in which after 8 weeks of treatment with D603, the content of tanshinone Ⅱ_A in the roots reached up to(1.42±0.59) mg·g~(-1). By the qRT-PCR analysis results, we found that D603 could improve the expression levels of some key genes(DXR, DXS, GGPP, HMGR, CPS) of tanshinone biosynthesis pathway in host plant S. miltiorrhiza, but the promoting effect mainly occurred in the early stage of the interaction, and the enzyme activity level decreased in varying degrees of the later stage. In summary, seed-associated endophyte P. herbarum D603 can promote the growth and root development of S. miltiorrhiza by producing hormones, promoting nutrient absorption and siderophore production, and promote the synthesis and accumulation of tanshinones by regulating the expression level of key genes in the synthetic pathway in S. miltiorrhiza.

[Cloning,subcellular localization and expression pattern analysis of transcription factor SmGRAS3 from Salvia miltiorrhiza].

GRAS transcription factors play important roles in the regulation of plant root growth and GA signaling. In this study,SmGRAS3 gene was cloned,which open reading frame was 2 247 bp,and encoding 748 amino acids. The physicochemical properties and structure of SmGRAS3 and its encoded protein were analyzed by bioinformatics software. This gene belongs to the SCL9 subfamily of the GRAS family,and its promoter sequence mainly contains the light response,stress response,and hormone response elements. It may interact with the GA signal pathway and anti-stress related proteins. The subcellular localization showed that SmGRAS3 protein was mainly located in the nucleus. The expression pattern analysis showed that the expression of Sm GRAS3 was the highest in the root and the lowest in the stem,and both light and low temperature could induce the high expression level of SmGRAS3. This study provides a foundation for further study on the roles of SmGRAS3 gene in the root growth and stress tolerance of Salvia miltiorrhiza.