Molecular cloning and functional characterization of 2,3-oxidosqualene cyclases from Artemisia argyi.

Artemisia argyi is a traditional medicinal and edible plant, generating various triterpenoids with pharmacological activities, such as anti-virus, anti-cancer, and anti-oxidant. The 2,3-oxidosqualene cyclase family of A. argyi offers novel insights into the triterpenoid pathway, which might contribute to the medicinal value of its tissue extracts. Nevertheless, the biosynthesis of active triterpenoids in Artemisia argyi is still uncertain. In this study, four putative OSC (2,3-oxidosqualene cyclase) genes (AaOSC1-4) were first isolated and identified from A. argyi. Through the yeast heterologous expression system, three AaOSCs were characterized for the biosynthesis of diverse triterpenoids including cycloartenol, ß-amyrin, (3S,13R)-malabarica-14(27),17,21-trien-3ß-ol, and dammara-20,24-dien-3ß-ol. AaOSC1 was a multifunctional dammara-20,24-dien-3ß-ol synthase, which yielded 8 different triterpenoids, including tricyclic, and tetracyclic products. AaOSC2 and AaOSC3 were cycloartenol, and ß-amyrin synthases, respectively. As a result, these findings provide a deeper understanding of the biosynthesis pathway of triterpenes in A. argyi.

In Vitro Anti-Toxoplasma Activity of Extracts Obtained from Tabebuia rosea and Tabebuia chrysantha: The Role of ß-Amyrin.

Toxoplasmosis is a parasitic disease caused by the protozoan Toxoplasma gondii that is highly prevalent worldwide. Although the infection is asymptomatic in immunocompetent individuals, it severely affects immunocompromised individuals, causing conditions such as encephalitis, myocarditis, or pneumonitis. The limited therapeutic efficacy of drugs currently used to treat toxoplasmosis has prompted the search for new therapeutic alternatives. The aim of this study was to determine the anti-Toxoplasma activity of extracts obtained from two species of the genus Tabebuia. Twenty-six extracts, 12 obtained from Tabebuia chrysantha and 14 from Tabebuia rosea, were evaluated by a colorimetric technique using the RH strain of T. gondii that expresses ß-galactosidase. Additionally, the activity of the promising extracts and their active compounds was evaluated by flow cytometry. ß-amyrin was isolated from the chloroform extract obtained from the leaves of T. rosea and displayed important anti-Toxoplasma activity. The results show that natural products are an important source of new molecules with considerable biological and/or pharmacological activity.

Anti-ulcerogenic Potential of Kalanchoë gastonis-bonnieri Extracts in Male ICR Mice Model of Ethanol-induced Gastric Ulcers.

Peptic ulcers (PU) are a breach in the mucosa of the digestive tract and are related to several factors including an altered immune system and an unbalanced diet. Current treatment carries to long-term complications; therefore, the use of medicinal plants is an alternative for several inflammatory diseases including ulcerative lesions. Kalanchoë gastonis-bonnieri, is a succulent plant and, has been used in traditional medicine against gastric ulcers, inflammation, and cancer among others. The main goal of this work was to analyze the anti-ulcerogenic potential of extracts from leaves of K. gastonis-bonnieri in an assay of ethanol-induced gastric ulcers. An ethanolic extract was obtained by maceration from fresh leaves of K. gastonis-bonnieri, and fractions were obtained through bipartition and chemical fractioning. The chemical characterization of the extract was made through HPLC, GC-MS, and NMR. Total extract and fractions showed an anti-ulcerogenic effect in specimens of male ICR mice with a gastric ulcer index (UI) of 3.27-5.47. The recorded effect is attributed to the presence of terpenoid compounds such as ß-Amyrin acetate, which showed antioxidant properties and lessened formations of ulcers induced by ethanol administration in mice stomach.

[Cloning and functional characterization of oxidosqualene cyclase gene for α-amyrin synthesis].

Ursolic acid has gradually attracted much attention due to its unique pharmacological activities and valuable market value in recent years. Currently, ursolic acid is mostly extracted from loquat leaves, but the plant extraction method has low yield and high cost, and chemical synthesis is not readily available, so the biosynthesis method provides a new source for ursolic acid. α-amyrin acts as the main precursor for the synthesis of ursolic acid, and its yield is positively correlated with ursolic acid yield. Oxidosqualene cyclase(OSC) belongs to a multigene family which can catalyze the common precursor 2,3-oxidosqualene to generate different types of triterpene backbones, and plays a decisive role in the synthesis of triterpenoids. However, there are fewer reported key genes catalyzing the synthesis of α-amyrin in medicinal plants, and the yield and proportion of α-amyrin in the catalyzed products have always been a focus of research. In this study, ItOSC2, MdOSC1, AaOSC2 and CrAS, four enzymes capable of catalyzing the production of α-amyrin from 2,3-oxidosqualene, were cloned from Iris tectorum, Malus domestica, Artemisia annua and Catharanthus roseus, subject to sequence alignment and phylogenetic tree analyses, and transformed into Saccharomyces cerevisiae as plasmids. After 7 days of fermentation, the yield and proportions of α-amyrin, ß-amyrin and ergosterol were measured. Finally, AaOSC2 with the best ability to catalyze the generation of α-amyrin was filtered out, providing a key gene element for the later construction of engineered yeast strains with high production of α-amyrin and ursolic acid.

OeBAS and CYP716C67 catalyze the biosynthesis of health-beneficial triterpenoids in olive (Olea europaea) fruits.

The bioactive properties of olive (Olea europaea) fruits and olive oil are largely attributed to terpenoid compounds, including diverse triterpenoids such as oleanolic, maslinic and ursolic acids, erythrodiol, and uvaol. They have applications in the agri-food, cosmetics, and pharmaceutical industries. Some key steps involved in the biosynthesis of these compounds are still unknown. Genome mining, biochemical analysis, and trait association studies have been used to identify major gene candidates controlling triterpenoid content of olive fruits. Here, we identify and functionally characterize an oxidosqualene cyclase (OeBAS) required for the production of the major triterpene scaffold ß-amyrin, the precursor of erythrodiol, oleanolic and maslinic acids, and a cytochrome P450 (CYP716C67) that mediates 2α oxidation of the oleanane- and ursane-type triterpene scaffolds to produce maslinic and corosolic acids, respectively. To confirm the enzymatic functions of the entire pathway, we have reconstituted the olive biosynthetic pathway for oleanane- and ursane-type triterpenoids in the heterologous host, Nicotiana benthamiana. Finally, we have identified genetic markers associated with oleanolic and maslinic acid fruit content on the chromosomes carrying the OeBAS and CYP716C67 genes. Our results shed light on the biosynthesis of olive triterpenoids and provide new gene targets for germplasm screening and breeding for high triterpenoid content.

Aided-efflux and high production of ß-amyrin realized by ß-cyclodextrin in situ synthesized on surface of Saccharomyces cerevisiae.

As a plant-derived pentacyclic triterpenoid, ß-amyrin has been heterogeneously synthesized in Saccharomyces cerevisiae. However, ß-amyrin is intracellularly produced in a lower gram scale using recombinant S. cerevisiae, which limits the industrial applications. Although many strategies have been proven to be effective to improve the production of ß-amyrin, the intracellularly accumulation is still a challenge in reaching higher titer and simplifying the extraction process. To solve this problem, the amphiphilic ß-cyclodextrin (ß-CD) has been previously employed to aid the efflux of ß-amyrin out of the cells. Nevertheless, the supplemented ß-CD in the medium is not consistent with ß-amyrin synthesis and has the disadvantage of rather high cost. Therefore, an aided-efflux system based on in situ synthesis of ß-CD was developed in this study to enhance the biosynthesis of ß-amyrin and its efflux. The in situ synthesis of ß-CD was started from starch by the surface displayed cyclodextrin glycosyltransferase (CGTase) on yeast cells. As a result, the synthesized ß-CD could capture 16% of the intracellular ß-amyrin and improve the total production by 77%. Furthermore, more strategies including inducing system remodeling, precursor supply enhancement, two-phase fermentation and lipid synthesis regulation were employed. Finally, the production of ß-amyrin was increased to 73 mg/L in shake flask, 31 folds higher than the original strain, containing 31 mg/L of extracellular ß-amyrin. Overall, this work provides novel strategies for the aided-efflux of natural products with high hydrophobicity in engineered S. cerevisiae.

Chemical Composition and Antimicrobial Effect of Western Ethiopian Propolis.

Propolis or bee glue is commonly named as a natural resinous mixture produced by honeybees (Apis mellifera) from substances collected from parts of plants, buds, and exudate. The result of the ethyl acetate - methanol (3 : 2) volume by volume fraction yielded a total of two compounds namely betulinic acid and ß-amyrin isolated from Bodji Dirmaji and Fincha'a district propolis, respectively. The crude ethanolic extract was portioned with the different solvent systems by increasing the polarities in the following order of hexane, ethyl acetate, and methanol. Column chromatographic method on normal silica gel was used to isolate the compounds. The structures of the compounds were characterized using 1D NMR techniques. The study revealed that western Ethiopian propolis was rich in saponins, tannins, flavonoids, steroids, triterpenes, and glycosides. The antibacterial activity for the isolated compound (betulinic acid) showed the highest inhibition for S. aureus (11.2±1.6), E. coli (17.7±1.1), and A. niger (12.6±1.2) mm.

Molecular Cloning and Functional Characterization of Oxidosqualene Cyclases from Panax vietnamensis.

Panax vietnamensis is a valuable medicinal resource with promising preclinical applications. Ginsenosides, which are triterpenoids, are the primary active components in P. vietnamensis. Oxidosqualene cyclases (OSCs) catalyze the formation of the basic skeleton of triterpenes from 2,3-oxidosqualene, which is a crucial step in the biosynthesis of triterpenoids. The OSCs involved in triterpenoid biosynthesis in P. vietnamensis have not yet been characterized. Four OSC genes (PvOSC1-4) were cloned from P. vietnamensis and functionally characterized via heterologous expression in yeast. Transgenic yeast expressing PvOSC1, PvOSC3, and PvOSC4 produced the corresponding products ß-amyrin, cycloartenol, and dammarenediol-II, respectively. PvOSC1, PvOSC3, and PvOSC4 are monofunctional OSCs. In this study, we characterized three PvOSC genes, providing a better understanding of the biosynthesis of triterpenoids in P. vietnamensis and the multiple choices of plant OSCs for metabolic engineering in yeast and other hosts.

Antispasmodic Effect of Alstonia boonei De Wild. and Its Constituents: Ex Vivo and In Silico Approaches.

BACKGROUND: Alstonia boonei, belonging to the family Apocynaceae, is one of the best-known medicinal plants in Africa and Asia. Stem back preparations are traditionally used as muscle relaxants. This study investigated the antispasmodic properties of Alstonia boonei Stem back and its constituents. METHOD: The freeze-dried aqueous Stem back extract of A. boonei, as well as dichloromethane (DCM), ethyl acetate, and aqueous fractions, were evaluated for their antispasmodic effect via the ex vivo method. Two compounds were isolated from the DCM fraction using chromatographic techniques, and their antispasmodic activity was evaluated. An in silico study was conducted by evaluating the interaction of isolated compounds with human PPARgamma-LBD and human carbonic anhydrase isozyme. RESULTS: The Stem back crude extract, DCM, ethyl acetate, and aqueous fractions showed antispasmodic activity on high-potassium-induced (K+ 80 mM) contractions on isolated rat ileum with IC50 values of 0.03 ± 0.20, 0.02 ± 0.05, 0.03 ± 0.14, and 0.90 ± 0.06 mg/mL, respectively. The isolated compounds from the DCM fraction were ß-amyrin and boonein, with only boonein exhibiting antispasmodic activity on both high-potassium-induced (IC50 = 0.09 ± 0.01 µg/mL) and spontaneous (0.29 ± 0.05 µg/mL) contractions. However, ß-amyrin had a stronger interaction with the two proteins during the simulation. CONCLUSION: The isolated compounds boonein and ß-amyrin could serve as starting materials for the development of antispasmodic drugs.

Cuticular Waxes and Cutin in Terminalia catappa Leaves from the Equatorial São Tomé and Príncipe Islands.

This study presents for the first time an analysis of the content and chemical composition of the cuticular waxes and cutin in the leaves of the widespread and important tropical species Terminalia catappa. The leaves were collected in the equatorial Atlantic islands of São Tomé and Príncipe, in the Gulf of Guinea. The epicuticular and intracuticular waxes were determined via dichloromethane extraction and their chemical composition via GC-MS analysis, and the content and monomeric composition of cutin were determined after depolymerization via methanolysis. The leaves contained an epidermal cuticular coverage of 52.8 µg cm-2 of the cuticular waxes (1.4% of mass) and 63.3 µg cm-2 (1.5% of mass) of cutin. Cuticular waxes include mainly n-alkanols and fatty acids, with a substantial proportion of terpenes in the more easily solubilized fraction, and sterols in the more embedded waxes. Cutin is mostly constituted by C16 fatty acids and dihydroxyacids, also including aromatic monomers, suggesting a largely linear macromolecular arrangement. The high proportion of triacontanol, α-amyrin, ß-amyrin, germanicol, and lupeol in the easily solubilized cuticular fraction may explain the bioactive properties attributed to the T. catappa leaves via the popular medicine, which allows us to consider them as a potential source for the extraction of these compounds.

Phytochemical and Biological Investigation of an Indigenous Plant of Bangladesh, Gynura procumbens (Lour.) Merr.: Drug Discovery from Nature.

Gynura procumbens (Lour.) Merr. (Family: Asteraceae) is a tropical Asian medicinal plant found in Thailand, China, Malaysia, Indonesia, and Vietnam. It has long been utilized to treat a variety of health concerns in numerous countries around the world, such as renal discomfort, constipation, diabetes mellitus, rheumatism, and hypertension. The chemical investigation resulted in the isolation and characterization of six compounds from the methanol (MeOH) extract of the leaves of Gynura procumbens, which were identified as phytol (1), lupeol (2), stigmasterol (3), friedelanol acetate (4), ß-amyrin (5), and a mixture of stigmasterol and ß-sitosterol (6). In-depth investigations of the high-resolution 1H NMR and 13C NMR spectroscopic data from the isolated compounds, along with comparisons to previously published data, were used to clarify their structures. Among these, the occurrence of Compounds 1 and 4 in this plant are reported for the first time. The crude methanolic extract (CME) and its different partitionates, i.e., petroleum ether (PESF), chloroform (CSF), ethyl acetate (EASF), and aqueous (AQSF) soluble fractions, were subjected to antioxidant, cytotoxic, thrombolytic, and anti-diabetic activities. In a DPPH free radical scavenging assay, EASF showed the maximum activity, with an IC50 value of 10.78 µg/mL. On the other hand, CSF displayed the highest cytotoxic effect with an LC50 value of 1.94 µg/mL compared to 0.464 µg/mL for vincristine sulphate. In a thrombolytic assay, the crude methanolic extract exhibited the highest activity (63.77%) compared to standard streptokinase (70.78%). During the assay for anti-diabetic activity, the PESF showed 70.37% of glucose-lowering activity, where standard glibenclamide showed 63.24% of glucose-reducing activity.

ß-Amyrin ameliorates diabetic nephropathy in mice and regulates the miR-181b-5p/HMGB2 axis in high glucose-stimulated HK-2 cells.

Diabetic nephropathy (DN) is a diabetic complication that can cause renal failure. ß-amyrin has been identified to possess anti-diabetic property. This study was designed to evaluate the potential role of ß-amyrin in DN and its underlying mechanism. Streptozotocin-induced diabetic mice were used as the in vivo model, and high glucose (HG)-stimulated human proximal tubular HK-2 cells were utilized as the in vitro model. Renal histological changes in mice were assessed by hematoxylin-eosin and periodic acid-Schiff staining. HK-2 cell viability and apoptosis were detected by Cell Counting Kit-8 assay and flow cytometry analysis, respectively. ß-amyrin was found to ameliorate kidney injury in DN mice and suppressed inflammatory response as well as apoptosis of HG-stimulated HK-2 cells. miR-181-5p expression in murine renal tissues and HK-2 cells was detected by in situ hybridization (ISH) and fluorescence in situ hybridization (FISH). MiR-181b-5p, a previously identified target for diabetic kidney disease, was downregulated in renal tissues and HG stimulated HK-2 cells, and ß-amyrin induced the upregulation of miR-181b-5p. Binding relationship between miR-181b-5p and high mobility group box 2 (HMGB2) was confirmed by luciferase reporter assay. MiR-181b-5p bound to 3' untranslated region of HMGB2 to suppress its expression. As shown by immunohistochemical staining and immunofluorescence staining, HMGB2 was upregulated in the in vivo and in vitro models of DN, and ß-amyrin induced the downregulation of HMGB2. Moreover, HMGB2 overexpression neutralized the suppressive effects of miR-181b-5p elevation on the inflammatory response and apoptosis of HG-treated HK-2 cells. Overall, ß-amyrin ameliorates DN in mice and suppresses inflammatory response and apoptosis of HG-stimulated HK-2 cells via the miR-181b-5p/HMGB2 axis.

Potential Efficacy of ß-Amyrin Targeting Mycobacterial Universal Stress Protein by In Vitro and In Silico Approach.

The emergence of drug resistance and the limited number of approved antitubercular drugs prompted identification and development of new antitubercular compounds to cure Tuberculosis (TB). In this work, an attempt was made to identify potential natural compounds that target mycobacterial proteins. Three plant extracts (A. aspera, C. gigantea and C. procera) were investigated. The ethyl acetate fraction of the aerial part of A. aspera and the flower ash of C. gigantea were found to be effective against M. tuberculosis H37Rv. Furthermore, the GC-MS analysis of the plant fractions confirmed the presence of active compounds in the extracts. The Mycobacterium target proteins, i.e., available PDB dataset proteins and proteins classified in virulence, detoxification, and adaptation, were investigated. A total of ten target proteins were shortlisted for further study, identified as follows: BpoC, RipA, MazF4, RipD, TB15.3, VapC15, VapC20, VapC21, TB31.7, and MazF9. Molecular docking studies showed that ß-amyrin interacted with most of these proteins and its highest binding affinity was observed with Mycobacterium Rv1636 (TB15.3) protein. The stability of the protein-ligand complex was assessed by molecular dynamic simulation, which confirmed that ß-amyrin most firmly interacted with Rv1636 protein. Rv1636 is a universal stress protein, which regulates Mycobacterium growth in different stress conditions and, thus, targeting Rv1636 makes M. tuberculosis vulnerable to host-derived stress conditions.

[Cloning and bioinformatics analysis of ß-amyrin synthase in Dipsacus asper].

Dipsaci Radix is one of the commonly used Chinese medicinal materials in China, with a long history. It has the medicinal activities of nourishing liver and kidney, recovering from broken sinews, and treating bone fracture. Triterpenoid saponins are the main functional ingredients of Dipsacus asper. ß-Amyrin synthases(ß-AS) as a superfamily of oxidosqualene cyclases(OSCs) can catalyze the construction of the skeleton structure of oleanane-type triterpenoid saponins. There are only a few studies about the ß-AS in D. asper, and the catalytic mechanism of this enzyme remains to be explored. To enrich the information of ß-AS, according to the transcriptome sequencing results, we cloned DaWß-AS gene from D. asper into a specific vector for heterologous expression in Escherichia coli. In the meantime, real-time PCR was performed to analyze the relative expression of DaWß-AS in four different tissues of D. asper. The results of RT-qPCR showed DaWß-AS had the highest expression level in leaves. Bioinformatics results indicated that DaWß-AS had a conserved domain of PLN03012 superfamily, belonging to the cl31551 superfamily. There was no transmembrane domain or signal peptide in DaWß-AS. This study provides a scientific basis for revealing the biological pathways of triterpenoid saponins in D. asper, which will facilitate the biosynthesis of the associated saponins and afford reference for the cultivation and development of high-quality resources of D. asper.

ß-Amyrin Synthase1 Controls the Accumulation of the Major Saponins Present in Pea (Pisum sativum).

The use of pulses as ingredients for the production of food products rich in plant proteins is increasing. However, protein fractions prepared from pea or other pulses contain significant amounts of saponins, glycosylated triterpenes that can impart an undesirable bitter taste when used as an ingredient in foodstuffs. In this article, we describe the identification and characterization of a gene involved in saponin biosynthesis during pea seed development, by screening mutants obtained from two Pisum sativum TILLING (Targeting Induced Local Lesions IN Genomes) populations in two different genetic backgrounds. The mutations studied are located in a gene designated PsBAS1 (ß-amyrin synthase1), which is highly expressed in maturing pea seeds and which encodes a protein previously shown to correspond to an active ß-amyrin synthase. The first allele is a nonsense mutation, while the second mutation is located in a splice site and gives rise to a mis-spliced transcript encoding a truncated, nonfunctional protein. The homozygous mutant seeds accumulated virtually no saponin without affecting the seed nutritional or physiological quality. Interestingly, BAS1 appears to control saponin accumulation in all other tissues of the plant examined. These lines represent a first step in the development of pea varieties lacking bitterness off-flavors in their seeds. Our work also shows that TILLING populations in different genetic backgrounds represent valuable genetic resources for both crop improvement and functional genomics.

Histochemical and Phytochemical Analysis of Lamium album subsp. album L. Corolla: Essential Oil, Triterpenes, and Iridoids.

The aim of this study was to conduct a histochemical analysis to localize lipids, terpenes, essential oil, and iridoids in the trichomes of the L. album subsp. album corolla. Morphometric examinations of individual trichome types were performed. Light and scanning electron microscopy techniques were used to show the micromorphology and localization of lipophilic compounds and iridoids in secretory trichomes with the use of histochemical tests. Additionally, the content of essential oil and its components were determined using gas chromatography-mass spectrometry (GC-MS). Qualitative analyses of triterpenes carried out using high-performance thin-layer chromatography (HPTLC) coupled with densitometric detection, and the iridoid content expressed as aucubin was examined with spectrophotometric techniques. We showed the presence of iridoids and different lipophilic compounds in papillae and glandular and non-glandular trichomes. On average, the flowers of L. album subsp. album yielded 0.04 mL/kg of essential oil, which was dominated by aldehydes, sesquiterpenes, and alkanes. The extract of the L. album subsp. album corolla contained 1.5 × 10-3 ± 4.3 × 10-4 mg/mL of iridoid aucubin and three triterpenes: oleanolic acid, ß-amyrin, and ß-amyrin acetate. Aucubin and ß-amyrin acetate were detected for the first time. We suggest the use of L. album subsp. album flowers as supplements in human nutrition.

α-Amyrin and ß-Amyrin Isolated from Celastrus hindsii Leaves and Their Antioxidant, Anti-Xanthine Oxidase, and Anti-Tyrosinase Potentials.

Celastrus hindsii is a popular medicinal plant in Vietnam and Southeast Asian countries as well as in South America. In this study, an amount of 12.05 g of an α-amyrin and ß-amyrin mixture was isolated from C. hindsii (10.75 g/kg dry weight) by column chromatography applying different solvent systems to obtain maximum efficiency. α-Amyrin and ß-amyrin were then confirmed by gas chromatography-mass spectrometry (GC-MS), electrospray ionization-mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR). The antioxidant activities of the α-amyrin and ß-amyrin mixture were determined via 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,20-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays with IC50 of 125.55 and 155.28 µg/mL, respectively. The mixture exhibited a high potential for preventing gout by inhibiting a relevant key enzyme, xanthine oxidase (XO) (IC50 = 258.22 µg/mL). Additionally, an important enzyme in skin hyperpigmentation, tyrosinase, was suppressed by the α-amyrin and ß-amyrin mixture (IC50 = 178.85 µg/mL). This study showed that C. hindsii is an abundant source for the isolation of α-amyrin and ß-amyrin. Furthermore, this was the first study indicating that α-amyrin and ß-amyrin mixture are promising in future therapies for gout and skin hyperpigmentation.

Structural elucidation and in vivo anti-arthritic activity of ß-amyrin and polpunonic acid isolated from the root bark of Ziziphus abyssinica HochstEx. A Rich (Rhamnaceae).

Two natural products, compounds 1 and 2 were isolated from the root bark of Ziziphus abyssinica for the first time and were structurally elucidated as ß-amyrin and polpunonic acid, respectively. Both compounds were further subjected to an in vivo study in rats to evaluate their anti-arthritic potency. Compared to the arthritic control group, rats treated with different doses of 1 or 2 (3, 10, and 30 mg/kg) exhibited significantly higher total change in body weight as well as lower arthritic scores and total change in paw edema and erythema. Histopathological examinations of the hind paws of the rats further demonstrated the beneficial effects of both compounds as they significantly reversed cartilage erosion, subchondral cyst, and Weichselbaum's lacunae formation. Evidence of bone remodeling was also observed in all groups of rats treated with 1 or 2. Hematological and serum biochemical parameters were not significantly affected by treatment of 1 or 2. Taken together, the results from the present study suggest potential therapeutic benefit of ß-amyrin and polpunonic acid in rheumatoid arthritis and related inflammatory disorders.

[CbCYP716A261, a New ß-Amyrin 28-Hydroxylase Involved in Conyzasaponin Biosynthesis from Conyza blinii].

Conyzasaponins produced by the traditional Chinese herb Conyza blinii are oleanane-type saponins with a wide range of biological activities. Here, we identified a gene, designated CbCYP716A261, encoding a ß-amyrin 28-hydroxylase in conyzasaponins biosynthesis. Ten full putative CYP sequences were isolated from Conyza blinii transcript tags. The CbCYP716A261 gene product was selected as the putative ß-amyrin 28-hydroxylase by phylogenetic analysis and transcriptional activity analysis of methyl jasmonate-treated Conyza blinii. To identify the enzymatic activity, we performed enzymatic activity experiments in vitro and in vivo. The HPLC results revealed that CbCYP716A261 catalyzes the hydroxylation of ß-amyrin at the C-28 position to yield oleanolic acid. Our findings provide new information about the conyzasaponin biosynthesis pathway and widen the list of isolated ß-amyrin 28-hydroxylases.

[Regulation of ß-mercuryl alcohol metabolic flow in Saccharomyces cerevisiae cells].

In this study, citrate synthase gene(CIT2), and malate synthase gene(MLS1) were successfully knocked out in ß-amyrin-producing yeast cells by using CRISPR/CAS9. The promoter of phosphoglucose isomerase gene(PGI1) was replaced by that of cytochrome c oxidase subunit Ⅶa(Cox9)to weaken its expression, aiming to channel more carbon flux into the NADPH-producing pathway. The fermentation results showed that CIT2 deletion had no effect on the ß-amyrin production. Compared with the control strain, the production of ß-amyrin was increased by 1.85 times after deleting MLS1, reaching into 3.3 mg·L~(-1). By replacing the promoter of PGI1, the ß-amyrin yield was 3.75 times higher than that of the control strain, reaching up to 6.7 mg·L~(-1). This study successfully knocked out the CITT2 and MLS1 genes and weakened the PGI1 gene by using CRISPR/CAS9, which directly influenced the production of ß-amyrin and provided some reference for the the metabolic engineering of triterpernoid producing strain.