Elucidation of the complete biosynthetic pathway of the main triterpene glycosylation products of Panax notoginseng using a synthetic biology platform

UDP-glycosyltransferase (UGT)-mediated glycosylation is a widespread modification of plant natural products (PNPs), which exhibit a wide range of bioactivities, and are of great pharmaceutical, ecological and agricultural significance. However, functional annotation is available for less than 2% of the family 1 UGTs, which currently has 20,000 members that are known to glycosylate several classes of PNPs. This low percentage illustrates the difficulty of experimental study and accurate prediction of their function. Here, a synthetic biology platform for elucidating the UGT-mediated glycosylation process of PNPs was established, including glycosyltransferases dependent on UDP-glucose and UDP-xylose. This platform is based on reconstructing the specific PNPs biosynthetic pathways in dedicated microbial yeast chassis by the simple method of plug-and-play. Five UGT enzymes were identified as responsible for the biosynthesis of the main glycosylation products of triterpenes in Panax notoginseng, including a novel UDP-xylose dependent glycosyltransferase enzyme for notoginsenoside R1 biosynthesis. Additionally, we constructed a yeast cell factory that yields >1 g/L of ginsenoside compound K. This platform for functional gene identification and strain engineering can serve as the basis for creating alternative sources of important natural products and thereby protecting natural plant resources.     

三七皂苷R1对大鼠缺血心肌VEGF、bFGF的影响

目的:探讨三七皂苷R1对大鼠缺血心肌VEGF、bFGF的影响。方法:选择雄性Wistar大鼠39只,建立心肌梗死(AMI)模型,术后24h存活大鼠随机分为药物组(n=13)、对照组(n=13),另设假手术组(n=8)。药物组给予三七皂苷R1水溶液(2.5 mg·kg-1·d-1)腹腔注射、对照组及假手术组给予等体积生理盐水腹腔注射,用药4周。于实验终点处死大鼠,心肌组织取材,Ⅷ因子染色计数微血管数(MVC)及微血管密度(MVD),免疫组织化学法观察缺血心肌VEGF、bFGF蛋白的表达。结果:药物组及对照组MVC、MVD均高于假手术组,且药物组高于对照组(P0.05);大鼠缺血心肌药物组及对照组VEGF、bFGF蛋白表达均高于假手术组(P0.05),且药物组高于对照组(P0.05)。结论:三七皂苷R1促进大鼠缺血心肌血管再生同时可上调缺血心肌VEGF、bFGF蛋白水平。     

Six new dammarane-type triterpene saponins from the processed leaves of Panax notoginseng

Six new dammarane-type triterpenoid saponins, notoginsenosides SFt₅–SFt₁₀ (1–6) were isolated from the processed leaves of Panax notoginseng (Burk.) F.H.Chen (Araliaceae), together with eight known notoginsenosides (7–14), fourteen known ginsenosides (15−28), two known vinaginsenosides (29−30), and one known gypenoside (31). Their structures were established by detailed spectroscopic analysis (NMR, UV, IR, HRESI-MS) and acidic hydrolysis. Four compounds notoginsenoside SFt₁ (7), ginsenosides Rg₅ (17), C-Mc (23) and 20(R)–Rg₃ (25) have significant protective effects against L-glutamate-induced SH–SY5Y nerve injury (10 μM).     

三七地下部分皂甙成分的HPLC比较研究

应用HPLC定量分析方法,对三七(Panax notoginseng)地下部位的皂甙成分进行分析,通过比较人参皂甙Rg1,Rb1,Re,Rd和三七皂甙R1等5种主要皂甙成分和总皂甙的含量变化,探讨不同部位和组织中皂甙成分的分布规律。结果表明在三七药材的不同商品等级中,人参皂甙Rg1和Rb1的含量以主根60头为最高,5个主要皂甙的总含量也明显高于其他的等级;根茎的生物产量只为全根的18%,皂甙含量占25%以上;主根和根茎中韧皮部的生物产量和总皂甙的含量均高于木质部;二年生三七的生物产量及皂甙含量均较三年生三七低得多。不同表型三七的皂甙组成也有区别。     

High-level sustainable production of the characteristic protopanaxatriol-type saponins from Panax species in engineered Saccharomyces cerevisiae

The Chinese medicinal plant Panax notoginseng has been traditionally used to activate blood flow and circulation, and to prevent blood stasis. P. notoginseng contains protopanaxatriol (PPT)-type saponins as its main active compounds, thus distinguishing it from the other two famous Panax species, P. ginseng and P. quinquefolius. Ginsenoside Rg1 (Rg1), notoginsenoside R1 (NgR1), and notoginsenoside R2 (NgR2) are three major PPT-type saponins in P. notoginseng and possess potential cardiovascular protection activities. However, their use in medical applications has long been hampered by the lack of sustainable and low-cost industrial-scale preparation methods. In this study, a PPT-producing yeast chassis strain was designed and constructed based on a previously constructed and optimized protopanaxadiol (PPD)-producing Saccharomyces cerevisiae strain, and further optimized by systemically engineering and optimizing the expression level of its key P450 biopart. Rg1-producing yeast strains were constructed by introducing PgUGT71A53 and PgUGT71A54 into the PPT chassis strain. The fermentation titer of Rg1 reached 1.95 g/L. A group of UDP-glycosyltransferases (UGT) from P. notoginseng and P. ginseng were characterized, and were found to generate NgR1 and NgR2 by catalyzing the C₆–O-Glc xylosylation of Rg1 and Rh1, respectively. Using one of these UGTs, PgUGT94Q13, and the previously identified PgUGT71A53 and PgUGT71A54, the biosynthetic pathway to produce saponins NgR1 and NgR2 from PPT could be available. The NgR1 cell factory was further developed by introducing PgUGT94Q13 and a heterologous UDP-xylose biosynthetic pathway from Arabidopsis thaliana into the highest Rg1-producing cell factory. The NgR2-producing cell factory was constructed by introducing PgUGT71A54, PgUGT94Q13, and the UDP-xylose biosynthetic pathway into the PPT chassis. De novo production of NgR1 and NgR2 reached 1.62 g/L and 1.25 g/L, respectively. Beyond the realization of artificial production of the three valuable saponins Rg1, NgR1, and NgR2 from glucose, our work provides a green and sustainable platform for the efficient production of other PPT-type saponins in engineered yeast strains, and promotes the industrial application of PPT-type saponins as medicine and functional foods.     

Protective effects of the notoginsenoside R1 on acute lung injury by regulating the miR-128-2-5p/Tollip signaling pathway in rats with severe acute pancreatitis

Notoginsenoside R1 (NG-R1), the extract and the main ingredient of Panax notoginseng, has anti-inflammatory effects and can be used in treating acute lung injury (ALI). In this study, we explored the pulmonary protective effect and the underlying mechanism of the NG-R1 on rats with ALI induced by severe acute pancreatitis (SAP). MiR-128-2-5p, ERK1, Tollip, HMGB1, TLR4, IκB, and NF-κB mRNA expression levels were measured using real-time qPCR, and TLR4, Tollip, HMGB1, IRAK1, MyD88, ERK1, NF-κB65, and P-IκB-α protein expression levels using Western blot. The NF-κB and the TLR4 activities were determined using immunohistochemistry, and TNF-α, IL-6, IL-1β, and ICAM-1 levels in the bronchoalveolar lavage fluid (BALF) using ELISA. Lung histopathological changes were observed in each group. NG-R1 treatment reduced miR-128-2-5p expression in the lung tissue, increased Tollip expression, inhibited HMGB1, TLR4, TRAF6, IRAK1, MyD88, NF-κB65, and p-IκB-α expression levels, suppressed NF-κB65 and the TLR4 expression levels, reduced MPO activity, reduced TNF-α, IL-1β, IL-6, and ICAM-1 levels in BALF, and alleviated SAP-induced ALI. NG-R1 can attenuate SAP-induced ALI. The mechanism of action may be due to a decreased expression of miR-128-2-5p, increased activity of the Tollip signaling pathway, decreased activity of HMGB1/TLR4 and ERK1 signaling pathways, and decreased inflammatory response to SAP-induced ALI. Tollip was the regulatory target of miR-128-2-5p.     

三七皂苷单体Rg1对低氧高二氧化碳肺动脉平滑肌细胞p38MAPK表达的影响

目的:研究三七皂苷单体Rg1对低氧高二氧化碳肺动脉平滑肌细胞(PASMCs)p38MAPK表达的影响。方法:分离、纯化SD大鼠PASMCs,实验用2至5代细胞,实验分六组:常氧组(N组),低氧高二氧化碳组(H组),DM-SO对照组(HD组),Rg1干预组(RgL、RgM、RgH组)。采用Western blot检测磷酸化p38MAPK蛋白表达,RT-PCR检测p38MAPK mRNA的表达。结果:Westernblot、RT-PCR结果显示,HD组p-p38MAPK蛋白和p38MAPK mRNA表达明显高于N组(P<0.01),RgL、RgM、RgH组不同程度抑制了p-p38MAPK蛋白和p38MAPK mRNA和的表达(P<0.01),并呈剂量依赖关系。结论:三七皂苷单体Rg1对低氧高二氧化碳条件下PASMCs有保护作用,其机制可能与抑制p38MAPK的表达有关。     

p38 MAPK and ERK1/2 pathways are involved in the pro-apoptotic effect of notoginsenoside Ft1 on human neuroblastoma SH-SY5Y cells

Aims

This study aims to investigate the effect and the mechanisms of notoginsenoside Ft1, a natural compound exclusively found in P. notoginseng, on the proliferation and apoptosis of human neuroblastoma SH-SY5Y cells.

Main methods

CCK-8 assay was used to assess the cell proliferation. Flow cytometry was performed to measure the cell cycle distribution and cell apoptosis. Hoechst 33258 staining was conducted to confirm the morphological changes of apoptotic cells. Protein expression was detected by western blot analysis and caspase 3 activity was measured by colorimetric assay kit.

Key findings

Among the saponins examined, Ft1 showed the best inhibitory effect on cell proliferation of SH-SY5Y cells with IC₅₀ of 45 μM. Ft1 not only arrested the cell cycle at S, G₂/M stages, but also promoted cell apoptosis, which was confirmed by Hoechst 33258 staining. Further studies demonstrated that Ft1 up-regulated the protein expressions of cleaved caspase 3, phospho-p53, p21, and cyclin B1, but down-regulated that of Bcl-2. Moreover, Ft1 enhanced the phosphorylation of ERK1/2, JNK and p38 MAPK. However, the phosphorylation of Jak2 and p85 PI3K was reduced by Ft1. Inhibitors of p38 MAPK and ERK1/2 but not JNK abrogated the up-regulated protein expressions of cleaved caspase 3, p21 and down-regulated protein expression of Bcl-2 as well as elevated caspase 3 activity induced by Ft1.

Significance

Ft1 arrested the proliferation and elicited the apoptosis of SH-SY5Y cells possibly via p38 MAPK and ERK1/2 pathways, which indicates the potential therapeutic effect of it on human neuroblastoma.     

三七皂苷R1 对肝纤维化大鼠TGF-beta1/Smad3信号的影响

目的:探讨SD大鼠肝纤维化后肝组织及血清中转化生长因子-β1(Transforming Growth Factor-β1,TGF-β1)及Smad3的表达和变化,以及三七皂苷R1对肝纤维化的保护作用。方法:72只健康雄性SD大鼠分为对照组、二甲基亚硝胺(NDMA)组和三七皂苷R1组,再按不同时间点分为1、2、4周,3个亚组,每个亚组8只动物。NDMA组采用NDMA 2 m L/kg腹腔注射,三七皂苷R1组同时静脉注射三七皂苷R1,剂量为100 mg/kg体重,对照组注射等量的生理盐水。在各组的不同时间点采用RT-PCR及ELISA技术检测肝组织及血清中TGF-β1、Smad3的表达及变化。结果:1、TGF-β1、Smad3 m RNA及蛋白在各组中均有表达。2、对照组各时间点比较均无统计学意义(P>0.05)。NDMA组中,随着损伤时间的延长,TGF-β1、Smad3 m RNA及蛋白的表达逐渐上调,且各时间点与对照组比较有统计学意义(P<0.05)。而三七皂苷R1组TGF-β1、Smad3 m RNA及蛋白在各时间点均较NDMA组表达下调,有统计学意义(P<0.05)。结论:1、TGF-β1/Smad3信号参与了肝纤维化的发生和发展过程,且随损伤的逐渐加重,表达越高。2、三七皂苷R1可降低肝组织中TGF-β1/Smad3信号的表达,减轻肝细胞的纤维化,发挥保护肝组织损伤的作用。     

三七根的微量成分（1）

从三七根中分到１个新的微量皂甙,命名为三七皂甙Ｒ７,其结构经光谱分析和化学方法证明为人参二醇－３－Ｏ－β－Ｄ－葡萄吡喃糖甙;此外,还分到１个已知的炔烯类化合物,人能炔三醇。