Biotransformation of Saponins by Endophytes Isolated from Panax notoginseng

The biotransformation of the major saponins in Panax notoginseng, including the ginsenosides Rg1, Rh1, Rb1, and Re, by endophytes isolated from P. notoginseng was studied. One hundred and thirty‐six endophytes were isolated and screened for their biotransformational abilities. The results showed that five of the tested endophytes were able to transform these saponins. These five strains were identified based on their ITS or 16S rDNA sequences, which revealed that they belonged to the genera Fusarium, Nodulisporium, Brevundimonas, and Bacillus genera. Ten transformed products were isolated and identified, including a new compound 6‐O‐[α‐L ‐rhamnopyranosyl‐(1→2)‐β‐D ‐glucopyranosyl]‐20‐O‐β‐D ‐glucopyranosyldammarane‐3,6,12,20,24,25‐hexaol ( 3 ), and nine known compounds, compound K ( 1 ), ginsenoside F2 ( 2 ), vinaginsenoside R13 ( 4 ), vinaginsenoside R22 ( 5 ), pseudo‐ginsenoside RT4 ( 6 ), (20S)‐protopanaxatriol ( 7 ), ginsenoside Rg1 ( 8 ), vinaginsenoside R15 ( 9 ), and (20S)‐3‐O‐β‐D ‐glucopyranosyl‐6‐O‐β‐D ‐glucopyranosylprotopanaxatriol ( 10 ). This is the first study on the biotransformation of chemical components in P. notoginseng by endophytes isolated from the same plant.     

Relationship between anti-fibrotic effect of Panax notoginseng saponins and serum cytokines in rat hepatic fibrosis

The aim of this study was to investigate the relationship between anti-fibrotic effect of Panax notoginseng saponins (PNS) and serum cytokines in rat hepatic fibrosis. Hepatic fibrosis induced by carbon tetrachloride (CCl₄) was studied in animal models using SD rats. Liver index, serum alanine amino transferase (ALT), aspartate amino transferase (AST), transforming growth factor-β1 (TGF-β1), tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10) were measured, respectively. Liver index and the degree of liver fibrosis were also determined. Our results showed that the levels of ALT, AST and liver index in PNS-treated group were markedly lower than those in model group. PNS therapy also significantly attenuated the degree of hepatic fibrosis, collagen area and collagen area percent in liver tissue. Furthermore, the levels of serum TGF-β1, TNF-α and IL-6 were strikingly reduced in PNS-treated group compared with model group while the production of IL-10 was up-regulated. These findings demonstrate that PNS has certain therapeutic effects on hepatic fibrosis probably by immunoregulating the imbalance between pro-fibrotic and anti-fibrotic cytokines.     

Impact of external calcium and calcium sensors on ginsenoside Rb1 biosynthesis by Panax notoginseng cells

The effects of external calcium concentrations on biosynthesis of ginsenoside Rb1 and several calcium signal sensors were quantitatively investigated in suspension cultures of Panax notoginseng cells. It was observed that the synthesis of intracellular ginsenoside Rb1 in 3-day incubation was dependent on the medium Ca2+ concentration (0-13 mM). At an optimal Ca2+ concentration of 8 mM, a maximal ginsenoside Rb1 content of 1.88 +/- 0.03 mg g(-1) dry weight was reached, which was about 60% and 25% higher than that at Ca2+ concentrations of 0 and 3 mM, respectively. Ca2+ feeding experiments confirmed the Ca2+ concentration-dependent Rb1 biosynthesis. In order to understand the mechanism of the signal transduction from external Ca2+ to ginsenoside biosynthesis, the intracellular content of calcium and calmodulin (CaM), activities of calcium/calmodulin-dependent NAD kinase (CCDNK) and calcium-dependent protein kinase (CDPK), and activity of a new biosynthetic enzyme of ginsenoside Rb1, i.e., UDPG:ginsenoside Rd glucosyltransferase (UGRdGT), in the cultured cells were all analyzed. The intracellular calcium content and CCDNK activity were increased with an increase of external Ca2+ concentration within 0-13 mM. In contrast, the CaM content and activities of CDPK and UGRdGT reached their highest levels at 8 mM of initial Ca2+ concentration, which was also optimal to the ginsenoside Rb1 synthesis. A similar Ca2+ concentration-dependency of the intracellular contents of calcium and CaM and activities of CCDNK, CDPK, and UGRdGT was confirmed in Ca2+ feeding experiments. Finally, a possible model on the effect of external calcium on ginsenoside Rb1 biosynthesis via the signal transduction pathway of CaM, CDPK, and UGRdGT is proposed. Regulation of external Ca2+ concentration is considered a useful strategy for manipulating ginsenoside Rb1 biosynthesis by P. notoginseng cells.     

复合酶解法提取三七皂苷的实验研究

以三七提取液中总皂苷的含量和提取物得率为指标,考察了乙醇回流法、渗漉法、纤维素酶解法、果胶酶解法、复合酶解法的优劣,并采用单因素法和四因素（纤维素酶用量、果胶酶用量、酶解温度、乙醇浓度）三水平正交设计法对复合酶解法提取工艺条件进行优选,得到如下较理想的提取工艺条件：纤维素酶用量为15U／g（生药）、果胶酶用量为140U／g（生药）,酶解pH值为4．5,酶解温度为50℃,乙醇浓度为80％,提取时间为2．5h。所得三七提取液中总皂苷的含量为12．01％,提取物得率为35．82％。     

三七皂苷对缺氧复氧心肌细胞损伤保护作用的研究

目的：研究三七皂苷对纯化培养大鼠乳鼠心肌细胞缺氧/复氧损伤的保护作用及机制。方法：采用纯化培养的心肌细胞建立缺氧/复氧损伤模型,测定细胞凋亡率、caspase-3、乳酸脱氢酶（LDH）、丙二醛（MDA）、超氧化物歧化酶（SOD）含量。结果：与正常组比较,模型组LDH、MDA含量、caspase-3活性及细胞凋亡率明显升高（P＆lt;0.01）,SOD活性明显降低（P＆lt;0.01）;三七皂苷组降低LDH、MDA含量、caspase-3活性和细胞凋亡率,提高SOD活性,与缺氧/复氧组比较各实验指标差异均具有显著性（P＆lt;0.05）。结论：三七皂苷对缺氧/复氧心肌细胞损伤有保护作用,作用机制与清除氧自由基,抗脂质过氧化及降低细胞凋亡率有关。     

Protopanaxadiol 6-hydroxylase and its role in regulating the ginsenoside heterogeneity in Panax notoginseng cells

Various structure-similar plant secondary metabolites like ginseng saponins (ginsenosides) possess different or even totally opposite biological activities. Intentional manipulation of the ginsenoside heterogeneity in cellular biosynthesis is of great interest and significance [Zhong and Yue (2005); Adv Biochem Eng Biotechnol 100:53-88]. In this work, CO-binding spectra of microsomes prepared from the suspended cells of Panax notoginseng showed increases in absorption at 450 nm compared with the control without CO sparging, and protopanaxadiol 6-hydroxylase (P6H), a new enzyme catalyzing the conversion of ginsenoside aglycone protopanaxadiol into protopanaxatriol, was found. P6H was dependent on NADPH and molecular oxygen. The enzymatic reaction was inhibited by carbon monoxide and partially reversible upon illumination with blue light, and sensitive to cytochrome P450 inhibitors. The results supported the contention that P6H was a cytochrome P450-dependent hydroxylase, whose catalytic product was confirmed to be protopanaxatriol by HPLC-MS. Induction of P6H activity by phenobarbital, a cytochrome P450 inducer, was observed. A maximal activity of P6H was obtained with addition of 0.5 mM phenobarbital on day 4 of shake-flask cultivation. The maximum content of protopanaxatriol-type ginsenosides (Rg(1) and Re, Rg group) and the maximum ratio of the content of protopanaxatriol: protopanaxadiol reached 6.88 +/- 0.21 mg g(-1) dry weight and 7.0, respectively, which was about 1.4 and 2.0-fold that of respective controls (without addition of phenobarbital). Oxidative burst was also observed in the cell cultures with addition of phenobarbital. P6H was concluded as a key enzyme in regulating Rg-group ginsenoside biosynthesis in P. notoginseng cells.     

基于UPLC法测定离体大鼠及人源肠道菌对三七中人参皂苷代谢的差异

为探究人与大鼠肠道菌群对三七水煎液中三醇型人参皂苷Rg1、Re及二醇型人参皂苷Rb1、Rd体外代谢的差异性及发现其代谢产物原人参二醇PPD与原人参三醇PPT,实验利用UPLC方法测定三七水煎液分别与人、大鼠肠道菌群在厌氧条件下共培养24h后的孵育液中4种皂苷的含量及代谢产物PPD与PPT的含量。结果表明三七中含有三醇型人参皂苷Rg19.4500mg/g、Re1.8872mg/g,二醇型人参皂苷Rb18.5816mg/g、Rd1.9456mg/g。与人源肠道菌共培养后,三七中含有的二醇型、三醇型人参皂苷含量显著降低,重要的是,在培养液中检测到代谢产物PPD和PPT的存在,含量分别为0.2136mg/g及0.0344mg/g,与大鼠肠道菌共培养后,三七中含有的二醇型皂苷含量有轻微降低,而三醇型皂苷含量未见明显变化,但有少量PPT(0.0184mg/g)的生成。由此可见:在体外条件下,三七水煎液中人参皂苷会被人肠道菌群降解生成代谢产物PPD和PPT,而大鼠肠道菌群的降解产物却仅有PPT生成,二者存在种属差异。     

Isolation of four high-purity dammarane saponins from extract of Panax notoginseng by centrifugal partition chromatography coupled with evaporative light scattering detection in one operation

Introduction – Centrifugal partition chromatography (CPC), as a continuous liquid–liquid partition chromatography with no solid support matrix, combined with evaporative light scattering detection (ELSD) was employed for systematic separation and purification of weak‐chromophoric saponins from a highly valued and important traditional Chinese herbal medicine, Panax notoginseng. Objective – To separate and isolate high‐purity saponins from extract of Panax notoginseng using CPC‐ELSD with a simple and low toxicity solvent system. Methodology – Samples were preparaed by extracting the root material with acetone, treated with n‐butanol and then freeze‐dried. CPC‐ELSD was applied in the separation and detection of notoginsenoside and ginsenosides from extract of Panax notoginseng using a solvent system composed of ethyl acetate–n‐butanol–water (1:1:2, v/v/v). The saponins were analysed and identified by their retention time with high‐performance liquid chromatography (HPLC) coupled with ELSD, as well as electrospray ionisation tandem mass spectrometry (ESI‐MSⁿ ) in the negative and positive ion modes with the authentic standards. Results – A total of 9.6 mg of notoginsenoside R₁, 67.8 mg of ginsenoside Rg₁, 2.3 mg of Re and 286.5 mg of Rb₁ were purified from 487.2 mg of n‐butanol extract of P. notoginseng. The purities of obtained saponins in a single run were assessed to be over 98% by HPLC‐ELSD. Conclusion – CPC‐ELSD was proved to be a very fast and efficient tool for separation of high‐purity dammarane saponins. Copyright © 2011 John Wiley & Sons, Ltd.     

三七总皂苷原粉和超微粉的理化性质比较研究

通过对药用植物三七总皂苷原粉和超微粉的粉体粒度、电位、显微结构、红外光谱、溶解速度这几项理化性质的研究,判定两种粉体的优劣,为三七总皂苷超微粉的市场推广提供依据。实验结果表明,三七总皂苷原粉经纳米化处理后成为三七总皂苷超微粉,其化学结构没有发生变化,但显微结构从条状的晶体变为由纳米球紧密排列的不规则形态,其粉体平均粒径也从1 122.4 nm缩小到153.4 nm,完成了从微米到纳米的转变,其水溶液也变为稳定的胶体溶液。运用高效液相色谱法,在模拟人体环境的条件下,发现三七总皂苷超微粉比三七总皂苷原粉早1 min完全溶解。说明三七总皂苷超微粉比三七总皂苷原粉颗粒更小,更易溶于水,更易与人体吸收。     

Fusarium sacchari对三七茎叶中有效成分生物转化条件的优化

利用从种植人参的土壤中分离、筛选的稀有菌种Fusarium sacchari,对三七茎叶中的主要有效成分三七叶苷进行生物转化,以3种抗肿瘤活性成分20(S)-原人参二醇-20-O-β-D-吡喃葡萄糖苷(C-K)、20(S)-原人参二醇-20-O-β-D-吡喃木糖苷(1→6)-β-D-吡喃葡萄糖苷(C-Mx)和20(S)-原人参二醇-20-O-α-L-呋喃阿拉伯糖基(1→6)-β-D-吡喃葡萄糖苷(G-Mc)的总生成量为考查指标,通过因子转化实验确定最佳转化条件为:培养基初始pH值6、底物加入量40 mg、装液量30 ml,30℃、160 r.min-1转化6 d.该方法可提高三七茎叶的利用率和经济效益.     

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。     

三七总皂甙和银杏叶提取物预防急性氧中毒的实验研究

目的:研究三七总皂甙和银杏叶提取物对急性氧中毒的预防作用及其可能机制.方法:分别给小鼠连续腹腔注射三七总皂甙和银杏叶提取物5 d后,在500kPa高压氧中暴露60 min,观察惊厥潜伏期、惊厥次数、惊厥间隔时间等指标;另外测定高压氧暴露15 min后脑组织中活性氧单位、脂质过氧化物、一氧化氮、谷胱甘肽的含量和过氧化氢酶、谷胱甘肽过氧化物酶、单胺氧化酶的活性.结果:三七总皂甙和银杏叶提取物可以明显延长氧惊厥潜伏期和惊厥间隔时间,减少惊厥次数;降低高压氧暴露后脑组织中脂质过氧化物、一氧化氮的含量,使活性氧单位、谷胱甘肽含量和谷胱甘肽过氧化物酶活性保持在较高的水平;对过氧化氢酶和单胺氧化酶活性的影响则不显著.结论:三七总皂甙和银杏叶提取物可以有效预防急性氧中毒,其机制可能与它们的抗氧化活性有关.     

Improved production of ginseng polysaccharide by adding conditioned medium to Panax notoginseng cell cultures

By adding 50% (v/v) filtered culture broth to fresh MS medium, the specific growth rate of Panax notoginseng was increased from 0.046 d–1 to 0.068 d–1, and the polysaccharide production and productivity reached 1.21 g l–1 and 61 mg/(ld), respectively, which were 1.3- and 2.3-fold of the control. Further supplementation of the conditioned medium with sucrose, ammonium, nitrate and phosphate gave a cell density of 13.7 g l–1 and a specific growth rate of 0.086 d–1. Polysaccharide production was 1.65 g l–1 and the productivity was 78 mg/(ld).     

Simple and efficient preparation of ginsenoside (S)-Rg2 from ginsenoside Re by biotransformation with Cellulosimicrobium sp. 21

A novel ginsenoside-hydrolyzing strain was isolated from ginseng-cultivation soil in Changbai Mountain (China). The strain was identified as Cellulosimicrobium sp. 21 by 16S rDNA sequencing. Using the β-glucosidases secreted from Cellulosimicrobium sp. 21, protopanaxatriol-type ginsenoside Re was converted to the highly active neuroprotective molecule (S)-Rg2 by removal of the C-20-glucopyranosyl residue. The α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranose at the C-6 position of Rg2 was not further attacked by Cellulosimicrobium sp. 21, so the transformation shows high specificity. To simplify the transformation and product-preparation process, a simple and efficient transformation system was developed in a phosphate buffer system instead of organic media. The optimum conditions for transforming ginsenoside Re into Rg2 by Cellulosimicrobium sp. 21 were determined through single-factor experiments and response surface methodology. Under the optimized conditions: transformation buffer, 50 mM phosphate buffer, at pH: 7.00; temperature: 27.6°C; substrate concentration: 0.50 mg/ml; biotransformation period: 12 h; the biotransformation efficiency reached 89.8% (molar ratio) in 2-L reaction system. This simple biotransformation with high specificity and efficiency has potential for use in Rg2 preparation in the pharmaceutical industry.     

Efficient elicitation of ginsenoside biosynthesis in cell cultures ofPanax notoginseng by using self-chemically-synthesized jasmonates

A series of fluorine and hydroxyl containing jasmonate derivatives, which were chemically synthesized in our institute, were investigated for their effects on the biosynthesis and heterogeneity of ginsenosides in suspension cultures ofPanax notoginseng cells. Compared to the control (without addition of elicitors), 100 μM of each of the jasmonate was added on day 4 to the suspension cultures ofP. notoginseng cells. It was observed that, jasmonates greatly enhanced the ginsenoside content and the ratio of Rb group to Rg group (i.e. (Rb₁+Rd)/(Rg₁+Re)) in theP. notoginseng cells. Some of the synthetic jasmonates, such as pentafluoropropyl jasmonate (PFPJA), 2-hydroxyethyl jasmonate (HEJA) and 2-hydroxyethoxyethyl jasmonate (HEEJA), could promote the ginsenoside content to 2.55±0.11, 3.65±0.13 and 2.94±0.06 mg/100 mg DW, respectively, compared to that of 0.64±0.06 mg/100 mg DW for the control and 2.17±0.04 mg/100 mg DW by the commercially available methyl jasmonate (MJA); and they could change the respective Rb: Rg ratio to 1.60±0.04, 1.87±0.01 and 1.56±0.05, compared to that of 0.47±0.01 for the control and 1.42±0.06 by MJA. The results suggest that suitable esterification of MJA with fluorine or hydroxyl group could increase the elicitation activity to induce plant secondary metabolism. The information obtained from this study is useful for hyper-production of heterogeneous products by plant cell cultures.     

Dammarane-type triterpene extracts of Panax notoginseng root ameliorates hyperglycemia and insulin sensitivity by enhancing glucose uptake in skeletal muscle

Skeletal muscle is an important organ for controlling the development of type 2 diabetes. We discovered Panax notoginseng roots as a candidate to improve hyperglycemia through in vitro muscle cells screening test. Saponins are considered as the active ingredients of ginseng. However, in the body, saponins are converted to dammarane-type triterpenes, which may account for the anti-hyperglycemic activity. We developed a method for producing a dammarane-type triterpene extract (DTE) from Panax notoginseng roots and investigated the extract’s potential anti-hyperglycemic activity. We found that DTE had stronger suppressive activity on blood glucose levels than the saponin extract (SE) did in KK-A^y mice. Additionally, DTE improved oral glucose tolerance, insulin sensitivity, glucose uptake, and Akt phosphorylation in skeletal muscle. These results suggest that DTE is a promising agent for controlling hyperglycemia by enhancing glucose uptake in skeletal muscle.     

Immunological adjuvant effect of ginsenoside Rh4 from the roots of Panax notoginseng on specific antibody and cellular response to ovalbumin in mice

Ginsenoside Rh(4) (1), a saponin isolated from the roots of Panax notoginseng (Burk.) F. H. Chen, was evaluated for its haemolytic activity and adjuvant potential on specific antibody and cellular response to ovalbumin (OVA) in mice. Compound 1 showed a slight haemolytic effect, its concentration inducing 50% of the maximum haemolysis (HD(50) value) being 407+/-12 microg/ml using a 0.5% suspension of red blood cells. Compound 1 significantly increased the concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and OVA-induced splenocyte proliferation in OVA-immunized mice especially at a dose of 25 microg (P<0.05, P<0.01, or P<0.001). The OVA-specific serum IgG, IgG1, and IgG2b antibody levels were also significantly enhanced by 1 at a dose of 25 microg compared to the OVA control group (P<0.05 or P<0.01). Moreover, the enhancing effect of 1 on the OVA-specific IgG2b antibody responses to OVA in mice was more significant than that of Alum (Al(OH)(3) gel; P<0.01). These results suggest that 1 could be safely used as adjuvant with low or non-haemolytic effect.