β-Glycosidase-assisted bioconversion of ginsenosides in purified crude saponin and extracts from red ginseng (Panax ginseng C. A. Meyer)

Major ginsenosides in ginseng (Panax ginseng) and its products are highly glycosylated, hence poorly absorbed in the gastrointestinal tract. β-Glycosidase-assisted deglycosylation of pure ginsenosides was peformed to study bioconversion mechanisms. Ginsenoside standard compounds, crude saponin, and red ginseng extracts were incubated with β-glycosidase (0.05% w/v, 55°C). β-Glycosidase has a broad specificity for β-glycosidic bonds, hydrolyzing the β-(1→6), α-(1→6), and α-(1→2) glycosidic linkages. The final metabolite of protopanaxadiol ginsenosides was Rg3 while the metabolite of protopanaxatriol ginsenosides was Rh1. β-Glycosidase treatment of red ginseng extracts resulted in a decrease in the amounts of Rb1, Rc, Re, and Rg2 after 24 h, whereas levels of the less glycosylated Rd, Rb1, Rg, Rg3, Rg1, and Rh1 forms increased. When crude saponin was incubated with β-glycosidase for 24 h, levels of Rb1, Rc, Re, and Rg1 decreased while levels of Rd, Rg3, and Rh1 increased as deglycosylated ginsenosides.     

Changes in ginsenosides and antioxidant activity of Korean ginseng (Panax ginseng C.A. Meyer) with Heating Temperature and Pressure

This study was carried out to investigate the changes of ginsenoside compositions and antioxidant activity of fresh ginseng induced by thermal processing at different temperatures (25, 100, 121, and 150°C), pressure (0.1, 10, 20, and 30 MPa), and soaking solvents (water and ethanol). The levels of ginsenosides were similar trend with the pressure of 0.1–30 MPa, while there were significantly differences in heated ginseng with heating temperature and soaking solvent. When water and ethanol was used, the ginsenoside compositions significantly changed at 100 and 121°C, respectively, and it was rapidly decreased at 150°C. After heating, the level of 3 ginsenosides (Re, Rf, and Rg1) decreased and that of 5 other ginsenosides [Rb1, Rb2, Rb3, Rc, and Rg2(S)] increased up to 121°C compare to raw ginseng. Ginsenoside F2, F4, Rg2(R), Rk3, Rh4, Rg3(S), Rg3(R), Rk1, and Rg5, which was absent in raw ginseng, was detected in heated ginseng. Especially, ginsenoside Rg3(S), Rg3(R), Rk1, and Rg5 were remarkably produced after thermal processing. After heating, the phenolic compounds (1.43–11.62 mg/g), 50% inhibition concentration (IC₅₀) value (1.48–3.11 mg/g), and ABTS radical scavenging activity (0.66–9.09 mg AA eq/g) of heated ginseng were increased.     

Ginseng fermented by mycotoxin non-producing <Emphasis Type="Italic">Aspergillus niger</Emphasis>: ginsenoside analysis and anti-proliferative effects

Korean ginseng was fermented using Aspergillus niger (A. niger) FMB S494 and mycotoxins such as ochratoxin and fumonisin were not detected in the fermented ginseng. Protopanaxadiol-type ginsenosides such as glycosylated forms of Rb1, Rb2, Rc, and Rd decreased to 0 while compound K (cK) increased from 0 to 9 × 10⁴ ppm in the extract of fermented ginseng. Protopanaxtriol-type ginsenosides such as Re and Rg1 decreased from 7.1 × 10⁴ to 3.0 × 10⁴ ppm and 6.8 × 10⁴ to 4.6 × 10⁴ ppm, respectively. Rg2 and Rh1 increased from 0 to 1.9 × 10⁴ ppm and 0 to 2.7 × 10⁴ ppm, respectively. We can demonstrate that A. niger was more inclined to transform protopanaxadiol-type ginsenosides. Moreover, fermented ginseng extract showed a dramatically enhanced anti-proliferative effect on human HT-29 cell line with a minimum effective concentration of about 1 µg/mL, which might be attributed to the high degree of biotransformation of ginsenosides, especially the high output of ginsenoside cK.     

Use of Lactobacillus rossiae DC05 for bioconversion of the major ginsenosides Rb1 and Re into the pharmacologically active ginsenosides C-K and Rg2

Rb1 and Re are the major ginsenosides in protopanaxadiol and protopanaxatriol with contents of 38.89 and 13.34%, respectively. β-Glucosidase-producing food grade Lactobacillus rossiae DC05 was isolated from kimchi using esculin-MRS agar and an enzyme of L. rossiae DC05 was used for bioconversion of the major ginsenosides Rb1 and Re. Strain DC05 showed strong activity in converting ginsenosides Rb1 and Re into the minor ginsenosides compound-K and Rg2, respectively. Within 4 days, 100% of ginsenoside Rb1 was decomposed and converted into C-K, while 85% of Re was decomposed and converted into Rg2 after 6 days of incubation. The biosynthesis rate of ginsenoside C-K was 72.88%, and the biosynthesis rate of Rg2 was 53.94%. Strain DC05 hydrolyzed ginsenosides Rb1 and Re along the pathway Rb1→Rd→F2→CK and the pathway Re→Rg2, respectively. The optimum temperature and pH of the enzyme were 30°C and 7.0, respectively.     

高效液相色谱法同时测定人参制剂中20 种人参皂苷方法的建立

为了更加有效评价人参制剂生产质量,建立了一种同时测定人参制剂中20 种人参皂苷的高效液相色谱方法。结果表明,20 种人参皂苷Rg1、Re、Rg2、Rg3、Rg5、Rf、F1、F2、Rc、Rd、Rb1、Rb2、Rb3、Rh2、compound K、20（R）-Rh1、Rk3、Rh4、原人参二醇及原人参三醇均得到良好分离,线性关系良好（R≥0.999 2）。该方法快捷简便、稳定可靠,能够精确全面检测分析人参皂苷含量,对于人参加工品及其制剂的质量控制更为全面准确可行。     

Biotransformation of ginsenoside Rd in the ginseng extraction residue by fermentation with lingzhi (Ganoderma lucidum)

Ginseng and lingzhi (Ganoderma lucidum) both are valuable traditional Chinese medicines and have been extensively utilised in functional foods and traditional medicines in many Asian countries. However, massive quantity of ginseng residue is produced after extraction of ginseng which still contains a lot of bioactive compounds such as ginsenosides. The goal of this study was to reuse the American ginseng extraction residue as the fermentation medium of G. lucidum to produce bioactive ginsenoside enriched biotransformation products. The changes of ginsenosides in the fermentation products were analysed during fermentation. Our results showed that after 30 days of fermentation, ginsenoside Rg1, Rd, and compound K (CK) significantly increased, especially Rd, while other ginsenosides (Re, Rb1 and Rc) decreased during fermentation. Ginsenoside Rd is the major ginsenoside in the final fermentation product. Furthermore, the biotransformation of ginsenosides was the major reaction in this fermentation process.     

蒸参水中人参皂苷的鉴别及测定

以红参加工副产物“蒸参水”为原料,分别采用薄层色谱和紫外-可见分光光度法对人参皂苷进行定性及定量检测,并对紫外-可见分光光度法进行方法学考察.结果表明,蒸参水中含有人参皂苷Re,Rgl,人参总皂苷(以Re计)在0.035mg～0.350mg间线性关系良好,总皂苷含量为186.08mg/100g.     

Retention of Ginsenosides in Dried Ginseng Root: Comparison of Drying Methods

North American ginseng (Panax quinquefolius) has a long history of use and is currently a commercially reliable natural health commodity. Ginsenosides or triterpene saponins are generally regarded as bioactive constituents for several observed health effects associated with ginseng. North American ginseng was dried using 3 different drying techniques to assess the ginsenoside content of prepared extracts. Drying methods included freeze‐drying (FD), air‐drying (AD), and vacuum microwave‐drying (VMD) of ginseng root. High‐performance liquid chromatography (HPLC) analysis showed that FD ginseng processing gave greater (P≥ 0.05) amounts of the fingerprint ginsenosides Rg1 (28 ± 0.9 mg/g, dry weight) and Re (45 ± 0.1) compared with AD (Rg1 19 ± 0.7, Re 29 ± 0.1) and VMD (Rg1 22 ± 0.8, Re 24 ± 0.1); whereas, VMD produced greater amounts of Rb1 (83 ± 0.1) and Rd (13 ± 0.0) than FD (Rb1 62 ± 0.1, Rd 9 ± 0.1) and AD (Rb1 69 ± 0.1, Rd 5 ± 0.0), respectively. Total ginsenoside content was similar for FD and VMD and was the lowest (P≥ 0.05) for AD. Electrospray mass spectrometry (ESI‐MS) analysis showed a total of 12 compounds detected in FD ginseng compared with 10 compounds in ginseng dried by both VMD and AD. Our results support the fact that FD and VMD drying methods of North American ginseng can improve both extraction efficiency and actual retention of individual ginsenoside in root material.     

Physicochemical and in vitro digestion characteristics of size-different red ginseng powders

The aims of the present study were to prepare different-sized red ginseng powders and investigate the particle size effect on the release property of ginsenosides in in vitro digestion conditions. Ultrafine powder showed bimodal particle size distribution with a large peak at around 100 μm and small peak at around 10 μm, differently from fine powder showing unimodal distribution at 100 μm. The specific surface areas of fine- and ultrafine powders were 48.72 ± 6.41 and 86.74 ± 5.96 m²/g, respectively. Time-dependent release property of the powders in the simulated gastrointestinal fluids was determined by quantifying ginsenoside Rg1 released. The initial and final concentrations of ginsenoside Rg1 released was higher in ultrafine powder than fine one. It is expected that particle size reduction and corresponding increase in the specific surface area have a potential to improve the release of ginsenosides in the gastrointestinal tract and enhance the chances to be absorbed in human body.     

Ultra high pressure extraction (UHPE) of ginsenosides from Korean Panax ginseng powder

To elucidate the potential of ultra high pressure (UHP) processing on ginseng, effect of UHP on extraction yield, crude saponin content, and ginsenoside contents of ginseng powder was investigated. Ginseng slurries (70, 80, and 90% moisture content) were put into a retortable pouch then hermetically sealed. These mixtures were pressurized at room temperature up to 600 MPa for 5–15 min. UHP ginseng showed relatively higher extraction yield (312.2–387.1 mg) and amounts of crude saponins (19.3–32.6 mg/g ginseng) than control ginseng (189.9 and 17.5 mg/g ginseng, respectively). Correlation coefficient between extraction yield and crude saponin content was relatively low (R²=0.2908). In high performance liquid chromatography (HPLC) analysis, amounts of measured total ginsenosides (Rb1, Rb2, Rc, Rd, Re, and Rg1) increased with UHP processing but pressure level and pressing time did not proportionally influence the ginsenosides content. This work shows a potential of UHP processing on extraction of ginseng powder and provides basic information on UHP extraction of ginseng powder.     

微生物转化人参皂苷Re为人参皂苷Rh1的研究

人参皂苷Re是人参的主要药理活性成分,具有抑制癌细胞增长、阻止癌细胞转移及保护神经等重要生理作用,稀有人参皂苷Rh1在抗癌方面的疗效更为显著。为获得较高含量的Rh1,微生物转化是目前较为有效途径。该研究从人参种植土中筛选可转化常量人参皂苷Re为稀有人参皂苷Rh1的目的菌株S329,以西洋参提取物为反应底物进行微生物发酵实验,利用高效液相色谱（HPLC）法对人参皂苷Re及其发酵产物进行分析。结果表明,通过对菌株形态学观察和18S rDNA测序分析,且通过在NCBI数据库上的Blast比对分析,鉴定高效菌株S329属于溜曲霉（Aspergillus tamarii）,人参皂苷Re转化人参皂苷Rh1的转化率为27.65%。     

人参果浆中人参皂苷Re的提取和生物转化

为了充分利用人参资源,本文以加工厂废弃的人参果浆为原料,分析人参果浆中的皂苷含量和组成,并从中提取人参皂苷Re,以人参皂苷Re为底物,采用人参自身酶为催化剂,生物转化得到人参皂苷Rg2组。结果表明,人参加工厂废弃果浆的干品中,皂苷含量为6.21%(W/W),其中人参皂苷Re的含量为55.1%(W/W)。从果浆的干品中提取纯化得到了人参皂苷Re,得率为2.4%(W/W)。人参皂苷Re生物转化制备得到人参皂苷Rg2组,得率为65%(W/W)。经高效液相色谱(HPLC)及超高效液相-四级杆飞行时间串联质谱(UPLC-Q-TOF-MS)分析得出,人参皂苷Rg2组由20(S)-Rg2、20(R)-Rg2、Rg4和Rg6组成,本论文为人参加工厂废弃果浆的综合利用提供了理论依据。     

人参-虫草双向固体发酵过程中人参皂苷含量变化研究

研究人参-冬虫夏草双向固体发酵过程中主要人参皂苷的组成和含量变化,为发酵产物的质量控制和开发利用提供理论依据。采用高效液相色谱-质谱联用（HPLC-MS）技术对发酵过程及产物中人参皂苷进行定性定量分析。结果表明,人参-虫草双向固体发酵产物中共鉴定出11种皂苷。其在200～1 500 ng/mL范围内线性关系良好（R2＞0.999）,加标回收率范围为95.13%～105.04%,相对标准偏差（RSD）为0.85%～2.36%,精密度、稳定性、重复性试验结果的RSD均＜2.6%,表明该检测方法精密度、准确度、稳定性及重复性良好。人参-虫草双向固体发酵过程中人参皂苷Rg1、Re、Rb1含量明显降低,人参皂苷Rh1、Rg2、Rd、Rg3、F2含量明显增加。     

超高效液相色谱-四极杆静电场轨道阱高分辨质谱法测定米炒人参炮制前后皂苷成分及其裂解规律的研究

目的 建立超高效液相色谱-四极杆静电场轨道阱高分辨质谱法（ultra performance liquid chromatography-quadrupole-orbitrap-mass spectrometry, UPLC-Q-Orbitrap-MS）检测分析方法。方法 采用Supelco C18色谱柱,以乙腈-0.1%甲酸水溶液梯度洗脱,应用电喷雾离子源（ESI electro-spray ionization）,负离子全扫描模式采集一、二级质谱数据,扫描范围为150~2000 m/z。结合质谱数据库及相关文献信息,运用X Calibur2.2软件对米炒人参中皂苷类成分进行鉴定。以6种人参皂苷Re、Rg1、Rb1、Rc、Rb2、Rb3进行模拟炮制,确定皂苷类成分裂解产物,明确皂苷成分的裂解规律。结果 从人参中检测出14个成分,鉴定出13种人参皂苷成分;米炒人参中检测出23个成分,鉴定出20种人参皂苷成分。通过比较人参米炒前后的皂苷类成分,发现米炒人参中存在人参中未检测到的8种稀有人参皂苷20(S)-Rg2、20(S)-Rh1、20(R)-Rh1、F2、20(S)-Rg3、20(R)-Rg3、20(S)-Rs3、20(R)-Rs3。模拟炮制结果表明,人参皂苷Re脱去C-20糖基,转化为稀有人参皂苷20(S)-Rg2;人参皂苷Rg1脱去C-20位糖基,转化为稀有人参皂苷20(S)-Rh1、20(R)-Rh1;人参皂苷Rb1、Rb2、Rb3、Rc脱去C-20或C-3位糖基,转化为稀有人参皂苷20(S)-Rg3、20(R)-Rg3或F2。结论 人参经米炒后,稀有人参皂苷成分增加,产生的稀有皂苷为原型皂苷发生苷键裂解而获得,模拟炮制可作为其裂解规律研究的有效方法。     

Metabolite profiling of ginsenoside Re in rat urine and faeces after oral administration

Following oral administration of ginsenoside Re, the compound and its metabolites were identified and quantified in rat urine and faeces by liquid chromatography coupled with triple quadrupole mass spectrometry (LC–MS/MS). Ginsenoside Re (200 mg/kg) was orally administered to rats by gastric intubation, and urine and faeces samples were then collected during the next 24 h using metabolic cages. Samples were prepared by solid phase extraction and analysed by LC–MS/MS. The precursor-product ion pairs used for LC–MS/MS analysis were: m/z 945 → 475 for ginsenoside Re, 799 → 637 for ginsenoside Rg1, 783 → 475 for ginsenoside Rg2, 637 → 475 for ginsenosides Rh1 and F1, 475 → 391 for protopanaxatriol, and 779 → 641 for digoxin (internal standard). The major ginsenosides excreted in urine were ginsenosides Re and Rg1, and only minimal amounts of ginsenosides Rg2 and Rh1 were found. Greater amounts of ginsenoside metabolites were detected in the faeces samples; biotransformation to ginsenoside Rg1 was predominant but further deglycosylated metabolites including ginsenoside F1 and protopanaxatriol were additionally detected. The total recovery of ginsenosides over 24 h was approximately 46%.     

蛹虫草产β-葡萄糖苷酶与α-L-阿拉伯呋喃糖苷酶活性分析及转化人参皂苷Rg1与Rc应用

通过鉴定筛选得到1株高产β-葡萄糖苷酶的长白山虫草属真菌蛹虫草。研究碳源、氮源及pH值对菌株产β-葡萄糖苷酶、α-L-阿拉伯呋喃糖苷酶、虫草酸与生物量的影响规律,从而获得高产生物活性物质的培养条件,并对蛹虫草转化人参皂苷Rg1与Rc的路径与转化率进行了研究。采用紫外检测法测定β-葡萄糖苷酶与α-L-阿拉伯呋喃糖苷酶活力,超高效液相色谱-四极杆飞行时间质谱法鉴定转化产物中人参皂苷的组成,利用高效液相色谱法测定虫草素含量及人参皂苷的转化率。结果表明：蛹虫草在碳源为纤维二糖、氮源为牛肉膏、pH 8、培养120 h条件下有较高β-葡萄糖苷酶活力（（74.70±0.09）U/mL）。在碳源为乳糖、氮源为蛋白胨、pH 4、培养72 h条件下有最高的α-L-阿拉伯呋喃糖苷酶活力（（11.55±0.01）U/mL）。蛹虫草转化人参皂苷Rg1的路径为Rg1→Rh1和Rg1→F1,转化Rc路径为Rc→Rd→Rg3→CK和Rc→CMc。经过168 h的转化,人参皂苷Rg1转化率为54.9%,Rc转化率达到83.44%。本研究为提高药食两用真菌蛹虫草生物转化人参皂苷效率提供了理论基础,也为蛹虫草和人参食品、药...     

产β-葡萄糖苷酶酵母菌的分离鉴定及其在人参皂苷Rg3转化中的应用

筛选产β-葡萄糖苷酶的微生物及其在转化人参皂苷Rg3中的应用。kefir粒发酵人参后,采用改良七叶苷琼脂培养基筛选产β-葡萄糖苷酶的微生物,并结合菌落形态以及26S rDNA D1/D2区核酸序列鉴定。结果发现1 株在七叶苷琼脂培养基上呈黑色水解斑的微生物,经分子生物学鉴定,该菌种为马克斯克鲁维酵母（Kluyveromyces marxianus）。在单因素试验基础上,应用Box-Behnken设计原理,设计人参-水质量比、发酵时间、接种量3因素3水平响应面试验,优化马克斯克鲁维酵母发酵人参,转化人参皂苷Rg3的发酵条件,建立回归模型。优化后的发酵条件为人参-水质量比1∶2.65、接种量2.94%、发酵时间3?d,该条件下人参皂苷Rg3含量为3.31?mg/g,转化率为248%。该结论为全组分人参发酵食品工业化生产提供理论数据。     

Chemical conversion of ginsenosides in puffed red ginseng

The effects of puffing process on chemical conversion of ginsenosides, extraction yields and crude saponin contents in red ginseng were investigated. To reach a maximum extraction yield, puffed red ginseng took only 8 h, while non-puffed red ginseng required at least 20 h showing extraction yields of 45.7 g solid extract/100 g sample and 44.5 g solid extract/100 g sample, respectively. Extraction yield increased slightly with increasing puffing pressure. Puffed red ginseng showed higher crude saponin contents (201.0-219.0 mg/g extract) than non-puffed one (161.7-189.0 mg/g extract). As the puffing pressure increased, minor ginsenosides (Rg3, F2, Rk1 and Rg5) increased but the contents of major ginsenosides (Rb1, Rb2, Rc, Rd, Re and Rg1) decreased. These results indicated that a puffing process may provide an effective method to reduce the extraction time, improve the extraction yield and increase the crude saponin content of red ginseng.     

高效液相色谱法测定农田人参中9种人参皂苷单体含量

为评价农田人参质量,建立同时测定农田人参中9种人参皂苷单体含量的方法。采用反相高效液相色谱法(reversed phase-high performance liquid chromatography,RP-HPLC)对农田人参与伐林人参中9种人参皂苷单体含量进行比较分析,色谱条件:色谱柱(4.6mm×150mm,5μm),乙腈(A)-水(B)为流动相,梯度洗脱[0min(18%A)→24min(22%A)→26min(26%A)→30min(32%A)→50min(33.5%A)→55min(38%A)],流速为1.0mL/min,检测波长203 nm,柱温35℃。结果表明:农田人参含有与伐林人参相同种类的9种人参皂苷Rg 1、Re、Rf、Rg2、Rb1、Rc、Rb2、Rb3、Rd;6年生农田人参9种皂苷含量均高于6年生伐林人参,但除Rg1含量差异显著外(P<0.05),其他8种皂苷含量均不显著(P>0.05);4年生农田人参除Rg1、Rf显著高于4年生伐林人参(P<0.05)外,其他7种皂苷含量与4年生伐林人参差异均不显著(P>0.05)。农田人参中Rgl、Rb1的含量、Rgl和Re含量之和、Rgl和Re含量之和均超过中国药典、欧洲药典与美国药典的要求。     

Monitoring of roasting-induced changes in ginsenoside composition of ginseng (Panax ginseng C.A. Meyer)

This study was intended to roast freeze-dried ginseng in order to determine the effect of roasting conditions on major ginsenosides by monitoring their changes using response surface methodology. As the roasting temperature and time increased, the contents of ginsenoside Re, Rg₁, Rf, Rb₁, Rc, Rb₂, and Rd tended to decrease but that of ginsenoside Rg₃ increased, reaching the maximum content (0.96 mg/g) at 189.99°C and 20.29 min as compared to the initial value (0.01 mg/g). The total ginsenoside content was estimated to be increased from 4.30 to 5.19 mg/g at 140.17°C and 27.51 min. It was found that the roasted ginseng has different ginsenoside compositions from raw ginseng. Therefore, further studies are needed to investigate the functional and biological properties of roasted ginseng as compared to the conventional white and red ginseng.