Ultrasound‐assisted extraction of ginsenosides in supercritical CO2 reverse microemulsions

Ultrasound‐assisted extraction of ginsenosides from ginseng in supercritical CO₂ reverse microemulsions formed by bis(2‐ethylhexyl) sodium sulfosuccinate (AOT) was studied. Prior to extraction the ginseng was soaked in water for 12 h. It was found that ultrasound significantly enhanced supercritical CO₂ reverse microemulsion extraction. The ginsenoside extraction yield from supercritical CO₂ reverse microemulsion with ultrasound of 20 kHz, 15.2 W cm^?2 and 3/6 s was 2.63 times that without ultrasound at 24 MPa extraction pressure, 45 °C extraction temperature, 4 h extraction time, 5 MPa separation pressure, 55 °C separation temperature and 2 L h^?1 CO₂ flow rate with 140 mL of 0.07 mol L^?1 AOT/ethanol. The maximum extraction yields of ginsenosides from different concentrations of reverse microemulsions were obtained at different ultrasonic intensities. The extraction yield with 20 kHz ultrasound was higher than that with 38 kHz ultrasound at suitably low intensity; however, it was lower at high intensity. The yield improvement may be basically attributed to the mechanical and thermal effects of ultrasound. Copyright © 2006 Society of Chemical Industry     

三七原料药材与注射用血栓通（冻干）中皂苷类成分对比分析及转移规律研究

本研究采用高效液相色谱（HPLC）法和高效液相色谱�串联四极杆飞行时间质谱（HPLC-Q-TOF MS）法研究三七原料药与注射用血栓通（冻干）中的皂苷类成分。通过对比三七原料药材与注射用血栓通（冻干）中的共有峰,以及各皂苷类成分的峰面积比例,探讨由三七原料药材到注射用血栓通（冻干）过程中各皂苷类成分的转化损失。采用Waters XBridge C18色谱柱（4.6 mm×250 mm×5 μm）,柱温40 ℃,流速0.35 mL/min,流动相为0.01%甲酸-水（A）-0.01%甲酸-乙腈（B）,进行梯度洗脱。其中,HPLC法采用UV检测器,检测波长203 nm;HPLC-Q-TOF MS法在ESI负离子模式下进行数据采集。在该分析条件下,各皂苷类成分分离良好,各色谱峰对称性较高。三七原料药材与注射用血栓通（冻干）中皂苷类成分对比分析结果表明,两者中均含有三七皂苷R1,人参皂苷Rg1、Re、Rg2、Rb1、Rd、F2以及七叶胆苷ⅩⅦ,其峰面积比值分别为2.55、6.88、3.02、9.75、31.30、15.68、15.98、714.34,表明各皂苷类成分由原料药材到最终制剂的转移率存在较大差别,其中转移率最高的成分为三七皂苷R1,而生产过程中损失较大的物质为七叶胆苷ⅩⅦ。     

三七叶总皂苷酶解产物抗抑郁活性的评价

本文研究了三七叶总皂苷酶解产物EP-3的抗抑郁药效。采用强迫游泳、悬尾、获得性无助、利血平拮抗等行为模型以及甩头实验、奔跑实验、打斗实验进行评价。强迫游泳实验,EP-3可以使强迫游泳小鼠的不动时间缩短43.39~52.00%,具有明显的抗抑郁活性。获得性无助实验,EP-3能显著减少获得性无助小鼠的逃避失败次数,测定的第2 d EP-3给药组逃避失败次数减少了16.56~29.96%,测定的第3 d减少了20.06~35.28%,第4 d为29.63~62.82%,呈现出随给药时间延长,作用增强的特点。EP-3可以明显逆转利血平诱导的小鼠肛温下降和拮抗小鼠眼睑下垂,一定程度上缓解小鼠运动不能症状。此外,EP-3显著增加5-HTP诱导的小鼠甩头次数,减少可乐定诱导的小鼠打斗行为,提高L-Dopa诱导的小鼠奔跑速度。但是EP-3对悬尾实验小鼠的不动时间没有影响（P>0.05）。EP-3具有良好的抗抑郁作用,其作用机制可能与5-HT、NE和DA等单胺能神经递质或其受体有关。     

全自动固相萃取-紫外可见分光光度法测定保健食品中总皂苷含量

目的 应用全自动固相萃取-紫外可见分光光度法测定保健食品总皂苷的含量。方法 采用固相萃取柱填料为Amberlite-XAD-2大孔树脂和中性氧化铝的Sepline全自动固相萃取装置, 对保健食品中的总皂苷富集纯化, 通过70%乙醇回收, 以香草醛-高氯酸为显色剂进行显色, 采用紫外分光光度计(λ＝560 nm)进行测定。结果 总皂苷在0.038~0.268 mg的浓度范围内线性关系良好(r=0.9997), 在不同类型保健食品中的平均加标回收率均在98.3%~102.4%, 相对标准偏差为03%~5.4% (n=6)。结论 本方法简便、准确、灵敏度高、重复性好, 可用于不同类型保健食品中总皂苷的含量测定, 对产品进行质量控制。     

同时测定不同人参加工产品中20 种人参皂苷的UPLC-PDA方法开发和验证

建立一种简单、有效、精密和准确的超高效液相色谱方法评价不同人参加工产品的质量,同时快速测定20 种人参皂苷Rg1、Re、Rf、20(S)-Rg2、20(R)-Rg2、Rb1、Rc、Ra1、Rb2、Rb3、Rd、Rk3、F2、20(S)-Rg3、20(R)-Rg3、Compound K（CK）、Rg5、20(S)-Rh2、20(R)-Rh2和protopanaxadiol（PPD）。采用二极管阵列检测器和ACQUITY UPLC BEH-C18（2.1 mm×50 mm,1.7 μm）色谱柱,以乙腈-水为流动相,流速0.3 mL/min,柱温30 ℃,梯度洗脱。20 种人参皂苷在31 min内可达到良好的分离,考察方法的线性范围、回收率、日内和日间精密度。在本方法条件下,线性关系良好,相关系数R2均大于0.998,日内相对标准偏差不大于4.65%,日间相对标准偏差不大于4.88%,回收率为85.71%～108.50%。方法检出限为0.81～3.10 μg/mL,方法定量限为2.88～10.00 μg/mL。本方法快速、可靠,已成功用于不同人参加工产品包括保鲜参、红参和白参中20 种人参皂苷的分析检测,有效揭示不同人参加工产品中人参皂苷含量水平的显著变化,可用于鲜人参及其加工产品中活性化合物的分析和质量控制。     

Cu~(2+)对多粘类芽孢杆菌增殖及其转化人参皂苷的影响

目的:研究Cu~(2+)对多粘类芽孢杆菌增殖及转化人参皂苷的影响。方法:采用酶标仪和高效液相色谱仪分别测定加入Cu~(2+)后多粘类芽孢杆菌的增殖及转化人参皂苷情况。结果:Cu~(2+)促进多粘类芽孢杆菌增殖适宜质量浓度为10~50 mg/L,Cu~(2+)质量浓度为30 mg/L时促进多粘类芽孢杆菌增殖效果最好,增殖时间最短,与对照组相比提前1 d,生物量显著增加;转化体系中Cu~(2+)质量浓度为10~50 mg/L时,促进多粘类芽孢杆菌转化人参皂苷,Cu~(2+)质量浓度为30 mg/L时,多粘类芽孢杆菌转化人参皂苷能力最强;但Cu~(2+)质量浓度超过100 mg/L时反而抑制多粘类芽孢杆菌转化人参皂苷,并且随Cu~(2+)质量浓度增大而抑制作用增强。结论:Cu~(2+)质量浓度为30 mg/L时,可促进多粘类芽孢杆菌增殖及其转化人参皂苷。     

CRISPRi-Guided Metabolic Flux Engineering for Enhanced Protopanaxadiol Production in Saccharomyces cerevisiae

Protopanaxadiol (PPD), an aglycon found in several dammarene-type ginsenosides, has high potency as a pharmaceutical. Nevertheless, application of these ginsenosides has been limited because of the high production cost due to the rare content of PPD in Panax ginseng and a long cultivation time (4–6 years). For the biological mass production of the PPD, de novo biosynthetic pathways for PPD were introduced in Saccharomyces cerevisiae and the metabolic flux toward the target molecule was restructured to avoid competition for carbon sources between native metabolic pathways and de novo biosynthetic pathways producing PPD in S. cerevisiae. Here, we report a CRISPRi (clustered regularly interspaced short palindromic repeats interference)-based customized metabolic flux system which downregulates the lanosterol (a competing metabolite of dammarenediol-II (DD-II)) synthase in S. cerevisiae. With the CRISPRi-mediated suppression of lanosterol synthase and diversion of lanosterol to DD-II and PPD in S. cerevisiae, we increased PPD production 14.4-fold in shake-flask fermentation and 5.7-fold in a long-term batch-fed fermentation.     

高效液相色谱-电喷雾电离质谱法分析人参皂甙

利用反相高效液相色谱-电喷雾电离质谱(HPLC-ESI/MS)方法,分离分析了人参皂甙。液相色谱采用乙腈-水流动相进行梯度洗脱,质谱法利用电喷雾电离质谱(ESI/MS)研究了3种人参皂甙的一级质谱(主要给出分子量)和二级、三级质谱(提供碎片离子信息),并通过质谱图的差异对其结构进行了鉴别,建立了高效液相色谱-质谱联用法分析人参皂甙的方法。     

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.     

不同林型、产地、参龄及坡向对林下参20 种单体皂苷含量的影响

采用同时测定20?种人参皂苷高效液相色谱法评价不同林型、产地、参龄及坡向对林下参皂苷含量的影响。结果表明,不同林型、产地、参龄及坡向林下参均含有14?种人参皂苷单体;与其他3?种林型比较,樟子松林型下栽培的林下参单体皂苷Rg1、Re、Rf、Rb1、Rb2、Rb3、Rg5含量与20?种单体皂苷加和值最高;与其他5?个产地相比,抚松县露水河镇林下参单体皂苷Rg1、Rf含量最高;3?种不同年生林下参比较,15?a生林下参单体皂苷Rg1、Rf、Rb1含量最高,5?a生林下参单体皂苷Compound?K、原人参二醇含量最高;3?种不同坡向相比,阳坡林下参单体皂苷Rb2、Rb3含量最高,阴坡林下参单体皂苷Rh2含量最高。不同林型、产地、参龄及坡向林下参皂苷种类相同但含量不同,5～15?a生林下参均达到2015年《中国药典》要求。