Supercritical Fluid Extraction of Saponins from Ginseng

Introduction Ginseng（ Panax ginseng C. A. Meyer, Araliaceae） is one of the most valuable Chinese crude drugs and has been used widely for over 2000 years. Studies have demonstrated that ginseng can act on the central nervous system, the cardiovascular system and the endocrine system; it can enhance immune function and metabolism; it possesses a biomodulation action, anticancer effect, anti-stress and anti-ageing activities, and so on.

Total saponins of Panax ginseng effects on proliferation and differentiation of human embryonic neural stem cells and in a Parkinson's disease mouse model

BACKGROUND： Total saponins of Panax ginseng （TSPG） exhibits neuroprotection against Parkinson＇s disease in the substantia nigra. OBJECTIVE： To investigate the effects of TSPG on human embryonic neural stem cells （NSCs） proliferation and differentiation into dopaminergic neurons using in vitro studies, and to observe NSC differentiation in a mouse model of Parkinson＇s disease, as well as behavioral changes before and after transplantation. DESIGN, TIME AND SETTING： In vitro neural cell biology trial and in vivo randomized, controlled animal trial were performed at the Institute of Basic Medical Sciences, Chongqing Medical University between September 2004 and December 2007. MATERIALS： TSPG （purity 〉 95%） was isolated, extracted, and identified by Chongqing Academy of Chinese Materia Medica. Recombinant human basic fibroblast growth factor （bFGF） and recombinant human epidermal growth factor （EGF） were purchased from PeproTech, USA. A total of 25 C57/BL6J mice, aged 18-20 weeks were included. Twenty were used to establish a Parkinson＇s disease model with i.p. injection of MPTP （1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine） and TSPG alone or combined with interleukin-1 （IL-1）-treated NSCs prior to transplantation into the corpus striatum. The remaining five mice were pretreated for 3 days with TSPG prior to MPTP injection, serving as the TSPG prevention group. METHODS： Primary NSCs were isolated, cultured and purified from embryonic cerebral cortex. Immunocytochemistry was employed to detect specific antigen expression in the NSCs. In vitro experiment： （1） to induce proliferation, NSCs were treated with TSPG, EGF＋bFGF, or TSPG＋EGF＋bFGF, respectively; （2） to induce dopaminergic neuronal differentiation, NSCs were treated with TSPG, IL-1, or TSPG＋IL-1, respectively. MAIN OUTCOME MEASURES： In vitro experiment： the effects of TSPG on NSCs proliferation were evaluated with flow cytometry and MTT assay. Tyrosine hydroxylase expression was determined by immunocytochemistry assay to observe effects of TSPG on dopaminergic neuronal differentiation. In vivo experiment： differentiation of grafted NSCs in the mouse brain was determined by immunohistochemical staining. Behavioral changes were evaluated by spontaneous activity frequency, memory function, and score of paralysis agitans. RESULTS： （1） NSCs were cultured and passaged for more than three passages. Immunocytochemistry revealed positive nestin staining, as well as neurofilament protein and glial fibrillary acidic protein. （2） TSPG significantly increased NSC proliferation, in particular when combined with EGF and bFGF, which was twice as effective as FGF or bFGF alone. TSPG also induced dopaminergic differentiation in NSCs, in particular when TSPG was added together with IL-1, resulting in an effect five times greater than that of IL-1 alone. （3） At day 30 following transplantation, most NSCs in the TSPG prevention group differentiated into dopaminergic neurons, and the scores of paralysis agitans, spontaneous activity, and memory function were significantly increased compared with TSPG alone or TSPG＋IL-1 groups （P 〈 0.05）. CONCLUSION： TSPG stimulated NSC proliferation, in particular when combined with FGF and bFGF. TSPG significantly induced dopaminergic neuronal differentiation of NSCs, and the effect was greater when combined with IL-1. In addition, TSPG greatly improved behavior in the Parkinson＇s disease mouse model following NSC transplantation. Following NSC transplantation, TSPG pretreatment exhibited superior efficacy over either TSPG alone or TSPG in combination with IL-1, in terms of behavioral improvements in the Parkinson＇s disease mouse model.

Simultaneous Determination of Saponins in Radix Glycyrrhizae, Notoginseng and Ginseng by High Performance Liquid Chromatography

To establish a method for determining five saponins(notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1 and ammonium glycyrrhizinate) in Glycyrrhizae, Notoginseng and Ginseng, the high performance liquid chromatography with diode array detector(HPLC-DAD) method was applied to an Inertsil ODS-SP column(4.6 mm×250 mm, 5 μm) with a mobile phase consisting of acetonitrile-0.05% phosphoric acid in a gradient elution manner. The flow rate was 1.0 mL/min. The column temperature was 30 ℃ and the detection wavelengths were 203 nm and 237 nm, respectively. The linear ranges were 0.700,0—7.000,0 μg for R1(r=1.000,0), 0.751,1— 7.511,4 μg for Rg1(r=1.000,0), 0.677,2—6.771,6 μg for Re(r=1.000,0), 0.733,9—7.339,1 μg for Rb1(r= 1.000,0), and 0.540,0—5.399,8 μg for ammonium glycyrrhizinate(r=0.999,9), respectively. In addition, their average recoveries were 100.28%, 105.83%, 104.09%, 99.36% and 98.54%, respectively. The relative standard deviations(RSDs) of precision, reproducibility and recovery were all less than 1.5%. The results indicate that the method is simple, accurate and reproducible so that it can be used for the simultaneous determination of the five saponins in Chinese patent medicines containing the three kinds of herbs.

Determination of Saponins in Leaf of Panax Ginseng C. A. Mey. by High Performance Liquid Chromatography

A high performance liquid chromatography（HPLC） with UV detection was established for simultaneous determination of saponins in the leaf of Panax ginseng C. A. Mey. Nine ginsenosides（Rbl, Rb2, Rb3, Rc, Rd, F1, F2, F3, F5） and notoginsenoside Fe（NFe） were studied. Among the saponins, the ginsenosides F1, F2, F3, F5 and NFe were determined by HPLC-UV method for the first time. The determination of the ginsenosides via the HPLC-UV method was performed on a reversed-phase C18 column with gradient elution in 40 min. The linearity, precision, accuracy, and detection limit for determining the saponins were studied and the samples from different areas in China were analyzed. The HPLC-ESI-MS was used to identify the saponins. The results indicate that the HPLC-UV provided a good accuracy, reproducibility and sensitivity for the determination of the ten saponins.

A NEW TRITERPENE SAPONIN FROM THE STEMS AND LEAVES OF PANAX GINSENG

a new triterpene compound was isolated from the stems and leaves of Panax ginseng C. A. Meyer and established as 3β, 6α, 12β-trihydroxy-dammar-20(21), 24-diene-6-0-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside on the basis of spectral analysis and chemical evidence.

Research progress on chemical diversity of saponins in Panax ginseng

Saponins,the major bioactive components of Panax ginseng C.A.Mey.,are gradually emerging as research hotspots owing to the possession of various pharmacological activities.This review updates the ginsenosides list from P.ginseng and the steam-processed ginseng(red ginseng and black ginseng)up to 271 by June of 2024,encompassing 243 saponins from different parts of P.ginseng(roots,stems,leaves,flowers,berries,and seeds),103 from red ginseng,and 65 from black ginseng,respectively.Among 271 saponins,there are a total of 249(1–249)dammarane type(with a–z subtypes)tetracyclic triterpene saponins reported from each part of P.ginseng and steam-processed ginseng,two(250–251)lanostane type tetracyclic triterpene saponins identified from red ginseng,18(252–269)oleanane type pentacyclic triterpenoid saponins discovered from each part of P.ginseng and steam-processed ginseng,and two(270–271)ursane type pentacyclic triterpenoid saponins reported from red ginseng.Overall,this review expounds on the chemical diversity of ginsenosides in various aspects,such as chemical structure,spatial distribution and subtype comparison,processed products,and transformation.This facilitates more indepth research on ginsenosides and contributes to the future development of ginseng.

Effects of steaming process on rare saponins and efficacy of Panax ginseng, Panax notoginseng and Panax quinquefolium

The steamed Panax ginseng C. A. Mey., Panax notoginseng(Burk.) F. H. Chen, and Panax quinquefolium L. not only facilitate storage, but also increase the content of rare saponins and enhance their clinical application value. The traditional steaming process has high energy consumption, low efficiency, and lacks standardized operating procedures and unified standards. This paper retrieves the research literature on the steaming process parameters, rare saponin increments, and efficacy enhancement results of ginseng plants. By summarizing the effects of different steaming processes on rare saponins and pharmacodynamics in P. ginseng, P. notoginseng and P. quinquefolium, we explored new steaming methods and established a new quality evaluation system to provide guarantee for the effectiveness and safety of clinical medication and provide scientific basis for its rational use.

高效液相色谱-串联四极杆质谱法测定人参组培不定根中11种皂苷成分

建立高效液相色谱-串联四极杆质谱(HPLC-MS/MS)法定量分析人参组培不定根中11种皂苷成分。样品经粉碎研磨,以70%甲醇水溶液为溶剂进行超声提取,离心过滤后测定。采用C_(18)色谱柱(100 mm×2.1 mm,1.8μm),用水和乙腈进行梯度洗脱,流量为0.3 mL/min,柱温为35℃。质谱采用电喷雾电离源,负离子扫描,多反应监测模式进行检测。11种皂苷的质量浓度在0.1~10μg/mL(Rb1为0.2~10μg/mL)范围内和响应强度线性相关,相关系数均大于0.995,各皂苷的定量限为0.001~0.010 g/kg,加标回收率为90.43%~97.82%,相对标准偏差为1.93%~6.33%(n=6)。该方法操作简单,分析时间短,灵敏度高,准确可靠,适用于人参组培不定根中多种皂苷类成分的同时测定。

人参不定根总皂苷的提取工艺优化及其抗氧化与抗疲劳作用

目的:探究人参不定根总皂苷(ginseng adventitious roots total saponins,GARS)的最佳提取工艺及抗氧化和抗疲劳作用。方法:利用乙醇回流法提取GARS,通过正交试验考察乙醇浓度、料液比、提取温度、提取时间对GARS含量的影响;以1,1-二苯基-2-三硝基苯肼(DPPH)、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS+)、3-氧代-2-苯基-4,4,5,5-四甲基咪唑啉-1-氧(PTIO)、羟自由基(·OH)、超氧阴离子自由基(O_(2)^(-)·)清除能力及还原力作为测定抗氧化活性指标;通过测定小鼠力竭游泳和爬杆时间,测定肌/肝糖原、乳酸(LD)、尿素氮(BUN)含量及乳酸脱氢酶(LDH)活性来判定GARS抗疲劳能力。结果:通过正交试验得到最佳提取工艺条件为乙醇浓度70%、料液比1:30 g/mL、提取温度70℃、提取时间40 min,得到GARS含量为107.85 mg/g;GARS对DPPH、ABTS^(+)、PTIO、OH、O_(2)^(-)自由基都有一定程度清除作用,且具有还原性,其清除能力和还原性与浓度呈正比;GARS能够显著延长小鼠力竭游泳和爬杆时间(P<0.05),显著增加肌/肝糖原含量(P<0.05),降低LD、BUN含量,提高LDH活性,并与其浓度呈正比。结论:GARS提取的最优条件为乙醇浓度70%、料液比1:30 g/mL、提取温度70℃、提取时间40 min,可为GARS产业化生产提供依据,且GARS具有一定的抗氧化和抗疲劳作用,可为明确人参不定根营养成分和活性物质提供理论支持,为开发人参不定根药食同源的相关产品提供技术参考。

林下西洋参药材质量评价研究

目的研究云南禄劝林下栽培西洋参的质量,为林下西洋参种植提供数据支撑。方法通过观察西洋参的外观性状和薄层色谱特征,测定水分、醇溶性浸出物、重金属、有机氯类农药、20种单体皂苷、总皂苷和多糖含量,多指标评价林下西洋参质量。结果林下西洋参外观性状、薄层鉴别、水分、醇溶性浸出物均符合2020年版《中国药典》规定;重金属含量远低于药典标准;22种常见有机氯类农药均未检出。人参皂苷Rg1+Re+Rb1含量为2.14%~2.75%,总皂苷含量为4.40%~5.47%,多糖含量为3.43%~5.45%。结论林下西洋参药材质量符合2020年版《中国药典》规定,值得进一步推广种植。

参玫五味粉中总皂苷与多糖的提取及其体外活性研究

试验旨在探究参玫五味粉中总皂苷与多糖联合提取的最佳工艺条件及其体外活性。试验以参玫五味粉总皂苷与多糖的提取质量分数为考察指标,通过对提取溶剂浓度和浸泡时间的优化确定最佳提取工艺,测定参玫五味粉总皂苷提取物与多糖提取物对羟基自由基的清除率及对亚硝酸盐的抑制率,探索其体外抗氧化和抗肿瘤活性。结果显示:在最佳提取工艺条件下,联合提取4根层析柱中总皂苷的提取率依次为92.65%、98.99%、102.68%、104.31%,出膏率为10.00%,干浸膏中总皂苷含量为34.99 mg/g;多糖的提取率依次为93.57%、98.20%、100.77%、101.27%,出膏率为12.55%,干浸膏中多糖含量为19.20 mg/g。参玫五味粉总皂苷与多糖提取物清除羟基自由基的IC_(50) 值均大于对照VC,表明两者具有体外抗氧化活性,且具有一定的亚硝酸盐抑制作用。研究表明,柱层析联合提取法同时提取参玫五味粉总皂苷与多糖,提取率高且操作简便易行,可为畜牧业饲料添加剂的发展提供参考价值。

人参不定根中皂苷对小鼠脂质代谢的影响研究

目的研究人参不定根皂苷对小鼠脂质代谢的影响。方法将小鼠随机分成5个实验组(共50只,每组10只),分别为正常组(生理盐水)、对照组(生理盐水)、人参不定根皂苷低(50 mg/kg)、中(100 mg/kg)、高(200 mg/kg)剂量组,连续灌胃30 d,测定小鼠血脂、肝损伤和抗氧化指标。结果与高脂对照模型组相比,人参不定根皂苷高剂量组甘油三酯、总胆固醇显著降低(P<0.05)、低密度脂蛋白胆固醇极显著降低(P<0.01);中、高剂量组谷草转氨酶和谷丙转氨酶含量极显著降低(P<0.01);中、高剂量组总超氧化物歧化酶活性极显著升高(P<0.01)、丙二醛含量极显著降低(P<0.01)。结论人参不定根皂苷能够促进高脂饲料诱导高脂血症小鼠机体代谢,降低血脂,修复肝损伤,降低过氧化损伤,增强抗氧化能力。

HPLC结合化学计量学分析研究人参和三七不同部位丙二酰基人参皂苷和中性皂苷的差异性

建立HPLC法同时测定人参和三七不同部位丙二酰基人参皂苷和中性皂苷的含量,并采用聚类分析和主成分分析的化学计量学方法分析丙二酰基人参皂苷和中性皂苷含量,以对人参和三七地上和地下药用部位进行分类。结果表明人参中性皂苷和丙二酰基人参皂苷在人参和三七不同部位中存在着显著的差异。在人参的地下部位中,丙二酰基人参皂苷的含量均略高于中性人参皂苷;在人参的地上部位,丙二酰基人参皂苷含量低于中性人参皂苷;然而丙二酰基人参皂苷不论在三七地下部位还是地上部位含量均较低。聚类分析和主成分分析结果显示,12批人参和三七样品分成4类,人参地下部位R1〜R4聚为一类,地上部位R5〜R6聚为一类;三七地下部位S1〜S4聚为一类,地上部位S5〜S6聚为一类。此方法灵敏度高,稳定可靠,可为人参和三七不同部位的准确鉴别提供科学依据。

SPE-UPLC联用快速检测保健品中人参皂苷Rh2成分

目的:基于固相萃取与超高液相色谱联用技术,建立一种快速准确检测保健品中人参皂苷Rh2成分的方法,为市售含有人参皂苷Rh2成分的保健品的质量评价提供参考。方法:样品预处理后经甲醇超声提取,中性氧化铝SPE小柱净化,筛选合适的洗脱剂进行HLB-SPE小柱分离纯化,采用WatersT3(100 mm×2.1 mm,1.8μm)色谱柱,乙腈-水为流动相梯度洗脱,PDA全波长扫描,流速0.3 mL·min^(-1)。结果:SPE-UPLC法测得人参皂苷Rh2浓度在9.7~99.8μg·mL^(-1)与其峰面积线性关系良好(r=0.999),平均回收率为99.20%。结论:该方法准确、简便、快速,并能有效去除辅料干扰,稳定性高,可用于含有人参皂苷Rh2有效成分的保健食品的质量控制。

不同光质激光对人参生长的影响

近年来,随着半导体激光器技术的不断进步,在农业领域应用的激光技术备受关注。激光光源具有单色性强、电光转换效率高、使用成本低等优势,为提高农作物质量、增加产量提供了新的技术支持。为了研究不同光质激光对人参生长、生理生化指标以及多糖、皂苷积累等方面的影响,选取二年生人参载子进行试验。从展叶期到叶片枯萎前的每日上午7:00—11:00,下午14:00—18:00分别利用全波长LED灯、红色激光灯(R3)、蓝色激光灯(B3)、混合激光灯(R2B1、R3B2)(光量子通量密度分别为4.48μmol/(m^(2)·s),4.61μmol/(m^(2)·s))补光,针对不同光质补光的影响,本研究设立了不补光作为空白对照组,研究发现,人参的净光合作用能力、叶片的气孔导度等都有所改善。特别是LED处理组的净光合速率表现最为突出,而蓝光处理组则在蒸腾速率和胞间CO_(2)浓度方面取得了最显著的效果提升。R3B2处理组气孔导度最大。就生长情况而言,人参在红蓝混合光处理下表现最佳,相比于对照组,蓝光能够显著提高植株的高度、增加叶片的长度和宽度,LED补光组根重、叶重均高于其他处理;从营养物质积累方面来看,蓝光处理下多糖含量最高,红光处理下皂苷含量积累普遍高于蓝光处理组,说明红光促进人参皂苷含量的积累,R2B1处理组Rg1、Rb1皂苷含量最高,远高于其他处理,R3B2处理时Re皂苷含量与红光处理组结果相当,高于其他4个处理。在未来的研究中,我们将选取最适宜人参生长及营养物质积累的激光补光条件,为缩短人参生长周期、提质增效而提供可行性方法。

Effects of Ginseng Fruit Saponins on Serotonin System in Sprague-Dawley Rats with Myocardial Infarction, Depression, and Myocardial Infarction Complicated with Depression

Background： Our previous studies have demonstrated that the levels of 5-hydroxytryptamine （5-HT） and 5-HT 2A receptor （5-HT2AR） in serum and platelet were associated with depression and myocardial infarction （MI）, and pretreatment with ginseng fruit saponins （GFS） before MI and depression had an effect on the 5-HT system. In this study, the effects of GFS on the 5-HT system in the Sprague-Dawley （SD） rats with MI, depression, and MI ＋ depression were evaluated. Methods： A total of eighty SD rats were allocated to four groups： MI, depression, MI ＋ depression, and control groups （n = 20 in each group）. Each group included two subgroups （n = 10 in each subgroup）： Saline treatment subgroup and GFS treatment subgroup. The levels of 5-HT, 5-HT2AR, and serotonin transporter （SERT） were quantified in serum, platelet lysate, and brain tissue through the enzyme-linked immunosorbent assay method, respectively. Results： Compared with those in the saline treatment subgroups, the levels of 5-HT in serum and platelet lysate statistically significantly increased in the GFS treatment subgroups of MI, depression, and MI ＋ depression groups （serum： all P = 0.000; platelet lysate： P = 0.002, 0.000, 0.000, respectively）. However, the 5-HT levels in brain homogenate significantly decreased in the GFS treatment subgroups compared with those in the saline treatment subgroups in MI and depression groups （P = 0.025 and 0.044 respectively）, and no significant difference was observed between saline and GFS treatment subgroups in MI ＋ depression group （P = 0.663）. Compared with that in GFS treatment subgroup of control group, the 5-HT2AR levels in the platelet lysate significantly decreased in GFS treatment subgroups of MI, depression, and MI ＋ depression groups （all P = 0.000）. Compared to those in the saline treatment subgroups, the serum SERT levels significantly decreased in the GFS treatment subgroups in MI, depression, and MI ＋ depression groups （P = 0.009, 0.038, and P = 0.001, respectively）, while the SERT levels of platelet lysate significantly decreased in GFS treatment subgroup of MI group （P = 0.000）, significantly increased in GFS treatment subgroup of depression group （P = 0.019）, and slightly changed in GFS treatment subgroup of MI ＋ depression group （P = 0.219）. No significant changes for SERT levels in brain homogenate could be found between the saline and GFS treatment subgroups in MI, depression, and MI ＋ depression groups （P = 0.421, 0.076 and P = 0.642）. Conclusions： This study indicated that GFS might inhibit the reuptake of 5-HT from serum to platelet according to decreased 5-HT2AR in platelet and SERT in serum and platelet. The change of 5-HT in serum after GFS treatment was inconsistent with that in the brain. It seemed that GFS could not pass through the blood-brain barrier to affect the central serotonergic system.

人参发酵产物中总皂苷的纯化研究

以人参总皂苷为评价指标,考察洗脱液体积、上样液质量浓度、上样液体积等因素,测定纯化过程的最佳参数。结果表明,最佳的提纯工艺是采用D101大孔树脂,湿法填装,药液的质量浓度0.8 g/mL,上样时所用的速度2 BV/h,静置1 h吸附药液,洗脱时先用蒸馏水将未吸附杂质洗去,再用5 BV的75%乙醇溶液洗去有效成分。最终确定的纯化工艺既简单又高效,可以适用于大生产。

人参酵素皂苷含量的分析研究

人参酵素是以人参作为主要原料,经过发酵产生的一种红棕色的液体,不但可以预防多种疾病,而且在美容方面发挥着非常重要的作用。人参是营养价值极高的一种植物资源,具有非常强大的滋补能力,研究发现,人参中可以分离出多种皂苷和多糖,它们是人参生理活性的物质基础。文章主要研究人参酵素中皂苷含量,分别使用p H试纸和p H计测定人参酵素的酸碱度;使用分光光度计在400~700 nm波长之间对人参皂苷Re进行光谱分析,得到550 nm是其最大吸收波长,然后在550 nm的波长下以人参皂苷Re作为标准品测定其吸光度,再计算人参酵素中的皂苷含量;pH计测出人参酵素呈酸性,pH为3.68;人参总皂苷在0~100 uL范围内线性关系相对良好,S_(RSD)(相对标准差)为2.1%(n=3),结果显示人参酵素中皂苷含量为0.2 mg/mL。由以上实验结果可知,酵素是一种酸性的液体,易于保存。人参皂苷是人参酵素中的主要活性物质,保留了人参的生物活性。

Effect of Total Saponins of Ginseng Leaves on Immunity in Aged Rats

Objective: To explore the effect of total saponins of Ginseng leaves(GS) on the functions ofneutrophil and lymphocyte in the aged rats of 20 months. Methods: Three groups of aged rats were injected intraperiton-eally with doses of 12. 5 mg/kg, 25 kg/mg and 50 mg/kg of GS once daily for 3 days, the declinedimmunity of spleen lymphocytes (proliferation of spleen lymphocyte, phagocytosis), superoxide anion and nitricoxide formation were studied and were compared with those of young rats (2 months). Results: The declinedimmunity of spleen lymphocytes were improved dose-dependently after GS injected. Conclusion: The results indicate that GS may enhance neutrophil and lymphocyte mediated non--specific and specific defense function inaged rats.

The Effects of Total Saponins of Panax Ginseng on Hematopoietic Progenitor Cells in Healthy Humans and Aplastic Anemia Patients

Ginseng is said to have beneficial effects on anemia. The proliferation effects of totalsaponins of Panax ginseng (TSPG) on hematopoietic progenitor cell in healthy individuals and 29 patientswith aplastic anemia (AA) were observed through bone marrow cultures of burst forming unit-erythroid(BFU-E) , colony forming unit-erythroid (CFU-E) and colony forming unit-granulocyte/macrophage (CFU-GM) in vitrcacompared with methyltestosterone (MT). The results suggest TSPG might prompt the prolif-eration of normal progenitor cellS at a concentration of 20 g/ml. The numbers of BFU-E ,CFU-E and CFU-GM increased by 37. 8±2.9 % , 31. 4±2. 9 % and 33. 3± 4. 0 % respectively over the controls ; further-more TSPG was still useful to BFU-E,CFU-E growth without Epo in vitro, although the colony nurnberswere much lower. Otherwise MT was useless to CFUGM. Of the 29 patients with AA, 14 who respondedto MT showed sensitivity to TSPG in marrow culture (the rising rate of colony formation exceeded 30 % ) ,but immune-mediated AA (patient's peripheral blood mononucleated cell suppressed normalhematopoiesis) and stem cell decreased AA (few of colonies were formed) showed almost no expressionfor TSPG activity because of the immunological suppression system and the absence of progenitors.