超声辅助提取红花中主要活性成分的工艺优化

采用HPLC-UV测定红花提取物中黄酮类成分含量,确定评价指标,通过单因素试验设计,筛选红花活性成分提取方法;利用Box-Behnken试验设计原理,以花瓣中主要黄酮类成分为响应值,以单因素结果所选因素为自变量,建立响应面分析试验模型,优化提取工艺。红花花瓣中主要黄酮类成分为羟基红花黄色素A和山奈酚-3-O-芸香糖苷,占比85%以上;红花活性成分提取方法应采用超声提取法,响应面优化的最佳提取条件为:料液比0.1∶25 g·mL-1、超声温度70℃、超声时间45 min、溶剂为57%甲醇、超声功率177 W,在该最优条件下红花黄色素A提取率为1.74%,山奈酚-O-β-芸香糖苷提取率为1.53%。该方法羟基红花黄色素A提取率比药典中提取方法显著提高,其余黄酮类成分提取率也呈极显著增加。     

红花的化学成分及DPPH自由基清除活性研究

从红花中分离得到14个化合物,通过波谱数据和理化性质分别鉴定为:异光黄素(1)、(2S)-4',5,6,7-四羟基二氢黄酮-6-O-β-D-葡萄糖苷(2)、新红花苷(3)、山柰酚(4)、山柰酚-3-O-β-D-葡萄糖苷(5)、山柰酚-3-O-β-芸香糖苷(6)、6-羟基山柰酚(7)、6-羟基山柰酚-3-O-β-D-葡萄糖苷(8)、槲皮素(9)、对羟基苯甲酸(10)、对羟基桂皮酸(11)、尿嘧啶(12)、腺嘌呤(13)、β-谷甾醇(14),其中化合物1首次从红花属植物中分离得到。采用TLC-DPPH生物自显影法筛选单体化合物及红花醇提物各萃取部位的自由基清除活性,结果表明乙酸乙酯萃取物及化合物4、9、11具有明显的DPPH自由基清除活性。     

经典恒温法预测红花的有效期

通过经典恒温法进行加速试验,采用HPLC测定羟基红花黄色素A在各红花样品中的含量,根据Arrhenius指数定律推测出红花中羟基红花黄色素A含量变化规律,研究不同温度时间下红花中羟基红花黄色素A的稳定性,并预测红花在阴凉条件(20℃)下的有效期为1.20年。说明红花在高温条件下稳定性差,因此需在阴凉条件下贮存。     

红花绿绒蒿的非生物碱成分

本文对红花绿绒蒿的非生物碱成分进行了研究。分离并用波谱法鉴定了的其中6个成分为:21α-羟基熊果酸(1),肉豆蔻酸2,3-二羟基丙酯(2),豆甾醇(3),β-香树精(4),熊果酸(5)及胡萝卜甙(6)。     

基于HPLC-ESI-TOF/MS法分析测定乌天麻和红天麻中化学成分的研究

建立高效液相色谱-电喷雾飞行时间质谱(HPLC-ESI-TOF/MS)联用技术用于指认乌天麻和红天麻中的化学成分,并对天麻素、对羟基苯甲醇、对羟基苯甲醛、腺苷、巴利森苷A、4,4'-二羟基二苄基醚6种成分进行含量测定。采用Agilent 1120高效液相系统,YMC-PEAK ODS-A column(250 mm×4.6 mm,5μm)色谱柱,流动相为0.1%甲酸水溶液(A)-甲醇溶液(B),梯度洗脱:0~5 min,5%B;5~65 min,5%~40%B;65~80 min,40%~100%B;分析时间80 min;体积流量1 m L/min;柱温25℃;进样量50μL。最终指认了天麻提取物中的15种成分,其中天麻素、对羟基苯甲醇、对羟基苯甲醛、腺苷、巴利森苷A、4,4'-二羟基二苄基醚6种成分在线性范围内均具有良好的线性关系(r≥0.9991);平均回收率在94.90%~99.81%之间,RSD2.40%。在不同品种的天麻饮片中,6种成分的量存在差异,红天麻各成分含量稍高于乌天麻,一级乌天麻各成分含量高于二级乌天麻。同一品种天麻中,巴利森苷类成分含量较高,腺苷和4,4'-二羟基二苄基醚含量均较低。建立的HPLC测定方法分离效果与重复性好、快速、简便,为天麻饮片的质量控制提供参考。     

秃叶黄皮树叶子的化学成分研究(英文)

从秃叶黄皮树叶子(Phellodendron chinensevar.glabriusculumSchneid)分离到7个化合物,经波谱解析鉴定为6-O-乙酰基黄柏苷(1),6-O-乙酰基二氢黄柏苷(2),(2R)-4′,5-二羟基-7-O-β-D-吡喃葡萄糖基-8-异戊烯基-二氢黄酮(3),黄柏苷(4),2-O-β-D-吡喃葡萄糖基-6-羟基-苯甲酸苄酯(5),柑橘素C(6),3-羰基齐墩果烷(7),其中化合物1～3,5～7为首次从秃叶黄皮树叶子中分离得到。     

利用HPLC建立对比筛选法测定红花中色素金橙Ⅱ

利用高效液相色谱法,分别检测并对比红花药材中羟基红花黄色素A和金橙Ⅱ的浓度,从而建立对比筛选红花样品中金橙Ⅱ的方法。结果在46个红花样品中,推测有26个可疑阳性样品;而通过对比筛选法能将可疑阳性样品数减至2个。经HPLC-MS法确证了对比筛选法的结果。检测羟基红花黄色素A(HSYA)的色谱条件为:甲醇-乙腈-0.01%磷酸溶液(体积比为26∶2∶72)为流动相。检测金橙Ⅱ的色谱条件为:乙腈-0.025 mol/L溶液(40∶60)为流动相。此法相比于常规只检测红花中金橙Ⅱ的方法,筛选效率与精确度更高,并且更经济、适应性更广。     

黑果枸杞中花色苷的提取与结构鉴定

采用紫外-可见光谱法并结合高效液相色谱-电喷雾串联质谱对黑果枸杞中花色苷的组成及结构进行了鉴定。结果显示:(1)黑果枸杞花色苷在0.1%盐酸-甲醇溶液中呈紫红色表明花色苷中的主要成分可能是飞燕草色素、牵牛花色素、锦葵色素及其衍生物中的一种或几种;向提取溶液中加入5%Al Cl3甲醇溶液后无红移现象表明花色苷结构B环上无邻位酚羟基;A440nm/Aλmax比值小于20%表明该色素是3,5位均带有糖苷键的双取代花色苷;在304 nm处有一最大吸收峰表明该色素分子内含有酰基;色素水解后主要生成葡萄糖。由上述可初步推测出黑果枸杞色素中主要为酰基化的锦葵色素-3,5-二葡萄糖苷;(2)经紫外分光光度法、质谱和文献报道综合分析鉴定出黑果枸杞中含有8种花色苷,分别是:飞燕草素-3-O-葡萄糖苷、芍药素-3-O-葡萄糖苷、矮牵牛素-5-O-葡萄糖苷、矮牵牛素-3-O-(6-O-对香豆酰)芸香糖苷-5-O-葡萄糖苷、锦葵色素-3-O-(6-O-对香豆酰-3-O-乙酰)-5-O-二葡萄糖苷、飞燕草素-3-O-(6-O-乙酰)葡萄糖苷、锦葵色素-3-O-(6-O-对香豆酰)葡萄糖苷和锦葵色素-3,5-二葡萄糖苷,其含量依次为0.86%、1.17%、2.38%、11.79%、68.35%、0.63%、5.24%、9.58%。花色苷是黑果枸杞中重要的组成成分,该试验可为黑果枸杞的质量控制提供依据。     

三七总皂苷酶水解产物成分研究

为了研究葡萄糖苷酶催化三七提取物的水解产物中主要皂苷成分。采用色谱法从三七提取物水解产物中分离纯化得到11个皂苷成分。利用波谱解析确定了它们的结构,分别鉴定为20(S)-原人参二醇-20-O-β-D-吡喃木糖基-(1→6)-β-D-吡喃葡萄糖基-(1→6)-β-D-吡喃葡萄糖苷(1),以及10个已知的皂苷成分分别为:人参皂苷compound K(2)、3β,12β,20(S),25-四羟基达玛-23-烯-20-O-β-D-吡喃葡萄糖苷(3)、3β,20(S)-二羟基达玛-24-烯-12β,23β-环氧-20-O-β-D-吡喃葡萄糖苷(4)、3β,12β,20(S)-三羟基-25-过氧羟基达玛-23-烯-20-O-β-D-吡喃葡萄糖苷(5)、人参皂苷F1(6)、人参皂苷Rg1(7)、人参皂苷Rg2(8)、人参皂苷Mc(9)、20(S)-原人参二醇-3-O-β-D-吡喃木糖基-(1→2)-β-D-吡喃葡萄糖基-20-O-β-D-吡喃葡萄糖苷(10)和人参皂苷Re(11)。其中化合物1为新化合物,化合物3~5和10为首次从三七中被分离得到。     

米碎花的酚苷类化合物及其鼻咽癌细胞毒活性

为了解米碎花(Eurya chinensis)的化学成分及其生物活性,运用多种色谱技术从其乙醇提取物分离得到11个化合物,并对化合物进行体外抗鼻咽癌细胞增殖活性评价。经波谱数据分析,分别为异落新妇苷(1)、3,5,7-三羟基色原酮-3-O-α-L-鼠李糖苷(2)、1-O-反式-桂皮酰基-β-D-葡萄糖(3)、1-O-(4-羟基苯乙基)-6-O-反式-桂皮酰基-β-D-葡萄糖(4)、eutigoside D (5)、1-O-(3,4-二羟基苯乙基)-6-O-反式-香豆酰基-β-D-葡萄糖(6)、eutigoside A (7)、1-O-(4-羟基苯乙基)-6-O-反式-咖啡酰基-β-D-葡萄糖(8)、grayanoside A (9)、1-O-(4-羟基苯乙基)-6-O-(4-羟基苯甲酰基)-β-D-葡萄糖(10)、3-O-β-D-葡萄糖基-4-羟基-苄基苯甲酸酯(11)。其中,化合物4为首次从天然来源获得,化合物2～4和8～11均为首次从该属植物中分离得到。MTT法表明,化合物10具有中等抑制5-8F细胞增殖活性。     

Hydroxysafflor yellows alleviate thrombosis and acetaminophen-induced toxicity in vivo by enhancing blood circulation and poison excretion

BackgroundHydroxysafflor yellow A (HSYA) from the flower of Carthamus tinctorius (Safflower) has been reported to have various pharmacological effects. However, little is known about the bioactivities of other chemical constituents in Safflower and the relationship between enhancement of blood circulation and hepatoprotection by HSYA.PurposeThe present research was to evaluate the antithrombotic and hepatoprotective activities of HSYA and C, examine their mechanisms of actions, including influence on the excretion velocity of acetaminophen, and the relationship between the antithrombotic, hepatoprotective, and other bioactivities.MethodsThe hepatoprotective activities were examined by acetaminophen (APAP)-induced zebrafish toxicity and carbon tetrachloride (CCl₄)-induced mouse liver injury. The concentrations of APAP in zebrafish and APAP that was excreted to the culture media were quantified by UHPLC-MS. The anti-thrombosis effect of HSYA and C were examined by the phenylhydrazine (PHZ)-induced zebrafish thrombosis.ResultsHSYA and HSYC showed robust protection on APAP-induced toxicity and PHZ-induced thrombosis. The hepatoprotective effects of HSYA and C were more potent than that of the positive control, acetylcysteine (61.7% and 58.0%, respectively, vs. 56.9% at 100 µM) and their antithrombosis effects were more robust than aspirin (95.1% and 86.2% vs. 52.7% at 100 µM). HSYA and C enhanced blood circulation, rescued APAP-treated zebrafish from morphological abnormalities, and mitigated APAP-induced toxicity in liver development in liver-specific RFP-expressing transgenic zebrafish. HSYC attenuated CCl₄-induced mouse liver injury and regulated the levels of HIF-1α, iNOS, TNF-α, α-SMA, and NFκB in liver tissues. HSYA was also protective in a dual thrombotic and liver toxicity zebrafish model. By UHPLC-MS, HSYA accelerated the excretion of APAP.ConclusionHSYA and C are the bioactive constituents of Safflower that are responsible for the herbal drug's traditional use in promoting blood circulation to remove blood stasis. Safflower and its chalcone constituents may protect from damage due to exogenous or disease-induced endogenous toxins by enhancing the excretion velocity of toxins.     

Isoxazole carboxylic acids as protein tyrosine phosphatase 1B (PTP1B) inhibitors

Guided by X-ray crystallography, we have extended the structure-activity relationship (SAR) study on an isoxazole carboxylic acid-based PTP1B inhibitor (1) and more potent and equally selective (>20-fold selectivity over the highly homologous T-cell PTPase, TCPTP) PTP1B inhibitors were identified. Inhibitor 7 demonstrated good cellular activity against PTP1B in COS 7 cells.     

Identification of AFLP fragments linked to hydroxysafflor yellow A in Flos Carthami and conversion to a SCAR marker for rapid selection

Hydroxysafflor yellow A (HSYA), an important active compound in treating focal cardiac and cerebral ischemia, is uniquely present in flower petals of Carthamus tinctorius. In this study, inheritance and molecular marker analyses for HSYA trait in safflower were carried out. HSYA contents in parents, cross hybridized F₁ and F₂ individuals were analyzed by high performance liquid chromatography. Results revealed that the presence/absence of HSYA was controlled by one major nuclear gene termed HSya. A total of 48 AFLP primer combinations were screened, and bulked segregant analysis was performed by preparing two pools of 10 present-HSYA and ten absent-HSYA plants selected from the 498 individuals of the F₂ segregating population. Four AFLP markers, AFLP-5, AFLP-7, AFLP-15 and AFLP-16, were identified to be closely associated with HSya. Of those, AFLP-16 was the closest to HSya, estimated at about 9.4 cM in genetic distance. The dominant AFLP-16 marker was converted into a simple sequence characterized amplified region marker based on the sequence information of the cloned flanking regions of the AFLP fragment and was designated as SCM16. Our result has direct application for marker-assisted selection of quality breeding in safflower.     

Amphiphilic and hydrophilic nature of sheep and human platelet phosphotyrosine phosphatase forms.

To date, although at least 75 different PTPases (protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) have been identified, those detected in platelets are rather scarce. Based on previous results from our laboratory, we investigated the existence of new PTPases in platelets. Triton X-114 phase partitioning of Triton X-100-solubilized human and sheep platelet membranes allowed PTPase to be recovered in the detergent-rich (40-35%, respectively) and -poor phases (60-65%, respectively). Sedimentation analyses of both phases from the sheep species revealed hydrophilic 6S and 3.7S, and amphiphilic 7.5S and 10.3S PTPase forms. Sedimentation analyses of human platelet membrane-associated or cytosolic PTPase revealed hydrophilic 6.7S and 4.3S, and amphiphilic 5.5S and 10.8S forms, or hydrophilic 4S, 5.9S and 6.9S forms, respectively. Western blot analysis using monoclonal antibodies (MoAb) against human PTP1B, PTP1C, PTP1D and RPTPalpha (mouse anti-human PTPase MoAbs) showed that RPTPalpha was not present in platelets and that the PTP1C type and PTP1D type (but probably not the PTP1B type) were expressed in sheep species. Immunoblots also revealed that all PTPases detected were mainly membrane-associated, with similar percentages of cellular distribution in both species. All PTPases were mainly recovered in the detergent-poor phases from the Triton X-114 phase partitioning, although PTP1D from human species was also significantly present (30%) in the detergent-rich phase. Additionally, all PTPases sedimented within the same PTPase peak in sucrose gradients (sedimentation coefficients around 4S). These findings indicate that amphiphilic and hydrophilic PTPases different from PTP1B, PTP1C, PTP1D or RPTPalpha, with higher sedimentation coefficients and with higher activity when O-phosphotyrosine or a synthetic peptide phosphorylated on tyrosine were used as substrates, are present in platelets.     

Identification of flavonolignans from Silybum marianum seeds as allosteric protein tyrosine phosphatase 1B inhibitors

Protein tyrosine phosphatase 1B (PTP1B) is an attractive molecular target for anti-diabetes, anti-obesity, and anti-cancer drug development. From the seeds of Silybum marianum, nine flavonolignans, namely, silybins A, B (1, 2), isosilybins A, B (3, 4), silychristins A, B (5, 6), isosilychristin A (7), dehydrosilychristin A (8), and silydianin (11) were identified as a novel class of natural PTP1B inhibitors (IC₅₀ 1.3 7–23.87?µM). Analysis of structure–activity relationship suggested that the absolute configurations at C-7" and C-8" greatly affected the PTP1B inhibitory activity. Compounds 1–5 were demonstrated to be non-competitive inhibitors of PTP1B based on kinetic analyses. Molecular docking simulations resulted that 1–5 docked into the allosteric site, including α3, α6, and α7 helix of PTP1B. At a concentration inhibiting PTP1B completely, compounds 1–5 moderately inhibited VHR and SHP-2, and weakly inhibited TCPTP and SHP-1. These results suggested the potentiality of these PTP1B inhibitors as lead compounds for further drug developments.     

Residues distant from the active site influence protein-tyrosine phosphatase 1B inhibitor binding

Regions of protein-tyrosine phosphatase (PTP) 1B that are distant from the active site yet affect inhibitor binding were identified by a novel library screen. This screen was based on the observation that expression of v-Src in yeast leads to lethality, which can be rescued by the coexpression of PTP1B. However, this rescue is lost when yeast are grown in the presence of PTP1B inhibitors. To identify regions of PTP1B (amino acids 1-400, catalytic domain plus 80-amino acid C-terminal tail) that can affect the binding of the difluoromethyl phosphonate (DFMP) inhibitor 7-bromo-6-difluoromethylphosphonate 3-naphthalenenitrile, a library coexpressing PTP1B mutants and v-Src was generated, and the ability of yeast to grow in the presence of the inhibitor was evaluated. PTP1B inhibitor-resistant mutations were found to concentrate on helix alpha7 and its surrounding region, but not in the active site. No resistant amino acid substitutions were found to occur in the C-terminal tail, suggesting that this region has little effect on active-site inhibitor binding. An in-depth characterization of a resistant substitution localizing to region alpha7 (S295F) revealed that this change minimally affected enzyme catalytic activity, but significantly reduced the potency of a panel of structurally diverse DFMP PTP1B inhibitors. This loss of inhibitor potency was found to be due to the difluoro moiety of these inhibitors because only the difluoro inhibitors were shifted. For example, the inhibitor potency of a monofluorinated or non-fluorinated analog of one of these DFMP inhibitors was only minimally affected. Using this type of library screen, which can scan the nearly full-length PTP1B sequence (catalytic domain and C-terminal tail) for effects on inhibitor binding, we have been able to identify novel regions of PTP1B that specifically affect the binding of DFMP inhibitors.     

Molecular dynamics simulation integrating the inhibition kinetics of hydroxysafflor yellow A on α-glucosidase

Inhibition of α-glucosidase has attracted the attention of researchers due to its connection to type-2 diabetes. Hydroxysafflor yellow A (HSYA) extracted from Carthamus tinctorius L. is a natural antioxidant used in traditional Chinese medicine. In this study, the effect of HSYA on α-glucosidase was evaluated using inhibitory kinetics based on the antioxidant properties of HSYA and by performing computational simulation integration methods. HSYA reversibly inhibited α-glucosidase in a competitive inhibition manner and the evaluated kinetic parameters were IC₅₀ = 1.1 ± 0.22 mM and K_i = 1.04 ± 0.23 mM, respectively. The results of spectrofluorimetry showed that the inner hydrophobic regions of α-glucosidase, which are mostly in the active site, were exposed to the surface with increasing HSYA concentrations, indicating that the inactivation of α-glucosidase by HSYA was accompanied by regional unfolding. The molecular dynamics simulations indicated that the four rings of HSYA interact with four residues such as G217, A278, H279, and G280 at the entrance of the active site. Our study provides insight into the inhibition of α-glucosidase and the accompanying structural changes by HSYA. Based on its α-glucosidase-inhibiting effect and its potential as a natural antioxidant, HSYA is a potential agent for treating α-glucosidase-associated type-2 diabetes.     

Kinetics of reduction of tyrosine phenoxyl radicals by glutathione

Erythropoietin (Epo) is crucial for promoting the survival, proliferation, and differentiation of mammalian erythroid progenitors. The central role played by tyrosine phosphorylation of erythropoietin receptor (EpoR) in Epo-cell activation has focused attention on protein tyrosine phosphatases (PTPs) as candidates implicated in the pathogenesis of the resistance to therapy with human recombinant Epo. Prototypic member of the PTP family is PTP1B, which has been implicated in the regulation of EpoR signaling pathways. In previous reports we have shown that PTP1B is reciprocally modulated by Epo in undifferentiated UT-7 cell line. However, no information is available with respect to the modulation of this phosphatase in non-Epo depending cells or at late stages of erythroid differentiation. In order to investigate these issues we induced UT-7 cells to differentiate and studied their PTP1B expression pattern. Simultaneous observations were performed in TF-1 cells which can be cultured either with GM-CSF, IL-3 or Epo. We found that Epo induced PTP1B cleaveage in TF-1 and differentiated UT-7 cells. This pattern of PTP1B modulation may be due to an increased TRPC3/TRPC6 expression ratio which could explain the larger and sustained calcium response to Epo and calpain activation in Epo treated TF-1 and differentiated UT-7 cells.     

Regulation of the insulin antagonistic protein tyrosine phosphatase 1B by dietary Se studied in growing rats

Protein tyrosine phosphatase 1B (PTP1B) is a key enzyme in the counterregulation of insulin signaling, and its physiological modulation depends on H2O2 and glutathione (GSH). Se via GSH peroxidases (GPxs) and its specific metabolism is involved in the removal of H2O2 and in the regulation of GSH metabolism. Recent results from animal trials and epidemiological studies with humans have shown that a high GPx1 activity or a permanent surplus of Se may promote the development of obesity and diabetes. Our nutrition physiological study with 7 x 7 growing rats was carried out to examine if PTP1B is modulated by Se supplements and, thus, may represent one trigger mediating these undesirable metabolic effects of Se. One group of rats was fed an Se-deficient diet for 8 weeks. The diets of the other six groups contained Se as selenite or selenate according to the recommendations (0.20 mg/kg diet) and at two supranutritional levels (1.00 and 2.00 mg/kg diet). All Se-supplemented animals featured a significantly higher body weight (6-14%) compared to their Se-deficient companions. Expression and activity of GPx1 in the liver of Se supplemented animals was 10- and 70-fold higher compared to Se deficiency. The detailed study of PTP1B regulation using an enzymatic assay and Western Blot analysis with an antibody against protein glutathionylation revealed that PTP1B was significantly up-regulated by both a maximization of GPx1 activity and by increasing dietary Se supply, reducing its inhibition via glutathionylation. Selenate effected a stronger PTP activation compared to selenite. In conclusion, our results suggest that the modulation of PTP1B activity may represent one plausible mechanism by which a long-term intake of Se supplements exceeding the requirements can promote the development of obesity and diabetes and needs further intensive investigation.