Pharmacognostic Identification of Hedyotis auricularia and Mitracarpus villosus

[Objectives]To conduct the pharmacognostic identification of Hedyotis auricularia and Mitracarpus villosus in Guangxi and provide a scientific basis for their identification.[Methods]The characteristics of original plants were studied by origin identification method;the properties and characteristics were studied by character identification method;and the microscopic features of the roots,stems,leaves and medicinal powder of H.auricularia and M.villosus in Guangxi were studied by paraffin method and powder slicing method.[Results](i)Origin identification.H.auricularia:Leaves leathery,apex acuminate,base cuneate;petiole shorter;cyme axillary;corolla hairy at throat;fruit indehiscent at maturity;testa black after drying.M.villosus:Leaf apex short pointed,base attenuate,blade sessile;flowers small,clustered in axillary;fruits dehiscent by lid at or below middle at maturity,seeds dark brown.(ii)Character identification.Fracture surface of H.auricularia uneven,white in outer layer and sepia in inner layer.Fracture surface of M.villosus hollow,uneven and white.(iii)Microscopic identification.H.auricularia:Root phloem thick,cambium visible,duct cells quasi-polygonal,large;rays obvious.Stem transection quasi-circular square,often with non-glandular hairs on epidermis;calcium oxalate raphides present in leaf parenchymal cells.Power grayish brown,starch granules single-grained;calcium oxalate raphides frequent,calcium oxalate clustered crystals occasional;catheter spiral,rarely annular,stomata infinitive.M.villosus:Root parenchyma cells with scattered calcium oxalate raphides,calcium oxalate clustered crystals and brownish red substances visible.Stem transection quasi-square,edge angle with 4 short narrow wings.Powder brown,simple starch granules numerous,compound starch granules also present;calcium oxalate raphides numerous,calcium oxalate clustered crystals and calcium oxalate square cubic crystals also present;catheter spiral,stomata paracytic.[Conclusions]The above transaction microscopic characteristics of the roots,stems and leaves and powder characteristics can be used as the identification features of H.auricularia and M.villosus.

Anti-tumor Effect and Protective Effect on Chemotherapeutic Damage of Water Soluble Extracts from Hedyotis diffusa

Bai-Hua-She-She-Cao Hedyotis diffusa Willd. (Ru-biaceae) is a medicinal herbwidely distributed in northeast Asian countries. In traditional Chinese medicine, it has the effectof 'clearing away heat and toxic material, promoting blood circulation and removing blood stasis'.It is a well known Chinese folk-medicine used for the treatment of appendicitis, sore throat, mumps,acne, sebo-rheic dermatitis and various kinds of tumors, such as tumors of digestive tract,carcinoma of liver. It was reported that the MeOH extract of H. diffusa demonstrated a significantantitumor activity and ursolic acid succeeded in being isolated from the MeOH extract as an activecomponent . Shan BN, et al suggested that the direct aqueous extract of H. diffusa hadimmuno-modulating activity and antitumor activity in vitro through stimulating the immune system tokill or engulf tumor cells. But regarding anti-tumor activity in vivo of water soluble extracts fromH. diffusa, no detail was reported. Therefore, we prepared water soluble extracts (H_1 and H_2)from H. diffusa and evaluated their anti-tumor property in vivo experiments as well as protectiveeffect on chemo-therapeutic damage.

Establishing a Quality Control System of Hedyotis corymbosa

Objective Hedyotis corymbosa(Shui Xian Cao,水线草,HC)is the main antitumor medicine in China and other southeast Asian countries.A comprehensive quality control system of HC was established in this study,as the current system was ineffective.Methods Microscopy was used to observe the characteristics of the powder and cross-sectional tissue.Thin layer chromatography(TLC)was employed to separate HC from Hedyotis diffusa(Bai Hua She She Cao,白花蛇舌草,HD).Simultaneously,high-performance liquid chromatography(HPLC)was used to establish the characteristic spectrum of HC and determine the concentrations of hedyotiscone A.Regarding the issue of systematicness,concentrations of water,total ash,acid-insoluble ash,heavy metals(including lead,arsenic,mercury,cadmium and copper)and pesticide residue were measured by the methods described in the 2015 edition of Chinese Pharmacopoeia.Results Calcium oxalate needle crystal bundles and oil drops were observed in the cross-sectional tissue.Three characteristic blue fluorescent spots were observed in the HC samples by TLC.There were five distinct characteristic peaks in HC but not in HD,and hedyotiscone A was the sharpest characteristic peak.The generated linear equation of hedyotiscone A was Y=3.15×108X+312.04(r2=1.000)between 0.00005270 and 0.002108 mg/mL,and the average recovery was 100.42%.Precision[relative standard deviation(RSD)0.92%],repeatability(RSD 0.14%),and recovery(RSD 0.85%)met the requirements for quantitative analysis.Hedyotiscone A concentrations ranged from 0.00017%~0.00113%across different habitats.Concentrations of water,ash and acid-insoluble ash were 8.80%~11.2%,7.00%~15.3%and 0.440%~7.70%,respectively.Concentrations of Pb,As,Hg,Cd and Cu were 1.60~4.60,0.650~1.60,0.0300~0.100,0.240~0.690 and 10.1~16.6μg/g,respectively.Pesticide residues were less than 0.00100 ppm.Conclusion The quality control system can effectively distinguish HC from HD,so as to comprehensively control the quality of HC,to provide references for the clinical use of HC.

Physiological Effect and Quality Influences of Hedyotis diffusae Extract on Tobacco Infected by TMV

The extract from Hedyotis diffusae was used to treat tobacco plants infected by TMV before and after incidence. The effect of the changes of some physiological and biochemical indices related with the virus-resistance in tobacco leaves on tobacco quality ware determined. The resuits showed that H. diffusae extract could remarkably increase the total phenol content in tobacco leaves ,strength the activity of phenylalanine ammonialyase （ PAL） , improve the membrane permeability and tobacco quality. Prevention treatment was better than therapy treatment. In conclusion, H. diffusae extract could induce plants to produce disease resistance and strength tha antiviral capability of tobacco against TMV.

Antitumor effects and compatibility mechanism of Hedyotis Diffusa-Sculellaria Barbata herb pair

OBJECTIVE Hedyotis diffusa Willd and Scutellaria barbata D Don are most commonly used herb pair for cancer treatment in traditional Chinese medicine.This study aimed to evaluated the in vitro and in vivo antitumor effects and the compatibility mechanisms of Hedyotis diffusa-Sculellaria barbata herb pair.METHODS Hedyotic diffusa extract,Scutellariae Barbatae extract,and herb pair extract were prepared and the component were detected by UPLC-Q-TOF-MS.The effects of different concentrations of Hedyotic diffusa extract,Scutellariae Barbatae extract,and herb pair extract were detected using MTS assay.The in vivo antitumor effects were evaluated in Panc28 pancreatic cells bearing nude mice model.The compatibility mechanism of Hedyotis diffusa-Sculellaria barbata herb pair were evaluated based on the network pharmacology,using TCMSP,Cytoscape 3.6.1 software,and DAVID 6.8.The anticancer mechanism were further validated in vitro.RESULTS Both the MTS and in vivo results showed that herb pair extract showed more obvious inhibitory effects on cancer cells compared to each individual.A total of 37 active components were selected from Hedyotis diffusa and Sculellaria barbata,33 kinds of active ingredients are involved in their anti-tumor effects.58 cancer-related targets and 66 KEGG pathways were identified.The potential targets for the herb pair might be prostaglandin G/H synthase 2(PTGS2),HSP90,EGFR,72 ku typeⅣcollagenase,PPAR-γ,et al.In vitro validation result showed that compatibility mech⁃anisms was related with HSP90,EGFR related pathways.CONCLUSION The result of the study preliminarily verified the basic anti-tumor pharmacological effects of Hedyotis diffusa-Sculellaria barbata herb pair,and lays a solid foundation for further studies on the anti-tumor mechanism of the herbal pair.

Effect of Polysaccharides from Hedyotis diffusa on Immune Cells of Small Intestinal Mucosa of Chicks

[Objectives]This study aimed to investigate the effect of polysaccharides from Hedyotis diffusa on the immune cells of the intestinal mucosa of chicks.[Methods]Total 200 seven-day-old AA chicks were evenly and randomly divided into four groups:control group and three treatment groups.The chicks in the control group(group I)were fed basal diet,and those in the treatment groups were fed with basal diet supplemented with 0.4(group II),0.8(group III)and 1.2(group IV)g/kg H.diffusa polysaccharides(OPS),respectively.At the age of 14,28 and 42 d,10 chicks from each of the groups were sacrificed,respectively for counting of intraepithelial lymphocytes(IELs)and lamina propria secretory IgA(SIgA)positive cells in small intestinal mucosa.[Results]At the age of 14 d,the duodenal and jejunal IEL and SIgA positive cell counts in groups II,III and IV were significantly greater than those in group I(P<0.05).At the age of 28 d,the jejunal IEL count of group IV was significantly lower than those of groups I,II and III(P<0.05);and there was no significant difference in IEL or SIgA positive cell count between group I and the other groups(P>0.05).At the age of 42 d,the duodenal and jejunal IEL counts in group II were significantly greater than those in groups I,III and IV(P<0.05),while the differences in duodenal and jejunal IEL count between groups III,IV and I were insignificant(P>0.05).The duodenal SIgA positive cell counts in the treatment groups were insignificantly different from that in group I(P>0.05).The jejunal SIgA positive cell count in group II was significantly greater than those in groups I,III and IV(P<0.05).There was no significant difference in jejunal SIgA positive cell count between groups III and IV and group I(P>0.05).[Conclusions]OPS at 0.4 g/kg can promote the proliferation of immune cells in the small intestine mucosa of 14-42-day-old AA chicks,and it is recommended to be added to the feed.

In-vitro Antibacterial Effect of Hedyotis hedyotidea Ethanol Extract

[Objectives]This study was conducted to investigate the antibacterial effect of the extract from Hedyotis hedyotidea(DC)Merr.[Methods]Through in-vitro antibacterial tests,the antibacterial effects of H.hedyotidea ethanol extract on Escherichia coli,Staphylococcus aureus and Bacillus subtilis were analyzed.[Results]The extract had a low-susceptibility inhibitory effect to the three kinds of bacteria,and the minimal inhibitory concentrations(MICs)corresponding to S.aureus,E.coli and B.subtilis were 200,175 and 150 mg/ml,respectively.The minimal bactericidal concentrations(MBCs)corresponding to E.coli and S.aureus were both 500 mg/ml.[Conclusions]This study provides theoretical support for the wide application of H.hedyotidea.

Microscopic Characteristics and Routine Detection of Hedyotis hedyotidea(DC.)Merr.

[Objectives]To study the microscopic characteristics and routine detection of Hedyotis hedyotidea(DC.)Merr.[Methods]The microscopic characteristics of the root,stem cross section and the whole plant powder of H.hedyotidea were studied by the methods of slide-shaft section,biological staining and microscopic imaging;the routine detection items such as moisture,ash,acid-insoluble ash and extract were detected and analyzed in accordance with the requirements of Chinese Pharmacopoeia.[Results]The microscopic characteristics of the root,the stem and the whole plant powder of H.hedyotidea were obtained.The moisture content of 10 batches of H.hedyotidea samples was in the range of 4.25%-7.90%,the water-soluble extract is in the range of 15.08%-22.52%,the total ash was in the scope of 8.27%-10.45%,and the acid-insoluble ash was in 0.13%-0.95%.The proposed water content of H.hedyotidea should not exceed 10.00%,the water-soluble extract should not be less than 12.00%,the total ash content should not exceed 13.00%,and the acid-insoluble ash content should not exceed 1.20%.[Conclusions]The results of this experiment can provide a reference for the quality control and quality standard of H.hedyotidea.

Anti-tumor activity of Hedyotis diffusa Willd. in mice

The whole plant with root ofHedyotis diffusa Willd. （AE） has been demonstrated to have anti-inflammatory, antioxidant, and anti-bacterial activities. In our study, we aim to examine the anti-tumor effect of alcoholic extract of AE in mice implanted with sarcoma S180 cells （SBT mice）. We also compared the immune system function and the life span of SBT mice with that of control mice. We found that AE displayed a significant inhibitory effect on solid tumor growth, as well as increased the life span. At the dose of 10 mg/kg body weight, both tumor weight and volume were decreased significantly. We also measured several immune fimction markers, inlcuding spleen index （SI）, thymus index （TI） and parameters of hematological. We observed no reduce in these markers, indicatine that AE could inhibit tumor ~rowth without affectino immune function in SBT ,nice.

One New Iridoid Glycoside from Hedyotis tenelliflora

Objective To study the chemical constituents of Hedyotis tenelliflora.Methods The compounds were isolated by chromatographic separation technology.The structures were identified on the basis of chemical and spectral data.Results Four iridoid glycosides were isolated from the whole plant of H.tenelliflora.On the basis of the chemical and spectral methods,their structures were elucidated as teneoside C(1),harpagoside(2),harpagide(3),and asperulosidic acid(4).Conclusion Compound 1 is a new compound,and compounds 2 and 3 are isolated from H.tenelliflora for the first time.

Inhibition of vascular endothelial growth factor expression by Chinese medicine of Hedyotis diffusa Willd herbal compounds

The Hedyotis diffusa Willd herbal compounds(HDWHCs)are commonly used as Chinese medicine to treat cancer patients with established clinical therapeutic efficacy in China.However,the underlying mechanisms remain to be elucidated.In this study,we used freeze-dried powder of the water extracts of HDWHCs to investigate the potential mechanisms of HDWHCs in cancer treatment.HDWHCs treatment significantly inhibited vascular endothelial growth factor(VEGF)mRNA levels and VEGF transcriptional activation in cancer cells.HDWHCs also had a remarkable inhibitory effect on the expression of hypoxia-inducible factor 1alpha(HIF-1alpha).Forced expression of HIF-1αrestored VEGF transcriptional activation inhibited by HDWHCs,indicating that HDWHCs suppressed VEGF expression through decreasing HIF-1alpha expression.Moreover,HDWHCs inhibited cyclooxygenase-2(COX-2)expression,and overexpression of HIF-1alpha restored HDWHCs’inhibitory effect on COX-2 at transcriptional level.These findings may provide better understanding of HDWHCs’anti-cancer mechanism in cancer treatment.

Ethyl Acetate Fraction in Hedyotis Diffusa Willd Inhibits T Cell Proliferation to Improve the Pathogenesis of Systemic Lupus Erythematosus

Background:Abnormal proliferation of T cells plays an essential role in the pathogenesis of Systemic lupus erythematosus(SLE).The pharmaceutical effect of Hedyotis Diffusa Willd(HDW)on SLE has been investigated previously.Nevertheless,the biomedical mechanism is still left unclear.Objective:This study has been arranged to evaluate the therapeutic effect of the ethyl acetate fraction of HDW(EAHDW)on lupus mice and explore the potential therapeutic mechanism.Methods:EAHDW was prepared with 80%ethanol reflex extraction followed by successive extraction,and ana-lyzed with HPLC and UPLC-Q/TOP-MS.The potential targets and STAT3 affinity regulators were predicted with network pharmacology.The pharmaceutic effect of EAHDW was studied with MRL/lpr mice.Cytokines and au-toantibodies were quantified with ELISA assays.The pathological damage of glomerulus and STAT3 expression in the kidney was detected with histochemical and immunohistochemical techniques.The cell cycle properties in cell proliferation were identified with the flow cytometry.The western blot and dual-Luciferase reporter assay were applied to evaluate translational and transcriptional activity of STAT3,respectively.Results:In this study,the extraction ratio of EAHDW was 2.7±1%,in which 19 ingredients were identified.Network pharmacological analysis showed that the target genes of EAHDW were highly focused on influencing the abnormal T cell proliferation in SLE.EAHDW showed the beneficial effects on pathological changes and STAT3 expression in the glomerulus of lupus mice,and the levels of cytokines and autoantibodies in serum.In cytological study,EAHDW treatment attenuated the transcription and phosphorylation of STAT3,which inhibited T cell proliferation by prolonged S-phase of the cell cycle.A total of 5 compounds in EAHDW exhibited high docking affinity to the DNA-binding site of STAT3.Conclusion:EAHDW could reduce the inflammatory response and inhibit the proliferation of T cells by interfering with the STAT3 signaling pathway,thereby playing a therapeutic effect on SLE.

微波辅助酶法提取白花蛇舌草多糖的工艺优化

为优化微波辅助酶法提取白花蛇舌草(Hedyotis diffusa Willd.)多糖工艺。在单因素试验基础上,应用中心组合设计,采用二次多项式逐步回归分析对提取工艺条件进行优化,确定微波辅助酶法提取白花蛇舌草多糖工艺最佳条件为酶解时间2.9 h,酶解温度60℃,酶用量0.9%,p H 6.0;按此工艺条件,提取3次,白花蛇舌草多糖平均含量可达31.02%。

紫外分光光度法测定攀茎耳草中总黄酮含量

[目的]测定攀茎耳草总黄酮含量。[方法]以芦丁为对照品,用超声辅助提取法提取攀茎耳草中的总黄酮,再用紫外分光光度法测定总黄酮含量。[结果]在0.011～0.110 mg/ml范围内芦丁浓度与吸光度有良好的线性关系,回归方程为Y=18.015X+0.020,相关系数R2=0.999 4;攀茎耳草中总黄酮的相对百分含量为1.609%,精密度试验RSD为1.10%。[结论]该方法操作简便、结果可靠,能为攀茎耳草药用价值的开发及扩大资源利用提供科学的依据。

微波辅助酶法提取白花蛇舌草总黄酮的工艺研究

为优化微波辅助酶法提取白花蛇舌草(Hedyotis diffusa)总黄酮工艺,在单因素试验基础上,应用中心组合设计,采用二次多项式逐步回归分析方法对提取工艺条件进行了优化,确定微波辅助酶法提取白花蛇舌草总黄酮工艺最佳条件为酶解时间1.5 h、酶解温度45℃、酶用量1.4%、p H 5.5,按此工艺条件进行验证,白花蛇舌草总黄酮提取率可达4.42%。

紫外-可见光分光光度法测定攀茎耳草中总生物碱

采用碱浸、超声辅助氯仿萃取法提取攀茎耳草（Hedyotis scandens Roxb.）干燥根皮中的总生物碱,并以小檗碱为对照品,采用紫外分光光度法测定其相对含量。结果表明,小檗碱对照品在0-53.33μg/mL范围内有良好的线性关系（R2=0.999 4,RSD=0.253%）。该方法操作简便、结果可靠,为攀茎耳草开发提供了一定的科学参考。

乙醇-磷酸氢二钾双水相体系萃取白花蛇舌草总黄酮的工艺研究

利用乙醇-磷酸氢二钾双水相体系对白花蛇舌草(Hedyotis diffusa)总黄酮进行萃取分离。通过单因素实验分别考察了m(K2HPO4)、超声时间及超声温度对白花蛇舌草总黄酮萃取效果的影响,并用响应面法优化了萃取工艺。经优化后,白花蛇舌草总黄酮的最佳萃取条件为:m(K2HPO4)2.0g,超声时间37min,超声温度57℃。在此条件下,白花蛇舌草总黄酮的萃取率可高达99.45%。本方法精密度良好,准确度较高,可为白花蛇舌草总黄酮的高效萃取提供参考。

白花蛇舌草总黄酮提取工艺及其抗氧化活性研究

研究白花蛇舌草(Hedyotis diffusa Willd)总黄酮的提取工艺及其体外抗氧化活性。以白花蛇舌草总黄酮提取率为评价参数,通过单因素试验、正交试验优化提取工艺;测定了总还原力及Fe2+螯合力、羟自由基和超氧阴离子清除能力研究白花蛇舌草总黄酮的抗氧化活性。结果表明,用95%乙醇与白花蛇舌草粉末按40∶1(m L∶g),在90℃下提取180 min,此时提取率最高,达(2.22±0.01)%。体外抗氧化活性结果显示白花蛇舌草总黄酮具有较强的总还原力,Fe2+螯合率最高为90%,羟自由基和超氧阴离子的清除率分别达79%、77%。白花蛇舌草总黄酮具有显著的体外抗氧化活性,具有进一步开发利用的价值。

艳丽耳草的二型花柱及异型自交不亲和系统

异型花柱是一种受遗传因素控制的花型多态性现象,包括二型花柱和三型花柱两种类型。本文以茜草科艳丽耳草(Hedyotispulcherrima)为实验材料,通过对其野外居群的花型、花部形态及花粉特征等观察,发现艳丽耳草野外居群同时存在长花柱型花和短花柱型花,长/短花柱型花的数量比例为1:1。两型花具有精确的交互式雌雄异位特征,并且该特征与花冠长度相关性显著。长/短花柱型花的柱头裂片长度、花粉大小及淀粉含量等具有二型性。花粉体外培养时花粉萌发率及花粉管生长速率无显著性差异。人工授粉后,艳丽耳草长/短花柱型花型间异交花粉管生长形态正常,授粉24h后花粉管均已进入子房。而长/短花柱型花在自交及型内异交下均表现为不亲和,花粉管生长停止于柱头,花粉管顶端累积胼胝质并膨大。艳丽耳草没有无融合生殖现象,型间人工辅助异交授粉结实率为100%,显著高于自然结实率。本研究结果表明,艳丽耳草是典型的二型花柱植物,并具有异型自交不亲和系统。

白花蛇舌草提取物诱导人胃癌MKN-45细胞凋亡

为探究白花蛇舌草提取物对人胃癌MKN-45细胞凋亡的影响。用不同浓度的白花蛇舌草提取物处理MKN-45细胞,然后在光学显微镜和激光共聚焦显微镜下观察数量和形态。流式细胞仪检测细胞凋亡率,RT-qPCR和Western Blot检测凋亡相关基因Bax和Bcl-2的m RNA和蛋白表达水平。结果表明,不同浓度的白花蛇舌草提取物处理48 h后,在显微镜下观察到细胞体积缩小、细胞核裂解和染色质形态变化。细胞凋亡率随着药物浓度的增加而增加,用30μg/mL白花蛇舌草提取物处理后细胞凋亡率达到23.6%,Bax基因表达水平显著增加,Bcl-2基因表达水平显著降低。综上所述,白花蛇舌草提取物可诱导人胃癌MKN-45细胞凋亡,具有潜在的医学和药用价值。