Anti-fatigue Effect of Water Extract from Centella asiatica(L.)Urban on Mice

[Objectives]To study the anti-fatigue effect of water extract from Centella asiatica(L.)Urban on mice.[Methods]After intragastric administration of low,medium and high concentration of water extract from Centella asiatica(L.)Urban for 14 d,the rotarod time and the content of serum lactic acid in mice were determined,respectively.[Results]Compared with the control group,the rotarod time of mice in the low and medium concentration groups was significantly prolonged(P<0.05),but there was no significant difference between the control group and the high concentration group;the content of serum lactic acid in the medium concentration group was significantly lower than that in the control group(P<0.01),but there was no significant difference between low concentration group and high concentration group and the control group.[Conclusions]A certain concentration of water extract from Centella asiatica(L.)Urban had a good anti-fatigue effect.

The Stimulatory Effects of the Antimicrobial Agents Bavistin, Cefotaxime and Kanamycin on <i>In Vitro</i>Plant Regeneration of <i>Centella asiatica</i>(L.)—An Important Antijaundice Medicinal Plant

Antimicrobial agents such as bavistin, cefotaxime and kanamycin were evaluated for their effects on the rapid shoot regeneration from nodal explants of Centella asiatica (L.). Filter sterilized bavistin (250 mg/L) was augmented alone and in combination with cytokinins such as BAP and TDZ into the media to trace the effect on regeneration. On this basis, the potential use of bavistin (150 mg/L) along with BAP (2.0 mg/L) was evaluated which showed the maximum shoot number (6.6) and shoot length (4.4 cm) respectively. Cefotaxime at the concentration of 100 μM/L was found to be effective to obtain the maximum shoot number formation (5.8) with the regeneration frequency (90%). Kanamycin at the concentration of 80 μM/L induced maximum shoot regeneration (5.12). Kanamycin at 100 μM/L or at higher concentrations reduced the shoot regeneration. The best rooting response was noticed when in vitro regenerated microshoots were transferred to the rooting medim which was supplemented with IBA (2.0 mg/L). This combination generates 90% of regeneration frequency and maximum number of roots per shoot (14.2) and high root length (4.2 cm). The rooted plants were acclimatized and transferred to field for survivalance. The addition of antibiotics was found to be more effective and safer for using since their effects on regeneration were found to be negligible.

A new triterpene and a saponin from Centella asiatica

A new triterpene and a saponin, named 2α,3β23-trihydroxyurs-20-en-28-oic acid （1） and 2α,3β,23-trihydroxyurs-20-en-28-oic acid O-α-L-rhanmopyranosyl-（1→4）-O-β-D-glucopyranosyl-（1→6）-O-β-D-glucopyranosyl ester （2）, have been isolated from the aerial part of CenteUa asiatica. Their structures were elucidated by spectral methods, including 2D-NMR spectra.

Antifertility properties ofCentella asiatica ethanolic extract as a contraceptive agent: Preliminary study of sperm proteomic

Objective:To investigate the antifertility properties ofCentella asiatica L. ethanolic extract and identify the sperm proteomic changes in treated male rats.Methods: A total of 32 male Sprague-Dawley rats were divided into two groups: control group (n=16) received distilled water and treatment group (n=16) which received 300 mg/kg ofCentella asiatica L. extract for 42 d. After the treatment period, the number of implantation sites was recorded and the sperm proteomic changes were analysed by 2D gel electrophoresis. In addition, the expression of protein spots was quantified by MALDI-TOF analysis.Results:Centella asiatica L. extract resulted in low number of implantation sites in the treatment group (100.00±2.82) compared to the control group (183.00±2.14). The percentage of infertile male rats in the treatment group was higher (43.75%) compared to the control group (18.75%). Proteomic analysis showed the expression of protein spots identified in the treatment group decreased with 234 spots compared to the control group with 282 spots.Conclusions:The results revealed that there was antifertility activity in the male rats with the administration ofCentella asiatica L. ethanol extract and the identified proteins could provide understanding on the adverse effect ofCentella asiaticain male reproductive system.

Vegetative Growth and Molecular Identification of Fusarium equiseti Isolated from Wilt Disease of Centella asiatica L. in Bangladesh

Centella asiatica (L.) is commonly known as Thankuni plant and has ethnobotanical importance in Bangladesh. Present experiment was conceded to investigate the wilt disease of C. asiatica, vegetative growth and molecular characterization of pathogenic fungi. Pathogenic fungus, Fusarium equiseti was identified as a causal agent of wilt disease in C. asiatica. The effect of culture media on the mycelial growth of F. equiseti showed the highest (89.25 mm) on potato dextrose agar (PDA) medium followed by carrot agar (CA) medium and the lowest growth (40.25 mm) was measured in HA medium. The optimal temperature and pH for mycelial growth of F. equiseti were 30&deg;C and 7, respectively. The genetic variation of the selected species of fungi, the internal transcribed spacer (ITS) region was amplified using ITS4 and ITS5 primers and sequenced. The PCR product of the ITS region of F. equiseti was 535 bp. Phylogenetic tree of thirty-seven strains of Fusarium sp. based on the nucleotide sequences of the ITS region using the neighbor-joining method with 1000 bootstrapping indicated that 98% - 100% identity with MN886590.1 JUF0046 (F. equiseti). ITS sequences are generally constant, or show little variation within species, but vary between species in a genus. The ITS region is relatively short and can be easily amplified by PCR using universal single primer pairs. Genetic distance exhibited high level of similarity with identical ITS sequences. To date, no published research articles are found on the molecular identification of F. equiseti, the causal agent of fusarium wilt disease of C. asiatica in Bangladesh.

HPLC-ELSD法测定积雪草中积雪草苷及羟基积雪草苷的含量

积雪草提取物通常被用作化妆品原料,有美白、抗氧化等功效,发挥此功效的主要物质有积雪草苷、羟基积雪草苷。采用高效液相色谱-蒸发光散射检测(HPLC-ELSD)技术,建立了测定积雪草中积雪草苷和羟基积雪草苷含量的方法。选用Unitary C18色谱柱(4.6×250 mm,5μm),以乙腈-水为流动相,梯度洗脱,流速1 mL/min,柱温为30℃,采用Agilent 1260 InfinityⅡ蒸发光散射检测器(漂移管温度70℃,载气流速1.6 L/min)。研究表明,积雪草苷和羟基积雪草苷质量浓度分别在100~702 mg/L,105~738 mg/L范围内,峰面积与质量浓度线性关系良好,相关系数分别为0.9996和0.9997。积雪草苷和羟基积雪草苷平均加样回收率分别为101.20%,98.30%。相对标准偏差(RSD)均在3%以内。方法简单,准确度高,分离效果好,干扰少,可用于积雪草中积雪草苷和羟基积雪草苷的含量测定。

积雪草药材HPLC指纹图谱的研究

目的:研究积雪草药材的高效液相色谱指纹图谱,为科学评价及有效控制药材质量提供可靠的方法。方法:利用高效液相色谱法,梯度洗脱,测定了16个不同产地的积雪草药材样品。色谱柱为Alltech C18柱(250 mm×4.6 mm5,μm),流动相为乙腈-水,流速为1.0 mL/min,检测波长为205 nm。结果:建立了积雪草药材的HPLC指纹图谱,并检测了16批积雪草药材样品,标定了15个共有峰,通过与对照品的保留时间比对及LC-ESI-MSn进行结构解析,其中5～10号峰分别鉴定为羟基积雪草苷、积雪草苷、槲皮素、山萘酚、羟基积雪草酸、积雪草酸。经相似度计算,各产地药材之间相似性较差。结论:该方法准确可靠,重现性好,可作为控制积雪草药材内在质量的标准。

基于LC-MS积雪草不同部位中化学成分分析

为获取积雪草不同部位中的化学成分及其相对含量,以积雪草叶片、茎和根为材料,利用LC-MS技术进行非靶向检测并进行代谢组学分析。结果显示:(1)积雪草中共检测出440种代谢产物,其中氨基酸及其衍生物、生物碱、类脂化合物、糖与苷类以及萜类等5类化合物数量较多,分别有60、54、52、50和49种,还检测到21种有机酸,15种甾族化合物,12种黄酮类化合物,此外还有极少数的简单苯丙素类、维生素及木质素、香豆素和挥发油类物质;(2)对积雪草中2种重要的成分积雪草苷和积雪草酸分析发现,在3个部位中积雪草酸相对含量均明显高于积雪草苷,且均在叶片中含量最高,根中最低;(3)12种黄酮类代谢产物中漆树黄酮等4种物质在积雪草根、茎、叶中相对含量接近;山奈酚等3种代谢产物在叶片中的相对含量显著高于根和茎,其中山奈酚在其叶中相对含量最高;橙皮素、黄芩素、根皮苷及黄芪苷总体相对含量较低,其中以黄芩素和根皮苷最低,其余几种黄酮类物质在积雪草不同部位中的相对含量也存在一定差异。

Prophylactic effects of asiaticoside-based standardized extract of Centella asiatica(L.) Urban leaves on experimental migraine: Involvement of 5HT1A/1B receptors

The present study aimed at evaluation of prophylactic efficacy and possible mechanisms of asiaticoside(AS) based standardized extract of Centella asiatica(L.) Urban leaves(INDCA) in animal models of migraine. The effects of oral and intranasal(i.n.) pretreatment of INDCA(acute and 7-days subacute) were evaluated against nitroglycerine(NTG, 10 mg·kg-1, i.p.) and bradykinin(BK, 10 μg, intra-arterial) induced hyperalgesia in rats. Tail flick latencies(from 0 to 240 min) post-NTG treatment and the number of vocalizations post-BK treatment were recorded as a measure of hyperalgesia. Separate groups of rats for negative(Normal) and positive(sumatriptan, 42 mg·kg-1, s.c.) controls were included. The interaction of INDCA with selective 5-HT1 A, 5-HT1 B, and 5-HT1 D receptor antagonists(NAN-190, Isamoltane hemifumarate, and BRL-15572 respectively) against NTG-induced hyperalgesia was also evaluated. Acute and sub-acute pre-treatment of INDCA [10 and 30 mg·kg-1(oral) and 100 μg/rat(i.n.) showed significant anti-nociception activity, and reversal of the NTG-induced hyperalgesia and brain 5-HT concentration decline. Oral pre-treatment with INDCA(30 mg·kg-1, 7 d) showed significant reduction in the number of vocalization. The anti-nociceptive effects of INDCA were blocked by 5-HT1 A and 5-HT1 B but not 5-HT1 D receptor antagonists. In conclusion, INDCA demonstrated promising anti-nociceptive effects in animal models of migraine, probably through 5-HT1A/1B medicated action.

积雪草多糖制备、结构表征及其抗氧化活性

该文采用热水浸提法提取积雪草多糖,并对其进行结构表征和抗氧化活性研究。经过提取纯化得到积雪草多糖[polysaccharide of Centella asiatica(L.)Urban,CAP],相对分子质量为1.81×106Da,其中总糖含量为(91.35±1.12)%,蛋白质含量为(0.72±0.08)%,糖醛酸含量为(16.88±0.53)%,无还原糖;CAP在溶液中的平均粒径为(325.13±6.05)nm,Zeta电位为(-15.24±0.38)mV,结果表明CAP是一种酸性阴离子多糖;离子色谱表明CAP主要由岩藻糖、鼠李糖、阿拉伯糖、半乳糖、葡萄糖、木糖、甘露糖、葡萄糖醛酸、古罗糖醛酸组成,其摩尔比为0.01∶0.14∶0.44∶0.69∶1.00∶0.03∶0.02∶0.06∶0.11。傅里叶变换红外光谱和核磁共振波谱检测表明CAP是一种含有α-糖苷键和β-糖苷键的吡喃型多糖,并检测出5种糖残基,异头氢/碳信号分别为δ 5.36/99.93、5.26/109.45、5.20/107.62、4.60/104.57、4.45/103.65;抗氧化试验表明CAP清除ABTS+·的效果较好,而清除·OH的效果较差。

积雪草皂苷生物合成基因筛选与表达量分析

目的:筛选积雪草皂苷生物合成相关的关键酶基因,并利用荧光定量PCR(qRT-PCR)技术进行验证。方法:采用HPLC对积雪草不同部位进行皂苷含量测定;利用转录组学、KEGG富集分析等手段挖掘积雪草皂苷生物合成关键酶基因;采用qRT-PCR技术验证挖掘出的关键酶基因在不同组织中的表达模式,并使用皮尔逊(Pearson)法分析关键酶与皂苷类成分的相关性。结果:在根、茎、叶、叶柄、果实这5个不同组织中,叶的积雪草皂苷含量最高;挖掘遴选出积雪草皂苷生物合成相关的关键酶基因4个,分别为氧化鲨烯环化酶(CaOSC1、CaOSC2)、鲨烯环化酶(CaSE)、鲨烯合酶(CaSS);这4个关键酶基因均在叶中表达量最高。结论:积雪草不同组织的皂苷含量与4个关键酶基因表达量关系紧密,该结果为今后探究积雪草皂苷生物合成与积累的调控机制积累了必要的研究基础。

积雪草化学成分研究

目的研究积雪草Centella asiatica的化学成分。方法采用各种色谱技术进行分离纯化,通过理化性质和波谱分析进行结构鉴定。结果从积雪草中共分离得到14个化合物,其结构鉴定为β-谷甾醇(Ⅰ)、二十六醇辛酸酯(Ⅱ)、山柰酚(Ⅲ)、槲皮素(Ⅳ)、胡萝卜苷(Ⅴ)、香草酸(Ⅵ)、丁二酸(Ⅶ)、积雪草酸(Ⅷ)、羟基积雪草酸(Ⅸ)、ter-minolic acid(Ⅹ)、积雪草苷(Ⅺ)、积雪草二糖苷(Ⅻ)、羟基积雪草苷、积雪草苷-B。结论化合物为一新化合物;羟基积雪草酸和terminolic acid、羟基积雪草苷和积雪草苷-B为两对同分异构体。

酶法提取积雪草苷

采用酶法提取积雪草中的积雪草苷,对酶的种类、用量、积雪草与溶剂水的料液质量比、酶解液初始pH、酶解温度、酶解时间等因素进行了考察,结合正交实验设计筛选出酶解最优工艺参数,并将酶法提取结果与其他方法比较.研究结果表明,优化的提取条件为纤维素酶与底物积雪草质量比m(E)∶m(S)=1∶50,料液质量比为(积雪草与溶剂水的质量比)1∶25,pH 6.0,酶解温度40℃,酶解时间1.5 h.与传统提取方法相比,酶法提取速率快,工艺条件温和,得率高,是一种良好的提取方法.

积雪草提取物对抑郁模型动物行为的影响

目的观察积雪草提取物对抑郁大鼠、小鼠干预后行为学的改变。方法 60只Wistar大鼠随机分为6组,每组1 0只,分别为对照组、模型组、氟西汀组、积雪草提取物高剂量组(成分10.08mg+成分24.48 mg/kg),中剂量组(成分10.04mg+成分22.24mg/kg),低剂量组(成分10.02 mg+成分21.12 mg/kg)。采用慢性不可预知性温和应激刺激制备大鼠抑郁模型,糖水消耗量检测积雪草提取物抗抑郁效应。60只昆明小鼠,随机分为6组,分组情况同上。自主活动、强迫游泳、悬尾实验检测积雪草提取物对小鼠抑郁样行为的影响。结果积雪草提取物高、中、低剂量组能够明显增加大鼠糖水消耗量,与模型组比较(P<0.01)。积雪草提取物高、中、低剂量组能明显缩短悬尾实验和强迫游泳小鼠不动时间。结论积雪草提取物对大、小鼠抑郁样行为有一定的改善作用。

高效液相法测定积雪草配方颗粒中积雪草苷的含量

目的:建立测定积雪草配方颗粒剂中积雪草苷的HPLC含量分析方法。方法:采用C18柱,RP-HPLC法,水-乙腈为流动相,梯度洗脱,检测波长205 nm。结果:积雪草苷在0.02-0.51 mg·mL-1范围内,峰面积与浓度呈线性关系,r=0.9999,高、中、低浓度的平均回收率分别为99.6%,101.7%,102.2%;RSD分别为2.9%,1.7%,4.9%(n=3)。结论:该方法简便,结果准确,适用于积雪草配方颗粒剂的质量控制。

肠杆菌FM-1强化积雪草修复镉污染土壤机理

分析了肠杆菌FM-1的耐镉性及促生特性,并研究了肠杆菌FM-1对矿山型和非矿山型积雪草镉富集及根际土壤pH值的影响.结果表明,肠杆菌FM-1对镉有较强的耐受性,其分泌的吲哚乙酸含量,铁载体A/Ar值和溶磷能力分别达到(72.85±0.62)mg/L,0.21±0.01和(143.33±2.13)mg/L,表明肠杆菌FM-1具有良好的促生能力.将肠杆菌FM-1接种于采自广西柳州泗顶铅锌矿周边4种类型的土壤中,均不同程度提高了矿山型和非矿山型积雪草对镉的富集.其中,下游区土壤接种肠杆菌FM-1后,矿山型积雪草茎和叶中镉含量分别增加了87.90%～161.84%和21.85%～76.42%,非矿山型叶片中的镉含量增加了5.84%～35.20%.研究表明肠杆菌FM-1促进了积雪草对镉的富集.同时,肠杆菌FM-1的添加在一定程度上影响了积雪草根际土壤的pH值,显著降低了下游区矿山型积雪草根际土壤中的pH值.

4种中药体外抗结核分枝杆菌H_(37) RV的研究

目的研究中药夏枯草、积雪草、筋骨草和青蒿素的体外抗结核分枝杆菌H37RV的活性。方法采用改良罗氏固体培养基试管培养法进行活性检测。结果夏枯草醇提物抗结核效果明显;积雪草苷片、筋骨草及青蒿素均无效。结论夏枯草醇提物抗结核菌效果优于水提物;积雪草醇提物有微弱抑制结核杆菌活性,但其有效成分可能不是积雪草苷;青蒿素及二氢青蒿素无抑制结核分枝杆菌的活性。

民族药届招觅的生药学鉴定

目的对多民族常用的植物药届招觅进行生药学鉴定,为其鉴别及应用提供科学依据。方法采用原植物、性状、显微鉴别的方法。结果届招觅在原植物、性状、显微等方面具有专属性的特征。结论通过原植物、性状、显微鉴别能够很好地鉴定该药材。

制备型高效液相色谱法从积雪草提取物中分离纯化积雪草甙和羟基积雪草甙对照品

积雪草甙和羟基积雪草甙是积雪草及其相关制品质量控制的两个指标成分,本研究利用制备型高效液相色谱从积雪草提取物中同时分离纯化得到这两个成分。对制备色谱的流动相组成、流速、进样量和检测波长等制备参数进行了优化。采用的色谱柱为C18柱(50mm×200mm,5μm);流动相为甲醇-水(体积比为60∶40),流速100mL/min;二极管阵列检测器在220nm检测;进样体积为1.5mL。在20min的运行时间内,积雪草甙和羟基积雪草甙与干扰成分得到很好的分离,产品纯度达到98%以上。此方法具有快速高效、产品纯度高的特点,可以用于制备积雪草甙和羟基积雪草甙对照品。

积雪草提取纯化工艺的研究

目的研究积雪草的提取纯化工艺。方法采用正交试验进行工艺优化,薄层色谱扫描法测定羟基积雪草苷含量。结果提取工艺最佳条件为:水提取2次,第1次加10倍量水提取2h,第2次加8倍量水提取1.5h。纯化工艺最佳条件为壳聚糖用量0.4mL/g,药液浓缩比例1.2:1,絮凝温度70℃。结论本方法可节省原料,缩短工时,降低成本,利于工业化大生产。