基于质量综合评价的积雪草干燥方法筛选研究

目的探讨不同干燥方式对药材质量的影响,筛选积雪草适宜干燥方法。方法分别采用热风干燥、晒干、晒半干后热风干燥、阴干、微波以及真空干燥对积雪草全草鲜品进行干燥加工,并测定干燥时间和折干率;参考药典方法对积雪草药材性状特征、鉴别、检查、浸出物含量进行分析;采用HPLC法测定样品中积雪草苷、羟基积雪草苷、积雪草酸、羟基积雪草酸、山柰酚-3-O-芸香糖苷、山柰酚和槲皮素的含量;并采用加权评分法进行多指标综合评价排序。结果不同干燥方法耗时差异大,其中晒干、阴干和50℃真空干燥超过100 h,微波干燥低至1.5 h;折干率均值为24.83%;不同干燥方法对药材性状影响主要反映在色泽和气味,其中50～70℃热风干燥色泽较佳,为浅绿色,80、85℃热风干燥和微波干燥气味也发生了显著变化;水分和灰分含量虽有一定差异,但均符合药典标准;干燥方法对浸出物也有一定影响,晒干方式的浸出物含量达到最大,为45.70%,阴干方式的浸出物含量最小,为29.67%;HPLC法测定的7个有效成分含量总和最高的是较为温和的干燥方式阴干,为83.032 mg/g,最低的为75℃热风干燥,为40.982 mg/g;羟基积雪草苷和积雪草苷总和含量最高的为80℃热风干燥,最低的为75℃热风干燥;加权评分排在前3位的分别为70℃热风干燥、晒半干后50℃热风干燥、50℃热风干燥,85℃热风干燥排在末位。结论积雪草产地的适宜干燥方法为70℃热风干燥。     

HPLC-CAD法分离测定三金片中同分异构体积雪草苷B和羟基积雪草苷

目的建立一种高效液相色谱-电喷雾检测器(HPLC-CAD)法分离测定三金片中积雪草苷B和羟基积雪草苷2个同分异构体含量的方法。方法采用Thermo AcclaimTM C30色谱柱,甲醇-乙腈-水(35∶8∶57)为流动相,柱温35℃,CAD气化温度为50℃,采集频率为10 Hz,分离测定积雪草苷B和羟基积雪草苷的含量。结果积雪草苷B和羟基积雪草苷线性关系良好,r分别为0.999 8、0.998 4,线性范围分别为0.240 7～2.406 9、0.573 1～5.730 8μg,平均加样回收率分别为100.26%、98.32%,RSD分别为0.69%、1.78%;11批样品中积雪草苷B为1.65～3.18 mg/g,羟基积雪草苷为3.49～7.90mg/g。结论该方法简便、灵敏、准确度高,能有效分离测定积雪草苷B和羟基积雪草苷2个同分异构体,可用于三金片现行药典标准的提高完善。     

积雪草人工种植技术的初步研究

目的研究积雪草Centella asiatica人工种植技术。方法以积雪草产量及药材中积雪草苷和羟基积雪草苷的总量为评价指标,考察土壤类型、种植密度、基肥、施肥方式及荫蔽度等不同因素对种植效果的影响。结果积雪草适合种植于泥炭土-珍珠岩(1∶1)中,种植密度为20 cm×20 cm,基肥为有机肥/复合肥(1∶1),基肥用量为7.5 g/m2,定植4个月后施尿素(18 g/m2)、5个月后施复合肥(45 g/m2),适宜荫蔽度为75%。结论本实验研究可为建立积雪草GAP种植基地奠定基础。     

Madecassoside Ameliorates Bleomycin‐Induced Pulmonary Fibrosis in Mice by Downregulating Collagen Deposition

This study aimed to explore the protective effects of madecassoside (Mad), a triterpenoid saponin isolated from Centella asiatica herbs, on experimental pulmonary fibrosis (PF) and underlying mechanisms. PF model was established in mice by endotracheal instillation with bleomycin (5 mg/kg). Mice were orally administered with Mad (10, 20, 40 mg/kg) and prednisone (5 mg/kg) for 7 or 21 days. Mad (20, 40 mg/kg) significantly improved lung pathological changes and reduced collagen deposition. In the aspect of collagen synthesis, Mad (20, 40 mg/kg) reduced the expressions of α‐smooth muscle actin and transforming growth factor‐β1 (TGF‐β1), and inhibited the phosphorylations of Smad2 and Smad3 in the lung tissues. However, in vitro, Mad showed little effect on TGF‐β1‐induced phosphorylation of either Smad2 or Smad3 in primary mouse lung fibroblasts. Moreover, Mad (20, 40 mg/kg) attenuated oxidative damage and inflammation presented at the early stage of PF, evidenced by reduced total leukocytes in the bronchoalveolar lavage fluid, decreased myeloperoxidase activity and malondialdehyde level, and increased super‐oxide dismutase activity and glutathione level in lung tissues. On the other hand, Mad (40 mg/kg) elevated the matrix metalloproteinase 1/tissue inhibitor of metalloproteinase 1 ratio in lung tissues of PF mice mainly by downregulating tissue inhibitor of metalloproteinase 1 expression. The present study demonstrated that Mad can ameliorate PF by preventing the deposition of extracellular matrix, which might be achieved mainly through attenuating inflammation and oxidative stress and consequent TGF‐β1 overexpression. Copyright © 2014 John Wiley & Sons, Ltd.     

手性流动相添加剂法测定积雪草中积雪草总苷的量及其总苷特征图谱

目的 以β-环糊精为流动相添加剂,拆分积雪草药材及其总苷中羟基积雪草苷和积雪草苷,建立羟基积雪草苷和积雪草苷的定量测定方法,同时建立积雪草总苷的高效液相色谱特征图谱。方法 采用 Dikma Diamonsil^TM C₁₈ (250 mm×4.6 mm, 5 μm) 色谱柱;流动相为乙腈-2 mmol/L β-环糊精溶液 (24∶76);柱温 30 ℃;体积流量 1.0 mL/min;检测波长 205 nm;理论塔板数按积雪草苷峰计算应不低于 4 000。结果 β-环糊精的加入很好地分离积雪草药材及其总苷中的羟基积雪草苷和积雪草苷,满足了定量分析的要求,其中有效成分羟基积雪草苷和积雪草苷的线性范围分别为 0.181～18.1 μg (r=0.999 98) 和 0.195～19.5 μg (r=0.999 98) ,积雪草药材的平均加样回收率 (n=9) 分别为 101.2% 和 101.6%,积雪草总苷的平均加样回收率 (n=9) 分别为 100.3% 和 99.3%,11 批次的积雪草总苷样品的高效液相色谱特征图谱中确定 6 个共有峰作为定性的指标峰。结论 本法简便快速、结果准确、重现性好,可用于积雪草药材及其总苷的质量控制。     

Induction of keratinocyte migration by ECa 233 is mediated through FAK/Akt,ERK, and p38 MAPK signaling

Centella asiatica is widely considered the most important medicinal plant for treating and relieving skin diseases. Recently developed standardized extract of Centella asiatica ECa 233 has demonstrated positive effects on wound healing of incision and burn wound in rats. However, knowledge associated with wound healing mechanism of ECa 233 was scare. Therefore, this study aimed to investigate the effect and underlying molecular mechanisms of ECa 233 on the migration of a human keratinocyte cell line (HaCaT) using scratch wound healing assay. Formation of filopodia, a key protein in cell migration as well as signaling pathways possibly involved were subsequently assessed. It was found that HaCaT cell migration was significantly enhanced by ECa 233 in a concentration‐ and time‐dependent manner. The filopodia formations were accordingly increased in exposure to ECa 233 at concentrations of 0.1–100 μg/ml. Furthermore, ECa 233 was found to significantly upregulate the expression of Rac1 and RhoA and to induce phosphorylation of FAK and Akt as well as ERK and p38 MAPK. Taken all together, it is suggestive that ECa 233 induces cell migration and subsequently promotes wound healing activity, through the activation of FAK, Akt, and MAPK signaling pathways thereby supporting the role of ECa 233 to be further developed for the clinical treatment of wound.     

羟基积雪草苷对氧化应激条件下黑素细胞的保护作用及其诱导自噬机制的研究

目的：积雪草是一种传统中医药物,具有很多医学作用,如创伤愈合和抗氧化。中医也发现它可用于白癜风复色和炎症后色素沉着。本研究评估羟基积雪草苷对氧化应激下人黑素细胞作用效果及机制。方法：正常的人黑素细胞予 0.01 mmol/L 过氧化氢处理,再予不同浓度的羟基积雪草苷（0、10、50、100 μg/mL）。评估人黑素细胞树突的回缩速率,流式细胞术及细胞内游离钙浓度([Ca2+]i)检测线粒体膜电位（MMP）,透射电子显微镜(TEM)观察线粒体超微结构,吖啶橙染色检测自噬,蛋白质印迹检测自噬体形成的指标LC3-Ⅱ/LC3-Ⅰ 的比值。结果：在过氧化氢引发的氧化应激条件中,羟基积雪草苷在一定浓度下抑制了黑素细胞树突的回缩,提高了膜电位并减少了 [Ca2+]i的积累,减弱了线粒体的损伤,增强了自噬活动。结论：人黑素细胞遭受氧化应激损伤时,羟基积雪草苷可通过自噬激活起到抗氧化作用。因此,羟基积雪草苷可成为氧化应激引起的白癜风的有效治疗手段。     

Madecassoside induces apoptosis of keloid fibroblasts via a mitochondrial‐dependent pathway

Centella asiatica herb is a frequently prescribed drug in southeastern Asia and China that can simultaneously facilitate wound healing and prevent scar formation. The active constituents and underlying mechanisms responsible for these biphasic actions remain unknown. Previous studies in our laboratory demonstrated that madecassoside, the main active triterpene saponin in C. asiatica herbs, was able to treat trauma‐caused scars in rabbit ear and facilitate burn wound healing in mice. As the formation and progression of keloid scars is closely related to the excessive proliferation and insufficient apoptosis of dermal fibroblasts, the effects of madecassoside on the proliferation and apoptosis of keloid fibroblasts (KFs) were examined in the present study. Primary KFs, originating from human earlobe keloids, were purified and cultured, and then treated with increasing concentrations of madecassoside (10, 30, and 100 µM) for 48 h. Madecassoside inhibited the proliferation of KFs in a time‐and concentration‐dependent manner, and induced KF apoptosis as revealed by Hoechst 33258 staining and flow cytometry analysis. Furthermore, madecassoside activated caspase‐9 and caspase‐3 rather than caspase‐8, depolarized the mitochondrial membrane potential, and regulated expression of Bcl‐2 family members in KFs. These findings suggest that madecassoside induced the apoptosis of KFs through a mitochondrial‐dependent pathway. Drug Dev Res 72: 315–322, 2011. © 2010 Wiley‐Liss, Inc.     

积雪苷霜软膏质量标准研究

目的对积雪苷霜软膏原标准进行修订和提高。方法将原标准中薄层色谱鉴别项下的"显相同的蓝褐色斑点"修订为"显相同颜色的斑点"。建立了以乙腈-2mmol.L-1倍他环糊精溶液(24∶76)为流动相的HPLC含量测定方法。结果羟基积雪草苷和积雪草苷线性范围分别是0.8573~12.8589μg、0.8607~12.9102μg,二者平均回收率分别为97.6%和98.8%,RSD为0.48%和1.4%。结论所用方法简单、专属性强,适用于积雪苷霜软膏含量控制。     

高效液相色谱法测定三金胶囊中积雪草苷和羟基积雪草苷含量

目的建立高效液相色谱含量测定方法,替代原薄层扫描法测定三金胶囊中积雪草苷的含量,以更好地控制该制剂质量。方法色谱柱为Spherisorb ODSC l8柱（250mmx4．6mffl,5仙m）柱,流动相为乙腈－2mmol／LB－环糊精溶液（25：75）,检测波长为205nm,流速为1．0mL／min。结果积雪草苷和羟基积雪草苷质量浓度线性范围分别为16．86～101．18,17．89～107．33Ixg／mL,平均回收率分别为97．36％和97．99％,RSD分别为1．65％和1．43％。结论高效液相色谱法灵敏、准确、重现性好,比薄层扫描法能更好地控制三金胶囊的质量。