Cytotoxic and aromatic constituents from Salvia miltiorrhiza

As part of an ongoing study of traditional Chinese medicinal plants, the root tissue of Salvia miltiorrhiza was further investigated for its chemical constituents. Five naturally occurring products along with 13 known constituents were isolated from an ethyl acetate-soluble portion of its ethanol extract. Their structures were elucidated by means of spectroscopic methods. Some selected compounds were also evaluated for biological activity.     

Protection of a polysaccharide from Salvia miltiorrhiza, a Chinese medicinal herb, against immunological liver injury in mice

This study was designed to evaluate the hepatoprotective effects of S. miltiorrhiza polysaccharides (SMPS) in immunological liver injury induced by Bacille-Calmette-Guerin (BCG) and lipopolysaccharide (LPS) in mice. SMPS effectively improved the liver index, spleen index and thymus index, reduced the serum levels of alanine aminotransferase, aspartate aminotransferase and nitric oxide, and restored liver homogenate contents of tumor necrosis factor-alpha and interleukin-1beta. The histopathological analysis suggested that SMPS reduced the degree of liver injury. The results suggest that SMPS play a protective role against immunological liver injury, which may have important implications for our understanding on the immunoregulatory mechanisms of polysaccharides.     

Antioxidant and cardioprotective effects of Danshensu (3-(3, 4-dihydroxyphenyl)-2-hydroxy-propanoic acid from Salvia miltiorrhiza) on isoproterenol-induced myocardial hypertrophy in rats

Myocardial hypertrophy has been linked to the development of a variety of cardiovascular diseases, and is a risk factor for myocardial ischemia, arrhythmias, and sudden cardiac death. The objective of the present study was to evaluate the cardioprotective effects of Danshensu (DSS), a water-soluble active component of Danshen, on cardiac hypertrophy in rats. We are the first to report that DSS reversed Cx43 down-regulation in ventricular tissue. Cardiomyopathy in rats was produced using isoproterenol (Iso) treatment (2.5 mg/kg/d, s.c.) for seven days. DSS (3 and 10 mg/kg/d, i.p.) and Valsartan (Val) (10 mg/kg, i.g.) were administered on days 4-7 of Iso-treatment. Heart weight index, hemodynamic parameters, and ECG II parameters were monitored and recorded; protein expression of left ventricular connexin 43 (Cx43) and the activity of the redox system were assayed, and arrhythmias were produced using a coronary ligation/reperfusion procedure. The results demonstrated that DSS treatment significantly decreased heart weight/body weight (HW/BW) and left ventricular weight/body weight (LVW/BW) ratios. The protective role of DSS against Iso-induced myocardial hypertrophy was further confirmed using ECG. The incidences of ventricular tachycardia and ventricular fibrillation (VT, VF) and arrhythmic scores were higher in the model group and were suppressed by DSS. DSS decreased the serum and myocardium levels of creatine kinase, lactate dehydrogenase, and malondialdehyde (CK, LDH, and MDA) and increased serum activity of superoxide dismutase (SOD) in a dose-dependent manner. Cx43 expression in the left ventricle was down-regulated, and there was significant oxidative stress in this model of cardiomyopathy. DSS reversed the down-regulated Cx43 protein levels and showed potent anti-oxidative activities and cellular protection. These data demonstrate that DSS can prevent cardiac I/R injury and improve cardiac function in a rat model of hypertrophy, the effects partially resulting from antioxidants and the protection from Cx43 expression.     

Comparative antioxidant-capacity and -content of leaves, bulbs, roots, flowers and fruit of Gethyllis multifolia L. Bolus and G. villosa Thunb. species

The total polyphenol, oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), radical cation scavenging ability, flavonol and flavanone contents were measured in the leaves, bulbs, roots, flowers and fruit (dry weight) of two natural populations of Gethyllis multifolia (Kukumakranka) and Gethyllis villosa. The flowers and fruit of G. multifolia and G. villosa showed higher, and in some cases significantly (P < 0.05) higher antioxidant activities when compared to the leaves, bulbs and roots. This, however, was not true for the flavanone content in both species. The total polyphenol content in the fruits of G. multifolia (21.54 mg GAE/g) and G. villosa (27.64 mg GAE/g) were found to be in agreement with those of raisins (28.30 mg GAE/g), blueberries (24 mg GAE/g) and strawberries (15.40 mg GAE/g). The FRAP values of G. multifolia flowers (76.66 μmole AAE/g) and fruit (91.51 μmole AAE/g) were found to be significantly (P < 0.05) higher than those of the other plant parts (16.76 to 39.08 μmol AAE/g). On the other hand, the flowers (590.23 μmol TE/g) and fruit (741.16 μmol TE/g) of G. villosa revealed a significantly (P < 0.05) higher ORAC when compared to the other plant parts (251.25 to 410.60 μmol TE/g). A strong correlation was evident in the fruit of both species between the total polyphenols and FRAP (r = 0.95), ORAC (r = 0.95) and flavonol content (r = 0.79).     

丹参环阿屯醇合酶基因克隆及表达分析

以丹参cDNA为模板,克隆了丹参环阿屯醇合酶(cycloartenol synthase,CAS)基因的cDNA序列(SmCAS),对其序列进行生物信息学分析,并采用实时荧光定量PCR方法研究了该基因在丹参不同器官及不同胁迫处理下的表达模式。结果显示:该基因全长2 346bp,包含2 271bp开放阅读框,编码756个氨基酸。预测其编码蛋白分子量为86.16kD,具有氧化鲨烯环化酶超家族典型的DCTAE结构域和QW结构域。该基因推测的氨基酸序列与人参、田七、积雪草、甘草、拟南芥的相似性分别为83%、84%、83%、81%和80%。SmCAS基因在丹参根、茎、叶、花中均有表达,在花中表达量最高;而且SmCAS基因能够响应ABA、低温和干旱的诱导。     

Protective effects of Danshensu from the aqueous extract of Salvia miltiorrhiza (Danshen) against homocysteine-induced endothelial dysfunction

Homocysteine (Hcy) is a by-product of methionine metabolism. An imbalance of Hcy in the body may lead to hyperhomocysteinemia, a condition with elevated Hcy concentration in blood that may be one of the risk factors responsible for the development of several vascular diseases (thromboembolism, atherosclerosis, stroke, vascular diseases and dementia). Radix Salvia miltiorrhiza (Danshen), a well-known Chinese medicinal herb that can activate and improve blood microcirculation, is noticeable for its beneficial effect in treating cardiovascular diseases. The present study is to demonstrate the protective effect of Danshen extract against the homocysteine-induced adverse effect on human umbilical vein endothelial cell (HUVEC). Homocysteine (5 mM) not only decreased the cell viability but also caused the disruption of capillary-like structure formation in vitro. The protective effect of Danshen aqueous extract and its active compounds on endothelial cell function were demonstrated through an in vitro tube formation assay, which mimics the new blood vessel formation. To identify the active components in the aqueous extract of Danshen, the content was characterized by instrumental analysis using high performance liquid chromatography with diode array detector (DAD) and electrospray tandem mass spectrometry (ESI-MS/MS). Interestingly, Danshen extract and its pure compounds showed different effectiveness in protecting HUVEC against Hcy-induced injury according to the following descending order: Danshen aqueous extract, 3-(3,4-dihydroxy-phenyl)-2-hydroxy-propionic acid (Danshensu), protocatechuic acid, catechin and protocatechualdehyde. We believed that such findings might provide evidence in understanding the beneficial effects of Danshen on the cardiovascular system.     

低温处理对北美冬青叶片抗氧化能力的影响

为了解低温对北美冬青(Ilex verticillata)生长的影响,对2年生扦插苗经低温处理(-6℃)后的生长和抗氧化能力进行了研究。结果表明,随着低温处理时间的延长,北美冬青叶片的叶绿素/类胡萝卜素比值和叶绿素b含量增加,而其他色素含量都呈下降趋势;相对电导率(REC)和H_2O_2含量(72 h除外)显著增加。低温处理对超氧化物歧化酶(SOD)活性影响不大,却显著降低CAT的活性。低温处理过氧化物酶(POD)活性显著提高,48 h活性达到最高;而抗坏血酸过氧化物酶(APX)活性在处理12 h达最高,然后下降。还原型抗坏血酸(As A)、还原型谷胱甘肽(GSH)含量均呈先升高再下降的趋势。因此,北美冬青在短时间内(≤24 h)能够忍受低温(-6℃),抗氧化防御系统在低温初期起关键的作用,但随时间延长(72 h)其抗氧化能力减弱。     

Extraction, preliminary structural characterization, and antioxidant activities of polysaccharides from Salvia miltiorrhiza Bunge

Four polysaccharides were extracted from Salvia miltiorrhiza Bunge using hot water, ultrasonic, alkali, and enzyme methods. Preliminary structural characterization was conducted by physicochemical property, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) analyses. Antioxidant activities against 2,2-diphenyl-1-picryl-hydrazyl (DPPH), hydroxyl, and superoxide radicals were also evaluated. The physicochemical property analysis indicated the identicalness of the polysaccharide indices obtained by the hot water and ultrasonic methods. The indices obtained by the alkali and enzyme methods were significantly different. The FTIR spectra revealed the general characteristic absorption peaks of the four polysaccharides. The SEM images demonstrated significant differences in the surface features of the different polysaccharides. The antioxidant activity assay revealed the significant antioxidant activities of three polysaccharides. Overall, the polysaccharides from S. miltiorrhiza Bunge may have potential applications in the medical and food industries.     

Effect of yeast elicitor and salicylic acid on the fluctuation of phytohormone contents in Ti-transformed Salvia miltiorrhiza cell cultures

When Ti transformed Salvia miltiorrhiza cells werecultured in a MS-NH₄ medium (MS without ammonium nitrate, containing30 g/L sucrose) at 25 °C in darkness for 18d, the total tanshinone (cryptotanshinone and tashinone IIA)contents in cultures were 12.23 mg/L and 15.07 mg/Lfor yeast elicitor (4 g/L), and yeast elicitor plus 200mol/L salicylic acid (SA) treated cultures, respectively,whereas only trace amounts of tanshinone were detected in the control or SAtreated cells. To explore the hormonal background concerning these phenomena,endogenous phytohormones were determined using ELISA kits. We found that ABA andiPAs contents in yeast elicitor plus SA treated cell cultures were increased 2.8to 9.8-fold and 3.6 to 5.8-fold respectively, while contents of GA₁and IAA were decreased by 13.2%–56.9% and 34.8%–74.6% respectively.This suggests that higher levels of ABA and iPAs combined with lower levels ofGA₁ and IAA inhibit the growth of cells, then probably stimulate thetanshinone production.     

Antioxidant activity and protective effect on DNA cleavage of extracts from Cistus incanus L. and Cistus monspeliensis L.

The genus Cistus includes many typical species of Mediterranean flora; Cistus species are used as antidiarrheics, as general remedies for treatment of various skin diseases in folk medicine and as anti-inflammatory agents. These species contain flavonoids that are considered to be chain-breaking antioxidants. In this work, we have investigated the effects of crude aqueous extracts from Cistus incanus and Cistus monspeliensis on DNA cleavage and their free-radical scavenging capacity. In addition, their effect on lipid peroxidation in rat liver microsomes was evaluated. These extracts showed a protective effect on DNA cleavage and a dose-dependent free-radical scavenging capacity; Cistus monspeliensis was more active than Cistus incanus; these results were confirmed by a significant inhibition of lipid peroxidation in rat liver microsomes. The experimental evidence, therefore, suggests that because of their antioxidant activity these extracts may offer excellent photoprotection for skin and may be useful in the treatment of human diseases where oxidative stress plays a key role. This revised version was published online in July 2006 with corrections to the Cover Date.     

Water-soluble polysaccharides from Salvia officinalis L. possessing immunomodulatory activity

A water-soluble polysaccharide complex (A) composed of galactose (17.9%), 3-O-methyl-galactose (3.0%), glucose (15.5%), mannose (8.3%), arabinose (30.4%), xylose (7.6%), fucose (2.6%), rhamnose (6.7%), and uronic acids (8.0%) has been isolated from the aerial parts of sage (Salvia officinalis L.) by cold water extraction. It showed a broad molecular-mass distribution pattern (Mw approximately 2000-93,000) with a predominance of polymers with Mw< 10,000. Ion-exchange chromatography of A afforded six polymeric fractions (A1-A6) in which arabinogalactans associated with galacturonan and/or rhamnogalacturonan backbones prevail. Sage polysaccharides were examined for their immunomodulatory activity in the comitogenic thymocyte test which is interpreted as being an in vitro correlate of adjuvant activity. The acidic polysaccharide fractions A2, A3 and A4 exhibited the highest mitogenic and comitogenic activities of all fractions tested, and relatively high SI(comit)/SI(mit) ratios approximately 3 indicate potential adjuvant properties of these polysaccharides.     

Purification of lipase from Cunninghamella verticillata by stepwise precipitation and optimized conditions for crystallization

Lipase from the oil-mill waste isolate Cunninghamella verticillata was purified by stepwise precipitation using acetone, as a sequel to our earlier conventional column chromatographic method [Gopinath et al. (2002)World Journal of Microbiology and Biotechnology 18, 449–458]. The yield of purified lipase was approx. 4-fold higher than by the previous method and the purified lipase was obtained with 70–80% acetone saturations. The enzyme was resolved as a single band with homogeneity both by native and by SDS–PAGE. The optimum condition for the lipase to crystallize was 5 g of enzyme in 0.05 M sodium phosphate buffer (pH 6.5) with 5 mM FeCl₂ and 10% 2-methyl 2,4-pentanediol (MPD).These authors equally contributed to this work     

Abietane diterpenoid dimers from the roots of Salvia prionitis

Three abietane diterpenoid dimers, bisprioterones A-C (1-3), were isolated from roots of the Chinese folk medicinal plant Salvia prionitis Hance (Labiatae). Compounds 1-3 possessed two different abietane diterpenoid skeleta, which were linked via either a C-C single bond (1 and 2) or an ether bridge (3). Their structures were elucidated by analysis of 1D and 2D NMR spectroscopic data. The structure of 1 was further confirmed by a single-crystal X-ray diffraction determination.     

Induction of phytoalexin formation in crown gall and hairy root culture of Salvia miltiorrhiza by methyl viologen

Methyl viologen (MV) (20–150 M), a generator of superoxide anion (O₂ ^–), but not hydrogen peroxide (H₂O₂) (10 M–2 mM) triggered the formation of cryptotanshinone (a phytoalexin) in cultures of both crown galls and hairy roots of Salvia miltiorrhiza. MV also inhibited the biomass formation and decreased the contents of phenolic acids in both cultures whereas H₂O₂ did not. In addition, MV and yeast elicitor induced cryptotanshinone formation synergistically only in crown gall cultures. Treatment of the cultures with 3.3 M diphenylene iodonium, an inhibitor of NAD(P)H oxidase, did not exhibit any detrimental effect on the yeast elicitor-induced cryptotanshinone formation in hairy root cultures whereas 1 M diphenylene iodonium was inhibitory on yeast elicitor-induced cryptotanshinone formation in crown gall cultures.     

放线菌Act12与腐植酸钾配施对丹参生长及其根域微生态的影响

探讨生防放线菌菌剂与腐植酸钾配施对丹参生长及其根域微生态的影响。以常规移栽处理为对照,研究小区试验中放线菌菌剂与腐植酸钾不同配施比例下对丹参生长、产量及抗根结线虫侵染的影响;并采用稀释平皿涂抹法测定丹参根区土壤、根表土壤、根外土壤及根系中细菌(B)、真菌(F)与放线菌(A)的数量,同时对优势细菌、真菌和放线菌进行了分子生物学鉴定,研究放线菌菌剂与腐植酸钾配施处理下丹参根域微生态变化。研究结果表明:1配施能增强菌剂对丹参的促生效果。菌剂与腐植酸钾配施T20处理丹参出苗率较对照提高8.7%,收获时的死亡率较对照减少39.0%;茎叶鲜质量、根鲜质量、单株根鲜质量、根干质量以及单株根干质量分别较对照增加6.1%、28.6%、11.1%、36.3%以及9.0%。2可以调整丹参植株根域土壤微生态平衡,降低有害微生物数量,增加有益微生物数量,改善微生物区系。在丹参根表土壤中,菌剂与腐植酸钾配施处理B/A值较对照降低78.4%,A/F值较对照增加95.0%。在丹参根系内,菌剂与腐植酸钾配施处理细菌数量较对照增加195.0%,未检测到真菌和放线菌存在。3在放线菌处理丹参根区、根表土壤中,有6株优势菌可能对丹参生长及抗病有益:3株优势细菌分别为硝基愈疮木胶节杆菌(Arthrobacter nitroguajacolicus)、放射型根瘤菌(Rhizobium radiobacter)和弗雷德里克斯堡假单胞菌(Pseudomonas frederiksbergensis);3株优势放线菌分别为淀粉酶产色链霉菌(Streptomyces diastatochromogenes)、砖红链霉菌(S.lateritius)和卡伍尔链霉菌(S.cavourensis)。有2株优势菌疑为有害微生物:优势细菌为耐寒短杆菌(Brevibacterium frigoritolerans),优势放线菌为肿痂链霉菌(S.turgidiscabies)。这2种菌对其他作物的有害作用已有报道。4对丹参根结线虫侵染有强烈抑制作用,可使田间根结线虫侵染率降低49.3%。生防放线菌与腐植酸钾配施处理后能明显促进丹参生长,提高丹参产量及抗病虫能力,调节丹参根域微生态平衡。     

Phytochemical profile and antioxidant activity of extracts of the peruvian peppertree Schinus areira L. from Chile

The Andean tree Schinus areira L. has multiple traditional uses, from the treatment of bronchitis and rheumatic diseases to menstrual cycle regulation and wound healing. With reported hypotensive, analgesic, antitumoral and anti-inflammatory properties, it acts predominantly against diseases related to oxidative stress. This study focuses on the antioxidant activity and phytochemical profile of the extracts of Schinus areira L.Serial extraction of the fruits was performed both by maceration and by Soxhlet. Total phenols and flavonoids were measured using the Folin-Ciocalteu method and AlCl₃, respectively. In vitro antioxidant activity was determined by FRAP and DPPH.Results were similar for both extraction methods. Primary metabolites detected included carbohydrates, proteins and amino acids; secondary metabolites included tannins, flavonoids, saponins, steroids and triterpenes. Antioxidant activity was confirmed for ethyl acetate, methanolic and aqueous extracts. The methanolic extract had both the highest polyphenol content (>195 mg GAE/ g dry weight) and the highest antioxidant activity [EC₅₀ > 476 μg/mL; >273 mg AA/g dry weight (DPPH); >301 mg AA/ g dry weight (FRAP)]. The extract does not produce macrophage cytotoxicity in RAW 264.7, which is indicated by an average cytotoxicity of 2% over 24 h.Our study serves as a starting point for future research on the pharmacological properties of Schinus areira L.     

丹参bHLH转录因子基因SmMYC2的克隆和表达分析

MYC2是植物茉莉酸类激素响应途径中的核心转录因子,在植物防御反应、次生代谢调控及生长发育过程中均有重要的调控作用。基于丹参转录组和基因组survey序列,本研究克隆了丹参转录因子MYC2的基因序列,并命名为Sm MYC2(Gen Bank No.KJ945636)。Sm MYC2基因的c DNA序列长度为1910 bp,开放阅读框(ORF)的长度为1809 bp,编码602个氨基酸,无内含子;该基因编码蛋白与烟草、番茄等植物的MYC2蛋白具有较高的同源性;Sm MYC2蛋白无跨膜结构域、信号肽等结构。实时荧光定量PCR检测结果显示,Sm MYC2在丹参的根、茎、叶、花中均有表达,并且在根和茎中的表达量更高;此外,该基因表达受茉莉酸甲酯、光和机械损伤的诱导,但受赤霉素的抑制。本实验结果为进一步探讨Sm MYC2基因在丹参中的生物学功能奠定了基础。     

Arcobacter ellisii sp. nov., isolated from mussels

As part of a study carried out for detecting Arcobacter spp. in shellfish, three mussel isolates that were Gram-negative slightly curved rods, non-spore forming, showed a new 16S rDNA-RFLP pattern with a specific identification method for the species of this genus. Sequences of the 16S rRNA gene and those of the housekeeping genes rpoB, gyrB and hsp60 provided evidence that these mussel strains belonged to an unknown genetic lineage within the genus Arcobacter. The similarity between the 16S rRNA gene sequence of the representative strain (F79-6^T) and type strains of the other Arcobacter species ranged between 94.1% with A. halophilus and 99.1% with the recently proposed species A. defluvii (CECT 7697^T). DDH results between strain F79-6^T and the type strain of the latter species were below 70% (53 ± 3.0%). Phenotypic characteristics together with MALDITOF mass spectra differentiated the new mussel strains from all other Arcobacter species. All the results indicate that these strains represent a new species, for which the name Arcobacter ellisii sp. nov. with the type strain F79-6^T (=CECT 7837^T = LMG 26155^T) is proposed.     

加拿大一枝黄花提取物的抗氧化活性研究

为进一步开发和利用加拿大一枝黄花(Solidago canadensis),该研究采用氯化铝显色法和福林酚法测定加拿大一枝黄花乙醇提取物及其不同极性萃取物中的总黄酮和总酚的含量;以抗坏血酸(Vitamin C,Vc)和二丁基羟基甲苯(BHT)为阳性对照,应用2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)、1,1-二苯基苦基苯肼(DPPH)自由基清除体系、铁离子还原能力(FRAP)法和抗氧化能力指数(ORAC)法研究其体外抗氧化活性。结果表明:乙酸乙酯萃取物中的总黄酮(202.45 mg·g~(-1))和总酚(485.94 mg·g~(-1))含量最高,且其抗氧化活性最强,并强于阳性对照Vc(P0.05)。因而,加拿大一枝黄花乙酸乙酯萃取物将有可能成为一种潜在的天然高效抗氧化剂,具有广泛的应用前景。