Studies On The Chemical Constituents And Bioactivities Of Bletilla Striata

Bletilla striata（Thunb.）Reichb.f.is one of the most commonly used traditional Chinese herb medicine.The tuber of this plant（Bai-Ji）has been widely used under the guidance of traditional Chinese medical theory due to its properties,such as astringency and hemostasis,detumescence and promoting granulation.For clinical therapy Bai-Ji is mostly used for hemoptysis,hematemesis,nosebleed,hemafecia,traumatic bleeding,carbuncle,thermal injury,rhgdi mnus et pedis and anal fissure,and so on.Modern pharmacological studies have implied that Bai-Ji had various bioactives,including anti-microbial,healing wounds,anticancer,anti-fibrosis,antioxidant,antiulcer and hemostasis.The purpose of this study was to classify constituents of Bletilla striata systematically,and to ascertain the relationship between the chemical constituents and their bioactivity.The EtOH extract of the roots and stems of Bletilla striata was suspended in H₂O,and then sequentially partitioned with petroleum ether,Et OAc,and n-Bu OH.The four fractions obtained were further isolated using various column chromatographic techniques.As results,30 compounds were obtained and identified by chromatograms（silica gel,ODS,and sephadex LH-20）and spectrum analysis（MS,1D-NMR and 2D-NMR）,including:7-hydroxy-2-methoxy-phenanthrene-3,4-dione（1）,7,2′,6′-trihydroxy-3,5′,7′-trimeth oxy-[4,10′]biphenanthrenyl-1,2-dione（2）,3-（（2′,6′-dihydroxy-2′,7′-dihydrooxepin-3′-yl）oxy）-2H-pyran-6-carbaldehyde（3）,2-hydroxymethyl-2-isopropylmalonic acid（4）,Bletillasterol A（5）,6-hydroxy-1,5,7-trimethoxyphenanthren-2-ol（6）,4-O-（6’-O-glucosyl-4”-hydroxybenzoyl）-4-hydroxybenzyl alcohol（7）,2-hydroxy succinic acid（8）,palmitic acid（9）,4-hydroxybenzylamine（10）,DL-α-palmitin（12）,5-（hydroxymethyl）-2-furaldehyde（11）,brugnanin（13）,4-（Hydroxymethyl）phenylβ-D-glucopyranoside（14）,2-methyl anthraquinone（15）,2-methoxy-4-carboxyphenol（17）,stigmasta-5,22-dien-3β-ol（16）,（3β）-3-{[2-O-（6-deoxy-α-L-mannopyranosyl）-β-D-galactopyranosyl]oxy}olean-12-en-28-oic acid（18）,2-hydroxy-1,3-dioxol-2-yl-2-hydroxyacetate（19）,（4-hydroxyphenyl）methanesulfonic acid（20）,β-sitosterol（21）,1-（4’-hydroxybenzyl）-4,8-dimethoxyphenanthrene-2,7-diol（22）,2,4-dimethoxy phenanthrene-3,7-diol（23）,4-hydroxybenzoic acid（24）,cycloneolitsol（25）,hexacosyl（2E）-3-（4-hydroxy-3-methoxyphenyl）acrylate（26）,cyclobalanone（28）,β-sitosteryl-palmitate（27）,militarine（29）,1,8-Dihydroxy-3-methoxy-6-methyl-9,10-anthraquinone（30）.Among which,5 compounds were new.Compounds were subsequently evaluated for cytotoxicity test against three human tumor cell lines（A549,MCF-7,HT-29）and HUVEC.Compound 12,11,1,23,5,18and 7 exhibited obvious activities against human tumor cells.The IC₅₀value for the seven compounds,being similar to that for cisplatin,makes them as promising antiproliferative agents against cancer.Compounds 11,1,5,6,12,23,18 and 7 were subjected to cell cycle analysis with A549 cells.The results obtained revealed that compounds 12,23,18 and 7 could induce G0/G1 phase arrest effectively after treatment for 24 hours.Compound 11 and 1 had a more strong capacity to arrest A549 cell cycle in the G0/G1 phase,while there was an increase in the G2/M phase,accompanied by a decrease in the G0/G1 phase for Compounds 5 and a significant increase in the sub-G0/G1 phase and the G0/G1 phase,and an increase in the G2/M phase for compound 18 following treatment.Furthermore,there were marked increases in ROS formation after treatment of compound 1 for 24 hours,with an approximately 4-fold increase,when compared with those of the unexposed control goup.Moreover,Compound 5,18,6 and 7 could obviously inhibit ROS production in A549 cells after treatment for 24 h.Then the method of MTT was used to examine the effects of compounds isolated from Bletilla striata on proliferation of HUVEC.The experimental data showed that compound 14,20,21 and 9 could promote HUVEC proliferation in a dose-dependent manner except compound 21.There was a significant increase in the S phase,following treatment with compound 9 and 14,suggesting that it might be associated with promoting the cell proliferation.All compounds exhibited weak bacteriostatic action with MIC>100μg/m L for in vitro antimicrobial test.Investigation on pharmacological acitivities of polysaccharides from Bletilla Striata:polysaccharides from Bletilla Striata displayed that these polysaccharides could promote burn wound healing,inidating that it might be associated with level reduction of LPS,TLR4 and related proinflammatory cytokines TNF-αand IL-6 levels.