Advances in the Study of Anoectochilus Roxburghii Extracts and Prospects for Cosmetic Application Potentials

Cosmetics made from natural ingredients are becoming more and more common because they contain bioactive compounds that provide many health benefits,are more environmentally friendly and sustainable.Anoectochilus roxburghii(AR)is a precious Chinese herb in China,possessing bioactive components including flavonoids,polysaccharides,volatile oils,kinsenoside,etc.Due to its unique bioactivity and high safety,the prospect of development and utilization is very broad.In this paper,the extraction,separation and purification techniques of AR’s flavonoid,polysaccharides,volatile oils and kinsenoside are briefly summarized.Meanwhile,we discusses their conformational relationships in cosmetics with antioxidant,whitening,antibacterial and soothing effect,and looks forward to the study of Anoectochilus roxburghii in the field of cosmetics,with a view to providing references to the development and utilization of this resource in the field of cosmetics.

Tissue Culture Rapid Propagation and Transplantation Techniques of Anoectochilus roxburghii (Wall) Lind.

[Objectives]To screen the tissue culture rapid propagation formula suitable for each tissue culture stage of Anoectochilus roxburghii( Wall) Lind. [Methods] The stem segments of Fujian A. roxburghii were used as explant to study the tissue culture rapid propagation and transplantation techniques. The comparative experiment was carried out to study the effects of different hormone concentrations on the induction of stem segments,proliferation of cluster buds,rooting and seedling hardening of A. roxburghii,and study the effects of transplantation matrix on the transplantation of A. roxburghii. [Results]MS + 0. 5 mg/L NAA + 2 mg/L BA + 20 g/L sucrose + 6 g/L agar was suitable for induction of stem segments of A. roxburghii; MS + 0. 5 mg/L NAA + 2 mg/L BA + 1 mg/L KT + 25 g/L sucrose + 6 g/L agar was most suitable for proliferation of cluster buds of A. roxburghii; MS + 1. 0 mg/L IBA + 1. 0 mg/L NAA + 1 g/L activated carbon + 50 g/L mashed banana + 25 g/L sucrose + 6 g/L agar was most suitable for rooting and seedling hardening of A. roxburghii; using peat soil: fine sand( 3∶ 1) as transplantation matrix,the survival rate was the highest. [Conclusions] The experiment results are expected to provide references for factory production of A. roxburghii.

Research Progress on Rapid Breeding Technology of Anoectochilus roxburghii Seedlings

As a traditional precious Chinese herbal medicine,Anoectochilus roxburghii has the cooling and detoxifying effects,and can nourish yin to reduce pathogenic fire,and has anti-inflammatory and analgesic effects. In this paper,we summarized the research progress in detail about the biological characteristics,resource distribution,composition,medicinal value and rapid seedling breeding technology concerning the rare Chinese herbal medicine A. roxburghii,in order to provide a reference for the in-depth study and rational development and utilization of A.roxburghii.

Combined analysis of transcriptomics and metabolomics revealed complex metabolic genes for diterpenoids biosynthesis in different organs of Anoectochilus roxburghii

Objective:Diterpenoids with a wide variety of biological activities from Anoectochilus roxburghii,a precious medicinal plant,are important active components.However,due to the lack of genetic information on the metabolic process of diterpenoids in A.roxburghii,the genes involved in the molecular regulation mechanism of diterpenoid metabolism are still unclear.This study revealed the complex metabolic genes for diterpenoids biosynthesis in different organs of A.roxburghii by combining analysis of transcriptomics and metabolomics.Methods:The differences in diterpenoid accumulation in roots,stems and leaves of A.roxburghii were analyzed by metabonomic analysis,and its metabolic gene information was obtained by transcriptome sequencing.Then,the molecular mechanism of differential diterpenoid accumulation in different organs of A.roxburghii was analyzed from the perspective of gene expression patterns.Results:A total of 296 terpenoid metabolites were identified in the five terpenoid metabolic pathways in A.roxburghii.There were 38,34,and 18 diterpenoids with different contents between roots and leaves,between leaves and stems,and between roots and stems,respectively.Twenty-nine metabolic enzyme genes with 883 unigenes in the diterpenoid synthesis process were identified,and the DXS and FDPS in the terpenoid backbone biosynthesis stage and CPA,GA20ox,GA3ox,GA2ox,and MAS in the diterpenoid biosynthesis stage were predicted to be the key metabolic enzymes for the accumulation of diterpenoids.In addition,14 key transcription factor coding genes were predicted to be involved in the regulation of the diterpenoid biosynthesis.The expression of genes such as GA2ox,MAS,CPA,GA20ox and GA3ox might be activated by some of the 14 transcription factors.The transcription factor NTF-Y and PRE6 were predicted to be the most important transcription factors.Conclusion:This study determined 29 metabolic enzyme genes and predicted 14 transcription factors involved in the molecular regulation mechanism of diterpenoid metabolism in A.roxburghii,which provided a reference for the further study of the molecular regulation mechanism of the accumulation of diterpenoids in different organs of A.roxburghii.

Screening for differentially expressed genes in Anoectochilus roxburghii (Orchidaceae) during symbiosis with the mycorrhizal fungus Epulorhiza sp.

Mycorrhizal fungi promote the growth and development of plants, including medicinal plants. The mechanisms by which this growth promotion occurs are of theoretical interest and practical importance to agriculture. Here, an endophytic fungus （AR-18） was isolated from roots of the orchid Anoectochilus roxburghii growing in the wild, and identified as Epulorhiza sp. Tissue-cultured seedlings of A. roxburghii were inoculated with AR-l 8 and co-cultured for 60 d. Endotrophic mycorrhiza formed and the growth of A. roxburghii was markedly promoted by the fungus. To identify genes in A. roxburghii that were differentially expressed during the symbiosis with AR-18, we used the differential display reverse transcription polymerase chain reac- tion （DDRT-PCR） method to compare the transcriptomes between seedlings inoculated with the fungus and control seedlings. We amplified 52 DDRT-PCR bands using 15 primer combinations of three anchor primers and five arbitrary primers, and nine bands were re-amplified by double primers. Reverse Northern blot analyses were used to further screen the bands. Five clones were up-regulated in the symbiotic interaction, including genes encoding a uracil phosphoribosyltransferase （UPRTs; EC 2.4.2.9） and a hypothetical protein. One gene encoding an amino acid transmembrane transporter was down-regulated, and one gene encoding a tRNA-Lys （trnK） and a maturase K （matK） pseudogene were expressed only in the inoculated seedlings. The possible roles of the above genes, especially the UPRTs and marK genes, are discussed in relation to the fungal interaction. This study is the first of its type in A. roxburghii.

Kinsenoside:A Promising Bioactive Compound from Anoectochilus Species

Kinsenoside is a main active component isolated from plants of the genus Anoectochilus,and exhibits many biological activities and pharmacological effects,including hepatoprotective,anti-hyperglycemic,anti-hyperliposis,anti-inflammatory,vascular protective and anti-osteoporosis effects and so on,which is contributing to its promising potency in disease treatments.This review aims to recapitulate the pharmacological functions of kinsenoside,as well as its source,extraction,identification,quantitative analysis,pharmacokinetics,synthesis and patent information.The data reported in this work can confirm the therapeutic potential of kinsenoside and provide useful information for further new drug development.

A Novel Flavonoid Glucoside from Anoectochilus roxburghii （Wall,） Lindl.

Eight compounds were isolated from the ethyl acetate- and n-butanol-soluble fractions of the ethanollc extract of the whole plant of Anoectochllus roxburghll（Wali.） Lindi. （Orchidaceee）. On the basis of spectroscopic methods, the structures of these compounds were elucidated as quercetin-7-O-β-D-[6＂-O-（trans-feruloyi）]- giucopyranosids （compound 1）, 8-C-p-HydroxybenzyiquerceUn （compound 2）, isorhamnetin-7-O-β-D- giucopyranoside （compound 3）, isorhamnetin-3-O-β-D-giucopyranoside （compound 4）, kaempferoi-3-O-β-D- giucopyranoside （compound 5）, kaempferoi-7-O-β-D-giucopyranoside （compound 6）, 5-hydroxy-3＇,4＇,7- trimethoxyfiavonoi-3-O-β-D-rutinoside （compound 7）, and isorhamnetin-3-O-β-D-rutinoside （compound 8）. Of the compounds isoisted, compound 1 was a new flavonoid giucoside and exhibited strong scavenging activity against the 1,1-diphenyi-2-picryihydrazyi free radical, whereas the ethanolic extract showed weak activity. Compounds 2-8 were obtained from this family for the first time.

不同栽培模式对金线莲产量和品质的影响

为优化金线莲(Anoectochilus roxburghii)人工栽培技术,采用正交设计试验,研究不同栽培基质、不同密度、不同肥料对金线莲产量和有效成分的影响。结果表明,栽培基质、种植密度、肥料对金线莲多糖、氨基酸、维生素C影响极显著;栽培基质和种植密度对灰分含量影响显著;肥料对Ca含量影响显著。综合3个因素对金线莲有效成分和经济产量的影响,以栽培基质为泥炭土∶椰糠∶沙=4∶2∶1,栽培密度3 cm×3 cm,肥料管理以2号肥花宝5号1 000倍液+氨基酸1 000倍液,更能促进金线莲经济效益和品质的提高。

Rapid Propagation of Three Herbal Species in a Newly Developed Temporary Immersion System

[ Objeelive] This study aimed to develop a rapid propagation method in a novel temporary immersion bioreactor system （TIS） for herbal plantlets com- pared with solid culture method. [ Method ] Three herbal species, including Dendrobium candidum Wall. Ex IJndl （D. candidum）, Anoectochilus roxburghii （Wall.）Lindl. （A. roxburghii） and Lilium davidii var. unicolor （L. davidii）, were used and tested by TIS against solid culture method. [Result] When the two culture methods were compared, the multiplication rate of D. candidum in TIS was found to be 1 ： 24.71, which was 6.55 times to those of solid culture. The multiplication rate of A. roxburghii was higher than those of the solid culture, but the plantlets was poorer than those of solid culture at the last phase in bioreactor culture, under the culture condition tested. The multiplication rate in TIS ofL. davidii was 1：17.23 whilst the rate was only 1：4.45 on solid culture, resulting larger bulbs than those in the solid culture. [ Conclusion] The TIS designed in our study could provide a potential mean for industrial production of plandets. However, the parameters vary greatly among different species, and it is to be optimized according to plant species.

不同培养基与环境条件对金线莲幼苗生长的影响

利用不同浓度的NAA和6-BA的MS培养基对金线莲(Anoectochilus roxburghii)幼苗生长的影响进行对比试验,在不同培养环境条件下进行培养,分析总结了在不同浓度培养基和不同培养环境对金线莲幼苗生长的显著差异。

金线兰黄酮相关基因的克隆与生物信息学分析

为了对金线兰查尔酮合成酶CHS和黄酮醇合成酶FLS进行基因的克隆和生物学信息分析,本研究参照测序得到的金线兰CHS与FLS基因的序列,设计特异性引物进行扩增。利用软件,对金线兰CHS和FLS蛋白的生物信息进行预测分析。结果显示,金线兰CHS基因的CDS总长度1 185 bp,编码394个氨基酸,金线兰FLS基因的CDS总长度1 035 bp,编码344个氨基酸。金线兰CHS蛋白与FLS蛋白皆是酸性、亲水性、无信号肽、非分泌、非跨膜蛋白,亚细胞定位在其他细胞器上,丝氨酸磷酸化位点具有调控二者构象变化的重要作用。在Blast上进行序列比对,发现金线兰CHS序列和FLS蛋白序列与一些兰科植物的相似度很高,前者序列同源性均高达85%以上,后者同源性高达82%和81%,构建的进化树显示了他们较近的亲缘关系。推测得到的CHS序列即为金线兰查尔酮合成酶的编码序列,FLS蛋白序列即为金线兰黄酮醇合成酶的编码序列。本研究为进一步研究金线兰中黄酮相关基因及其调控提供一定参考。