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.

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.

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.

Comparison of Differences in Total Flavonoids between Leaves and Whole Plants of Anoectochilus roxburghii

[Objectives] To compare the differences in the total flavonoids between leaves and whole plants of Anoectochilus roxburghii.[Methods] The total flavonoids in the tissue cultured leaves and whole plants of A.roxburghii were extracted by microwave-assisted extraction.The content of total flavonoids in the extracts was determined by UV spectrophotometry.[Results]The results of the content determination showed that there were significant differences in the flavonoid content between the leaves and the whole plants of A.roxburghii.[Conclusions]The content of total flavonoids in tissue cultured leaves was higher than that of whole plants.Thus,in the development and utilization of A.roxburghii,tissue cultured leaves can be used to take the place of the whole plants.

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.

Analyses on Community Composition and Diversity of Endophytic Bacteria in Different Tissues of Anoectochilus roxburghii

[Objective] This study aimed to investigate the diversity of community composition and diversity of endophytic bacteria in Anoectochilus roxburghii.[Method] By using Illumina MiSeq high-throughput sequencing technology, the 16S rDNA-V4 region of endophytic bacteria in ^4. roxburghii was sequenced. Qiime and Mothur software packages were used to sort and count the number of sequences and operational taxonomic units （OTUs） and analyze the species abundance and distribution, Alpha diversity and differences of species abundance among samples. [Result] The number of effective sequences/OTUs for each sample was 110485/247 （stem） and 98524/259 （leaf） , respectively. The rarefaction curve showed that adequate sampling was performed. The number of OTUs had almost reached a saturation point. Endophytic bacteria in A. roxburghii mainly belonged to the genera Acidovorax, Pseudomonas, Sphingomonas and Halomonas. [Conclusions] Illumi-na MiSeq high-throughput sequencing technology provides more accurate and scientific data resources for the study of endophytic bacteria in 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.

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.

不同基质对金线莲栽培的影响

以野生金线莲[Anoectochilus roburghii(Wall.)Lindl]为试验材料,研究不同基质对野生金线莲栽培驯化的影响,通过分析金线莲叶长、叶宽、叶间距、株高及叶片数等指标,以期筛选出适宜的基本培养基质。结果表明,4种基质中以椰糠的栽培效果最好,其叶长、叶宽、叶间距、株高均显著优于其他3组,不同栽培基质之间,叶长、叶宽、叶间距和株高差异显著。研究结果可为金线莲迁地保护与栽培提供技术参考和理论依据。

鼎湖山金线兰人工栽培技术研究

[目的]对鼎湖山金线兰的人工栽培技术进行研究。[方法]以组培幼苗为材料,比较自然栽种条件下5种基质处理对植株生长的影响,研究鼎湖山金线兰人工种植的适生条件。[结果]泥炭土-珍珠岩(4∶1,V/V)或泥炭土-沙子-木屑(3∶1∶1,V/V/V)混合基质适宜于金线兰的生长,幼苗成活率达到97.6%～94.3%,植株高度、单株叶片数和叶质量状况良好。结合试验地小环境自然气候变化情况,初步得出鼎湖山金线兰栽培适温为23～28℃,相对湿度在90%左右,适生pH约为4.75,栽培时设置遮阳网,基质不宜过湿。[结论]该试验对鼎湖山金线兰的人工栽培技术进行研究,为鼎湖山金线兰的进一步开发利用提供了依据。

GC-MS法对金线兰挥发油化学成分的分析

利用索氏提取法和超声辅助提取法,分别通过非极性溶剂(石油醚)、极性溶剂(乙醇)来提取金线兰[Anoectochilus raxburghii(Wall.)Lindl]挥发油,并采用气相色谱-质谱联用法测定化合物组成。结果表明,石油醚索氏提取法效果最佳,共检出34种成分,鉴定出26种化合物,占挥发油总量的86.3%,主要成分为叶绿醇32.823%,油酸28.759%,植物醇5.271%,油酸酰胺4.797%,反式角鲨烯3.484%,植酮2.787%,2-单棕榈酸甘油1.789%。该结果将为金线兰的进一步开发提供基础数据。

福建金线兰多倍体的诱导和倍性鉴定

[目的]对福建金线兰多倍体进行诱导和倍性鉴定。[方法]以福建金线兰为材料,以秋水仙素为诱变剂,分别用浸泡法和涂抹法处理福建金线兰试管苗不定芽,进行多倍体诱导,并在显微镜下观察鉴定其染色体的倍性。[结果]以浓度0.05%秋水仙素为诱变剂,用涂抹法处理72 h的效果较佳,加倍率为35%;对多倍体材料进行染色体鉴定发现,二倍体染色体为2n=40条,四倍体为4n=80条。[结论]该方法可以培育福建金线兰多倍体,能提高其移栽成活率和亩产量。

金线莲中槲皮素的超声提取及HPLC法测定

以金线莲（Anoectochilus roxburghii）为研究对象,采用超声波法提取,用高效液相色谱法测定了金线莲中槲皮素的含量。在单因素试验基础上,对乙醇体积分数、料液比、超声时间、超声功率4个因素进行了4因素3水平的正交试验。结果表明,槲皮素在0.000 5-0.010 0 mg/mL范围内呈良好线性关系,加样回收率为84.61%-85.90%。金线莲中槲皮素的最佳提取工艺条件为乙醇体积分数80%、料液比1∶25（g∶mL）、超声功率420 W、超声时间30 min。在此工艺条件下,金线莲中槲皮素的提取率为0.011 0%。试验对金线莲中有效成分含量的测定具有参考价值。

荔波喀斯特林下金线莲种植气候因子分析

利用荔波县历史气象资料,对比金线莲生长所需的气候条件,探讨该地区的喀斯特林下对金线莲栽培的适宜性。结果表明荔波县气候因子能满足金线莲生长所需的气候条件;在喀斯特林下可以培育出药性好、药物产量高的优良金线莲植物品种,建议在当地推广种植金线莲。

兴仁金线莲丛生芽诱导增殖研究

[目的]筛选兴仁金线莲丛生芽的诱导和增殖的最佳培养基。[方法]用野生兴仁金线莲植株茎段作外植体,以MS为基本培养基,并添加不同浓度的6-BA和NAA,诱导丛生芽,筛选最佳培养基配方。[结果]MS是兴仁金线莲快繁最佳基本培养基;2.0～3.0 mg/L的6-BA有利于丛生芽的诱导;1.0 mg/L 6-BA+0.1 mg/L NAA有利于芽的生长;最佳蔗糖浓度为25 g/L。[结论]MS+2 mg/L 6-BA+0.2 mg/L NAA+25 g/L蔗糖为丛生芽增殖的最佳培养基配方。

喀斯特林下石灰岩上金线莲种植技术研究

通过对金线莲在喀斯特林下石灰岩上进行仿野生栽培试验和探究,并对金线莲在喀斯特林下石灰岩上的人工种植的技术进行总结,发现喀斯特林下种植金线莲技术有利于提高珍稀濒危药用植物金线莲的快速繁育,同时可以节约土地资源,加快石漠化治理,让珍稀濒危药用植物金线莲和少数民族的传统医药得到有效的保护和延续。

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。

筛选金线莲种质资源的通用DNA条形码序列及鉴定其混伪品

本研究对32份金线莲(Anectochilus roxburhii)种质资源的四个DNA条形码(ITS, rbcL, psbA-trnH,matK)基因进行PCR扩增及测序,从基因库下载了金线莲及其3种主要混伪品的29条序列,分析比较各序列的差异位点、信息位点、GC含量等,并进行种内和种间的遗传距离计算,构建基于K2P模型的NJ系统发育树,综合比较不同序列的鉴定能力,最终从四个DNA条形码候选序列中筛选出可用于金线莲资源鉴定的通用序列。结果显示,ITS和psbA-trnH基因序列的种内、种间的变异位点差异和遗传距离差异较大,相比rbcL和matK序列具有显著优势,且ITS基因不仅能够快速而准确区分金线莲及其混伪品,而且能够在种内水平上区分金线莲的不同品种。因此,ITS基因序列是较适合鉴别金线莲资源的理想条形码。所得结论与前人关于金线莲的有效成分的研究结果进行了关联,发现不同品系的金线莲所含有效成分差异较大。因此,本研究可以为金线莲的功能产品研发、优良品种选育、药理药效研究等方面提供帮助,尤其在金线莲资源选择上提供了较为理想的方法,研究具有一定的应用前景和科学价值。