New Methods Of Spectroscopy And Sensing For Quality Identification Of Dendrobium

Dendrobium is a perennial epiphytic herb of the genus Dendrobium of the family Orchidaceae,a traditional rare and endangered Chinese herb in China,also known as immortal herb,soul-returning herb,thousand gold herb,soft gold,etc.It has the efficacy of benefiting stomach,nourishing Yin and clearing heat,and has high medicinal value and healthcare efficacy.Dendrobium has a medicinal history of more than two thousand years,and the medicinal dendrobiums in China include D.huoshanense（Dendrobium huoshanense C.Z.Tang et S.J.Cheng）,D.officinale（Dendrobium officinale Kimura et Migo）,D.nobile（Dendrobium nobile Lindl.）and other species,mainly distributed in Anhui,Zhejiang,Yunnan,Guizhou and Guangxi province.In recent years,due to excessive mining,environmental pollution and other reasons,Dendrobium resources have been in an endangered state.In addition,the price of Dendrobium in the market is high,resulting in confusion in the origin of Dendrobium,falsification and mislabeling,which seriously damage the legitimate rights and interests of consumers and the safety of medicine.In addition,the composition of Dendrobium is complex,containing polysaccharides,alkaloids,flavonoids flavonoids and phenols and other compounds,which belong to multi-factor complex system,and the quality is affected by geographical environment,climate,farming methods and so on.The traditional analysis methods are difficult to achieve rapid identification.Therefore,it is urgent to establish a kind of identification method for rapid detection of Dendrobium quality.In this paper,Dendrobium species of different varieties,origins and cultivation methods were used as the research objects,and modern instrumental analysis methods such as high performance liquid chromatography-high resolution mass spectrometry,fluorescence spectroscopy,ultraviolet spectroscopy,near-infrared and mid-infrared spectroscopy were used,combined with chemical analysis methods for the quality difference factors of Dendrobium from different producing areas and varieties were screened.At the same time,a variety of nano materials were designed and prepared as signal amplification or sensing units to construct array sensing.Using the response characteristics of active components in Dendrobium and nano materials in different degrees,a variety of spectral identification methods and visual identification methods based on nano materials were constructed.（1）Firstly,Dendrobium from different origins（Zhejiang,Yunnan）and varieties（Dendrobium Huoshan,Dendrobium nobile and Dendrobium officinale）were used as the research objects,and the content of polysaccharides,total sugars,flavonoids and total phenols in Dendrobium was obtained by rapid determination of kits.,and the antioxidant activity in vitro was determined by the ABTS method.Analysis of variance showed that there were significant differences in the contents of polysaccharides,total sugars,flavonoids and total phenols in Dendrobium from different origins and varieties（P<0.05）.Among them,Dendrobium Huoshan has the highest content of total sugar,polysaccharide and flavonoids,and has the strongest antioxidant activity,and Dendrobium goldenchai has the highest total phenolic content.The Pearson correlation analysis showed that the total sugar content of Dendrobium from different origins and varieties had a very significant positive correlation with the antioxidant activity（r=0.884）.In different varieties of Dendrobium,the difference component with important contribution was screened and obtained total sugar.Among them,the difference components obtained by screening between Dendrobium Huoshan and Dendrobium nobile were total sugars and polysaccharides,indicating that sugar compounds are the main difference components that distinguish Dendrobium Huoshan,Dendrobium officinale and Dendrobium nobile.In Dendrobium officinale from different productions,the differential components with important contributions were screened and obtained total sugars,polysaccharides and total phenols.It indicated that sugars and phenolic compounds were the main different components in Dendrobium officinale from different productions.（2）On the basis of studying the differences of active components such as Dendrobium polysaccharides and total phenols,in order to further screen the quality difference factors other than common active components such as polysaccharides and total phenols,high-performance liquid chromatography-high-resolution mass spectrometry was used.The chemical composition information of Dendrobium was obtained by HPLC-HRMS.The non-targeted metabolomics automatic analysis strategy Ant DAS＿MS2 independently developed by our group was used for the preliminary screening and identification of differential compounds,and then the quality differential factors were screened by chemometric analysis.In different varieties of Dendrobium,a total of 5 quality difference factors were screened out,and the contribution values were ranked from large to small as Biochanin A>Rutin>Dihydroresveratrol>Ferulic tyramine>Resveratrol.Among them,biochanin A,dihydroresveratrol,ferulic tyramine and resveratrol have the highest content in Dendrobium Huoshan,and the content of rutin in Dendrobium officinale is the highest.Three quality difference factors were selected from Dendrobium officinale from different origins,namely ferulic tyramine,plant sphingosine and dihydrosphingosine.（3）On the basis of the above differential component analysis,it is clear that there are significant differences in phenolic compounds with fluorescence properties in different varieties of Dendrobium.Firstly,a method based on excitation emission matrix fluorescence spectroscopy（EEM）and chemometrics is proposed to quickly identify Dendrobium Huoshan and 6 cheap adulterated Dendrobium species.Three groups of components with chemical significance were obtained by parallel factor analysis（PARAFAC）.The excitation/emission wavelength of component 1 was 260/395 nm,mainly including rutin,naringin and other flavonoids;Component 2（295/345 nm）is mainly composed of phenolic compounds,such as vanillic acid,syringic acid,protocatechuic acid,etc;Component 3（390/425 nm）is mainly coumarin compounds.Partial least squares discriminant analysis（PLS-DA）,linear discriminant analysis（LDA）,k nearest neighbor（KNN）and n-dimensional partial least squares discriminant analysis（n-pls-da）were used to classify Dendrobium Huoshan and other six cheap Dendrobium species,respectively.The results show that k NN and N-PLS-DA have the best classification performance,with 100%accuracy on both training sets and prediction sets,and the highest sensitivity and specificity（100%）.（4）Secondly,because the cultivation mode and growth year of Dendrobium Huoshan have a great impact on its quality,but the difference of active components between them is small,the single spectral method based on fluorescent components is difficult to achieve accurate identification.In order to accurately identify the cultivation methods and growth years of Dendrobium Huoshan,four different cultivation methods（greenhouse cultivation,understory rock,understory grid and live tree epiphyte cultivation）and four growth years（1 year,2 years,3 years and 4 years）of Dendrobium Huoshan as the research object,a method based on multi-feature fusion spectrum of nano-effect was designed to identify the cultivation methods and growth years of Dendrobium Huoshan.Firstly,a zinc porphyrin（Zn TPP）was designed and synthesized to amplify the difference signal of spectrum after reaction with Dendrobium Huoshan,so as to obtain more effective information.After screening of important variables,4727 spectral variables were obtained,and data fusion was performed to obtain nano-effect feature fusion spectra,which were combined with chemometrics to discriminate.The results show that the PLS-DA model can accurately discriminate the growth year of Dendrobium Huoshan,and the accuracy of the training set and prediction set is 100%.OPLS-DA can achieve 100%accurate discrimination between greenhouse cultivation and three imitation wild cultivation.Finally,the growth year of Dendrobium Huoshan was predicted by partial least squares regression（PLSR）model.The results showed that the PLSR model had good linearity and accuracy,and the corrected root mean square error（RMSEC）and verified root mean square error（RMSEV）were 0.1478 and 0.1951,respectively.（5）Finally,in order to realize the instrument-free detection of Dendrobium quality,taking Dendrobium officinale as an example,Dendrobium officinale and the adulterated variety Dendrobium nobile from different origins（Anhui,Zhejiang,Yunnan）were selected as the research objects.A number of noble metal nanomaterials were designed and synthesized as colorimetric probes,and metal ions(Zn²⁺,Cr³⁺,Cu²⁺)were used as sensing elements,and a three-channel visual array sensor was constructed for quality identification of Dendrobium officinale.The sensor array consists of channel 1:purple Ag NPRs-Zn²⁺,channel 2:blue Ag NPRs-Cr³⁺and channel 3:red Au NPs-Cu²⁺.The detection mechanism is based on the different binding affinity between active components and sensing elements in Dendrobium officinale from different producing areas,resulting in a series of color changes and UV-Vis spectral response,which can be used as the"fingerprint information"of each Dendrobium officinale sample.Finally,it is discriminated and analyzed by partial least squares discrimination（PLS-DA）.The results show that compared with the original UV-Vis absorption spectrum,the accuracy of training set and prediction set based on nano-effect UV-Vis absorption spectrum is improved from 82.1%and 81.3%to 100%and 100%.In addition,when discriminated based on RGB values,the accuracy rates of single-channel 1-3 training set and prediction set are 91.3%and72.3%,100%and 81.8%,100%and 90.9%,respectively.The accuracy of the training set and prediction set obtained by the fusion of the three channels is increased to100%,which realizes the simultaneous detection of Dendrobium officinale samples from different origins and adulterated varieties.The method is simple,intuitive and fast,which can realize instrument-free analysis,and has potential application value in the rapid identification of Chinese medicinal materials.