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Background Dendrobium huoshanense (DHS) is a typical traditional Chinese medicine with unique medical and high economic values; however, it may easily be adulterated with cheaper alternatives (e.g. Dendrobium henanese, DHN), because of their similar appearances and tastes. Objective In this study, adulteration of DHN in DHS was detected by near infrared (NIR) spectroscopy combined with chemometric methods. Methods By performing partial least squares (PLS) analysis, PLS multivariate methods including partial least-squares discriminant analysis (PLS-DA), and partial least-squares regressions (PLSR) were applied to the obtained spectral data to build models. The PLS-DA model was employed to differentiate between pure DHS samples and those adulterated with DHN. Results The R2 value obtained for the PLS-DA model was 0.4898 with an RMSEP error of 0.1554, resulting in a 100% accuracy of validation sample sets. Similarly, a PLSR model was also developed to quantify the amount of DHN adulterant in DHS samples. Experimental results indicated that the good performance of the multiplicative scattering correction (MSC) model is the better model showing a prediction performance of RMSEP of 2.38 and R2 of 0.9946. Conclusions These results suggest that the combination of NIR spectroscopy and chemometric method provides a fast, simple and reliable method for detecting adulteration of DHS. Highlights The method of classification allowed identification of both authentic and adulterated DHS samples. Comparison of six different techniques for spectra preprocessing to improve quantitative model performance was obtained with MSC derivative spectra. The method can detect most of the current DHS adulterations in the Chinese market.

In this study, the potentiality of applying attenuated total reflectance near-infrared (ATR-NIR) and attenuated total reflectance mid-infrared (ATR-MIR) techniques combined with a partial least squares (PLS) regression technology to quantify the total polyphenols (TPs) in Dendrobium huoshanense (DHS) was investigated and compared. The real TP contents in the DHS samples were analysed using methods of reference. The capability of the two IR spectroscopic techniques to quantify the TPs in DHS was assessed by the root-mean-square error of calibration (RMSEC) and determination coefficients (R2 ). The results showed that both NIR and MIR might be used as a fast and simple tool to replace traditional chemical assays for the determination of the TP contents in DHS, and the best NIR model showed slightly better prediction performance [root-mean-square error of prediction (RMSEP): 0.307, R2 : 0.9122, ratio performance deviation (RPD): 4.43] than the best MIR model (RMSEP: 0.440, R2 : 0.9069, RPD: 3.09). Results from this study indicated that both the NIR and MIR models could be used to quantify the TP in DHS, and ATR-NIR appeared to be the more predominant and more robust technique for the quantification of the TP in DHS.

Dendrobium huoshanense (DHS) has long been used to make tea drink, soup, and porridge to protect eye and liver in many Southeast Asian countries for centuries. As a rare and endangered functional food, adulteration in DHS with visually similar but cheaper and more accessible plants such as Dendrobium henanense (DHN) because of their similarities in morphology has become prevalent in the market. In this study, the Attenuated Total Reflectance Fourier transform Infrared Spectroscopy (ATR-FTIR) combined with chemometric methods was established to detect fraudulent addition in DHS with DHN. The partial least squares (PLS) models based on the ATR-FTIR files of DHS mixed with different proportions of DHN were built under cross validation and tested with different independent data sets. To reduce the variables’ lack of information and increase the accuracy of the model, different wavelength selection methods including Moving Window Partial Least Squares (MW-PLS), Monte Carlo-uninformative variable elimination (MC-UVE), and interval random frog (iRF) were compared.The results showed that iRF performed the most perfectly with the number of latent variables (nLVs = 7), the lowest Root Mean Square Error of Cross-Validation (RMSECV = 7.37), and the maximum determination coefficients (R2 = 0.9721). The excellent performance of the model was proved by the low RMSEP value of 6.44% and the high R2 value of 0.9556. The developed method could rapidly quantify the adulteration DHN in DHS, and our study might provide an efficient and great potential technique tool for the rapid, green, low-cost, and nondestructive identification and quantification for DHS adulterated with DHN.

A phenylhexyl isothiocyanate (PITC) precolumn derivatization quantitative analysis of multicomponents by a single marker (QAMS) strategy for the simultaneous analysis of 20 free amino acids (FAA) in Dendrobium huoshanense is proposed. The method was validated by the linearity, limit of detection (LDO), and limit of quantitation (LOQ), recovery, precision, and stability. The results showed that when applying the established method, the LOQ of the FFAs was lower than 1 ng/ml except threonine (1.32 ng) and cysteine (1.16 ng). The QAMS investigation revealed that, using any one of the 20 FAAs as the reference internal standard, no significant differences were observed between the external standard method and the QAMS method for the quantification of FAAs in D. huoshanense by PITC precolumn derivatization [The relative standard deviation (RSD, %) by QAMS and ESM were all below 5%]. HPLC fingerprint investigation combined with similar analysis (the similarity values for S1-S25 were >0.875) and quality fluctuation analysis showed that the cultivation environment might have a great effect on the accumulation of FAAs in D. huoshanense. Overall, our study showed that we might increase the accuracy and scope of the simultaneous quantification of multicomponents using the QAMS technique by being derivatized with a strong UV absorbing group, and QAMS combined with chromatographic fingerprinting can be considered good quality criteria for the quality control of D. huoshanense and may provide analytical technical support for research on Maillard Reaction during the further processing of D. huoshanense.

A rapid pressurized capillary electrochromatography (pCEC) method has been successfully developed for the simultaneous determination of 16 phytohormones in Dendrobium huoshanense. Effects of wavelength, mobile phase, the flow rate, pH value, concentration of buffer and applied voltage were investigated, respectively. The results showed that the 16 phytohormones could be baseline-separated rapidly in less than 21 min on a reversed-phase EP-100–20/45–3-C18 capillary column (total length of 45 cm, effective length of 20 cm, diameter of 100 μm, ODS packing inside for 3 μm) with ACN/5.0 mM ammonium acetate (containing 0.05% formic acid, pH = 3) as the mobile phase using gradient elution mode as follows: 0.1–10.0 min 40%ACN,10–15.0 min 70%ACN, 15.0–20 min 80% ACN, 20–21.0 min 80% ACN at a flow rate of 0.12 mL/min, applied voltage of −5 kV and a UV detection wavelength of 210 nm. The method validation howed that the established method is precise and stability, and the RSDs of intra- and inter-day precision based retention time and peak area were all below 5%. Employed the established method, in our experimental conditions, total 6 endogenous hormones including IAA, IBA, NAA, GA, ABA, t-Z were detected in D. huoshense. However, a relative larger amount of exogenous hormone 2,4-D (25.3 ~ 4.2 μg/kg) and 6-BA (79.5 ~ 35.4 μg/kg) were detected in 1 ~ 4 year old cultivated D. huoshense, suggesting there were still a certain amount of exogenous hormone residue in tissue-cultured D. huoshanese though they had been transplanted to field cultivation from the test-tube plantlets for several years.

Ptaquiloside (PTA) is a carcinogenic norsesquiterpene glucoside existing in Pteridium aquilinum, the young fronds of which are widely used as a wild vegetable in China. To ensure food safety, it is extremely important to establish a fast and accurate method for the quantification of PTA in P. aquilinum. This paper aimed to establish a qualification and quantification method for PTA in P. aquilinum based on a solid-phase extraction (SPE) pre-concentrating technique followed by TOF-MS/MS and HPLC-DAD analysis. The results showed that the developed method can qualify and quantify the PTA in P. aquilinum with an average recovery of 87.0% for PTA. The method is repeatable extra- and inter- days, with RSD values lower than 5%. A total of 18 P. aquilinum samples from different places of China were analyzed by the established method. The contents of PTA in the investigating samples varied from 5.61 ± 0.50–4250 mg kg−1 in dry weight, which was mostly lower than those of cases reported abroad. This study presents the characterization and quantitative assay of PTA in P. aquilinum from different places of China using direct TOF-MS/MS and HPLC-DAD quantification combined with solid-phase extraction (SPE) pre-concentrating technique, which can be employed for the safety evaluation of the food derived from P. aquilinum.

Plant tissue culture technique is widely used in the conservation and utilization of rare and endangered medicinal plants and it is crucial for tissue culture stocks to obtain the ability to produce similar bioactive components as their wild correspondences. In this paper, a headspace gas chromatography–mass spectrometry method combined with chemometric methods was applied to analyze and evaluate the volatile compounds in tissue-cultured and wild Dendrobium huoshanense Cheng and Tang, Dendrobium officinale Kimura et Migo and Dendrobium moniliforme (Linn.) Sw. In total, 63 volatile compounds were separated, with 53 being identified from the three Dendrobium spp. samples. Different provenances of Dendrobium s had characteristic chemicals and showed remarkable quantity discrepancy of common compositions. The similarity evaluation disclosed that the accumulation of volatile compounds in Dendrobium samples might be affected by their provenance. Principal component analysis showed that the first three components explained 85.9% of data variance, demonstrating a good discrimination between samples. Gas chromatography–mass spectrometry techniques, combined with chemometrics, might be an effective strategy for identifying the species and their provenance, especially in the assessment of tissue-cultured Dendrobium quality for use in raw herbal medicines.

An attenuated total reflectance Fourier transform infrared spectroscopy method combined with partial least square regression was established to rapidly quantify the total polysaccharide and its major monosugars in Dendrobium huoshanense. The results showed that the optimal predictive methods were the models established by the infrared spectroscopy files pretreated by standard normal transformation combined with the second derivative; the accuracy of the models selecting wavelength regions based on the assignments of Fourier transform infrared spectroscopy signals of polysaccharide was obviously higher than the models selecting wavelength regions suggested by Thermofisher Quantity Analyst software and other possible wavelength region selecting modes. The external validation and the complete external validation confirmed the robustness and reliability of the developed attenuated total reflectance Fourier transform infrared model. Our study might provide an efficient technique tool for the rapid, green, low-cost, and nondestructive quantification of the total polysaccharide and the main monosaccharides in Dendrobium huoshanense and other rich-in-polysaccharide plant food or medicines.

The accumulating of pharmaceutical chemicals in medicinal plants would greatly be affected by their ages and establishing a fast quality-identification method to evaluate the similarity of medicinal herbs at different cultivated ages is a critical step for assurance of quality and safety in the TCM industry. In this work, tri-step IR macro-fingerprinting and 2D-COS IR spectrum techniques combined with statistical pattern recognition were applied for discrimination and similarity evaluation of different ages of tissue-cultured and wild Dendrobium huoshanense C. Z. Tang et S. J. Cheng as well as Dendrobium henanense J.L.Lu et L.X Gao. Both tissue-cultured and wild D. huoshanense were easily differentiated from D. henanense by FTIR and SD-IR spectra, while it's quite difficult to discriminate different cultivated years of the three investigated Dendrobiums. In 2D-COS IR spectra, 1–5 auto-peaks with different indensity and positions were located in the region 1160–1030 cm−1 of the twelve Dendrobium samples and thus could be used to identify Dendrobium samples at different ages. Principle component analysis (PCA) of synchronous 2D-COS data showed that the twelve samples were effectively identified and evaluated. The results indicated that the tri-step infrared macro-fingerprinting combined with PCA method was suitable to differentiate the cultivated ages of Dendrobiums with species and orgins rapidly and nondestructively.

The FTIR method was applied to evaluate the similarity of tissue-cultured and wild Dendrobium huoshanense C.Z. Tang et S.J. Cheng, Dendrobium officinale Kimura et Migo and Dendrobium moniliforme (Linn.) Sw and discriminate different Dendrobium species, especially D. huoshanense and its main goldbrick Dendrobium henanense J.L. Lu et L.X. Gao. Despite the general pattern of the IR spectra, different intensities, shapes and peak positions were found in the IR spectra of these samples, especially in the range of 1800–600 cm −1 , which could be used to discriminate them. The methanol, aqueous extracting procedure and the second derivative transformation obviously enlarged the tiny spectral differences among these samples. The similarity evaluation based on the IR spectra and the second derivative IR spectrum revealed that the similarity of the methanol extracts between tissue-cultured and wild Dendrobiums might be lower than that between different Dendrobium species. The similarities of the powders and aqueous extracts between tissue-cultured and wild Dendrobiums were higher than those between different Dendrobium species. The further principal component analysis showed that the first three components explained 99.7%, 87.7% and 85.1% of data variance for powder, methanol extract and aqueous extract, respectively, demonstrating a good discrimination between samples. Our research suggested that the variations of secondary metabolites between different origins of the investigated Dendrobiums might be higher than what we had supposed. Tissue culture techniques were widely used in the conversation of rare and endangered medicinal amedica, however, our study suggested that the chemical constituents of tissue-cultured plants might be quite different from their wild correspondences.

A rapid, green, low cost and nondestructive attenuated total reflection near infrared (ATR NIR) method was developed to quantify the total polysaccharide and the main monosaccharides mannose and glucose in Dendrobium huoshanense. Total 100 D. huoshanense samples from different places were analyzed using ATR NIR method. Potential outlying samples were initially removed from the collected NIR data using the PCA-Mahalanobis distance method. Spectral data preprocessing was studied in the construction of a partial least squares (PLS) model and six different signal pretreatment methods, including multiplicative scattering correction (MSC), standard normal transformation (SNV), first and second derivatives, the combination of MSC with the first derivative, and the combination of SNV with the first derivative, were compared. The results showed that the best signal pretreatment method was the spectral data pretreated by SNV combined with the first derivative due to it showed the lowest root-mean-square error of cross-validation (RMSECV), highest R 2 for both the polysaccharide and its main monosaccharides. In order to improve the performance of the model, the pretreated full spectrum was calculated by different wavelength selection method. The results showed that the optional wavelength selection model was the one simultaneously selecting the NIR wavelength ranges 7500–5750 cm −1 , 5250–4700 cm −1 , 4450–4300 cm −1 and 4200–4100 cm −1 because of the lowest RMSECV and the highest R 2 among the ten wavelength selection models. The external validation and the complete external validation confirmed the robustness and reliability of the developed NIR model. The contents of the total polysaccharide and the main monosaccharides are the essential quality assessment criterion for plant medicines while their traditional quantification methods involved sample destruction, tedious sample processing and non-environmentally friendly pretreatment, therefore, our study might provide an efficient technique tool for the rapid, green and nondestructive quantification of the total polysaccharide and the main monosaccharides for D. huoshanense and other rich-in-polysaccharide plant medicines.

A water-soluble polysaccharide TC-DHPA4 with a molecular weight of 8.0 × 105 Da was isolated from tissue-cultured Dendrobium huoshanense by anion exchange and gel permeation chromatography. Monosaccharide analysis revealed that the homogeneous polysaccharide was made up of rhamnose, arabinose, mannose, glucose, galactose and glucuronic acid with a molar ratio of 1.28:1:1.67:4.71:10.43:1.42. The sugar residue sequence analysis based on the GC-MS files and NMR spectra indicated that the backbone of TC-DHPA4 consisted of the repeated units:→6)-β-Galp-(1→6)-β-Galp-(1→4)-β-GlcpA-(1→6)-β-Glcp-(1→6)-β-Glcp-(→. The sugar residue sequences β-Glcp-(1→)-α-Rhap-(1→3)-β-Galp-(1→, β-Glcp-(1→4)-α-Rhap-(1→3)-β-Galp-(1→, β-Galp-(1→6)-β-Manp-(1→3)-β-Galp-(1→, and α-l-Araf-(1→2)-β-Manp-(1→3)-β-Galp-(1→ were identified as the branches attached to the C-3 position of (1→6)-linked galactose in the backbone.

Dendrobium huoshanense is a rare traditional Chinese medicinal herb, and the anti-tumor activity of its polysaccharides is a research hotspot in traditional Chinese medicine resources domain. This study aims to explore the material basis for the anti-tumor activity of polysaccharide. D. huoshanense was used as raw material in the experiment, and the different protein components were obtained through low salt solution extraction and ammonium sulfate fractional precipitation. Then glycoprotein components were determined by SDS-PAGE electrophoresis staining, and were further isolated and purified by DEAE ion column and Sephadex gel column. At the same time, MTT assay was used in detecting the cytotoxicity of different products on HepG2 cells in vitro. As a result, three kinds of glycoprotein components RG1, RG2, RG3 with relative molecular mass of 22.5, 19.8, 15.6 kDa were gained, and the IC₅₀ of three compounds on human liver cancer cell HepG2 was 534.23 mg•L⁻¹, meanwhile IC₅₀ of single glycoprotein component RG1, RG2 was 432.96, 413.91 mg•L⁻¹ respectively, and glycoprotein component RG3 had no cytotoxicity on HepG2 cells. All in all, the experiment results suggested that two kinds of glycoproteins components with relative molecular mass of 22.5, 19.8 kDa may be one of the material basis for anti-tumor activity of D. huoshanense polysaccharide, and they had a synergistic effect.

To establish a PMP-HPCE method for comparing the monosaccharides of polysaccharide in tissue-cultured and wild Dedrobium huoshanese and Dedrobium moniliforme as well as wild Dedrobium henanese, in order to investigate the similarities of their bioactive components.The PMP-monosaccharides of polysaccharide from the five investigated Dedrobium samples were separated by HPCE on a fused silica capillary column(100 cm x 50 µm) at 25 °C with 350 mmol/L BAS (adjusted to pH 10 with 1.0 mol/L NaOH) as running buffer for 34 min. The applied voltage was 20 kV and the detection wavelength was set at 250 nm.Total six monosaccharides including xylose, glucose, mannose, galactose, galacturonic acid and ribose were detected in the five Dendrobiurms samples and the similarity coefficients between the ten batches of the same Dendrobium species were all above 0. 98,while remarkable dissimilarity were exhibited among species and different resources.PMP-HPCE technique combined with chemometrics is simple, convenient, precise, reproducible and proved to be an effective strategy for identifying the species and origins, especially in the quality assessment of Dendrobium stems.

GC-MS method combined with FTIR techniques by the analysis of polysaccharide was applied to evaluate the similarity between wild (W) and tissue-cultured (TC)Dendrobium huoshanense(DHS),Dendrobium officinale(DO), andDendrobium moniliforme(DM) as well as 3 wildDendrobiumspp.:Dendrobium henanense(DHN),Dendrobium loddigesii(DL), andDendrobium crepidatum(DC). Eight monosaccharides involving xylose, arabinose, rhamnose, glucose, mannose, fructose, galactose, and galacturonic acid were identified in the polysaccharide from eachDendrobiumsample while the contents of the monosugars varied remarkably across origins and species. Further similarity evaluation based on GC-MS data showed that thercorvalues of different origins of DHS, DO, and DM were 0.831, 0.865, and 0.884, respectively, while thercorvalues ranged from 0.475 to 0.837 across species. FTIR files of the polysaccharides revealed that the similarity coefficients between W and TC-DHS, DO, and DM were 88.7%, 86.8%, and 88.5%, respectively, in contrast to the similarity coefficients varying from 57.4% to 82.6% across species. These results suggested that the structures of polysaccharides between different origins of the investigatedDendrobiumsmight be higher than what we had supposed.

To compare the composition and content of alkaloid of Dendrobium huoshanense tissue-culture seedling and wild plant.A comparative evaluation on the quality was carried out by HPLC and TLC methods including the composition and the content of alkaloids.Remarkable variation existed in the two kinds of Dendrobium huoshanense. For the tissue-culture plant, only two alkaloids were checked out by both HPLC and TLC while four alkaloids were observed in the wild plant. The alkaloid content of tissue-culture seedling and wild plant was(0. 29 ± 0. 11)%o and(0. 43 ± 0. 15) %o,respectively.Distinguished difference is observed in both composition and content of alkaloids from the annual shoots of different provenances of Dendrobium huoshanense. It suggested that the quality of tissue-culture seedling of Dendrobium huoshanense might be inconsistent with the wild plant. Furthermore, the established alkaloids-knock-out HPLC method would provide a new research tool on quality control of Chinese medicinal materials which contain unknown alkaloids.