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Development, Identification, and Application of a Germplasm Specific SCAR Marker for Dendrobium officinale Kimura et Migo.

ABSTRACT: The stems of Dendrobium officinale have been used as a rare and valuable Chinese tonic medicine, known as "Tiepi Fengdou", since the Qing dynasty. Because of the increased market demand and continued exploitation of this plant, the reserves of wild D. officinale resources have been depleted, and D. officinale products on the market are being increasingly adulterated. Such changes have strongly affected the sustainable utilization of this valuable medicinal plant resource and the development of related industries. In this study, a species-specific DNA marker was developed for the rapid and accurate authentication of D. officinale. In total, 36 start codon-targeted (SCoT) polymorphism primers were screened in 36 definite Dendrobium species, and a distinct species-specific DNA amplicon (SCoT13-215) for D. officinale was obtained. After the sequence was cloned and sequenced, a sequence-characterized amplified region marker was developed (named SHF/SHR) and validated through PCR amplification of all 38 Dendrobium samples. The marker's specificity for D. officinale was confirmed through the consistent amplification of a clear 197-bp band. This SCAR marker can be used to rapidly, effectively, and reliably identify D. officinale among various Dendrobium species and may play an important role in ensuring the quality of medicinal preparations and protecting the germplasm of this important medicinal species.

SUBMITTER: Zheng K

PROVIDER: S-EPMC8160518 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Dendrobium officinale Kimura et Migo is a famous plant with a high medicinal value which has been recorded in the Chinese Pharmacopoeia (2020 Edition). The medicinal properties of D. officinale are based on its chemical composition. However, there are no reports on how different cultivation methods affect its chemical composition. In order to reveal this issue, samples of the D. officinale were collected in this study through tree epiphytic cultivation, stone epiphytic cultivation, and greenhouse cultivation. Polysaccharides were determined by phenol sulfuric acid method and secondary metabolites were detected by the UPLC-MS technique. In addition, with regards to metabolomics, we used multivariate analyses including principal component analysis (PCA) and orthogonal partial least squares analysis (OPLS-DA) to screen for differential metabolites which met the conditions of variable importance projection values >1, fold change >4, and p < 0.05. The differential metabolites were taken further for metabolic pathway enrichment analysis, which was based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and validated by antioxidant activity. Comparing the three groups of samples according to the standards of the ChP (2020 edition), the results showed that the polysaccharide content of the samples from stony epiphytic cultivation and greenhouse cultivation was significantly higher than that of the samples from live tree epiphytic cultivation. Metabolomic analysis revealed that there were 185 differential metabolites among the 3 cultivation methods, with 99 of the differential metabolites being highest in the stone epiphytic cultivation. The results of the metabolic pathway enrichment analysis showed that the different cultivation strategies mainly effected four carbohydrate metabolic pathways, five secondary metabolite synthesis pathways, six amino acid metabolic pathways, one nucleotide metabolism pathway, three cofactor and vitamin metabolism pathways, and one translation pathway in genetic information processing. Furthermore, D. officinale from stone epiphytic cultivation which had the best antioxidant activity was implicated in differential metabolite production. This study revealed the effects of different cultivation methods on the chemical composition of D. officinale and also provided a reference for establishing the quality control standards to aid its development and utilization.

Project description:ObjectiveThe present study aimed to investigate the potential mechanism of Dendrobium officinale (D. officinale) on colorectal cancer and the relevant targets in the pathway using a network pharmacological approach.Methods(1) We identified the major bioactive components of D. officinale by UPLC-ESI-MS/MS and established the in-house library by using the literature mining method. (2) Target prediction was performed by SwissADME and SwissTargetPrediction. (3) A protein-protein interaction (PPI) network and component-target-pathway network (C-T-P network) were constructed. (4) The GO pathways and the KEGG pathway enrichment analysis were carried out by the Metascape database. (5) Molecular docking was performed by AutoDock software. (6) A series of experimental assays including cell proliferation, cell invasion and migration, and TUNEL staining in CRC were performed in CRC cell lines (HT-29, Lovo, SW-620, and HCT-116) to confirm the inhibitory effects of D. officinale.Results(1) In total, 396 candidate active components of D. officinale were identified by UPLC-ESI-MS/MS and selected from the database. (2) From OMIM, GeneCards, DrugBank, and TTD databases, 1,666 gene symbols related to CRC were gathered, and (3) 34 overlapping gene symbols related to CRC and drugs were obtained. (4) These results suggested that the anti-CRC components of D. officinale were mainly apigenin, naringenin, caffeic acid, γ-linolenic acid, α-linolenic acid, cis-10-heptadecenoic acid, etc., and the core targets of action were mainly ESR1, EGFR, PTGS2, MMP9, MMP2, PPARG, etc. (5) The proliferation of muscle cells, the regulation of inflammatory response, the response of cells to organic cyclic compounds, and the apoptotic signaling pathway might serve as principal pathways for CRC treatment. (6) The reliability of some important active components and targets was further validated by molecular docking. The molecular docking analysis suggested an important role of apigenin, naringenin, PTGS2, and MMP9 in delivering the pharmacological activity of D. officinale against CRC. (7) These results of the evaluation experiment in vitro suggested that D. officinale had a strong inhibitory effect on CRC cell lines, and it exerted anti-CRC activity by activating CRC cell apoptosis and inhibiting CRC cell migration and invasion.ConclusionThis study may provide valuable insights into exploring the mechanism of action of D. officinale against CRC.

Project description:Dendrobium officinale extract shows potent anti-fatigue effects; however, the active substance responsible for these effects remains undetermined. A glucomannan with a huge molecular size of 730 kDa, called DOP, was identified as the unique authentication marker of this expensive herb. DOP exhibited immunomodulating effects on macrophages and lymphocytes in our previous study. Clinical reports also showed that people with fatigue syndrome have a disturbed immune system. Because DOP is the unique and dominant component of D. officinale, we hypothesize that DOP may also have anti-fatigue activity. The present study aims to evaluate the anti-fatigue activity of DOP on BALB/c mice, with Rhodiola rosea extract as a positive control. DOP and Rhodiola rosea extract were orally administered at doses of 50 mg/kg and 100 mg/kg, respectively, for four weeks, and the anti-fatigue activity of DOP on BALB/c mice was evaluated using the weight-loaded swimming test. The contents of lactic dehydrogenase (LDH), creatine phosphokinase (CK), triglyceride (TG), blood urea nitrogen (BUN), superoxide dismutase (SOD), malondialdehyde (MDA), lactic acid (LD), and glutathione peroxidase (GSH-Px) in serum, glycogen of liver and gastrocnemius muscle were also determined. Their effects on variability of T cells and B cells were determined by using tetrazolium compound (MTS) method. The weight-loaded swimming exercise caused fatigue syndrome, mainly including the decreases of serum SOD/GSH-Px and gastrocnemius glycogen, as well as the increases of LDH, BUN, MDA, CK, TG, and LD in serum. All of these indicators of fatigue were inhibited to a certain extent by both DOP and Rhodiola rosea extract; however, the effects of DOP were much stronger than those of Rhodiola rosea extract. Compared to the positive control, mice dosed with DOP showed increases in endurance, body weight, and food intake. Furthermore, DOP-feeding mice significantly increased the cell variability of T lymphocytes and B lymphocytes, compared with that of mice in control group. This study indicates that the unique and dominant polysaccharide DOP of D. officinale has stronger anti-fatigue activity than Rhodiola rosea extract. As such, DOP has promising potential for pharmaceutical development into health products to reduce fatigue.

Project description:Introduction: Numerous studies have demonstrated that the stems of D. officinale have the effect of lowering blood glucose, but the leaves of D. officinale have seldom been investigated. In this study, we mainly studied the hypoglycemic effect and mechanism of D. officinale leaves. Methods: Initially in vivo, male C57BL/6 mice were administered either standard feed (10 kcal% fat) or high-fat feed (60 kcal% fat) along with either normal drinking water or drinking water containing 5 g/L water extract of D. officinale leaves (EDL) for 16 weeks, and changes in body weight, food intake, blood glucose, etc., were monitored weekly. Next in vitro, C2C12 myofiber precursor cells which were induced to differentiate into myofibroblasts and cultured with EDL to detect the expression of insulin signaling pathway related proteins. HEPA cells were also cultured with EDL to detect the expression of hepatic gluconeogenesis or hepatic glycogen synthesis related proteins. Eventually after separating the components from EDL by ethanol and 3 kDa ultrafiltration centrifuge tube, we conducted animal experiments using the ethanol-soluble fraction of EDL (ESFE), ethanol-insoluble fraction of EDL (EIFE), ESFE with a molecular weight of >3 kDa (>3 kDa ESFE), and ESFE with a molecular weight of <3 kDa (<3 kDa ESFE) for intensive study. Results: The results in vivo revealed that the mice fed the high-fat diet exhibited significantly decreased blood glucose levels and significantly increased glucose tolerance after the EDL treatment, whereas the mice fed the low-fat diet did not. The results in vitro showed that EDL activated the expression of protein kinase B (AKT), the phosphorylation of AKT, and the expression of downstream GSK3β in the insulin signaling pathway. EDL treatment of HEPA cells confirmed that EDL did not affect hepatic gluconeogenesis or hepatic glycogen synthesis. In the experiment of studying the composition of EDL, we found that the >3 kDa ESFE displayed the effect of lowering blood glucose. In summary, the effect of EDL in lowering blood glucose may bethanole achieved by activating the insulin signaling pathway to increase insulin sensitivity, and the main functional substance was contained within the >3 kDa ESFE. Discussion: The findings of this study represent a reference point for further exploration of the hypoglycemic effects of D. officinale leaves and may assist in both the identification of new molecular mechanisms to improve insulin sensitivity and the isolation of monomeric substances that lower blood glucose. Furthermore, the obtained results may provide a theoretical basis for the development of hypoglycemic drugs with D. officinale leaves as the main component.

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Development, Identification, and Application of a Germplasm Specific SCAR Marker for Dendrobium officinale Kimura et Migo.

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