Tibetan Medicinal Plant Research Articles

Identification of New Compounds from the Roots of Rubia tibetica Hook. f.

Rubia tibetica Hook. f is a traditional Tibetan medicinal plant, with its roots serving as the primary medicinal part. Two new compounds, Rubiaxylm C and E (1 and 3), and one new natural compound, Rubiaxylm D (2) were isolated from R. tibetica, along with five known compounds (4-8). The structures of the previously undescribed compounds were elucidated by HR-ESIMS, 1D/2D NMR spectroscopic data and electronic circular dichroism calculations. Compound 1 is a rare anthrone substituted with a carbonyl group at position 1. Furthermore, all isolated compounds were tested for their cytotoxicity against HepG2 cell lines.

Factors contributing to organelle genomes size variation and the intracellular DNA transfer in Polygonaceae

The use of complete organelle genomes, including chloroplast and mitochondrial genomes, is a powerful molecular method for studying biological evolution and gene transfer. However, in the case of Polygonaceae, an important family with numerous edible, medicinal, and ornamental species, the mitochondrial genomes of only three species have been sequenced and analyzed. In this study, we present the mitochondrial and chloroplast genomes of two important Tibetan medicinal plants, Bistorta viviparum and B. macrophyllum. All the organelle genomes are assembled into a single circular structure and contain a common set of 32 protein-coding genes (PCGs). Some genes such as rps2 and ndhF were found to have high nucleotide polymorphism (Pi) in the chloroplast genomes, while cox1, mttB and rps12 showed pronounced Pi values in the mitochondrial genomes. Furthermore, our analysis revealed that most chloroplast genes and mitochondrial PCGs in Polygonaceae plants are under purifying selection. However, a few genes, including the chloroplast gene psaJ and the mitochondrial genes ccmFc, atp8 and nad4, showed positive selection in certain Polygonaceae plants, as indicated by a Ka/Ks ratio greater than one. Structural variation analysis revealed a wealth of differences between the mitochondrial genomes of five Polygonaceae species, with a particularly notable large-scale inversion observed between Reynoutria japonica and Fallopia aubertii. Furthermore, an analysis of the homologous sequences in the chloroplast and mitochondrial genomes revealed that the rps7 has been transferred from the chloroplast to the mitochondrial genome in all five Polygonaceae species. Finally, ecological niche models were constructed for B. viviparum and B. macrophyllum, indicating that mean annual temperature and altitude are the main climatic factors influencing the distribution of both species. Although the current distribution of B. viviparum is significantly wider than that of B. macrophyllum, projections suggest that the optimal growth ranges of both species will expand in the future, with B. macrophyllum potentially exceeding B. viviparum. This study not only contributes to the plastid genome database for Polygonaceae plants, but also provides theoretical insights into the adaptive evolution of these plants.

De Novo Synthesis of Anticholinergic Hyoscyamine and Scopolamine in Nicotiana benthamiana Based on Elucidating Tropane Alkaloid Biosynthetic Pathway of Anisodus luridus

Anisodus luridus, a perennial herb belonging to the genus Anisodus of the Solanaceae family, is an important Tibetan medicinal plant that produces pharmaceutical tropane alkaloids (TAs) including hyoscyamine and scopolamine. Its high yield of hyoscyamine makes A. luridus a valuable plant source for commercially producing TAs. In this study, we conduct homologous gene research across transcriptome data of different tissues together with functionally tested sequences in Atropa belladonna as a reference and identify 13 candidate genes for TAs biosynthesis in A. luridus. The results show that these 13 TAs biosynthesis genes identified in A. luridus were highly conserved in terms of sequence similarity and gene expression patterns compared to A. belladonna, suggesting that the two species may share the same biosynthetic pathway for TAs biosynthesis. Furthermore, scopolamine was detected in Nicotiana benthamiana leaves when these 13 enzymes were co-expressed in N. benthamiana, which confirmed that these 13 TAs biosynthesis genes are involved in the biosynthesis of TAs. The results of our study not only systematically elucidate the tropane alkaloid biosynthetic pathway of A. luridus, but also realize the de novo synthesis of TAs in N. benthamiana for the first time. It is now possible to make N. benthamiana a potential source for TAs production through synthetic biology techniques.

Assessment of Population Genetic Diversity of Medicinal Meconopsis integrifolia (Maxim.) Franch. Using Newly Developed SSR Markers.

Meconopsis integrifolia is an endangered Tibetan medicinal plant with significant medicinal and ornamental value. Understanding its genetic diversity and structure is crucial for its sustainable utilization and effective conservation. Here, we develop a set of SSR markers based on transcriptome data to analyze the genetic diversity and structure of 185 individuals from 16 populations of M. integrifolia. The results indicate that M. integrifolia exhibits relatively high genetic diversity at the species level (the percentage of polymorphic bands PPB = 91.67%, Nei's genetic diversity index He = 0.2989, Shannon's information index I = 0.4514) but limited genetic variation within populations (PPB = 12.08%, He = 0.0399, I = 0.0610). The genetic differentiation among populations is relatively high (the coefficient of gene differentiation GST = 0.6902), and AMOVA analysis indicates that 63.39% of the total variation occurs among populations. This suggests that maintaining a limited number of populations is insufficient to preserve the overall diversity of M. integrifolia. Different populations are categorized into four representative subclusters, but they do not cluster strictly according to geographical distribution. Limited gene flow (Nm = 0.2244) is likely the main reason for the high differentiation among these populations. Limited seed and pollen dispersal abilities, along with habitat fragmentation, may explain the restricted gene flow among populations, highlighting the necessity of conserving as many populations in the wild as possible.

Insights into the molecular mechanism underlying cold-induced flavonoids biosynthesis in callus of a Tibetan medicinal plant Saussurea laniceps

Insights into the molecular mechanism underlying cold-induced flavonoids biosynthesis in callus of a Tibetan medicinal plant Saussurea laniceps

The Chrysosplenium sinicum genome provides insights into adaptive evolution of shade plants

Chrysosplenium sinicum, a traditional Tibetan medicinal plant, can successfully thrive in low-light environments for long periods of time. To investigate the adaptive evolution of shade plants in low-light environments, we generated a chromosome-scale genome assembly (~320 Mb) for C. sinicum by combining PacBio sequencing and Hi-C technologies. Based on our results, gene families related to photosynthesis and cell respiration greatly expanded and evolved in C. sinicum genome due to intracellular DNA transfer from organelle genome to nuclear genome. Under positive selective pressure, adaptive evolution of light-harvesting complex II (LHCII) component protein CsLhcb1s resulted in the expansion of threonine residues at the phosphorylation site of STN7 kinase, potentially establishing a crucial genomic foundation for enhancing C. sinicum’s adaptability in low-light environments. Through transcriptome and metabolome analysis, we identified chrysosplenol and chrysosplenoside as predominant flavonoid metabolites of C. sinicum and predicted their synthesis pathways. In addition, analysis of alternative splicing (AS) revealed that AS events help regulate state transition and flavonoid biosynthesis. The present study provides new insights into the genomes of shade plants exposed to low-light conditions and adaptive evolution of these genomes; in addition, the results improve our current knowledge on the biosynthetic and regulatory processes of chrysosplenol and chrysosplenoside.

Screening inhibitors against the Ef-Tu of Fusobacterium nucleatum: a docking, ADMET and PBPK assessment study.

The oral pathogen Fusobacterium nucleatum has recently been associated with an elevated risk of colorectal cancer (CRC), endometrial metastasis, chemoresistance, inflammation, metastasis, and DNA damage, along with several other diseases. This study aimed to explore the disruption of protein machinery of F. nucleatum via inhibition of elongation factor thermo unstable (Ef-Tu) protein, through natural products. No study on Ef-Tu inhibition by natural products or in Fusobacterium spp. exists till todate. Ef-Tu is an abundant specialized drug target in bacteria that varies from human Ef-Tu. Elfamycins target Ef-Tu and hence, Enacyloxin IIa was used to generate pharmacophore for virtual screening of three natural product libraries, Natural Product Activity and Species Source (NPASS) (n = 30000 molecules), Tibetan medicinal plant database (n = 54 molecules) and African medicinal plant database (n > 6000 molecules). Peptaibol Septocylindrin B (NPC141050), Hirtusneanoside, and ZINC95486259 were prioritized from these libraries as potential therapeutic candidates. ADMET profiling was done for safety assessment, physiological-based pharmacokinetic modeling in human and mouse for getting insight into drug interaction with body tissues and molecular dynamics was used to assess stability of the best hit NPC141050 (Septocylindrin B). Based on the promising results, we propose further in vitro, in vivo and pharmacokinetic testing on the lead Septocylindrin B, for possible translation into therapeutic interventions.

Using UAVRS and deep learning to conduct resource surveys of threatened Tibetan medicinal plants in the Qinghai-Tibet Plateau

Gentiana szechenyii Kanitz. and Gentiana veitchiorum Hemsl. are two wild medicinal plants widely used in Tibetan medicine. In recent decades, their wild populations have declined rapidly due to persistent over-harvesting of their flowers. Because their flowers are small and dense, it is difficult to rely on manual counting under wild conditions. The combination of deep learning and unmanned aerial vehicle remote sensing (UAVRS) is a new method for plant surveys. We trained 12 of the most advanced or widely used YOLO models on a custom dataset to achieve quantitative detection of both plant flowers in UAVRS images. The accuracy, precision and recall of G. szechenyii Flower (GSF) detection can reach 97.00%, 90.40% and 95.40%, respectively (based on YOLOv7). Similarly, those of G. veitchiorum Flower (GVF) detection can reach 93.40%, 91.30% and 90.60%, respectively (based on YOLOv5n), and the highest mAP can reach 94.90% (based on YOLOv5m). Based on the detection results, it is calculated that the total yield of dried GSF and GVF that can be harvested as medicinal materials in study area ranges from 1.88 to 2.10 g·m−2 and 4.42–4.62 g·m−2, respectively. The results show that deep learning and UAVRS can be used for quantitative detection of GSF and GVF, which is helpful for further research and protection of these two plants.

Sequence Analysis of the Plastomes of Two Tibetan Medicinal Plants of the Family Papaveraceae.

With the rapid development of next-generation sequencing technology, more plants plastomes have been sequenced, further advancing species identification and phylogenetic studies. However, there are a few studies on the genetic and phylogenetic analysis of the plastomes of Dicranostigma lactucoides Hook. f. et Thoms. and Hypecoum leptocarpum Hook. f. et Thoms. In this study, we sequenced and analyzed the plastomes of Dicranostigma lactucoides Hook. f. et Thoms. and Hypecoum leptocarpum Hook. f. et Thoms., and conducted a phylogenetic analysis using 13 related species. The results showed that the plastomes of both D. lactucoides and H. leptocarpum had a typical tetrad structure, with sizes of 166,819 bp and 163,282 bp, respectively. We annotated 133 genes for D. lactucoides and 120 genes for H. leptocarpum. A total of 72 and 43 simple repetitive sequences were detected in D. lactucoides and H. leptocarpum, respectively. Codon preference analysis showed that the relative usage frequency of codons and the relative abundance of synonymous codons used were the same for both plastomes. Nucleotide polymorphism analysis identified seven variant loci with high nucleotide diversity (Pi) values, all located in the large single copy (LSC) region. Inverted repeat (IR) boundary analysis revealed differences in gene types and locations on both sides of the boundary, except for the small single copy/inverted repeat a (SSC/IRa) boundary. The phylogenetic analysis showed the species clustered into two major groups, one with five genera (Hypecoum, Corydalis, Papaver, Meconopsis, and Dicranostigma) and the other with two genera (Coreanomecon; and Hylomecon). Comparative analysis of the plastome genomic characteristics and phylogeny of D. lactucoides and H. leptocarpum laid the foundation for identifying the above two species and the phylogenetic study and comprehensive exploitation of the Papaveraceae.

2D-HPLC-MS Technology Combined with Molecular Network for the Identification of Components in Tibetan medicine Aconitum pendulum.

In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate chemical composition of the Tibetan medicinal plant APB. Through the implementation of 2D-HPLC, enhanced separation of complex mixtures was achieved, enabling the isolation of individual compounds for subsequent analysis. The molecular network approach further aided in elucidating structural relationships among these compounds, contributing to the determination of potential bioactive molecules. This integrated strategy efficiently identified a wide array of chemical components present within the plant. The findings revealed a diverse spectrum of chemical constituents within APB, including alkaloids, among others. This research not only advances understanding of the phytochemical profile of this traditional Tibetan medicine but also provides valuable insights into its potential therapeutic properties. The integration of 2D-HPLC-MS and molecular network proves to be a powerful tool for systematically exploring and identifying complex chemical compositions in herbal medicines, paving the way for further research and development in the field of natural product discovery.

Exploring the hypolipidemic effects of bergenin from Saxifraga melanocentra Franch: mechanistic insights and potential for hyperlipidemia treatment

ObjectiveThe goal of this study was to explore the hypolipidemic effects of bergenin extracted from Saxifraga melanocentra Franch (S. melanocentra), which is a frequently utilized Tibetan medicinal plant known for its diverse bioactivities. Establishing a quality control system for black stem saxifrage is crucial to ensure the rational utilization of its medicinal resources.MethodsA one-step polyamide medium-pressure liquid chromatography technique was applied to isolate and prepare bergenin from a methanol extract of S. melanocentra. A zebrafish model of hyperlipidemia was used to investigate the potential hypolipidemic effects of bergenin.ResultsThe results revealed that bergenin exhibited substantial hypo efficacy in vivo. Specifically, bergenin significantly reduced the levels of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-c) while simultaneously increasing high-density lipoprotein cholesterol (HDL-c) levels. At the molecular level, bergenin exerted its effects by inhibiting the expression of FASN, SREBF1, HMGCRα, RORα, LDLRα, IL-1β, and TNF while promoting the expression of IL-4 at the transcriptional level. Molecular docking analysis further demonstrated the strong binding affinity of bergenin to proteins such as FASN, SREBF1, HMGCRα, RORα, LDLRα, IL-4, IL-1β, and TNF.ConclusionsFindings indicate that bergenin modulates lipid metabolism by regulating lipid and cholesterol synthesis as well as inflammatory responses through signaling pathways associated with FASN, SREBF1, and RORα. These results position bergenin as a potential candidate for the treatment of hyperlipidemia.

Current status of research on endophytes of traditional Tibetan medicinal plant and their metabolites.

The Qinghai-Tibet Plateau, known as the "Third Pole of the World," has a rich variety of medicinal plants that play an important role in the field of medicine due to its unique geographical environment. However, due to the limited resources of Tibetan medicinal plants and the fragility of the ecological environment of the Qinghai-Tibet Plateau, more and more Tibetan medicinal plants are on the verge of extinction. As a reservoir of biologically active metabolites, endophytes of medicinal plants produce a large number of compounds with potential applications in modern medicine (including antibacterial, immunosuppressive, antiviral, and anticancer) and are expected to be substitutes for Tibetan medicinal plants. This paper reviews 12 Tibetan medicinal plants from the Qinghai-Tibet Plateau, highlighting the diversity of their endophytes, the diversity of their metabolites and their applications. The results show that the endophytes of Tibetan medicinal plants are remarkably diverse, and the efficacy of their metabolites involves various aspects, such as antioxidant, anti-disease and anti-parasitic. In addition, conservation measures for the resources of Tibetan medicinal plants are summarised to provide a reference for an in-depth understanding of the endophytes of Tibetan medicinal plants and to stimulate the scientific community to bioprospect for the endophytes of Tibetan medicinal plants, as well as to provide ideas for their rational exploitation.

A neutral heteropolysaccharide from Halenia elliptica D. Don: Extraction, structural characterization, antioxidant and antiaging activities

Halenia elliptica D. Don (H. elliptica), which is also known as “heijicao” and “luanehuamao” in China, is recognised as a valuable Tibetan medicinal plant with polysaccharides as the main active ingredient. However, studies on the polysaccharides isolated from H. elliptica are few. A polysaccharide (HEPN-1) with a molecular weight of 10.80 kDa was mainly composed of Gal, Ara, Man, Glc, Rha and Fuc in a molar ratio of 25.56:24.52:4.58:3.37:2.62:1.00. Structural analysis showed that HEPN-1 had a backbone mainly consisting of 4-β-Galp, 3,6-β-Galp and 3,4,6-β-Galp and branched chains that contained two arabinan (R1 and R2) and two heteropolysaccharide (R3 and R4) side chains. The branching degree of HEPN-1 was 0.52. Within the range of doses (75–300 μg/mL), HEPN-1 increased the enzyme activity of SOD, CAT and GSH-Px and decreased the MDA level in H2O2-induced RAW 264.7 cells in a dose-dependent manner. After 6 weeks of intragastric administration, 300 mg/kg HEPN-1 considerably improved the learning and memory deficits in mice and the antioxidant enzyme system. Moreover, the MDA formation in D-gal-induced aging mice was inhibited, possibly partly via the activation of the PI3K/Akt and Nrf2/HO-1 signalling pathways. Therefore, HEPN-1 could serve as a potential natural antioxidant to prevent aging.

Insights into the mechanism underlying UV-B induced flavonoid metabolism in callus of a Tibetan medicinal plant Mirabilis himalaica

Mirabilis himalaica is an important Tibetan medicinal plant in China. However, it has become a rare and class I endangered Tibetan medicine plant. Therefore, the use of callus to propagate germplasm resources is of great significance. We found that the flavonoid content of M. himalaica callus increased continuously with the extension of UV-B treatment. Multi-omics profiles were used to reveal the co-expression patterns of gene networks of flavonoid metabolism in M. himalaica callus during UV-B radiation. Results showed that five medicinal metabolics, including geranin, eriodictyol, astragalin, isoquercetin, pyrotechnic acid, and one anthocyanin malvide-3-O-glucoside were identified. The transcriptome data were divided into 46 modules according to the expression pattern by WGCNA (weighted gene co-expression network analysis), of which the module Turquoise had the strongest correlation with six target metabolites. We found that seven structural genes and twenty-five transcription factors were related to the metabolism of flavonoid synthesis, among which the structural genes CHI, C4H and UGT79B6 had strong co-expression relationships with the 6 target metabolites. WRKY42, WRKY7, bHLH128 and other transcription factors had strong co-expression relationships with multiple structural genes. Consequently, these findings suggest callus grown under UV-B treatment could be an effective alternative medical resource of M. himalaica, which is valuable for conservation and usage of this wild and endangered plant.

Microbial diversity patterns in the root zone of two Meconopsis plants on the Qinghai-Tibet Plateau.

In the extreme alpine climate of the Qinghai-Tibet Plateau (QTP), plant growth and reproduction are limited by extremely cold temperatures, low soil moisture, and scarce nutrient availability. The root-associated microbiome indirectly promotes plant growth and plays a role in the fitness of plants on the QTP, particularly in Tibetan medicinal plants. Despite the importance of the root-associated microbiome, little is known about the root zone. This study used high-throughput sequencing to investigate two medicinal Meconopsis plants, M. horridula and M. integrifolia, to determine whether habitat or plant identity had a more significant impact on the microbial composition of the roots. The fungal sequences were obtained using ITS-1 and ITS-2, and bacterial sequences were obtained using 16S rRNA. Different microbial patterns were observed in the microbial compositions of fungi and bacteria in the root zones of two Meconopsis plants. In contrast to bacteria, which were not significantly impacted by plant identity or habitat, the fungi in the root zone were significantly impacted by plant identity, but not habitat. In addition, the synergistic effect was more significant than the antagonistic effect in the correlation between fungi and bacteria in the root zone soil. The fungal structure was influenced by total nitrogen and pH, whereas the structure of bacterial communities was influenced by soil moisture and organic matter. Plant identity had a greater influence on fungal structure than habitat in two Meconopsis plants. The dissimilarity of fungal communities suggests that more attention should be paid to fungi-plant interactions.

The total iridoid glycoside extract of Lamiophlomis rotata Kudo induces M2 macrophage polarization to accelerate wound healing by RAS/ p38 MAPK/NF-κB pathway.

Lamiophlomis rotata (Benth.) Kudo (L. rotata), a Tibetan medicinal plant, is used to treat "yellow-water diseases", such as skin disease, jaundice and rheumatism. Our previous study showed that the iridoid glycoside extract of L. rotata (IGLR) is the major constituent of skin wound healing. However, the role of IGLR in the biological process of trauma repair and the probable mechanism of the action remain largely unknown. To investigate the role of IGLR in the biological process of trauma repair and the probable mechanism of the action. The role of IGLR in wound healing was investigated by overall skin wound in mice with Hematoxylin and Eosin (H&E) and Masson trichrome staining. The anti-inflammatory, angiogenesis-promoting and fibril formation effects of IGLR were visualized in wound skin tissue by immunofluorescence staining, and the proinflammatory factors and growth factors were assayed by real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Macrophages, dermal fibroblasts, and endothelial cells were cultured to measure the direct/indirect interaction effects of IGLR on the proliferation and migration of cells, and flow cytometry was employed to assess the role of IGLR on macrophage phenotype. Network pharmacology combined with Western blot experiments were conducted to explore possible mechanisms of the actions. IGLR increased the expression of CD206 (M2 markers) through the RAS/p38 MAPK/NF-κB signaling pathway during wound injury in vivo and in vitro. IGLR suppressed the inflammatory cytokines iNOS, IL-1β and TNF-α in the early stage of wound healing. During the proliferation step of wound repair, IGLR promoted angiogenesis and fibril formation by increasing the expression of VEGF, CD31, TGF-β and α-SMA in wound tissue, and similar results were verified by RT-PCR and ELISA. In a paracrine mechanism, the extract promoted the proliferation of dermal fibroblasts, and endothelial cells were founded by the conditioned medium (CM). IGLR induced M2 macrophage polarization in the early stage of wound healing; in turn, IGLR played a key role in the transition from inflammation to cell proliferation during the biological process of wound healing.

Chromosome-Level Genome Assembly of Herpetospermum pedunculosum (Cucurbitaceae)

This study presents a chromosome-level reference genome assembly of a traditional Tibetan medicinal plant, Herpetospermum pedunculosum belonging to the Cucurbitaceae family. Following a combined PacBio high-fidelity sequencing and Hi-C analysis, a final H. pedunculosum genome assembly, 804.11 Mb in length was obtained, 90.45% of which was anchored into ten pseudochromosomes with a contig N50 of 24.39 Mb. In addition, 579.55 Mb repetitive sequences and 23,924 high-confidence protein-coding genes were annotated. Phylogenetic analysis revealed that H. pedunculosum was sister to a clade formed by cucumber, zucchini, and wax gourd. Further whole-genome duplication analysis revealed no recent polyploidization event in the H. pedunculosum genome. The high-quality H. pedunculosum genome presented here will be highly useful in investigating the molecular mechanisms underlying the biosynthesis of its active compounds and adaptation strategies to the extreme environment. It will also provide great insights into comparative genomic studies of Cucurbitaceae and flowering plants.

Comparative study on flavonoids from Tibetan medicinal plants Saussurea species using HPLC-DAD-ESI-MS

Three kinds of “Snowlotus” including Saussurea involucrate, Saussurea medusa and Saussurea laniceps had been prescribed as folk medicine interchangeably in Tibet and Uygur, China. To verify the feasibility of this phenomenon, a HPLC-DAD-ESI-MS method was developed in this study to determine the flavonoid compounds from three Saussurea species qualitatively and quantitatively. The LODs and LOQs ranged from 0.0263–0.1172 µg/mL and 0.0862–0.3867 µg/mL, respectively. The relative standard deviation (RSD × 100%) of the validation for the instrument precision was below 2.03%, while the RSD × 100% values for the method precision were below 5.73%, respectively. The recoveries ranged from 92.98–101.79%. These results further demonstrated that this method was precise and practical for determining flavonoid compounds from three Saussurea species. In this study, 14 kinds of constituents were simultaneously identified according to the ESI-MS data and previous studies. The contents of total flavonoids and eight components were determined with spectrophotometry and HPLC-DAD methods, respectively. The results demonstrated that the total flavonoids in S. involucrate had the hightest value and there were some differences in the content of eight flavonoids. For S. involucrate, a proportion of rutin, luteolin-7-O-glucoside, and hispidulin were detected. For S. laniceps, Arctiin, apigenin, and arctigenin were the major flavonoid compounds. For S. medusa, seven types of compounds were detected and arctiin presented the highest value, whereas apigenlin exhibited the lowest value. This comparative study may promote the differentiation in use of three kinds of snowlotus.

Berberis dictyophylla F. inhibits angiogenesis and apoptosis of diabetic retinopathy via suppressing HIF-1α/VEGF/DLL-4/Notch-1 pathway

Ethnopharmacological relevanceXiao Bopi (XBP, སྐྱེར་བའི་བར་ཤུན།), as a classical Tibetan medicinal plant in China, which derived from the stem bark of Berberis dictyophylla F., has the function of “clearing heat and decreasing mKhris-pa”. And it traditionally is utilized to treat the diabetes mellitus and its complications, such as diabetic retinopathy (DR). However, its underlying mechanisms remain unclear. Aim of the studyThe purpose of this study aimed to explore the microvascular protection of water extract of XBP against the spontaneous retinal damage of db/db mice. Meanwhile, the underlying mechanisms of XBP on angiogenesis and apoptosis were further interpreted. Materials and methodsWe firstly used high-performance liquid chromatography to detected the representative chemical ingredients in the water extract of XBP. The DR model of db/db mice was then randomly divided into five groups: model group, calcium dobesilate (0.23 g/kg) group, and the water extract of XBP (0.375, 0.75 and 1.5 g/kg, respectively) groups. After 8 weeks of continuous administration, the parameters including body weight, fasting blood glucose, oral glucose tolerance test and insulin tolerance test were measured. The pathological changes and abnormal angiogenesis of the retina were detected by optical coherence tomography, HE, periodic acid–Schiff staining and transmission electron microscopy. Simultaneously, molecular docking was used to predict the potential connections between representative ingredients in XBP and angiogenesis/apoptosis-related proteins. The level of angiogenesis-related proteins and gene hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth (VEGF), delta-like ligand 4 (DLL-4) and Notch-1 were estimated by immunofluorescence analyses and real time-PCR. Further, TUNEL staining and immunofluorescence analyses were performed to investigate the apoptotic phenomenon and the expression of Bax, Bcl-2, Apaf-1, Cyto-c and cleaved caspase-3 and cleaved caspase-9 in the retina. ResultsPhytochemical analysis revealed that magnoflorine, jatrorrhizine, palmatine and berberine were principally representative ingredients in XBP. The results demonstrated that XBP effectively increased glucose tolerance and insulin sensitivity, whereas no effect on body weight of DR mice. Moreover, retinal thickening, pathological and retinal ultrastructure changes in DR mice were evidently ameliorated by XBP. The molecular docking results demonstrated that the main components of XBP and the protein of angiogenesis and apoptosis had a potential bind. XBP restrained the gene and protein levels of HIF-1α, VEGF, DLL-4 and Notch-1 in retina. Additionally, the TUNEL-positive cell rate and the down-regulated proteins of Bax, Apaf-1, Cyto-c, cleaved Caspase-3 and cleaved Caspase 9 and increased Bcl-2 level were revised by XBP. ConclusionsTo sum up, the results suggested that XBP against DR could attribute to alleviating angiogenesis and apoptosis by suppressing the HIF-1α/VEGF/DLL-4/Notch-1 pathway. This evidence sheds a new light on the potential mechanisms of XBP in the treatment of DR.

Selection and Validation of Reference Genes for RT-qPCR Analysis in Tibetan Medicinal Plant Saussurea Laniceps Callus under Abiotic Stresses and Hormone Treatments.

Real-time quantitative PCR (RT-qPCR) is an important technique for studying gene expression analysis, but accurate and reliable results depend on the use of a stable reference gene. This study proposes to test the expression stability of candidate reference genes in the callus of Saussurea laniceps, a unique Tibetan medicinal plant. Based on the S. laniceps callus transcriptome, eleven candidate reference genes, including TUA2, TUB3, TUB8, TIF3B1, TIF3H1, ELF5A, PP2AA2, UEV1D, UBL5, UBC36, and SKIP1), were validated for RT-qPCR normalization in the callus under abiotic stress (salt, cold, and UV) and hormone treatments (abscisic acid, MeJA, and salicylic acid). The stability of the candidate genes was evaluated in all the samples of S. laniceps. Comprehensive analysis of all samples showed that the best reference genes were UBC36 and UBL5. ELF5A and TIF3B1 were ranked as the most stable genes in the sample sets under abiotic stress. For hormone stimulation, UBC36 and TIF3H1 genes had the best stability. This study provides useful guidelines and a starting point for reference gene selection for expression analysis using RT-qPCR techniques in S. laniceps.