Extraction and characterization of hepatoprotective polysaccharides from Anoectochilus roxburghii against CCl4-induced liver injury via regulating lipid metabolism and the gut microbiota.

Anoectochilus roxburghii polysaccharides exhibit notable hepatoprotective effects, but the underlying substance basis and mechanisms remain unknown. In this study, four new polysaccharides named ARP-1a, ARP-1b, ARP-2a and ARP-2b, were isolated from A. roxburghii. Their structural characteristics were systematically analyzed using HPGPC, HPLC, GC-MS, IR and NMR analysis. ARP-1a, the leading polysaccharide isolated from A. roxburghii, was further evaluated for its hepatoprotective effects on acute liver injury mice induced by CCl4. ARP-1a significantly reduced the serum ALT, AST, TNF-α, IL-1ß and IL-6 levels, liver MDA content, and increased the SOD and CAT activities and GSH level in liver. H&E staining revealed that ARP-1a pretreatment could markedly relieve liver injury. Further mechanism exploration indicated that ARP-1a could relieve CCl4-induced oxidative damage through activating the Nrf2 signaling. In addition, metabolomics, lipidomics and 16S rRNA amplicon sequencing were used to elucidate the underlying mechanisms of ARP-1a. Multi-omics analysis indicated that ARP-1a exerted hepatoprotective effect against CCl4-induced acute liver injury by regulating lipid metabolism and modulating the gut microbiota. In conclusion, the above results suggest that ARP-1a can be considered a promising and safe candidate for hepatoprotective drug, as well as a potential prebiotic for maintaining intestinal homeostasis and promoting human intestinal health.

Anoectochilus roxburghii Extract Extends the Lifespan of Caenorhabditis elegans through Activating the daf-16/FoxO Pathway.

As a significant global issue, aging is prompting people's interest in the potential anti-aging properties of Anoectochilus roxburghii (A. roxburghii), a plant traditionally utilized in various Asian countries for its purported benefits in treating diabetes and combating aging. However, the specific anti-aging components and mechanisms of A. roxburghii remain unclear. This study aims to investigate the anti-aging effects and mechanisms of A. roxburghii extract E (ARE). Caenorhabditis elegans (C. elegans) were exposed to media containing different concentrations of ARE whose superior in vitro radical scavenging capacity was thus identified. Lifespan assays, stress resistance tests, and RT-qPCR analyses were conducted to evaluate anti-aging efficacy, reactive oxygen species (ROS) levels, antioxidant enzyme activity, and daf-16, sod-3, and gst-4 levels. Additionally, transcriptomic and metabolomic analyses were performed to elucidate the potential anti-aging mechanisms of ARE. Fluorescence protein assays and gene knockout experiments were employed to validate the impacts of ARE on anti-aging mechanisms. Our results revealed that ARE not only prolonged the lifespan of C. elegans but also mitigated ROS and lipofuscin accumulation, and boosted resistance to UV and heat stress. Furthermore, ARE modulated the expression of pivotal anti-aging genes including daf-16, sod-3, and gst-4, facilitating the nuclear translocation of DAF-16. Significantly, ARE failed to extend the lifespan of daf-16-deficient C. elegans (CF1038), indicating its dependency on the daf-16/FoxO signaling pathway. These results underscored the effectiveness of ARE as a natural agent for enhancing longevity and stress resilience to C. elegans, potentially to human.

Anoectochilus roxburghii polysaccharide reduces D-GalN/LPS-induced acute liver injury by regulating the activation of multiple inflammasomes.

BACKGROUND: Acute liver injury (ALI) is a serious syndrome with a high mortality rate due to viral infection, toxic exposure, and autoimmunity, and its severity can range from mildly elevated liver enzymes to severe liver failure. Activation of the nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is closely associated with the development of ALI, and the search for an inhibitor targeting this pathway may be a novel therapeutic option. Anoectochilus roxburghii polysaccharide (ARP) is a biologically active ingredient extracted from Anoectochilus roxburghii with immunomodulatory, antioxidant, and anti-inflammatory bioactivities and pharmacological effects. In this study, we focused on D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced acute liver injury by ARP through inhibition of NLRP3 inflammasome. METHODS: An inflammasome activation model was established in bone marrow-derived macrophages (BMDMs) to investigate the effects of ARP on caspase-1 cleavage, IL-1ß secretion, and ASC oligomerization in inflammasomes under different agonists. We used the D-GalN/LPS-induced acute liver injury model in mice, intraperitoneally injected ARP or MCC950, and collected liver tissues, serum, and intraperitoneal lavage fluid for pathological and biochemical indexes. RESULTS: ARP effectively inhibited the activation of the NLRP3 inflammasome and had an inhibitory effect on non-classical NLRP3, AIM2, and NLRC4 inflammasomes. It also effectively inhibited the oligomerization of apoptosis-associated speck-like protein (ASC) from a variety of inflammatory vesicles. Meanwhile, ARP has good therapeutic effects on acute liver injury induced by D-GaIN/LPS. CONCLUSION: The inhibitory effect of ARP on a wide range of inflammasomes, as well as its excellent protection against acute liver injury, suggests that ARP may be a candidate for acute liver injury.

Phytochemical Research and Anti-Inflammatory Activities of the Ethanol Extract from Anoectochilus Roxburghii (Wall.) Lindl.

Anoectochilus roxburghii is a well-known and valuable traditional Chinese herb due to various medicinal and functional benefits. In-depth investigation is necessary to discover active ingredients and expand its application. In this study, four new compounds (1-4) along with ten known compounds (5-14) were isolated from the ethanol extract of A.roxburghii. Their structures were elucidated by spectroscopic data interpretation. The isolates were screened for their inhibitory activities on the production of NO in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Among them, compounds 5, 6, 9, 10, 12, 13 and 14 exhibited significant anti-inflammatory activity through inhibiting the release of NO.

Physiological and transcriptomic analyses reveal the regulatory mechanisms of Anoectochilus roxburghii in response to high-temperature stress.

BACKGROUND: High temperatures significantly affect the growth, development, and yield of plants. Anoectochilus roxburghii prefers a cool and humid environment, intolerant of high temperatures. It is necessary to enhance the heat tolerance of A. roxburghii and breed heat-tolerant varieties. Therefore, we studied the physiological indexes and transcriptome of A. roxburghii under different times of high-temperature stress treatments. RESULTS: Under high-temperature stress, proline (Pro), H2O2 content increased, then decreased, then increased again, catalase (CAT) activity increased continuously, peroxidase (POD) activity decreased rapidly, then increased, then decreased again, superoxide dismutase (SOD) activity, malondialdehyde (MDA), and soluble sugars (SS) content all decreased, then increased, and chlorophyll and soluble proteins (SP) content increased, then decreased. Transcriptomic investigation indicated that a total of 2740 DEGs were identified and numerous DEGs were notably enriched for "Plant-pathogen interaction" and "Plant hormone signal transduction". We identified a total of 32 genes in these two pathways that may be the key genes for resistance to high-temperature stress in A. roxburghii. CONCLUSIONS: To sum up, the results of this study provide a reference for the molecular regulation of A. roxburghii's tolerance to high temperatures, which is useful for further cultivation of high-temperature-tolerant A. roxburghii varieties.

Effects of compound Anoectochilus roxburghii (Wall.) Lindl. oral liquid on relative metabolic enzymes and various biochemical indices in Wistar rats with isoniazid-induced liver injury.

Isoniazid (INH) is the first-line anti-tuberculosis drug in clinical practice, and its main adverse effect is drug-induced liver injury (DILI). This study aimed to investigate the hepatoprotective effect of Compound Anoectochilus roxburghii (Wall.) Lindl. Oral Liquid (CAROL) and to provide a new strategy for the search of potential drugs against INH-induced liver injury in Wistar rats. Animal experiment was based on INH (100 mg/kg) induced liver injury to explore the intervention effects of CAROL at doses of 1.35, 2.70, and 5.40 mL/kg. LC-QTOF-MS/MS was used to identify hepatoprotective components in CAROL and its' exposed components in rat serum. The hepatoprotective effect of CAROL was evaluated by pathological observation of rat liver tissue and changes in levels of biochemical indices and cytokines in serum or liver tissue. Of the 58 hepatoprotective components identified, 15 were detected in the serum of rats with liver-injured treated by high-dose CAROL. Results of animal experiments showed that the levels of various biochemical indexes and cytokines were significantly reversed with CAROL intervention. In particular, the expression level of cytokeratin-18 and high-mobility group box 1, as specific and sensitive indicators of DILI, was significantly reduced in the serum of rats with CAROL intervention compared with the INH model group. The same reversal was observed in the levels of TBIL, ALP, ALT, and AST in serum, as well as in the levels of TNF-α, IL-6, SOD, and MDA in liver tissue. For INH-metabolizing enzymes, an evident expression inhibition was observed in N-acetyltransferase 2 and glutathione S-transferases with CAROL intervention, which may be the key to controlling INH hepatotoxicity. CAROL has a favorable hepatoprotective effect on INH-induced liver injury. This study takes the first step in studying the hepatoprotective mechanism of CAROL against INH hepatotoxicity and provides reference for wider clinical applications.

Biochemical and Transcriptome Analysis Reveals Pigment Biosynthesis Influenced Chlorina Leaf Formation in Anoectochilus roxburghii (Wall.) Lindl.

Anoectochilus roxburghii (Wall.) Lindl is a perennial herb of the Orchidaceae family; a yellow-green mutant and a yellow mutant were obtained from the wild type, thereby providing good material for the study of leaf color variation. Pigment content analysis revealed that chlorophyll, carotenoids, and anthocyanin were lower in the yellow-green and yellow mutants than in the wild type. Transcriptome analysis of the yellow mutant and wild type revealed that 78,712 unigenes were obtained, and 599 differentially expressed genes (120 upregulated and 479 downregulated) were identified. Using the Kyoto Encyclopedia of Genes and Genomes pathway analysis, candidate genes involved in the anthocyanin biosynthetic pathway (five unigenes) and the chlorophyll metabolic pathway (two unigenes) were identified. Meanwhile, the low expression of the chlorophyll and anthocyanin biosynthetic genes resulted in the absence of chlorophylls and anthocyanins in the yellow mutant. This study provides a basis for similar research in other closely related species.

The evaluation of the anti-cancer effects of Anoectochilus roxburghii on hematological cancers in vitro.

The cause of hematological cancers is the uncontrolled proliferation of hematopoietic and lymphoid tissues, and chemotherapy is used to treat cancer. However, adverse side effects of chemotherapy are common. Therefore, the use of plant extracts as a method for treating cancer is becoming increasingly popular. Anoectochilus roxburghii (wall.) Lindl. (A. roxburghii) is one of the original sources of the valuable medicinal plants known as the king medicine and the golden grass. This study investigated the potential anticancer effect of A. roxburghi (AR) on JURKAT, MM1S, THP1 and U266 cells. To test the cytotoxic and apoptotic effects of AR, hematological cancer cells were exposed to increasing doses of AR (0.1-0.5 µg/µl). The spectrophotometric MTT assay and the flow cytometric Annexin V staining were used to examine the viability and apoptosis of the cells, respectively. qRT-PCR was used to determine the expression levels of the apoptosis-related genes BAD, BAX, BIM and BCL-2. Our results show that AR treatment decreased cell viability and induced apoptosis in each cell line. Our RT-PCR data showed that AR significantly increased the expression levels of the pro-apoptotic BAX gene in JURKAT and MM1S cells, whereas it significantly increased the expression levels of both BAX and BIM in U266 cells. This is the first study to investigate how AR modulates apoptosis in hematological cancer cells. As a result, AR therapy may be a promising treatment modality for the treatment of cancer.

Mechanism of the Effect of Compound Anoectochilus roxburghii (Wall.) Lindl. Oral Liquid in Treating Alcoholic Rat Liver Injury by Metabolomics.

Purpose: Compound Anoectochilus roxburghii (Wall.) Lindl oral liquid (CAROL) is often as a hepatoprotective agent. The present study aimed to elucidate the protective mechanism of CAROL against alcoholic liver injury in rats by untargeted metabolomics combined with multivariate statistical analysis. Methods: An alcoholic liver disease model was established in sprague-dawley (SD) rats by gavage of alcohol, and CAROL treatment was administered. The hepatoprotective effect of CAROL was evaluated by examining liver tissues changes and detecting biochemical index activities and cytokines in serum and liver homogenates. The metabolites in serum samples were examined using ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) and multivariate statistical analysis to screen for differentially expressed metabolites and Kyoto Encyclopedia of Genes and Genomes (KEGG) to assess potential metabolic pathways. Results: CAROL has the potential to downregulate inflammation levels and alleviate oxidative stress. The differential metabolites are mainly engaged in riboflavin metabolism, arginine and proline metabolism, phenylalanine, tyrosine and tryptophan biosynthesis metabolism, phenylalanine metabolism, pyrimidine metabolism, and vitamin B6 metabolism to achieve hepatoprotective effects. Conclusion: CAROL may exhibit beneficial hepatoprotective effects by reducing inflammation, mitigating oxidative stress, and modulating metabolites and their metabolic pathways.This study has important implications for advancing the clinical application of CAROL.

Flavonoid profile of Anoectochilus roxburghii (Wall.) Lindl. Under short-term heat stress revealed by integrated metabolome, transcriptome, and biochemical analyses.

Global warming severely threatens plant growth, and could lead to yield reduction. Although findings suggest that flavonoids play important roles in biological process in plants, their response to heat stress in Anoectochilus roxburghii (Wall.) Lindl. remains unclear. Here, we aimed to examine the flavonoid profile of A. roxburghii under heat stress and assess the effect of exogenous application of quercetin on heat stress tolerance. Metabolome analysis showed that quercetin, tricetin, isorhamnetin, scutellarein, and 4',7-Isoflavandiol were the main upregulated flavonoids in A. roxburghii, based on variable importance in the projection >1 and with fold change >2. Determination of the concentrations of the flavonoids using a standard curve revealed that quercetin, kaempferol, and isorhamnetin contents increased by 8.24-, 7.55-, and 5.01-fold, respectively, during heat stress, whereas rutin concentration decreased from 83.04 to 80.89 mg/kg (dry weight). Additionally, transcriptome analysis indicated increased expression of several genes in flavonoid biosynthesis pathways, including phenylalanine ammonia-lyase and chalcone synthase. Moreover, exogenous application of quercetin improved the antioxidant capacity and physiological parameters, including photosynthetic rate and chlorophyll content, of A. roxburghii under heat stress. Overall, the flavonoid profile of A. roxburghii under short-term heat stress was characterized based on integrated metabolomic, transcriptomic, and biochemical analyses, providing new insights for improving the biological value of A. roxburghii.

Effects of dietary supplementation of Anoectochilus roxburghii extract (ARE) on growth performance, abdominal fat deposition, meat quality, and gut microbiota in broilers.

The broiler industry frequently encounters 2 common problems: excessive deposition of abdominal fat and poor quality of meat. However, there are limited nutritional manipulation strategies to address these issues. While Anoectochilus roxburghii (Wall.) Lindl., a traditional Chinese herb, has been shown to have multiple beneficial effects in humans, its potential roles in broiler chickens remain unexplored. In this study, the effects of dietary supplementation with Anoectochilus roxburghii extract (ARE) on growth performance, abdominal fat deposition, meat quality, blood indices, and gut microbiota were investigated in yellow-feather broiler chickens. A total of 90 twenty-one-day-old yellow-feather broilers were randomly divided into 3 treatments, and each treatment included 5 replicates with 6 birds per replicate. Birds were fed a basal diet supplemented with 0, 0.15, or 0.30% ARE for 6 wk. The results showed that the inclusion of ARE in the diet did not have any significant effect on meat yield (P > 0.05). However, it did lead to a reduction in abdominal fat deposition and an improvement in meat quality (P < 0.05). Mechanistically, the addition of ARE inhibited lipid biosynthesis and enhanced lipid breakdown in both the liver and adipose tissue of the broilers. Furthermore, ARE supplementation increased the antioxidase activities in the muscle and serum of the broilers (P < 0.05). In addition, the supplementation of ARE optimized the diversity and composition of the cecal microbiota, particularly by lowering the ratio of Firmicutes to Bacteroidetes (P < 0.05). Moreover, the abundance of some bacteria that were positively correlated with abdominal fat deposition was reduced by ARE, and vice versa (P < 0.05). Collectively, the results suggest that ARE is a promising candidate as a feed additive for reducing abdominal fat deposition and improving meat quality in the broiler industry.

Serum metabolome profiling, network pharmacology analysis, and experimental validation of Anoectochilus roxburghii in the treatment of carbon tetrachloride-induced liver injury.

Anoectochilus roxburghii (Wall.) Lindl. (AR) has been traditionally used to treat inflammatory diseases, but the specific mechanism underlying its hepatoprotective effect remains unclear. Here, serum metabolomics and network pharmacology were employed to investigate the hepatoprotective mechanism of AR. Thirty male Sprague-Dawley rats were divided into six groups: normal, model, positive, high-dose AR, middle-dose AR, and low-dose AR. The positive group received therapeutic doses of silibinin, whereas the AR-treated groups received different doses of AR extract once daily. After 10 days of intragastric administration, the rats were intraperitoneally injected with a 50% CCl4 olive oil solution (2 mL/kg) to induce liver injury. Serum and liver samples were obtained, and GC-MS was utilized to monitor changes in serum metabolome. The levels of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and hydrooxproline in serum significantly increased in the model group. On the contrary, AR-treated group showed a significant decrease in the levels of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and hydrooxproline. Histopathological observation also revealed that the extent of liver injury was alleviated in the AR-treated group. Fifty differential metabolites were identified, suggesting that AR may prevent liver damage by modulating carbohydrate and amino acid metabolism.

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.

Isolation, structural and bioactivities of polysaccharides from Anoectochilus roxburghii (Wall.) Lindl.: A review.

Anoectochilus roxburghii (Wall.) Lindl. (A. roxburghii), a valuable herbal medicine in China, has great medicinal and edible value. Polysaccharides, as one of the main active components of A. roxburghii, comprise glucose, arabinose, xylose, galactose, rhamnose, and mannose in different molar ratios and glycosidic bond types. By varying the sources and extraction methods of A. roxburghii polysaccharides (ARPS), different structural characteristics and pharmacological activities can be elucidated. ARPS has been reported to exhibit antidiabetic, hepatoprotective, anti-inflammatory, antioxidant, antitumor, and immune regulation activities. This review summarizes the available literature on the extraction and purification methods, structural features, biological activities, and applications of ARPS. The shortcomings of the current research and potential focus in future studies are also highlighted. This review provides systematic and current information on ARPS to promote their further exploitation and application.

Two polyamines -responsive WRKY transcription factors from Anoectochilus roxburghii play opposite functions on flower development.

Anoectochilus roxburghii is a rare and precious plant with medicinal and healthcare functions. Embryo abortion caused the lack of resources. Polyamine promoted its flowering and stress resistance in our previous study. But the mechanism remains unclear. The WRKY transcription factor family has been linked to a variety of biological processes in plants. In this study, two WRKY TFs (ArWRKY5 and ArWRKY20) of A. roxburghii, which showed significant response to Spd treatment, were identified and functionally analyzed. Tissue specific expression analyzation showed both of them mostly present in the flower. And ArWRKY5 expressed highest in the flower bud stage (-1 Flowering), while ArWRKY20 showed the highest expression in earlier flower bud stage (-2 Flowering) and the expression gradually decreased with flowering. The transcriptional activation activity assay and subcellular localization revealed that ArWRKY5 and ArWRKY20 were located in the nucleus and ArWRKY20 showed transcriptional activity. The heterologous expression of ArWRKY5 in Arabidopsis thaliana showed earlier flowering, while overexpression of ArWRKY20 delayed flowering. But the OE-ArWRKY20 lines had a robust body shape and a very significant increase in the number of rosette leaves. Furthermore, stamens and seed development were positively regulated by these two ArWRKYs. These results indicated that ArWRKY5 and ArWRKY20 not only play opposite roles in the floral development, but also regulate the plant growth and seed development in A. thaliana. But their specific biological functions and mechanism in A. roxburghii need to be investigated further.

Three new compounds from Anoectochilus roxburghii (Wall.) Lindl.

In this study, three new compounds, roxburic acid A (1) and two flavone glycosides isorhamnetin-3-O-α-L-rhamnosyl-(1→6)-ß-D-glucopyranose-(1→3)-ß-D-glucopyranoside (2), and kaempferol-7-O-ß-D-glucopyranosyl-(1→3)-ß-D-glucopyranoside (3) were isolated from an ethanol extract of the fresh Anoectochilus roxburghii (Wall.) Lindl., together with 10 known compounds (4-13). The structures of these compounds were comprehensively characterized by HR-ESI-MS, 1H NMR, 13C NMR, and 2 D-NMR. The DPPH free radical scavenging activity of the isolated compounds was evaluated, and the results showed that kaempferol-7-O-ß-D-glucopyranosyl- (1→3) -ß-D-glucopyranoside (3) and rutin (11) has the potential antioxidant activity with IC50 values of 139 µg/mL and 22.5 µg/mL respectively.

Effect of Burkholderia ambifaria LK-P4 inoculation on the plant growth characteristics, metabolism, and pharmacological activity of Anoectochilus roxburghii.

Background: Plant growth-promoting bacteria (PGPB) represents a common biological fertilizer with remarkable effect in improving crop production and environmental friendliness. Methods: In the present work, we presented a detailed characterization of plant morphology and physiology, metabolism, and pharmacological activity of A. roxburghii between Burkholderia ambifaria LK-P4 inoculation and un-inoculation (CK) treatment by routine analytical techniques (include microscopy and enzymatic activity assays and so on) coupled with metabolomics approaches. Results: Morphological and physiological results showedthat the P4 bacteria could significantly increase plant stomatal density, freshweight, survival rate,and the content of total flavonoids in leaves but reducethe amount of free amino acid. Furthermore, metabolite data showed that fatty acids (linoleic acid, linolenic acid, stearic acid) and active substance (kyotorphin and piceatannol) were specifically up-regulated in P4 inoculation. It was also demonstrated that the differential metabolites were involved in citrate cycle, glyoxylate and dicarboxylate metabolism, and biosynthesis of unsaturated fatty acids pathway. In addition, pharmacological efficacy found that A. roxburghii under P4 inoculation can significantly decrease (p < 0.05) blood glucose levels and protect the organs of mice with similar effect of Glibenclamide tablets. Conlusion: Overall, it can be seen that the exogenous P4 bacteria can promote the growth and increase content of special metabolites in A. roxburghii. This study provided theoretical basis and supported for the high-yield and high-quality bionic cultivation of A. roxburghii.

A comparative study of characteristics in diploid and tetraploid Anoectochilus roxburghii.

Artificial induction of polyploidy is an efficient technique for improving biological properties and developing new varieties of many plants. In this study, we analyzed and compared differences in characteristics (morphological and biological) of diploid and tetraploid Anoectochilus roxburghii plants. We found significant differences between tetraploid plants and their diploid counterparts. The tetraploid plants exhibited dwarfing and stockiness. They were also bigger and had more voluminous roots and larger stomata than the diploid plants. Moreover, the biochemical analyses showed that the contents of some amino acids and minerals elements were significantly higher in tetraploid plants. The chlorophyll content of the leaves exhibited no definitive changes, but the photosynthetic performance was higher in the tetraploid plants. In addition, contents of major bioactive compounds, such as kinsenoside and some flavonoids, were enhanced in tetraploids. This is the first detailed analysis of characteristics in diploid and tetraploid A. roxburghii plants. The results may facilitate breeding programs with the species.

Induction, Proliferation, Regeneration and Kinsenoside and Flavonoid Content Analysis of the Anoectochilus roxburghii (Wall.) Lindl Protocorm-like Body.

Anoectochilus roxburghii (Wall.) Lindl has been used in Chinese herbal medicine for treating various ailments. However, its wild resources are endangered, and artificial cultivation of the plant is limited by the low regeneration rate of conventional propagation methods. The lack of A. roxburghii resources is detrimental to the commercial production of the plant and kinsenoside, which is unique to Anoectochilus species. To develop highly efficient methods for A. roxburghii micropropagation and find alternative resources for kinsenoside production, we created an induction, proliferation, and regeneration of PLBs (IPR-PLB) protocol for A. roxburghii. We also analyzed the kinsenoside and flavonoid contents during the induction and proliferation of PLBs. The best media of IPR-PLB for PLB induction and proliferation (secondary PLB induction and proliferation), shoot formation, and rooting medium were Murashige and Skoog (MS) + 3 mg/L 6-benzylaminopurine (6-BA) + 0.5 mg/L naphthaleneacetic acid (NAA) + 0.8 mg/L zeatin (ZT) + 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2, 4-D), MS + 3 mg/L 6-BA + 0.5 mg/L NAA, and MS + 0.5 mg/L NAA, respectively. On these optimized media, the PLB induction rate was 89 ± 2.08%, secondary PLB induction rate was 120 ± 5%, secondary PLB proliferation rate was 400 ± 10% and 350 ± 10 % in terms of the quantity and biomass at approximately 1 month, shoot induction rate was 10.5 shoots/PLB mass, and root induction rate was 98%. All plantlets survived after acclimation. Darkness or weak light were essential for PLB proliferation, and light was crucial for PLB differentiation on these optimized media. The kinsenoside contents of PLBs and secondary PLBs were 10.38 ± 0.08 and 12.30 ± 0.08 mg/g fresh weight (FW), respectively. Moreover, the peak kinsenoside content during the proliferation of secondary PLBs was 34.27 ± 0.79 mg/g FW, which was slightly lower than that of the whole plant (38.68 ± 3.12 mg/g FW). Two flavonoids exhibited tissue- or temporal-specific accumulation patterns, and astragalin accumulated exclusively during the first 2 weeks of cultivation. The IPR-PLB protocol for A. roxburghii may facilitate the efficient micropropagation of A. roxburghii plants. Furthermore, the PLBs are a good alternative resource for kinsenoside production.

Study on Quality Control of Compound Anoectochilus roxburghii (Wall.) Lindl. by Liquid Chromatography-Tandem Mass Spectrometry.

Compound Anoectochilus roxburghii (Wall.) Lindl. (A. roxburghii) oral liquid (CAROL) is a hospital preparation of A. roxburghii and Ganoderma lucidum (G. lucidum), which have hepatoprotective effects. Eight active components (five nucleosides/nucleobases and three triterpenoid acids) in CAROL, A. roxburghii, and G. lucidum were simultaneously detected by high-performance liquid chromatography−tandem mass spectrometry (LC−MS/MS). The multiple reaction monitoring (MRM) mode was applied for the detection of analytes. These eight compounds were separated well within 12 min and quantified using the internal standard working curve method. The method showed good linearity (R2 > 0.9935) and high sensitivity (limit of detection = 0.29 ng/mL). The analyte recovery ranged from 85.07% to 97.50% (relative standard deviation < 3.31%). The content of the target analytes in four batches of CAROL, and the raw materials of G. lucidum and A. roxburghii from the five regions was determined using this method. The contents of guanosine and ganoderic acid A in four batches of oral liquid were high and stabilized and could be recommended as quality markers (Q-marker) for CAROL. Simultaneous qualitative and quantitative analysis of nucleosides and triterpenoid acids in CAROL, A. roxburghii, and G. lucidum by LC−MS/MS based on the MRM model was reported for the first time. The proposed method provides a sensitive, rapid, and reliable approach for the quality control of Chinese medicinal products.