Anoectochilus Roxburghii Research Articles

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.

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-1β 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.

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.

Comparative Metabolome and Transcriptome Analyses of the Regulatory Mechanism of Light Intensity in the Synthesis of Endogenous Hormones and Anthocyanins in Anoectochilus roxburghii (Wall.) Lindl.

To explore the regulatory mechanism of endogenous hormones in the synthesis of anthocyanins in Anoectochilus roxburghii (Wall.) Lindl (A. roxburghii) under different light intensities, this study used metabolomics and transcriptomics techniques to identify the key genes and transcription factors involved in anthocyanin biosynthesis. We also analyzed the changes in and correlations between plant endogenous hormones and anthocyanin metabolites under different light intensities. The results indicate that light intensity significantly affects the levels of anthocyanin glycosides and endogenous hormones in leaves. A total of 38 anthocyanin-related differential metabolites were identified. Under 75% light transmittance (T3 treatment), the leaves exhibited the highest anthocyanin content and differentially expressed genes such as chalcone synthase (CHS), flavonol synthase (FLS), and flavonoid 3'-monooxygenase (F3'H) exhibited the highest expression levels. Additionally, 13 transcription factors were found to have regulatory relationships with 7 enzyme genes, with 11 possessing cis-elements responsive to plant hormones. The expression of six genes and two transcription factors was validated using qRT-PCR, with the results agreeing with those obtained using RNA sequencing. This study revealed that by modulating endogenous hormones and transcription factors, light intensity plays a pivotal role in regulating anthocyanin glycoside synthesis in A. roxburghii leaves. These findings provide insights into the molecular mechanisms underlying light-induced changes in leaf coloration and contribute to our knowledge of plant secondary metabolite regulation caused by environmental factors.

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

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. An inflammasome activation model was established in bone marrow-derived macrophages (BMDMs) to investigate the effects of ARP on caspase-1 cleavage, IL-1β 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. 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. 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.

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

BackgroundHigh 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.ResultsUnder 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.ConclusionsTo 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.

ArPAOs from A. roxburghii showed essential roles in polyamine mediating water stress tolerance

Anoectochilus roxburghii is a precious Chinese herb with excellent pharmacological effects. When meet stress, plants produced polyamines (PAs) which are identified in active tissues and play crucial roles in stress responses. Nevertheless, the molecular mechanism of PAs responding to stress in A. roxburghii were largely unknown. Here, for the first time, three PAOs in A. roxburghii were identified from transcriptome data and functionally analyzed. Expression analysis was performed using fluorescence quantitative PCR and eventually, transgenic Arabidopsis thaliana were created to confirm the biological function of ArPAOs. It was found that the highest expression was in young flower buds and the expression levels of three ArPAO genes in various tissues were also different, which indicated their different catalytic preferences and functions. Under the treatment of exogenous spermine and spermidine, the expression of three ArPAOs fluctuated and the overall expression level increased. Overexpression (OE) of ArPAOs could enhance water stress tolerance through mediating polyamine catabolism. It might because the excessive expression of ArPAOs reduce the content of spermine and spermidine, creating a negative feedback regulation upstream and causes plants manufacturing PAs which reinforce water stress tolerance. Taken together, the study firstly reported the overexpression of ArPAOs enhance plant water stress tolerance and regulate the content of polyamines. This work will provide a vital theoretical foundation for uncovering the operation and regulatory mechanism of ArPAOs in promoting plant development and managing stress.

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.

Untargeted metabolomics in Anectocillus roxburghii with habitat heterogeneity and the key abiotic factors affecting its active ingredients.

Anoectochilus roxburghii is a rare, endangered herb with diverse pharmacological properties. Understanding the main metabolite types and characteristics of wild A. roxburghii is important for efficiently utilizing resources and examining quality according to origin. Samples were collected from the main production areas across five regions in Fujian Province, China. An untargeted metabolomics analysis was performed on the entire plants to explore their metabolic profiles. We utilized UPLC-MS/MS to specifically quantify eight targeted flavonoids in these samples. Subsequently, correlation analysis was conducted to investigate the relationships between the flavonoids content and both the biological characteristics and geographical features. A comprehensive analysis identified a total of 3,170 differential metabolites, with terpenoids and flavonoids being the most prevalent classes. A region-specific metabolite analysis revealed that the Yongchun (YC) region showed the highest diversity of unique metabolites, including tangeretin and oleanolic acid. Conversely, the Youxi (YX) region was found to have the smallest number of unique metabolites, with only one distinct compound identified. Further investigation through KEGG pathway enrichment analysis highlighted a significant enrichment in pathways related to flavonoid biosynthesis. Further examination of the flavonoid category showed that flavonols were the most differentially abundant. We quantified eight specific flavonoids, finding that, on average, the YX region exhibited higher levels of these compounds. Correlation analysis highlighted a significant association between flavonoids and habitat, especially temperature and humidity. Untargeted metabolomics via LC-MS was suitable for identifying region-specific metabolites and their influence via habitat heterogeneity. The results of this study serve as a new theoretical reference for unique markers exclusively present in a specific sample group.

Anoectochilusroxburghii extract down-regulates METTL3 to reduce RNA m6A on WNT pathway and inhibit melanoma cancer cell metastasis.

Characterized by high invasiveness and expansive growth, melanoma progression is intricately regulated, with RNA m6A modifications playing a pivotal role. Among these regulators, METTL3 modulates the migratory capabilities of melanoma cells, specifically by modifying the RNA m6A of UCK2. The extract of Anoectochilusroxburghii, a plant known for its broad-spectrum antitumour properties, has been shown to effectively curb the invasion and migration of melanoma cells and impede tumour growth. The primary mechanism involves the downregulation of METTL3 expression. By doing so, the Anoectochilus roxburghii extract reduces the m6A level on the mRNA of UCK2, accelerating UCK2 mRNA degradation and, consequently, decreasing UCK2 expression. This process, in turn, suppresses gene expression within the Wnt/β-catenin pathway, contributing to its antimelanoma effects.

Characterization of Anoectochilus roxburghii Bioactive Compounds and Its Inhibition on the Metabolism‐Related Enzyme Activities In Vitro

In this study, the Anoectochilus roxburghii (A. roxburghii) was studied for its chemical composition and biological activities. The first aim of this work was to isolate and purify compounds from A. roxburghii using Sephadex LH‐20 and HPLC. The identification of seven compounds was achieved, with 5,4′‐dihydroxy‐3,3′,7‐trimethoxy‐flavone and 5‐hydroxy‐3,4‐dimethyl‐5‐pentylfuran‐2(5H)‐one being isolated from A. roxburghii for the first time. The second aim was to describe its inhibition on the metabolism‐related enzyme activities in vitro. Further, 3,5‐dihydroxy‐7,3′,4′‐trimethoxyflavone and 5‐hydroxy‐3,4‐dimethyl‐5‐pentylfuran‐2(5H)‐one showed inhibitory activity on xanthine oxidase, while isorhamnetin‐3‐O‐rutinoside (IC50 = 87.45 μg/mL) may be potential inhibitors of α‐amylase. Molecular docking analysis revealed that isorhamnetin‐3‐O‐rutinoside exhibited the highest binding affinity towards PL. In addition to scientifically expanding the compound library of A. roxburghii, the discovery is also a vital reference for finding new insights in the field of medication research, especially as natural metabolism‐related enzyme inhibitors.

Molecular insights into the mechanisms of a leaf color mutant in Anoectochilus roxburghii by gene mapping and transcriptome profiling based on PacBio Sequel II

Plants with partial or complete loss of chlorophylls and other pigments are frequently occurring in nature but not commonly found. In the present study, we characterize a leaf color mutant ‘arly01’ with an albino stripe in the middle of the leaf, which is an uncommon ornamental trait in Anoectochilus roxburghii. The albino “mutant” middle portion and green “normal” leaf parts were observed by transmission electron microscopy (TEM), and their pigment contents were determined. The mutant portion exhibited underdevelopment of plastids and had reduced chlorophyll and other pigment (carotenoid, anthocyanin, and flavonoid) content compared to the normal portion. Meanwhile, comparative transcript analysis and metabolic pathways mapping showed that a total of 599 differentially expressed genes were mapped to 78 KEGG pathways, most of which were down-regulated in the mutant portion. The five most affected metabolic pathways were determined to be oxidative phosphorylation, photosynthesis system, carbon fixation & starch and sucrose metabolism, porphyrin and chlorophyll metabolism, and flavonoid biosynthesis. Our findings suggested that the mutant ‘arly01’ was a partial albinism of A. roxburghii, characterized by the underdevelopment of chloroplasts, low contents of photosynthetic and other color pigments, and a number of down-regulated genes and metabolites. With the emergence of ornamental A. roxburghii in southern China, ‘arly01’ could become a popular cultivar due to its unique aesthetics.

The improvement of kinsenoside in wild-imitated cultivation Anoectochilus roxburghii associated with endophytic community

Due to the low content of active ingredients and poor efficacy in herbal medicine in modern large-scale planting conditions, a growing number of methods for improving the content of active ingredients have been studied, such as improving planting techniques, developing and applying microbial engineering bacteria. In recent years, most studies have focused on improving the quality of Anoectochilus roxburghii through conventional farming methods. However, the role of endophytes in improving the quality of herbal medicine was often ignored. Although some studies have shown that microorganisms can regulate Anoectochilus roxburghii growth, there are still few studies on their regulation of plant active ingredients in plants. In this study, endophytic community and active ingredient content under the three culture patterns were examined through amplicon sequencing and high performance liquid chromatography-evaporative light scattering detector, and the correlation between the abundance of endophytes and kinsenoside content was analyzed. The results indicated a significant alteration in the diversity and network complexity of endophytic community within Anoectochilus roxburghii under the three culture conditions. After being cultivated in a wild-imitated environment for 3 months, the microbial composition of wild-imitated cultivated Anoectochilus roxburghii differentiated and converged toward that of wild-tending planted Anoectochilus roxburghii. Correlation analyses showed that the diversity of bacterial community has a significant positive relationship with the content of kinsenoside, and Burkholderia-Caballeronia-Paraburkholderia is the key taxa affecting kinsenoside content. In addition, acetylacetyl-CoA reductase, a key enzyme for the synthesis of kinsenoside, was significantly enriched in wild-imitated cultivated and wild tending cultivated Anoectochilus roxburghii. The study proposed that the medicinal quality of wild-imitated cultivated and wild tending cultivated Anoectochilus roxburghii, with high content of kinsenoside, which is likely attributed to the Burkholderia-Caballeronia-Paraburkholderia and acetylacetyl-CoA. This study provides a basis for the improvement and sustainable utilization of Anoectochilus roxburghii resources.

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 (0.1-0.5µg/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.

Identification of flavonoids in Anoectochilus roxburghii and in vitro inhibition of key enzymes relevant to metabolic syndrome

The traditional Chinese herb Anoectochilus roxburghii (A. roxburghii) is well-known for its medicinal value, but few studies have reported its ability to combat metabolic syndrome activities in vitro. In this study, the extraction of seven compounds from A. roxburghii using 70% ethanol, RP-C18, thin layer chromatography (TLC), Sephadex LH-20, high performance liquid chromatography (HPLC) and subsequent identification of their structures through nuclear magnetic resonance (NMR). Among these compounds, kaempferol-3-O-rutinoside and isorhamnetin-3-O-α-l-rhamnopyranoside-7-O-β-d-glucopyranoside were obtained from A. roxburghii for the first time. Results showed that those compounds inhibited key enzymes involved in metabolic syndrome in vitro. Molecular docking and binding free energy calculations were employed to elucidate the inhibition mechanism of these compounds, while SwissADME was used to assess their drug-like characteristics. Notably, 9-hydroxy-megastigma-4,7-dien-3-one-9-O-β-d-glucopyranoside has the potential as an α-amylase inhibitor for the first time. This research enriches the compound library of A. roxburghii and provides new ideas for medication research and the development of metabolic syndrome.

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

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. 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. 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. 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.

In situ neutral desorption-extractive electrospray ionization mass spectrometry reveals red-blue light promoted the accumulation of amino acids and polyphenols in Anoectochilus roxburghii

Light quality affects plant leaf growth and its metabolism of amino acids and polyphenols. Anoectochilus roxburghii has been used for food, medicine, and ornamental purposes. To obtain the optimal light quality for improved accumulation of metabolites in A. roxburghii in vitro. The seedings were cultured under the white light (W), blue light (B), red light (R), red: blue light = 1:2 (R1B2), red: blue light = 1:1 (R1B1), red: blue light = 2:1 (R2B1). Twenty compounds in raw leaves were in situ rapidly identified within 20 s by neutral desorption-extractive electrospray ionization mass spectrometry (ND-EESI-MS) without any sample pretreatment, including eight amino acids, eleven polyphenols, and kinsenoside. Meanwhile, leaf area and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were evaluated. Our results showed that leaf area and antioxidant enzyme activities of the R2B1 were significantly higher than B or R group. The red-blue light increased the content of isoleucine, histidine, serine, arginine, and promoted the accumulation of caffeic acid, ferulic acid, quercetin, myricetin, kaempferol, and rutin in phenylpropane metabolism, especially in the R2B1, which could be used as the optimal light quality. ND-EESI-MS could enhance our understanding of the effect of light quality on the accumulation of amino acids and polyphenols in A. roxburghii.

Exogenous spermidine enhanced the water deficit tolerance of Anoectochilus roxburghii by modulating plant antioxidant enzymes and polyamine metabolism

Water stress is an important factor affecting herb quality and yield and exogenous spermidine (Spd) addition can enhance dehydration tolerance of plants. Anoectochilus roxburghii (Wall.) Lindl is a perennial herb widely used in medicine, health care, and beauty industry, yet, its dehydration resistance remains largely unknown. In this view, the current research aimed at elucidating the drought resistance mechanism of A. roxburghii under PEG-induced water stress at different levels (10%, 20%, and 30% PEG6000). In particular, the morphological, physiological, and biochemical changes occurring in leaves treated with (0.5 mM, 5 mM) or without Spd were discussed under moderate water stress (20% PEG6000). Results showed that water stress significantly inhibited the growth of A. roxburghii by decreasing the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), arginine decarboxylase (ADC), S-adenosylmethionine decarboxylase (SAMDC), and polyamine oxidase (PAO). The contents of soluble protein, soluble proline, hydrogen peroxide (H2O2), malondialdehyde (MDA), and the relative conductivity (REC) of leaves increased by 27.32%, 31.37%, 68.84%, 103.61%, and 63.64%, respectively. Under dehydration condition, foliar spraying Spd effectively improved plant growth with the contents of soluble protein and soluble proline increased, while the H2O2, REC, and MDA contents decreased. The SOD activity of 0.5 mM and 5 mM treatments were 1.13 and 1.01 times higher than only 20% PEG treatment, respectively. Similarly, the POD activity of 0.5 mM and 5 mM treatments increased by 52.82% and 34.11%, while the CAT activity of 0.5 mM and 5 mM treatments increased by 34.39 and 33.15 U/mgprot, respectively. In addition, exogenous Spd application significantly increased the key enzyme activities of polyamine metabolism (ADC, SAMDC, and PAO) under moderate water stress. In conclusion, exogenous Spd addition with low concentration (0.5 mM) improved the resistance of A. roxburghii against water stress by increasing the antioxidant enzymes activities and endogenous polyamines contents. This study helps to the understanding of dehydration resistance of A. roxburghii and provide guidelines for its cultivation under water deficit condition.

Kinsenoside from Anoectochilus roxburghii (Wall.) Lindl. suppressed oxidative stress to attenuate aging-related learning and memory impairment via ERK/Nrf2 pathway

Ethnopharmacological relevanceAnoectochilusroxburghii (Wall.) Lindl. (AR), as an exceptionally valuable traditional Chinese medicine, has been widely used to treat hepatitis, cancer, diabetes, etc. But, the effects and the primary functioning element of AR on attenuating aging and aging-related learning and memory degradation has not yet been explored. Aim of the studyThis study aimed at exploring the protective property of aqueous extract of AR (AEAR) on alleviation of aging and aging-related learning and memory impairment in vivo, and further investigating the main active ingredient and mechanism of AEAR. Materials and methodsD-galactose (D-gal) induced aging mice and HT22 cells exposed with L-Glutamic acid (Glu) were used as in vivo and in vitro model, separately. The effects of AEAR on aging and aging-related learning and memory degradation were explored by using morris water maze test, immunohistochemistry staining, biochemistry assay, etc. The effects and mechanism of AEAR and Kinsenoside (Kin) on antioxidation in vitro were investigated by cell viability assay, biochemistry assay, qRT-PCR, western blotting and molecular docking studies. ResultsTreatment with AEAR (containing 69.52 ± 0.85% Kin, i.g.) for 63 days, alleviated low growth rate, abnormal brain, liver and thymus index, and decline in learning and memory capability of aging mice. Meanwhile, AEAR inhibited the decreased activities of SOD and GSH-PX, the decline in the ratio of GSH to GSSG, and the increase of MDA in both serum and brain, and also promoted the Nrf2 nuclear translocation in brain of aging mice induced by D-gal. The effects of AEAR on alleviating abnormal physiological characteristics, attenuating learning and memory impairment, and inhibiting oxidative stress in aging mice was similar to or even better than that of Vc. In HT22 cells exposed with Glu, Kin increased the cell viability, up-regulated the activities of SOD and GSH-PX, enhanced the ratio of GSH to GSSG, and down-regulated MDA, which was superior to AEAR. Kin up-regulated the ratio of p-ERK1/2 to ERK1/2, promoted the Nrf2 nuclear translocation and its downstream target genes, i.e. HO-1, NQO-1, GCLC and GCLM expression at the mRNA and protein levels, which were consistent with AEAR. Further, molecular docking results also confirmed that Kin had strong binding energy with ERK1 and ERK2. ConclusionThe present study indicated that Kin could alleviate the oxidative stress in aging mice via activating the ERK/Nrf2 signaling pathway, in order to attenuate aging and aging-related learning and memory impairment, as the main active ingredient of AR.

Kinsenoside mitigates myocardial ischemia/reperfusion-induced ferroptosis via activation of the Akt/Nrf2/HO-1 pathway

Ischemia-induced myocardial infarction is regarded as one of the major killers of humans worldwide. Kinsenoside (KD), a primary active ingredient derived from Anoectochilus roxburghii, shows antioxidant and vascular protective properties. Myocardial ischemia/reperfusion (I/R) injury is associated with oxidative damage and could be regulated by KD. However, its targets and the exact mechanism by which it operates remains unclear. The aim of this study was to investigate the role of KD in myocardial I/R injury and to define the mechanism by which it works. We established both myocardial I/R model in vivo and hypoxia/reoxygenation (H/R) cardiomyocyte model in vitro in this study. KD can attenuate I/R-induced myocardial injury in vivo and inhibit H/R-induced injury in vitro in a dose-dependent manner. KD increased mitochondrial membrane potential, SOD activity, and GSH activity in cardiomyocytes, whereas MDA accumulation, iron accumulation, and Mito-ROS production were decreased. We intersected differentially expressed genes (DEGs) from RNA-seq results with ferroptosis-related genes, and found KD significantly downregulated COX2 expression and upregulated GPX4 expression. These findings were further confirmed by Western blot analysis. Additionally, KD increased AKT phosphorylation and Nrf2 translocation into the nucleus, as well as HO-1 expression. When Akt or Nrf2 were inhibited in the KD group, the anti-ferroptosis properties of KD were nullified. Thus, Kinsenoside may exert anti-ferroptosis effect in myocardial I/R injury by decreasing mitochondrial dysfunction and increasing anti-oxidation through the Akt/Nrf2/HO-1 signaling pathway, suggesting it could be used as a potential therapeutic agent for myocardial reperfusion injury.