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 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.

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.

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.

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.

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.

UNASSIGNED: Plant growth-promoting bacteria (PGPB) represents a common biological fertilizer with remarkable effect in improving crop production and environmental friendliness.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

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.

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.

Anoectochilus roxburghii and Anoectochilus formosanus, belong to the Anoectochilus genus, have been used for Chinese herbal drugs as well as health food. Phenylalanine ammonia-lyase (PAL), the key enzyme in primary metabolism and phenylpropanoid metabolism, produces secondary metabolites (flavonoids) in plants, which are beneficial for the biosynthesis of phenylpropanoid metabolites.
The PAL genes were cloned from A. formosanus and A. roxburghii according to our previous transcriptomic analysis. The PALs were introduced into pCAMBIA2300-35S-PAL-eGFP to generate 35S-PAL-eGFP. The constructs were further used for subcellular localization and transgenic Arabidopsis. The expression of AfPAL and ArPAL under precursor substance (L-Phe), NaCl, UV, and red-light were analyzed by real-time quantitative PCR (RT-qPCR).
AfPAL and ArPAL , encoding 2,148 base pairs, were cloned from A. formosanus and A. roxburghii. The subcellular localization showed that the ArPAL and AfPAL were both localized in the nucleus with GPF. Quantitative RT-PCR analysis indicated that the ArPAL and AfPAL genes function in the phenylalanine pathway as well as response to induced conditions. Overexpression of the AfPAL and ArPAL could increase flavonoids and anthocyanin content in the transgenic Arabidopsis.
The results suggest that AfPAL and ArPAL play a crucial role in the flavonoid biosynthesis in Anoectochilus. Also, our study provides new insights into the enrichment of secondary metabolites of traditional Chinese medicines A. formosanus and A. roxburghii, which can improve their medicinal active ingredients and be used for drug discovery in plants.