Ipomoea obscura Represents a New Host of Phytoplasma Belonging to 16SrII Group Associated with Witches'-Broom Symptoms in China.

Phytoplasmas are phloem-limited plant pathogenic prokaryotes which can not be cultured in vitro. The pathogens could cause various plant symptoms such as witches'-broom, virescence, and leaf yellows. Ipomoea obscura is a valuable plant species belonging to the family Convolvulaceae, mainly used as a traditional Chinese medicine used to treat diseases such as dehydration and diuresis. In western countries it is commonly referred to as 'obscure morning glory'. During 2020 to 2021, plants showing abnormal symptoms including witches'-broom, internode shortening, and small leaves were found in Hainan Province, a tropical island of China. Approximately 30 % of I. obscura plants in the sampling regions which spanned 400 acres, showed symptoms. In order to identify the associated pathogen, six symptomatic samples and three asymptomatic samples were collected and total DNA were extracted from 0.10 g fresh plant leaf tissues using CTAB DNA extraction method. 16S rRNA and secA gene fragments, specific to phytoplasmas, were PCR amplified using primers R16mF2/R16mR1 and secAfor1/secArev3. The target PCR bands were obtained from the DNA of six symptomatic samples, whereas not from the DNA of the asymptomatic samples. The PCR products of phytoplasma 16S rRNA and secA gene obtained from the diseased samples were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). The 16S rRNA and secA gene sequences identified in the study were all identical with the length of 1330 bp (GenBank accession: OR625212) and 720 bp (OR635662) respectively. According to methods and protocols of phytoplasma identification and classification (Wei and Zhao, 2022), the phytoplasma strain identified in the study was described as Ipomoea obscura witches'-broom (IoWB) phytoplasma, IoWB-hnld strain. The partial 16S rRNA gene sequence of IoWB showed 100 % sequence identity over the full 1330 bp sequence to phytoplasmas belonging to 16SrII group like cassava witches'-broom phytoplasma (KM280679). The BLAST search of the 720 bp partial secA gene fragment of IoWB showed 100% sequence identity for the full sequence to phytoplasmas belonging to 16SrII group like 'Sesamum indicum' phyllody phytoplasma (OQ420657). RFLP analysis based on the 16S rRNA gene using iPhyClassifier demonstrated that the IoWB strain was a member of 16SrII-A subgroup with the similarity coefficient 1.00 to the reference phytoplasma strain (L33765). Phylogenetic analysis based on 16S rRNA and secA genes by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value indicated that IoWB-hnld was clustered into one clade with the phytoplasmas belonging to 16SrII group, with 98% and 100% bootstrap value separately. To our knowledge, this is the first report that Ipomoea obscura can be infected by phytoplasmas belonging to 16SrII-A subgroup in China. This report adds to the host range of 'Ca. Phytoplasma aurantifolia', documenting the symptoms on I. obscura which will assist in monitoring and control of the associated pathogen.

Xueliankoufuye Suppresses Microglial Activation with Inflammatory Pain by Blocking NF-κB Signaling Pathway.

Xuelian, as a traditional Chinese ethnodrug, plays an important role in anti-inflammation, immunoregulation, promoting blood circulation, and other physiological functions. It has been prepared into different traditional Chinese medicine preparations for clinical use, with xuelian koufuye (XL) being widely used to treat rheumatoid arthritis. However, whether XL can relieve inflammatory pain and its analgesic molecular mechanism are still unknown. The present study explored the palliative effect of XL on inflammatory pain and its analgesic molecular mechanism. In complete Freund's adjuvant (CFA)-induced inflammatory joint pain, oral XL dose-dependently improved the mechanical withdrawal threshold of inflammatory pain from an average value of 17.8 g to 26.6 g (P < 0.05) and high doses of XL significantly reduced inflammation-induced ankle swelling from an average value of 3.1 cm to 2.3 cm compared to the model group (P < 0.05). In addition, in carrageenan-induced inflammatory muscle pain rat models, oral XL dose-dependently improved the mechanical withdrawal threshold of inflammatory pain from an average value of 34.3 g to 40.8 g (P < 0.05). The phosphorylated p65 was inhibited in LPS-induced BV-2 microglia and spinal cord of mice in CFA-induced inflammatory joint pain within a value of 75% (P < 0.001) and 52% reduction (P < 0.05) on average, respectively. In addition, the results showed that XL could effectively inhibit the expression and secretion of IL-6 from an average value of 2.5 ng/ml to 0.5 ng/ml (P < 0.001) and TNF-α from 3.6 mg/ml to 1.8 ng/ml with IC50 value of 20.15 µg/mL and 112 µg/mL respectively, by activating the NF-κB signaling pathway in BV-2 microglia (P < 0.001). The above-given results provide a clear understanding of the analgesic activity and mechanism of action not found in XL. Considering the significant effects of XL, it can be evaluated as a novel drug candidate for inflammatory pain, which establishes a new experimental basis for expanding the indications of XL in clinical treatment and suggests a feasible strategy to develop natural analgesic drugs.

Neuroprotective effect of Ziziphi Spinosae Semen on rats with p-chlorophenylalanine-induced insomnia via activation of GABAA receptor.

Ziziphus jujuba var. spinosa (Bunge) Hu ex H.F.Chow [Rhamnaceae; Ziziphi Spinosae Semen (ZSS)] has attracted extensive attention as the first choice of traditional Chinese medicine in the treatment of insomnia. However, recent studies on the sleep-improving mechanism of ZSS have mainly focused on the role of single components. Thus, to further reveal the potential mechanism of ZSS, an assessment of its multiple constituents is necessary. In this study, ZSS extract (ZSSE) was obtained from ZSS via detailed modern extraction, separation, and purification technologies. The chemical constituents of ZSSE were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS). For in vivo experiments, a rat model of insomnia induced by p-chlorophenylalanine (PCPA) was established to investigate the potential effect and corresponding mechanism of ZSSE on improving sleep. Hematoxylin-eosin staining (HE) results revealed that the drug group showed prominent advantages over the model group in improving sleep. Moreover, the brain levels of γ-aminobutyric acid (GABA), glutamic acid (Glu), 5-hydroxytryptamine (5-HT), and dopamine (DA) were monitored via enzyme-linked immunosorbent assay (ELISA) to further study the sleep-improving mechanism of ZSSE. We found that sleep was effectively improved via upregulation of GABA and 5-HT and downregulation of Glu and DA. In addition, molecular mechanisms of ZSSE in improving sleep were studied by immunohistochemical analysis. The results showed that sleep was improved by regulating the expression levels of GABA receptor subunit alpha-1 (GABAARα1) and GABA acid receptor subunit gamma-2 (GABAARγ2) receptors in the hypothalamus and hippocampus tissue sections. Therefore, this work not only identified the active ingredients of ZSSE but also revealed the potential pharmacological mechanism of ZSSE for improving sleep, which may greatly stimulate the prospective development and application of ZSSE.

Nano-selenium alleviates cadmium-induced cerebellar injury by activating metal regulatory transcription factor 1 mediated metal response.

This study aims to investigate the role of metal regulatory transcription factor 1 (MTF1)-mediated metal response in cadmium (Cd)-induced cerebellar injury, and to evaluate the antagonistic effects of nano-selenium (Nano-Se) against Cd toxicity. A total of 80 chicks (1 d old, male, Hy-Line Variety White) were randomly allocated to 4 treatment groups for 3 months: the control group (fed with a basic diet, n = 20), the Nano-Se group (basic diet with 1 mg/kg nano-Se 1 mg/kg Nano-Se in basic diet, n = 20), the Nano-Se + Cd group (basic diet with 1 mg/kg Nano-Se and 140 mg/kg CdCl2, n = 20) and the Cd group (basic diet with 140 mg/kg CdCl2 , n = 20). The results of the experiment showed that the Purkinje cells were significantly decreased with their degradation and indistinct nucleoli after Cd exposure. Moreover, exposure to Cd caused a significant accumulation of Cd and cupper. However, the contents of Se, iron, and zinc were decreased, thereby disturbing the metal homeostasis in the cerebellum. The Cd exposure also resulted in high levels of malondialdehyde (MDA) and down regulation of selenoprotein transcriptome. Furthermore, the expressions of MTF1, metallothionein 1 (MT1), MT2, zinc transporter 3 (ZNT3), ZNT5, ZNT10, zrt, irt-like protein 8 (ZIP8), ZIP10, transferrin (TF), ferroportin 1 (FPN1), ATPase copper transporting beta (ATP7B), and copper uptake protein 1 (CTR1) were inhibited by Cd exposure. However, all these changes were significantly alleviated by the supplementation of Nano-Se. This study proved that Cd could disorder metal homeostasis and induce oxidative stress, whereas Nano-Se could relieve all these negative effects caused by Cd via activating the MTF1-mediated metal response in the cerebellum of chicken.

Preparation and Characterization of Methyl Jasmonate Microcapsules and Their Preserving Effects on Postharvest Potato Tuber.

Potato tubers tend to sprout during long-term storage, resulting in quality deterioration and shortened shelf life. Restrictions on the use of chlorpropham, the major potato sprout suppressant, have led to a need to seek alternative methods. In this study, the effects of methyl jasmonate (MeJA) solutions and MeJA microcapsules on sprouting and other key quality attributes of the potato tuber were investigated. The results showed that the MeJA solution was most effective at 300 µmol L-1 according to TOPSIS analysis. To prepare MeJA microcapsules, the optimal formulation is with 0.04% emulsifier, 2.5% sodium alginate, 0.5% chitosan and 3% CaCl2. Compared to 300 µmol L-1 MeJA solution, MeJA microcapsules consumed a lower dose of MeJA but demonstrated a better retaining effect on the overall quality attributes of potato tubers. MeJA microcapsules are promising agents for the preservation of postharvest potato tubers.

Carica papaya Represents a New Host of 16SrI-B Subgroup Phytoplasma Associated with Yellow Symptoms in China.

Carica papaya Linn, belonging to the Caricaceae family, is an economic and medicinal plant, which is widely cultivated in tropical and subtropical countries (Soib et al., 2020). Beginning in 2021, abnormal symptoms of Carica papaya exhibiting leaf yellow, crinkle and leaflet were found in Wanning city of Hainan Province, China. The diseased symptoms of the plant, with about 20 % incidence in the sampling regions, were suspected to be induced by phytoplasma, a phloem-limited and could not be cultured in vitro prokaryotic pathogen. Total DNAs were extracted from 0.10 g fresh leaves of symptomatic or asymptomatic Carica papaya using CTAB DNA extraction method (Doyle and Doyle, 1990). PCR reactions were performed using primers R16mF2/R16mR1 (Gundersen and Lee, 1996), secAfor1/secArev3 (Hodgetts et al., 2008) and AYgroelF/AYgroelR (Mitrovic et al., 2011) specific for phytoplasma 16S rRNA, secA and groEL gene fragments. PCR products of the 16S rRNA, secA and groEL gene target fragments of phytoplasma were obtained from the DNA of eight diseased Carica papaya samples whereas not from the DNA of the asymptomatic plant samples. The PCR amplicons of the three genes were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Shanghai, China) and the sequences data were deposited in GenBank. The 16S rRNA, secAgroEL gene of phytoplasma was in length of 1326 (GenBank accession: OL625608), 716 (OL630087) and 1300 (OL630088) bp separately, putatively encoding 238 (secA) and 432 (groEL) amino acids sequence. The phytoplasma strain was named as Carica papaya yellow phytoplasma (CpY), CpY-hnwn strain. A blast search based on 16Sr RNA gene of CpY-hnwn showed 100 % sequence identity with that of 16SrI aster yellows group members (16SrI-B subgroup), such as Onion yellows phytoplasma strain OY-M (AP006628), Chinaberry witches'-broom phytoplasma (CWB) strain CWB-hnsy1 (KP662119) and CWB-hnsy2 (KP662120), Rapeseed phyllody phytoplasma isolate RP166 (CP055264). RFLP analysis based on the 16S rRNA gene fragment of CpY-hnwn was performed by the interactive online phytoplasma classification tool iPhyClassifier (Zhao et al., 2009) indicated that the phytoplasma strain is a member of 16SrI-B subgroup. Blast search based on secA gene of CpY-hnwn showed 100 % sequence identity with that of CWB strains CWB-gdgz (KP662182), CWB-jxnc (KP662180) and CWB-fjya (KP662178) belonging to 16SrI-B subgroup. Blast search based on groEL gene of CpY-hnwn showed 99.77 % sequence identity with that of mulberry dwarf phytoplasma (AB124809) and 99.69 % sequence identity with that of Onion yellows phytoplasma strain OY-M (AP006628) and Rapeseed phyllody phytoplasma isolate RP166 (CP055264). Phylogenetic analysis based on the 16S rRNA gene fragments performed by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value (Kumar et al., 2016; Felsenstein, 1985) indicated that the CpY-hnwn phytoplasma strain was clustered into one clade with the phytoplasma strains of OY-M (AP006628), RP166 (CP055264), CWB-hnsy1 (KP662119), CWB-hnsy2 (KP662120) and areca palm yellow leaf (KF728948), with 100 % bootstrap value. To our knowledge, this is the first report that a 16SrI-B subgroup phytoplasma infects Carica papaya in Hainan Province, a tropical island of China. Carica papaya was previously reported to be infected by 16SrXII-O subgroup phytoplasmas in Nigeria (Kazeem et al., 2021), 16SrII-U subgroup in Hainan Province of China (Yang et al., 2016). The findings in this study indicated that one plant couldthe phytoplasmas belonging to different 16Sr groups, which would be beneficial to the specific detection of the pathogens and the epidemic monitoring of the related diseases. References: Doyle, J.J. and Doyle, J.L. 1990. Focus 12: 13-15. Felsenstein, J. 1985. Evolution 39: 783-791. Gundersen, D.E. and Lee, I.M. 1996. Phytopath. Medit. 35: 144-151. Hodgetts, J., et al. 2008. Int. J. Syst. Evol. Microbiol. 58: 1826-1837. Kazeem, S.A., et al. 2021. Crop Prot. 148: 105731. Kumar, S., et al. 2016. Mol. Biol. Evol. 33: 1870-1874. Mitrovic, J., et al. 2011. Ann. Appl. Biol. 159: 41-48. Soib, H.H., et al. 2020. Molecules, 25: 517. Yang, Y., et al. 2016. Int. J. Syst. Evol. Microbiol. 66: 3485-3491. Zhao, Y., et al. 2009. Int. J. Syst. Evol. Microbiol. 59: 2582-2593.

Traditional Chinese Medicine Decoctions Improve Longevity Following Diagnosis with Stage IV Esophageal Squamous Cell Carcinoma: A Retrospective Analysis.

OBJECTIVE: Traditional Chinese medicine (TCM) is an ancient form of personalized medicine and may improve morbidity and mortality in patients with esophageal cancer. This retrospective study aimed to evaluate the utility of TCM in the treatment of stage IV esophageal squamous cell carcinoma (SCC). METHODS: We collected the medical records of patients with stage IV SCC admitted to Henan Provincial Hospital of Traditional Chinese Medicine and Linzhou Hospital of Traditional Chinese Medicine between July 2017 and June 2020. We used univariate and multivariate analyses to determine if the use of TCM improved patient prognosis. Moreover, cluster analysis was used to classify the patients according to TCM syndrome type and identify the most frequently used combinations of remedies. RESULTS: After that 402 patients were included in PSM, of which 196 (48.8%) were treated with traditional Chinese medicine. TCM prolonged the survival time of patients with stage IV esophageal SCC (P=0.084), and was an independently associated with prognosis as demonstrated by Cox multivariate regression analysis [risk ratio (RR) =0.543, 95% confidence interval (CI): 0.390-0.755, P<0.001]. Association analysis revealed 75 cases (38.26%) had obstruction of phlegm and qi syndrome, 53 cases (27.04%) had phlegm and blood stasis syndrome, 38 cases (19.39%) had yang-qi deficiency syndrome, and 30 cases (15.31%) had heat retention and fluid consumption syndrome. CONCLUSION: Treatment with TCM derived therapies may increase the survival time of patients with stage IV esophageal SCC. Since these patients were diagnosed with different TCM syndromes, individualized TCM therapy is essential for improving symptoms and survival.

Immediate acupuncture with GB34 for biliary colic: protocol for a randomised controlled neuroimaging trial.

INTRODUCTION: As the main manifestation of gallstone disease, biliary colic (BC) is an episodic attack that brings patients severe pain in the right upper abdominal quadrant. Although acupuncture has been documented with significance to lead to pain relief, the immediate analgesia of acupuncture for BC still needs to be verified, and the underlying mechanism has yet to be covered. Therefore, this trial aims first to verify the immediate pain-alleviation characteristic of acupuncture for BC, then to explore its influence on the peripheral sensitised acupoint and central brain activity. METHODS AND ANALYSIS: This is a randomised controlled, paralleled clinical trial, with patients and outcome assessors blinded. Seventy-two patients with gallbladder stone disease presenting with BC will be randomised into a verum acupuncture group and the sham acupuncture group. Both groups will receive one session of immediate acupuncture treatment. Improvements in patients' BC will be evaluated by the Numeric Rating Scale, and the pain threshold of acupoints will also be detected before and after treatment. During treatment, brain neural activity will be monitored with functional near-infrared spectroscopy (fNIRS), and the needle sensation will be rated. Clinical and fNIRS data will be analysed, respectively, to validate the acupuncture effect, and correlation analysis will be conducted to investigate the relationship between pain relief and peripheral-cerebral functional changes. ETHICS AND DISSEMINATION: This trial has been approved by the institutional review boards and ethics committees of the First Teaching Hospital of Chengdu University of Traditional Chinese Medicine, with the ethical approval identifier 2019 KL-029, and the institutional review boards and ethics committees of the First People's Hospital of Longquanyi District, with the ethical approval identifier AF-KY-2020071. The results of this trial will be disseminated through peer-reviewed publications and conference abstracts or posters. TRIAL REGISTRATION NUMBER: CTR2000034432.

Compound traditional Chinese medicine dermatitis ointment ameliorates inflammatory responses and dysregulation of itch-related molecules in atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease accompanied with itchy and scaly rash. Compound traditional Chinese medicine dermatitis ointment (CTCMDO) consists of a mixture of extracts from five plants, which had been used in AD treatment due to good anti-inflammatory and anti-allergic effects. MATERIALS AND METHODS: In this study, high-performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometer (LC/MS) were performed to analyze the active ingredients of CTCMDO in detail and to establish its HPLC fingerprint. Furthermore, the anti-inflammatory and antipruritic activities of CTCMDO were studied in the treatment of DNCB-induced AD in mice. RESULTS: A total of 44 compounds including phenylpropionic acid compounds, alkaloid compounds, curcumin compounds and lignans were identified via combined HPLC and LC/MS. A fingerprint with 17 common peaks was established. In AD-like mice, DNCB-induced scratching behavior had been suppressed in the treatment of CTCMDO in a dose-dependent manner. Furthermore, the detailed experimental results indicated that the AD can be effectively improved via inhibiting the production of Th1/2 cytokines in serum, reversing the upregulation of substance P levels of itch-related genes in the skin, and suppressing the phosphorylation of JNK, ERK, and p38 in the skin. CONCLUSION: This work indicated that CTCMDO can significantly improve AD via attenuating the pathological alterations of Th1/2 cytokines and itch-related mediators, as well as inhibiting the phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB).

Diosmetin has therapeutic efficacy in colitis regulating gut microbiota, inflammation, and oxidative stress via the circ-Sirt1/Sirt1 axis.

Diosmetin (3',5,7 -trihydroxy-4'-methoxy flavone) is a natural flavonoid compound in the citrus species, it exhibits a variety of pharmacological activities, but little is known of its effects on colitis. In this study we evaluated the therapeutic effects of diosmetin on mouse models of chronic and acute colitis. Chronic colitis was induced in mice by drinking water containing 3% dextran sulfate sodium (DSS) from D0 to D8, followed by administration of diosmetin (25, 50 mg · kg-1 · d-1) for another 8 days. Acute colitis was induced by drinking water containing 5% DSS from D0 to D7, the mice concomitantly received diosmetin (25, 50 mg · kg-1 · d-1) from D1 to D7. During the experiments, body weight and disease activity index (DAI) were assessed daily. After the mice were sacrificed, colon tissue and feces samples were collected, and colon length was measured. We showed that in both models, diosmetin administration significantly decreased DAI score and ameliorated microscopic colon tissue damage; increased the expression of tight junction proteins (occludin, claudin-1, and zonula occludens-1), and reduced the secretion of proinflammatory cytokines IL-1ß, IL-6, TNF-α, and Cox-2 in colon tissue. We found that diosmetin administration remarkably inhibited colon oxidative damage by adjusting the levels of intracellular and mitochondrial reactive oxygen species, GSH-Px, SOD, MDA and GSH in colon tissue. The protection of diosmetin against intestinal epithelial barrier damage and oxidative stress were also observed in LPS-treated Caco-2 and IEC-6 cells in vitro. Furthermore, we demonstrated that diosmetin markedly increased the expression of Nrf2 and HO-1 and reduced the ratio of acetylated NF-κB and NF-κB by activating the circ-Sirt1/Sirt1 axis, which inhibited oxidative stress and inflammation in vivo and in vitro. Diosmetin reversed the effects of si-circSirt1 and si-Sirt1 in LPS-treated Caco-2 and IEC-6 cells. When the gut microbiota was analyzed in the mouse model of colitis, we found that diosmetin administration modulated the abundance of Bacteroidetes, Actinobacteria, Cyanobacteria and Firmicutes, which were crucial for inflammatory bowel disease. Our results have linked colitis to the circ-Sirt1/Sirt1 signaling pathway, which is activated by diosmetin. The results imply that diosmetin may be a novel candidate to alleviate DSS-induced colitis and can be a lead compound for future optimization and modification.

Intervention Effect of Dance Therapy Based on the Satir Model on the Mental Health of Adolescents during the COVID-19 Epidemic.

BACKGROUND: To investigate the intervention effect of dance therapy based on the Satir Model on the mental health of adolescents with depression during the COVID-19 epidemic. SUBJECTS AND METHODS: A total of 62 adolescents with depression were selected using Symptom Checklist 90 and randomly divided into two groups according to the matching of male and female participants; the experiment group had 32 members and the control group had 30 members. The experiment group received group psychological intervention and dance therapy based on the Satir Model, whereas the control group was not given any intervention. RESULTS: After the intervention, the scores of the experiment group in anxiety and depression are lower than those prior to intervention (p<0.01) and of the control group (p<0.01); the scores of the experiment group in life satisfaction, psychological resilience and their dimensions are higher than those prior to intervention (p<0.01) and higher than those of the control group (p<0.01). CONCLUSIONS: The combination of group intervention and dance therapy based on the Satir Model is a feasible method to effectively alleviate adolescents' anxiety and depression, promote their life satisfaction and psychological resilience, and thus improve their mental health.

Mechanism of Microbial Metabolite Leupeptin in the Treatment of COVID-19 by Traditional Chinese Medicine Herbs.

Coronavirus disease 2019 (COVID-19) has caused huge deaths and economic losses worldwide in the current pandemic. The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is thought to be an ideal drug target for treating COVID-19. Leupeptin, a broad-spectrum covalent inhibitor of serine, cysteine, and threonine proteases, showed inhibitory activity against Mpro, with a 50% inhibitory concentration (IC50) value of 127.2 µM in vitro in our study here. In addition, leupeptin can also inhibit SARS-CoV-2 in Vero cells, with 50% effective concentration (EC50) values of 42.34 µM. More importantly, various strains of streptomyces that have a broad symbiotic relationship with medicinal plants can produce leupeptin and leupeptin analogs to regulate autogenous proteases. Fingerprinting and structure elucidation using high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS), respectively, further proved that the Qing-Fei-Pai-Du (QFPD) decoction, a traditional Chinese medicine (TCM) formula for the effective treatment of COVID-19 during the period of the Wuhan outbreak, contains leupeptin. All these results indicate that leupeptin at least contributes to the antiviral activity of the QFPD decoction against SARS-CoV-2. This also reminds us to pay attention to the microbiomes in TCM herbs as streptomyces in the soil might produce leupeptin that will later infiltrate the medicinal plant. We propose that plants, microbiome, and microbial metabolites form an ecosystem for the effective components of TCM herbs. IMPORTANCE A TCM formula has played an important role in the treatment of COVID-19 in China. However, the mechanism of TCM action is still unclear. In this study, we identified leupeptin, a metabolite produced by plant-symbiotic actinomyces (PSA), which showed antiviral activity in both cell culture and enzyme assays. Moreover, leupeptin found in the QFPD decoction was confirmed by both HPLC fingerprinting and HRMS. These results suggest that leupeptin likely contributes to the antiviral activity of the QFPD decoction against SARS-CoV-2. This result gives us important insight into further studies of the PSA metabolite and medicinal plant ecosystem for future TCM modernization research.

Insight Into Polysaccharides From Panax ginseng C. A. Meyer in Improving Intestinal Inflammation: Modulating Intestinal Microbiota and Autophagy.

Polysaccharides from Panax ginseng C. A. Meyer (P. ginseng) are the main active component of P. ginseng and exhibit significant intestinal anti-inflammatory activity. However, the therapeutic mechanism of the ginseng polysaccharide is unclear, and this hinders the application for medicine or functional food. In this study, a polysaccharide was isolated from P. ginseng (GP). The primary structure and morphology of the GP were studied by HPLC, FT-IR spectroscopy, and scanning electron microscopy (SEM). Further, its intestinal anti-inflammatory activity and its mechanism of function were evaluated in experimental systems using DSS-induced rats, fecal microbiota transplantation (FMT), and LPS-stimulated HT-29 cells. Results showed that GP modulated the structure of gut microbiota and restored mTOR-dependent autophagic dysfunction. Consequently, active autophagy suppressed inflammation through the inhibition of NF-κB, oxidative stress, and the release of cytokines. Therefore, our research provides a rationale for future investigations into the relationship between microbiota and autophagy and revealed the therapeutic potential of GP for inflammatory bowel disease.

The protective effect of nnano-selenium against cadmium-induced cerebellar injury via the heat shock protein pathway in chicken.

Cadmium (Cd) is one of the toxic environmental heavy metals that poses health hazard to animals due to its toxicity. Nano-Selenium (Nano-Se) is a Nano-composite form of Se, which has emerged as a promising therapeutic agent for its protective roles against heavy metals-induced toxicity. Heat shock proteins (HSPs) play a critical role in cellular homeostasis. However, the potential protective effects of Nano-Se against Cd-induced cerebellar toxicity remain to be illustrated. To investigate the toxic effects of Cd on chicken's cerebellum, and the protective effects of Nano-Se against Cd-induced cerebellar toxicity, a total of 80 male chicks were divided into four groups and treated as follows: (A) 0 mg/kg Cd, (B) 1 mg/kg Nano-Se (C) 140 mg/kg Cd + 1 mg/kg Nano-Se (D) 140 mg/kg Cd for 90 days. We tested heat shock protein pathway-related factors including heat shock factors (HSFs) HSF1, HSF2, HSF3 and heat shock proteins (HSPs) HSP10, HSP25, HSP27, HSP40, HSP60, HSP70 and HSP90 expressions. Histopathological results showed that Cd treatment caused degradation of Purkinje cells. In addition, HSFs and HSPs expression decreased significantly in the Cd group. Nano-Se co-treatment with Cd enhanced the expression of HSFs and HSPs. In summary, our findings explicated a potential protective effect of Nano-Se against Cd-induced cerebellar injury in chicken, suggesting that Nano-Se is a promising therapeutic agent for the treatment of Cd toxicity.

Divanillyl sulfone suppresses NLRP3 inflammasome activation via inducing mitophagy to ameliorate chronic neuropathic pain in mice.

BACKGROUND: Chronic neuropathic pain is a frequent sequel to peripheral nerve injury and maladaptive nervous system function. Divanillyl sulfone (DS), a novel structural derivative of 4,4'-dihydroxydibenzyl sulfoxide from a traditional Chinese medicine Gastrodia elata with anti-nociceptive effects, significantly alleviated neuropathic pain following intrathecal injection. Here, we aimed to investigate the underlying mechanisms of DS against neuropathic pain. METHODS: A chronic constrictive injury (CCI) mouse model of neuropathic pain induced by sciatic nerve ligation was performed to evaluate the effect of DS by measuring the limb withdrawal using Von Frey filament test. Immunofluorescence staining was used to assess the cell localizations and expressions of Iba-1, ASC, NLRP3, and ROS, the formation of autolysosome. The levels of NLRP3-related proteins (caspase-1, NLRP3, and IL-1ß), mitophagy-related proteins (LC3, Beclin-1, and p62), and apoptosis-related proteins (Bcl-XL and Bax) were detected by Western blotting. The apoptosis of BV-2 cell and caspase activity were evaluated by flow cytometry. RESULTS: DS significantly alleviated the neuropathic pain by increasing the mechanical withdrawal threshold and inhibiting the activation of NLRP3 in CCI-induced model mice. Our findings indicated that DS promoted the mitophagy by increasing the LC3II and Beclin 1 and decreasing the levels of p62 protein in BV-2 cell. This is accompanied by the inhibition of NLRP3 activation, which was shown as inhibited the expression of NLRP3 in lysates as well as the secretion of mature caspase-1 p10 and IL-1ß p17 in supernatants in cultured BV-2 microglia. In addition, DS could promote mitophagy-induced improvement of dysfunctional mitochondria by clearing intracellular ROS and restoring mitochondrial membrane potential. CONCLUSION: Together, our findings demonstrated that DS ameliorate chronic neuropathic pain in mice by suppressing NLRP3 inflammasome activation induced by mitophagy in microglia. DS may be a promising therapeutic agent for chronic neuropathic pain.

Occurrence of Phytoplasma Belonging to 16SrII Group Associated with Witches'-broom Symptoms in Emilia sonchifolia in Hainan Island of China.

Emilia sonchifolia is a medical plant belonging to the family of Asteraceae, mainly used as a traditional Chinese medicine with the function of anti-inflammatory, analgesic, antibacterial and so on. During October to November 2020, the plants showing abnormal symptoms including witches'-broom, internode shortening, leaf chlorosis and leaflet were found in Hainan province, a tropical island of China. The total DNA of the plant samples were extracted using 0.10 g fresh plant leaves using CTAB method. PCR reactions were performed using primers R16mF2/R16mR1 and secAfor1/secArev3 specific for phytoplasma 16S rRNA and secA gene fragments. The target productions of the two gene fragments of phytoplasma were detected in the DNA from three symptomatic plant samples whereas not in the DNA from the symptomless plant samples. The two gene fragments of the DNA extracted from the symptomatic plant samples were all identical, with the length of 1324 bp 16S rRNA and 760 bp secA gene sequence fragments, putatively encoding 253 (secA) amino acids sequence. The phytoplasma strain was named as Emilia sonchifolia witches'-broom (EsWB) phytoplasma, EsWB-hnda strain. To our knowledge, this was the first report that Emilia sonchifolia witches'-broom disease was caused by the phytoplasma belonging to16SrII-V subgroup in Hainan island of China, with close relationship to 16SrII peanut witches'-broom group phytoplasma strains infecting the plants like peanut, Desmodium ovalifolium and cleome from the same island of China and cassava from Viet Nam.

Antioxidant, Anti-Inflammatory, and Cytotoxic Properties and Chemical Compositions of Filipendula palmata (Pall.) Maxim.

Filipendula palmata (Pall.) Maxim. remains unexplored and underutilized resources with a high potential to improve human health. In this study, a new ursane-type triterpenoid, namely, 2α, 3ß-dihydroxyurs-12-en-28-aldehyde (compound 10), and other 23 known compounds were isolated. 5 triterpenoids (compounds 6, 8, and 10-12), 11 flavonoids (compounds 13-15 and 17-24), 6 phenolic compounds (compounds 1, 2, 4, 5, 9, and 16), 2 sterols (compounds 3 and 7) were isolated from the aqueous solution extract of the aerial parts of F. palmata. The structures of all compounds were elucidated by the use of extensive spectroscopic methods such as infrared spectroscopy (IR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), 1H-NMR, and 13C-NMR. The solvent extractions of ethyl acetate fraction were evaluated for antioxidant activities using DPPH (2, 2-diphenyl-1-picrylhydrazyl) and ABTS+ (2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) methods. The anti-inflammatory effects of the compounds were evaluated in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. The extract cytotoxicity on the cancer cell lines MCF-7, HeLa, 4T1, and A549 was determined by MTT assay. As a result, compounds 10, 11, and 12 exhibited better antioxidant activity compared to the other compounds. Compounds 8-24 had different inhibitory effects on the release of NO, TNF-α, and IL-6 in LPS-stimulated RAW 264.7 cells. The new compound has shown a significant inhibiting effect on cancer cells, and the cell inhibition rate increased in a dose-dependent manner. Further research to elucidate the chemical compositions and pharmacological effects of F. palmata is of major importance towards the development and foundation of clinical application of the species.

Waltheria indica is a New Host of Phytoplasma Belongs to 16SrI-B Subgroup Associated with Virescence Symptoms in China.

Waltheria indica L. is a kind of medicinal plants belonging to the family of Sterculiaceae distributed in China, which extracts with many active compounds used for treatment of rheumatism and sore pains (Hua et al., 2019). During September to November 2020, the plants showing abnormal symptoms including floral virescence, leaf chlorosis and leaflet, as shown in Fig.1, were found in Dingan county of Hainan province, China, with about 70% incidence. The disease symptoms which were suspected to be infected by the phytoplasma, a phloem-limited cell-wall-less prokaryotic pathogen could not be cultured in vitro, severely impacted Waltheria indica growth resulting in financial loss and ecological damage in the location. For identification of the causal pathogen, the total DNA of symptom or symptomless Waltheria indica samples were extracted using 0.10 g fresh plant tissues using CTAB method. PCR reactions were performed using primers R16mF2/R16mR1 (Lee et al., 1993) and AYgroelF/AYgroelR (Mitrovic et al., 2011) specific for phytoplasma 16S rRNA and groEL gene fragments. The target productions of the two gene fragments of phytoplasma were detected in the DNA from four symptomatic plant samples whereas not in the DNA from the symptomless plant samples. The PCR productions were sequenced and the data were deposited in GenBank. The two gene fragments of the DNA extracted from the symptom plant samples were all identical, with the length of 1340 bp 16S rRNA (GenBank accession: MW353909) and 1312 bp groEL (MW353709) gene sequence fragments, putatively encoding 437 (groEL) amino acids sequence. The phytoplasma strain was named as Waltheria indica virescence (WiV) phytoplasma, WiV-hnda strain. A Blast search based on the 16S rRNA gene fragment of WiV-hnda phytoplasma strain revealed the highest level of sequence identities (99.85%) with that of 16SrI aster yellows group members (16SrI-B subgroup), such as Onion yellows phytoplasma strain OY-M (AP006628) from Japan (Oshima et al., 2004); Periwinkle virescence phytoplasma strain PeV-hnhk (KP662136), Chinaberry witches'-broom phytoplasma strain CWB-hnsy1 (KP662119) and CWB-hnsy2 (KP662120), all the strains from Hainan island of China (Yu et al., 2017). A Blast search based on the groEL gene sequence fragment of WiV-hnda indicated 99.92% sequence identity with that of 16SrI aster yellows group members (16SrI-B subgroup) such as Onion yellows phytoplasma strain OY-M (AP006628). Homology and phylogenetic analysis by DNAMAN 5.0 and MEGA 7.0 software indicated that the phytoplasma strains of WiV-hnda, OY-M, PeV-hnhk, CWB-hnsy1 and CWB-hnsy2 were clustered into one clade based on the 16S rRNA gene fragments. WiV-hnda, OY-M and Aster yellow witches'-broom (AYWB) (CP000061) phytoplasma strains were clustered into one clade based on the groEL gene fragments. To our knowledge, this was the first time that Waltheria indica virescence disease induced by 16SrI-B subgroup phytoplasma strain was reported in China. Genetic analysis showed that WiV-hnda was closely related to the phytoplasma strains causing Onion yellows in Japan, Periwinkle virescence and Chinaberry witches'-broom disease in China.

Recent progress in polysaccharides from Panax ginseng C. A. Meyer.

Panax ginseng C. A. Meyer (P. ginseng) has a long history of medicinal use and can treat a variety of diseases. P. ginseng contains a variety of active ingredients, such as saponins, polypeptides, volatile oils, and polysaccharides. Among them, saponins have always been considered as the main components responsible for its pharmacological activities. However, more and more studies have shown that polysaccharides play an indispensable role in the medicinal value of ginseng. Modern biological and medical studies have found that ginseng polysaccharides have complex structural characteristics and diverse biological activities, such as immune regulation, anti-tumor, antioxidant, hypoglycemic, and anti-radiation functions, among others. Additionally, the structural characteristics of ginseng polysaccharides are closely related to their activity. In this review, the research background, extraction, purification, structural characteristics, and biological activities of ginseng polysaccharides from different parts of P. ginseng (roots, flowers stems and leaves, and berries) under different growth conditions (artificially cultivated ginseng, mountain ginseng, and wild ginseng) are summarized. The structural characteristics of purified polysaccharides were reviewed. Meanwhile, their biological activities were introduced, and some possible mechanisms were listed. Furthermore, the structure-activity relationship of polysaccharides was discussed. Some research perspectives for the study of ginseng polysaccharides were also provided.

Platycodon grandiflorum extract: chemical composition and whitening, antioxidant, and anti-inflammatory effects.

Whitening cosmetics have a large market scale and broad development prospects, while whitening products of traditional Chinese medicine have always been a research hotspot. In this study, the whitening active extract of Platycodon grandiflorum (PGE) was isolated and purified for the first time, and the whitening activity mechanism and chemical composition of PGE were elucidated. A total of 45 components were identified via high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis, including arbutin, syringin, chlorogenic acid, platycoside E, platycodin D3, baicalin, platycodin D, and luteolin. The scavenging rates of PGE toward DPPH and ABTS free radicals were 98.03% and 84.30%, respectively. The inhibition rate of PGE toward tyrosinase was up to 97.71%. The PGE had significant anti-inflammatory effects on RAW264.7 macrophages stimulated by lipopolysaccharide (LPS) and had significant inhibition effects on tyrosinase and melanin generation of B16F10 cells stimulated by α-MSH. The results showed that the PGE achieved a synergistic whitening effect by inhibiting the activation of oxygen free radicals on tyrosinase, antioxidation, anti-inflammatory effect, enzyme activity, and melanin generation. As a whitening agent extracted from natural plants, PGE has great potential in the research and development of plant whitening cosmetics, which lays a foundation for the further development and utilization of Platycodon grandiflorum resources and also provides a theoretical basis for the development of green and organic whitening cosmetics.