PI3K/AKT mediated De novo fatty acid synthesis regulates RIG-1/MDA-5-dependent type I IFN responses in BVDV-infected CD8+T cells.

Bovine viral diarrhea virus (BVDV) has caused massive economic losses in the cattle business worldwide. Fatty acid synthase (FASN), a key enzyme of the fatty acid synthesis (FAS) pathway, has been shown to support virus replication. To investigate the role of fatty acids (FAs) in BVDV infection, we infected CD8+T lymphocytes obtained from healthy cattle with BVDV in vitro. During early cytopathic (CP) and noncytopathic (NCP) BVDV infection in CD8+ T cells, there is an increase in de novo lipid biosynthesis, resulting in elevated levels of free fatty acids (FFAs) and triglycerides (TG). BVDV infection promotes de novo lipid biosynthesis in a dose-dependent manner. Treatment with the FASN inhibitor C75 significantly reduces the phosphorylation of PI3K and AKT in BVDV-infected CD8+ T cells, while inhibition of PI3K with LY294002 decreases FASN expression. Both CP and NCP BVDV strains promote de novo fatty acid synthesis by activating the PI3K/AKT pathway. Further investigation shows that pharmacological inhibitors targeting FASN and PI3K concurrently reduce FFAs, TG levels, and ATP production, effectively inhibiting BVDV replication. Conversely, the in vitro supplementation of oleic acid (OA) to replace fatty acids successfully restored BVDV replication, underscoring the impact of abnormal de novo fatty acid metabolism on BVDV replication. Intriguingly, during BVDV infection of CD8+T cells, the use of FASN inhibitors prompted the production of IFN-α and IFN-ß, as well as the expression of interferon-stimulated genes (ISGs). Moreover, FASN inhibitors induce TBK-1 phosphorylation through the activation of RIG-1 and MDA-5, subsequently activating IRF-3 and ultimately enhancing the IFN-1 response. In conclusion, our study demonstrates that BVDV infection activates the PI3K/AKT pathway to boost de novo fatty acid synthesis, and inhibition of FASN suppresses BVDV replication by activating the RIG-1/MDA-5-dependent IFN response.

Fingerprint analysis of dang-gui-Si-Ni decoction and its anticoagulant activity in vivo-in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Dang-Gui-Si-Ni (DGSN) decoction is a classic prescription in the clinical practice of traditional Chinese Medicine (TCM). DGSN decoction is often used to relieve symptoms of cold coagulation and blood stasis recorded by Treatise on Febrile Diseases (Shang Han Lun) and treat Raynaud's disease, dysmenorrhea, arthritis, migraine in TCM clinic. Accumulated evidences have suggested that this diseases are related to microcirculation disturbance. However, the anticoagulant activity and underlying mechanisms of DGSN decoction responsible for the therapeutic not well understood. AIM OF THE STUDY: The fingerprint and anticoagulant activity in vivo-in vitro of DGSN decoction were evaluated to strengthen the quality control and activity study of formulas. MATERIALS AND METHODS: The chemical components of DGSN decoction were analyzed by HPLC and its fingerprint similarity were evaluated by "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software (2012 Edition)". The anticoagulant activity of DGSN decoction was assessed by measuring four coagulation factors (PT, TT, APTT, FIB) in vitro. Zebrafish thrombosis model induced by punatinib was established to evaluate the activity of improving microvascular hemodynamics in vivo. Quantitative real-time polymerase chain reaction (q-PCR) were adopted to compare the changes in the RNA expression levels of coagulation factor II (FII), VII (FVII), IX (FIX) and X (FX) in zebrafish thrombosis model. RESULTS: The fingerprint similarity evaluation method of DGSN decoction was established. The results showed that 18 samples had higher similarity (S1-S18 > 0.878). Pharmacodynamic results showed that DGSN decoction could extend PT, TT and APTT, and reduce FIB content in vitro. Meanwhile, it markedly enhanced the cardiac output and blood flow velocity at low dosage (500 µg mL-1) in vivo. q-PCR data demonstrated that DGSN decoction (500 µg mL-1) could downregulate the RNA expression of FII, FVII, FIX and FX. Interestingly, there were a bidirectional regulation of FII, FIX and FX in a certain concentration range. In general, DGSN decoction can significantly improve hemodynamics and downregulate coagulation factors, and the results were consistent both in vitro - in vivo. CONCLUSION: The fingerprint study provide a new perspective for improving the quality control of DGSN decoction. DGSN decoction possess anticoagulant activity by regulating multiple coagulation factors simultaneously. Thus, it has the potential to develop into the novel raw material of anticoagulant drugs.

Responses of root-associated fungal community structure of mycorrhizal plants to nitrogen and/or phosphorus addition in a subtropical forest.

Root-associated fungi play a vital role in maintaining nutrient absorption and health of host plants. To compare the responses of root-associated fungal community structures to nitrogen (N) and/or phosphorus (P) additions across differential mycorrhizal types, we collected roots of nine plant species belonging to three mycorrhizal types (arbuscular mycorrhiza, ectomycorrhiza, and ericoid mycorrhiza) under control and N and/or P addition treatments from a subtropical forest, and detected the diversity and community composition of fungi inhabiting roots through the high-throughput sequencing technique. The results showed that root-associated fungal communities of all nine plant species were mainly composed of Basidiomycota and Ascomycota. The relative abundance of Ascomycota and Basidiomycota was significantly lower and higher under the P addition than that under control, respectively. The relative abundance of Ascomycota of ericoid mycorrhizal trees was significantly higher than those of arbuscular mycorrhizal and ectomycorrhizal trees, while the relative abundance of Basidiomycota was significantly lower than the other two mycorrhizal types. Compared with the control, P addition significantly reduced the α-diversity and changed community composition of root-associated fungi across different mycorrhizal plant types, while no effect of N addition or mycorrhizal type was observed. Compared with the control and N addition treatments, NP addition caused root-associated fungal communities of all plants becoming integrally divergent. In addition, the fungal communities of ectomycorrhizal mycorrhizal trees became apparently convergent in comparison with those of arbuscular and ericoid mycorrhizal trees under the NP addition. Collectively, our results highlighted that P was a critical factor influencing community structures of tree root-associated fungi in subtropical forest soils. This study would enhance our understanding of the responses and maintenance mechanisms of plant root-associated fungal diversity under global environmental changes in the subtropical region.

Essential oil of Ligusticum chuanxiong Hort. Regulated P-gp protein and tight junction protein to change pharmacokinetic parameters of temozolomide in blood, brain and tumor.

ETHNOPHARMACOLOGICAL RELEVANCE: The existence of the blood-brain barrier/blood tumor barrier (BBB/BTB) severely restricts the effectiveness of anti-tumor drugs, thus glioma is still an incurable disease with a high fatality rate. Chuanxiong (Ligusticum chuanxiong Hort., Umbelliferae) was used as a messenger drug to increase the distribution of drugs in brain tissue, and its application in Chinese herbal formula for treating glioma was also the highest. AIM OF THE STUDY: Our previous researches showed that essential oil (EO) of chuanxiong could promote temozolomide (TMZ) entry into glioma cells in vitro and enhance TMZ-induced anticancer efficiency in vivo, and therefore, the aim of this study was to investigate whether EO could increase the concentration accumulation of TMZ in brain or tumor of C6 glioma rats and the related mechanisms. MATERIALS AND METHODS: The pharmacokinetics were conducted in C6 glioma rats by administering either TMZ alone or combined with EO through oral routes. TMZ concentration in blood, brain and tumor was detected using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and then pharmacokinetic parameters were calculated. The changed expressions of P-gp protein, tight junction occludin, claudin-5 and zonula occludens-1 (ZO-1) in brain of glioma rats were studied by Western blot to clarify the mechanism. Finally, the chemical composition of EO was analyzed by gas chromatography-massspectrometry (GC-MS). RESULTS: The results showed that EO significantly affected the pharmacokinetic parameters such as Tmax, Cmax and CL (p < 0.01), but did not significantly change the AUC(0→∞) of TMZ in blood (p > 0.05). However, EO markedly improved the AUC(0→∞)of TMZ in brain and tumor (p < 0.01). The calculate drug targeting index was greater than 1, indicating that EO could promote the distribution of TMZ to the brain and tumor. Western blot analysis showed that EO significantly inhibited the expression of P-gp, tight junction protein claudin-5, occludin and ZO-1. And meanwhile, the expressions of P-gp, claudin-5 and occludin also markedly down-regulated in EO-TMZ co-administration treatment. GC-MS analysis of the TIC component of EO was (E)-Ligustilide (36.93%), Terpinolene (7.245%), gamma-terpinene (7.225%) etc. CONCLUSION: EO could promote the distribution of TMZ in the brain and tumor of C6 glioma rats, which may attribute to down-regulate the expression of P-gp, claudin-5 and occludin.

Sesquiterpene dimers from Chloranthus fortunei and their protection activity against acute lung injury.

Chloranthus fortunei (family Chloranthaceae), a perennial herb, widely distributed in south China with an altitude of 170-340 m. The whole plants were used as an anti-inflammatory agent for the treatment of cough, arthritis and tumor. Five previously unreported compounds fortulactones A-E were isolated from the aerial part of Chloranthus fortunei. Their structures were elucidated using 1D/2D NMR and HRESIMS and their absolute configuration were determined using the ECD excitron chirality method. All isolates were tested for inhibitory effects on the NO production of liposaccharide (LPS)-induced RAW 264.7 macropahges. The most potent compound 1 was further evaluated its protective activity against LPS stimulated A549 cells, the ELISA kits results showed the abnormal states of MDA and SOD were corrected to a certain extent. Meanwhile, the pro-inflammatory cytokine, such as TNF-α, IL-6 and IL-1ß were also attenuated. In conclusion, these results showed that 1 exhibited therapeutic potential for ameliorating ALI.

[Comparison of chemical components between aerial and underground parts of Coptis chinensis based on UPLC-Q-TOF-MS~E technology].

The ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS~E) technology was employed to compare the chemical components between the aerial and underground parts of Coptis chinensis samples from different batches. According to the retention time, molecular ion peak, and LC-MS~E fragment information of the reference substances and available literature, we identified a total of 40 components. Thirty-three and 31 compounds were respectively identified in the underground part(taproots) and the aerial part(stems and leaves) of C. chinensis. Among them, 24 compounds, including alkaloids(e.g., berberine and jatrorrhizine) and phenolic acids(e.g., chlorogenic acid, quinic acid, and tanshinol), were common in the two parts. In addition, differential components were also identified, such as magnoline glucoside in the underground part and(±) lariciresionol-4-ß-D-glucopyranoside in the aerial part. The analysis of fragmentation pathways based on spectra of reference substances indicated the differences among samples of different batches. Furthermore, we performed the principal component analysis(PCA) for the peak areas of C. chinensis in different batches. The results showed that the underground part and the aerial part were clearly clustered into two groups, indicating that the chemical components contained in the two parts were different. Furthermore, the results of partial least squares discriminant analysis(PLS-DA) identified 31 differential compounds(VIP value>1) between the underground part and the aerial part, mainly including alkaloids, phenolic acids, lignans, and flavonoids. This study proves that C. chinensis possesses great development potential with multiple available compounds in stems and leaves. Moreover, it sheds light on for the development and utilization of non-medicinal organs of C. chinensis and other Chinese medicinal herbs.

[Evaluation and optimization of content determination method of Chrysanthemi Flos in Chinese Pharmacopoeia].

This study discovered that the resolution of 3,5-O-dicaffeoylquinic acid(isochlorogenic acid A) in the content determination method of Chrysanthemi Flos in Chinese Pharmacopoeia(ChP)(2020 edition) was poor, which affected accurate quantification. We tested the method in ChP with chromatographic columns of seven brands to clarify the problems in the existing method, optimized the chromatographic conditions by adjusting the mobile phase composition and elution ratio and replacing the chromatographic column packing, and carried out the reproducibility assay for the new method. The two methods were compared for the content determination results of Chrysanthemi Flos prepared from six different varieties. As evaluated by the resolution based on different chromatographic columns of seven brands, the existing method failed to separate isochlorogenic acid A and isochlorogenic acid D well. The peaks of the two components were not completely separated on three chromatographic columns, and isochlorogenic acid A and isochlorogenic acid D generated a co-effluent peak in the other four columns. Isochlorogenic acid A and isochlorogenic acid D could be completely separated under the optimized chromatographic conditions. The difference in the peak areas of isochlorogenic acid A+isochlorogenic acid D obtained by the optimized method and the method in ChP was not significant, with deviation less than 3.0%, which further proved that the result measured by the method in ChP was the co-effluent of isochlorogenic acid A and isochlorogenic acid D. The optimized method can ensure the accurate quantification of isochlorogenic acid A. The existing content determination method of Chrysanthemi Flos has the problem of poor resolution. It is recommended to revise the chromatographic conditions for the content determination method of Chrysanthemi Flos to improve the resolution of isochlorogenic acid A and ensure its accurate quantification.

Functional connectivity changes during migraine treatment with electroacupuncture at Shuaigu (GB8).

OBJECTIVE: To investigate the changes in the functional connectivity (FC) in the right insula between migraine without aura (MWoA) and healthy controls by using resting-state functional magnetic resonance imaging (rs-fMRI), and to observe the instant alteration of FC in MWoA during electroacupuncture (EA) stimulation at Shuaigu (GB8). METHODS: A total of 30 patients with MWoA (PM group) and 30 healthy controls (HC group) underwent rs-fMRI scans. The PM group underwent a second rs-fMRI scan while receiving EA at GB8. The right insula subregions, including the ventral anterior insula (vAI), dorsal anterior insula (dAI) and posterior insula (PI), were selected as the seed points for FC analysis. RESULTS: Aberrant FC, including dAI with right postcentral gyrus, PI with left precuneus, was found among PM before EA (PMa), PM during EA (PMb) and HC. Meanwhile, decreased FC between dAI and the right postcentral gyrus was found in the PMa compared to the HC and PMb. Increased FC between the PI and left precuneus was found in the PMa compared to the HC and PMb. Correlation analysis showed that the FC value of the right postcentral gyrus in PMa was negatively correlated with the scores of Hamilton Rating Scale for Depression and Hamilton Rating Scale for Anxiety. The FC value of the left precuneus in PMa was positively correlated with the visual analogue scale score. CONCLUSION: The alteration of FC between the right insula subregions and multiple brain regions may be an important index for MWoA. EA at GB8 was able to adjust the FC between the right insula subregions and parietal lobe, namely, the right dAI and right postcentral gyrus, and the right PI and left precuneus, thereby rendering an instant effect in the management of MWoA.

[Effect of acupuncture on migraine without aura: a resting-state functional magnetic resonance study].

OBJECTIVE: To observe therapeutic effect of acupuncture on migraine without aura and the changes of brain functional connectivity (FC) using resting-state functional magnetic resonance imaging (rs-fMRI). METHODS: Thirty-four patients with migraine without aura were included into an observation group and treated with acupuncture at Baihui (GV 20), Xuanlu (GB 5), Shuaigu (GB 8), Taiyang (EX-HN 5), etc. Using G6805-Ⅱ electric acupuncture apparatus, the ipsilateral Fengchi (GB 20) and Shuaigu (GB 8) were connected and stimulated with continuous wave, 2 Hz in frequency and 0.1 mA to 1.0 mA in current intensity, depending on patient's tolerance. Acupuncture stimulation lasted 20 min each time, twice weekly (at the interval>2 days). A total of 6 weeks of treatment (12 times) was required. Matching the gender and age as those of the observation group, 16 healthy subjects were recruited into a control group and no any intervention was provided. The headache days, the score of visual analogue scale (VAS) for headache severity, the total score of headache symptoms, the score of migraine-specific quality of life questionnaire (MSQ), the score of self-rating anxiety scale (SAS), and the score of self-rating depression scale (SDS) were compared before and after acupuncture, and the clinical efficacy was assessed in the observation group. The data of the resting-state functional magnetic resonance were collected in the observation group before and after treatment as well as the control group at baseline. The periaqueductal gray (PAG) was taken as the seed to analyze the effect of acupuncture on the brain FC and the correlation between FC and VAS scores, headache days in the patients of migraine without aura. RESULTS: After treatment, the headache days, VAS score, the total score of headache symptoms, SAS score and SDS score were all reduced (P<0.01); and the scores of the restrictive, preventive, and emotional functional domains of the MSQ were increased (P<0.01) in the observation group compared with those before treatment. The total effective rate was 94.1% (32/34). Compared with the control group, FC between PAG and the right cerebellum Ⅷ was decreased in the observation group before treatment (P<0.05). FC of PAG with the bilateral cerebellum Ⅷ and the left precuneus was increased in the observation group after treatment compared with those before treatment (P<0.05). In the observation group, the FC intensity of PAG and the right cerebellum Ⅷ was negatively correlated with VAS score (r =-0.41, P<0.05) before treatment, while the FC intensity of PAG and the left precuneus was positively correlated with the improvement in headache days (r =0.40, P<0.05) after treatment. CONCLUSION: Acupuncture is effective on migraine without aura. The brain functional connectivity is abnormal in the patients. The effect onset of acupuncture is obtained probably by regulating the abnormal brain regions and activating brain regions relevant with pain and emotions.

Discovery of potential pharmacodynamic ingredients of Dang-Gui-Si-Ni decoction based on absorbed ingredients and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: The Dang-Gui-Si-Ni (DGSN) decoction as a classic prescription has been widely used for thousands of years in the clinical practice of traditional Chinese medicine (TCM). Especially in recent years, the potential efficacy of TCM for the treatment of Raynaud's syndrome has attracted great attention as there are still no specific remedies for this disease. However, the active constituents and underlying mechanisms responsible for the therapeutic benefits are not well understood, which makes it difficult to ensure quality control or to design research and drug development strategies. To identify the potential pharmacodynamic ingredients (PPIs) of TCM will help to achieve suitable process control procedures for industrial production and large-scale manufacturing. AIM OF THE STUDY: In the present study, we propose a multi-dimensional qualitative analysis method combining water-decoction spectra, in-vitro intestinal absorption spectra, in-vivo plasma spectra, and molecular docking of components to quickly identify the PPIs for the DGSN decoction of TCM. MATERIALS AND METHODS: Water-based decoctions of DGSN were prepared in accordance with the clinical use registered in ancient books. Ultra-high-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UHPLC-Q/TOF-MS) coupled with computerized modelling activity screening was used to quickly identify the PPIs of the DGSN decoction. Bioactive compounds absorbed in vitro were identified using the everted intestinal sac model from rats and compounds absorbed in vivo were confirmed in portal vein blood samples obtained following oral administration in rats. Molecular docking validation experiments were adopted to predict the binding activity to coagulation factors I, II, VII, X, and IX. The active components were further confirmed by pharmacodynamics analysis. The anticoagulant activity of the DGSN decoction was verified using rat models. RESULTS: Thirty-one compounds were identified in the DGSN decoction. According to the in vivo experiments, 22 compounds that could be absorbed in vivo were detected by the everted intestinal sac model in rats. This model greatly reduces the scope of PPIs and is easy to perform. Ten compounds were detected in the portal vein blood in rats. The compounds detected in plasma provide stronger evidence supporting the PPIs. Molecular docking in vitro experiments indicated that 7 compounds exhibited better binding activity with coagulation factors I, II, VII, X, and IX. The animal experiments confirmed that the DGSN decoction could improve the microcirculation, providing indirect proof of anticoagulant activity suggested by the molecular docking studies. Finally, based on the multi-dimensional methods, 9 potential compounds present in the DGSN decoction were identified as PPIs (i.e., ferulic acid, paeoniflorin, albiflorin, chlorogenic acid, cryptochlorogenic acid, liquiritin, liquiritin apioside, cinnamaldehyde and glycyrrhizic acid). CONCLUSION: Overall, this study combined the water-decoction spectra, intestinal absorption spectra in vitro, plasma spectra in vivo, and molecular docking studies to establish a multi-dimensional qualitative analysis method of the DGSN decoction. Meanwhile, 9 compounds in DGSN decoction were identified as PPIs using this method, and are proposed for application as quality standards for complex TCM prescriptions.

[Herbal textural research, morphologic characteristics and DNA barcoding of botanical origins of Alismatis Rhizoma].

Alismatis Rhizoma(Zexie) is a commonly used traditional Chinese medicine, and it is separated into "Chuan Zexie"(Sichuan and Hubei provinces), "Jian Zexie"(Fujian and Jiangxi provinces) and "Guang Zexie"(Guangxi province) according to different producing areas. Alisma plantago-aquatica and A. orientale were listed as the original plants of Alismatis Rhizoma in different editions of Chinese Pharmacopoeia(Ch.P), respectively. The botanical origins of Alismatis Rhizoma caused much controversy during a period of time. This study aimed to define the botanical origins of Alismatis Rhizoma from different producing areas, and supply scientific evidence for Ch. P 2020 edition. In this paper, we summarized the descriptions of original plants and producing areas of Alismatis Rhizoma in ancient literatures. Flowers and fruits of original plants of Alismatis Rhizoma were collected from different typical areas, and compared with the morphological description of two species from Alisma genus in the Flora of China. Thirty-nine batches of leaves from 8 different areas were identified using DNA barcoding technology. The results showed that original plants of Alismatis Rhizoma from different areas could be distinguished from each other based on morphological characteristics and molecular characteristics. Then, "Jian Zexie" was identified as A. orientale, while "Chuan Zexie" and "Guang Zexie" were identified as A. plantago-aquatica. In conclusion, combining with herbal textural research, morphologic characteristics, DNA barcoding technology and market situation, this paper recommended that the botanical sources of Alismatis Rhizoma could be revised as Alisma orientale(Sam.) Juzep. and Alisma plantago-aquatica Linn. in the Ch. P 2020 edition.

[Regional differences analysis of Alismatis Rhizoma based on UPLC characteristic chromatogram and determination of eight terpenoids].

An ultra-performance liquid chromatography(UPLC) method integrating characteristic chromatogram and eight terpenoids determination has been established for comparing the differences of Alismatis Rhizoma(Zexie) from different product areas. Thirty-seven batches of crude drugs and thirty batches of prepared slices of Alismatis Rhizoma were analyzed. The obtained data were analyzed by similarity evaluation, principal components analysis(PCA) and partial least squares discriminant analysis(PLS-DA). There were three main characteristic peaks in the characteristic chromatograms, and alisol B 23-acetate(S) was selected as the reference. Compared with the S peak, the relative retention times of the other two characteristic peaks were 0.55(alisol) and 0.77(alisol B), respectively. Peak areas and the ratio of alisol B to alisol B 23-acetate could be used to distinguish Alismatis Rhizoma from different geographical origins. The samples were divided into three groups by PCA and PLS-DA based on the content determination results, and they were "Jian Zexie"(Fujian and Jiangxi provinces), "Chuan Zexie"(Sichuan and Hubei provinces), and "Guang Zexie"(Guangxi province). The contents of chemical components in samples from different producing areas were notably different. For example, the contents of alisol A and alisol A 24-acetate were significantly higher in "Guang Zexie" compared with "Jian Zexie" and "Chuan Zexie"(P<0.000 1). The contents of alisol B and alisol C were significantly higher in "Chuan Zexie" compared with "Jian Zexie"(P<0.000 1). Combining the characteristic chromatograms and quantitative analysis of eight terpenoids, this study showed that the relative contents of components and their ratios were notable different in samples from different regions, but types and numbers of chemical compositions were basically similar. The results of this study illustrated the regional differences of Alismatis Rhizoma and their components characteristics, and provided references for authentication and quality control of Alismatis Rhizoma.

[Qualitative and quantitative research on nucleosides of Alismatis Rhizoma from different regions].

Ten compounds, including nucleosides and amino acids were identified by UPLC-Q-TOF-MS. HPLC fingerprints on these compounds in Alismatis Rhizoma were established for the first time. The comparisons of Alismatis Rhizoma from different regions were conducted by the similarity evaluation and hierarchical cluster analysis(HCA). Meanwhile, the HPLC-DAD method for the content determination of five nucleosides was also established. The results showed that the similarities of Alismatis Rhizoma collected from Sichuan and Fujian provinces were above 0.96, whereas they were less than 0.87 in those from Guangxi province. The results of HCA showed the samples from Sichuan and Fujian were gathered in the same group, all samples from Guangxi in another group, which indicated the similarities between samples from Sichuan and Fujian in nucleosides and they were different from the samples from Guangxi. The total contents of five nucleosides were revealed, of which samples from Sichuan and Fujian were 0.81-1.30 mg·g~(-1) followed a descending order of vernine>cytidine>uridine>adenine>adenosine, and from Guangxi were 0.35-0.50 mg·g~(-1) with the sequences of uridine>adenine>vernine>cytidine>adenosine. The nucleosides contents of samples from Sichuan and Fujian were both higher than that from Guangxi. For samples from Sichuan and Fujian, the former was slightly higher, except for adenine. These results would be helpful to reveal the bioactive constituents in aqueous extract and provided important evidences for the quality control of Alismatis Rhizoma.

[Recommendations on quality standards of Alismatis Rhizoma in Chinese Pharmacopoeia(2020 edition)].

The present research was launched to improve the quality standards of Alismatis Rhizoma and supply scientific evidence and recommendations for the quality control of Alismatis Rhizoma in Chinese Pharmacopoeia(Ch. P) 2020 edition. The contents of water, total ash, heavy metals and deleterious element, pesticide residues and alcohol-soluble extract were analyzed according to the methods listed in the volume Ⅳ of Ch. P 2015 edition. Alisol B 23-acetate, alisol C 23-acetate and reference herbs were used to identify Alismatis Rhizoma by TLC method, which was developed by using a mixture of dichloromethane-methanol(15∶1) as developing solvent on silica gel GF_(254 )precoated plates. In HPLC method, alisol B 23-acetate and alisol C 23-acetate were separated with acetonitrile-water as the mobile phase and detected at 208 nm and 246 nm, respectively. Thirty-seven batches of crude drugs, thirty batches of prepared slices and nineteen batches of salt prepared slices of Alismatis Rhizoma were determined according to the methods established. The quality standards established based on the research results were specific and repeatable, and suitable for the quality evaluation of Alismatis Rhizoma. We recommended that the botanical sources, TLC examination, alcohol-soluble extract of salt prepared slices and content determination should be revised in the Ch. P 2020 edition.

[Advances in studies on chemical compositions of Alismatis Rhizoma and their biological activities].

Alismatis Rhizoma is a traditional Chinese medicine, which was widely used in clinical prescriptions and proprietary Chinese medicine. Over 220 compounds have been isolated from it, including triterpenoids, sesquiterpenoids, diterpenoids, polysaccharides, nitrogen compounds, phenylpropanoids, flavones and sterides. The pharmacological studies show that Alismatis Rhizoma exhibits diuretic, anti-urolithiatic, anti-hyperlipidemia, antidiabetics, antitumor, antioxidative, anti-inflammatory, anti-complementary activities, etc. In this review, the chemical compositions and its pharmacological activities of Alismatis Rhizoma in recent 50 years were summarized. The authors hope to provide references for further study, development and utilization of Alismatis Rhizoma.

[Rolling needle pricking-cupping therapy and traditional pricking-cupping therapy for cervical spondylosis of neck type: a randomized controlled trial].

OBJECTIVE: To compare the clinical efficacy between rolling needle pricking-cupping (RNP-C) and traditional pricking-cupping (TP-C) for cervical spondylosis of neck type. METHODS: A total of 96 patients with cervical spondylosis of neck type were randomly divided into an RNP-C group, a TP-C group and an electroacupuncture (EA) group, 32 cases in each group. Each group was treated with EA at Jingbailao (EX-HN 15), Fengchi (GB 20), Dazhui (GV 14), Jianjing (GB 21) and ashi points with continuous wave and 2 Hz of frequency; each EA treatment lasted for 20 min, once every 3 to 5 days, totaling 6 treatments. On the basis of EA treatment, the patients in the TP-C group were treated with bloodletting by seven-star needle, followed by fire cupping; the patients in the RNP-C group were treated with bloodletting by rolling needle, followed by fire cupping. The treatment was given once a week for 4 weeks. The follow-up was 1 month. Before treatment, 2 and 4 weeks into treatment and follow-up, the Northwick Park neck-pain questionnaire (NPQ) and visual analogue scale (VAS) scores were evaluated. The acupuncture pain degree was recorded at the first treatment and 2 and 4 weeks into treatment. The efficacy was evaluated after 4 weeks of treatment. RESULTS: Compared before treatment, the scores of NPQ and VAS in each group were all reduced at 2 and 4 weeks into treatment and follow-up (P<0.05). The scores of NPQ in the TP-C group and the RNP-C group were lower than those in the EA group at 2 and 4 weeks into treatment (P<0.05). The scores of VAS in the TP-C group and the RNP-C group were lower than those in the EA group at 2 and 4 weeks into treatment and follow-up (P<0.05). The differences of NPQ and VAS scores between the TP-C group and the RNP-C group at each time point after treatment were not significant (P>0.05). The acupuncture pain degree in the RNP-C group was lower than that in the TP-C group (P<0.05). The total effective rates were 79.3% (23/29) in the TP-C group and 75.0% (24/32) in the RNP-C group, which was superior to 63.3% (19/30) in the EA group (P<0.05), but there was no statistical significance between the TP-C group and the RNP-C group (P>0.05). CONCLUSION: TP-C and RNP-C could both improve the cervical pain symptoms in patients with cervical spondylosis of neck type, and improve the overall function of the cervical spine, and the curative effect is similar.

Homoharringtonine regulates the alternative splicing of Bcl-x and caspase 9 through a protein phosphatase 1-dependent mechanism.

BACKGROUND: Homoharringtonine (HHT) is a natural alkaloid with potent antitumor activity, but its precise mechanism of action is still poorly understood. METHODS: We examined the effect of HHT on alternative splicing of Bcl-x and Caspase 9 in various cells using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The mechanism of HHT-affected alternative splicing in these cells was investigated by treatment with protein phosphatase inhibitors and overexpression of a protein phosphatase. RESULTS: Treatment with HHT downregulated the levels of anti-apoptotic Bcl-xL and Caspase 9b mRNA with a concomitant increase in the mRNA levels of pro-apoptotic Bcl-xS and Caspase 9a in a dose- and time-dependent manner. Calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), significantly inhibited the effects of HHT on the alternative splicing of Bcl-x and Caspase 9, in contrast to okadaic acid, a specific inhibitor of PP2A. Overexpression of PP1 resulted in a decrease in the ratio of Bcl-xL/xS and an increase in the ratio of Caspase 9a/9b. Moreover, the effects of HHT on Bcl-x and Caspase 9 splicing were enhanced in response to PP1 overexpression. These results suggest that HHT-induced alternative splicing of Bcl-x and Caspase 9 is dependent on PP1 activation. In addition, overexpression of PP1 could induce apoptosis and sensitize MCF7 cells to apoptosis induced by HHT. CONCLUSION: Homoharringtonine regulates the alternative splicing of Bcl-x and Caspase 9 through a PP1-dependent mechanism. Our study reveals a novel mechanism underlying the antitumor activities of HHT.

Resin glycosides from the seeds of Ipomoea muricata and their multidrug resistance reversal activities.

Resin glycosides represent an important chemotaxonomic marker of the Convolvulaceae family and possess multidrug resistance (MDR) reversal activity. In our recent study, nine previously undescribed resin glycosides, Calonyctins B-J, were isolated from the seeds of Ipomoea muricata. Their structures with the absolute configuration were established on the basis of comprehensive spectroscopic analysis and chemical methods. Among these, Calonyctins F-I possessed a skeleton in which the aglycone moiety and the oligosaccharide core were linked by a 3-hydroxy-2-methylbutanoic acid moiety to form a 25-membered macrocyclic structure. Calonyctins E, J, and muricatic acid C methyl ester were non-cytotoxic but enhanced the cytotoxicity of vincristine by 2.5-407.1 fold at 25 µM in KB/VCR cells. Calonyctin E was the most active one.

Effectiveness of meibomian gland massage combined with topical levofloxacin against ocular surface flora in patients before penetrating ocular surgery.

OBJECTIVE: To investigate the bacterial profile in the conjunctiva and meibomian glands in patients before penetrating ocular surgeries, and to compare the anti-bacterial efficacy of 0.5% levofloxacin and its combination with meibomian gland massage. DESIGN: Hospital-based, case-control study. PARTICIPANTS: Two hundred and twenty-six eyes from 226 patients with non-infective ocular diseases and scheduled for penetrating ocular surgeries. METHODS: Tested eyes were administered topical 0.5% levofloxacin (4 times daily) for 2 days. Among them, 91 eyes received meibomian gland massage before levofloxacin application. Samples from the conjunctival sac and meibomian glands were collected for aerobic and anaerobic cultures. MAIN OUTCOME MEASURES: Culture-positivity and bacterial strains. RESULTS: Before treatment, aerobes and anaerobes were cultured from 38.5% and 11.0% of the conjunctival samples respectively, compared with 38.5% and 8.8% in the meibomian secretions respectively. Staphylococcus epidermidis and Propionibacterium acnes were the commonest isolated aerobe and anaerobe. Two-day application of levofloxacin reduced aerobic growth to 29.6% in the conjunctiva and 19.3% in the meibomian glands. It had no effect on the anaerobes in these regions (13.3% in the conjunctiva and 10.4% in the meibomian glands). Combined levofloxacin with meibomian gland massage further reduced aerobic growth to 19.8% in the conjunctiva and 11.0% in the meibomian glands. It also drastically decreased anaerobic growth in the meibomian glands (1.1%). CONCLUSIONS: Meibomian glands carrying considerable bacteria should be considered as a potential source of contamination in ocular surgery. Meibomian gland massage shows additional anti-bacterial effects to topical levofloxacin and could be recommended as a complementary preoperative prophylaxis.