Objective To optimize the extraction, separation and purification process of Actinoside E. Methods Single factor experiment combined with orthogonal test was used to determine the optimal extraction process of Actinoside E using its content as an index. The extracts were separated and purified by optimizing the chromatographic conditions of macroporous resin, silica gel and ODS column. Results 25 times amount of 55% ethanol with heating reflux at 95℃ for one hour were used as the optimal extraction process of Actinoside E. The optimum separation and purification process was as follows: D101 macroporous resin column was eluted with 7 BV of 50% ethanol, silica gel column was eluted with 5 BV of ethyl acetate-ethanol（10∶1）and 50% methanol eluted fraction was purified repeatedly by ODS column to obtain Actinoside E. The transfer rate of Actinoside E in the whole process was 53.70%, the yield was 0.35%, and the purity was 99.9%. Conclusion The process is stable and viable, which can provide material foundation for the development and utilization of Actinoside E.

ObjectiveTo investigate the effect of folic acid-modified crebanine polyethylene glycol-polylactic acid hydroxyacetic acid copolymer（PEG-PLGA） nanoparticles（FA-Cre@PEG-PLGA NPs， hereinafter referred to as NPs） combined with ultrasonic irradiation on subcutaneous tumor of liver cancer in Kunming（KM） mice. MethodsEighty-four healthy male KM mice were utilized to establish a subcutaneous tumor model of mouse hepatocellular carcinoma with H22 cells， then mice were randomly divided into model group， placebo group， hydroxycamptothecin group（8 mg∙kg^-1）， low， medium and high dose crebanine raw material groups（2， 2.5， 3 mg∙kg^-1， hereinafter referred to as the low， medium and high dose crebanine groups， respectively）， low， medium and high dose NPs groups（2， 2.5， 3 mg∙kg^-1）， and low， medium and high dose NPs combined with ultrasonic irradiation groups（2， 2.5， 3 mg∙kg^-1， hereinafter referred to as the low， medium and high dose combination groups， respectively）. The corresponding doses of drugs were administered via tail vein injection， the model group received no treatment， while the placebo group was injected with an equivalent amount of normal saline. Dosing was conducted for a total of 10 times on alternate days. The body mass of the mice was monitored， and parameters such as body mass change rate， thymus index， spleen index， tumor volume， tumor weight， relative tumor growth rate（T/C）， and tumor inhibition rate（TGI） were calculated. Pathological changes in liver and kidney tissues as well as the tumor were observed by hematoxylin-eosin（HE） staining. Additionally， the levels of aspartate aminotransferase（AST）， alanine aminotransferase（ALT）， blood urea nitrogen（BUN） and creatinine（CREA） in serum of mice were detected by biochemical method. Furthermore， the effect of ultrasound on the distribution of NPs in subcutaneous tumors of mouse hepatocellular carcinoma was observed by in vivo imaging technique. ResultsAmong different treatment methods， the combination of NPs and ultrasound irradiation had the best therapeutic effect. Compared with the model group， the body mass growth rates of mice in the medium and high combination groups decreased， while the thymus index and spleen index increased， but there was no statistically significant difference in serum AST， ALT， BUN and CREA levels， indicating that NPs combined with ultrasound irradiation had little effect on the normal physiological state of the body， oth groups had TGI>40% and T/C<60%， indicating a clear anti-tumor effect. Pathological analysis showed that compared with the NPs groups， the combination groups exhibited varying degrees of necrosis in tumor cells， accompanied by less damage to the liver and kidneys. In vivo imaging of small animals showed that compared with the high dose NPs group， the high dose combination group had stronger tumor targeting ability（P<0.01）. ConclusionNPs combined with ultrasonic irradiation can not only effectively targeted the drug to the tumor site， inhibit the subcutaneous tumor growth of mouse liver cancer， but also decrease damage to liver and kidney tissues.

Objective To establish the functional constipated mouse model by compound diphenoxylate, and explore the anti-constipation effect of black garlic polysaccharide. Methods Mouse small intestine ink propulsion experiment and mouse defecation experiment were carried out respectively. The mice in each experiment were randomly divided into blank group, model group, positive group and black garlic polysaccharide （0.25, 0.5, 1 g/kg） groups. Mice in blank group and model group were given distilled water, and in positive group were given lactulose oral solution. Compound diphenoxylate （5 mg/kg） was intragastric administrated after 1 week of administration, and small intestine propulsion experiment and defecation experiment were conducted respectively. Results Compared with model group, intestinal propulsion rate of black garlic polysaccharide groups was significantly increased and first dejection time was significantly shorten, and the number, weight and fecal water content increased significantly at 6 h in middle and high dose groups. Conclusion Black garlic polysaccharide could promote intestinal propelling, shorten defecation time and increase fecal water content.

OBJECTIVE To investigate the targeting effect of folic acid-modified crebanine nanoparticles （FA-Cre@PEG- PLGA NPs， hereinafter referred to as “NPs”） combined with ultrasound irradiation on M109 cells in vitro and in vivo after administration， and explore the anti-tumor mechanism. METHODS CCK-8 assay was used to detect the inhibitory effect of NPs combined with ultrasound irradiation on the proliferation of M109 cells， and the best ultrasound time was selected. Using human lung cancer A549 cells as a control， the targeting of NPs combined with ultrasound irradiation to M109 cells was evaluated by free folic acid blocking assay and cell uptake assay. The effects of NPs combined with ultrasound irradiation on the migration， invasion， apoptosis， cell cycle and reactive oxygen species （ROS） levels of M109 cells were detected by cell scratch test， Transwell chamber test and flow cytometry at 1 h after 958401536@qq.com administration； the changes of mitochondrial membrane potential （MMP） were observed by fluorescence inverted microscope. A mouse subcutaneous tumor model of M109 cells was constructed， and the in vivo tumor targeting of NPs combined with ultrasound irradiation was investigated by small animal in vivo imaging technology. RESULTS NPs combined with ultrasound irradiation could significantly inhibit the proliferation of M109 cells， and the optimal ultrasound time was 1 h after administration. The free folic acid could antagonize the inhibitory effect of NPs on the proliferation of M109 cells， and combined with ultrasound irradiation could partially reverse this antagonism. Compared with A549 cells， the uptake rate of NPs in M109 cells was significantly higher （P＜0.01）， and ultrasound irradiation could promote cellular uptake. NPs combined with ultrasound irradiation could inhibit the migration and invasion of M109 cells and block the cell cycle in the G0/G1 and G2/M phases. Compared with control group， the apoptosis rate of M109 cells and ROS level were increased significantly （P＜0.01）， while the MMP decreased significantly （P＜0.01） in the different concentration （100， 200， 300 μg/mL） groups of M109 cells. Compared with the mice in non-ultrasound group， the fluorescence intensity and tumor-targeting index of the tumor site in the 0 h ultrasound group were significantly enhanced （P＜0.05 or P＜0.01）. CONCLUSIONS NPs combined with ultrasound irradiation have a strong targeting effect on M109 cells in vitro and in vivo， the anti-tumor mechanism includes inhibiting cell migration and invasion， blocking cell cycle， and inducing apoptosis.

Objective Fragrant sachets are items with significant Chinese cultural characteristics and have multiple application values, with their medicinal value being an important aspect. Especially in recent years, with the successive outbreaks of SARS, MERS, COVID-19, the medicinal effect of traditional Chinese medicine sachets has been increasingly valued and widely used.However, there are no relevant standards for traditional Chinese medicine sachets at the national, industry, local, or organizational levels, which is not conducive to the healthy development of the industry. To establish standards for traditional Chinese medicine sachets to lead the development of the industry. Methods Based on the review of the current application status of traditional Chinese medicine sachets, a study on the quality standards of traditional Chinese medicine sachets was conducted through investigation and research, data collection, drafting of standard drafts, soliciting opinions, review and approval, and standard verification. Results The first group standard of traditional Chinese medicine sachet in China: Technological specification of traditional Chinese medicine sachet (powder core), which ensures the scientificity, progressiveness, rationality and practicability of the production of the standard of traditional Chinese medicine sachet. Conclusion The established group standard for traditional Chinese medicine sachets are practical, safe, reliable, and easy to implement, providing technical references for the inheritance and promotion of traditional Chinese medicine sachets.

Objective To explore the protective effect of hydroalcoholic extract of Portulaca oleracea L.(POHA)on ulcerative colitis(UC)and bone loss in mice.Methods The C57BL/6 mice were treated with dextran sulfate sodium(DSS)to establish UC model.A total of 50 mice were randomly assigned to including control group,DSS group,mesalazine(MS)group,low dose of POHA(POHAL)group,or high dose of POHA(POHAH)group.The control group freely drank drinking water,while the DSS,MS,POHAL and POHAH groups drank drinking water containing DSS for 8 weeks.Since the 2nd week,the control group and DSS group were given normal saline by gavage.The MS group was given MS(100 mg/kg)by gavage.The POHAL group and POHAH group were given POHA(1 000 mg/kg and 2 000 mg/kg)by gavage,respectively.Body weight and disease activity index(DAI)were recorded and calculated every 2 d.On the 56th day,the colon weight index,liver index,and spleen index were calculated,and the histological changes of colon were observed.Serum levels of bone metabolism markers and microstructure parameters of femur were detected.Results Compared with the control group,the DSS group showed significantly increased DAI score,colon weight index,liver index,and spleen index(all P＜0.01).The DSS group exhibited significant pathological damage in colon tissues and significantly increased serum levels of osteocalcin,C-terminal peptide of collagen type Ⅰ,and tartrate-resistant acid phosphatase 5b(P＜0.01).The bone loss was significant in the DSS group,manifested by markedly decreased bone mineral density(BMD),bone tissue volume to tissue volume ratio(BV/TV),trabecular bone number(Tb.N),and trabecular bone thickness(Tb.Th),and markedly increased bone surface to bone volume ratio(BS/BV)and trabecular bone separation(Tb.Sp)(P＜0.05 or P＜0.01).Compared with the DSS group,the BMD,BV/TV,Tb.N and Tb.Th of the femur in the MS group and POHAH group of mice were all increased(P＜0.05 or P＜0.01),the BS/BV all decreased(P＜0.05 or P＜0.01),and the Tb.Sp all decreased without significant differences(all P＞0.05).The above bone microstructure parameters in the POHAL group showed no significant differences compared with those in the DSS group(all P＞0.05).Conclusion POHA has protective effect on DSS-induced UC and bone loss,and the mechanism may be related to the inhibition of hyperactive bone metabolism.

Objective To screen the pharmacodynamic material basic components of Artemisiae Annuae Herba and study its antioxidant activity in vitro by investigating the spectrum-effect relationship between the HPLC fingerprints of 11 batches of Artemisiae Annuae Herba(dried aerial part of Artemisia annua L.).Methods The determination was performed on Aglient C18 column(250 mm×4.6 mm,5 μm)with mobile phase consisted of 0.2%phosphoric acid solution-Methanol(gradient elution)at the flow rate of 1.0 ml/min.The column temperature was indoor temperature,and detection wavelength was 220 nm,with sample size of 10 μl.Using isochlorogenic acid A as reference,HPLC fingerprints of 11 batches of samples were determined.The common peaks of 11 batches of samples were identified and recorded through TCM chromatographic fingerprint similarity evaluation system(2012 edition).Using scavenging rate of DPPH and ABTS free radical as pharmacodynamic indicators of antioxidant effects,SIMCA 14.1 analysis software was used for PLSR to establish the spectra-effect relationship.Results There were 48 common peaks on 11 batches of sample,11 components were identified as scopoletin,scoparone,isochlorogenic acid B,A,C,luteolin,apigenin,chrysosplenetin,artemisinin,artemisetin and artemisinic acid.The scavenging activity of 11 batches of samples to DPPH and ABTS free radicals was detected.The spectrum-effect relationship showed that isochlorogenic acid A,B,C and scoparone were positively associated with its antioxidant capacity,and variable projection value was greater than 1.It was suggested that these components were the material basis of antioxidant effect in Artemisiae Annuae Herba.Conclusion This study investigates the antioxidant capacity of different substances in Artemisiae Annuae Herba in vitro,and proves that isochlorogenic acid A,B,C and scoparone play a major role for the antioxidant capacity.

Objective To explore the effect of Bajitianwan(BJTW)formula on bone formation of Aβ-injured osteoblasts and its mechanism.Methods Osteoblasts isolated from neonatal 24-hour Wistar rats were used for the study,and osteoblasts were subjected to damage with Aβ1-42 oligomers,and pharmacological intervention was performed with the aqueous extract of BJTW formula.The MTT assay,alkaline phosphatase(ALP)activity assay,catalase(CAT)activity assay,superoxide dismutase(SOD)activity assay,glutathione(GSH)activity assay and malondialdehyde(MDA)activity assay were carried out respectively.The expression levels of bone morphogenetic protein 2(BMP2),osteogenic specific transcription factor(RUNX-2)and osteoprotective protein(OPG)were detected by Western blotting.After confirming the effect of BJTW formula on Aβ-injured osteoblasts,the network pharmacology method was used to predict the potential pathways.Results The BJTW formula significantly promoted the proliferation of Aβ-injured osteoblasts,increased ALP,SOD and GSH activity,inhibited MDA activity,and promoted the expression of bone formation-related proteins BMP2,RUNX-2 and OPG.Network pharmacological analysis showed that the effect of ameliorating of Aβ-injured osteoblasts by BJTW formula was mainly mediated by AGE-RAGE,PI3K-Akt,MAPK and neuroactive ligand-receptor interaction signaling pathways.Conclusion In this study,the effect of BJTW formula on improving the osteoblasts damaged by Aβ was confirmed for the first time,and its related mechanism was explored based on network pharmacology method.The results lay a strong foundation for the clinical application of traditional formula BJTW against osteoporosis.

Objective To explore the otherness of orthotopic injection of cell suspensions and transplantation of tumor tissue blocks to establish orthotopic implantation models of hepatocellular carcinoma in mice,and to provide a technical reference for the establishment of an orthotopic implantation model.Methods Healthy KM mice were divided into four groups:group A,direct injection of H22 cells;group B,direct injection of H22 ascitic cells;group C,transplantation of tissues;and group D,direct injection of saline.Activity and weight changes were observed regularly in each group and survival times were recorded.Liver tumor formation,tumor size,abdominal organ adhesion degree,and metastasis were observed in all groups.B-ultrasound imaging was performed,concentrations of alpha fetoprotein(AFP)and abnormal prothrombin(DCP)were detected,and liver histopathological changes were detected by hematoxylin and eosin staining.Results Mice molding operation time in groups A,B,and C were(3.36±0.44)min,(3.30±0.41)min,and(5.68±0.65)min,respectively.After modeling for 25 days,the rates of model formation in groups A,B,and C were all 100.0%.Severe abdominal adhesions occurred in 40.0%of mice in group A and 60.0%in group B,but in no mice in group C or D.Ascites occurred in 40.0%,100.0%,and 0.0%and abdominal wall tumors in 30.0%,60.0%,and 0.0%of mice in groups A,B,and C,respectively,while 40.0%of mice in group B also had liver metastasis.B-ultrasound imaging,detection of serum AFP and DCP levels,and histopathological result showed smooth liver margins,uneven echo and slightly lower echo mass,maintained high AFP and DCP secretion,and large numbers of inflammatory cells and tumor cells in mice in groups A,B,and C.Conclusions At day 25,all three methods can thus be used to establish orthotopic transplantation models of HCC.Among these,inj ection of cell suspensions demonstrated the advantage of simplicity in operation and the presence of multiple metastatic nodules within the liver,compared to transplantation of tumor tissue.Conversely,transplantation of tumor tissue showed the advantage of causing less impact on the abdomen and other organs when compared to inj ection of cell suspensions.

Objective To explore the effect and mechanism of Bajitianwan on preventing D-galactose (D-gal)-induced osteoblast bone loss. Methods Osteoblasts isolated from 24 h old Wistar rats were injured by D-gal and intervened with Bajitianwan extract. The osteoblastic proliferation and differentiation were determined by MTT and alkaline phosphatase (ALP), respectively. The cell reactive oxygen species (ROS) levels were detected by DCFH-DA fluorescent probes. The expression of cellular oxidation-related protein nuclear factor erythroid 2-related factor 2 (Nrf2), phosphorylated protein kinase B (p-AKT), protein kinase B (AKT), heme oxygenase-1 (HO-1), and NADPH quinone oxidoreductase 1 (NQO1) were detected by Western blotting. The intranuclear expression of Nrf2 protein was measured by immunofluorescence. Results Bajitianwan extract had significantly increased the osteoblastic proliferation and differentiation, and significantly reduced the intracellular ROS level. Bajitianwan extract had activated the PI3K/AKT pathway via activating the phosphorylation of AKT in osteoblasts, and promoted NQO1 and HO-1 expression. In addition, Bajitianwan had significantly promoted the expression of Nrf2 in the nucleus of osteoblasts, activating Nrf2 signaling pathway, and further promoted bone formation. Conclusion This study confirmed that Bajitianwan could prevent D-gal injured osteoblastic bone loss for the first time. The mechanism might be related to the regulation of oxidative stress associated PI3K/AKT and Nrf2 signaling pathway.