[Objective] Ursolic acid and oleanolic acid in Acantopanax senticosus (Rupr. et Maxim.) Harms were extracted with the ultrasonic technology. A determination method of ursolic acid and oleanolic acid was established with the high performance liquid chromatograph and photodiode array detector. [Method]The chromatographic column, Nova-Pak C18 (3.9?300 mm, 4 ?m), methanol-water (88∶12,V/V), mobile phase with 0.8 mL/min flow rate, the detected wave length (210nm),and the column temperature(25 ℃) were adopted. [Results] The calibration curve of ursolic acid was linear under the content of 0.136 ~1.224 ?g and the correlation coefficient was 0.9995. The calibration curve of oleanolic acid was linear under the content of 0.052 ~0.468 ?g and the correlation coefficient was 0.9994. The average recovery of ursolic acid was 98.1 % and RSD was 1.1 % (n=5). The average recovery of oleanolic acid was 97.5 % and RSD was 1.0 % (n=5). [Conclusion]The content determination of HPLC is accurate and reliable, and can be used to analyse quantificationally ursolic acid and oleanolic acid in Acantopanax senticosus (Rupr. et Maxim.) Harms.

OBJECTIVE:To further exploit the active components from Loquat leaf and its gruffs.METHODS:The chief components in loquat leaf and its gruffs were extracted by ultrasound extraction and Soxhlet extraction,respectively,the sepa?ration of ursolic acid and other constituents was performed by HPLC,and the relative amounts of which were determined and compared.RESULTS:Soxhlet extraction is superior to ultrasound extraction in the extraction of loquat leaf and8components in gruffs.Under the same extraction method,the content of ursolic acid in loquat leaves is equivalent to that of gruffs.Besides,the contents of6components in gruffs were above those in loquat leaves.CONCLUSION:The essential component-triterpenic acid in loquat leaves suffered no loss after extraction with water,the active components in gruffs can be further exploited.

AIM:To observe the contents of ursolic acid and oleanolic acid in Liuwei Dihuang Pill(Radix Rehmanniae praeparata,Rhizoma Dioscoreae,Fructus Corni,Rhizoma Alismatis,etc.) from different manufacturers and explore their effects on quality of Liuwei Dihuang Pill.METHODS:Ursolic acid and oleanolic acid in Liuwei Dihuang Pill were determined by HPLC-PAD method using Kromasil C_ 18 column(4.6 mm?250 mm,5 ?m) as chromatographic column,methanol-water-phosphate(88∶12∶0.02) as the mobile phase at the rate of 1.0 mL/min.RESULTS:6.462-0.718 ?g of ursolic acid and 3.132-0.348 ?g of oleanolic acid presented a good linear relationship.The average recoveres were 99.73% and 97.59%,RSD were 1.5% and 1.7%,respectively.There were significant differences in the contents of ursolic acid and oleanolic acid in Liuwei Dihuang Pill from four different manufacturers.CONCLUSION:The method is simple,rapid and feasible to set up determination of ursolic acid and oleanolic acid in Liuwei Dihuang Pill by HPLC-PAD.Although the contents of ursolic acid and oleanolic acid have significant differences in different manufactures,the results show good consistency in the content variation of the two acids.

To use Agrobacterium rhizogenes-induced hairy roots to create new germplasm of Dianthus caryophyllus, we transformed D. caryophyllus with A. rhizogenes by leaf disc for plant regeneration from hairy roots. The white hairy roots could be induced from the basal surface of leaf explants of D. caryophyllus 12 days after inoculation with A. rhizogenes ATCC15834. The percentage of the rooting leaf explants was about 90% 21 days after inoculation. The hairy roots could grow rapidly and autonomously in liquid or solid phytohormone-free MS medium. The transformation was confirmed by PCR amplification of rol gene of Ri plasmid and silica gel thin-layer chromatography of opines from D. caryophyllus hairy roots. Hairy roots could form light green callus after cultured on MS+6-BA 1.0-3.0 mg/L + NAA 0.1-0.2 mg/L for 15 days. The optimum medium for adventitious shoots formation was MS + 6-BA 2.0 mg/L + NAA 0.02 mg/L, where the rate of adventitious shoot induction was 100% after cultured for 6 weeks. The mean number of adventitious shoot per callus was 30-40. The adventitious shoots can form roots when cultured on phytohormone-free 1/2 MS or 1/2 MS +0.5 mg/L NAA for 10 days. When the rooted plantlets transplanted in the substrate mixed with perlite sand and peat (volume ratio of 1:2), the survival rate was above 95%.
Agrobacterium ; Chromatography, Thin Layer ; Culture Media ; Dianthus ; growth & development ; Plant Growth Regulators ; Plant Leaves ; Plant Roots ; growth & development ; Plants, Genetically Modified ; Rhizobium ; Tissue Culture Techniques ; Transformation, Genetic

Objective To establish antigen capture ELISA methed to detect Kaposi's sarcoma-as-sociated herpesvirus(KSHV)antigen,and to evaluate its feasibility for clinical application.Methods The BALB/c mice and New Zealand white rabbits were injected with purified recombinant KSHV gpK8.1 proteins to prepare the monoclonal antibody(McAb)and polyclonal antibody(PcAb)anti-gpK8.1,respectively.A new antigen capture ELISA method was established for KSHV antigen detection.The detection reproducibili-ty as well as the sensitivity and specificity of this new assay were determined by the optimization test,which antibody pairs were analyzed to choose the best coating antibody and detecting antibody.The 3 KSHV posi-tive patients sera and 257 patients sera from sexually transmitted disease,cancers or gynecological diseases were detected with this assay to evaluate its value for clinical application.Results When the McAb as coat-ing antibody at concentration of 5 μg/ml and PcAb as detecting antibody at concentration of 1.6μg/ml were selected,the highest P/N value could be obtained.The sensitive analysis of this test could detect recombi-nant KSHV gpK8.1 antigen of 31.28 ng/ml.Meanwhile,it is highly specific to detect KSHV antigen with-out cross reaction to Epstein-Barr vims(EBV),herpes simplex virus(HSV)-1 or HSV-2.All of three KSHV-positive sera and 4 sera from 257 clinical samples were positive with this new assay.which indicated that it could be used for capturing KSHV antigen.Conclusion A sensitive and specific McAb-based anti-gen capture ELISA method to detect KSHV antigen were established successfully.It is of great potential val-ue to develop reagent for KSHV clinical serologic dingnosis.

Objective To study the radiosensitizing effect of nano-gold nano-silver particles in hepatocellular carcinoma cells (HepG2) in vitro and the possible mechanisms.Methods MTT assay and clonogenic assay were performed to determine the killing effect of nano-gold and nano-silver particles in HepG2 cells.Flow-cytometry was used to measure cell apoptosis and cell cycle distribution.Western blotting was used to measure the expression of Caspase-3,Bax and Bcl-2.ELIASA was used to determine the content of catalase (CAT),superoxide dismutase (SOD),and total glutathione (GSH).Results Nano-gold and nano-silver particles inhibited the proliferation of HepG2 cells with IC50 of 6.51 μg/ml and 2.47 μg/ml,respectively.Nano-gold and nano-silver particles significantly enhanced the radiosensitivity of HepG2 cells.Obtained by Dq,the SER of 1/5 IC50 nano-gold and nano-silver particles were 1.37 and 1.48,and 1/10IC50 with 1.11 and 1.09.Nano-gold and nano-silver particles increased the expression of Caspase-3 and Bax and reduced the exprcssion of Bcl-2.CAT,SOD and total GSH were significantly reduced.Conclusions Nano-gold and nano-silver particles can enhance the radiation sensitivity of HepG2 cells.Specific sensitizing mechanism may be the activation of the mitochondrial apoptosis pathway and the induction of reactive oxygen species in apoptotic pathways,which ultimately induces apoptosis.

Object To research and choose the best technology c onditions for extracting ursolic acid from Sambucus chinensis Li ndl. Methods According to physicochemical character of ursolic aci d, orthogonal design and tests of extract technology for ursolic acid were ca rried on, and four factors were chosen such as concentration, qu an tity of ethanol, extracting time, concentration of clearing agent, and three lev els of each factor were used for orthogonal design and test. Results That was the best technology condition that ursolic acid was extracted by means of ethyl alcohol (90%) of 7 times as much as raw materials, and it is heat ed 2 times (each 1 h), and the concentration of clearing agent was 3%. Conclusion That is a better technology for industrial producti on because it is advanced and rational, practical and feasible.

Objective To investigate the effect of resveratrol (Res) on paraquat (PQ)-induced acute lung injury (ALI) and mortality in mice and the mechanism of nuclear factor-κB (NF-κB) inflammatory pathway. Methods Sixty-eight healthy male ICR mice with grade SPF were enrolled, among them 20 mice were used for mortality observation (n = 10), and other 48 were used for determination of related parameters (n = 6). The mice were randomly divided into four group s: normal saline (NS) control group, Res control group, PQ group and PQ + Res group. The mice in the latter two groups were subdivided into 6, 24, 72 hours subgroups. The PQ poisoning model of mice was reproduced by one injection of 30 mg/kg PQ intraperitoneally. The mice in PQ + Res group were given 60 mg/kg Res intraperitoneally on the contralateral side after PQ injection. The mice were sacrificed at 6, 24, 72 hours after PQ poisoning, and lung tissue was harvested. The serum levels of tumor necrosis factor-α (TNF-α), interleukins (IL-6 and IL-1β) were determined by enzyme linked immunosorbent assay (ELISA). The pathological changes in lung tissue were observed with electron microscopy. Apoptosis cells in the lung were identified by terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) for the estimation of apoptosis rate. The protein expression of NF-κB p65 was determined by Western Blot. Results Compared with PQ group, the death number of mice at 48, 72, 96 hours in PQ + Res group was slightly decreased (0 vs. 2, 2 vs. 5, 4 vs. 6) but without statistically significant difference (all P > 0.05). Under electron microscope, the lung injury in PQ group was severer than that in NS control group, and Res was found to be able to alleviate the lung injury. Compared with NS control group [(2.45±0.61)%], the apoptosis rate at 6 hours in PQ group was significantly increased [(8.42±1.48)%], and peaked at 72 hours [(21.23±3.47)%]. Res could decrease the apoptosis rate after PQ poisoning [6 hours: (5.56±1.31)% vs. (8.42±1.48)%, 24 hours: (11.14±2.07)% vs. (16.88±2.96)%, 72 hours: (13.28±2.32)% vs. (21.23±3.47)%, all P < 0.05]. The serum levels of TNF-α, IL-6, and IL-1β, and NF-κB p65 in lung tissue were all markedly increased after PQ poisoning, and they were significantly decreased after Res intervention as compared with those of PQ group [TNF-α (ng/L): 2.62±0.29 vs. 4.06±0.74 at 6 hours, 3.98±0.41 vs. 6.79±0.80 at 24 hours, 5.06±0.75 vs. 11.00±0.75 at 72 hours; IL-6 (ng/L): 14.19±1.54 vs. 16.55±1.24 at 6 hours, 13.21±1.37 vs. 19.73±0.85 at 24 hours, 13.72±0.56 vs. 22.45±0.72 at 72 hours; IL-1β (ng/L): 8.54±1.64 vs. 12.59±0.66 at 6 hours, 10.15±0.29 vs. 16.24±1.03 at 24 hours, 16.14±0.70 vs. 19.55±0.56 at 72 hours; 6-hour NF-κB p65: (1.34±0.07) folds vs. (1.86±0.11) folds when the expression in NS control group was represented as 1, all P < 0.05]. Conclusions Res cannot lower the mortality in mice with PQ poisoning, but it seems to be able to attenuate PQ-induced ALI and cell apoptosis. The mechanism responsible for the latter maybe the inhibitive effect of Res on NF-κB p65 translocation and cytokines production.

Aim To study the effects of extracellular acidosis on articular chondrocytes pyroptosis and its possible mechanisms. Methods Primary articular chondrocytes were incubated in different pH and NAC. The expression of proinflammatory cytokines IL-1β, IL-18, ASC, NLRP3, caspase-1 were detected by Western blot and real-time PCR. The state of pyropto-sis was identified by AO/EB staining and LDH con-tents. The expression of ROS was observed by DCFH-DA, and ELISA was used to detect the IL-1β,IL-18 in cultured supernatants. Results Compared with the normal cell, extracellular acidosis could increase the expression of IL-1β, IL-18, ASC, NLRP3 and caspase-1 , upregulate the fluorescence intensity of in-tercellular ROS, accompanied with the promoted release of LDH. Moreover, it is observed that extra-cellular acidosis could also induce chondrocytes death by AO/EB staining. NAC,the scavenger of ROS could inhibit these effects of extracellular acidosis on chon-drocytes. Conclusion Extracellular acidosis may in-duce chondrocyte pyroptosis via upregulating the intra-cellular ROS content.

Objective To explore the clinical efficacy and safety of umbilical cord derived mesenchymal stem cells transplantation(UC-MSCT)for patients with refractory systemic lupus erythematosus (SLE).Methods Twelve patients with refractory SLE were enrolled in this study.UC-MSCs(≥106/kg cell number)were infused intravenously for each patient. The clinical manifestations and laboratory parameters were compared before and after MSCT. Results The twelve patients were followed up for one to twenty-six months after MSCT.The systemic lupus erythematosus disease activity index(SLEDAI)score decreased from 18±4 to 10±4 one month after MSCT(n=12,P＜0.01)and then decreased to 7±4 at three month follow-up.Nine patients showed improvement of 24 h proteinuria[(2103±749)mg vs(3359±1248)mg,P＜0.01]one month after MSCT.Further improvement of 24 h proteinuria was observed in eight patients[(1427±616)mg vs(3342±1333)mg,P＜0.01]at three months post MSCT.Serum creatinine of five patients decreased significantly and ten patients showed an increase of serum albumin. Serum complement C3 increased in three patients and four patients showed obvious amelioration of hematological abnormalities. There was no transplantation related complications for all the patients. Conclusion UC-MSCT is effective and safe for refractory SLE,but further observation is required to evaluate its long term efficacy.