Isoprenoid flavonoids isolated from Sophora davidii and their activities induces apoptosis and autophagy in HT29 cells.

Four previously undescribed isoprenoid flavonoids (2-5) were isolated from Sophora davidii, along with five known analogues. The structures of the compounds were established through comprehensive analysis of spectroscopic data, including HRESIMS, 1D and 2D NMR, and absolute configurations determined by theoretical calculations, including ECD and NMR calculation. The cytotoxic effects of the isolated compounds on human HT29 colon cancer cells were evaluated using the MTT assay, compound 1 exhibited cytotoxicity against human HT29 colon cancer cells with an IC50 value of 8.39 ± 0.09 µM. Studies conducted with compound 1 in HT29 cells demonstrated that it may induce apoptosis and autophagy in HT29 by promoting the phosphorylation of P38 MAPK and inhibiting the phosphorylation of Erk MAPK.

[Effect of Electroacupuncture Intervention on Gastrointestinal Motility and Expression of Insulin-like Growth Factor 1 and Its Receptor Proteins in Gastric Antrum in Diabetic Gastroparesis Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) intervention of "Zusanli"(ST 36), etc. on gastrointestinal motility and levels of insulin-like growth factor 1 (IGF-1) and IGF-1 receptor (IGF-1 R) proteins in the gastric antrum in diabetic gastroparesis (DGP) rats, so as to study its mechanism underlying improvement of DGP. METHODS: Fifty SD rats, half male and half female, were randomly allocated to control, model, EA acupoint, EA non-acupoint and medication (metoclopramide) groups (n=10 rats/group). The DGP model was induced by 2% streptozotocin (STZ, i.p.i.,55 mg/kg) and high fat-sugar forage for 8 weeks. EA (10 Hz/50 Hz, 2 mA) was applied to "Zusanli"(ST 36), "Liangmen"(ST 21), "Sanyinjiao"(SP 6) and non-acupoints (about 5 mm lateral to ST 36, ST 21 and SP 6, respectively) for 20 min, one daily for 15 days, and 1.7% metocloppramide (1 mL/100 g) was given (gavage) to rats of the medication group, once daily for 15 days. After the treatment, the rats' general conditions were scored, the blood glucose level was determined using a glucometer, and the gastrointestinal mobility was evaluated by measuring the gastric emptying rate (GER) and the intestinal propulsion rates (IPR) marked by oral-infused phenol red solution (50 mg/dL). The immunoactivity levels of IGF-1 and IGF-1 R of gastric antrum tissue were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: After modeling, the symptom integrative score, the blood glucose, IGF-1 and IGF-1 R levels were significantly increased (P<0.01), and the GER and IPR were notably decreased relevant to the control group (P<0.01). After EA stimulation of ST 36, ST 21 and SP 6, the symptom score, blood glucose, and IGF-1 and IGF-1 R levels of the gastric antrum were significantly reduced, while the GER and IPR were considerably increased (P<0.05,P<0.01). Except the GER and IPR were considerably increased in the medication group(P<0.05,P<0.01), no significant changes were found in the symptom integrative score, the blood glucose, IGF-1 and IGF-1 R levels in both EA non-acupoint and medication groups relevant to the model group (P>0.05),the GER and IPR in the EA non-acupoint group (P>0.05). Compared with the EA acupoint group, the symptom integrative score, IGF-1 and IGF-1 R levels were significantly increased in both EA non-acupoint and medication groups(P<0.05,P<0.01). CONCLUSIONS: EA stimulation can improve gastrointestinal motility and lower blood glucose in DGP rats, which may be closely associated with its effectiveness in reducing IGF-1 and IGF-1 R immunoactivity levels in gastric antrum.

[Effects of Electroacupuncture on Ultrastructure of Interstitial Cells of Cajal and Stem Cell Factor-kit Signal Pathway of Gastric Antrum in Diabetic Gastroparesis Rats].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on gastrointestinal motility and the ultrastructure of interstitial cells of Cajal (ICC) and the expressions of c-kit receptor protein and stem cell factor (SCF) mRNA in diabetic gastroparesis (DGP) rats, so as to explore its mechanism. METHODS: Fifty SD rats were randomly divided into normal, model, acupoint, non-acupoint and metoclopramide groups (n＝10 rats/group). DGP model was established by intraperitoneal injection of streptozotocin (STZ, 2%), and raised with high-sugar high-fat diet irregularly. EA (sparse-dense, 10 Hz/50 Hz, 2 mA, 20 min) was applied at "Zusanli" (ST 36), "Liangmen" (ST 21) and "Sanyinjiao" (SP 6), and the corresponding non-acupoints of the 3 acupoints, daily for 15 days. The rats in metoclopramide group received intragastric administration of metoclopramide (1.7%, 1 mL/100 g) for 15 days, once a day. Blood sugar was determined with One Touch blood glucose test paper. The gastric emptying rate (GER) and the intestinal propulsion rate (IPR) were measured by intragastric phenol red. The ultrastructure of ICC was detected by transmission electron microscopy. The expression levels of c-kit receptor protein and SCF mRNA of gastric antrum were examined respectively by Western blot and RT-PCR. RESULTS: Compared with the normal group, the blood glucose significantly increased in the model group (P<0.01), while the GER, IRP and the expression level of SCF mRNA in the gastric antrum significantly decreased (P<0.01), and the ultrastructure of ICC appeared apoptosis-like changes. The blood glucose of the EA group was obviously decreased compared with that of the model group (P<0.05); the GER and IRP significantly increased(P<0.05, P<0.01); the expression level of SCF mRNA increased (P<0.01), the number of ICC increased and its ultrastructure was repaired. There was some relief on ICC ultrastructure in the acupoint group compared with that in the non-acupoint group; and SCF mRNA increased (P<0.05). There was no significant difference on c-kit receptor expression among all the modeling groups (P＞0.05). CONCLUSIONS: EA at ST 36, etc. can regulate the blood glucose and improve gastrointestinal emptying in DGP rats. The mechanism may be related to up-regulating SCF mRNA, repairing ICC ultrastructure, restoring the pacing function, and improving gastrointestinal motility.

[Effects of rutaecarpine on inflammatory cytokines in insulin resistant primary skeletal muscle cells].

It is now well established that inflammation plays an important role in the development of numerous chronic metabolic diseases including insulin resistance (IR) and type 2 diabetes (T2DM). Skeletal muscle is responsible for 75% of total insulin-dependent glucose uptake; consequently, skeletal muscle IR is considered to be the primary defect of systemic IR development. Our pre- vious study has shown that rutaecarpine (Rut) can benefit blood lipid profile, mitigate inflammation, and improve kidney, liver, pan- creas pathology status of T2DM rats. However, the effects of Rut on inflammatory cytokines in the development of IR-skeletal muscle cells have not been studied. Thus, our objective was to investigate effects of Rut on inflammatory cytokines interleukiri (IL)-1, IL-6 and tumor necrosis factor (TNF)-α in insulin resistant primary skeletal muscle cells (IR-PSMC). Primary cultures of skeletal muscle cells were prepared from 5 neonate SD rats, and the primary rat skeletal muscle cells were identified by cell morphology, effect of ru- taecarpine on cell proliferation by MTT assay. IR-PSMC cells were induced by palmitic acid (PA), the glucose concentration was measured by glucose oxidase and peroxidase (GOD-POD) method. The effects of Rut on inflammatory cytokines IL-1, IL-6 and TNF-α in IR-PSMC cells were tested by enzyme-linked immunosorbent assay (ELISA) kit. The results show that the primary skeletal muscle cells from neonatal rat cultured for 2-4 days, parallel alignment regularly, and cultured for 7 days, cells fused and myotube formed. It was shown that Rut in concentration 0-180. 0 µmol x L(-1) possessed no cytotoxic effect towards cultured primary skeletal muscle cells. However, after 24 h exposure to 0.6 mmol x L(-1) PA, primary skeletal muscle cells were able to induce a state of insulin resistance. The results obtained indicated significant decrease (P < 0.05 to P < 0.001) IL-1, IL-6 and TNF-α production by cultured IR-PSMC cells when incubating 24 hours with Rut, beginning from 20 to 180.0 µmol x L(-1). IL-1, IL-6 and TNF-α in the Rut treated groups were dose-dependently decreased compared with that in the IR-PSMC control group. Our results demonstrated that the Rut promoted glucose consumption and improved insulin resistance possibly through suppression of inflammatory cytokines in the IR-PSMC cells.

[SSR information in Erigeron breviscapus transcriptome and polymorphism analysis].

OBJECTIVE: The SSR information in the transcriptome of Erigeron breviscapus was analyzed in this study, in order to further develop new functional genes SSR markers laid a solid foundation. METHOD: SSR loci were searched in all of 52,060 unigenes by using est_timmer. Perl program and SSR primers were designed by Primer3. Furthermore, 36 pairs of primers were randomly selected for the polymorphism analysis on 13 Erigeron breviscapus plants collected from different places. RESULT: A total of 3639 SSRs were found in the transcriptome of Erigeron breviscapus, distributed in 3260 unigenes with the distribution frequency of 6.99%. Di-nucleotide repeat was the main type, account for as much as 34.41% of all SSRs, followed by mono-nucleotide (31.41%) and tri-nucleotide repeat motif (28.08%). The di-nucleotide repeat motifs of AT/AT and AC/GT were the predominant repeat types (28.71%). The tri-nucleotide repeat motifs of AAT/AT was the predominant repeat types (7.94%). For validation the availability of those SSR primers, we randomly selected 36 pairs of primers for PCR amplification. Among them, 34 pair primers (94.44%) produced clear and reproductive bands, 19 pair primers showed polymorphism (52.78%), and 13 Erigeron breviscapus plants were divided into 2 groups. CONCLUSION: There are numerous SSRs in Erigeron breviscapus transcriptome with high frequency and various types, this will provide abundant candidate molecular markers for genetic diversity study and genetic map in this plant.

[Probe excavations about using gene chip to identify original plants in Chinese pharmacopoeia based on NCBI sequence database].

OBJECTIVE: Based on the DNA fragments of medicinal plants of NCBJ database, the DNA Probe,which can be used to identify original plants in the Chinese Pharmacopoeia (2010 edition), was got. METHODS: First of all, get the Latin name of the original plants by collating the Chinese Pharmacopoeia. Next,download the medicinal plants' DNA fragments from the NCBI database, including ITS, matK, rbcL, psbK-psbI and trnH-psbA, then design probe by using Array Designer 4. 2. Finally, analyze each probe's versatility in the same kind of original plant and conservatism in different kinds of original plants by using Matlab, then determine the specificity of the probe. RESULTS: Regarding the Latin name of 586 original plants in the Chinese Pharmacopoeia (2010 edition) and the above five gene fragments as retrieval condition, 7 613 sequences were downloaded from NCBI, then 315 436 probes were got in total by analyzing. What's more, after analyzing versatility and conservatism of the probes,13 814 specific probes were got. Furthermore,in theory, 376 kinds of original plants could be detected. Because there existed the lack of related gene fragments in the NCBI database,or the sequences were short of specificity,210 species of original plants which were involved in the Chinese Pharmacopoeia didn't receive the corresponding probe. CONCLUSION: The results of the study can provide the further development of medicinal plants' identification chip with vital information support,and the excavation methods of probe can be widely used. Furthermore,the results of the study indicate the original plants which need sequencing importantly in the future.

[Cloning and bioinformation analysis of flavone synthase II gene of Erigeron breviscapus].

OBJECTIVE: Erigeron breviscapus is a medicinal plant with the most developmental potential in Yunnan province, which is belongs to Erigeron genus of Compositae family. Scutellarin, the main active component of Erigeron breviscapus is one of flavone 7-O-glucuronide derivatives, its biosynthesis pathway is still not clear. METHOD: Full length cDNA encoding flavone syhthase II gene in E. breviscapus was cloned in this study using R-PCR, 3'-RACE and 5'-RACE. RESULT: The opening reading frame of FS II cDNA of E. breviscapus is 1 557 bp long and encoding 518 amino acids, designed as EbFS II, which is highly homologous with FS II of Compositae species, like Callistephus chinensis, Cynara cardunculus var. scolymus, Gerbera hybrida, Dahlia pinnata and Lobelia erinus. CONCLUSION: Phylogenetic analysis showed that EbFS II might has the function of directly converting flavanone to flavone.

[Simultaneous determination of chlorogenic acid, scutellarin, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid in different parts of Erigeron breviscapus by high-performance liquid chromatography].

OBJECTIVE: A high-performance liquid chromatographic (HPLC) method was developed for simultaneous determination of chlorogenic acid, scutellarin, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid in different parts of Erigerontis Herba. METHOD: The four constituents were measured on an Agilent Zorbax SB-C18 column (4.6 mm x 450 mm, 5 microm) with a gradient elution of acetonitrile (A) -0.3% phosphoric acid solution (B) (0-10 min, 12%-15% A, 10-32 min, 15% A, 32-33 min, 15%-20% A, 33-50 min, 20%-22% A) at wavelength of 335 nm and 327 nm, and a flow rate of 1.0 mL x min(-1) and the column temperature was 30 degrees C. RESULT: Linearity of each standard was established in the concentration range of 0.050 1-1.002 microg for chlorogenic acid, 0.165 9-3.318 microg for chlorogenic acid, 0.049 7-0.994 microg for 3,5-dicaffeoylquinic acid, 0.048 7-0.974 p.g for 4,5-dicaffeoylquinic acid respectively, with correlation coefficient r > 0.999 6. Average recoveries (n = 6) of 4 compounds were 98.53% with a RSD of 0.94%, 99.68% with a RSD of 0.49%, 98.78% with a RSD of 1.1%, 99.06% with a RSD of 0.81%, respectively. CONCLUSION: The developed method is simple, accurate, and precise, it can be used for the quantitative analysis of Erigeron breviscapus.

[Genetic diversity and group consistency of breeding strains of Erigeron breviscapus determined by AFLP marker].

OBJECTIVE: To analyze the genetic diversity and breeding strains of the E. breviscapus germplasms, in order to provide theoretical information for Erigeron breviscapus breeding. METHOD: The genetic diversity and genetic structure were assayed to six germplasm resource of E. breviscapus which collected from Yunnna with 11 pairs primers and AFLP molecular marker. RESULT: Six hundred and four amplification bands among 636 DNA bands were from six accession of E. breviscapus, which are about 82.40% of total bands. The six germplasms could be divided into three group at the 0. 706 similarity coefficient level. The first category include QS-1, QS-2 and Dali, Shilin, Kunming population. The second category included wild population of Qiubei. The third category included several sample from different district. The mean genetic similarity coefficient of QS-1 and QS-2 was bigger, genetic similarity coefficient range was smaller, hereditary character was more stable. Molecular system clustering analysis showed that the geographical origin of the same part had relative polymerization phenomenon and its genetic relationship was close. Qiubei was a single group possibly relating to the specific genetic basis. CONCLUSION: The analysis of genetic diversity of E. breviscapus by AFLP marker is reliable. The systematic E. breviscapus breeding is feasible.

[Identification model of cultivated and wild Chinese medical herbs erigeron breviscapus with near-infrared spectroscopy].

Forty three cultivated and wild Chinese medical herbs erigeron breviscapus were scanned on two Fourier transform near-infrared spectroscopy instruments. Twenty samples were used to set up the BP-NN models and the others were used to validate the models. Fifteen principal components, whose variance contribution rate is above 99%, were collected as input nodes for BP-NN models. The correct identification rates of calibration samples were 100% for the models on both the two instruments, and the correct identification rates of validation samples were 100% and 95.7%, irrespectively. The results showed that using NIR to fast detect cultivated and wild Chinese medical herbs erigeron breviscapus was feasible.