Preparation，characterization，in vitro drug release property and cytotoxicity of Periplaneta americana extract-loaded spider fibroin membrane / 中国药房

OBJECTIVE To prepare spider fibroin membrane loaded with Periplaneta americana extract， and investigate its characterization， in vitro drug release property and cytotoxicity. METHODS Using natural spider silk collected from Chilobrachys guangxiensis as raw material， P. americana extract as model drug， the drug-loaded spider fibroin membrane （hereinafter referred to as drug-loaded membrane） was prepared by solvent casting method. The material matrix spider fibroin membrane without P. americana extract （hereinafter referred to as blank membrane） was prepared with same method. The membrane structure was characterized by static water contact angle， Fourier infrared chromatography， X-ray diffraction and scanning electron microscopy from different angles； drug release characteristics in artificial saliva were simulated in vitro to evaluate the drug sustained-release performance. MTT assay was adopted to validate the cytotoxicity of drug-loaded membrane. RESULTS The drug-loaded membrane was prepared， and the static water contact angle was less than 90°， which was less than that of blank membrane. The drug-loaded membrane showed the characteristic absorption peak to polypeptide of P. americana extract at 1 500-1 700 cm－1. X-ray diffraction and scanning electron microscopy also proved that the drug was successfully loaded into the pellicle. The release time of the pellicle in artificial saliva was more than 200 min. The MTT test results showed that the cell proliferation rates of blank membrane and drug-loaded membrane were 84.6% and 79.4% （both greater than 70%）， respectively， without significant potential cytotoxicity. CONCLUSIONS Drug-loaded membrane prepared with natural spider silk has a certain sustained-release effect in artificial saliva， which can be further developed as a drug sustained-release carrier with excellent biological characteristics and biocompatibility.

Periplaneta americana extract CⅡ-3 induces senescence of leukemia K562 cells via SIRT1/mTOR signaling pathway / 中国中药杂志

This study aims to investigate the role of slient mating-type information regulation 2 homolog 1(SIRT1)/tuberous sclerosis complex 2(TSC2)/mammalian target of rapamycin(mTOR) signaling pathways in the Periplaneta americana extract CⅡ-3-induced senescence of human leukemia K562 cells. K562 cells were cultured in vitro and treated with 0(control), 5, 10, 20, 40, 80, and 160 μg·mL~(-1) of P. americana extract CⅡ-3. Cell counting kit-8(CCK-8) and flow cytometry were employed to examine the proliferation and cell cycle of the K562 cells. Senescence-associated β-galactosidase stain kit(SA-β-gal) was used to detect the positive rate of senescent cells. Mitochondrial membrane potential was detected by flow cytometry. The relative mRNA level of telomerase reverse transcriptase(TERT) was determined by fluorescence quantitative PCR. The mRNA and protein levels of SIRT1, TSC2, and mTOR were determined by fluorescence quantitative PCR and Western blot, respectively. The results showed that CⅡ-3 significantly inhibited the proliferation of K562 cells and the treatment with 80 μg·mL~(-1) CⅡ-3 for 72 h had the highest inhibition rate. Therefore, 80 μg·mL~(-1) CⅡ-3 treatment for 72 h was selected as the standard for subsequent experiments. Compared with the control group, CⅡ-3 increased the proportion of cells arrested in G_0/G_1 phase, decreased the proportion of cells in S phase, increased the positive rate of SA-β-Gal staining, elevated the mitochondrial membrane potential and down-regulated the mRNA expression of TERT. Furthermore, the mRNA expression of SIRT1 and TSC2 was down-regulated, while the mRNA expression of mTOR was up-regulated. The protein expression of SIRT1 and p-TSC2 was down-regulated, while the protein expression of p-mTOR was up-regulated. The results indicated that P. americana extract CⅡ-3 induced the senescence of K562 cells via the SIRT1/mTOR signaling pathway.

Comparative study of the oil repellent activity of Copaifera officinalis Linnaeus and Copaifera reticulata Ducke front nymphs of Periplaneta americana Linnaeus

Abstract To control urban pests, especially cockroaches of the Periplaneta americana species, various pesticides have been developed that are increasingly potent and effective. However, the unrestrained application of pesticides has had negative consequences, such as the disappearance of some useful insect species and, consequently, the appearance of new pests, both in the countryside and cities. Due to the current scenario, it was necessary to search for new alternatives for the control of these insects. Among the species studied, Copaíba stood out. The oils were analyzed using GC-MS, b-caryophyllene and a-bergamotene being the predominant compounds. Repellency tests were performed with three different concentrations of C. officinalis and C. reticulata, 500 μg/mL, 250 μg/mL and 125 μg/mL, in triplicate. It can be observed that the oil of C. officinalis was more repellent to the nymphs at concentrations of 500 μg/mL and 250 μg/mL, however, when the behavior in nymphs exposed to the concentration of 125 μg/mL was compared, it was noted that C. reticulata oil was more repellent at this concentration. Copaifera has shown promising activity as a repellent against arthropods owing to the complex chemical composition of its oils.

Study on Periplaneta americana Polypeptide PAE2 in Reversing Multidrug Resistance for Liver Cancer / 中国实验方剂学杂志

Objective:To investigate the effect and mechanism of PAE₂， a polypeptide of <italic>Periplaneta americana， </italic>in reversing multidrug resistance （MDR） for liver cancer <italic>in vivo</italic>. Method:Balb/c-nude mice were inoculated with HepG2 and HepG2/ADM cells under the armpits to establish animal models of liver cancer sensitive strains and animal models of MDR respectively. After successful modeling， the nude mice were randomly divided into normal group， HepG2 model group， HepG2/ADM model group， sorafenib group （positive drug control group， <italic>ig</italic> 30 mg·kg^-1）， HepG2/ADM+PAE₂ （<italic>iv</italic>） low， medium and high dose groups （50， 100， 200 mg·kg^-1）， HepG2/ADM+PAE₂ （<italic>ig</italic>） low， medium， and high dose groups （50， 100， 200 mg·kg^-1）， skim cream group （<italic>ig</italic> 200 mg·kg^-1）， and CⅡ-3 group （<italic>ig</italic> 200 mg·kg^-1）， all of which received corresponding drug treatment. The body weight and tumor volume of nude mice were measured and recorded every 2 days. The next day after the last administration， tumor tissues of nude mice were taken to record the tumor weight. The effect of <italic>P. americana </italic>polypeptide PAE₂ on permeability-glycoprotein（P-gp）， lung resistance protein（LRP） ， breast cancer resistance protein（BCRP）， protein kinase C（PKC）， glutathione S-transferase-π（GST-π）， topo-isomerase typeⅡ（ToPoⅡ）， multidurg resistance gene 1（MDR1）and Multidrug resistance-associated proteins（MRP1） of the protein level and gene level expression in tumor tissues were determined by immunohistochemistry （IHC） and real-time quantitative polymerase chain reaction （Real-time PCR）. In addition， both oral and intravenous administration groups were set up at the same time for preliminary study on the basic pharmacokinetic characteristics of <italic>P. americana </italic>polypeptide PAE₂. Result:After the successful modeling， the body weight of the nude mice was significantly lower than that in the normal mice（<italic>P</italic><0.05）. After treatment with corresponding drugs， the body weight increased to a certain extent， but it was still not as good as the normal nude mice. In <italic>iv</italic> administration， the medium-dose <italic>P. americana </italic>polypeptide PAE₂ showed the best anti-tumor effect as compared with the model group （<italic>P</italic><0.05）， while in oral administration， the anti-effect increased with the increase of the dose， so the high-dose group showed the best effect （<italic>P</italic><0.05）. Preliminary crude extract CII-3 had no obvious anti-tumor effect， and skim cream showed a certain anti-tumor effect （<italic>P</italic><0.05）. <italic>P. americana </italic>polypeptide PAE₂ had certain effects on MDR related proteins and enzymes<italic> in vivo</italic>， mainly by inhibiting the expression of LRP and BCRP in tumor tissues and affecting the expression of these related proteins and genes to different degrees to inhibit intracellular drugs outflow， thereby promoting tumor apoptosis， and the effect was superior to that of the <italic>P. americana</italic> crude extract CⅡ-3 and skim cream. Conclusion:<italic>P. americana</italic> polypeptide PAE₂ may reduce the drug efflux， promote intracellular drug accumulation and apoptosis by affecting the expression of related proteins and enzymes that mediate multidrug resistance， thereby exerting a reverse effect on HepG2/ADM cells Balb/c MDR in nude mice.

Advances in Research on Active Ingredients and Related Pharmacology of Periplaneta americana / 中国实验方剂学杂志

Periplaneta americana is one of the important basic medicinal materials of traditional Chinese medicine "fei lian". The traditional functions mainly include promoting blood circulation， sore muscles， diuresis， spleen and phlegm. Because of its exact curative effect， proprietary Chinese medicines， which are mainly used as raw materials， are widely used in clinical practice， especially in the repair of various wounds. The drug has not been included in the Chinese Pharmacopoeia. The local standard is only based on the alanine content of total amino acids. The physiologically active small peptides， nucleosides， proteins and other substances have not been obtained. Qualitative or quantitative control. In recent years， peptide monomers isolated from the P.americana， such as antimicrobial peptides， neuropeptides， and diuretic peptides， have strong pharmacological activities such as antibacterial， antitumor， and muscular neurotrophic， and dihydroisocoumarins are also irritating. Dermal Dermal fibroblasts produce collagen. Based on this， this paper uses CNKI， Wanfang Database and Pubmed Database to search the relevant research literatures of P.americana from 1984 to 2019， and systematically analyzes the current research of P.americana from three aspects： chemical composition-pharmacological action-clinical application. Interpretation provides reference for the further development of the drug and the development of more specific and stable quality control standards for its proprietary Chinese medicines.

Phenotypic Characterization and Antibiotic Susceptibility Profile of Coagulase Positive Staphylococci from The External Surfaces of Hospital Cockroaches (Periplaneta americana)

A study to phenotypically characterize and determine the antibiogram of coagulase positive Staphylococci (CoPS) from the external surfaces of hospital cockroaches (Periplaneta americana) was conducted using standard microbiological methods. Out of the 50 cockroaches collected from various hospitals in Uyo, sixty-two percent (n = 31) had coagulase positive Staphylococci which consisted of Staphylococcus aureus (44.0 %; n = 22) and Staphylococcus intermedius (18.0 %; n = 9). The CoPS isolates showed 100% resistance to Penicillin, Tetracycline, Clindamycin and 80.6% sensitivity to Amoxicillin-clavulanate. The CoPS showed multiple antibiotic resistances to ≥ 3 antibiotics, with 60 % exhibiting resistance to 6 antibiotics. Out of the 80 % (n = 31) of the multidrug resistant CoPS that were sensitive to Amoxicillin-clavulanate, none of them showed production of beta lactamase. The cockroaches bore multiple antibiotic resistant CoPS on their external surfaces and their contact can initiate contamination of patients' food. Pest control measures in hospital are hereby recommended to minimize cockroach related infections

Partial purification and characterization of antimicrobial peptide from the hemolymph of cockroach Periplaneta americana

The partial purification and characterization of antimicrobial peptide (AMP) from the hemolymph ofcockroach, Periplaneta americana, was studied. Hemolymph was drawn from cockroach and the AMP waspurified on a sephadex G-75 gel filtration column. The gel filtration showed two peaks, I and II, and only peakII showed five active fractions, namely, 12, 13, 14, 15, and 16, of antimicrobial activity. Fraction 13 showedthe highest microbial Inhibition concentration (MIC) and a single protein band on SDS-PAGE(sodium dodecylsulfate–polyacrylamide gel electrophoresis) with a molecular mass of 60.2 kDa. Purified antimicrobial proteinexhibited the highest antimicrobial activity against Escherichia coli at 30°C, pH 6.0, and 5 mM calcium ion.The AMP showed higher activity of MIC against lipopolysaccharides and β-1,3 glucan during 5 hours ofexposure. The study concludes that the AMP from hemolymph was effective against microbes or was able torecognize the molecular pattern of microorganisms

Partial purification and characterization of lectin from serum of American cockroach, Periplaneta americana

The partial purification and characterization of haemagglutinin (lectin) were carried out from the hemolymphof the adult American cockroach, Periplaneta americana. The hemolymph was drawn from cockroach andlectin was purified by a single-step method, using ammonium sulfate (NH4)2SO4 salt fractionation and gelfiltration. Gel filtration showed two peaks. The Hemagglutination Activity (HA) was observed in the 20thfraction of the second peak. The purified lectin showed a molecular weight of 26.8kDa on Sodium DodecylSulfate-Poly Acrylamide Gel Electrophoresis. The purified lectin showed an increase in HA at pH 7.5 and,subsequently, a sharp decline at pH 8. This indicates that HA was specific to a certain pH level. Similarly, anincrease in HA was observed until 30°C, followed by a decline at 40°C. This indicates the heat labile nature oflectin. The HA showed a higher specificity to divalent Ca2+ and showed no specificity for Ba2+. It also showeda higher inhibition for sugar D-galactose and a least inhibition for D-lactose. The HA to vertebrate blood groupshowed a highest activity to goat Red Blood Cells (RBCs). The study concludes that carbohydrate-bindingspecific lectin is important for recognition of the cell surface carbohydrate of invading pathogens

Optimization of processing methods for deodorizing Periplaneta americana based on subjective and objective smell evaluation combined with analysis of volatile components / 中草药

Objective: To screen a processing method suitable for deodorizing of Periplaneta americana (PA) through subjective and objective evaluation combined with changes in volatile components, and explore the mechanism of deodorization. Methods: Raw, vinegar-processed, wine-processed, wheat bran-processed products of PA were prepared respectively. Volunteer sensory evaluation combined with electronic nose system was used to evaluate the odor difference between raw and processed products to screen a better processed product. HS-SPME-GC-MS and ROAV were used to analyze the key odorous components of PA. The peak area normalization method combined with multivariate statistical analysis were used to analyze the difference of volatile components between raw and processed products to explore the mechanism of processing. Results|| Volunteer scores showed that the average scores of raw, vinegar-processed, wine-processed, wheat bran-processed products were 3.38, 1.25, 2.88, and 3.04, respectively. The results of the electronic nose showed that the Euclidean distance between the raw and vinegar-processed, wine-processed, wheat bran-processed products were 7.34, 3.77 and 1.60, respectively, but the direction of scatter of vinegar-processed product was opposite to that of wine-processed product and wheat bran-processed product, suggesting that the mechanism of deodorization may be different. Vinegar processing was determined as the optimum method for deodorizing by comprehensive analysis of subjective and objective evaluation data. The key odor components of PA were 3-methyl butanal, hexanal, nonyl aldehyde, heptaldehyde, decyl aldehyde, phenyl acetaldehyde, (E,E)-2,4-nonadienal, 2-pentylfuran, (+)-limonene, and myristic aldehyde. The PCA result of volatile components showed that four kinds of processed products can be clearly distinguished, and there were differences in volatile components and content. There were seven differential chemical components between raw and vinegar-processed products, including hexanal, palmitic acid, oleic acid, acetic acid, ethyl oleate, ethyl palmitate, and ethyl linoleate. The content of vinegar-processed products was lower than that of raw products, especially the key odorous component hexanal. Conclusion: The vinegar processing is a better method to improve the odor of PA, and its mechanism may be associated with reducing odorous components such as hexanal.

Discrimination of Periplaneta americana and other insectivorous herbs by FTIR spectroscopy combined with chemometrics / 中草药

Objective: To establish a method for the identification of the Periplaneta americana and other insectivorous herbs (Scorpio and Hirudo) based on infrared spectroscopy combined with chemometrics, and to provide a basis for the identification of the P. americana. Methods: Fourier transform infrared spectroscopy was used to collect the infrared spectrum data of three kinds of insect medicine powders. After the second order derivation of the obtained spectral data, the ordinate verticalization method and standardization method were used to optimize the spectrum. The spectral data was further analyzed by chemometrics, such as hierar chicalcluster analysis (HCA), principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). Results: There were differences in the infrared fingerprints of the P. americanas, Scorpios, Leeches. The absorption peaks of the P. americanas at 1 711, 1 410, and 712 cm-1 were obvious. The absorption peaks of the Scorpios at 1 753, 1 400, 1 168, and 717 cm-1 were obvious. The absorption peaks of the Leeches at 1 558, 1 457, 1 400, and 669 cm-1 were obvious, And the leeches have no absorption peak at 1 753-1 711 cm-1. The peak shape of the three insectivorous herbs was significantly different at 1 800-1 700 cm-1. In the second derivative spectrum, the positions of the main peaks are the same, but the intensity of the common peaks is different. Using the HCA analysis method, it was found that the three insectivorous herbs could be quickly distinguished. The PCA and PLS-DA analysis methods were used to find that the three insectivorous herbs were distributed in different regions. Conclusion: Infrared spectroscopy combined with chemometrics can easily and quickly identify the P. americanas and other insectivorous herbs (Scorpios and Leeches), and provide reference for the quality control and evaluation of P. americanas.

Study on the Mechanism of Periplaneta americana Extract Reversing Multi-drug Resistance of Human Hepato- cellular Carcinoma HepG 2/ADM Cells / 中国药房

OBJECTIVE：To study the mechanism of Periplaneta americana extract degreasing cream and CⅡ-3（shorted for “degreasing cream ”and“CⅡ-3”）reversing the multi-drug resistance of human HepG 2/ADM cells. METHODS ：MTT assay was used to investigate the toxicity effects of different concentrations of sorafenib （positive control ），degreasing cream and C Ⅱ-3 on HepG2/ADM cells ，then IC 20 was calculated. The experiment was divided into sensitivity drug ，drug-resistance group ，sorafenib group，degreasing cream group and C Ⅱ-3 group. HepG 2 cells were included in sensitivity group ，and HepG 2/ADM cells were included in the latter 4 groups. Sensitivity group and drug-resistance group were treated with routine medium ，and other 3 groups were treated with relevant medicine （IC20 as drug concentration ）. The content of ADM in HepG 2/ADM cells was determined by Laser scanning confocal microscopy. The expression of apoptosis-related protein as Bcl- 2 and Cleaved-Caspase- 9 p37 were detected by Western blotting assay. RT-qPCR and immunocytochemistry were adopted to detect mRNA and protein expressions that related to multidrug resistance [P-gp （expression produce of MDR1 gene），LRP，BCRP] and that related to enzyme-mediated multidrug resistance pathway （GST-π and Topo Ⅱ）. RESULTS ：The IC 20 of degreasing cream ，CⅡ-3 and sorafenib were （2.40±0.16）， （200.44±27.52），（18.00±1.82）μg/mL，respectively. Compared with sensitivity group ，the protein expressions of Bcl- 2，P-gp， LRP，BCRP and Topo Ⅱ，the mRNA expressions of MDR 1， LRP，BCRP and GST-π were increased significantly in drug resistance group （P＜0.05 or P＜0.01）. Compared with @qq.com drug-resistance group ，the mRNA and protein expression of MDR1 mRNA and LRP ，BCRP，GST-π were significantly decreased in degreasing cream group and C Ⅱ-3 group（P＜ 0.05 or P＜0.01）；the protein expression of Bcl- 2 and the mRNA expression of Topo Ⅱ were significantly decreased （P＜0.01）， while the protein expression level of Cleaved-Caspase- 9 p37 was significantly increased in C Ⅱ -3 group （P＜0.05）. CONCLUSIONS：Degreasing cream and C Ⅱ-3 can reverse multidrug resistance of HepG 2/ADM cells by reducing drug efflux ， promoting cell apoptosis ，reducing the mRNA and protein expression of multi-drug resistance gene as well as gene in enzyme-mediated multi-drug resistance pathway. The effect of C Ⅱ-3 is better than that of degreasing cream.

Study on the Improvement Effects of Periplaneta americana Extract Ento-A on Rats with Damp-heat Ulcerative Colitis / 中国药房

ABSTRACT OBJECTIVE：To study the improvement effect of Periplaneta americana extract Ento-A on damp-heat ulcerative colitis（UC）model rats. METHODS：Totally 70 rats were randomly divided into normal control group（n＝8）and modeling group （n＝62）. The damp-heat UC model was induced in modeling group by high sugar，high fat，spicy diet combined with 2，4， 6-trinitrobenzene sulfonic acid enema. 48 modelrats were randomly divided into model control group，mesalazine group （300 mg/kg），Changyanning group（300 mg/kg）and Ento-A low-dose，medium-dose and high-dose（50，100，200 mg/kg，calculated by the extract），with 8 rats in each group. Normal control group and model control group were given normal saline intrsgastrically，and other groups were given relevant medicine intragastrically，once a day，for consecutive 14 days. After last administration， disease activity index （DAI）， colonic mucosal injury index （CMDI） and histopathological score （HS）of rats were determined. The spleen index，liver index and colon index in rats were determined. The serum levels of IL-8，IL-17，SOD and MDA，colonic levels of IL-2，PGE2 and MPO were detected by ELISA. RESULTS：Compared with normal control group，the DAI score，CMDI score，HS score，colonic index，the serum levels of IL-8，IL-17 and MDA， colonic levels of MPO and PGE2 were increased significantly（P＜0.01）；serum level of SOD and colonic level of IL-2 were decreased significantly （P＜0.01）. Compared with model control group，DAI score，CMDI score，serum levels of IL-17 and MDA，colonic levels of PGE2 were decreased significantly in Ento-A high-dose groups（P＜0.05 or P＜0.01），while serum level of SOD and colonic level of IL-2 were increased significantly（P＜0.01）. CMDI score and HS score，serum levels of IL-8，IL-17 and MDA，colonic levels of PGE2 and MPO were decreased significantly in Ento-A medium-dose group（P＜0.05 or P＜0.01）， while colonic level of IL-2 was increased significantly（P＜0.01）. HS score，serum levels of IL-17 and MDA，colonic levels of MPO and PGE2 were decreased significantly in Ento-A low-dose group（P＜0.05 or P＜0.01），while serum level of IL-2 was increased significantly（P＜0.01）. CONCLUSIONS：P. americana extract Ento-A may play improvement effect on damp-heat UC rats by regulating immune system balance and reducing inflammatory damage.

Effects of Periplaneta americana Extract YS-F on the Proliferation and Apoptosis of Human Non-small Cell Lung Cancer A 549 Cells / 中国药房

OBJECTIVE：To investigate the effects of Periplaneta americana extract on the proliferation and apoptosis of human non-small cell lung cancer A 549 cells as well as its possible mechanism. METHODS ：The dry bodies of P. americana were soaked with 90％ ethanol and eluted with gradient water-methanol by polyamide column chromatography. The 20％，30％，40％， 50％，60％，70％，80％，90％ methanol elution sites （YS-A-H）were obtained. MTT method was used to screen the active site ， and the inhibition rate of different doses of active site was detected. Flow cytometry was adopted to detect cell apoptosis ，cell cycle and mitochondrial membrane potential of cells after treated with different doses of active site. RESULTS ：Half inhibition concentrations of YS-A-H were （95.25±8.42），（129.93±7.24），（221.28±12.68），（275.39±14.87），（276.76±16.32），（31.90± 5.34），（163.15±6.97），（122.81±8.36）μg/mL，respectively. YS-F had the strongest activity. After treated with 3，9，27，81 μg/mL YS-F for 24，48，72 h，cell proliferation inhibitory rate was increased significantly at different time points ；after treated for 48，72 h，that was significantly higher than same group after treated for 24 h；after 72 h treatment ，that was significantly higher than same group after 48 h treatment （P＜0.01）. There was no significant effect of 24 h treatment of 3 μg/mL YS-F and 72 h treatment of 9 μg/mL YS-F on the percentage of cells in the late stage of necrosis，24 h treatment of 3 μg/mL YS-F on the percentage of cells in G2/M phase and 48 h treatment of 3 μg/mL YS-F on the reduction rate of mitochondrial membrane potential（P＞0.05）. The percentage of cells in the early stage of apoptosis ，the late stage of apoptosis and the early stage of necrosis ，the late stage of necrosis，as well as the percentage of cells in the Sub-G 0/G1 and S phase at each time point were significantly increased in other different doses groups ，while the percentage of cells in G 0/G1 and G 2/M phase was decreased significantly （P＜0.01）. In each dose group，the percentage of cells in the early stage of apoptosis ，the late stage of apoptosis and the early stage of necrosis ，the late stage of necrosis （except for the percentage of cells in the late stage of necrosis treated with YS-F 9 μg/mL for 72 h）and the percentage of cells in Sub-G 0/G1 phase，G2/M phase （except for YS-F 27，81 μg/mL for 48 h）after treated for 48，72 h were significantly higher than same group after 24 h of treatment ；the percentage of cells in G 0/G1 phase，S phase and G 2/M phase （except for YS-F 9 μg/mL for 48 h）after treated for 48，72 h were significantly lower than same group after 24 h of treatment （P＜0.01）；the percentage of cells in the early stage of apoptosis ，the late stage of apoptosis and the early stage of necrosis ，the late stage of necrosis （except for the percentage of cells in the late stage of apoptosis and early stage of necrosis when treated with YS-F 27 μg/mL for 72 h，the percentage of cells in the late stage of necrosis when treated with YS-F 3，9 μg/mL for 72 h were decreased significantly ）and the percentage of cells in S phase （except for YS-F 3 μg/mL for 72 h）and Sub-G 0/G1 phase after treated for 72 h were significantly higher than same group after 48 h of treatment ，while the percentage of cells in G 0/G1 and G 2/M phase were significantly lower than same group after 48 h of treatment （P＜0.01）. After treated with YS-F 9，27，81 μg/mL for 48 h，the reduction rate of cell mitochondrial membrane potential was increased significantly ；YS-F 27，81 μg/mL groups were significantly higher than YS-F 9 μg/mL group，and YS-F 81 μg/mL group was significantly higher than YS-F 27 μg/mL group. CONCLUSIONS：YS-F can inhibit the proliferation and promote the apoptosis of A 549 cells by preventing cell transformation from S phase to G 2/M phase ，and reducing mitochondrial membrane potential ，in time-dependent or dose-dependent manner.

Preparation of Periplaneta americana Thermosensitive Hydrogel and Investigation of Its Wound Healing Effect on Diabetic Rats / 中国实验方剂学杂志

Objective:To prepare Periplaneta americana thermosensitive hydrogel and investigate its effect on wound healing in diabetic rats. Method:Taking N-isopropylacrylamide (NIPAM) and acrylic acid (AAc) as monomers, thermosensitive poly(NIPAM-co-AAc) [P(NIPAM-co-AAc)] polymeric material was prepared by free radical polymerization, then thermoresponsive copolymer P(NIPAM-co-AAc)-g-HA was synthesized by conjugating P(NIPAM-co-AAc) to hyaluronic acid (HA). The structure and lower critical solution temperature (LCST) of the graft copolymer were characterized by proton nuclear magnetic resonance spectroscopy (1H-NMR) and ultraviolet spectrophotometry (UV). P. americana thermosensitive hydrogel was prepared by dialysis method, and it was characterized by scanning electron microscope (SEM), rotation rheometer and thermogravimetric analyzer to observe section structure, rheological properties and thermal stability. Differential scanning calorimetry, X-ray diffraction and Fourier transform infrared spectroscopy were employed to identify the inclusion of P(NIPAM-co-AAc)-g-HA temperature sensitive material for P. americana extract, and to investigate the effect of P. americana thermosensitive hydrogel on wound healing in diabetic rats, and the rate of wound healing was calculated by Image-Pro Plus 6.0 software. Hematoxylin-eosin (HE) and Masson staining were used to observe the pathological changes of the wounds of rats in each group. Result:P(NIPAM-co-AAc)-g-HA temperature sensitive material was successfully synthesized, its LCST was between 29 ℃ and 31 ℃, it had a dense and uniform porous structure and could uniformly include P. americana extract. Pharmacodynamic studies showed that P. americana thermosensitive hydrogel group had the best effect on promoting wound healing, its infiltration degree of inflammatory cells was significantly reduced, collagen and fibroblasts arranged neatly and compactly, and the density of neovascularization was significantly increased by comparing with the model group. Conclusion:P. americana thermosensitive hydrogel can effectively promote wound healing of diabetic rats and overcome the shortage of marketed P. americana liquid preparations, this paper can provide a reference for the development of P. americana extract preparations to promote wound healing in diabetic patients.

Study on Antitumor Mechanism of Periplaneta americana Extract C Ⅱ-3 on MFC Tumor-bearing Mice Based on 1H-NMR Metabonomics / 中国药房

OBJECTIVE：To preliminarily study the antitumor mechanism of Periplaneta americana extract C Ⅱ-3 on MFC tumor-bearing mice. METHODS ：Balb/c mice were randomly divided into model group （normal saline 20 mL/kg）and C Ⅱ-3 group （200 mg/kg），with 6 mice in each group. MFC cell suspension （0.2 mL）was injected under the right armpit of mice. On the next day，mice were given relevant medicine intragastrically ，once a day ，for consecutive 10 d. 24 h after the last administration ，Based on the measurement of tumor size ， 1H-NMR technology combined with unsupervised PCA ，supervised PLS-DA and OPLS-DA were used to compare metabolic spectrum of liver tissue from tumor-bearing mice of 2 groups，to analyze differential metabolites and to explore the potential antitumor mechanis m of C Ⅱ -3. RESULTS ：Compared with model group ，the tumor body was significantly reduced in tumor-bearing mice of C Ⅱ-3 group. There were differences in 1H-NMR spectra between the 2 No.81960712）； groups. According to unsupervised PCA ，supervised PLS-DA and OPLS-DA ，totally six potential differential metabolites ，as glycogen （increased），pyruvate （decreased），arginine （de- creased），hydroxyproline （increased），inosine （increased） and niacinamide （increased），were identified in the liver tissue，which were mainly attributed to the metabolism of arginine ，energy and nucleic acid. CONCLUSIONS：The anti tumor effect of C Ⅱ-3 may be related to the regulation of arginine metabolism ，energy metabolism and nucleic acid metabolism.

Effect of Periplaneta Aamericana Polypeptide on Angiogenesis / 中国实验方剂学杂志

Objective: To investigate the effect of the Periplaneta Americana polypeptide on the angiogenesis. Method:Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and cell scratch assay were used to observe effect of different concentration (6.25,12.5,25,50,100 mg·L-1) of the Periplaneta Americana polypeptide, CⅡ-3 and skimmed cream on the proliferation and migration of human umbilical vein endothelial cells (HUVECs), and a normal group and a thalidomide group were also established in this study. The tubule formation assay was used to detect the effect of different concentration (25, 50, 100 mg·L-1) of the Periplaneta Americana extracts on the formation of tubules in HUVECs cells. The adhesion between HepG2 cells and HUVECs cells was observed by cell adhesion assay. The expression of vascular endothelial growth factor (VEGF) proteins in HUVECs was detected by immunocytochemical staining and enzyme linked immunosorbent assay (ELISA). Result:MTT results showed that the Periplaneta Americana polypeptide could inhibit the proliferation of HUVECs in a dose-dependent manner (PPPPPPPPPConclusion:The Periplaneta Americana polypeptide can inhibit the invasion, metastasis and tube formation of HUVECs, and down-regulate the expression of VEGF in HUVECs. The effect of Periplaneta Americana polypeptide is better than CⅡ-3 and skimmed cream, and the among the polypeptide, the effect of PAP-2 is superior to the other two.

Analysis on Odorous Components in Different Processed Products of Periplaneta americana by HS-SPME-GC-MS / 中国实验方剂学杂志

Objective:To analyze the odorous components and their contents in raw products, wine-processed products, vinegar-processed products and wheat bran-processed products of Periplaneta americana. Method:Headspace solid-phase microextraction (HS-SPME) was used to extract the volatile components from different processed products, the chemical compositions were analyzed by gas chromatography-mass spectrometry (GC-MS), and the relative contents of each component was calculated by peak area normalization method. Result:A total of 41, 32, 40 and 47 components were respectively identified from raw, wine-processed, vinegar-processed and wheat bran-processed products of P. americana, involving a total of 13 common components. Conclusion:The odorous components in the raw products are mainly derived from aldehydes, alcohols, amines, hydrocarbons and other volatile substances. Odorous components can be reduced effectively and flavoring substances can be increased by wine, vinegar and wheat bran processing. This study provides a scientific basis for the further study of correcting odor of P. americana, it also provides a reference for analysis and correction of odor of animal medicines.

Optimization of polysaccharide preparation and comprehensive utilization of wound healing in residue of Periplaneta americana / 中草药

Objective :To optimize the extraction of polysaccharide from the residue of Periplaneta americana by response surface method and study the effect of P. americana polysaccharide (PAP) on wound healing, so as to provide ideas for the comprehensive utilization of P. americana residues. Methods On the basis of single factor experiment, Box-Behnken response surface method was used to design the best extraction process. And the monosaccharide composition of polysaccharide was analyzed. Polysaccharide concentrations of 0.1, 0.05, and 0.025 g/mL were applied to the wound injury model mice. The wound healing rate was calculated, and hematoxylin-eosin (HE) and Sirius Red staining were used to evaluate the wound healing degree. Results :The optimum extraction technology of PAP were as follows: The concentration of NaOH was 0.02 mol/L; And the extraction time was 1.65 h; The extraction temperature was 62 ℃. The transfer rate of polysaccharide extracted by this process was 13.68%. The polysaccharide of the P. americana consisted of seven monosaccharides of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and fucose. Wound healing experiment showed that wound healing rate of the polysaccharide group on day 3, 6, 9, and 14 was higher than that of the model group, and HE and Sirius Red staining showed that the polysaccharide could promote the formation of collagen and granulation tissue, thus promoting the wound healing. Conclusion: The optimized extraction process of PAP from P. americana residues by response surface method was reliable, with high extraction rate. And PAP could promote wound healing in mice, suggesting that the polysaccharide from P. americana residues could be used as wound related products.

Proteolytic Enzymes Screening for Enzymolysis Preparation Technology of Active Parts of Periplaneta americana against Liver Fibrosis / 中国药房

OBJECTIVE： To optimize the proteolytic enzymes for enzymolysis technology of degreasing ointment from Periplaneta americana， and to improve the extraction rate and activity of anti-liver fibrosis active part from P. americana. METHODS： Using degreasing ointment of P. americana as control， ninhydrin method and folin-ciocalteu method were used to investigate the hydrolysis degree of trypsin （TR）， pepsin （PE）， alkaline protease （AL）， papain （PA） and neutral protease （NE） to the degreasing ointment. Macroporous resin isolation and purification method was used to investigate the yield of elution part from hydrolyzate， with 50％， 60％， 70％， 95％ ethanol as eluting solvents. Inhibition test in vitro of rat hepatic stellate cells HSC-T6 was performed， and anti-liver fibrosis activity of elution part from hydrolyzate was investigated. RESULTS： The hydrolysis degree of PA and NE were 14.15％ and 15.70％， showing strong enzymatic hydrolysis ability. The yield of 95％ ethanol elution part from PA， NE and AL hydrolyzate were （0.73±0.04）％，（0.65±0.01）％ and（0.64±0.05）％， improving 30.36％， 16.07％， 14.29％ compared with degreasing ointment without enzyme. Results of inhibition test in vitro showed that inhibitory rate of 50％， 60％， 70％ ethanol elution parts isolated and  purified from hydrolyzate had a low inhibition rate or a growth-promoting effect on HSC-T6 cells. Inhibition rates of 95％ ethanol elution parts to HSC-T6 cells were all more than 20％. IC50 of 95％ ethanol elution part isolated and purified from PA and NE hydrolyzate for 24-72 h were 94.5-112.3 and 117.1-120.0 μg/mL， which were lower than that （116.1-123.0 μg/mL） of degreasing ointment without enzyme. CONCLUSIONS： PA is the best hydrolyzate for enzymolysis technology of active parts against liver fibrosis in degreasing ointment from P. americana， followed by NE and AL； PE and TR， which have poor effect， are not suitable for the enzymatic hydrolysis technology.

Study on Anti-tumor Effects of Periplaneta americana Polypeptide PAP- 2 on H 22 Tumor-bearing Mice / 中国药房

OBJECTIVE： To investigate anti-tumor effects of Periplaneta americana polypeptide PAP-2 on H22 tumor-bearing mice. METHODS： The mice tumor-bearing model was established by subcutaneous injection of ascites of H22 hepatocellular carcinoma mice via axilla. 70 mice were randomly divided into model group （normal saline）， 5-FU group （positive drug control， 20 mg/kg）， P. americana extract skimmed cream group （200 mg/kg， calculated by extract）， CⅡ-3 group （polypeptide isolated from skimmed cream as main active ingredient， 200 mg/kg， calculated by extract） and polypeptide PAP-2 high-dose， medium-dose and low-dose groups （isolated from CⅡ-3， 200， 100， 50 mg/kg， calculated by monomer）， with 10 mice in each group. The mice in the 5-FU group were given intraperitoneal injection once every other day， while the mice in the other groups were given intragastric administration once a day， the administration cycle was 10 d. After medication， the changes of tumor were observed and the organs （spleen， thymus and liver） index were measured. Histopathological changes of tumor tissue were observed after HE staining. The contents of VEGF， IL-1β and IL-4 in serum were determined by ELISA. RESULTS： Skimmed cream， CⅡ-3 and different doses of PAP-2 could inhibit the growth of tumor in tumor-bearing mice to different extent and increase organ index， and PAP-2 showed a dose-effect relationship. The tumor inhibition rate （38.95％） of PAP-2 high dose group was significantly higher than those of skimmed cream group and CⅡ-3 group （P＜0.05）， which was close to that （40.87％） of 5-FU group （P＞0.05）. Spleen index， thymus index and liver index of mice in PAP-2 high dose group were significantly those of model group and CⅡ-3 group （P＜0.05）； and the liver index of mice in PAP-2 high dose group was significantly higher than that of skimmed cream group （P＜0.05）. In addition， PAP-2 could decrease the serum contents of VEGF and IL-4， and increased serum content of IL-1β， with high dose group showed significant difference compared with model group （P＜0.05）； the serum content of IL-1β of mice in PAP-2 high dose group was significantly higher the that of skimmed cream group and CⅡ-3 group （P＜0.05）， serum contnet of IL-4 in PAP-2 high dose group was significantly lower the  that of skimmed cream group and CⅡ-3 group （P＜0.05）， but the serum content in which was significantly lower than that of skimmed cream group and CⅡ-3 group（P＜0.05）. CONCLU- SIONS： P. americana polypeptide PAP-2 it has a certern anti-tumor effects on H22 tumor-bearing mice， and its can increase the index of organs of H22 tumor-bearing mice， decrease the contents of VEGF and IL-4 in serum， increase the content of IL-1β in serum.