[Effect of animal medicines for "extinguishing wind to arrest convulsions" on central nervous system diseases].

The human health is seriously affected by central nervous system(CNS) diseases, but the pathogenesis of CNS diseases is still not completely clear. Currently, the drugs used to treat CNS diseases are mainly receptor modulators and neurotransmitter inhibitors, which have serious side effects; and there are short of drugs for treating CNS diseases clinically. Studies suggest that animal medicines mainly include protein, polypeptide and small-molecule compounds, and have such pharmacological effects in calming, resisting convulsions and improving brain tissues. Plenty of studies suggest that animal medicines usually have a strong activity and good curative effect on these diseases, with a promising prospect in research and development of drugs treating CNS diseases. Based on systematic reviews of literatures, this paper summarizes active ingredients and main pharmacological effects of animal medicines in "extinguishing wind to arrest convulsions" for the CNS diseases, epilepsy and cerebral ischemia, and discusses their study value and application prospects. The results showed that the studies of protein and peptides were relatively simple, and some animal medicines were still blank. The authors believed that amino acids and small molecular compounds should be transferred to oligopeptide, advanced protein extraction and separation techniques shall be adopted for identifying the protein polypeptide composition structure and studying the efficacy, and the methods of biological technology were used to develop peptide biological products for the treatment of CNS diseases. This paper could provide ideas and reference for developing animal medicine products for the treatment of CNS diseases.

Characterization and optimization of lyophilization and storage conditions of Leech saliva extract from the tropical leech Hirudinaria manillensis.

The medicinal Malaysian leeches have been used in traditional medicine to treat many different ailments. In this study, leech saliva extract (LSE) was collected from the medicinal Malaysian leech Hirudinaria manillensis. Gel electrophoresis of LSE was carried out to estimate the peptide and protein molecular weights of its content. Results showed that LSE contains more than 60 peptides and proteins with molecular masses ranging from 1.9-250kDa. Thrombin time assay in vitro was employed to assess the collected LSE antithrombin activity. First, to study its stability, LSE was lyophilized under the following different conditions: pre-freezing temperature, type of container and lyophilization cycle. Pre-freezed LSE sample at -20°C and lyophilized for 24 hours retained about 100-95% of its original biological activities. Second, the LSE antithrombin activity was monitored for a period of six months. Storage temperature, type of the container and photosensitivity effects on antithrombin activity of the lyophilized (solid state) and non-lyophilized (liquid state) were investigated. Results showed that storage temperature drastically affected the biological activity of LSE with -20 °C as the optimum temperature. Samples stored at ambient temperature and +4 °C were light photosensitive and adversely affected when stored in polypropylene tubes. Lyophilized samples were more stable than non-lyophilized ones over the period of study. To sum up, in order to have a biologically active stock of LSE, it has to be lyophilized for no more than 24 hours following freezing at -20°C and has to be stored at -20°C in glass tubes protected from light.

[Apoptotic mechanism of gecko crude peptides on human liver carcinoma cell line HepG2].

OBJECTIVE: To investigate the effect of Gecko crude peptides (GCPS) on human liver carcinoma HepG2 cells and its mechanism. METHODS: MTT assay was used to analyze the effect of the GCPS on the proliferation of HepG2 Cell; Nucleus change of HepG2 treated with GCP was observed by Hoechst33258 fluorescence staining, and BAX and BCL-2 were detected with western-blot assay. RESULTS: GCPS could inhibit the proliferation of HepG2 Cell in a time and dosage dependent way, and its half-maximal inhibitory concentration (IC50) was 1.2 mg/mL; HepG2 pretreated with GCPS showed apoptotic morphological changes. GCPS (1.6 mg/mL, 0.8 mg/mL) could decrease the expression of BCL-2 protein, and increase the expression of BAX protein. CONCLUSION: GCPS can inhibit the proliferation of HepG2 cell. The mechanism may be related to the induction apoptosis of HepG2.

[Research on the protein C-activating property and influencial factors of protein C activator isolated from the venom of Agkistrodon halys].

OBJECTIVE: To investigate the protein C-activating property and experimental conditions that may affect the activities of protein C activator (PCA-SV). METHODS: PCA-SV's functional characteristic, possible anticoagulant and amidase activities were measured by method of APTT and that of specific chromogenic substrate respectively in various experimental conditions. RESULTS: PCA-SV can specifically act on protein C. The pH-optimum and temperature-optimum for the action of PCA-SV were 6.0 - 7.0 and 37 - 40 degrees C respectively. The anticoagulants (heparin, sodium citrate) had no effect on the activities. CONCLUSION: PCA-SV is a new protein C-activating reagent that has high potency and specificity.

Neuronal peptide (enkephalin) receptors in the ear artery of the rabbit.

Methionine-enkephalin methylester (MEM) and leucine-enkephalin (LE) inhabit the vasoconstrictor responses of the rabbit ear artery to nerve stimulation by acting on a specific neuronal peptide (enkephalin)-receptor insensitive to opiate agonists. The tetrapeptide: H-Tyr-Gly-Phe-Leu-OCH3 is ineffective. This is the first instance of enkephalins acting in an organ devoid of receptors. In a new test for the analysis of opiate receptors, MEM (ID50=6.9 X 10(-9) M) was a potent inhibitor of transmission. The presence was shown of opiate receptors in the brain which were insensitive to high i.v. or intraventricular doses of enkephalins. It is concluded that enkephalins are not natural ligands to the opiate receptors, but that some of the receptors confuse these structures because of similar characteristics which determine the binding of both opiates and peptides.

Human isolated small intestine: motor responses of the longitudinal muscle to field stimulation and exogenous neuropeptides.

(1) Longitudinal muscle strips from the human small intestine (jejunum/ileum) responded to electrical field stimulation (1-50 Hz) with frequency-related primary contractions which were largely atropine- (3 microM) sensitive. When the tone was raised by addition of galanin (0.3-1 microM), prostaglandin (PG) E2 (1-10 microM) or neurokinin A (NKA, 0.1 microM), a frequency-related relaxation was evident which was potentiated by atropine. All the responses to field stimulation were abolished by tetrodotoxin (1 microM), thus indicating their neural origin. (2) The atropine-sensitive primary contraction to field stimulation was virtually abolished by omega conotoxin fraction GVIA (CTX, 0.1-0.3 microM) while the relaxations were CTX-resistant. The field stimulation-induced relaxations, which were observed in the presence of atropine and guanethidine (3 microM), were also unaffected by apamin (0.1 microM). (3) NKA and substance P (SP) produced a concentration- (1 nM-1 microM for both peptides) related contraction, NKA being about 53 times more potent than SP. [Pro9]SP sulphone and [MePhe7]-NKB, selective agonists of the NK-1 and NK-3 receptor, respectively, were barely effective. On the other hand, [beta Ala8]NKA(4-10), a selective NK-2 receptor agonist, had a potent contractile activity, similar to that of NKA. (4) Galanin (1 nM-1 microM) produced an atropine- and tetrodotoxin-resistant concentration-related contraction of longitudinal muscle of human isolated small intestine. The response to galanin did not show any sign of fading and was particularly suitable to study the evoked relaxations.(ABSTRACT TRUNCATED AT 250 WORDS)

[Recovery effect of cardiomyopeptidin fractions and fraction addition on cardiac muscle cells in rats damaged by adriamycin].

OBJECTIVE: To investigate the recovery effect of cardiomyopeptidin fractions and fraction addition on the cardiac muscle cells in rats damaged by adriamycin. METHODS: Observing the activity of the succinic dehydrogenase which is at mitochondrion in the cells damaged by adriamycin with MTT. RESULTS: Five fractions have all promoted the activating effect of the enzyme, the action of PI being higher than the others. Fraction addition has also promoted the activating effect of the enzyme, but without additive effect. CONCLUSION: The recovery effect of cardimyopeptidin depends on the interplay among the fractions.

[Purification of small peptide from Agkistrodon blomhoffii Bioe and its inhibition on platelet aggregation].

This is the first report to purify a new peptide SV-PP-1 from Agkistrodon blomhoffii Bioe. SV-PP-1 has molecular weight of 1234.616 Da and can inhibit on platelet aggregation. SV-PP-1 significantly inhibited the ADP-induced platelet aggregation. It has been found up to now that SV-PP-1 is the smallest peptide inhibiting on platelet aggregation from snake venom.

[Purification and characteristics of polypeptide with inhibition of platelet aggregation from Agkistrodon blomhoffii Bioe].

A polypeptide, SV-PP-2, was purified from Agkistrodon blomhoffii Bioe by HPLC C18 column. The SV-PP-2 has molecular weight of 23339.00 Da, and can inhibit the ADP-induced platelet aggregation in dose-dependent manner. There have been no such polypeptides reported yet which has inhibition of platelet aggregation and similar molecular weight to SV-PP-2.

[A comparison of chemical composition and bioactivity of polypeptides from velvet antlers of Cervus nippon Temminck and Cervus elaphus Linnaeus].

OBJECTIVE: To compare the chemical composition and bioactivity of polypeptides(PPs) isolated from velvet antlers of sika deer (Cervus nippon Temminck) and red deer (Cervus elaphus Linnaeus). METHOD: The two kind of polypeptides were isolated from the above mentioned velvet antlers with same technology. The chemical composition was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF mass spectrometry. Stimulant activity of cells proliferation was measured by [3H] TdR incorporation into DNA. RESULT: The graphs of SDS-PAGE and MALDI-TOF MS of velvet antler polypeptides (VAPPs) from Chinese and New zealand red deer were very similar, but there were obvious difference in respect of graph between sika deer and red deer. VAPPs 25-50 mg.L-1 showed marked proliferation-promoting activity for rabbit costed chondrocytes, either sika deer or red deer. However, the activity of sika deer VAPPs 12.5 mg.L-1 for epidermal cells was weaker than that of red deer (12.5 mg.L-1). CONCLUSION: The chemical property and bioactivity of VAPPs from sika deer and red deer are significantly different.

Polypeptide from Chlamys farreri attenuates murine thymocytes damage induced by ultraviolet B.

AIM: Polypeptide from Chlamys farreri (PCF, molecular mass is 879) is a new marine polypeptide compound isolated from Chlamys farreri. This study investigates the possible protective roles and the mechanism of PCF against ultraviolet B (UVB)-induced apoptosis in murine thymocytes. METHODS: The rate of apoptosis and caspase-3 activation was measured by flow cytometry. The expression of stress-response genes c-fos and c-jun was observed by RT-PCR. Western blot analysis was performed to determine the release of cytochrome c. RESULTS: It was found that UVB induced murine thymocyte death. The cells treated with UVB showed an increase in cytochrome c release, caspase-3 activity, as well as in the expression of c-fos and c-jun. In addition, all were involved in UVB-induced cell apoptosis. CONCLUSION: Our present observations pointed to the ability of PCF to avert UVB-induced apoptosis in thymocytes by modulating c-fos and c-jun expression, cytochrome c release, and the consequent activation of caspase-3, which were essential components of the UV-induced cell apoptotic pathway. The results suggested that PCF is a promising protective substance against UV radiation.

[Anti-inflammatory effects of pilose antler peptide].

Pilose antler peptide (PAP:MW:7200; amino acid residue:68) isolated from the antlers of Cervus nippon temminck 10 and 20 mg.kg-1 i.p. produced inhibitions towards acute and chronic inflammations in a dose-dependent manner. PAP reduced ascorbic acid and cholesterol contents in adrenal glands and decreased the serum hydrocortisone level of rats. The reduction of ascorbic acid and cholesterol contents were unaffected by the pretreatment of dexamethasone. PAP also showed an anti-inflammatory action on the swelling of carrageenan-induced hind paw in adrenalectomized rats. The results suggest that the anti-inflammatory effects of PAP do not depend absolutely on pituitary-adrenal system.

Protective effects of polypeptide from Chlamys farreri on Hela cells damaged by ultraviolet A.

AIM: To study the protective effect of polypeptide isolated from Chlamys farreri (PCF) on Hela cells damaged by ultraviolet A (UVA) in vitro. METHODS: Cell proliferation was determined by MTT method; intra-cellular free calcium [Ca2+]i and rates of apoptosis and death were measured by flow cytometry (FCM). RESULTS: PCF (0.5 %-2 %) enhanced the activities of glutathione peroxidase (GSH-px), superoxide dismutase (SOD), and catalase (CAT), and stimulated cell proliferation. The concentration of [Ca2+]i was increased while the amounts of MDA and the rates of apoptosis and death of the cells were decreased. The differences between the PCF groups and control group were significant (P<0.05, P<0.01). CONCLUSION: PCF protected Hela cells against damage by UVA via its anti-oxidative mechanisms.

Velvet antler polypeptides promoted proliferation of chondrocytes and osteoblast precursors and fracture healing.

AIM: To study the effects of velvet antler (VA) total polypeptides (VATP) and VA polypeptides, VAP-A, VAP-B, and VAP-C on proliferation of chondrocytes and osteoblast precusors. METHODS: Chondrocytes (rabbit and human fetus) and osteoblast precusors (chick embryo) were incubated in the culture medium containing VATP or VAP-A, VAP-B, and VAP-C. [3H]TdR incorporation into DNA was measured. Fracture healing-promoting action of VATP was determined in rats. RESULTS: VATP 50-200 mg.L-1 and VAP-B 12.5, 25, and 50 mg.L-1 showed most marked proliferation-promoting activity for rabbit costed chondrocytes and increased incorporation of [3H]TdR from (73 +/- 9) Bq (control group) to (272 +/- 55), (327 +/- 38), and (415 +/- 32) Bq, respectively (P < 0.01). The activity of VAP-A was weaker than that of VAP-B, and VAP-C had no activity. VATP 10 and 20 mg.kg-1 by local injection into the cross-section fracture area accelerated healing of radial fracture. The healing rate of VATP-treated group was higher (75%) than that of control group (25%) (P < 0.05). CONCLUSION: VATP accelerated fracture healing by stimulating proliferation of chondrocytes and osteoblast precursors.

Protective effect of polypeptide from Chlamys farreri on mitochondria in human dermal fibroblasts irradiated by ultraviolet B.

AIM: To study the effect of polypeptide from Chlamys farreri (PCF) on mitochondria of human dermal fibroblasts irradiated by ultraviolet B (UVB) in vitro. METHODS: Malondialdehyde (MDA) and antioxidant enzymes including superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were determined by biochemical methods. Mitochondrial transmembrane potential was measured by flow cytometry. Ultrastructure of fibroblasts was observed with transmission electron microscope. RESULTS: UVB (1.176 x 10(-4) J/cm(2)) induced mitochondria damage in dermal fibroblast and PCF (0.25%-1%) reduced the damage in a concentration-dependent manner. Furthermore, PCF also concentration-dependently maintained the stability of mitochondrial transmembrane potential. PCF was able to reduce the MDA formation caused by UVB, meanwhile increased the activities of SOD and GSH-PX. The differences among the PCF groups and UVB model group were significant (P<0.05, P<0.01). CONCLUSION: The UVB-induced mitochondria damage was alleviated by PCF in human dermal fibroblasts.

Inhibitory effect of polypeptides from Chlamys farreri on UVB-induced apoptosis and DNA damage in normal human dermal fibroblasts in vitro.

AIM: To investigate the effect of polypeptide from Chlamys farreri (PCF) on ultraviolet B (UVB)-induced apoptosis and DNA damage in cultured normal human dermal fibroblasts. METHODS: MTT assay was used to measure the viability of cells. Measurements of apoptosis and cytosolic free [Ca2+]i were performed with flow cytometry. The comet assay was employed to detect DNA damage in individual cell. RESULTS: PCF (0.25 %-1%) greatly enhanced the proliferative capacity of cultured fibroblasts irradiated by UVB (1.176 x 10(-4) J/cm2) and markedly reduced apoptosis and the level of DNA damage in a concentration-dependent manner. Meanwhile, PCF could decrease the cytosolic free [Ca2+]i (P<0.01, compared with UVB model). CONCLUSION: The inhibitory effect of PCF on UVB-induced photoaging is due to enhanced abating of UVB-injured DNA and UVB-induced apoptosis. Therefore, PCF can resist UV-induced aging development at the initiation stage.