Purification And Pharmacological Activity Of CTX1 From Naja Naja Cantor Venom

Background:Cytotoxin (Cadiotoxins CTXs) is an important cobra venom of a class activity of proteins from Naja atra Cantor venom, content of about 25% to 40% of total protein. CTXs, a basic polypeptide with stationary acid residues isolated fromNaja naja atra venom, has been reported to have cytotoxic activity. There can cause tissue(skeletal muscle, cardiac muscle and nerve tissue) exciting such as the polarization, dissolve tumor cells, cardiac toxicity and antibacterial activity. Different regions and different species of the snake venom contain different CTXs, and also the same snake venom contains 2 to 5 CTXs, their structure and biological activity of some differences. The structures and functions of biological toxins in today is one of the hot spots. The objective our researchs are to optimize the separation method from original Naja venom to improve the separation efficiency and purity; CTXs have a wide range of biological activities, such as analgesia, inhibition of tumor value, etc. In this study, we research the analgesic activity of CTX1 with hot plate and acetic acid writhing test. 125I isotope labeling to CTX1 was observed using the physiological conditions and under the conditions of morphine addiction, CTX1 distribution in mice, comparison of the two cases, their distribution within the brain tissue to study in different pathological conditions, CTX1 through the blood Brain barrier permeability. To further the study that the pharmacological effects of CTX1 basis.ObjectiveThe use of CM-Sepharose FF cation exchange gel column chromatography one step of for the purification of Naja atra venom. By RP-HPLC, internal standard method can identification the isolated monomer. We evaluae the acute toxicity and analgesic effects of Naja venom cytotoxin CTX1. In the modle of physiological conditions and the conditions of morphine addiction,we observed the CTX1 distribution in mice, compared to the two cases that CTX1 distribution within the brain tissue useing 125I isotope labeling.Methods:1. The chromatography flow rate optimization of Naja venom CTX1b. Naja atra snake venom separation and purification on the one-step. The original venom samples collected from Naja arta, useing of CM-Sepharose FF cation exchange gel column chromatography, adjusted for different flow rates on the separation and purification of snake venom components.c. Desalting with Sephadex G-25 Using molecular principle of sieve chromatography elution, the elution peak contains some of the venom protein and salt solution elution. Adjust the flow rate can improve the desalination efficiency of different outcomes.d. Purification of Naja venom Identification of single components with RP-HPLC, internal standard method for testing Naja atra venom purified monomer, by comparing the retention time, determine the composition of isolated monomers.2. Determinate the toxicity and analgesic activity of the CTX1a. Acute toxicology LD50 toxicity was detected with the method of Bliss. Determined by pretest from 0% to 100% lethal dose lethal dose. Starting from the high concentration, drug concentration 0.8 times followed by dilution, a total of five concentration gradient. Observed after administration of 48h, record the time of death and symptoms of poisoning.b. Acute pain experiment Using hot plate test and acetic acid writhing test measured analgesia activity of CTX1 physiological pain .3. The distribution of CTX1 in vivo mouse and the different ability of the permeability of BBB in two cases model mice.a. The establishment of morphine in mice With gradually increasing doses of morphine in mice model Observe the withdrawal symptoms and naloxone changes in body weight of mice to evaluate the success of addiction model.b. 125I isotope labeling CTX1 With ketamine oxidation, using Sephadex G50, the labeled and unlabeled CTX1 will be separated; with paper chromatography test the labeling efficiency of 125I.c. Vein injection of 125I-CTX1 though mice tail, and get the organ at different time, testing the cpm value of the organ. Compare the distribution in the mice of CTX1, through the comparison of different models to study the blood-brain barrier permeability.4. Statistical analysis By t test,χ2 test, SPSS statistical to analysis the data of experiment.Results:1. U seing of CM-Sepharose FF cation exchange gel column chromatography Naja atra snake venom separation and purification on the one-step. adjusted for different flow rates on the separation and purification of snake venom components. When the flow is 1.6ml/min, we obtained perfect separation results and high purity single snake venom.2. with the Method of RP-HPLC, the separated components SV6 compared with CTX1 internal standard, retention time consistent with the results to determine the SV6 and CTX1 as the same ingredients.(CTX1 is SV6 in the paper)3. CTX1 acute toxicity and analgesic activity The LD50 of CTX1 was 4.33±0.56 mg/kg. In the hot plate and acetic acid writhing test, CTX1 express analgesic activity, with a dose-dependent,and has high therapeutic index.4. Detection CTX1 distribution in the body. In the modle of physiological conditions, CTX1 the distribution of the highest in the kidney, brain distribution of small quantities; in morphine addiction model conditions, CTX1 distribution in the brain increased significantly.Conclusions:1. Separation and purification Naja atra snake venom on the one-step, the flow is 1.6ml/min, we obtained perfect separation results and high purity single snake venom.2. CTX1 of acute pain has good analgesic effect and lower toxicity than the original drug.3. Isotope tracer method, CTX1 under physiological conditions in the blood-brain barrier permeability less, but under the conditions of morphine addiction, CTX1 significantly increased the permeability of the blood-brain barrier.