| BackgroundHelicobacter pylori （Hp） infection was closely related to diseases, such as gastric cancer, chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue lymphoma, cardiovascular disease, autoimmune diseases, skin diseases. About 50% of the population in the world, when they were children, they have been infected with Hp. If they did not use antibiotic treatment, they would be infected continuely in life-long. However, Hp cause of the exact pathogenesis of peptic ulcer had not been fully clarified. Now people generlly recognized that Hp colonization in the gastric mucosa of local released toxic substances and a variety of enzymes leading to local inflammation of the gastric mucosa reflected, thereby causing tissue injury, in the acidic environment together with local and accelerated the formation of ulcers. So, the treatment of Hp eradication had become the key of peptic ulcer and Hp-related diseases.At home and abroad, the treatment of Hp eradication could be divided into the treatment to proton pump inhibitor or bismuth-based agent with clarithromycin, amoxicillin （or tetracycline）, metronidazole （or tinidazole） three kinds of antibiotics in the two species composition of triple therapy. Using first-line drugs to achieve 85-90% eradication rate, there were a considerable number of patients with treatment failure to eradicate. First of all, the emergence of drug-resistant strains Hp eradication was the main reason for treatment failure. Because of Hp’s own protective mechanisms, Hp could cause a wide range of antibiotic resistance in the role of antibiotics chromosome mutation. Secondly, the treatment programs of eradication of Hp were very complicated. And there were a lot of drug reactions. They leaded in patients with poor compliance. So it was an important reasons for treatment failure. I hoped the majority of patients with fewer drugs, small doses, side effects, a short course of treatment. Metronidazole was cheap and effective. They sured that it was the key to the drug of the treatment of Hp infection. Some studies had shown that nearly 30% of patients had been affected to adverse drug reactions, of which nausea and vomiting, poor taste and other gastrointestinal symptoms and diarrhea in all the most common adverse reactions. A recent study showed that 11.5% of patients was early termination of treatment, of which 2/3 was due to adverse drug reactions. In addition, multi-drug combination therapy was expensive, it also affected the treatment effect. Therefore, it was an important research direction that we developed new drugs to reduce side effects and increase compliance in order to enhance the efficacy of Hp eradication therapy.Montmorillonite （MMT） belongded to 2 : 1 type layered aluminosilicate. It had a unique dual-layer octahedral striated structure and composition, due to its larger surface area, in the 456-676m2 /g, it was more strong adsorption. Research confirmed that the roles of MMT itself were several in the gastrointestinal tract. After oral administration it could cover the surface of mucin adsorption to prevent a variety of inflammatory factors on the gastric mucosal damage, balance of normal microflora in the gastrointestinal tract probiotics, promote the role of local immune digestive tract, as well as the role of local hemostasis. MMT as the main ingredient in smecta, it was applied increasingly widespread in the clinical: Smecta was used to treat diarrhea as a drug. Now it is also applied to other diseases, including gastrointestinal ulcers. Clinical studies showed: MMT could be used instead of bismuth drug in combination with anti-Hp （or/and 1-2 kinds of antibiotics）. It was similar to the eradication rate of bismuth. Forever, it was no side effects. MMT could prevent Hp to adhere in the gastric epithelial cell. And 82% of the Hp could be fixed, inhibited, removed by 0.01% of MMT suspension. It was only in the digestive tract. It did not go into the circulatory system. After 6 hours later, it was removed together with the bacteria. Through the ultrasonic oscillation, stirring and other methods, different materials could be inserted into the interlayer of MMT, the layer spacing of MMT increased after MMT intercalated by the other material. The specific surface area also increased. It had great adsorption capacity strengthening. So it could be used as the carrier of antimicrobial agents. In this paper, metronidazole/MMT antimicrobial compounds was prepared by solution intercalation. Respectively, it was characterized by sustained-release and antimicrobial test to provide a theoretical basis for further study.Methods1. Study on preparation and characteristics of Metronidazole/MMT antibacterial compounds.Took a certain amount of precision that metronidazole dissolved in 50ml deionized water, adding to a certain amount of Na-MMT solution, adjusting the solution pH to 1.0, after 40min of ultrasonic oscillations, mixing constant temperature oil bath at 90℃a certain period of time, static purchase layered, filtration, washing, vacuum drying, grinding and over 200 mesh sieve, that was metronidazole/MMT antibacterial compounds, drying and preservation. X-ray diffraction was used to measure X-ray diffraction point of view, the test conditions for radiation CuKα, voltage 40KV, scan speed of 3°·min-1, step for 0.02°. Using Fourier transform infrared spectroscopy test, it scanned range 4000- 400cm-1, KBr pellet. By thermal gravimetric analysis TGA curve, testing conditions was the air atmosphere, heating rate of 20℃·min-1, gas flow rate of 20ml·min-1.2. Metronidazole/MMT antimicrobial compounds release experiment in vitro.Took a certain amount that metronidazole/MMT antimicrobial compounds were placed in 500ml conical bottle to artificial gastric and intestinal fluid medium for release, they would be placed in conical flask in constant temperature water bath oscillator with the speed 100r/min and temperature （37±0.5）℃. They were sampled at different times, filtered through 0.45μm microporous membrane filtration, and promptly 5ml was added to media, using UV absorption spectrophotometry to measure the value of in lieu of the standard curve to calculate the corresponding cumulative release rate, to drawing the cumulative release amount- time curve.3. Metronidazole/MMT antibacterial compounds antibacterial experiment.Required the preparation of PYG medium, poured into the Petri dish. Then the holes was digged after cooled. And the liquid of bacterial was uniformly applied to flat-panel. The holes in the plate would be evenly coated tablet bacilli to different doses of Na-MMT and metronidazole/MMT antibacterial compounds under soil holes. Plated culture plate、anaerobic indicator of anaerobic capacity、anaerobic production of airbags into anaerobic tank, sealing. Then they were trained three days in 35-37℃. The size of inhibitory loops were recorded in order to evaluation of metronidazole/MMT antibacterial activity of antibacterial compounds.4. StatisticalSPSS13.0 statistical software was used to analyze experimental data for the mean±standard deviation of that, bivariate correlation analysis and randomized single-factor analysis of variance （One-way ANOVA）. Further analysis used LSD method to test the level ofα= 0.05, comparing to difference between the groups. Results1. XRDX-ray diffraction showed that Na-MMT （d001） diffraction peak was 7.160°of 2θangle, layer spacing 1.2345nm. After intercalation, metronidazole/MMT antibacterial compounds （d001） plane diffraction peak compared with Na-MMT to the displacement significantly , the direction of migration to the small-angle. It was 6.780°of 2θangle, layer spacing increased to 1.3026nm. This indicated that metronidazole had been intercalated into Na-MMT lamellar structure. At the same time, the two samples were （06,33） diffraction peaks before and after intercalation, with d values of less than 1.51 nm. they reflected the structural characteristics of the body surface.2. FTIRNa-MMT and metronidazole/MMT antimicrobial compounds common feature of FTIR were in the 3620cm-1 of Al-OH stretching vibration and 1050-1030cm-1 range of strong Si-O-Si vibration absorption peak of the cytoskeleton. In the metronidazole/MMT antimicrobial compounds the infrared spectrum characteristic peaks was that N=O stretching vibration appeared in the 1550cm-1. Combined with the results of the XRD of metronidazole/MMT antimicrobial compounds, it indicated that metronidazole had been into the Na-MMT interlayer.3. TGATGA curves showed that Na-MMT had the larger weight loss before at 200℃. It was due to the loss of Na-MMT surface of adsorbed water and the interlayer combination of water. There was an obvious weight loss in 500-750℃, which was Na-MMT silicate crystals as a result of the-OH at a high temperature to form water off. Metronidazole/MMT antimicrobial compounds of TGA process could be divided into three stages: The first phase of the initial stage of heating in 20-150℃, the surface of Na-MMT interlayer adsorption of water and the combination of water were evaporated at this stage. It caused the weight loss of metronidazole/MMT antimicrobial compounds, weight loss rate of roughly 7.2% of total weight. The second stage was the temperature of 230-340℃, the stage was the dramatic weight loss drug phase. It was also the main stage of thermal decomposition, the total weight loss of about 11.3% weight. The third stage was in 500-750℃, the same was due to Na-MMT silicate crystal of the formation of -OH off at a high temperature, weight-loss rate of the total weight of about 3.6%.4. The results of Metronidazole/MMT antimicrobial compounds release in vitro.Metronidazole could be released from Metronidazole/MMT antimicrobial compounds in the artificial gastric and intestinal fluid. It released fast in 0-0.5 hours, but it did slowly after 0.5 hours. So Na-MMT could be seen as a types of sustained-release carrier. In artificial gastric juice and artificial intestinal fluid medium for the dissolution of the system respectively, in 0-0.5 hours its released reached 4.64mg and 4.24mg, the release rates of them were 9.27mg/h and 8.47mg/h. In 0.5-6.0 hours metronidazole can be uniform released of intercalation of Na-MMT, and the release reached 5.25mg and 5.68mg. Respectively, the release rates of them were 0.11 mg/h and 0.26mg/h. The volume of them was 47% and 55% of the amount of metronidazole of the intercalation Na-MMT.5. The results of Metronidazole/MMT antimicrobial compounds antibacterial in vitro.The rings of inhibition did not appear around Na-MMT, and they did not change with the increase in quality. But they could be observed around Metronidazole/MMT antimicrobial compounds. It demonstrated that Metronidazole/MMT had antibacterial activity. Then the size of rings complexed with a number of Metronidazole/MMT that gradually changed. Compared between the groups, they were significantly different （F = 40.816, P = 0.000）, which was no significant difference between the 30mg group and the 40mg group （P = 0.173）, and it showed good correlation （Spearman r = 0.915, P = 0.000）.ConclusionMetronidazole/MMT antimicrobial compounds was analyzed by by XRD, FITR and TGA for its characterization. It was confirmed that Metronidazole had been intercalated into the interlayer of Na-MMT. The total drug weight was 11.3%. Metronidazole could be slowly released to extend the release time by Metronidazole/MMT antimicrobial compounds. So it had a good characteristic of sustained-release. However, pH had a certain influence about the speed of their release. In 0-0.5 hours, the adsorption part of metronidazole of Metronidazole/MMT antimicrobial compounds released faster in the acidic environment than in the alkaline environment, but in 0.5-6.0 hours the instered between layers of metronidazole did more slowly in the acidic environment than in the alkaline environment. Metronidazole/MMT antimicrobial compounds that had better antibacterial activity could be further developed into sustained-release anti-infective drugs. |
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