| Kangfuxinye is a pure natural medicine of ethanol extract from the dried periplaneta Americana, with rich bioactive substances, polyhydric alcohols, peptides active substance, including 18 kinds of amino acids. To take it orally can cure stasisloodhinders, such as gastrorrhagia, duodenum, anabrosis, phthisis, adjuvant therapy of deficiency of yin, tuberculosis, etc. To take it for external use can cure anabrosis, trauma, burn, scald, bedsore, and other mucocutaneous hemorrhage. At present, it is widely applied in many disciplines of clinic, and has achieved good effect.Candida Albicans is widely applied in the nature. It is common to see in the oral cavity, the upper respiratory tract, intestinal tract and vagina. The quantity is small if the morbidity does not occur. When the immunity of the organism or immunity is reduced, the bacteria will be in mass propagation. The disease will come if the mutual restriction of the normal flora is imbalanced. Candida Albicans can cause many parts of the human body to suffer diseases. The mucosal candidiasis, mycotic stomatitis, angular cheilitis and denture stomatitis are quite common.In recent years, the nonstandard usage of the domestic antibiotic and wide clinical application of hormone and immunosuppressor lead to the dysbacteriosis and immunity reducing. Besides, the rate that the skin, mucous membrance and viscera infect the fungus is increased. The drug resistance of the fungus becomes stronger. The occurrence rate of the mouth mucous membrane candidiasis becomes much higher. The research shows that, among the infant thrush, bacterial angular cheilitis, denture stomatitis, candida leukoderma and chronic mucous membrane skin candida, the detection rate of the candida Albicans is above 50%. At present, the common therapeutic methods have applying some medicine to the pars or taking the antifungal drug orally. These methods have already generated drug resistance. Therefore, it is of great importance to develop and seek for new medicines. Kangfuxinye belongs to an extract from the natural insect – cockroach. Compared with these traditional western medicines, it has high safety, little side effect, and convenient usage. Besides, it is not easy to generate drug resistance. Therefore, to study the bacteriostasis effect of Kangfuxinye on the Candida albicans and the change of the drug resistance gene expression after taking the medicine plays an active role in curing the candidiasis of the stomatology department.Objective:Candida albicans is the most common fungal disease. It refers to various acute, sub-acute or chronic infection caused by the candida albicans. It always invades the skin and mucosa. More seriously, it even gives rise to the infection on the viscera or the whole body. The clinical symptom is quite complex. In recent years, with the clinical application of large-dose antibiotics, hormone, and immunosuppressor, and wide implementation of organ transplantation, the morbidity of Candida albicans has been gradually increased. Even, it may threaten the life and leads to more serious consequence.Kangfuxinye has the following effects, such as inhibiting the bacteria, antifungal, antiviral, growth promotion, analgesia, improving the minicirculation, immuno-enhancement, and antineoplastic. It has been widely applied in many disciplines clinically. Besides, it has been gradually carried out in the department of stomatology, and its safety and practicability have been verified from many aspects. At present, there are few researches related to its application on the antifungal of the department of stomatology. Moreover, there is no definite date about the concentration and dosage. Therefore, to study the minimal inhibitory concentration and the minimum bactericidal concentration of Kangfuxinye on the Candida albicans in vitro and the change of the drug resistance gene expression after taking the medicine is favorable for guiding standard clinical usage.Methods:1 Study on the minimal inhibitory concentration and the minimum bactericidal concentration of Kangfuxinye on the Candida albicans in vitro: adopt Candida albicans for the tested bacterial strain. Get the Candida albicans vaccinated on the Sabouraud fluid nutrient medium for constant temperature incubation for 24 hours, and achieve Candida albicans suspension with stroke-physiological saline solution. Use turbidity comparator and adjust to 0.5 Maxwell turbidity(1*106-5*106 cfu/ml). Adopt improved constant broth dilution method with constant quantity, and 8 tubes, among which, 2 tubes are regarded as the contrast tube and dilute the experiment liquid through multiple proportions. Extract 100μL Candida albicans suspension; add them to the positive contrast tube and Kangfuxinye tube in succession. Do not add them to the negative contrast tube. Put three tubes into 35℃ constant temperature incubation for 24 h. Get the bacteria vaccinated to the culture dish, place the experiment plate and contrast plate into 37℃constant temperature incubation for 48 h. Observe the growth status of the Candida albicans. Determine the minimal inhibitory concentration and the minimum bactericidal concentration of Kangfuxinye towards the biological film of the Candida albicans.2 Select the concentration of Kangfuxinye as the original concentration, 1:1, 1:2, 1:4, 1:8, 1:16, bacterial suspension and positive contrast bacterial suspension after 24 hours, select bacteria with the same quantity, use Maxwell®16 Cell LEV RNA Purification Kit, and proceed with extracting gross RNA according to the reagent instruction. Then, adopt reverse transcriptase polymerase chain reaction to detect the change of the drug-resistance gene CDR1 expression of Candida albicans before and after taking the medicine: melt the primer, vibrate all primers, centrifuge primer for short term, and then place on the low-temperature device. Place RT-PCR Enzyme Mix on the low-temperature device, and melt 5×RT-PCR Buffer, and mix them sufficiently. Centrifuge 5×RT-PCR Buffer for a short time and then place it on the low-temperature device. Get agarose gel electrophoresis buffer 0.5×TBE with the voltage of 100 V. Get the specimen quantity of 8μl, DNA molecular weight Maker(100bp) of 8μl and go through electrophoresis for 50 minutes. Put it under the ultraviolet gel-imaging system and observe the result on the full-auto gel imaging device. It turns out that the strip similar to the target gene segment molecule is positive.Results:1 When the concentration of Kangfuxinye is 1:1, 1:2, 1:4 and 1:8, it has bacteriostatic activity towards the Candida albicans. When the concentration is 1:16, it has no bacteriostatic activity. The concentration of the minimum bactericidal concentration is 1:1.the lowest bacteriostatic activity towards the Candida albicans is 1:8.2 The reverse transcriptase polymerase chain reaction result indicates that, the expression level of the related drug-resistance gene CDR1 reduces while the gradient of the drug concentration increases. When the concentration is 1:1 or higher, the expression level of m RNA is completely inhibited.Conclusions:1 This experiment adopts improved constant broth dilution method with constant quantity to measure that Kangfuxinye has certain antimicrobial activity towards the Candida albicans in vitro. the minimum bactericidal concentration is 1:1.The minimal inhibitory concentration is 1:8.2 After the reverse transcriptase polymerase chain reaction detects the Candida albicans, the drug-resistance gene expression is weakened, which indicates that kangfuxinye has certain inhibiting effect on the biological film of the Candida albicans. The expression level of the related drug-resistance gene CDR1 reduces while the gradient of the drug concentration increases. When the concentration is 1:1 or higher, the expression level of m RNA is completely inhibited. |
PDF Full Text Request