| Background and objectiveThe chronic pelvic inflammatory disease (CPID) refers to chronic inflammations of the female internal reproductive organs, surrounding connective tissue and pelvic peritoneum. It may be resulted from the acute pelvic inflammatory disease (PID) treated incompletely; it can also manifest as a mild, moderate chronic pelvic inflammatory disease due to the patient’s strong constitution or mild infection. According to the survey of the U.S. Centers for Disease Control and Prevention (CDC), more than150million women in the United States suffered from PID each year, the hospitalization costs amounted to about$1.06billion, and hundreds of PID patients died each year due to complications. In the United States, the infection rate of PID in adolescents is higher than that in adult women,15to24-year-old women are a high-risk population for the diseases, which are more common in African American and Hispanic women. In China, due to personal health and medical conditions, or indifferent aseptic concepts in minor gynecologic surgeries and family planning surgeries, in addition to that IUD are widely used when patients do not pay attention to personal hygiene, the incidence rate of pelvic inflammatory disease is very high. With the increasingly frequent international exchanges, the incidence of sexually transmitted diseases (STD) is increasing year by year in our country, thus it can also lead to an increase in PID.Since there are many disadvantages in the antibiotics and surgical treatments of CPID, traditional Chinese medicine (TCM) has become a hot spot in the treatment of CPID. A lot of literatures reported that TCM such as Penyikang mixture showed good characteristics of anti-inflammation and not easy to induce drug resistance, and had many advantages including safe and effective medication, small side effect and lower cost. Jingangteng is the root of smilax, a traditional Chinese herb which can promote blood circulation to remove blood stasis, commercially available Jingangteng capsules have been sold for more than10years, and the curative effects are remarkable. Therefore, there are higher academic values in the further exploration of Smilax being prepared into effective fractions and their anti-inflammation and immune regulation mechanism.In this study, through observing the degree of the mouse ear edema, the inhibition rate of rat cotton ball granuloma, the hematological indices and pathological changes in the models of the mouse ear edema, rat cotton ball granuloma and rat chronic pelvic inflammatory disease (CPID), the activities and roles of anti-inflammation and anti CPID of ethanol extract and three chemical fractions of Smilax china L, which are respectively ethyl acetate fraction, n-butanol fraction and water-soluble fraction, were screened to determine their single active fraction of anti-inflammation and anti-CPID, the fractions were separated and purified by column separation, HPLC were used to detect the contents. And through observing the changes in inflammatory cytokines and adhesions related indicators in the serum and uterine tissues of CPID model rats, the immunomodulatory mechanism of anti-inflammation and anti-adhesion of the active fractions on CPID was explored to make the enrichment degree of effective substance high, the pharmacodynamic material relatively clear, and the pharmacological activity relatively specific, reflecting the scientificity and modernization of traditional Chinese medicine, also providing experimental data for the further development of smilax anti-CPID monomer.Experimental methods and results1. Extraction and separation of Smilax:using L9(34) orthogonal design, taking the ethanol concentration, ethanol consumption and extraction time as investigation factors, taking maximum contents of the astilbin and engeletin measured by HPLC as investigation indexes, we screened the methods for ethanol extract and determined the optimal extraction process of Smilax. Hypersil ODS C18column was chosen; mobile phase:acetonitrile:0.04%phosphoric acid; detection wavelengths:290nm; column temperature:room temperature; flow rate:0.7ml·min-1. since the contents of astilbin and engeletin obtained by the orthogonal experiment were estimated by analysis of variance, there were no significant differences in the effects of various factors on the experimental results (P>0.05). Whereupon, the visual analysis of the experimental results were carried out with a comprehensive balance method, the optimal extraction process of Smilax was obtained:Smilax pieces were crushed into small particles, then70%ethanol was added, the extraction was carried out3times by ethanol-water1:8,1:5and1:5, and the extraction times were2h,2h and1h respectively. The filtrate was decompressed to recover the ethanol and concentrated until there was no alcohol flavor, after placed in the refrigerator for12h, it was drained and filtered, and the filtrate was finally collected and added with water at constant volume.2. Three chemical fractions of Smilax extract were separated by extraction method.The ethyl acetate fractions were extracted with the extracting solution of ethyl acetate, the drug solution was extracted again with n-butanol to obtain the n-butanol fractions, and the remaining drug solution were water-soluble fractions. Of which, ethyl acetate and n-butanol fractions were extracted7times with water-saturated ethyl acetate and water-saturated n-butanol at a1:1.25%phenol mucilage0.06ml was injected into the right uterus of rats to cause CPID in rats to observe the effects of high, medium and low doses of ethanol extract and three chemical fractions of Smilax china L (corresponding to4,2and1times the clinical equivalent dose respectively) on changes in hematology and histopathology T-phenotype of CPID model rats; xylene was used to induce mouse ear edema to observe the inbibitional effects of high, medium and low doses of Smilax chemical fractions (corresponding to4,2and1times the clinical equivalent dose respectively) on mouse ear edema and explore the therapeutical effects of Smilax chemical fractions on acute inflammations.30μl Xylene was smeared on the right ear of each mouse,whose left ear was used as a blank control,20min later the mice were killed by cervical dislocation, two circular ear pieces were taken with8mm diameter hole puncher at the same middle parts of left and right ear edges and weighed by an electronic balance to calculate the swelling degree and the swelling inhibition rate; rat cotton ball granuloma was induced to observe the inbibitional effects of high, medium and low doses of Smilax chemical fractions (corresponding to4,2and1times the clinical equivalent dose respectively) on rat cotton ball granuloma and explore the anti-chronic inflammatory effects of Smilax chemical fractions. Autoclaved cotton balls (each weighing50±1g) were infused into cefmetazole1mg/0.1ml with a syringe before use, and they were implanted subcutaneously into the rat left groins. Result:The ethanol extract of Smilax and the high, middle doses of the ethyl acetate fractions can significantly reduce the increased hemodynamic indexes of rats, there were significant differences (P<0.01), they could significantly inhibit the mouse ear swelling, there were statistically significant differences (P<0.01), but there were no obvious effects in the low dose group (P>0.05); there was a very significant difference in rat cotton ball granuloma in the high dose group of ethyl acetate (P<0.01), the ethanol extract of Smilax in high dose group and the ethyl acetate fractions in medium, low dose groups had significant differences in rat cotton ball granuloma (P<0.05). The rest groups, including n-butanol fraction and water-soluble fraction groups, had no significant effects of anti-inflammation and anti-CPID. The pathological morphological changes of rat uterine tissues were observed under microscope, the ethanol extract of Smilax and the ethyl acetate fractions could significantly reduce the chronic inflammatory cell infiltration, improve the hyperemia, edema and layer structures of uterine walls, most of the gland structures were clear and complete, among which, the medium dose effects were less obvious than the high dose effects, there were no obvious pathological changes in the low dose group. The inflammation improvement in high dose group of N-butanol was less obvious than that in the ethyl acetate fraction group; in the low-dose group, a large number of epithelial cells fell off into uterine cavity, the inflammatory cells covered the entire mucosal layer, scattered eosinophils were seen. The water-soluble fraction group was compared with the model control group under microscope, and there was no significant difference.3. The active fractions of Smilax were further separated and purified using polyamide column to collect the effective ingredients of total flavonoids. Using HPLC, chromatographic column:Hypersil ODS C18(4.6×250nm,5μm); mobile phase: methanol; flow rate:1ml·min-1; Column temperature:room temperature; detection wavelength:290nm; sample size:20μl. One single standard substance for determination of multiple constituents (One for M) was used in the initial detection of the active ingredient contents. By area normalization method, the mass of astilbin(μg) as abscissa and the peak area as ordinate, the standard curve was drawn, the regression equation:Y=5.561×105X-5.911×105, R2=0.9997, and the total flavonoids content in ethyl acetate fractions was determined as55.73%by peak area method.4. Further verification of therapeutic effects of the active fractions of Smilax on CPID: After Smilax active fractions were separated, extracted, extracted with ethyl acetate, and then purified by a polyamide resin, the content was increased, therefore, when designing the experiment, we reduced the administration doses for rats, the clinical equivalent dose was taken as the medium dose, the high, medium and low doses were respectively equal to2,1and1/2times the clinical equivalent dose.7day after rat models were constructed, the drugs were administered once daily for10consecutive days, on the11th day,24h after the last administration, the blood was collected from abdominal aorta to detect the blood count and hemorheological changes; the morphological changes in modeling side of the rat uterus were visually observed; the uterus was taken out and weighed to calculate the viscera index; bilateral uteruses were soaked in10%formaldehyde fixative, then were conventionally stained with HE, the pathological changes of the rat uteruses were observed under microscope. Results:the high and medium doses of active fractions of Smilax can significantly reduce rat hematological indices, including whole blood viscosity, plasma viscosity and white blood cells, there were very significant differences, and a significant difference respectively (P<0.01, P<0.01and P<0.05), the rat uterus viscera index was statistically significant, and the difference was significant (P<0.05), the low dose effect was not obvious. Pathological results showed that the degrees of inflammation of the rat uterus CPID models were partially improved in the low dose group of purified Smilax active fractions and the TCM positive control group, while the pathological damages of the rat uterus CPID models were improved to a great extent in the high and medium dose groups, in addition, the inflammatory damages were improved.5. The effects of the active fractions of Smilax on inflammatory cytokines and adhesion-related indicators in the serums and uterine tissues of rat CPID models were observed to explore the immunomodulatory mechanism of their anti-inflammatory and anti-adhesion effects on CPID. SD female rats were taken as experimental subjects,84rats were randomly divided into control group, sham operation group, model control group, groups with high, medium and low doses of Smilax active fractions, and Jingangteng capsules group. The enzyme-linked immunosorbent adsorption method (ELISA) was used to detect the inflammatory cytokine levels in the serums of rats in each group, including interleukin-1β (IL-1β) and interleukin-10(IL-10); the immunohistochemical method was used to detect the expression of tumor necrosis factor-a (TNF-a) and intercellular adhesion molecule-1(ICAM-1) in rat uterine tissue of each group; the real time-polymerase chain reaction (RT-PCR) was used to detect the expression of TNF-a mRNA and ICAM-1mRNA in rat uterus tissues. Results:the active fractions of Smilax could regulate the serum IL-1β and IL-10levels of rat models in varying degrees. Of which, there were highly significant differences in regulating the inflammatory cytokines IL-1β between the high, medium and low dose groups of Smilax active fractions and the model control groups (P<0.01); in regulating the inflammatory cytokine IL-10, the active fractions of Smilax in the high and medium dose groups could significantly increase its serum contents in rats, the differences were highly significant (P<0.01), there was a significant difference between the low dose group and the model control group (P=0.043,<0.05). The immunohistochemical method was used to detect the expression of adhesion related indicators such as TNF-a and ICAM-1in the uterine tissues of rat CPID models; the results revealed that the active fractions of Smilax in three dose groups made extremely significant inhibitions on the high expressions of TNF-a, while the active fractions of Smilax made inhibitory effects on the expression of ICAM-1, there was a highly significant difference in high dose group (P<0.01) and significant differences in the medium and low dose groups (P<0.05).The expressions of TNF-a mRNA and of ICAM-1mRNA in rat uterus were detected by RT-PCR method, the results showed that the expressions of TNF-a mRNA and ICAM-1mRNA were decreased in the drug administration groups compared with the model control groups, the differences were highly significant (P=0.000, P<0.001). Of which, the average levels of various indicators in the groups with high and medium doses of ethyl acetate fractions of Smilax had been close to those in the blank control groups (P=0.000,<0.001). At the same time, the experimental results of fluorescence quantitative PCR amplification showed that three gene amplification curves were smooth and stable; the S-shaped curve of the fluorescence absorption spectrum was intact, in line with the requirements of quantitative detection. The melting curve showed a single peak, the peak shape was sharp, and no other peak existed. That indicated that the melting temperature of each gene was homogeneous, the amplification products had good specificities, and no non-specific double-stranded DNA products or primer dimers were seen.ConclusionsThrough studying the pharmacodynamic effects of several chemical fractions of Smilax on CPID rat models, mouse ear edema models and rat cotton ball granuloma models, we found that the active fractions of Smilax with anti-inflammatory and anti-CPID actions were mainly ethyl acetate fractions. Then the active fractions were further separated and purified by polyamide adsorption method, and the total flavonoids content in ethyl acetate fractions was determined as55.73%. The anti-CPID actions of active fractions of Smilax were verified in CPID rat models, this explained that through promoting circulation and removing stasis, the active fractions of Smilax could alleviate the hypercoagulability and hyperviscosity state of blood in CPID rats, reduce the degree of adhesion in the rat uteruses, reduce capillary and cell membrane permeability, reduce inflammatory exudation, inhibit the proliferation of fibrous connective tissues, and promote inflammation absorption. In addition, by regulating inflammatory cytokines in rat serum, the active fractions of Smilax could reduce the pro-inflammatory cytokines and increase the anti-inflammatory cytokines, thereby enhance the body’s anti-inflammatory activity to restore the immune balance; by regulating the expression of adhesion-related indicators in rat uteruses,the active fractions of Smilax could reduce the excessive infiltration of inflammatory cells and the formation of inflammatory mediators, reduce the formation of mucosal adhesion, effectively inhibit the inflammations in CPID rat models and alleviate their pelvic adhesions. |
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