Effects of Berberine on Red Blood Cells of Patients with G6PD Enzyme Deficiency
|School||Southern Medical University,|
|Keywords||Berberine hydrochloride G6PD enzyme deficiency Human erythrocytes Hemolysis|
Background and Purpose glucose-6 - phosphate dehydrogenase (Glucose-6-phosphate dehydrogenase, G6PD) deficiency is a red blood cell enzyme deficiency. It was found in a half-century ago, is the most common sex-linked genetic diseases According to statistics, about four hundred million people worldwide suffer from the disease, this disease is often the malaria high incidence of thalassemia and abnormal hemoglobin disease endemic areas. Southern China and south-western provinces, autonomous regions (Guangdong, Guangxi, Yunnan and Sichuan), is reportedly high incidence, Guangdong Province G6PD deficiency incidence of about 6%. Patients often acute infection, the incidence of eating beans or contact oxidation Drugs incentives. Onset of clinical manifestations of acute hemolytic anemia and hyperbilirubinemia resulting cause of neonatal hyperbilirubinemia, even irreversible bilirubin encephalopathy or kernicterus one of the important reasons Coptis commonly used in traditional Chinese medicine, medicinal beginning set out in the \Modern research shows that the Coptis mainly containing alkaloid berberine (berberine Berberine) as the main ingredient. Coptis has many pharmacological effects, including antibacterial, antiviral, antiprotozoal, anti-fungal, anti-diarrhea, anti-cerebral ischemia, anti-inflammatory and immunomodulatory effects, and even anti-ulcer, anti-cancer effects. Chinese medicine since ancient times, neonatal taking Coptis Coptis law, Qing Dynasty royal family to the newborn taking traditional Fushou Dan openings containing Coptis. So far, the southern provinces of Anhui, Hunan, Jiangxi, Sichuan, Fujian, Guangdong, Guangxi, Taiwan folk still have to neonatal taking the Coptis prescription habits. Professor Huang Xuewen, Singapore is found in the study of disease of the newborn kernicterus, the ethnic Chinese neonatal jaundice incidence was significantly higher than Caucasians, and found these newborns or their mothers taking Coptis and other traditional Chinese medicine in the perinatal period, there history, during World War II, the U.S. military think blacks taking to combat malaria, primaquine and other drugs pathological process caused by acute hemolysis, thereby Coptis, such as traditional Chinese medicine may belong to oxidant drugs, induced by glucose 6 - phosphate dehydrogenase ( G6PD) defective neonatal hemolytic jaundice may. To this end, the Singapore Ministry of Health announced on October 6, 1978: The Singapore nationwide ban on the use and sale of coptis berberine-containing drugs. Requirements of the Drugs Advisory Committee of Singapore, Western methods of scientific experiments and presenting scientifically based research report. Since then, a lot of research about the Coptis such as traditional Chinese medicine will cause G6PD enzyme deficiency in patients with hemolytic this topic, but with mixed results. The Chinese scholars Linna Research found that therapeutic doses of herbs on erythrocyte osmotic fragility did not significantly affect the normal rat isolated blood; Coptis and berberine on experimental G6PD deficiency rat erythrocyte osmotic fragility of no significant impact to the pregnant mice taking Coptis and small the Bo alkali fetal serum total bilirubin (TBIL), alanine aminotransferase (ALT), hemoglobin quantification, compared to the control group did not find a statistically significant difference, suggesting that it can not cause hemolysis. Pediatrician Yang of the University of Hong Kong, Sichuan berberine, bezoar, flowers bloom, Yin Zhi Huang and other traditional Chinese medicine has aggravated the risk of neonatal jaundice. Wang Lei and other studies suggest that Coptis make G6PD enzyme deficiency combined dysentery infection (G6PD-BD) of erythrocyte osmotic fragility increased. Liao Huawei retrospective clinical analysis study reported maternal taking Coptis may have increased the the neonatal acute hemolytic possibility G6PD enzyme deficiency. Taiwan scholars Liao Changli think the the Coptis not only does not induce hemolysis and neonatal hyperbilirubinemia contrary to certain Tuihuang, consistent with the documented Huanglian Li bile role. Fok, the Coptis, such as traditional Chinese medicine with normal G6PD deficiency neonatal jaundice occurred independent of, nor the treatment and prevention of neonatal jaundice. The Coptis whether the occurrence of hemolysis in patients with G6PD deficiency can induce varying results, may vary with the object of study and research methods, may also be different with traditional Chinese medicine preparation, dose related. For example: 1, more neonatal clinical study influencing factors combined ABO hemolytic, such as infection, hypoxia; birth after a physiological hemolytic process, erythrocyte poor stability; otherwise reported that the neonatal period erythrocyte G6PD enzyme stability, the neonatal period may have had low G6PD enzyme, to return to normal 3 months after birth. Therefore, using neonatal red blood cells as a research object, influenced by many factors. 2, previous research object mainly to normal newborns lack of a control group, G6PD deficiency in children with the number of cases small, the lack of statistical significance. 3, the experimental study using erythrocyte G6PD activity interference the strong oxidant acetyl phenylhydrazine drugs (APH) for modeling drug Copy G6PD deficiency rat model, but drug modeling with actual patients, red blood cells have a certain gap, and choose to study indicators erythrocyte osmotic fragility in vitro, the indicators sensitive enough. Coptis for herbal medicines, different origins, different seasons and different refining processes are likely to affect the drug content of the active ingredient, Berberine Hydrochloride (berberine) (Berberine, Ber) the Huanglian in its core pharmacological effects mainly ingredients content of up to about 10%, the pharmacological indicators of stable, mature, easy to control, the traditional view that berberine oral absorption difference, and there have been reports confirmed this. Bao-Xin Li study found that rabbit oral berberine 50mg.kg-1, the peak plasma concentration 92.7ug.L-1; Baolihua research found that peak plasma concentration of healthy human oral 300mg berberine 0.39mg.L-1 ; fifties of the last century, was measured by fluorescence found human oral berberine hydrochloride 0.4g 30min after the blood concentration of 1 mg / L or less, but every 4h continuous medication, plasma concentration but not l high. Clinical berberine hydrochloride dosage of 0.1-0.3g, 3 times a day, and therefore the normal amount of oral berberine hydrochloride, peak plasma concentration less than 1 mg / L, and more. In this study, the proposed adult G6PD enzyme deficiency disease free state venous whole blood red blood cells, berberine hydrochloride as an experimental drug study, select the four drug concentrations were 0.1 mg / L and 0.5 mg / L and 1.0mg / L and 5.0mg / L, covering small doses to large doses. Observe the different concentrations of berberine hydrochloride on the G6PD enzyme deficiency in patients with red blood cell morphology, red blood cell count, plasma free hemoglobin, G6PD activity to explore possibility of berberine hydrochloride-induced red blood cell hemolysis in patients with G6PD enzyme deficiency, as well as the drug concentration and hemolytic relationship, trying Coptis ability to provide experimental evidence used in G6PD enzyme activity deficient patients. Research methods. Reagents and materials: standard preparation used in this experiment berberine hydrochloride (Berberine, Ber) purchased at the Guangzhou Institute for Drug Control, batch number 110713-200911, the berberine hydrochloride saline dubbed 25mg / L 5 mg / L and 2.5mg / L, 0.5mg / L concentration, determination of various concentrations of berberine hydrochloride solution PH value and osmotic pressure cryopreservation spare; G6PD activity and quantitative detection kit was purchased from limited side Guangzhou Medical Devices Company; neighbor - A benzidine reagent was purchased from Shantou Guanghua Chemical Factory; whole blood cell analyzer Model:-3000 2 GY2. study: the Nanfang Hospital outpatient examination center examination and G6PD activity was measured in healthy adults, age 18-55 years old, the kit normal reference value of 1.3 ~ 3.6KU / L, the research setting G6PD enzyme activity less 1.2KU / L or less satisfied as experimental subjects. G6PD decrease of 20 cases, G6PD enzyme activity range 0.01-1.OKU / L (average 0.39 ± 0.32KU / L), G6PD activity of 20 cases of normal, G6PD enzyme activity range the 1.93-3.15KU / L (mean 2.93 ± 0.30 KU / L), take whole blood 6ml, using ACD anticoagulant. Test packet: blood concentration of berberine hydrochloride were divided into 4 groups, respectively: Ber 0.1mg / L group, 0.5mg / L group, 1.0mg / L group, 5.0mg / L group, as well as drug blank control group (saline group) and the positive control group (distilled water group). The sample is divided into six parts, each 800ul, adding different concentrations of saline formulated Ber 200u1. Detection indicators saline and distilled water 200ul, 30 minutes after dosing. 4. Of different concentrations Ber red blood cell morphology: Take to be tested a drop of whole blood, placed on a slide, push piece. Switzerland staining, and red blood cell morphology was observed under the microscope of 10 * 40 times, camera mining Fig. 5 different concentrations Ber red blood cell count: use of whole blood cell analyzer, the test sample is placed in the whole blood cell analyzer pipette aspiration button, the instrument automatically generated results. The basic principle is to use the pressure in the both ends between the electrodes to produce electrical pulses, the size of the pulse amplitude distinguish different cells, and the same amplitude electrical pulses to count the number of particles can be obtained. 6 different concentrations Ber free hemoglobin: free hemoglobin (Free Hemoglobin, FHb) refers to the amount of hemoglobin in the blood plasma. Usually hemoglobin in red blood cells, when the destruction of red blood cells, hemoglobin release, hemolysis, increased plasma free hemoglobin. Take centrifugation the upper layer plasma to be detected in plasma, using o - A benzidine method for the determination of plasma free hemoglobin values, take three tubes respectively marked blank tube, standard pipe, test tube laboratory reagents, step-by-step to join, at 435 nm colorimetric to the blank tube zero (corresponding concentration of berberine hydrochloride solution), read each the solution absorbance Hutchison A value. Free hemoglobin (mg / L) = (At / As) * 100 (At To tube A value was measured, and As is standard pipe A value). 6.1 Different concentrations of Ber plasma and whole blood, plasma free hemoglobin: the pre-test results found that added Ber whole blood, plasma free hemoglobin level is not only not increased, but lower than the saline group, suggesting that Ber may have reduce the role of plasma free hemoglobin, the impact of hemolysis judgment. Imagine if the whole blood is not the destruction of red blood cells, plasma free hemoglobin in the same amount and the amount of free hemoglobin in whole blood, berberine hydrochloride were added to whole blood and plasma, plasma free hemoglobin is still the same, so the author venous whole The blood sample is divided into 10 parts, the top five were directly added to different concentrations of hydrochloric berberine other 5 After centrifugation, the supernatant plasma, plasma with different concentrations of berberine hydrochloride compare whole blood and plasma of both plasma free hemoglobin levels the differences detected the same way as before. Dynamic changes of plasma free hemoglobin level 6.2. Join Ber: pre-test results found that the addition of berberine hydrochloride plasma free hemoglobin decreased, as the understanding of this phenomenon is a short-acting or long-lasting impact of selected the Ber blood eventually The drug concentration of 5mg / L for the dosing concentration respectively after dosing, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours plasma free hemoglobin values, the same time, at different time points Let blank drug group to do the control group, with the former detection methods. Different concentrations of berberine hydrochloride plasma free hemoglobin 6.3. 6 hours after dosing: the pre-test results found, Ber plasma free hemoglobin was measured affect after dosing 6 hours to eliminate detected favorable understanding of plasma free hemoglobin values original condition, it was centrifuged at 30 minutes after dosing separated plasma, the plasma was placed in 4 ° C refrigerator, to detect the value of the free hemoglobin in the plasma after 6 hours, the detection method is same as before. 7 different concentrations of Ber G6PD activity: G6PD activity and quantitative detection reagent containing glucose-6 - phosphate (glucose-6-phosphate, G6P), the G6PD enzyme catalytic G6P generate the sample glucose 6 - phosphate ester (6-phosphogluconate, 6PG) the oxidized coenzyme II (NADP) into a reduced coenzyme II (NADPH), the rising speed detection 340nm absorbance of G6PD activity in the sample can be calculated. Pipette accurately draw 20ul hematocrit RBC Add 1ml lysate erythrocytes completely dissolved, taking 20ul dissolving liquid was added to the 500ul reagent, after mixing measured HCC-9906 semi-automatic biochemical analyzer. 8. Statistical methods in each group data using SPSS13.0 statistical software for analysis. Experimental data using the mean ± standard deviation X ± S, the two paired design measurement data comparison to determine the difference follow a normal distribution, a paired samples t-test, the difference does not follow a normal distribution, Wilcoxon one-sample test. Repeated measurement data of the two treatment groups using a repeated measures factor analysis of variance (reject the test of sphericity, Greenhouse-Geisser method), P lt; 0.05 was considered statistically significant difference. Results. Different concentrations of berberine hydrochloride on red blood cell morphology G6PD activity of normal: Compared with the saline group, adding different concentrations of brine berberine, red blood cell morphology did not change, while the distilled water group RBC biconcave disc-shaped structure to reduce red blood cell swelling, shrinkage, deformation of the red blood cells and red blood cell fragments increase; G6PD activity flaws groups: compared with the saline group, 0.1mg / L, 0.5mg / L concentrations of red blood cell morphology did not change significantly with increasing concentration above red blood cell morphology changes more significantly, the distilled water group than in G6PD activity of the normal group the destruction of red blood cells. Different concentrations of berberine hydrochloride on the red blood cell count G6PD activity of normal: Compared with the saline group, various concentrations of berberine hydrochloride on erythrocyte count the impact was not statistically significant (P = gt; 0.05), distilled water group red blood cell count was significantly reduced, and the difference was statistically significant (P = lt; 0.05); G6PD activity flaws groups: compared with the saline group, 0.1mg / L and 0.5mg / L, erythrocyte count the change was not statistically significant ( P gt; 0.05), and 1.0mg / L and 5.0mg / L group red blood cell count decreased its change was statistically significant (P lt; 0.05), distilled water group significantly reduced red blood cell count, and the difference was statistically significant (P = lt ; 0.05). 3 different concentrations of berberine hydrochloride on whole blood, plasma free hemoglobin levels berberine hydrochloride solution is added to the whole blood, compared with the saline group, found that regardless of the G6PD activity of the normal group or G6PD activity decreased plasma free hemoglobin were decreased (P lt; 0.05), a further downward trend with the increase of the concentration of the drug, the level of plasma free hemoglobin, plasma free hemoglobin and distilled water group were increased, and the difference was statistically compared with the saline group significance (P lt; 0.05). Berberine hydrochloride solution were added to whole blood and plasma, plasma free hemoglobin value comparison: G6PD activity of the normal group compared to dosing in whole blood and plasma, plasma free hemoglobin value difference was statistically significant (P gt; 0.05), but the lack of G6PD enzyme activity in whole blood, plasma free hemoglobin value plus Ber Ber higher than plasma plus group and 1.0mg / L and 5.0mg / L group difference was statistically significance (P lt; 0.05) . Free hemoglobin in whole blood, suggesting that the phenomenon of a red blood cell destruction. The dynamic changes of plasma free hemoglobin levels in the plasma Ber: To understand the Ber timeliness of plasma free hemoglobin measured values ??decrease can observe changes in hemoglobin values ??at different time points after dosing. Was found shortly after dosing plasma free hemoglobin values ??both began to fall to the dosing 60 minutes after bottoming out, after the gradual recovery six hours to dosing almost rebounded to the level before dosing, after no significant changes, suggesting berberine hydrochloride determination of plasma free hemoglobin decline of aging in less than six hours. 6 different concentrations of hydrochloric berberine 6 hours after dosing, plasma free hemoglobin: In order to exclude Ber within 6 hours of plasma free hemoglobin measured values ??to reduce the impact of six hours after dosing, plasma free hemoglobin values. The results showed that: compared with the saline, G6PD activity of the normal group increases with the concentration of berberine hydrochloride, plasma free hemoglobin value was no significant change (P gt; 0.05); lack of G6PD enzyme activity group with the concentration of berberine hydrochloride increases plasma free hemoglobin values ??tended to increase, compared with the saline group, 1.0mg / L group, 5.0mg / L concentration group difference was statistically significant (P lt; 0.05), the other group differences were not statistically significant ( P gt; 0.05). 7 different concentrations of berberine hydrochloride on the G6PD enzyme activity measured values: G6PD activity of the normal group: compared with the saline group, increases with the concentration of berberine hydrochloride was no significant change in G6PD activity of G6PD enzyme of distilled water group activity was significantly increased, and the difference was statistically significant (P lt; 0.05); lack of G6PD enzyme activity group: increases with the concentration of berberine hydrochloride G6PD enzyme activity tended to increase, compared with the saline group, 1.0 mg / L, 5.0mg / L concentration group difference was statistically significant (P lt; 0.05), and distilled water group activity increased, compared with the saline group, the difference was statistically significant (P lt; 0.05). Summary: G6PD enzyme deficiency in patients with red blood cells for the study to observe the effect of Coptis pharmacological ingredients --- berberine, 4 drug concentration in the patient's red blood cells, the conclusions are as follows: 1 plasma concentration is less than or equal to 0.5 mg / L, the G6PD enzyme deficiency red blood cells had no significant effect; plasma concentration of lmg / L and 5mg / L, erythrocyte G6PD enzyme deficiency may have an impact, suggesting that patients taking higher doses of Coptis alert induced the possibility of hemolysis. Found four concentrations of berberine hydrochloride may result in lower plasma free hemoglobin measured value, the higher the concentration, the more obvious decline; timeliness of berberine hydrochloride on plasma free hemoglobin within 6 hours, efficacy peak one hour after dosing, the phenomenon of unexplained, its mechanisms, and whether the role of folk Tuihuang worth further explore.