Effects Of Berberine On Red Blood Cells Of Patients With G6PD Enzyme Deficiency

Background and purposeGlucose-6--phosphate dehydrogenase (Glucose-6-phosphate dehydrogenase, G6PD) deficiency is a red blood cell enzyme deficiency. It was found in half a century ago, is the most common sex-linked genetic diseases, according to statistics of about four hundred million people worldwide suffering from the disease, the disease often high incidence of malaria, thalassemia and abnormal hemoglobin disease and other popular regions. South China and Southwest China's provinces, autonomous regions (Guangdong, Guangxi, Yunnan and Sichuan), etc. for the high incidence. Often in patients with acute infection, eating beans, or exposure to drugs and other incentives under oxidative disease. Onset of clinical manifestations of acute hemolytic anemia and the resulting hyperbilirubinemia, is caused by neonatal hyperbilirubinemia, and even irreversible bilirubin encephalopathy or kernicterus one of the important reasons.Coptis is a commonly used Chinese medicine, the medicine only contained in the "Shen Nong's Herbal Classic", bitter, cold, a purging fire, detoxification, heat, dampness of the function; for Fanre coma, upset, insomnia, vomiting, abdominal pain, diarrhea dysentery, mouth sores, eczema, burns, etc., as a top grade. Modern research shows that mainly contains alkaloids berberine to berberine (that is, berberine Berberine) as the main ingredient. Coptis has many pharmacological effects, including antibacterial, antiviral and antiprotozoal effect, antifungal, anti-diarrhea effects against cerebral ischemia, anti-inflammatory, immune regulation, and even anti-ulcer, cancer and other effects. Taking medicine since ancient times to the newborn's Coptis Coptis law, the Qing imperial family have taken to the newborn with berberine for the longevity of the tradition of Dan openings. So far, Anhui, Hunan, Jiangxi, Sichuan, Fujian, Guangdong, Guangxi, Taiwan and other southern provinces to the newborn is still taking civil Coptis prescription habits.Singapore Professor Huang Xuewen neonatal kernicterus in the study found during the disease, the incidence of neonatal jaundice ethnic Chinese was significantly higher than Caucasians, and found that these newborns or their mothers had taken off in the berberine and other perinatal medicine, Then think of that black U.S. troops during World War II as taking malaria drugs such as primaquine morpholine acute hemolysis caused by the pathological process, which may belong to that berberine oxidant drugs such as Chinese medicine, there are induced by glucose 6-phosphate dehydrogenase (G6PD) deficient neonates with jaundice possible. To this end, the Singapore Ministry of Health, the term Effective Drug Advisory Committee, headed by the recommendation, in the October 6,1978 announced:Singapore National ban on the use and trading of berberine and berberine containing drugs. Singapore's Drug Advisory Committee also requested, the use of Western methods of scientific experiments and presents a scientific study, but has not yet been resolved.Since then other traditional Chinese medicine on whether berberine can cause hemolysis in patients with G6PD enzyme defects were a lot of research on the subject, but mixed results.Taiwan scholars Liaochang Li and so that berberine not only does not induce haemolysis neonatal hyperbilirubinemia, the opposite effect with some jaundice, gall bladder and the role of recorded Huanglian Li line. Our study found that scholars Linna therapeutic dose of berberine on rat normal osmotic fragility of red blood cells had no effect; berberine and berberine on experimental osmotic fragility of erythrocyte G6PD defect and no significant effect, suggesting that it could not cause hemolysis; to taking berberine and berberine pregnant mice, the fetal serum total bilirubin (TBIL), alanine aminotransferase (ALT), hemoglobin quantification, compared with the control group did not find a statistically significant difference. Fok also that berberine, capillaries and other traditional Chinese medicine both treatment and prevention of neonatal jaundice is not with the normal neonatal jaundice and G6PD deficiencies unrelated to the occurrence of disease.Yang believes that the Hong Kong University pediatrician, Sichuan berberine, bezoar, flowers bloom, capillaries and other traditional Chinese medicine have increased the risk of neonatal jaundice. Wang Lei and other studies suggest that berberine can merge dysentery infection in G6PD enzyme deficiency (G6PD-BD) increased erythrocyte osmotic fragility. Liao Huawei by retrospective clinical analysis of pregnant women reported taking berberine may increase the G6PD enzyme deficiency in the newborn the possibility of acute hemolysis.Whether the current induced by berberine in patients with G6PD deficiencies or red blood cell hemolytic mixed findings may be related to different subjects and research methods, may also be Chinese medicine, different dose related. Review of Research Methodology:1, retrospective survey of clinical studies, the impact of many factors, can not rule out neonatal infection or other causes of hyperbilirubinemia.2, berberine taken directly to the neonatal preparations, the researches of the normal children based, G6PD small number of cases the lack of children, the lack of statistical significance. After birth due to a physiological process of hemolysis, has been reported that the neonatal period may have a transient G6PD enzyme deficiency, 3 months after birth, return to normal, that the neonatal red blood cell G6PD enzyme stability is poor, and neonatal effects many factors, such as ABO hemolysis, infection, hypoxia, etc., the use of neonatal red blood cells as research subjects, conditions not easily controlled.3, with G6PD activity of red blood cell interference of strong oxidant drugs acetyl phenylhydrazine (APH) as drug modeling, replication defective G6PD (G6PD activity decreased) rat model (G6PD rat), in vivo, in vitro administration, But the choice of the basic indicators for the in vitro osmotic fragility of red blood cells, and the rough indicator of whether or not sensitive enough hemolysis reaction.Berberine for the herbs, different origins, different seasons and different extraction process may affect their drug active ingredient content, and berberine (berberine that is) (Berberine, Ber) is to play its core Coptis the main component of pharmacological effects, content up to 10% of its pharmacological targets stable, mature, easy to control, the traditional view that poor oral absorption of berberine, and reports confirm this. Li Baoxin rabbits and other study found that oral administration of berberine 50mg.kg-1, peak plasma concentration 92.7ug.L-1; Baolihua other study found that healthy people after oral administration of 300mg peak plasma concentration of berberine 0.39mg.L-1; the fifties of last century was determined by fluorescence method and found that oral administration of berberine 0.4g,30min later, the blood concentration of 1 mg/L or so, but every 4h 5 continuous medication, blood levels rise seen high. Clinically, the amount of berberine 0.1～0.3g,3 times a day, so use the normal amount of berberine, the peak plasma concentration is less than 1 mg/L, shows the peak concentration of berberine in plasma after oral administration of multiple small at 1 mg/L.The study is to G6PD enzyme defects in adult patients with disease-free venous whole blood, red blood cells under the study, the use of berberine as an experimental drug, select the four drug concentrations were 0.1mg/L,0.5mg/L, 1.0mg/L and 5.0mg/L, covering low-dose, medium dose, high dose and large dose. Of different concentrations of berberine in patients with G6PD enzyme defects in red blood cell morphology, red blood cell count, hemoglobin, G6PD activity, plasma free hemoglobin, and to explore whether the induction of berberine in patients with G6PD enzyme defects in red blood cells hemolytic, and drug concentration the relationship between hemolysis, berberine can be applied to defects in patients with G6PD activity basis.Research Methods1. Reagents and materials:In this experiment, Berberine (Berberine, Ber) standard preparation was purchased in Guangzhou City Drug, lot number 110713-200911, paired with saline berberine 25mg/L,5mg/L,2.5mg/L,0.5mg/L concentration, determined the concentration of berberine solution PH value and osmotic pressure cryopreservation standby; G6PD activity in the quantitative detection kit purchased from the Guangzhou Municipal Bureau Fang Medical Devices Limited.2. Subjects:Southern Center for outpatient examination, inspection G6PD activity measured in healthy adults, aged 18-55 years, normal reference values Kit 1.3～3.6KU/L, this study set the G6PD activity of less than 1.2KU/L The following are included as subjects. Decreased in 20 cases of G6PD, G6PD activity range of 0.01～1.0KU/L (mean 0.39±0.32KU/L), G6PD activity of normal in 20 cases, G6PD activity range of 1.93～3.15KU/L (mean 2.93±0.30KU/L), whole blood obtained 6ml, use of ACD anticoagulant.3. Test subgroups:according to blood concentrations of berberine were divided into 4 groups, namely:Ber 0.1mg/L group,0.5mg/L group, 1.0mg/L group,5.0mg /L group, and drug control group (-saline group). The sample is divided into five equal parts, each 800ul, were prepared by adding different concentrations of saline Ber 200ul,30 minutes after dosing detection indicators.4. Different concentrations of Ber on the morphology of red blood cells:Take one drop of whole blood to be seized, placed in a slide, push the film. Stained with Switzerland and 40 times in 1* red blood cell morphology observed under the microscope, cameras mining map.5. Different concentrations of Ber on the red blood cell count:In this study, using whole blood cell analyzer, the test sample of whole blood cell analyzer placed in the suction tube, press suction button, the instrument automatically generated results. Basic working principle is to use the pressure generated between the electrodes at both ends of electrical pulses, pulse amplitude to distinguish between different cells, while the same rate of electrical pulses to count the number of particles can be obtained.6. Different concentrations of Ber on the impact of free hemoglobin:free hemoglobin (Free Hemoglobin, FHb) refers to the amount of hemoglobin in plasma. Usually present in red blood cell hemoglobin, when the red blood cell destruction, release of hemoglobin, the hemolysis, the plasma free hemoglobin increased. After centrifugation, the upper plasma was taken for inspection of plasma, the use of o-A method for the determination of benzidine in plasma free hemoglobin values, from 3 test tubes were marked with blank pipe, standard pipe, test tube, follow the steps to add laboratory reagents, at 435 nm colorimeter to reagent blank to zero, the solution absorbance reading recorded for the A values. Free hemoglobin (mg/L)=(At/As)* 100 (At A value for the determination of management, As for the standard tube A value).6.1. Different concentrations of Ber on plasma and whole blood, respectively, in plasma free hemoglobin:pre-test results showed that adding Ber whole blood, plasma free hemoglobin levels not only increased, but lower than the saline group, suggesting that there may Ber reduce the role of plasma free hemoglobin, the authors divided the sample of whole blood 10, the top 5 were directly added various concentrations of berberine, the other top 5 were obtained after centrifugation of plasma, and then different concentrations in the plasma berberine compared with the concentration of different dosing levels of plasma hemoglobin changes in methods, detection methods with the former.6.2. Join Ber plasma free hemoglobin level of dynamic change:pre-test results showed that after adding Berberine decreased plasma free hemoglobin, as the understanding of this phenomenon is short-term or long-term effects, the authors selected the final drug concentration in blood Ber to 5mg/L, respectively,10 minutes after dosing,30 minutes,1 hour,2 hours,4 hours,6 hours,12 hours,24 hours measurement of plasma free hemoglobin values, detection methods with the former.6.3.6 hours after dosing different concentrations of berberine effects on plasma free hemoglobin:To observe the dynamics of plasma free hemoglobin level changes found, Ber impact on the determination of plasma free hemoglobin in the 6 hours after dosing, after elimination of the dosing After 6 hours after detection, on the determination of plasma free hemoglobin immaterial to 30 minutes after dosing plasma centrifuge, the plasma stored at 4℃refrigerator for 6 hours of free plasma hemoglobin values, with the former detection method.7. Different concentrations of Ber on the activity of G6PD:G6PD activity in the quantitative detection reagent containing G6P, the sample generated in the G6PD enzymatic G6P 6PG while oxidized coenzymeⅡ(NADP) into a reduced coenzymeⅡ (NADPH) The detection rate of increase in 340nm absorbance, the sample can be calculated in G6PD activity. Draw with precision pipette 20ul hematocrit red blood cells by adding lml lysate, the red blood cells completely dissolved, take 20ul lysate by adding 500ul reagent, measured on the machine after mixing.8. statistical methodseach group were analyzed SPSS 13.0 statistical software. Experimental data were expressed as mean±standard deviation, paired comparison design to determine the number of measurement data are the difference between normal distribution, paired sample t test, the difference is not normal, using Wilcoxon one-sample test. Repeated measurements of two treatment groups using repeated measurement factor analysis of variance (refused test of sphericity, the use of Greenhouse-Geisser method), P<0.05 was considered statistically significant difference.Results1.Different concentrations of berberine on the morphology of red blood cells: compared with the saline group, after adding different concentrations of berberine salt, G6PD activity in red blood cells with normal morphology did not change; compared with the saline group, G6PD activity of defective 0.1mg/L,0.5mg/L red blood cell concentrations did not change shape, increasing the concentration of red blood cells with normal biconcave disc shape of reduced red blood cell swelling, shrinkage, deformation of red blood cells and red blood cell fragmentation increased.2. Different concentrations of berberine on the red blood cell count:Normal G6PD activity:compared with the saline group, the concentration of berberine on the red blood cell count was not significant (P value> 0.05), the distilled water group decreased red blood cell count, the difference was statistically significant (P value<0.05); G6PD activity defect groups:compared with the saline group, with the increased concentration of red blood cell count drops Ber, 0.1mg/L and 0.5mg/L red blood cell count group were not significantly changed (P> 0.05), and 1.0mg/L and 5.0mg/L group was significantly alter red blood cell count (P<0.05), red blood cell count reduction of the distilled water group, the difference was statistically significant (P value<0.05). 3. Different concentrations of berberine on the levels of plasma hemoglobin in whole blood levels of:whole blood after addition of berberine solution was found, compared with the saline group, regardless of normal G6PD activity in G6PD activity decreased or plasma free hemoglobin decreased (P<0.05). and with the increase of drug concentration, plasma free hemoglobin level was further decreased.4. Whole blood and plasma were added to the solution of berberine hydrochloride comparison of plasma free hemoglobin values:compared with the saline group, whole blood and plasma free hemoglobin in plasma after adding Ber decreased, and the difference was statistically significant (P<0.05); G6PD activity of normal whole blood and plasma dosing compared to the value of plasma free hemoglobin was no significant difference (P> 0.05), but the lack of G6PD activity in whole blood after adding Ber higher than the plasma free hemoglobin in plasma Canada Ber group, and 1.0mg/L and 5.0mg/L group the difference was statistically significant (P<0.05). That free hemoglobin in blood were increased, suggesting a red blood cell destruction.5. Ber added to plasma in plasma free hemoglobin level of dynamic change:To understand the Ber can reduce plasma free hemoglobin in the timeliness of measured values observed in the different time points after dosing hemoglobin changes. It was found shortly after dosing plasma free hemoglobin values began to decrease only to bottom out 60 minutes after dosing, gradually picked up to 6 hours after dosing almost recovered to the level before dosing, after which no further decrease, suggesting berberine determination of the plasma free hemoglobin value decreased in the time at 6 hours.6.6 hours after dosing different concentrations of berberine effects on plasma free hemoglobin:in order to exclude Ber within 6 hours in the plasma free hemoglobin values decrease the impact of the 6 hours after dosing plasma free hemoglobin values. The results showed that:Compared with saline, G6PD activity in the normal group with berberine concentration, plasma free hemoglobin values remained unchanged, the difference was not statistically significant (P> 0.05); G6PD deficient activity of Berberis with hydrochloric acid alkali concentration, plasma free hemoglobin values tends to increase, and compared with the saline group,5.0mg/L concentration group was significantly (P<0.05), 1.0mg/L group was significantly (P <0.05), other groups were not significantly different (P> 0.05).7. Different concentrations of berberine determined value of G6PD activity effects:Compared with saline, G6PD activity in the control group increased with the concentration of berberine, G6PD activity did not change; and the lack of G6PD activity in small groups with the hydrochloric acid Bo alkali concentration increases, G6PD activity showed an increasing trend, and compared with the saline group, 1.0mg/L concentration group was significantly (P<0.05),5.0mg/L concentration group difference was also statistically significant (P<0.05), other groups were not statistically significant (P> 0.05).Summary:G6PD enzyme defects in red blood cell as the research object, the choice of five red blood cell injury index, to observe the main pharmacological ingredient of Chinese medicine---berberine hydrochloride, in 4 patients when the concentration of red blood cells, the following conclusions:1. Blood drug concentration is less than equal to 0.5mg/L when the G6PD enzyme deficiency does not cause obvious damage to red blood cells; blood concentration of 1mg/L may have mild damage effect; blood concentration of 5mg/ L, G6PD enzyme deficiency may cause red cell damage was apparent, suggesting that patients taking large doses of berberine to be alert when the possibility of inducing hemolysis.2. Found four concentrations of berberine may also lead to lower plasma free hemoglobin values, the higher the concentration decreased more; berberine effects on plasma free hemoglobin duration of about 6 hours, the reasons for this phenomenon unknown, and whether its mechanism of jaundice related to the role of civil worth further study.