High Performance Liquid Chromatography Liver capsule content Schisandrin
【Abstract】 Objective To investigate the allicin on
Hemorheology and physiological characteristics of red blood cells. Method of producing a high viscosity rat model of acute
arterial blood, through the determination of blood viscosity and erythrocyte aggregation index change index, the nuclear pore membrane filtration Determination of erythrocyte filtration index, fluorescence polarization determination of red cell membrane fluidity, as well as red blood cell infiltration fragility test, comparison of allicin intraperitoneal injection group and the untreated group differences. Results with the untreated group, allicin high, medium and low-dose group and blood stasis syndrome in rats
increased whole blood viscosity were significantly lower effect (P <0.01, P <0.05); middle-and high-dose group on
plasma viscosity significantly reduce the effect (P <0.05), and these effects showed a definite dose-effect relationship;
high-dose group could significantly lower hematocrit (P <0.05), can significantly improve the deformability of red blood cells (P <0.05); in low-dose group, to reduce the role
of erythrocyte aggregation; each dose group the increase of osmotic fragility of red blood cells to improve role; each dose group on erythrocyte membrane fluidity reduction to improve the role, but not statistically significant. Conclusion of allicin can improve erythrocyte deformability and lower blood viscosity, improve blood rheology, whereas for
the prevention and treatment of blood stasis syndrome.
Key words allicin; acute blood stasis syndrome; Hemorheology; erythrocyte
Effects of Allicin on Hemorheology and Physiological
Properties of Erythrocytes in Acute Blood-stagnated Rats
Abstract: ObjectiveTo explore the effects of allicin on hemorheology and physiological properties of
erythrocytes.MethodsWe established the rat models of acute hyperviscosity and collected artery blood samples. Then we examined the hemorheology index and erythrocyte aggregation index by the viscosity methods, detected the erythrocyte filtration index by the nuclear pore film, determined erythrocyte membrane fluidity by the fluorescence polarization and performed erythrocyte osmotic fragility test. The
difference between the allicin intrapertoneal injection group and the untreatment group was observed.ResultsIn comparison with the untreatment group, all the high-, medium - and low-
dose groups of allicin treatment had a decline in the enhancement of whole blood viscosity in rats with blood stasis (P <0.01, P <0.05). In the high-and medium-dose groups, the
plasma viscosity significantly decreased (P <0.05) and had the dose-effect relationship. In the high-dose group, the
hematocrit greatly decreased (P <0.05) and the erythrocyte deformability increased (P <0.05). In the medium-and low-dose
groups, the erythrocyte aggregation index decreased. All dose of allicin had effect on the increase of erythrocyte osmotic fragility and the decrease of erythrocyte membrane fluidity,
but no significance was detected. ConclusionAllicin may increase the erythrocyte deformability, decrease the whole blood viscosity and improve the blood rheological properties and it can be used in the treatment of blood stasis.
Key words: Allicin; Acute blood stasis; Erythrocyte; Hemorheology
Allicin (Allicin) has anti-bacterial, anti-fungal, anti-
tumor and the role of pesticides, etc. . In recent years, found that there are anti-virus, enhance immune function,
scavenging free radicals, anti-oxidation, reducing blood
pressure, blood fat, anti-atherosclerosis, lowering blood
sugar, anti-arrhythmia and the role of convergence . At present the medical profession which have been widely used for prevention and treatment of many diseases. Their blood pressure, lipid-lowering, anti-atherosclerotic effect mainly
on hemorheology, while hemorheology studies, most focus on
platelet function, the pairs of red blood cell rheology studies small. This article focuses on studies of allicin on
erythrocyte rheology and other physiological characteristics.
1.1 Animals Wistar rats of either sex, weight (200 ? 20)
g, 3 ~ 4 months of age, purchased from Beijing Haidian Tom Lee Laboratory Animal Breeding Center.
1.2 Drugs and reagents produced in Shanghai Garlic injection Hefong Pharmaceutical Co., Ltd., 30 mg / 2 ml.
1.3 main instruments NXE 1-type cone-plate viscometer:
Chengdu Instrument Factory products. DXC 300 type nuclear
membrane red blood cell deformability analyzer: Shanghai Medical Instrument Factory products. 650 60 Fluorescence
Polarization Instrument: Japanese Hitachi products. Micro-
high-speed centrifuge: Aerospace Huaxing Aircraft Wheel Corporation products.
2.1 Animal Grouping and administration of 50 experimental animals were randomly divided into 5 groups: blank control
group (saline 2 ml * kg-1 * d-1), acute hyperviscosity model
group (saline 2 ml * kg -1 * d-1), allicin low-dose group (5
ml * kg-1 * d-1), the dose of allicin (10 ml * kg-1 * d-1),
allicin high-dose group ( 20 ml * kg-1 * d-1). In addition to
the blank control group, the remaining animals fed a daily tap water 5 ml / only consecutive 7 d, the 7th day subcutaneous injection of 0.1% concentration of adrenaline 0.08 ml/100 g body weight, injected 2 h, immersed in ice water in 5 min an interval of 4 h injection of the first two times, producing acute hyperviscosity model . After modeling of all animals in the first two days starting the daily intraperitoneally (ip) corresponding drugs a second consecutive medication 7 d.
2.2 The changes in blood flow indices and the
determination of viscosity, erythrocyte aggregation method .
2.2.1 Blood and anticoagulation after the last administration 2 h, intraperitoneal injection of pentobarbital
sodium 40 mg / kg of narcotic. Carotid artery blood, heparin
20 IU / ml anticoagulant.
2.2.2 Determination of whole blood viscosity and plasma viscosity to take anti-clotting 1.3 ml, at 37 ?,
respectively, blood samples were measured in the shear rate 230S-1, and 11.5 S-1 when the value of whole blood viscosity.
2000r/min centrifuge 15 min, separated plasma were measured in 115 S-1 when the plasma values.
2.2.3 hematocrit (Hct) determination of Wen's method. Capillary pipette drawn with 0.5 ml of anti-coagulation,
insert the tube at the bottom of Wen's hematocrit, so that
blood to the scale at only 10. Do not inject air bubbles. Blood samples will be injected into the centrifuge tube hematocrit tube placed inside the bucket, to 2 300 r / min centrifugation 30 min. Remove the PCV pipe, in the white blood
cell and red blood cell level of alignment boundaries was read out at the scale of values, that is, the percentage of hematocrit.
2.2.4 erythrocyte aggregation index calculated red blood cell aggregation index (ηre) is defined as the
ηre = ηb ηpηp × 1Hct
One ηb: the low shear rate whole blood viscosity; ηp:
plasma viscosity; Hct: hematocrit; ηre namely, the low shear
rate whole blood reduced viscosity, and its value depends on the low-shear-rate level of red blood cell aggregation, ηre
more large, indicating that the stronger the red blood cell aggregation.
2.3 Determination of red blood cell deformability nuclear pore membrane filtration method .
2.3.1 Preparation of erythrocyte suspensions take 2.2.1
anticoagulant 1.5 ml, 3 000 r / min centrifugation 10 min, suction out the plasma and white blood cells, to pH values of 7.35 in PBS buffer wash three times, were centrifuged to the
upper fluid. Preparation of the above-mentioned buffer 10% of
the red blood cell suspension.
2.3.2 Determination of hematocrit (Hct) approach with 2.2.3.
2.3.3 Determination of filtration time of red blood cell suspension was incubated at 37 ?, take 1ml erythrocyte
suspension and PBS buffer were placed in deformability instrument to measure the nuclear pore membrane filtration time tg, ts.
2.3.4 calculate red blood cell filtration index of erythrocyte filtration index (IF) is defined as: IF = tg-tsts
× 1Hct, red blood cell deformability worse, the more difficult holes through the membrane, its suspension tg longer filtration time, so filtration index IF the larger the worse,
said red blood cell deformability.
2.4 red blood cell osmotic fragility test  said that
taking 120 ? to constant weight of the analytically pure NaCl 1.000 g, plus a small amount of distilled water and dissolved in 100 ml volumetric flask using distilled water, fixed volume, was 171.1 mmol / L NaCl solution. To take 12 test tubes, by adding sodium chloride solution and distilled water, prepared concentration
41.0,47.9,54.7,61.6,68.4,75.2,82.1,88.9,95.8,102.6,109.4,116.3 mmol / L of NaCl solution. Carotid artery does not take anticoagulant blood 1 ml, through the No. 6 needles, needle bevel up, to each of these tubes by adding a drop of blood, gently shaken, put it aside at room temperature for 2 h, observe the results. When judging the results, starting with a high concentration began to observe the upper transparent solution appeared red are for the commencement of hemolysis
tubes, the solution for the transparent tube at the end of the red and red blood cell hemolysis completely disappeared in the full pipe. Recorded separately in their respective NaCl concentration. Concentration is higher than normal controls,
indicating increased osmotic fragility and vice versa decreases. Reposted elsewhere in the paper for free download http://
2.5 Determination of red cell membrane fluidity by fluorescence polarization method .
2.5.1 DPH (1,6 diphenyl 1,3,5 B triene)
solution, said preparation to take DPH4.05 mg, dissolved in 10 ml tetrahydrofuran, dubbed the concentration of 2 × 10-3mol /
L, with pH value of 7.35 in PBS buffer solution was diluted to 2 × 10-6mol / L.
2.5.2 Preparation of erythrocyte suspensions and fluorescent markers to take anti-clotting 1 ml, prepared
according to 2.3.1 of 0.4% erythrocyte suspension, 1 ml suspension obtained by adding DPH solution 1 ml, 25 ?
incubation 30 min, 3 000 r / min centrifugation 4 min, to the
upper fluid, washed with PBS buffer two times, both to the upper centrifugal liquid. Adding PBS buffer to make the final volume of 2 ml.
2.5.3 Determination of fluorescence polarization and calculation of pre-measured with 0.1 mol / L solution of
quinine sulfate calibration equipment. Using excitation wavelength 361 nm, emission wavelength 463 nm, incident and exit slit are used 10 nm, the polarizer when the excitation and emission light path optical axis of the polarizer parallel to each other, the measured fluorescence intensity ? 1, when
the two optical axis perpendicular to each other, the measured fluorescence intensity ? 2.
Degree of polarization (P) = (? 1 - ? 2) / (? 1 + ?
2). Greater degree of polarization, indicating that the
smaller the red cell membrane fluidity.
2.6 Statistical analysis The results are count data are amounting to ? s said that the statistical analysis used analysis of variance and q test, class rank and test data used.
3.1 Effect of Allicin on the impact of erythrocyte aggregation in acute high-viscosity model group compared with the control group, whole blood high shear and low shear viscosity, plasma viscosity, hematocrit were significantly higher (P <0.01 or P <0.05), tips acute copy the success of
stasis syndrome model. Compared with model group, allicin high, medium and low-dose group rats with blood stasis
syndrome high-shear whole blood viscosity were decreased by 14.43% (P <0.01), 9.71% (P <0.01), 4.15% (P <0.05) ; of low
shear whole blood viscosity were decreased by 15.89% (P <0.01), 14.27% (P <0.01), 6.86% (P <0.01); on plasma viscosity were decreased by 11.33% (P <0.05), 9.78% (P <0.05), 3.49% (P> 0.05), and these effects showed a definite dose-effect
relationship. Allicin can significantly reduce the high dose group hematocrit (P <0.05), reduce the degree of 6.47%; middle and low-dose group and this effect is not obvious. Allicin, the low-dose group, to reduce the role of erythrocyte aggregation, but not statistically significant. The results in Table 1.
Table 1 garlic on erythrocyte aggregation effects (abbreviated)
With the blank control group, * P <0.05; ** P <0.01; and acute high-viscosity model group, ? P <0.05; ? ? P <0.01
3.2 Effect of Allicin on the impact of red blood cell deformability results showed: acute high viscosity red blood cell filtration index model group than the control group significantly increased (P <0.05), shows that red blood cell deformability. The results in Table 2.
Table 2 Effect of Allicin on the impact of red blood cell deformability (omitted)
With the blank control group, * P <0.05, and acute high-
viscosity model group, ? P <0.05
3.3 Effect of Allicin on erythrocyte osmotic fragility of
high-viscosity model of acute hemolysis and fully hemolytic group began when the NaCl concentration slightly higher than the control group, indicating that the model group rats erythrocytes resistance to hypotonic salt solution is poor, increased osmotic fragility large, osmotic fragility to some extent reflects the red cell membrane viscoelasticity and affect red blood cell deformability. Allicin each dose group the increase of osmotic fragility of red blood cells to improve role, but there was no statistical significance. The
results in Table 3.
3.4 Effect of Allicin on erythrocyte membrane fluidity in model group of acute high viscosity of erythrocyte membrane was significantly higher degree of fluorescence polarization, indicating lower membrane fluidity. Allicin dose groups on
erythrocyte membrane fluidity decrease in the role of both improved, but no statistical significance. The results in Table 4.
Table 3 Effect of Allicin on osmotic fragility of red blood cells (omitted)
Table 4 garlic on erythrocyte membrane fluidity (omitted)
With the blank control group, * P