Effects Of Extraction Of Pomegranate Peel On Physiological Characteristics Of Blood Cells

Polyphenol compounds widely exist in leaves, fruit, seeds, roots of pomegranate, and thehighest content is in pomegranate peel, up to 10-21 %. Previous researches showed thatgranatum polyphenols have a variety of pharmacological effects such as anti-tumor, antioxidant,bactericidal , anti-inflammatory. However, it has not been reported the effects of granatumpolyphenols on physiological characteristics of blood cells. In this study, we preparedpolyphenols crude extracts from pomegranate peel by the method of solvent extraction indifferent temperature, and finally studied the effects of polyphenols on erythrocyte fragility,erythrocyte sedimentation rate, bleeding time and anticoagulation, in order to offer a new idea ondevelopment and utilization of granatum polyphenols medicinal value and antithrombotic drugs.The research was divided into four parts:1. Preparation of polyphenols crude extracts and except polyphenols extracts frompomegranate peel by using different extracting conditions. The extraction condition ofpolyphenols from pomegranate peel are as follows: 1:30 the ratio, 1time/7h, total 2 times anddissolvent of aqueous in low temperature, or dissolvent of ethanol at medium temperature1,medium temperature2, high temperature, respectively. In addition, the extraction condition ofexcept polyphenols from pomegranate peel is the ratio 1:20（w/v）, 3h, ethanol as well as a certaintemperature. Crude extracts yield rate = polyphenol crude extracts mass/ raw materialmass×100%, the gallic acid was used as a standard compound, the total polyphenol content ofpomegranate peel was quantitated by measurement of the absorbance at 765nm with Folin -Ciocalteu reagent, its pH value was determined by accurate pH paper. The results were asfollows: the rates of aqueous extract and ethanol extract in turn were 39.30%, 38.10%, 38.40%,42.10%; the pH value of aqueous extract was 3.8, ethanol extract’s was 4.1, the pH value was nodifference among alcohol extraction at different temperatures; the polyphenols purity of crudeextracts were 27.94%, 24.44%, 25.35%, 27.87%, respectively. In addition, polyphenols were notdetected in except polyphenols extract of pomegranate peel. 2. The influence of pomegranate peel polyphenols on erythrocyte fragility, erythrocytesedimentation rate, bleeding time and anticoagulation. It was studied that the influence ofgranatum polyphenols on blood coagulation by vitro （ 3.8% ） and intragastric （ig） （50mg/kg） twoadministration methods by test tube method; to observe the impact of polyphenols on thebleeding time, we determined the bleeding time of ear-vein by ig （50, 500mg/kg）; we conductedthe ESR by using Wechsler’s test with vitro administration（0.5, 5, 50mg/ml） in order to observethe effect of polyphenols on the ESR; to study the impact of polyphenols on erythrocyte fragility,we determined it by concentration gradient of NaCl. These results showed granatum polyphenolssignificantly prolonged coagulation time by vitro or ig administration, decreased ESR （P<0.01）;high-concentration polyphenols can significantly shorten BT（P<0.01） while low-concentrationpolyphenols had no significant effect on BT and erythrocyte fragility（P>0.05）, compared withcontrol group. The above results suggested granatum polyphenols in certain dosage range havethe effects of anticoagulation, increasing the suspension property of cells, shorten BT while noeffects on erythrocyte fragility.3.The establishment of rabbit carotid artery thrombus model induced by FeCl₃. Thehealthy animals were divided randomly into normal saline group（30min） and three FeCl₃groups（FeCl₃for 30min, FeCl₃for 15min and FeCl₃for 10min） after local treatment, each group had 3animals. 60%FeCl₃-soaked Whatman paper applied to the bilatera exposed common cartid arteryfor different times to observe the weight of thrombus, detect size and density of thrombus bypathological section, determine the extent of thrombosis in the rabbit carotid artery, then selectoptimum animals model for antithrombotic drug experiments. It was found that thrombus wasapproximate to obliterans treated by FeCl₃treatment for 15 min, and more sensitive for drugsresponsing to thrombosis.4.The effect of pomegranate peel polyphenols on thrombosis induced by FeCl₃inrabbit arteria carotis communis. The healthy animals were randomly divided into pathologicalslices group and ultrasonography group, pathological slices group was divided into 5 subgroups:control I group（ig of double distilled water （DDW）+NS 15min ）, control II group （ig ofDDW+FeCl₃15min ） , control III group（ig of polyphenols+NS 15min ）, experimental I group（ igof polyphenols+FeCl₃15min ）; experimental II group（ ig of except polyphenols extract+FeCl₃15min）, ultrasonography group was almost the same as pathological slices group, except decreasing control （?） group and increasing high-concentration experimental group itscorresponding control group, each subgroups had 3 animals. In pathological slices group, generalanesthesia was induced by urethane, separated the bilateral common carotid artery afterintragastric administration 1h, then induced by FeCl₃for 15min, fixed, sliced, HE staining; at last,observed thrombus density, arterial lumen diameter and arterial wall thickness. Inultrasonography group, detected blood flow velocity , resistance index and heart rate of eachgroup before and after treated with FeCl₃for 15min by two-dimensional ultrasound. The resultsshowed that granatum polyphenols made thrombus density decreased, arterial lumen diameterincreased （P<0.05）, and vessel wall thinning（P>0.05） in pathological slices group compared withcontrol group. It suggested that granatum polyphenols could induce the angiectasis, protectvascular endothelial and inhibit thrombosis; compared with pre-treatment, systolic peak velocitybecame quickly, end diastolic velocity had no obviously change, and resistance index （RI） wasnot significantly increased （ P> 0.05 ）, heart rate was not markedly increased（P>0.05） in controlII group; resistance index increased, blood flow and heart rate became faster in experimental Igroup, all of them had no obvious change; the result of experimental II group was similar tocontrol II group, it showed that granatum polyphenols had inhibition on arterial thrombosisinduced by FeCl₃.In a word, granatum polyphenols that was extracted in different condition have inhibitionon coagulation, the best one was extracted by alcohol at medium temperature. Granatumpolyphenols which was extracted by alcohol at medium temperature can increase cellularlsuspensibility, decrease BT of ear vein, except that ,it had the effect of reducing size and densityof carotid artery thrombus induced by FeCl₃,hemangiectasis, and decreasing peak velocity. Tosummarize, granatum polyphenols play the role of inhibition of thrombosis. The mechanism ofGranatum polyphenols antithrombotic needs further study.