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마늘에 關한 實驗的 硏究

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 Experimental studies on garlic. 
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[영문] Garlic has been much used as a seasoning in Korea. Since Cavallito et al halve isolated allicin, a volatile substance from garlic, many workers have investigated it. Cavallito et al have reported, in diluted allicin solution, an antibacterial action against staphylococcus aureus, cholera vibrio, dysentery and so on. Rao and Gangley have found that garlic had a bacteriostatic effect on streptococcus hemolytics, gram positive and negative bacilli and acid fast bacilli, independently, Dittmore has reported that a volatile substance from garlic injured tumor cells, and Euler et al have reported that a skin tumor disappeared from intramuscular injection of 1∼3mg of allicin. Dubra et al have reported that garlic juice had shown an inhibitory action for growth against Aspergillus nigar and A. oryzae in 1:200 diluted solution, and against fusarium in 1:1,600 dilution. Kashimoto et at have reported that the water soluble substance of garlic (not alcohol soluble) depressed blood pressure in the rabbit and that this water soluble substance initially stimulated, later inhibited, and finally paralysed the respiration in mice. Damrau et al have reported that garlic concentrates depressed blood pressure in cats, and that allyl sulfide, which is a main component of garlic, had also depressed blood pressure. Nishi and Chung halve produced anemia from garlic, independently, Chung has reported that administration of 10 g per kg of body weight for 15 days in dogs produced a decrease of hemoglobin and of erythrocytes. Ohotani has reported that when 0.5g of garlic was taken orally for six months, hemopeietic function was retarded, while on the other hand 0.2g stimulated it. Lee has found an inhibitory action on blood coagulation in garlic. The effective substance was reported to be non-volatile. These facts might indicate that garlic may injure tissues and cells, especially the enzyme systems in the body.To test this hypotheses, the present investigation is a study of the effects of allicin on the Krebs citric acid cycle and the electron transport chain which are the most important metabolic pathways ill the body. It is also a study of its purification, the physical and chemical properties of the anticoagulant from garlic, and its inhibitory mechanism on blood coagulation. MATERIALS AND METHODS Preparation of allicin: allicin was prepared by the modified method of Cavallito. The yield was 0.2∼0.5 ml of allicin from 100g of garlic. Preparation of tissue homogenates: the normal albino rat(120∼200g of body weight) was killed by decapitation, and the desired organ (liver or heart) was quickly excised and immediately one gram of the tissue was weighed accurately on a torsion balance. The weighed tissue was dropped into a homogenizer tube which contained 9 ml of distilled water or Krebs Ringer phosphate buffer, pH 7.4. This 10% homogenate was dilluted to an appropriate concentration. The nitrogen content of this homogenate was determined by the method of micro-Kjeldhal. Chemical materials: all common chemicals were of analytical reagent grade. Diaphorase and DPNH from Calbiochem Lab. and cytochrome c from Mann Research Lab. have been used. DPNH-cytochrome reductase was prepared from pigeon breast muscle by the method of Edelhoch. Assay : oxygen consumption in tissues, succinic dehydrogenase (methylene blue added),succinic oxidase (cytochrome c added instead of methylene blue), cytochrome oxidase, and xanthine oxidase were measured manometrically, Total volume of the tested system was 3.0 ml. Diaphorase, DPNH-cytochrome c reductase and lactic dehydrogenase were measured spectrophotometrically. Mitochondria preparation and measurement of mitochondria swelling : the liver mitochondria were isolated in 0.33 M sucrose solution by a slight modification of Schneider's method. They were washed twice and suspended in 0.33 M sucrose solution. Swelling was followed by the changes in the optical density at 520mμ. Influence on the capillary permeability : capillary Permeability was determined by Miles and Miles method. Blood anticoagulant substance from garlic (G. E.-Garlic Extract) was prepared by the precipitation of the substance soluble in acid-water (but not in ether or alcohol) with 95% ethanol. The yield was abort 150mg from 200g of garlic. Prothrombin time was determined by the Quick's method and fibrinolytic activity was measured by the Astrup's method. The rabbits in ranges of 1.5∼2.5kg body weight were used in vivo test. G.E. was hydrolysed with 6 normal hydrochloric acid, and quantitative analyses and paper chromatography of inositol were determined by Hubscher's method. RESULTS 1. Effects on oxygen consumption in various tissues : oxygen consumption was inhibited by 17.3∼85.9%, and there was difference in sensitivity of tissues to inhibition by allicin. The heart was most resistant to allicin. 2. Effects on Krebs citric acid cycle : the intermediary products in the Krebs citric acid cycle, such as citrate, α-ketoglutarate, succinate, fumarate and palate showed inhibition of oxidation (15.5∼76.8%) by 0.05% allicin. 3. Effects on electron transport chain : succinic oxidate was inhibited (73.7∼84.4%) and cytochrome oxidase (20∼36.5%) by 0.05% allicin. DPNH-cytochrome c reductase was not inhibited. Neither were succinic dehydrogenase and diaphorase but xanthine oxidase was inhibited by it. But the coenzyme of these enzymes are a flavoprotein. Lactic dehydrogenase having DPNH as coenzyme wart not inhibited. These facts indicated that flavoprotein and DPNH themselves did not react with allicin. Cytochrome oxidase activity, inhibited by allicin, was not returned by adding cystine, which would mean SH group of the enzyme did not react with allicin. Allicin was a competitive inhibitor of cytorhrome oxidate. 4. Effect on mitochondria swelling : the inhibited mitochondria swelling by allicin did not returned with ascorbate. 5. Effect on capillary permeability : allicin increased capillary permeability to trypan blue in the rat skin. Pyribenzamine, an antihistamine drug, did not reduce the increased permeability by allicin. This would mean that histamine liberation was not, involved in the mechanism. 6. Effect on blood coagulation : blood coagulation time was prolonged by G.E. and its potency was the same as with potassium oxalate. 0.5mg of G.E. inhibited completely 1 ml of blood. Plasma, obtained from 1 ml of blood containing 5mg of G.E., prolonged prothrombin time. Various amounts of calcium chloride were added to the plasma and prothrombin time was determined. With an increase of calcium chloride in the plasma containing G.E., the prothrombin time was shortened, but not that in oxalated plasma. At the level of 1.5 or 2.5mg of G.E. increasing amount of calcium chloride prolonged the prothrombin time. Antithrombin and fibrinolytic effects were shown. 10 mg of human fibrin was partially dissolved with 5 mg G.E. and completely dissolved with 10∼25mg of G.E. In vivo tests the rabbits were killed by the intravenous injection of 100∼150mg of G.E., and the blood from the heart of dead rabbits showed prolongation of blood coagulation time, but was within normal limit of prothrombin time. 70mg G.E. did not prolong coagulation or prothrombin time. 7. Physical and chemical properties of G.E. : G.E. was white solid and water soluble(but not ether or alcohol soluble). It showed hygroscopic, weak acid reaction and was positive in ninhydrin test. 1% solution of G.E. showed 38 osmolarity. G.E. sodium salt consisted of 18.7% of phosphorus, 0.2% of nitrogen, 15.7% of sodium and a trace of sulfur. The acid hydrolysate contained inositol, which was confirmed by the paper chromatography and the consuming of periodate. The ratio of inositol, phosphorus and sodium of G.E. sodium salt was 1:6 : 6.7. This substance may suggest to be photic acid. SUMMARY Effects of allicin on oxygen consumption in various tissues, electron transport chain, Krebs citric. acid cycle, mitochondria swelling and capillary permeability were studied. Also isolation, physical and chemical properties of blood anticoagulant from garlic and its mechanism of anticoagulation were investigated. 1. Allicin inhibited oxygen consumption in various tissues (liver, heart, or diaphragm),and among these the heart was most resistant to inhibition. 2. The oxidation of intermediary products in the Krebs citric acid cycle were inhibited by allicin in rat liver slices. 3. Allicin inhibited succinic oxidase, cytochrome oxidase and xanthine oxidase, but did not inhibit DPNH-cytochrome c reductase, succinic dehydrogenase, diaphorase or lactic dehydrogenase. On the basis of these facts, the allicin may act somewhere between cytochrome b and cytochrome oxidase. Additional cystine did not return allicin-inhibited cytochrome oxidase activity, and allicin was a competitive inhibitor of cytochrome oxidase. 4. No inhibitory effects were found on oxygen consumption of tissues, succinic oxidase and cytochrome oxidase in vivo test. 5. Allicin-inhibited mitochondria swelling was not returned by ascorbate in rat liver. 6. Allicin increased capillary permeability of trypan blue in the rat skin. Pyribenzamine, an antihistamine drug, was not blocked this action. 7. A blood anticoagulant substance was isolated from garlic, and its physical and chemical properties and its mechanism of anticoagulation were studied. a. Clotting time was prolonged and its potency was the same as with potassium oxalate. Prothrombin time was prolonged. b. Antithrombin and fibrinolytic effects were found but an anti-Ac-globulin effect was not shown. c. At the level of 1.5 or 2.5mg of the anticoagulant, increasing of the amount of calcium chloride still prolonged the prothrombin time. d. No effect was found in vivo tests. e. The anticoagulant consisted of 18.7% phosphorus, 0.2% nitrogen, 15.7% solium and a trace of sulfur. It also contained inositol. The ratio of inositol, phosphrous and sodium was 1:6:6.7. This anticoagulant may suggest to be phytic acid .
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