Endocrinol. Japon. 1970, 17 (5), 339,-348
Effects of Glycyrrhizin and Cortisone on Cholesterol Metabolism in the Rat
MASAHIROYAMAMOTO, NOZOMU TAKEUCHI, SATORUKOTANI AND AKIRA KUMAGAI The Third Department of Internal Medicine, School of Medicine, Osaka University, Fukushima-ku, Osaka
Synopsis
Effects of glycyrrhizin, an active principle of liquorice, on cholesterol metabolism, and its correlation with cortisone action were investigated using adrenalectomized rats.
(1) Incorporation of acetate-1-14C and mevalonate-5-3H into cholesterol of liver slices was greatly stimulated by glycyrrhizin pretreatment, i.e., 1-10mg esp. 1mg per 100g body weight per day intramuscularly for 5 days. Combined administration of glycyrrhizin with cortisone even weakened the stimulatory action of glycyrrhizin, although cortisone itself had a stimulatory action on cholesterogenesis. (2) Direct addition of glycyrrhizin in vitro to the incubation media containing liver slices of normal rats also increased cholesterogenesis at the concentration of 10-5-10-3M. (3) Excretion of intravenously injected cholesterol-4-14C into bile of bile duct-cannulated rats was much accelerated by
glycyrrhizin pretreatment, 2mg per 100g body weight per day intramuscularly for 5 days. Rates of increase of radioactivities excreted into bile by glycyrrhizin pretreatment were 4.5 times, taurocholic acid; 2.8 times, taurochenodeoxycholic acid; 2.7 times, cholesterol; and 3.7 times, total 14C, respectively. (4) Fecal excretion of intraperitoneally injected cholesterol-4-14C was also stimulated by glycyrrhizin pretreatment. Total 14C and total bile acids-14C were increased up to about 2 times, while non-saponifiable materials-14C and 3ƒÀ-OH-sterols-14C remained within 1.5 times. (5) Elimination of intravenously injected cholesterol-4-14C from plasma was accelerated by glycyrrhizin injection. (6) Glycyrrhizin treatment reduced plasma cholesterol and triglycerides levels of cholesterol diet fed rats.
Kumagai and his colleagues (Shibata, 1962) sone (Kumagai et al., 1966a), and the corti- previously reported that the administration of coid itself influenced the cholesterol metabo- a small amount of glycyrrhizin (1mg/kg body lism (Kumagai et al., 1966b). weight) to the rabbits fed a lanolin-rich diet, The present experiments, therefore, were inhibited the elevation of plasma cholesterol designed to investigate the effects of gly- level and the development of aortic atheroma. cyrrhizin on the cholesterol biosynthesis and They also showed that glycyrrhizin increased turnover in adrenalectomized rats in com- both bile acid excretion and cholesterol ex- parison with that of the corticosteroid, and cretion into the bile and feces. those showed that the glycyrrhizin stimulated Glycyrrhizin was considered to have corti- directly the hepatic cholesterogenesis both in coid-like action, presumably due to inhibitory vivo and in vitro, increased biliary and fecal effect on the degradation of corticosteroids in excretion of cholesterol and bile acids and the liver (Kumagai et al., 1957), and later it accelerated blood cholesterol turnover. was reported that glycyrrhizin had also in.- hibitory effects on metabolic actions of corti- Materials and Methods Received for publication October 5, 1970. Presented in part at the 43rd Annual Meeting of the Treatment of animals Japan Endocrinological Society, Osaka, March, 1970. Male rats of Wistar strain, weighing about 150g, Endocrinol.Japon. 340 YAMAMOTO et al. October 1970
were fed a commercial laboratory chow diet (Purina) Krebs-Ringer-phosphate buffer (pH7.4) added either ad libitum before and during the experimental period . 5ƒÊCi of sodium acetate-1-14C (specific activity 9.7 The animals were devided into the following 6 groups: mC/m mole, Daiichi Pure Chemicals Co., Tokyo) or OperationInjection 2.5ƒÊCi of DL—mevalonic acid-5-3H (specific activity (intramuscular) 3.75 mC/m mole, New England Nuclear Corp., Group I Non-adrenalectomized Saline Boston, Mass). and 20 moles of glucose. Incubation II Adrenalectomized (adx.) Saline was carried out for 2hr in a 95% 02-5% CO2 atmos- III Adx.Cortisone 1mg/100 phere at 37•Ž in a Dubnoff metabolic shaking in- g b.w./d., 5d. cubator. At the end of the incubation period, 2ml of IV Adx.Cortisone 1mg, 5% alcoholic KOH solution was added to each flask, Glycyrrhizin 1mg the content of which was hydrolyzed for 2hr at 65•Ž. V Adx.Cortisone 1mg, From the hydrolysates, non-saponifiable materials Glycyrrhizin 10mg were extracted 4 times with petroleum ether and the VI Adx.Glycyrrhizin 10mg extract was washed 5 times with water (Kumagai et Adrenalectomy were performed under ether anes- al., 1966b) and cholesterol fraction was separated by thesia. On the following day, all animals were given digitonization. The cholesterol fraction precipitated intramuscular injection of either saline, glycyrrhizin by digitonization was washed once with acetone- ether (1:2) mixture, twice with ether and dissolved in (Minophagen Pharmaceutical Co., Tokyo), cortisone acetate (Schering & Co., Berlin) or both glycyrrhizin 2ml of methanol. An aliqout of the methanol solution and cortisone, and the injection continued once a day was used for cholesterol determination. Another for the following 5 days. The daily amounts of drugs aliquot was assayed for the radioactivity using a used, were cortisone 1mg, glycyrrhizin 1mg or 10mg scintillator consisted of 5g PPO and 300mg POPOP in 1l of toluene by a Packard liquid scintillation per 100g of body weight of animals in water suspen- spectrometer model 3003 with correction of th sion. On the 6th day, i.e., 12hr after the last injection , all animals that had been fasted for the last 12hr* quenching by the channel ratio method. were sacrificed by decapitation. Blood was collected Student T test for statistical analysis was applied by cardiac puncture and the plasma was separated for for all values obtained in this report. cholesterol determination according to the method of Free sterols were regenerated from the digitonides Leffler (1960). Livers of the animals were taken out according to Sperry (1963). The precipitate of sterol immediately after sacrifice, perfused with cold saline digitonides was dissolved in pyridine and free sterols from portal vein and sliced with a blade for incubation were extracted with diethyl ether. After being dried studies. in vacuo, the free sterols were redissolved in chloro- The effect of glycyrrhizin treatment on hyper- form. Gas liquid chromatography of standard cho- cholesteremia and hyperlipemia of the rats produced lesterol, cholestanol, and lanosterol as well as that of by cholesterol feeding was investigated. Twelve rats tissue sterols was carried out in a F & M high efficiency eas chromatouranh model 402 (Hewlett-Packard F were fed a laboratory chow diet (Powdered "MF" of & M ScientificDiv.,Avondale,Pa.).The 6ft column Oriental Yeast Co., Tokyo) containing 1% cholesterol was packed with Gas-chrom S coated with 1% QF-1 and 0.5 % cholic acid for 2 weeks, during which rats . were intramuscularly injected with either 1 mg of The gas phase, argon. The column temperature, 200•Ž. A half of the effluent was trapped with Pasteur pipettes. glycyrrhizin per 100g body weight per day or saline. Triglycerides and non-esterified fatty acids (NEFA) The inside of the pipettes was washed with ether and were determined by the methods of Fletcher (1968) the radioactivities of corresponding peaks were de- and Dole (1956). termined (Table 5).
The in vitro incorporation of acetate-1-14C or The in vivo incorporation of acetate-1-14C into mevalonate-5-3H into cholesterol in the liver cholesterol in liver of bile duct-ligated rats (Table (Table 2, 3, 4 and 5) 6) Liver slices of about 250mg were prepared and Twelve rats, weighing about 150g, had their bile placed in each flask containing a 2.5ml of 0.1M ducts ligated immediately after adrenalectomy. Either 1mg or 10mg of glycyrrhizin per 100g of body weight was injected intramuscularly into 8 of them daily for * Fasting for 24hr suppressed hepatic cholester - 5 days and the other 4 rats served as control. ogenesis but almost no suppression was found by On the 6th day of treatment, 12hr after the last 12hrs' fasting. Fasting was needed to minimize the injection of glycyrrhizin, all animals were given in- effect of dietary cholesterol on hepatic cholester- travenously 10ƒÊCi of sodium acetate-1-14C per 100g ogenesis. (Table 2) body weight, and sacrificed by decapitation 30 min Vol.17, No.5 GLYCYRRHIZIN AND CHOLESTEROL METABOLISM 341
after the isotope injection. Cholesterol concentration chromatography on Silica gel H, 250ƒÊ thick. Develop- in the plasma and liver, as well as the incorporation ing solvent system was isoamyl alcohollacetic acid- of labeled acetate into both hepatic total lipids and water (18:5:2) according to Hara et al. (1964). Spots cholesterol were determined. One g of liver or one ml were detected with iodine vapor, the adsorbent was of plasma from the animals was treated with 25ml of scraped off. Bile acids were eluted with 2% acetic chloroform-methanol (2:1) mixture using a glass acid in methanol and cholesterol was eluted with homogenizer and the extract was washed twice with chloroform-methanol (1:1). Radioactivities of these water and twice with water-methanol-chloroform fractions were determined by a Nuclear Chicago liquid
(46: 47: 3) mixture and evaporated to dryness under scintillation counter Mark I. Counting efficiency and N2 at 40•Ž. An aliquot of the chloroform-methanol quenching were corrected by the external standard method. The excretory rate of each fraction into bile solution of the extract was used for the determination was calculated as follows: dpm in bile/dpm of cho- of radioactivity of total lipids. Saponification and lesterol-4-14C injected. cholesterol extraction were carried out, as mentioned
previously.
The excretion of injected cholesterol-4-14C and
In vitro effect of glycyrrhizin on cholesterogenesis its metabolites into feces (Table 9)
in normal rat liver slices (Table 7) Ten rats weighing about 150 g were devided into Livers of healthy normal rats were cut into slices. 2 groups. Daily injections of glycyrrhizin and saline Each slice weighing about 500mg was incubated in were carried out for 5 days as described in the experi- 2.5ml of 0.67M phosphate buffer containing 5ƒÊCi of mental procedures about the excretion of cholesterol sodium acetate-1-14C, various amounts of glycyrrhizin into bile. On the fourth day and 4 hr after the fourth
(0.01, 0.03, 0.1, 0.8 and 8mg per ml of incubation treatment of glycyrrhizin, 0.25ƒÊCi of cholesterol-4-14C mixture respectively and 20ƒÊ moles of glucose. per 100g body weight was intraperitoneally injected The incorporation of the labeled acetate into total and then feces were collected for 48hr. Feces was lipids and cholesterol was determined using the same homogenized in an equal weight of water and refluxed incubation method described above. Watery solution in 10 volumes of hot ethanol for 24hr. An aliquot of of glycyrrhizin was the gift from Minophagen Phar- the ethanol extract of feces was used for determination maceutical Co., Ltd., Tokyo. of the total 14C excreted. Another aliquot of the ethanol extract of the feces was made alkaline by addition of one fifth volume of 10N KOH and saponi- The excretion of injected cholesterol-4-14C and fied at 2 atmosphere for 2hr.The non-saponifiable
its metabolites into bile (Table 8) materials were extracted 3 times with 5 volumes of
Ten rats weighing about 150g were devided into 2 petroleum ether. The extract was mixed and washed groups. Five male rats were given intramuscular in- 4 times water. 3ƒÀ-Hydroxysterols were precipitated by jections of 2mg of glycyrrhizin per 100g body weight usual digitonin method and the precipitates were per day for 5 days. The other 5 rats were given saline washed with acetone-ether (1:2) and with ether. injection correspondingly. Three hr after the last After the extraction of the non-saponifiable materials, injection, bile ducts of all animals were cannulated the water phase was acidified to pH1 with concen- under hexobarbital anesthesia. One hour after the trated HCl and total bile acids were extracted 3 times operation, 0.25ƒÊCi of cholesterol-4-14C* (specific by 5 volumes of diethyl ether. The extract was mixed activity 30mC/m mole, The Radiochemical Centre, and washed 4 times with water. England) per 100g body weight was intravenously injected and then bile was collected for 24hr. An aliquot of bile was used for the determination The effect of glycyrrhizin and cortisone on the
of radioactivity of total 14C excreted. An equal volume disappearance of cholesterol-4-14C from the of ethanol was added to bile and centrifuzed. After peripheral blood (Table 10) the supernatant fraction was evaporated, the residue Adrenalectomized rats weighing about 150g were
was dissolved in ethanol-chloroform (5:2). Tauro- given intramuscular injections of either glycyrrhizin cholic acid, tauro-dihydroxy-cholanic acids, glyco- 10mg, cortisone 1mg per 100g body weight or saline cholic acid and sterols were separated by the thin-layer daily for 8 days. On the 6th day, all animals were intraperitoneally administered 1ƒÊCi of cholesterol- 4-14C per 100g body weight and were sacrificed on * The radiochemical purity of cholesterol-4-14C was the 8th day or 48hr after the isotope injection. Total checked by paper chromatography before use. The lipid fraction from one ml of the plasma of the animals purity was found to be 99.3%. Cholesterol was was used for determination of cholesterol radioactivity suspended in saline with Tween 80. which remained in the plasma. Endocrinol. Japon. 342 YAMAMOTO et al. October 1970
Table 1. Effects of glycyrrhizin and/or cortisone administration on the plasma lipid levels of normal diet fed adrenalectomized rats and of cholesterol diet fed non- adrenalectomized rats
1% cholesterol and 0.5% cholic acid diet fed
* Mean •} standard error ** ns: Non-significant Adx.: Adrenalectomized
Effects of the cortisone and/or glycyrrhizin ad- ministration on the in vitro incorporation of Results acetate-1-14C or mevalonic acid-5-3H into chole- sterol by the liver-slices of the adrenalectomized Effects of the cortisone and/or glycyrrhizin ad- rats (Table 2, 3, 4 and 5) ministration on the plasma cholesterol level of Rats of the cortisone-treated group (group III) normal and cholesterol diet fed rats (Table 1) showed a significant increase in the incorporation of There were no significant differences in the plasma acetate-1-14C into both non-saponifiable materials cholesterol levels among experimental groups fed a and total cholesterol, compared with the adrenalecto- normal diet. Hypercholesteremia and hypertriglyceri- mized rats (group II)(Table 3). Glycyrrhizin markedly demia produced by cholesterol feeding were much increased the incorporation of the labeled precursor improved by glycyrrhizin treatment. On the contrary, into both fractions, the higher extent being in the total no change was observed in NEFA. cholesterol fraction. In the rats of the glycyrrhizin- Vol.17, No.5 GLYCYRRHIZIN AND CHOLESTEROL METABOLISM 343
Table 2. The effect of fasting on hepatic cholester- was mainly derived of cholesterol in both adrenal- ogenesis from 5ƒÊCi of acetate-1-14C by liver slices ectomized controls and adrenalectomized, glycyrrhi- of non-adrenalectomized rats (250mg, 2hr) zin-treated rats as shown in Table 5. Table 4 shows the result of the incorporation into the two fractions using mevalonic acid-5-3H. Ad- renalectomy (group II) resulted in a decrease of the incorporation, while cortisone administration to ad- renalectomized rats (group III) restored or even in- creased the incorporation of the labeled mevalonic acid into total cholesterol. Glycyrrhizin treatment (group VI) caused a greater increase in the incorpo- ration than cortisone treatment and 5.7 and 47.5 fold * Mean+standard error ** Non-significant greater incorporation respectively into both non- *** p<0 .05 saponifiable and total cholesterol fractions was ob- tained by glycyrrhizin treatment compared with con- trols. The specific activity of total cholesterol of group treated group (group VI), the radioactivities in both VI was 33.0 times greater than that of group II. Thus fractions were respectively 48 times and 99 times both glycyrrhizin and cortisone influenced much more greater than those of the adrenalectomized rats (group the incorporation of mevalonate into the total cho- II), and the specific activity of the total cholesterol of lesterol fraction than into the non-saponifiable ma- the former group was 67.4 times higher than that of terials (Table 4). It might be suspected that glycyrrhizin the latter one. Through the combined treatment of and cortisone accelerated cholesterol biosynthesis via cortisone and glycyrrhizin (group IV & V), the in- various intermediates. corporation of labeled acetate into both fractions was between those in group III & VI. The radioactivity of 3ƒÀ-hydroxysterols precipitated The effect of the glycyrrhizin on the in vivo in- from the non-saponifiable materials as the digitonides corporation of acetate-1-14C into cholesterol in
Table 3. Effects of glycyrrhizin and/or cortisone administration on the incorporation of acetate-1-14C into non-saponifiable materials and total cholesterol by slices of livers (250mg) of adrenalectomized rats
* Mean standard error **ns: Non-significant Adx.: Adrenalectomized Endocrinol. Japon. 344 YAMAMOTO et al. October 1970
Table 4. Effects of glycyrrhizin and/or cortisone administration on the incorporation of mevalonic acid-5-3H into non-saponifiable materials and total cholesterol by slices of livers (250mg) of adrenalectomized rats
* Mean•}standard error ** ns: Non -significant Adx.: Adrenalectomized
Table 5. Sterol synthesis in vitro from acetate-1-14C (5ƒÊCi) by liver slices of control and gly- cyrrhizin-treated rats (250mg): Result of gas-liquid chromatographic analysis
Values are averages of 3 samples. the liver of adrenalectomized rats with bile duct A smaller amount of glycyrrhizin (0.03-0.8mg/ml ligation of incubation medium) increased hepatic cholester- As shown in the Table 6, the in vivo hepatic lipo- ogenesis and lipogenesis in vitro and maximum values genesis and cholesterogenesisalso increased through a were attained at 0.1mg/ml. The increase of hepatic small dose of glycyrrhizin, though the effect on the cholesterogenesis by the addition of glycyrrhizin in cholesterogenesis was not so remarkable as that ob- vitro was not so marked as that observed in the in vivo served in the adrenalectomized rats without bile duct experiment using rats pretreated with glycyrrhizin. ligation. The effect of glycyrrhizin administration on ex- The effect of the direct addition of glycyrrhizin cretion of intravenously injected cholesterol-4- to the incubation medium on the hepatic choles- 14C and its metabolites into bile terogenesis (Table 7) The radioactivities of bile acids-14C and choles- Vol.17, No.5 GLYCYRRHIZIN AND CHOLESTEROL METABOLISM 345
Table 6. The effect of glycyrrhizin administration on the in vivo incorporation of acetate—1-14C into total lipids and cholesterol in the liver of adrenalectomized, bile duct-ligated rats
* Mean•}standard error** Non-significant*** p<0.01**** p<0.05
Adx.: Adrenalectomized
Table 7. The effect of the direct addition of glycyrrhizin to the incubation medium on cholesterogenesis in vitro in liver slices of the rat
* Mean•}standard error** Non-significant*** p<0.05 ****p<0.01***** p<0.001
terol-14C excreted into bile after cholesterol-4-4C rats. injection were shown in Table 8. The volume of bile The biliary excrtion of radioactive cholesterol and collected for 24hr after isotope injection was not its metabolites was greater in the first 12hr of bile changed by glycyrrhizin treatment. Radioactivities of collection than in the second 12hr in both normal and total 14C, taurocholic acid, taurochenodeoxycholic glycyrrhizin-treated groups. acid and sterols excreted into bile, however, were much increased by glycyrrhizin pretreatment. The rate The effect of glycyrrhizin administration on ex- of increase by glycyrrhizin in radioactivity of total 14C cretion of intraperitoneally injected cholesterol- excreted was 3.7; of taurocholic acid, 4.5; tauro- 4-14C and its metabolites into feces chenodeoxycholic acid, 2.8; sterols*, 2.7, respectively. The radioactivities of bile acids-14C and sterols- The ratio of taurocholic acid vs. taurochenodeoxycho- 14Cexcreted into feces after cholesterol-4-14C injection lic acid vs. cholesterol expressed as radioactivities ex- were shown in Table 9. The weight of feces collected creted into bile of normal rats was 10:1:3, while it for 48 hr after radioactive cholesterol-4-14C injection increased up to 16:1:3 in that of glycyrrhizin-treated was not changed by glycyrrhizin treatment. Radio- activities of total 14C,total bile acids-14C,non-saponi- * Gas chromatographic analysis showed that this fiable materials-14C and 3β-OH-sterols-14C excreted fraction mainly consisted of cholesterol. into feces, however, were much increased by glycy- Endocrinol. Japon. 346 YAMAMOTO et al. October 1970
Table 8. The effect of glycyrrhizin administration on excretion of intravenously injected cholesterol-4-14C and its metabolites into bile collected for 24hr in bile duct-can- nulated rats
* Mean•}standard error** Non-significant*** p<0 .05**** p<0.005 ***** p<0 .001•õ Percentage of dpm excreted/dpm injected
Table 9. The effect of glycyrrhizin administration on excretion of intraperitoneally injected cholesterol-4-14C and its metabolites into feces of rats collected for 48 hr
* Mean•}standard error** Non-significant *** p<0 .05**** p<0.01 ***** p<0 .001 f Percentage of dpm excreted/dpm injected
rrhizin treatment. The rates of increase by glycyrrhizin In comparison with the radioactivity recovered in radioactivities of both total 14C and total bile acids- from the plasma of the control group, glycyrrhizin 14C were about 2 fold but those of non-saponifiable treatment significantly decreased the radioactivity. On
materials-14C and 3ƒÀ-OH—sterols-14C remained within the other hand, cortisone appeared to increase the 1.5 fold. The analytical data as to feces collected for radioactivity, but there were no statistical differences 96 hr after cholesterol-4-14C injection showed the between control animals and cortisone-treated ones. similar stimulatory effect of glycyrrhizin on the fecal excretion of cholesterol.
The effects of glycyrrhizin and cortisone on the Discussion
elimination of cholesterol-1-14C from blood
(Table 10) As we reported previously, cortisone in- Vol.17, No.5 GLYCYRRHIZIN AND CHOLESTEROL METABOLISM 347
Table 10. The effect of glycyrrhizin or cortisol administration on the elimination of cholesterol-4-14C from peripheral blood
* Mean•}standard error** Non-significant*** p<0.05 Adx.: Adrenalectomized
creased biliary excretion but slightly decreased may be explained by the fact that the liver fecal excretion of both bile acid and 3ƒÀ-OH- treated with a large dose contained more cho- sterols in the rats after intraperitoneal injec- lesterol than that treated with a small dose, tion of cholesterol-4-14C. Cortisone was also presumably due to less excretion of bile acids reported to accelerate gastrointestinal absorp- and cholesterol into the bile and feces (Shiba- tion of cholesterol-4-14C and cholic acid-4- ta, 1962). It is also conceivable that a smaller 14C . Incorporation of acetate-1-14C into rat stimulatory effect on cholesterogenesis ob- liver cholesterol in vivo was also increased by tained with combined administration of corti- the cortisone when determined 15 min after sone plus a large dose of glycyrrhizin com- labeled acetate injection (Kumagai et al., pared with a large dose of glycyrrhizin is due 1966b). Shibata (1962) demonstrated that a to an increased gastrointestinal absorption of small amount of glycyrrhizin increased both cholesterol by cortisone (Kumagai et al., bile acids and cholesterol excretion into both 1966b) and a relatively smaller stimulatory bile and feces in the rabbits fed a lanolin-rich effect of a large dose of glycyrrhizin on biliary diet. excretion of cholesterol and bile acids than a In this paper, it is reported that intramus- small dose of glycyrrhizin (Shibata, 1962). cularly administered glycyrrhizin and corti- As shown in table 6, when a high level of sone had a stimulatory effect on the hepatic cholesterol in both plasma and liver was cholesterogenesis in vitro in the rats, and the produced by bile duct ligation, in vivo in- effect was much greater in the glycyrrhizin- corporation of acetate into cholesterol did not treated than in the cortisone-treated. Total increase by glycyrrhizin treatment so much as cholesterol content in the liver of animals in the non-ligated animals. Cholesterol bio- treated with cortisone plus a small dose of synthesis was reported to be enhanced by the
glycyrrhizin did not differ from that of the bile duct ligation notwithstanding high cho- control animals. This may presumably be due lesterol and bile acid contents in both liver and to increased excretion of bile acids and cho- plasma (Fredrickson et al., 1954; Weis and lesterol into bile and feces, irrespective of the Dietschy, 1969). The ligation of the bile duct stimulated cholesterol biosynthesis in the liver masked the stimulatory effect of glycyrrhizin treated with cortisone plus glycyrrhizin. The on the biliary excretion of cholesterol and bile combined treatment of cortisone and gly- acids which might reflect on hepatic cho- cyrrhizin showed a difference in cholester- lesterogenesis. ogenesis in the liver depending on the amount The in vitro addition of glycyrrhizin stimu- of glycyrrhizin used, a smaller effect being lated cholesterogenesis in the liver to a small obtained with a larger dose of the drug. This extent. It could be suspected, therefore, that Endocrinol. Japon. 348 YAAMMOTO et al. October 1970 glycyrrhizin might be relatively easily acces- sible to the metabolic site within the cells or might affect some mediater(s) which originate Acknowledgement from the cell membrane and regulate cho- lesterogenesis. It is possible that the accumu- The authors are indebted to Prof. Yuichi Yama- mura, The Third Department of Internal Medicine, lation of metabolic products within the cells Osaka University for his helpful suggestion and to Miss in in vitro studies might cause the smaller Hiromi Katoh for her technical assistance' effect of glycyrrhizin on cholesterogenesis, compared with that shown in the experiment where glycyrrhizin was administered in vivo. References Direct demonstration was made in this paper as to the stimulatory effect of glycyrrhi- Dole, V. P.(1956). J. Clin. Invest. 35, 150. zin on the biliary and fecal excretion of cho- Eriksson, S.(1957). Proc. Soc. Exptl. Biol. lesterol and bile acids after labeled cholesterol Med. 94, 578. injection (Table 8 and 9). The rates of increase Fletcher, M. J.(1968). Clinica Chimica Acta by glycyrrhizin pretreatment in radioactivities 22, 393. excreted into bile were greater in taurocholic Fredrickson, D. S., A. V. Lond, B. T. Hinkel- acid than in either taurochenodeoxycholic man, H. S. Schneider and I. D. Frantz, Jr. acid or cholesterol. The rates of increase by (1954). J. Exp. Med. 99, 43. glycyrrhizin in radioactivities excreted into Hara, S., M. Takeuchi, M. Tachibana and G. feces were also larger in total bile acids than Chihara (1964). Chem. Pharm. Bull.(Tokyo) in cholesterol. 12, 483. It was reported that thyroxin which had a Kumagai, A., M. Otomo, S. Yano, N. Take- hypocholesteremic effect decreased cholic acid uchi, K. Nishino, H. Ueda and M. Kita- and increased chenodeoxycholic acid excretion mura (1957). Endocrinol. Japon. 5, 122. into bile (Eriksson 1957). It is conceivable, Kumagai, A., K. Nishino, M. Yamamoto, M. therefore, that the increasing effect of gly- Nanaboshi and Y. Yamamura (1960a). cyrrhizin on the biliary excretion of bile acids Ibid. 13, 416. might not be mediated by thyroxin. Kumagai, A., M. Yamamoto, S. Yano and Notwithstanding the stimulatory effect of Y. Yamamura (1966b). Ibid. 13, 46. glycyrrhizin on cholesterol biosynthesis, the Leffler, H. H., F. W. Sunderman and F. W. enhanced bile acid-formation, the increased Sunderman, Jr. Lipids and the steroid hor- biliary excretion of both cholesterol and bile mones in clinical medicine. J. B. Lippincott acids, the stimulated fecal excretion of cho- Co., Philadelphia, p.18 (1960). lesterol and bile acids and the acceleration of Shibata, N.(1962). Medical J. Osaka Univer- plasma cholesterol elimination might explain sity 12, 297. the cholesterol-lowering action of glycyrrhizin. Sperry, W. M.(1963). J. Lipid Res. 4, 221. Weis, H. J. and J. M. Dietschy (1969). J. Clin. Invest. 48, 2398.