Food Phytochemicals for Cancer Prevention II - American Chemical

Chapter 32

Biological Activities, Production, and Use of Chemical Constituents of Licorice

Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on December 13, 2014 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0547.ch032

Kenji Mizutani Department of Research and Development, Maruzen Pharmaceuticals Company Ltd., 22-1 Sanada, Takanishi-cho, Fukuyama 729-01, Japan

To further expand the application of licorice, chemical and biological studies on glycyrrhizin derivatives as well as phenolic compounds were carried out. A new sweetener, monoglucuronide of glycyrrhetinic acid (MGGA) inhibited mouse skin tumor promotion more effectively than glycyrrhetinic acid. Our studies on antioxidative activity, antimicrobial activity, enzyme inhibition and other biological activities of phenolic compounds are summarized.

Licorice has been used as a crude drug in both the East and West since ancient times. Its main active principle is glycyrrhizin, which consists of a glycyrrhetinic acid and two molecules of glucuronic acid (Figure 1). Glycyrrhizin has been used not only as a medicine but also as a food additive because of its sweetness and flavoring property. Recently, the utilization of licorice extract and its active principles has been developed further. Demand and Utilization of Licorice in Japan Licorice grows widely throughout China, Russia, the Middle East and Southern Europe. Japan imports its entire demand of licorice, mainly from China, Russia, Afghanistan and Pakistan. The total amount of licorice imported into Japan averages about nine thousand tons a year. There are five companies extracting licorice in Japan. Our company extracts more than half of all the licorice in Japan, five thousand tons per year. Present uses of licorice in Japan are shown in Tables I and II. Licorice root has been used as a Chinese medicine and as raw material to prepare extracts and active principles. The hydrophilic extract and its active principle, glycyrrhizin, are used in medicines, cosmetics, food additives, cigarette flavoring and so on. Glycyrrhetinic acid and its derivatives have been used in medicines and cosmetics. Recently the hydrophobic extract has been added to cosmetics and used as an antioxidant. Medicinal and cosmetic utilization is expanding — 60% of the licorice used by our company is extracted for use in medicines and cosmetics.

0097-6156/94/0547-0322$06.00/0 © 1994 American Chemical Society

In Food Phytochemicals for Cancer Prevention II; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


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Chemical Constituents of Licorice

Glycyrrhizin

Figure 1. Structure of glycyrrhizin, M G G A and glycyrrhetinic acid

Table I. Uses of Licorice in Japan Licorice root Medicine

Hydrophilic extract Glycyrrhizin and its salt

Oral Kampo (Chinese medicine) Cold remedy Antitussive Anti-inflammatory Antiulcer

Cosmetic

Oral Prep. Anti-inflammatory Antiulcer Antidotic Injections Hepatitis remedy Antidotic Antiallergic Eve Drops Anti-inflammatory External Prep.

Shampoo

Facial cream Lotion Tonic

Sweetener Flavor

Sweetener Flavor

Food

Sweetener Pigment

Other

Cigarette flavor Raw material (for extract) Compost (Extracted residue)

Toothpaste


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FOOD PHYTOCHEMICALS II: TEAS, SPICES, AND HERBS

Table II. More Uses of Licorice in Japan


Glycyrrhetinic acid and its derivatives Medicine

Oral Prep. Antiulcer External Prep. Dermatitis

Cosmetic

Facial cream Lotion

Hydrophobic extract

Facial cream

Antioxidant

Food

Structure-sweetness Relationship of Glycyrrhizin Derivatives and Production of a New Sweetener In Japan, glycyrrhizin and stevioside preparation are mainly employed as natural low calorie sweeteners. As for stevioside sweeteners, enzymic transglucosylated products are commercially used as better sweeteners than stevioside. By means of this transglucosylation, its bitter taste is removed and its sweetness-intensity is increased. Tanaka et al studied the sweetness and structure relationship of enzymic transglucosylated products of stevioside (/). They found that the number of sugar molecules and their positioning are important for sweetness-intensity. As for glycyrrhizin-sweeteners, a similar transglucosylated product was developed as an improved sweetener. But in this case, an increase in sweetness was not observed, in fact, its sweetness decreased. Kamiya et al studied the sweetness of glycyrrhizin (2). These results show that glycyrrhetinic acid-glycosides with fewer sugar molecules are sweeter and that sweetness-intensity varies depending on the type of bound sugars. We synthesized glycyrrhetinic acid-glycosides containing various sugars — glucose, galactose, xylose, arabinose, glucuronic acid, cellobiose — and evaluated their sweetness. In the case of gly Table III. Relative Sweetness Of cyrrhetinic acid-glycosides, monoVarious Glycosides of Glycyrrhetinic Acid glycosides were sweeter than diglycosides. Glycosides of glucose, Relative sweetness Glycosides xylose and glucuronic acid were (x sucrose) found to be superior in sweetness to glycyrrhizin. Of these glycosides, 71 β-Cellobioside monoglucuronide of glycyrrhetinic 218 β-D-Glucoside acid ( M G G A ) was the sweetest 100-120 β-D-Galactoside compound. M G G A is approxi 31-34 a-L-Arabinoside mately 5 times sweeter than glycyr 544 β-D-Xyloside rhizin and 941 times sweeter than β-D-Glucuronide (MGGA) 941 cane sugar (Table III). We have already devised a production method for deriving

Glycyrrhizin

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M G G A from glycyrrhizin using an enzymic reaction. Usually glycyrrhizin is hydrolyzed into glycyrrhetinic acid through M G G A using commercial β-glucuronidase. We screened enzymes from several hundred soil microorganisms which selectively hydrolyze glycyrrhizin to produce M G G A . A new enzyme from Cryptococcus magnus hydrolyzed glycyrrhizin to quantitatively produce M G G A . Our company is now producing M G G A using this method. Screening Tests of Inhibition of Two-stage Carcinogenesis with Glycyrrhizin and Its Derivatives It has been observed that glycyrrhetinic acid, an aglycone of glycyrrhizin effectively inhibits tumor promotion in mouse skin (3,4). As a primary screening test for anti tumor-promoting agents, we studied the inhibition capabilities of the aforementioned glycyrrhetinic acid-glycosides. The effects were evaluated by using a short-term in vitro assay of Epstein-Barr virus early antigen activation in Raji cells induced by the tumor promotor 12-0-tetradecanoylphorbol-13-acetate (TPA). Of the glycosides, M G G A showed the strongest inhibition ability, to our surprise, even superior to glycyrrhetinic acid (Table IV).

Table IV. Inhibitory Effects of Glycosides of Glycyrrhetinic Acid on Epstein-Barr Virus Activation Concentration (Mol ratio/TPA) Glycosides

1000

500

100

10

β-Cellobioside β-D-Glucoside β-D-Xyloside a-L-Arabinoside β-D-Glucuronide (MGGA)

19.6 11.3 10.4 38.9 0

(80) (80) (80) (80) (70)

53.8 51.5 42.6 63.5 41.6

81.6 75.4 71.8 91.6 74.2

100.0 95.7 93.4 100.0 93.1

Glycyrrhetinic acid Glycyrrhizin

15.6 (70) 26.4 (70)

54.3 63.5

100.0 82.3

100.0 100.0

a

a

Percent of control (percent viability)

Then we compared the inhibition capabilities of M G G A with those of glycyrrhizin and glycyrrhetinic acid in two-stage mouse skin carcinogenesis. Initiation of carcinogenesis was carried out by a single application of 7,12-dimethylbenz[a]anthracene (DMBA) on the back of the mice. The tumor promoter TPA (1.7 nmol) was applied twice a week starting one week after the initiation. The test compounds were applied topically 60 minutes before T P A application at a molar concentration 50 times that of TPA (85 nmol). Each experimental group used 15 mice, and the experiment was carried out for 20 weeks. Figure 2 shows the time course of mouse skin tumor formation. In the control group, the first tumor appeared during 6th week and all mice had tumors by the 9th week. In groups treated with test compounds, the first tumor appeared at



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week 8. Of the compounds tested, M G G A possessed the most inhibitory activity against skin tumor promotion. Inhibition by M G G A of the number of tumorbearing mice compared with control was 60% at week 10, 47% at week 15 and 13% at week 20. The average number of tumors per mouse in the control group was 9.5 at week 20, whereas that of those treated with M G G A was 4.7. From these results, M G G A is expected to be an excellent cancer preventive agent. We are currently furthering our experimentation into the prevention of lung cancer in mice. As previously mentioned, M G G A is about 5 times sweeter than glycyrrhizin and is more effective than glycyrrhetinic acid at inhibiting tumor promotion. While glycyrrhetinic acid is difficult to dissolve in water, M G G A is easily dissolved for use in medicines or foods. As shown in Table V, M G G A has no problem with acute toxicity, mutagenicity, or metabolism.

-· •Δ •• -0

TPA (1.7 nmol) Glycyrrhizin (85 nmol) Glycyrrhetinic acid (85 nmol) M G G A (85 nmol)

Weeks of promotion

Weeks of promotion

Figure 2. Inhibitory effects of glycyrrhizin, glycyrrhetinic acid and M G G A on D M B A-TPA tumor promotion.

Table V . Properties of M G G A Formula State Sweetness Solubility Acute toxicity Mutagenicity Metabolism

C36H54O10

White powder Glycyrrhizin χ 5 (sucrose χ 941) Soluble in water LD50 > 5000 mg/kg (p.o. in mice) Negative (by umu-test) Glycyrrhizin-like (by human intestinal flora)


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Studies on Antioxidative Activity, Antimicrobial Activity, Enzyme Inhibition and Other Biological Activities of Phenolic Compounds Recently, chemical and biological studies of licorice have expanded to include components of hydrophobic extracts. In order to find new uses for the hydrophobic extracts of licorice, our research group studied the biologically active principles of commercially available licorice. Because the licorice comes from a number of different origins, the hydrophobic components varied. We have previously reported the antioxidant and antimicrobial principles in Xinjiang licorice (5). The Xinjiang licorice was collected in Xinjiang Province of China and identified as Glycyrrhiza inflata. Recently, our group investigated its hydrophobic extracts in more detail (6-8). We isolated a total of 20 phenolic compounds. Of these compounds, licochalcone-B and -D showed more potent antioxidant capabilities than other compounds. As for antimicrobial activities, the main phenolic constituent, licochalcone-A inhibited the growth of gram-positive bacteria most effectively. In in vitro tests of enzyme inhibition, glycydione-C and licochalcone-A exhibited the most effective inhibitory abilities against glucosyl-transferase and tyrosinase, respectively. The anti-tyrosinase capability of licochalcone-A was very weak, however, when compared to glabridin, which was isolated from Russian-licorice. The anti-tyrosinase ability of glabridin will be described later. We have identified the antioxidant and antimicrobial components licocoumarone and glycycoumarin in Xibei licorice (9). Xibei licorice was imported from northwestern China and assigned the name Glycyrrhiza uralensis. Licocoumarone exhibited a potent antioxidant capacity. As for antimicrobial capability, both licocoumarone and glycycoumarin inhibited the growth of gram-positive bacteria and yeasts. From the Russian licorice botanically named Glycyrrhiza glabra, we isolated two main phenolic constituents, glabridin and glabrene (5). Glabrene showed the most potent antioxidant capacity. As for antimicrobial abilities, both glabridin and glabrene inhibited the growth of gram-positive bacteria, yeasts and fungi. Glabridin showed more significant growth-inhibition than glabrene. The most important discovery was the inhibition of tyrosinase by glabridin. In an in vitro anti-tyrosinase assay, the preparation containing 40% glabridin is 60 times and 270 times more effective than kojic acid and ascorbic acid, respectively. We have already observed that this preparation inhibits melanin synthesis and shows a more effective whitening activity for human skin than kojic acid and albutin which are now being used as whitening agents in Japan. Based on these results, we are producing some preparations of hydrophobic extracts from licorice (Table VI). Based on our ongoing studies, we expect further developments in the use of licorice extracts and identification their biologically active principles. Table VI. Products of hydrophobic extracts from licorice Product

Origin

Utilization

SANKANON-30 SANKANON-FC LICORICE E X T R A C T - P U LICORICE EXTRACT-PT

G. uralensis G. glabra G. inflata G. glabra

Antioxidant in vegetable oils Antioxidants in animal oils UV-absorbent in cosmetics Whitening agent in cosmetics


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Acknowledgments


I wish to express my thanks to Professor Mutsuo Kozuka, Kyoto Pharmaceutical University and to Dr. Harukuni Tokuda, Kyoto Prefectural University of Medicine for inhibitory experiments on Epstein-Barr virus activation and mouse skin carcino genesis. I would also like to thank my many co-researchers.

Literature Cited 1. Fukunaga, Y.; Miyata, T.; Nakayasu, N.; Mizutani, K.; Kasai, R.; Tanaka, O. Agric. Biol. Chem. 1989, 53, 1603. 2. Esaki, E.; Konishi, F.; Kamiya, S.Agric. Biol. Chem. 1978, 42, 1599. 3. Nishino, H.; Yoshioka, K.; Iwashima, Α.; Takizawa, H.; Konishi, S.; Okamoto, H.; Okabe, H.; Shibata, S.; Fujiki,H.; Sugimura, T.Jpn. J. Cancer Res. (Gann) 1986, 77, 33. 4. Yasukawa, K.; Takido, M . ; Matsumoto, T.; Takeuchi, M . ; Nakagawa, S. Oncology 1991, 48, 72. 5. Okada, K.; Tamura, Y.; Yamamoto, M.; Inoue, Y.; Takagaki, R.; Takahashi, K.; Demizu, S.; Kajiyama, K.; Hiraga, Y.; Kinoshita, T. Chem. Pharm. Bull. 1989, 37, 528. 6. Proc. 38th Annual Meeting Japanese Soc. Pharmacognosy, Kobe, Japan 1991, p 95 (Part l),p96 (Part 2). 7. Proc. 112th Annual Meeting Pharmaceu. Soc. Japan, Fukuoka, Japan 1992, p 202. 8. Demizu, S.; Kajiyama, K.; Hiraga, Y.; Kinoshita, K.; Koyama, K.; Takahashi, K.; Tamura, Y.; Okada, K.; Kinoshita, T. Chem. Pharm. Bull. 1992, 40, 392. 9. Demizu, S.; Kajiyama, K.; Takahashi, K.; Hiraga, Y.; Yamamoto, S.; Tamura, Y.; Okada, K.; Kinoshita, T. Chem. Pharm. Bull. 1988, 36, 3474. R E C E I V E D May 4, 1993


Food Phytochemicals for Cancer Prevention II - American Chemical

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