Distribution and constituents of the three kinds of
Licorice is the root and stolon of the genus Glycyrrhiza plants. Licorice has a long and storied history of use in both Eastern and Western cultures for over 4000 years. Licorice extracts and its principal component, glycyrrhizin, have been used extensively in foods, tobacco, and cosmetics and in both traditional and herbal medicines. Since its start-up in 1938, our company has been working on extracting and purifying the flavoring, sweetening, cosmetic, and medicinal constituents from licorice. At first, we were manufacturing licorice extracts for soy sauce. Recently, our company has developed new licorice products, such as antioxidative and antimicrobial products for foods from hydrophobic licorice extracts; whitening, antioxidative, and antityrosinase products for cosmetics from hydrophobic licorice extracts; antiaging products for cosmetics from licorice leaves; and some disease-suppression products for agriculture and fishery by water-soluble licorice flavonoids. This chapter presents the history of several kinds of food and cosmetic applications from many extracts and purified constituents from licorice plants in our company.
- Glycyrrhiza plants
Licorice (liquorice in British English, Gancao in Chinese, the leguminous plant (Fabaceae)) is a perennial plant grown in the Mediterranean region, the Middle East, Central Asia, Russia, and China. Licorice is the root and stolon of the genus
Licorice is applied to the root and stolon of some
|Distribution||Water soluble constituents||Hydrophobic flavonoids|
|China, Mongolia, Kazakhstan||Glycyrrhizin||Liquiritigein and its glycosides isoliquiritigenin and its glycosides||Glycycoumarin licocoumarone|
Middle East Central Asia
|Glycyrrhizin||Liquiritigein and its glycosides isoliquiritigenin and its glycosides||Glabridin glabrene|
|Xinjiang(China)||Glycyrrhizin||Liquiritigein and its glycosides isoliquiritigenin and its glycosides||Licochalcone A licochalcone B|
3. Application of licorice products for foods
Pontefract cakes are made of licorice extract, molasses, sugar, and flour in the Yorkshire town of Pontefract, England, during the seventeenth century. In the nineteenth century, it was used extensively for confectionery . Licorice extracts and glycyrrhizin have the following properties :
High-intensity sweetener, glycyrrhizin possesses about 200 times the sweetness potency of sucrose
Improving foam stabilization and head-forming characteristics
Masking effect of bitter aftertaste
Soften the saltiness
Depression of freezing point
Full-bodied umami and sweetening
Therefore, licorice extracts and glycyrrhizin are used as food additives in a variety of foods such as alcohol beverages, nonalcohol beverages, chewing gum, candy, chocolate, sweet snacks, ice cream, soy sauce, Japanese pickled vegetables, seafood delicacies, steamed fish paste, and sausages in Japan.
Research work by our company in 1994 resulted in a yeast that selectively hydrolyzed the terminal β-glucuronyl linkage of glycyrrhizin to yield glycyrrhetic acid 3-
|Comparable sucrose concentration (%)||In water||In 5% salt solution|
4. Application of licorice products for pharmaceuticals and cosmetics
Glycyrrhizin has also demonstrated antiviral, antimicrobial, anti-inflammatory, antiallergy, antiulcer, antitussive, hepatoprotective, and blood pressure-increasing effects
Therefore, licorice extract, glycyrrhizin, and its derivatives are extensively used in the preparation of cosmetics in Japan. Glycyrrhizin as well as licorice extracts, glycyrrhetin, stearyl glycyrrhetinate, and succinyloxy -glycyrrhetinate (carbenoxolone) are used in drugs, quasi-drugs, and cosmetics. Glycyrrhizin and its salts are used in eye drops, lotions, and tonics as they are soluble in water. On the other hand, glycyrrhetic acid and its derivatives are used in creams, milky lotions, and sun oils as they are soluble in oil.
5. Application of hydrophobic extract of licorice for cosmetics and foods
Recently, the species-specific flavonoids from hydrophobic extracts of
5.1. Efficacies of hydrophobic extract from
G. inflata (HPGI)
The primary active ingredient isolated and extracted from
We indicate several efficacies of hydrophobic extracts from
|Tests||Inhibitory effect (IC50: ppm)|
|Testosterone 5α-reductase activity||18.7|
|Phospholipase A activity||0.38|
|Antimicrobial activity against ||15.6 (MIC)|
(1) Inhibitory test of testosterone 5α-reductase. As shown in Table 4, in an assay of the inhibitory effect against testosterone 5α-reductase activity, HPGI demonstrated the potent inhibitory activity. The inhibitory ability was more effective than positive controls (ethynyl estradiol (IC50: 31.5 ppm) and benzyl peroxide (IC50: 129 ppm)).
(2) Inhibitory effect against androgen receptor.
In this assay, HPGI inhibited the binding of dihydrotestosterone on the receptor at a low concentration and its IC50 was 5.8 ppm (Table 4). From this result, it was indicated that HPGI has a binding ability in the androgen receptor and works an androgen antagonist.
(3) Inhibitory effect against lipase and phospholipase A2.
HPGI indicated the inhibition of lipase (IC50: 43.6 ppm) and phospholipase A2 (IC50: 0.38 ppm). In this case, the potency of HPGI against phospholipase A2 was remarkable.
(4) Superoxide dismutase (SOD) like activity.
The suppressant effect of HPGI on active oxygen generation was examined by the reduction of nitro blue tetrazolium (NBT) in a xanthine-xanthine oxidase system. In this assay, HPGI suppressed the generation of active oxygen at low concentration (IC50: 7.0 ppm).
(5) Antimicrobial activity against
In the hair follicle and the sebaceous gland,
HPGI has antimicrobial activity against
(6) Efficacy assessment in acne patient.
From the above-mentioned results
Therefore, we assessed the efficacy of HPGI in acne patients.
Twenty female acne patients received anti-acne gel containing HPGI. All patients applied anti-acne gel onto the whole face twice or three times daily for 2 weeks.
|Efficacy||No. of patients|
5.2. Efficacies of hydrophobic extracts from
G. glabra (HPGG)
The hydrophobic extract from
(1) Inhibitory effect of tyrosinase activity.
We found that HPGG and its constituents had inhibitory effects on tyrosinase activity by absorbance measurement. Their tyrosinase inhibition doses (IC50: mg/mL) were as follows: glabridin 0.0003, HPGG 0.031, glabrene 0.0046, hydroquinone 0.016, and ascorbic acid 0.21 (Table 6) . The latter two compounds are commonly known as depigmenting agents.
(2) Melanization assay by 14C-thiouracil uptake.
Melanization was assayed by the incorporation of 14C-thiouracil into B-16 melanoma cells. Melanization was inhibited by HPGG and glabridin dose-dependently, although glabridin more strongly inhibited it than HPGG (Figure 4) .
(3) Application in patients with melasma.
We have first synthesized an HPGG that contains 40% of glabridin and using this HPGG, 0.1 or 0.2% HPGG creams were made. An open study has been carried out with application of 0.1 or 0.2% HPGG cream twice a day in patients with melasma, senile pigment freckle, and postinflammatory pigmented lesions for 4 months. The efficacy was evaluated by measuring skin lightness (
|Disease||No. of cases|
|Chloasma||20||Before 58.28 ± 4.06 |
After 59.25 ± 3.61
|Postinflammatory pigmentation||6||Before 57.56 ± 3.44 NS|
After 58.22 ± 2.46
|Chloasma + PIP||7||Before 60.26 ± 2.09 NS|
After 60.62 ± 3.44
|Total||33||Before 58.57 ± 3.65 |
After 59.35 ± 3.39
|Disease||No. of case|
|Chloasma||12||Before 55.80 ± 3.09 |
After 57.37 ± 2.68
|Postinflammatory pigmentation||8||Before 56.34 ± 3.84 |
After 57.94 ± 3.06
|Senile pigment freckle||15||Before 55.75 ± 3.48 |
After 56.99 ± 2.63
|Total||35||Before 55.90 ± 3.34 |
After 57.33 ± 2.69
6. Application of licorice leaf extract for cosmetics
The aerial parts of licorice are less used in cosmetics. A few phytochemical investigations on the
We found that licorice extract from
6.1. Isolation of licorice leaf components
6.2. Effects of licorice leaf extract on mRNA expressions of ceramide-related enzymes
To examine the effects of plant extracts on ceramide synthesis, real-time quantitative RT-PCR analysis was performed on gene expressions of serine palmitoyltransferase long chain base subunit 1 (SPTLC1) and SPTLC2, which were two subunits of serine palmitoyltransferase (SPT) and acid sphingomyelinase (SMPD1). SPT is known to catalyze the rate-limiting step of de novo ceramideynthesis. Acid sphingomyelinase (SMPD1) is also known to convert sphingomyelin into ceramide and plays an important role in ceramide generation for permeability barrier function. Licorice leaf extract and 6-prenyl-naringenin showed the promoting activity on mRNA expressions of SPTLC1, SPTLC2, and SMPD1 in a dose-dependent manner (Table 9) .
These results indicate that licorice leaf extract may increase de nove biosynthesis of ceramide and hydrolysis of sphingomyelin to ceramide.
6.3. Effect of licorice leaf extract on ceramide production in skin-equivalent models and human skin
To examine whether licorice leaf extract has an efficacy on the production of ceramide in skin-equivalent models. The extract dramatically promoted the production of ceramide in skin-equivalent models (Figure 6) . For further research to determine the efficacy on the production of ceramide in human skin, 1% licorice leaf extract or placebo lotion was topically applied on healthy volunteers (
These results suggested that licorice leaf extract has an efficacy on the synthesis of ceramide.
6.4. Effects of licorice leaf extract on mRNA expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), a key enzyme on cholesterol biosynthesis
To examine whether licorice leaf extract has a promoting effect on other stratum corneum lipids, real-time quantitative RT-PCR analysis was performed on mRNA expression of HMGCR, the key enzyme for de novo cholesterol synthesis. Treatment with licorice leaf extract showed a significant increase in the expression of HMGCR mRNA by 150% compared with the controls (Figure 8(A)). Among three components tested, especially 6-prenyl-naringenin enhanced the expression of HMGCR mRNA in the dose-dependent manner (Figure 8(B)) . These results indicate that licorice leaf extract and its components may increase biosynthesis of cholesterol as well as ceramide. Further studies will be needed to determine the promoting activity on cholesterol synthesis.
6.5. Effects of licorice leaf extract on HA production
Gene expression of HAS3 that relates to hyaluronan biosynthesis was upregulated by the treatment of licorice leaf extract in the dose-dependent manner in normal human epidermal keratinocytes (NHEK) (Figure 9(A)). Three components tested also enhanced the hyaluronan synthase 3 (HAS3) mRNA expression (Figure 9(B)) . All-trans retinoic acid that is known as a stimulator of HA synthesis in NHEK also showed a strong increase in the expression of HAS3 mRNA by 400% compared with the control. To examine whether licorice leaf extract has a promoting activity on HA production, ELISA analysis was performed using cell culture supernatant. These results indicate that licorice leaf extract and its components have a potent stimulation activity for HA production.
These results indicate that licorice leaf extract may be a useful ingredient for skin hydration and barrier repair because of their ability to synthesize ceramide through the enhancement of mRNA expressions of SPT and SMPD1 and the increase of mRNA of HMGCR related to cholesterol biosynthesis and the increase of HA production through the enhancement of mRNA levels of HAS3 by its active principles.
7. Application of flavonoid-rich water-soluble licorice flavonoids (WSLF) for agriculture and fishery
Table 10 shows the flavonoid composition of water-soluble licorice flavonoids (WSLF) tested. WSLF contains about 10% of the total flavonoids and 10% of glycyrrhizin. General water extracts of licorice contain 2% of the total flavonoids and 10% of glycyrrhizin. WSLF tested has five times higher content of total flavonoids .
7.1. Control of some fungal foliage diseases of vegetables using WSLF
In vitro test of WSLF
The antifungal activity
|Plants||Diseases||Pathogen||% Inhibition of mycerial growth|
|100 μg/mL||1000 μg/mL|
|Seed and seedling rot||16||17|
|Corynespora target spot||34||67|
|Egg plant||Leaf mold||19||58|
|Sweet pepper||Frogeye leaf spot||11||42|
|Cucumber||Corynespora leaf spot||20||51|
|Melon||Gummy stem blight||50||50|
WSLE solution of 20 μL of spore solution and 20 μL was mixed on the slide maintained at 25°C for 20 h. The germinated spores were counted under a microscope. WSLE at 0.1 and 1% inhibited the germination of spores in three kinds of fungi (Table 12) .
|Plants||Diseases||Concentration (%)||Inhibition rate (%)|
|No. 1||No. 2|
|Sweet pepper||Frogeye leaf spot||0.1||48.4||50.1|
|Egg plant||Leaf mold||1||76.2||72.3|
|Cucumber||Corynespora Leaf spot||1||62.4|
7.1.2. Control of fungal foliage diseases
Control efficacy of WSLE against seven pathogens was evaluated in pot tests. WSLE solutions were sprayed onto young plants. After air-drying the solutions, the plants were artificially inoculated with the spore suspension on the test pathogen, and incubated at 25°C for a given period. Percent of disease control was assessed after the inoculation of 9–12 days by visually measuring the number of diseasing spot.
|Plant disease||No. of disease spot||Inhibition rate (%)|
7.2. Efficacy of WSLE on fish diseases
7.2.1. Antibacterial activity of WSLE against fish disease causing bacteria
The antibacterial activity of WSLE was examined by the agar dilution method, which ranged from 32 to 1024 μg/mL against 33 kinds of bacteria. As shown in Table 14, WSLE inhibited the growth of Gram-positive bacteria with MIC values of 128–512 μg/mL. Whereas of the Gram-negative bacteria 17 kinds of bacteria were sensitive and nine kinds of bacteria were insensitive to the inhibitory effect .
|Bacteria||MIC (μg/mL)||Bacteria||MIC (μg/mL)|
The MICs of constituents, liquiritigenin, and isoliquiritigenin are shown in Table 15. Isoliquiritigen demonstrated significant antibacterial activity against all bacteria tested. In contrast, liquiritigenin exhibited no antibacterial activity against six kinds of bacteria tested .
7.2.2. Effects of WSLE on nonspecific immune responses and disease resistance against
Edwardsiella tarda infection in Japanese flounder, Paralichthys olivaceus
184.108.40.206. Effects of WSLE on nonspecific immune responses in Japanese flounder,
Healthy Japanese flounder, each weighting about 56 g, was divided into three groups used in 0, 5, and 50 mg/kgBW/day of WSLE. Each diet was fed to three groups once a day for 2 weeks. After 1 and 2 weeks of feeding, five fishes from each group were randomly collected. Blood was drawn from the caudal vein and used for hemolytic and lysozyme activities. Hemolytic activity of WSLE-treated fish was significantly higher (
Leukocytes were collected from the head kidney and the intestinal tract and used for superoxide anion release and phagocytic activities. The production of the superoxide anion was quantified by the reduction of nitro blue tetrazolium (NBT). WSLE showed significant higher activity than the control group after 1 and 2 weeks (Figure 12) . The activity increased according to time in most groups. The production of the superoxide anion is a method for destroying intracellular bacteria. Phagocytic activities of head-kidney and intestinal tract leukocytes were determined under a microscope by the zymosan-NBT method. Supplementation of WSLE significantly (
220.127.116.11. Effects of WSLE on disease resistance against
Edwardsiella tarda infection in Japanese flounder, P. olivaceus
Healthy Japanese flounder, each weighing about 53 g, was divided into three groups of 33 fishes fed with 0, 5, and 10 mg/kgBW/day of WSLE, respectively. These three groups were fed with each supplementation diet once a day for 10 days. On the 10th day of feeding, these groups were injected intraperitoneally with 8.0 × 102 CFU of
The cumulative survival rate of the experimental fish following
Oral administration of WSLE caused enhancement in humoral (hemolytic and lysozyme) and cellular (phagocytic and superoxide anion release) activities. After 10 days of dietary treatment with WSLE, the fish were challenged by intraperitoneal injection with
Licorice has been used for pharmaceuticals, cosmetics, and food products as water-soluble licorice extract that contains glycyrrhizin, the primary constituent having sweet-taste and various biological activities. Recently, many studies have focused on the licorice ingredients except glycyrrhizin, about 300 phenolic compounds were found from licorice. We investigated licochalcone A extracted from
9. Future direction
As for licorice resources, licorice plants are widely found growing wild in regions along the Silk Road. However, according to the recent overharvesting of wild licorice, its habitants are severely disturbed and many of them are degraded or undergoing desertification, especially in China. Therefore, licorice cultivation has been undertaken in China. However, in the present glycyrrhizin and flavonoid content of the cultivated licorice is lower than wild one. In the aim of securing a stable source of licorice, we have to study to obtain the cultivated licorice with same quality of wild licorice.
Over the past half-century, we have been engaged in the development of licorice extracts and its components, and have been offering a number of useful and unique materials to our customers in medicinal, cosmetic, functionary food, and food industries. Elucidation of the constituents and biological activities of both underground and aerial parts in licorice plants have led to the development of many valuable licorice products for various industries. We are further expanding the potential of licorice.
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