**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 [22].


**Table 10.** Contents of flavonoids and saponin of water‐soluble licorice flavonoids (WSLF).

#### **7.1. Control of some fungal foliage diseases of vegetables using WSLF**

#### *7.1.1. In vitro test of WSLF*

The antifungal activity *in vitro* of WSLF was evaluated by culture tests using the PDA agar medium. The antifungal activity of WSLF among various pathogens causing fungal foliage diseases on vegetables was investigated. WSLF exhibited the antifungal activity against 15 fungi tested (**Table 11**) [23].


**Table 11.** The antifungal activity of WSLF.


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**) [23].

**Table 12.** Effect of WSLE on the germination of spores.

**Compounds Contents (%)**

Liquiritin 4–8 Isoliquiritin 1–3 Liquiritigenin 0.5–2.0 Isoliquiritigenin 0.5–2.0

26 Biological Activities and Action Mechanisms of Licorice Ingredients

Glycyrrhizin 8–13

**Table 10.** Contents of flavonoids and saponin of water‐soluble licorice flavonoids (WSLF).

**7.1. Control of some fungal foliage diseases of vegetables using WSLF**

The antifungal activity *in vitro* of WSLF was evaluated by culture tests using the PDA agar medium. The antifungal activity of WSLF among various pathogens causing fungal foliage diseases on vegetables was investigated. WSLF exhibited the antifungal activity against 15

**Plants Diseases Pathogen % Inhibition of mycerial growth**

Bakanae disease *Gibberella fujikuroi* 37 84 Sheath blight *Thanatephorus cucumeris* 16 71 Seed and seedling rot *Pythium graminicola* 16 17

Gray mold *Botrytis cinerea* 18 45 Corynespora target spot *Corynespora cassicola* 34 67 Leaf mold *Passalora fulva* 29 60

Anthracnose *Colletotrichum orbiculare* 11 71

Rice Blast *Magnaporthe grisea* 14 61

Tomato Late blight *Phytothora infestans* 18 55

Egg plant Leaf mold *Mycovellosiella nattrassii* 19 58 Sweet pepper Frogeye leaf spot *Cercospora capsici* 11 42 Cucumber Corynespora leaf spot *Corynespora cassicola* 20 51

Melon Gummy stem blight *Didymella bryoniae* 50 50 Spinach Fusarium wilt *Fusarium oxysporum f. spinaciae* 38 59 Potato Late blight *Phytothora infestans* 29 43

**100 μg/mL 1000 μg/mL**

**Flavonoids**

**Saponin**

*7.1.1. In vitro test of WSLF*

fungi tested (**Table 11**) [23].

**Table 11.** The antifungal activity of WSLF.

#### *7.1.2. Control of fungal foliage diseases in vivo*

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.

Control efficacy of WSLE among seven pathogens exhibited 80–100% at 1% (**Table 13**) [24]. In the pot test, WSLE showed excellent control of diseases caused by various pathogens.



**Table 13.** Efficacy of WSLE against 6 pathogens.

#### **7.2. Efficacy of WSLE on fish diseases**

#### *7.2.1. Antibacterial activity of WSLE against fish disease causing bacteria in vitro*

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 [25].



**Table 14.** Antibacterial activity of WSLE.

**Plant disease No. of disease spot Inhibition rate (%)**

WSLE 1% 4.0 97

28 Biological Activities and Action Mechanisms of Licorice Ingredients

WSLE 1% 0 100

WSLE 1% 17.5 97

*7.2.1. Antibacterial activity of WSLE against fish disease causing bacteria in vitro*

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

**Bacteria MIC (μg/mL) Bacteria MIC (μg/mL)**

*Carnobacterium pisciicola* 256 *Staphylococcus epidermidis* 512 *Nocardia asteroides* 256 *S. aureus* 256 *Bacillus cereus* 256 *Lactococcus garvieae* 128

*Aeromonas hydrophila* >1024 *Vibrio. damsela* 1024 *A. salmonicida* 1024 *V. fisheri* 128 *Flavobacterium columnare* 64 *V. fluviaris* >1024 *F. psychrophilum* 64 *V. carchariae* >1024 *Flexibacter maritimus* 256 *V. harveyi* 1024 *Pseudomonas chloraruphis* >1024 *V. ichthyoenteri* 512 *Photobacterium damsela* 1024 *V. ordalli* 256

Water 2541.0

Water 134.5

Water 31.1 **Sweet pepper frogeye leaf spot**

Water 520.0

to the inhibitory effect [25].

*B. brebis* 256

**Gram-positive bacteria**

**Gram-negative bacteria**

**Table 13.** Efficacy of WSLE against 6 pathogens.

**7.2. Efficacy of WSLE on fish diseases**

**Cucumber anthracnose**

**Cucumber downy mildew**

The MICs of constituents, liquiritigenin, and isoliquiritigenin are shown in **Table 15**. Isoli‐ quiritigen demonstrated significant antibacterial activity against all bacteria tested. In contrast, liquiritigenin exhibited no antibacterial activity against six kinds of bacteria tested [23].


**Table 15.** Antibacterial activities of constituents in WSLE.

*7.2.2. Effects of WSLE on nonspecific immune responses and disease resistance against Edwardsiella tarda infection in Japanese flounder, Paralichthys olivaceus*

#### *7.2.2.1. Effects of WSLE on nonspecific immune responses in Japanese flounder, P. olivaceus*

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 (*P* < 0.05) than that of the control fish after 1 and 2 weeks (**Figure 10**) [24]. On the other hand, lysozyme activity showed little change (**Figure 11**) [24].

**Figure 10.** Hemolytic activity of Japanese flounder serum.

**Figure 11.** Lysozyme activity of Japanese flounder serum.

Leukocytes were collected from the head kidney and the intestinal tract and used for super‐ oxide 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**) [25]. 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. Supplemen‐ tation of WSLE significantly (*p* < 0.05) enhanced the phagocytic activity after 1 and 2 weeks (**Figure 13**) [26].

**Figure 12.** NBT reduction activity of Japanese flounder leukocytes.

The History of Licorice Applications in Maruzen Pharmaceuticals Co., Ltd. http://dx.doi.org/10.5772/65962 31

**Figure 13.** Phagocytic activity of Japanese flounder leukocytes.

**Figure 10.** Hemolytic activity of Japanese flounder serum.

30 Biological Activities and Action Mechanisms of Licorice Ingredients

**Figure 11.** Lysozyme activity of Japanese flounder serum.

**Figure 12.** NBT reduction activity of Japanese flounder leukocytes.

(**Figure 13**) [26].

Leukocytes were collected from the head kidney and the intestinal tract and used for super‐ oxide 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**) [25]. 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. Supplemen‐ tation of WSLE significantly (*p* < 0.05) enhanced the phagocytic activity after 1 and 2 weeks

#### *7.2.2.2. 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 *E. tarda*.

The cumulative survival rate of the experimental fish following *E. tarda* intraperitoneal challenge is shown in **Figure 14** [27, 28]. The cumulative survival rate was high 48 and 44% when infected fish were fed with 5 and 10 mg/kgBW/day diet and low as 20% in 0 mg/kgBW/ day diet fed group, respectively.

**Figure 14.** Effect of WSLE administration on the survival of Japanese flounder experimentally infected with *E. tarda*.

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 *E. tarda*, WSLE‐treated fish demonstrated increased survival rate.

#### **8. Conclusion**

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 *G. inflata* and glabridin from *G. glabra* in particular. The primary active ingredient isolated and extracted from *G. inflata* is licochalcone A, an oxygenated retrochalcone, which has been associated with various biological properties such as an antioxidant, antimicrobial, as well as anti‐inflammatory. As a result, licochalcone A showed several activities such as inhibitory effects of testosterone 5α‐reductase, lipase, and phospho‐ lipase A2, as well as androgen receptor antagonist, antimicrobial and SOD‐like activities, which relate to skin care, especially the suppression of acne formation and development. On the basis of this evidence, a trial of licochalcone A with acne patients was carried out and the efficacy was demonstrated clinically. The primary active ingredient of *G. glabra* is glabridin, a preny‐ lated isoflavonoid, which is one of the most studies licorice flavonoids, a comprehensive literature survey linked to its bioactivities. Glabridin has inhibitory effects on tyrosinase activity, a key enzyme in the production of melanins and melanization using cultured B16 melanoma cells. An open study of glabridin with melasma patients was conducted. The efficacy was evaluated by measuring skin lightness before and after therapy. Glabridin significantly improved after the therapy not only in melasma but also in lesions. The aerial parts of licorice have received scant interest. The few phytochemical investigations on the *G. glabra* leaves have shown the presence some flavonoids that are not in the roots. We found licorice leaf extract and its component, 6‐prenyl‐naringenin upregulated both SPTLC1 and SMPD1 mRNA expression related to ceramide synthesis as well as HMGCR mRNA expression related to the cholesterol synthesis. In addition, licorice leaf extract stimulated ceramide production in skin‐equivalent models and human skin and promoted HA synthesis by a mechanism that involves upregulation of HAS3 mRNA expression. These results suggested that the licorice leaf extract may be a useful ingredient for skin hydration and barrier repair. There are few reports on licorice extract and glycyrrhizin used in agriculture and fishery. We examined flavonoid‐rich water soluble licorice extract (WSLE) in agriculture and fishery uses. In agriculture, WSLE suppressed hyphal elongation of 12 kinds of plant pathogenic fungi and zoospore release from the conidia. In the pot test, WSLE suppressed the number of lesions in six kinds of plant diseases. As a result, we suggested WSLE has the control effects on some fungal diseases of vegetables such as cucumber, tomato, and sweet pepper. In fishery, WSLE and isoliquiritigenin inhibited the fish disease caused by bacteria, especially Gram‐positive bacteria. Effects of WSLE on nonspecific immune response of Japanese flounder were investi‐ gated. Oral administration of 5 or 50 mg/kgBW/day of WSLE for 2 weeks showed some significant enhance in humoral (hemolytic and lysozyme activities) and cellular (super oxide anion release and phagocytic activities) activities. After 10 days dietary treatment with WSLE, the fishes were challenged by intraperitoneal injection with *E. tarda*, WSLE‐treated fish demonstrated increased survival rate.
