**6. Application of licorice leaf extract for cosmetics**

The aerial parts of licorice are less used in cosmetics. A few phytochemical investigations on the *G. glabra* leaves have reported the presence of several phenolic constituents that are not present in the root [18–20].

We found that licorice extract from *G. glabra* has antiaging effect on human skin [21]. Skin hydration is one of the most important claims in cosmetics as hydrated skin gives an impres‐ sion of healthy skin. Intracellular lipids in stratum corneum (SC), which are composed mainly of cholesterol, fatty acids, and ceramide, play a crucial role for both water‐holding and permeability barrier function in SC. Hyaluronan (HA) is also known to have high water‐ retaining capacity released to skin hydration, elasticity, and plasticity. It has been reported that HA decreased and was found to be changed in aged skin. Therefore, materials that can modulate ceramide and HA contents in SC could be very effective for aging skin.

#### **6.1. Isolation of licorice leaf components**

Ten components were isolated from 70% ethanol extract of licorice leaf from *G. glabra*. Three active components out of 10 are shown in **Figure 5** [19].

**Figure 5.** Structure of isolated active components from licorice leaf extract. (1) Pinocembrin, (2) 6‐prenyl‐naringenin, (3) angophorol.

#### **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 palmitoyltrans‐ ferase (SPT) and acid sphingomyelinase (SMPD1). SPT is known to catalyze the rate‐limit‐ ing 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‐de‐ pendent manner (**Table 9**) [19].


**Table 9.** Effects of licorice leaf extract on the mRNA levels of SPTLC, SPTLC2, and SMPD1.

**6. Application of licorice leaf extract for cosmetics**

22 Biological Activities and Action Mechanisms of Licorice Ingredients

present in the root [18–20].

angophorol.

pendent manner (**Table 9**) [19].

**6.1. Isolation of licorice leaf components**

active components out of 10 are shown in **Figure 5** [19].

The aerial parts of licorice are less used in cosmetics. A few phytochemical investigations on the *G. glabra* leaves have reported the presence of several phenolic constituents that are not

We found that licorice extract from *G. glabra* has antiaging effect on human skin [21]. Skin hydration is one of the most important claims in cosmetics as hydrated skin gives an impres‐ sion of healthy skin. Intracellular lipids in stratum corneum (SC), which are composed mainly of cholesterol, fatty acids, and ceramide, play a crucial role for both water‐holding and permeability barrier function in SC. Hyaluronan (HA) is also known to have high water‐ retaining capacity released to skin hydration, elasticity, and plasticity. It has been reported that HA decreased and was found to be changed in aged skin. Therefore, materials that can

Ten components were isolated from 70% ethanol extract of licorice leaf from *G. glabra*. Three

**Figure 5.** Structure of isolated active components from licorice leaf extract. (1) Pinocembrin, (2) 6‐prenyl‐naringenin, (3)

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 palmitoyltrans‐ ferase (SPT) and acid sphingomyelinase (SMPD1). SPT is known to catalyze the rate‐limit‐ ing 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‐de‐

**6.2. Effects of licorice leaf extract on mRNA expressions of ceramide-related enzymes**

modulate ceramide and HA contents in SC could be very effective for aging skin.

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**) [19]. 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 (*n* = 10). The amount of ceramide by the topical application of 1% licorice leaf extract lotion (1.58 μg/mg) was increased as compared to that of placebo lotion (0.56 μg/mg) (**Figure 7**) [19].

**Figure 6.** Effects of licorice leaf extract lotion on ceramide production. \**p* < 0.05 compared with the control.

**Figure 7.** Effects of licorice leaf extract lotion on ceramide production. Placebo lotion (A) and 1% licorice leaf extract lotion (B). Values were the mean SEM; *n* = 10.

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)**) [19]. These results indicate that licorice leaf extract and its components may increase biosyn‐ thesis of cholesterol as well as ceramide. Further studies will be needed to determine the promoting activity on cholesterol synthesis.

**Figure 8.** Effects of licorice leaf extract (A) and the components (B) on the mRNA level of HMGCR. \**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001, compared with the controls.

#### **6.5. Effects of licorice leaf extract on HA production**

**Figure 7.** Effects of licorice leaf extract lotion on ceramide production. Placebo lotion (A) and 1% licorice leaf extract

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**

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)**) [19]. These results indicate that licorice leaf extract and its components may increase biosyn‐ thesis of cholesterol as well as ceramide. Further studies will be needed to determine the

**Figure 8.** Effects of licorice leaf extract (A) and the components (B) on the mRNA level of HMGCR. \**p* < 0.05; \*\**p* < 0.01;

**reductase (HMGCR), a key enzyme on cholesterol biosynthesis**

lotion (B). Values were the mean SEM; *n* = 10.

24 Biological Activities and Action Mechanisms of Licorice Ingredients

promoting activity on cholesterol synthesis.

\*\*\**p* < 0.001, compared with the controls.

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)**) [19]. 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.

**Figure 9.** Effects of licorice leaf extract (A) and the components (B) on the mRNA level of HAS3 in NHEK. \**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001, compared with the control.

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.
