[논문]감초 신품종 및 약전 수재감초의 항알러지 효과 비교 연구

강윤미; 김원남; 진종식; 이종현; 장재기; 이정훈; 안효진

doi:10.6116/kjh.2020.35.5.13.

[국내논문] 감초 신품종 및 약전 수재감초의 항알러지 효과 비교 연구
The Comparative Study of Anti-allergic Effect by Glycyrrhiza New Varieties and Official Compendia 원문보기

大韓本草學會誌 = The Korea journal of herbology, v.35 no.5, 2020년, pp.13 - 21

강윤미 (상지대학교 한의과대학 약리학교실) , 김원남 (세명대학교 한의과대학 본초방제학교실) , 진종식 (전북대학교 환경생명자원대학 한약자원학과) , 이종현 (동덕여자대학교 약학대학 약학과) , 장재기 (농촌진흥청 국립원예특작과학원 약용작물과) , 이정훈 (농촌진흥청 국립원예특작과학원 약용작물과) , 안효진 (상지대학교 한의과대학 약리학교실)

Abstract ▼ AI-Helper

Objective : The genus Glycyrrhiza has been used in food and traditional herbal medicine. Many experimental studies reported that Glycyrrhiza species possess several pharmacological properties. Glycyrrhiza new varieties WONGAM and SINWONGAM have been developed by Korea Rural Development Administration doing research for registration on Ministry of Food and Drug Safety. During the evaluations about pharmacological effect of Glycyrrhiza new varieties WONGAM and SINWONGAM, we focused the anti-allergic effect in this study. Methods : We investigated the anti-allergic effect of WONGAM and SINWONGAM compared with Glycyrrhiza uralensis Fischer and G. glabra L. using anti-dinitrophenyl-immunoglobulin E (IgE)/human serum albumin-stimulated RBL-2H3 cells, phorbol 12-myristate 13-acetate plus calcium ionophore A23187-stimulated HMC-1 cells and compound 48/80-induced anaphylaxis mice model. We analyzed the effect on the expression of various cytokines, and IgE from mast cells and the underlying molecular mechanisms of WONGAM and SINWONGAM in presented models. Results : WONGAM and SINWONGAM showed the inhibitory effect on the histamine release from rat peritoneal mast cells or human mast cells without cytotoxicity. WONGAM and SINWONGAM blocked anaphylactic shock and decreased the IgE production. Furthermore, WONGAM and SINWONGAM inhibited the productions of TNF-α and IL-6 in compound 48/80-induced anaphylaxis mice model. Conclusion : These results indicated that WONGAM and SINWONGAM would have protect effect on allergic responses through the inhibition of allergic mediators and pro-inflammatory cytokines. This study may facilitate the development on Glycyrrhiza new varieties for allergy.

Keyword

표/그림 (6)

그림 Figure 1. Effect of four Glycyrrhiza species samples on cell viability on RBL-2H3 cells. RBL-2H3 cells were treated with different concentrations of Glycyrrhiza species samples for 24 h and their viability were determined using MTT assay. The data shown represent mean ± S.D. of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs non-treated cells.
그림 Figure 2. Effect of four Glycyrrhiza species samples on the histamine release in IgE-antigen complex-stimulated RBL-2H3 cells. RBL-2H3 cells were sensitized with anti-DNP IgE (50 ng/㎖). After overnight incubation, the cells were pretreated with samples for 1 h and then challenged with DNP-HSA (100 ng/㎖). Histamine release was measured using ELISA kit. The data shown represent mean ± S.D. of three independent experiments. ^###p < 0.001 vs the control group, ***p < 0.001 vs sensitized with anti-DNP-IgE and stimulated with DNP-HAS group.
그림 Figure 3. Effect of four Glycyrrhiza species samples on cell viability on HMC-1 cells. HMC-1 cells were treated with different concentrations of Glycyrrhiza species samples for 24 h and their viability were determined using MTS assay. The data shown represent mean ± S.D. of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs non-treated cells.
그림 Figure 4. Effect of four Glycyrrhiza species samples on the histamine release in PMACI-stimulated HMC-1 cells. Cells were treated with Glycyrrhiza species samples for 30 min prior to the addition of PMACI and the cells were further incubated for 12 h. Histamine release was measured using ELISA kit. The data shown represent mean ± S.D. of three independent experiments. ^###p < 0.001 vs the control group, *p < 0.05 and ***p<0.001 vs PMACI-treated group.
그림 Figure 5. Effects of four Glycyrrhiza species samples on compound 48/80-induced mortality and IgE serum level in a mouse model of anaphylactic shock. Mice were injected with Glycyrrhiza species samples, DSCG and PBS as a vehicle (n = 5 per group) for 1 h before compound 48/80 injection (24 ㎎/㎏ i.p.). (A) Survival rates of these mice were monitored for 1 h. (B) IgE serum levels were measured using ELISA kit. The data shown represent mean ± S.D. of three independent experiments. ^###P < 0.001 vs. the control group; *P < 0.05 and ***P < 0.001 vs. compound 48/80-treated group.
그림 Figure 6. Effects of four Glycyrrhiza species samples on compound 48/80-induced pro-inflammatory cytokines in a mouse model of anaphylactic shock. (A) TNF-α and (B) IL-6 production from serum were measured using ELISA kit. The data shown represent mean ± S.D. of three independent experiments. ^###P < 0.001 vs. the control group; **P < 0.01 and ***P < 0.001 vs. compound 48/80-treated group.

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제안 방법

To investigate whether Glycyrrhiza species samples were cytotoxic to the RBL-2H3 cells, the MTT assay was employed. Glycyrrhiza species samples at concentrations up to 1000 ㎍/㎖ exhibited no significant cytotoxicity to the RBL-2H3 cells after 24 h of incubation when compared with control (Figure 1).
To determine the allergy suppressive effect of Glycyrrhiza species samples in rat-derived mast cells, the release of histamine, indicator of degranulation, was examined. The release of histamine were high in the DNP-IgE/HAS group.
To register the Glycyrrhiza new varieties in the pharmacopoeia, the efficacy homogeneity with existing Glycyrrhiza species should be evaluated. Since the anti-allergic effect of the listed Glycyrrhiza species has already been known, the allergy biomarkers was evaluated to determine the effect of Glycyrrhiza new varieties on allergic reaction using in vitro and in vivo model in this study. The results obtained showed all Glycyrrhiza species samples have no cytotoxicity and reduced histamine release in RBL-2H3 cells and HMC-1 cells, but the level of histamine in group of the high concentration of G.

대상 데이터

HMC-1 cells were provided by Professor Jong-Sik Jin (Chonbuk University, Republic of Korea) and were grown at 37℃ in IMDM supplemented with 10 % FBS, penicillin (100 U/㎖) and streptomycin (100 ㎍/㎖) in a humidified atmosphere with 5 % CO₂. HMC-1 cells were seeded at a density of 1×10⁶ cells per well, treated with Glycyrrhiza species samples for 30 min at 37°C in humidified air with 5 % CO₂ and then stimulated with 40 nM of PMA and 1 ㎛ of A23187 (PMACI) at 37℃.
A total of 35 male ICR mice (6 weeks old; 20–25 g body weight) were obtained from Charles River Laboratories (Harlan Laboratories, Inc., Wilmington, MA, USA) and maintained under constant conditions at a temperature of 20–25℃, humidity of 40–60 % and a 12 h light/dark cycle.

데이터처리

Statistically signifi cant differences were compared using one-way analysis of variance and Dunnett’s post hoc test.
Survival was monitored for 1 h following the induction of anaphylactic shock. Survival data were analyzed using the Kaplan-Meier method and log rank test. Following the assessment of animal survival, blood was collected from the heart of each mouse to measure serum cytokine production.

이론/모형

Effect of four Glycyrrhiza species samples on cell viability on RBL-2H3 cells. RBL-2H3 cells were treated with different concentrations of Glycyrrhiza species samples for 24 h and their viability were determined using MTT assay. The data shown represent mean ± S.
Effect of four Glycyrrhiza species samples on cell viability on HMC-1 cells. HMC-1 cells were treated with different concentrations of Glycyrrhiza species samples for 24 h and their viability were determined using MTS assay. The data shown represent mean ± S.

성능/효과

The decoction was filtered, lyophilized and stored at 4℃. The yields of the dried extract from the starting crude of WONGAM, SINWONGAM, G. uralensis and G. glabra were 8.8 %, 9.13 %, 6.53 % and 7.71%, respectively. To prepare the sample for the in vitro experiment, the extract powder that resulted from the drying process was dissolved in distilled water.
Various concentrations of Glycyrrhiza species samples decreased cell viability as concentration increased, but cell viability remained higher than 80 % in HMC-1 cells. WONGAM, SINWONGAM, G. uralensis and G. glabra displayed the cell viability at 92.62 %, 86.41 %, 89.44 % and 87.02 % in the higher concentration, respectively. Thus, Glycyrrhiza species samples did not show cytotoxicity in tested concentration in HMC-1 cells (Figure 3).
The result showed that pretreatment with samples and DSCG markedly suppressed the overexpression of TNF-α and IL-6 in compound 48/80-induced anaphylaxis shock mice.
Since the anti-allergic effect of the listed Glycyrrhiza species has already been known, the allergy biomarkers was evaluated to determine the effect of Glycyrrhiza new varieties on allergic reaction using in vitro and in vivo model in this study. The results obtained showed all Glycyrrhiza species samples have no cytotoxicity and reduced histamine release in RBL-2H3 cells and HMC-1 cells, but the level of histamine in group of the high concentration of G. glabra was higher than other Glycyrrhiza species samples in DNP-IgE/HAS-stimulated RBL-2H3 cells. In anaphylaxis shock test, all Glycyrrhiza samples exhibited the protective effect against compound 48/80-induced anaphylactic shock, delaying the mortality and significantly inhibited the serum level of IgE.
In anaphylaxis shock test, all Glycyrrhiza samples exhibited the protective effect against compound 48/80-induced anaphylactic shock, delaying the mortality and significantly inhibited the serum level of IgE. In particular, SINWONGAM was superior in terms of attenuating the expressions of mediators, the levels of which were similar to those in Glycyrrhiza species listed in the Korean Pharmacopoeia. These results suggest that Glycyrrhiza new varieties prevent IgE-mediated mast cell activation and suppress the production of histamine and pro-inflammatory cytokines, thus inhibit allergic reactions such as anaphylaxis.
The main constituent of roots is glycyrrhizin, a triterpenoid saponin that is almost 50 times sweeter than sucrose, being the primary active ingredient. The new Glycyrrhiza varieties have a 3.96 % glycyrrhizin content, which is much higher than G. uralensis (1.90 %). While G.
This study showed that Glycyrrhiza new varieties WONGAM and SINWONGAM decreased the histamine release without cytotoxicity in both of rat peritoneal mast cell and human mast cell. WONGAM and SINWONGAM exhibited the protective effect against anaphylactic shock showing increasing the survival and suppressing IgE and cytokine production.

후속연구

As a result, the safety and anti-allergic effects of Glycyrrhiza new varieties were demonstrated to be homogeneous with the listed Glycyrrhiza species. To support this, further specific molecular mechanisms related to the components of Glycyrrhiza new varieties will be needed in future studies.

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