Insecticidal, tick-repellent and anti-inflammatory activities and safety properties of supercritical fluid extract from Chamaecyparis obtusa

Abstract

초임계유체추출(Supercritical fluid extraction)은 초임계 이산화탄소(Supercitical Carbon Dioxide)를 용매로 이용한 기술로 액체나 기체와 다른 초임계유체의 특성을 이용하여 추출 및 분리 공정에 적용 가능한 기술이다. 본 연구에서는 편백 (Chamaecyparis obtusa)을 초임계 유체 추출 (CO2 Supercritical Fluid Extraction) 장비를 이용하여 추출 온도, 압력과 시간에 따른 시료의 추출 수율을 측정하였으며, 각 추출물을 GC/MS를 이용하여 분석을 진행하였다. 또한, 초임계유체추출물 및 분석을 통해 조사된 단일성분을 집먼지진드기 2종에 대해 살비활성, 작은소참진드기의 기피력 및 독일바퀴에 대한 살충 및 훈증 독성을 평가 하였으며, 아세틸콜린저해 활성, 항염 및 안정성 평가를 진행하였다. 편백잎을 초임계유체 추출장비를 이용하여 압력 및 분쇄 입자크기의 조건을 주어 진행한 결과 압력이 높을수록, 분쇄 입자가 작을수록 추출수율은 높아졌다. 편백잎의 초임계유체추출물과 이들에 함유된 성분들 및 편백 가지의 용매 추출물의 순차분획물을 HPLC를 이용하여 살비활성본체를 분리하여 분광학적 분석을 통해 얻은 β-thujaplicin을 큰다리집먼지진드기(Dermatophagoides farinae)와 세로무늬먼지진드기(Dermatophagoides pteronyssinus)에 대해 살비력을 3종의 상용살비제인 benzyl benzoate, N, Ndietyl m-touamide(DEET), dibutyl phtalate를 비교약재로 선정하여, 섬유확산법을 통해 검정하였다. 큰다리먼지진드기의 경우 편백초임계추출물과 β-thujaplicin이 LD50값이 각각 3.42㎍/㎠, 4.02㎍/㎠ 였으며, 비교약재로 사용한 benzyl benzoate 4.31㎍/㎠, DEET 78.34㎍/㎠ 및 dibutyl phthalate 31.62㎍/㎠보다 높은 활성을 보였고, 세로무늬먼지진드기 역시 편백초임계추출물과 β-thujaplicin이 LD50값이 각각 4.12㎍/㎠, 3.84㎍/㎠를 보여 비교약재로 사용한 benzyl benzoate (4.21㎍/㎠), DEET (78.64㎍/㎠) 및 dibutyl phthalate (30.72㎍/㎠)보다 높은 활성을 보였다. 작은소참진드기 기피력 평가에서는 상용기피제인 icaridin과 N, N-dietyl m-touamide(DEET)를 각각 10%를 처리하여 침입저지법 통해 비교 조사하였다. 결과, 편백 초임계유체추출물은 4㎍/㎠ 농도에서 100%, α-terpinene과 α-terpineol은 3.2㎍/㎠ 농도에서 노출 후 120분 후, 각각 85.5%와 68.9%의 기피력을 나타낸 반면, icaridin과 N, N-dietyl m-touamide (DEET)은 96.1% 및 100%의 기피력을 나타냈다. 독일 바퀴 암컷에 대한 살충활성 평가는 4종의 상용살충제인 deltamethrin, dichlorvos, permethrin 및 propoxur를 비교 약재로 선정하여 직접접촉법 및 훈증독성을 평가하였으며, 편백 초임계유체추출물의 단일성분을 이용하여 독일바퀴 암컷 머리와 흉부의 아세틸콜린에스터라제(acetylcholinesterae, AChE) 저해 활성을 조사하였다. 접촉 시험에서는 편백 초임계추출물이 2.77 ㎍/㎠, phellandrene (0.28 ㎍/㎠), terpinene-4-ol (0.42 ㎍/㎠), α-thujone (0.09 ㎍/㎠)로 높은 활성을 보여, permethrin (0.05 ㎍/㎠), propoxur (0.18 ㎍/㎠)와 비슷한 농도 활성을 보였다. 하지만, 0.007~0.013 ㎍/㎠의 활성을 보인 deltamethrin과 dichlorvos에는 미치지 못했다. 훈증독성 평가 에서는 α-thujone (18.43 mg/L of air), linalool (26.20 mg/L of air)의 높은 활성을 보였지만 dichlorvos ( 0.07 mg/L of air)에는 미치지 못했다. 아세틸콜린에스터라제(acetylcholinesterae, AChE) 저해 활성을 조사에서는 α-Pinene, β-pinene, α-terpinene, limonene, linalool, terpinene-4-ol 및 α-terpineol이 AChE 저해활성(IC50, 0.38 ~ 0.98 mM)을 보였다. 편백 초임계유체추출물의 단일성분에 대한 항염 효과를 In vitro assay를 통하여 효능을 검증하였고, T cell에서의 세포증식, Macrophages에서의 NO, Mast cell에서의 β-hexosaminidase release 에 대한 효능검증 수행하였다. 결과 β-Pinene는 NO 생성억제, IL-6, TNF-α의 유전자 발현 및 생성을 억제 하였고, β-thujaplicin은 NO 생성억제, IL-6 유전자 발현 및 생성을 억제 하였으며, DNP-BSA로 유도되어진β-hexosaminidase release를 억제 및 염증성 cytokine인 IL-4, TNF-α의 유전자 발현 및 생성을 억제하였다. TPA(12-O-tetradecanoylphorbol-13-acetate)-induced inflammation 시험에서는 β-pinene, β-thujaplicin 모두 TPA에 유도 되어진 귀의 부종을 억제하였다. 편백 초임계유체추출물의 안전성 시험은 마우스와 랫드에 급성경구독성(LD50, > 5,000mg/kg) 및 급성경피독성(LD50, > 5,000 mg/kg)으로 나타나 저독성으로 판정되었다. 안점막자극성은 무자극성물질로 판정되어 매우 안전한 것으로 나타났다. 이상의 연구를 통해 편백을 소재로하여 초임계유체추출 기술을 적용한 최적 추출 조건을 확립 하였다. 3종(집먼지진드기, 작은소참진드기 및 독일바퀴)의 위생해충의 살충, 기피력 및 안정성 평가를 통해 기피제 및 훈증제로서 활용 할 수 있을 것으로 기대하며, 항염 평가를 통해 면역조절제로서의 가능성을 확일 할 수 있었다.

Supercritical fluid extraction (SFE) is a rapid, selective and convenient method for sample preparation prior to the analysis of compounds in the volatile product of plant matrices. In this study, inscticidal and repellency describes a laboratory study to examine the active constituent isolated from the C. obtusa and supercritical fluid extract from C. obtusa against house dust mites, hard tick and German cockroach. Also, effects of the insecticidal phytochemicals on AChE inhibition and action on a model aminergic receptor system of German cockroach were investigated. The acaricidal constituent of C. obtusa branch oil was identified as β-thujaplicin against adult D. farinae and D. pteronyssunus. The active constituent of the C. obtusa branch was identified as ɑ-thujaplicin (C10H12O2) by spectroscopic analyses. The toxicity of C. obtusa oil and β-thujaplicin and the acaricides to adult D. farinae was examined by the direct contact bioassay. As judged by 24 h LD50 values, acaricidal activity of C. obtusa oil (3.42 µg/cm2) and β-thujaplicin (4.02 µg/cm2) used was high than that of benzy benzoate (4.31 µg/cm2), dibutyl phthalate (31.62 µg/cm2) and DEET (78.34 µg/cm2). Also, The toxicity of C. obtusa oil and β-thujaplicin and the acaricides to adult D. pteronyssinus were examined by the direct contact bioassay. As judged by 24 h LD50 values, acaricidal activity of C. obtusa oil (4.12 µg/cm2) and β-thujaplicin (3.84 µg/cm2) used was high than that of benzy benzoate (4.21 µg/cm2), dibutyl phthalate (30.72 µg/cm2) and DEET (75.64, µg/cm2). These results indicate that acaricidal activity of C. obtusa likely results from by ɑ-thujaplicin. ɑ-Thujaplicin merits further study as potential house dust mite control agents or lead compounds. The repellency of C. obtusa oil was evaluated in comparison with the two commercial repellency (Icaridin and DEET) against H. longicornis. In filter-paper diffusion method, C. obtusa oil (3.2 µg/cm2) gave 100% repellency and icaridine (3.2 µg/cm2) gave 96.1 % repellency, respectively, at 120 min. The repellency of the 31 compounds against H. longicornis was compared with that of DEET at 3.2 mg/cm2. Responses varied according to the test compound and exposure time. α-Terpinene, terpinene-4-ol and α-terpineol gave 100% repellency 30 min post-treatment but 85.5, 56.4 and 68.9% repellency at 120 min. C. obtusa oil, α-terpinene, terpinene-4-ol and α-terpineol described merit further study as potential fumigants or leads for the control of H. longicornis. The toxicity of C. obtusa oil and 31 compounds against adult females of the German cockroach, Blattella germanica L., was examined using direct contact and vapor phase toxicity bioassays and compared with those of deltamethrin, dichlorvos, permethrin, and propoxur, four commonly used insecticides. As judged by the 24 h LC50 values, C. obutsa oil (2.77 mg/cm2) was less effective than propoxur (0.18 mg/cm2), dichlorvos (0.007 mg/cm2), deltamethrin (0.013 mg/cm2), or permethrin (0.05 mg/cm2). The insecticidal activity of 31 compounds used against adult female B. germanica As judged by the 24 h LC50 values, the adulticidal activity of ɑ-thujone (0.09 mg/cm2) was comparable to that of permethrin (0.05 mg/cm2). The toxicity of linalool and ɑ-terpineol ranging from 0.10 to 0.12 mg/cm2, were higher than that of propoxur (0.18 mg/cm2). In vapor phase toxicity tests, ɑ-terpineol (21.89 mg/L of air) was the most toxic fumigant followed by linalool (26.20 mg/L of air) and ɑ-thujone (36.22 mg/L of air) On the basis of 24 h LC50 values. ɑ-Terpineol described merit further study as potential fumigants or leads for the control of B. germanica. The German cockroach AChE inhibitory activity of 31 test compounds were tested. Base on IC50 values, α-Pinene (IC50, 0.38 mM) exhibited the strongest inhibiton. In the study, the constituent compounds of C. obutsa oil utilizing supercritical flulid extraction, examined their anti-inflammatory effect in vitro and in vivo. In vitro, Anti-inflammatory effects were assessed by measuring the levels of secretory proteins and mRNA of TNF-α, IL-4 and IL-6 production. The results showed that β-pinene induced nitric oxide production (LPS stimulated RAW 264.7 cells), down-regulates mRNA expression of inflammatory genes and pro-inflammatory cytokines, such as iNOS and IL-6. Also, β-thujaplicin suppressed the DNP-BSA induced β-hexosaminidase secretion in IgE-sensitized RBL-2H3 cells, down-regulates mRNA expression of inflammatory genes and pro-inflammatory cytokines, such as TNF-α and IL-4. Furthermore, an in vivo anti-inflammatory study was performed using a TPA-induced skin inflammation mouse model, and the results showed that β-pienen and β-thujaplicin reduced TPA-induced inflammation and attenuated the expression of iNOS in TPA-induced mouse skin tissue. Thus, β-pienen and β-thujaplicin demonstrated anti-inflammatory activity both in LPS-induced RAW 264.7 cells and TPA-stimulated mouse skin and may therefore serve as a potential anti-inflammatory agent. The safty evaluation was designed to determine the safety profile of C. obtusa oil utilizing supercritical flulid extraction, based on acute oral toxicity, primary dermal irritation and primary eye irritation studies. The acute oral LD50 of C. obtusa oil is greater than 5,000 mg/kg of body weight in rats. Also, the single-dose acute dermal LD50 of C. obtusa oil is greater than 5,000 mg/kg of body weight in both male and female rats. The primary dermal irritation index (PDII) of C. obtusa oil was calculated to be 1.8, thus classifying C. obtusa oil to be slightly irritating to the skin. In primary eye irritation studies, the maximum mean total score (MMTS) of C. obtusa oil was observed to be 0.33 and classifying C. obtusa oil to be minimally irritating to the eye. In conclusion, C. obtusa derived preparations containing active constituents described herein merit further study as potential insecticides for the control of acaricide against house dust mites, repellency aganist hard tick and cockroach for protecting humans from bites and nuisance caused by vectors. Also, presented that possible applications of essential oil from C. obtusa as a useful anti-inflammatory agent. Moreover, the anti-inflammatory effects of the major pharmacological components present in the essential oil of C. obtusa might accelerate the development of new drugs for various inflammatory diseases.