• Journal of Acupuncture Research
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• 제17권2호
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• pp.187-208
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• 2000

By the activation of ovary hormone, many morphological changes occur in the epithelial cell lines and muscle cells in rat uterus. These two cells in uterus are important to the implantation of embryo, maintaining pregnancy and starting parturition. One important change associated with the morphological change of these two cells in uterus is the change on prostaglandin(PG) metabolism. Its presence and synthesis in endometriurn and myometrium in uterus affects estrous cycle and the start of embryo implantation in uterus. It also performs as an important modulator in parturition. So the abnormally weak expression of PG causes difficulty during labor and over-expression causes pre-term labor. PG biosynthesis starts from either free or liberated arachidonic acids from membrane phospholipid by phospholipase. Such arachidonic acids are converted into PG catalyzed by Cyclooxygenase. Under normal physiological condition, Cyclooxygenase-1(COX-1) having 602 units of amino acids controls the synthesis of PG. It acts as a local hormone regulating vasomodulation of blood flow, flexible muscle movement, increasing the blood permeability and contributing the protective role in preserving integrity of the stomach lining and Cyclooxygenase-2 (COX-2) is induced by the inflammation, pregnancy and increased its expression until parturition. Lipid metabolite like PG is located in uterine and expression of COX-2 increased with pregnancy. Increased expression of COX proteins in epithelial cells and myometrial cells are told to increase the muscle contractility in uterus but decreased right after the labor in rat. It is a good sign indicating that COX proteins are deeply related to the start of labor. Currently, Several studies report the use of PG and COX-2 inhibitor as medication for controlled abortion or to prevent pre-term labor but they entail various side-effects. Our study proposed to suggest use of acupuncture as an another mediator to control abortion or pre-term labor without causing unnecessary side-effects by those medicines. Two acupuncture sites, LI-4 & SP-6 were selected due to their known efficacy. From the immunohistochemical staining of COX-2, normal expression of COX-2 protein in nonpregnant SD rat's uterus revealed that COX-2 protein was primarily detected in the lumina epithelial lining and in the epithelial cell lining contacting the stromal cells. High resolution optical microscopic scanning revealed distinguishable staining in the myometrial mucosa. LI-4 acupuncture administered nonpregnant rat's uterus showed strong expression for COX-2 in endometrium contacted with lumina epithelial lining of rat uterus and in myometrial mucosa. Stromal cells showed more staining than untreated nonpregnant rat's uterus and stronger staining in stromal cells contacting myometrial layer compared to untreated nonpregnant rat's uterus. SP-6 acupuncture administered nonpregnant rat's uterus showed weak expression for COX-2 in myometrial layers and stromal cells but no staining was visible in lumina epitheliai and glandular epithelial cells. Few stromal cells and myometrial mucosa were positively stained for COX-2. Pregnant SD rat's uterus was also immunostained for COX-2 expression after 18 days of pregnancy. Unlike to untreated nonpregnant rat's uterus, luminal epithelial cells were not positively stained for COX-2 but stronger staining for COX-2 was revealed in stromal cells. LI-4 acupunctured SD rat's uterus had very strong expression of COX-2 in luminal epithelial lining. Few stromal cells showed stronger positive COX-2 staining and myometrial layers also showed more expression than untreated pregnant rat. SP-6 acupuncture administered pregnant SD rat's uterus showed positive expression of COX-2 in epithelial cells of luminal mucosa layer but weaker than that of LI-4 acupuncture treatment's case. However, strong positive staining was revealed in stromal mucosa and myometrial layers. Virgin SD rat's uterus motility index during LI-4 acupuncture was 66.52 % (Prob〉T = 0.0197) compared to its motility before the acupuncture treatment but the motility index was slighdy elevated up to 79.58 % (Prob〉T = 0.1175) after the acupuncture. During the SP-6 acupuncture treatment for 30 minutes, uterus motility index was 90.52 % (Prob〉T = 0.1832) showing lesser decrement but consequently reached similar motility index decreasal to 79.95 % (Prob〉T = 0.0215) after the acupuncture treatment as LI-4 showed. LI-4 acupuncture tend to be a quick treatment to reducing the uterus motility in a virgin rat but eventually both two acupuncture administration created very similar reduction of uterus motility seeing the index after the both acupunctures. The uterus movement monitored during the LI-4 acupuncture administered for 30 minutes, Pregnant SD rat showed decreased motility down to 77.90 % (Prob〉 T = 0.0076) compared to uterus motility before the acupuncture and it continuously decreased down to 71.81 %(Prob〉T = 0.0214) after the removal of needle. The statistical analysis using paired t-test showed significance difference for both two motility indexs at =0.05. SP-6 acupuncture administered to pregnant SD rat also had similar pattern of decreasing uterus motility index down to 74.70 % (Prob〉T = 0.1730) during the initial 30 minutes acupuncture administration and it was continuously lowered to 71.52 % (Prob〉T = 0.0155) after the acupuncture. The paired t-test resuit for SP-6 suggest prompt response of uterus motility index to the SP-6 acupuncture treatment but consequently reached same level of inducing the motility reduction as LI-4 at =0.05 level.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2016년도 춘계학술대회 및 임시총회
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• pp.19-20
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• 2016

Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

• 한국식품위생안전성학회지
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• 제34권2호
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• pp.124-134
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• 2019

본 연구는 농산물 중 2019년 기준신설 예정농약 Broflanilide에 대한 공정시험법을 개발하고자 수행하였다. Broflanilide는 meta-diamide계 살충제로 중추신경계 억제성 신경전달물질인 gamma-aminobutyric acid (GABA) 수용체에 대하여 비경쟁적으로 작용하여 해충을 마비시키는 강한 살충작용을 하고 담배거세미나방 방제에 효과적이다. 잔류물의 정의는 모화합물 broflanilide로 규정하여 관리할 예정으로 향후 대사산물의 안전관리를 고려하여 broflanilide 및 대사산물의 동시시험법을 확립하고자 하였다. 아세토니트릴으로 추출 후 무수황산마그네슘 및 염화나트륨을 첨가하였고 원심분리하였다. 상등액을 취하여 PSA와 $C_{18}$ 흡착제를 사용해 간섭물질을 효과적으로 제거한 후 LC-MS/MS로 분석하는 시험법을 개발하였다. 개발된 broflanilide 시험법의 직선성은 결정계수($r^2$)가 0.99 이상으로 우수하였으며, 검출한계 및 정량한계는 각각 0.004, 0.01 mg/kg으로 높은 감도를 나타내었다. 대표 농산물 5품목(현미, 감자, 대두, 감귤, 고추)에 대하여 시험법 검증결과 평균 회수율(5반복)은 broflanilide는 90.7~113.7%, DM-8007은 88.2~105.0%, S(PFP-OH)-8007은 79.8~97.8%이었으며 RSD는 8.8% 이하로 정확성 및 반복성이 우수함을 확인할 수 있었다. 또한 외부실험기관인 서울지방식약청과의 실험실간 평균 회수율은 broflanilide의 경우 86.3~109.1%, DM-8007은 87.8~109.7%, S(PFP-OH)-8007은 78.8~102.1%으로 상대표준편차(RSD)는 모두 20.8%이하로 국제식품규격위원회 가이드라인(Codex Alimentarius Commission, CAC/GL 40-1993, 2003)의 잔류농약 분석 기준 및 식품의 약품안전평가원의 '식품등 시험법 마련 표준절차에 관한 가이드라인(2016)'에 적합한 수준임을 확인하였다. 개발된 시험법은 낮은 검출한계 및 정량한계, 우수한 직선성, 회수율 실험을 통한 정확성과 정밀성, 재현성 등이 입증되어 농산물 중 broflanilide의 잔류검사를 위한 공정시험법으로 활용되기에 적합한 것으로 판단된다.

• 한국토양비료학회지
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• 제7권2호
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• pp.71-97
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• 1974

답상태토양중(畓狀態土壤中)의 물질변화(物質變化)에 관(關)한 연구(硏究)는 이제까지 많이 실시(實施)되어 많은 성과(成果)를 올리고 있다. 그러나 이들 연구(硏究)의 대부분(大部分)이 실험실내(實驗室內)에서 실시(實施)된 비커실험(實驗), 혹(或)은 주사통실험(注射筒實驗)으로 그 결과(結果)를 야외(野外)의 답토양(畓土壤)에 적용(適用)시키는 것은 약간(若干)의 난점(難點)이 예상(豫想)된다. 토양중(土壤中)의 물질변화(物質變化)라고 하는 관점(觀點)에서 비커, 또는 주사통내(注射筒內)에 충전(充塡)된 답작토층토양(畓作土層土壤)과 야외(野外)의 답작토층토양(畓作土層土壤)과의 가장 중요(重要)한 차(差)는 후자(後者)에 수도근(水稻根)이 만연(蔓延)되어 있다는 것과 토양중(土壤中)에서 물의 이동(移動)이 있다는 것이다. 물의 침투(浸透)가 답상태(畓狀態) 작토층토양(作土層土壤)의 물질변화(物質變化)에 미치는 영향(影響)에 관(關)한 연구(硏究)는 상당(相當)히 많이 실시(實施)되어 그 실체(實體)가 명백(明白)해져 가고 있다. 한편 수도근(水稻根)의 존재(存在)가 답상태작토층토양(畓狀態作土層土壤)의 물질변화(物質變化)에 미치는 영향(影響)에 관(關)한 연구(硏究)는 몇개(個)의 보고(報告)가 있으나 그 결과(結果)는 상호(相互) 좋은 일치(一致)를 보이지 않고 있으며 수도근(水稻根)의 존재(存在)가 토양(土壤)의 물질변화(物質變化)에 미치는 기구(機構)는 추측(推測)의 영역(領域)을 벗어나지 못하고 있기 때문에 본연구(本硏究)는 실험(實險) I에서 수도재배(水稻栽培)가 답상태토양중(畓狀態土壤中)의 물질변화(物質變化)에 미치는 제효과(諸効果)를 확인(確認)하고 실험(實驗) II, III에서 이들 効果를 가져 온 기구(機構)를 해명(解明)할 목적(目的)으로 실험(實驗)하였든바 그 결과(結果)를 요약(要約)하면 다음과 같다. 1. 대조구(對照區)의 작토층토양중(作土層土壤中)의 물질변화(物質變化) 및 용탈과정(溶脫過程)은 비커, 주사통(注射筒), 투수실험등(透水實驗等)의 실내실험(室內實驗)에서 나타냈든 기본적(基本的)인 유형(類型)에 따라 이루어졌으며 수도근(水稻根)의 존재(存在)는 이와같은 물질변화(物質變化) 및 용탈과정(溶脫過程)을 현저(顯著)히 변화(變化)시키는 것이 아니며 단지야간(單只若干)의 변화(變化)를 주는 정도(程度)였다. 2. 실험(實驗)I에서 경수(莖數)와 침투수중(浸透水中)의 양(陽) ion. $Ca^{{+}{+}}$, $Mg^{{+}{+}}$, Fe, Mn 간(間)의 상관관계(相關關係)는 전부(全部) 고도(高度)의 유의성(有意性)을 나타내고 있어 수도근(水稻根)은 이들 ion들의 용탈(溶脫)을 촉진(促進)시킨다고 본다. 3. 가리(加里), 규산(珪酸), 인산(燐酸) 등(等)은 분얼(分蘖) 최성기(最盛期)부터 흡수(吸收)로 인(因)하여 감소(減少)하였으며 $NH_4$-N 는 검출(檢出)되지 안했다. 4. 실험(實驗)II 에 있어서 경수(莖數)와 침투수중(浸透水中)의 전(全) 양(陽)ion, $Ca^{{+}{+}}$, $Mg^{{+}{+}}$, $Fe^{{+}{+}}$, Fe, Mn 간(間)의 상관관계(相關關係)가 Mg을 제외(除外)하고 전부 고도(高度)의 유의성(有意性)을 나타내고 있어, 이와같은 현상(現象)도 수도근(水稻根)에 의하여 이들 양(陽)ion의 용해(溶解), 용탈(溶脫)이 촉진(促進)되었다고 보는 것이 타당(妥當)하다고 생각된다. 5. 경수(莖數)와 $HCO_3{^-}$ 간(間)의 상관관계(相關關係)는 고도(高度)의 유의성(有意性)을 나타내고 있어 수도근(水稻根)의 활성(活性)이 증가(增加)함에 따라 $HCO_3{^-}$ 도 증가(增加)함을 알았다. 6. 침투수중(浸透水中)의 $HCO_3{^-}$ 와 전(全) 양(陽) ion, $Ca^{{+}{+}}$, $Mg^{{+}{+}}$, $Fe^{{+}{+}}$, Fe, Mn 과의 상관관계(相關關係)는 고도(高度)의 유의성(有意性)이 인정(認定)되었으며 수도근(水稻根)에 의(依)하여 생성(生成)된 $HCO_3{^-}$ 는 $Ca^{{+}{+}}$, $Mg^{{+}{+}}$, $Fe^{{+}{+}}$, Fe, Mn 의 용탈(溶脫)을 촉진(促進)시키며 이들 양(陽) ion은 중탄산염(重炭酸鹽)의 형태(形態)로 용탈(溶脫)된다는 것을 시사(示唆)하는 결과(結果)로 보아진다. 7. 침투수중(浸透水中)의 철(鐵)은 거의 전부(全部)가 2가철(價鐵)이며 2가철(價鐵)과 $HCO_3{^-}$의 상관관계(相關關係)를 보면 고도(高度)의 유의성(有意性)이 인정(認定)되므로 철(鐵)은 중탄산철(重炭酸鐵)의 형태(形態)로 용탈(溶脫)된다고 보는 것이 타당(妥當)하지 않을까 한다. 8. 근권토양(根圈土壤)은 타(他)의 미소부위(微小部位)에 비(比)하여 2가철(價鐵)이 경시적(經時的)으로 감소(減少)하였으며 Glucose 함량(含量)이 2~3배(倍)나 많은 것은 수도근(水稻根)이 산소(酸素)를 분필(分泌)하고 근권토양(根圈土壤)을 산화(酸化)시키며 유기물(有機物)을 분필(分泌)하고 노화(老化)된 물질(物質)의 탈락(脫落) 등(等)에 의(依)하여 유기물(有機物)을 부화(富化)시킨다고 하는 기왕(旣往)의 보고(報告)와 잘 일치(一致)하고 있다. 9. 근권토양(根圈土壤)은 타부위(他部位)에 비(比)하여 ${\beta}$-Glucosidase와 Phosphotase의 활성(活性)이 강(强)한 것은 근권토양(根圈土壤)에 Glucose 함량(含量)이 많기 때문에 미생물(微生物)의 활동(活動)이 왕성한 데에 원인(原因)이 있다고 본다. 10. 침투수(浸透水)의 pH는 재배구(栽培區)가 시종(始終)낮으며 재배구(栽培區)의 Eh는 후기(後期)에 높았다. 끝으로 본(本) 연구(硏究)를 수행(遂行)함에 있어서 시종(始終) 지도(指導)하여 주신 동경대학(東京大學) 농학부토양학연구실(農學部土壤學硏究室) 고정강웅교수(高井康雄敎授)에게 심심(深甚)한 사의(謝意)를 표(表)하며 여러가지로 조언(助言)과 협조(協助)를 하여 주신 화전수덕조교수(和田秀德助敎授)를 비롯한 연구실(硏究室) 제위(諸位) 그리고 공시토양(供試土壤)과 종자(種子)를 제공(提供)하여 주신 동학부(同學部) 천전신일랑교수(川田信一郞敎授) 산기경우박사(山崎耕宇博士), 수도재배(水稻栽培)에 便宜(便宜)를 제공(提供)하여 주신 동학부(同學部) 웅택희구웅교수(熊澤喜久雄敎授), 평전희박사(平田熙博士)에게 감사(感謝)를 드린다.