연잎, 상엽, 건조 무 혼합 추출물의 지표성분 wogonin, quercetin 및 quercetin-3-O-glucuronide의 분석법 개발 및 검증

장길웅,박은영,최승현,최선일,조봉연,심완섭,한웅호,조현덕,이옥환 한국식품위생안전성학회 2018 한국식품위생안전성학회지 Vol.33 No.4

본 연구는 연잎, 상엽, 건조 무 혼합 추출물의 wogonin, quercetin, quercetin-3-O-glucuronide의 함량 분석 및 해당 표준물질들의 동시 분석법의 개발 및 검증을 실시하였다. 분석법은 특이성, 직선성, 정확성, 정밀성, 검출한계 및 정량한계를 통하여 분석법의 신뢰성을 검증했다. HPLC를 이용한 분석방법에서 표준용액의 머무름 시간과 연잎, 상엽, 건조 무 혼합 추출물 중 quercetin-3-O-glucuronide의 머무름 시간이 일치하였으며 동일한 spectrum을 나타내는 것을 확인해 분석법의 특이성을 검증하였다. Wogonin, quercetin 및 quercetin-3-O-glucuronide의 검량선은 상관계수 값이 wogonin, quercetin 및 quercetin-3-O-glucuronide 모두 0.9999로 우수한 직선성을 보여 분석에 적합함을 알 수 있었고 정밀성과 정확성은 농도를 아는 시료에 인위적으로 저농도, 중농도, 고농도의 표준물질을 첨가해 계산하였다. Wogonin, quercetin, quercetin-3-O-glucuronide의 정밀성은 일간 정밀성, 일내 정밀성으로 확인했으며 세 가지 성분들의 일간 정밀성은 각각 0.76~1.24%, 0.90~1.40%, 0.74~1.87% 수준으로 확인되었고, 일내 정밀성은 0.12~1.06%, 0.28~1.12%, 0.52~0.92%로 5% 이하의 우수한 정밀성을 보였다. 정확성의 측정 결과 wogonin, quercetin 및 quercetin-3-O-glucuronide의 일간 정확성은 99.99~107.97%, 99.96~115.88%, 99.73~111.91%를 나타냈고 일내 정확성은 100.36~108.88%, 99.77~114.81%, 99.91~112.44%로 우수한 수준의 정확성을 보였다. Wogonin, quercetin 및 quercetin-3-O-glucuronide의 검출한계는 각각 0.16 μg/mL, 0.09 μg/mL, 0.15 μg/mL로 측정됐으며 정량한계는 각각 0.48 μg/mL, 0.26 μg/mL, 0.45 μg/mL로 나타나 저농도에서도 검출이 가능함을 확인할 수 있었다. 분석법 검증결과, 확립된 분석법은 특이성, 직선성, 정밀성, 정확성, 검출한계 및 정량한계가 모두 우수한 분석법임이 검증됐으며, 검증된 분석법을 이용해 연잎, 상엽, 건조 무 혼합 추출물의 wogonin, quercein, quercetin-3-O-glucuronide의 함량을 분석한 결과 wogonin과 quercetin은 검출이 되지 않았지만 quercetin-3-O-glucuronide는 함량을 가지고 있는 것으로 확인됐다. 본 연구 결과 에탄올로 추출한 11번(EM11) 샘플에서 quercetin-3-O-glucuronide의 함량이 가장 높게 나왔으며 열수와 에탄올로 추출한 샘플들이 함유하고 있는 quercetin-3-Oglucuronide의 함량은 불검출~50.95 mg/dry weight g 수준으로 함유되어 있는 결과를 보였다. The aim of this study was to develop and validate an analytical method for determining the presence of wogonin, quercetin, and quercetin-3-O-glucuronide in extracts of Nelumbo nucifera, Morus alba L., and Raphanus sativus mixtures We evaluated the specificity, linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ) of analytical methods for wogonin, quercetin, and quercetin-3-O-glucuronide using high performance liquid chromatography. Our result showed that the correlation coefficients of the calibration curve for wogonin, quercetin, and quercetin-3-O-glucuronide were 0.9999. The LOD for wogonin, quercetin, and quercetin-3-O-glucuronide ranged from 0.09 to 0.16 and those for the LOQ ranged from 0.26 to 0.48 μg/mL. The inter-day and intra-day precision values of wogonin, quercetin, and quercetin-3-O-glucuronide ranged from 0.74 to 1.87 and from 0.28 to 1.12%, respectively. The inter-day and intra-day accuracies were 99.96~115.88% and 99.73~114.81%, respectively. Therefore, the analytical method was validated for the detection of wogonin, quercetin, and quercetin-3-O-glucuronide in extracts of Nelumbo nucifera, Morus alba L., and Raphanus sativus mixtures.

Anticonvulsant effect of wogonin isolated from Scutellaria baicalensis

Park, H.G.,Yoon, S.Y.,Choi, J.Y.,Lee, G.S.,Choi, J.H.,Shin, C.Y.,Son, K.H.,Lee, Y.S.,Kim, W.K.,Ryu, J.H.,Ko, K.H.,Cheong, J.H. North-Holland ; Elsevier Science Ltd 2007 european journal of pharmacology Vol.574 No.2

In previous studies, we identified sedative effects of Scutellaria baicalensis extracts and found that these extracts or their constituents may also have anticonvulsive effects. Wogonin is a natural product isolated from S. baicalensis, which possesses central nervous system effects such as anxiolytic and neuroprotective activities. In this study, we investigated the effects of wogonin on convulsion related behaviors, such as myorelaxation, motor coordination, and anticonvulsant effects of wogonin on chemical induced seizure and electroshock seizure in mice or rats. The effect of wogonin on membrane potential was also observed. Wogonin was intraperitoneally injected into mice or rats 30 min prior to testing. Animals treated with wogonin did not change locomotor activities as well as endurance times on the rota-rod, which indicates that wogonin did not cause a sedative and myorelaxation effect. Wogonin significantly blocked convulsion induced by pentylenetetrazole and electroshock but not convulsion induced by strychnine. Wogonin also significantly reduced the electrogenic response score, but flumazenil treatment reversed this decrease to the level of the control group. The wogonin treatment increased Cl<SUP>-</SUP>influx into the intracellular area as dose increased. Flumazenil and bicuculline treatment, however, inhibited the Cl<SUP>-</SUP> influx induced by wogonin. These results indicate that the anticonvulsive effects produced by wogonin were mediated by the GABAergic neuron.

Synthetic Wogonin Derivatives Suppress Lipopolysaccharide-Induced Nitric Oxide Production and Hydrogen Peroxide-Induced Cytotoxicity

Wanjoo Chun,Hee Jae Lee,Pil-Jae Kong,Gun Hee Lee,Il-Young Cheong,Haeil Park,Sung-Soo Kim 대한약학회 2005 Archives of Pharmacal Research Vol.28 No.2

Wogonin (5,7-dihydroxy-8-methoxyflavone) has been reported to exhibit a variety of biological properties including anti-inflammatory and neuroprotective functions. In this study, biological activities of diverse synthetic wogonin derivatives have been evaluated in two experimental cell culture models. Inhibitory activities of wogonin derivatives on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV2 microglial cells and on hydrogen peroxide (H2O2)-induced neuronal cell death in SH-SY5Y human neuroblastoma were examined. Wogonin derivatives such as WS2 and WS3 showed more potent suppressive activities on LPS-induced NO production and H2O2-induced cytotoxicity than wogonin itself. In addition, thiol substitution played a minor role in enhancing the activities of the derivatives. These findings may contribute to the development of novel anti-inflammatory and neuroprotective agents derived from wogonin.

Wogonin Induces Apoptosis by Activation of ERK and p38 MAPKs Signaling Pathways and Generation of Reactive Oxygen Species in Human Breast Cancer Cells

유지선,김안근 한국분자세포생물학회 2011 Molecules and cells Vol.31 No.4

Wogonin is a one of the bioactive compounds of Scutellaria baicalensi Georgi which has been shown to have antiinflammatory, anticancer, antiviral and neuroprotective effects. However, the underlying mechanisms by which wogonin induces apoptosis in cancer cells still remain speculative. Here we investigated the potential activation of MAPKs and generation of reactive oxygen species (ROS) by wogonin on MCF-7 human breast cancer cells. These results showed that wogonin induced mitochondria and death-receptor-mediated apoptotic cell death, which was characterized by activation of several caspases, induction of PARP cleavage, change of antiapoptotic/pro-apoptotic Bcl-2 family member ratios and cleavage of Bid. We also found that generation of ROS was an important mediator in wogonin-induced apoptosis. Further investigation re-vealed that wogonin activated ERK and p38 MAPKs, which was inhibited by N-acetyl cysteine (NAC), a ROS scavenger, indicating that wogonin-induced ROS are associated with MAPKs activation. These data demonstrate that wogonin may be a novel anticancer agent for treatment of breast cancer

Wogonin inhibits osteoclast formation induced by lipopolysaccharide

Jang, Sungil,Bak, Eun Jung,Kim, Minyoung,Kim, Jin Moon,Chung, Won-Yoon,Cha, Jeong-Heon,Yoo, Yun-Jung John Wiley Sons, Ltd. 2010 Phytotherapy research Vol.24 No.7

<P>To evaluate the inhibitory activity of wogonin against lipopolysaccharide (LPS)-induced bone resorption, we investigated the effect of wogonin on osteoclastogenesis induced by LPS. Wogonin inhibited LPS-induced osteoclastogenesis in co-cultures of mouse calvaria-derived osteoblasts and bone marrow-derived pre-osteoclasts. Wogonin also suppressed osteoclastogenesis in LPS-injected mouse calvaria. In osteoblasts, the upregulation of receptor activator of nuclear factor-&kgr;B (RANKL) expression and the downregulation of osteoprotegerin (OPG) expression by LPS were inhibited by wogonin. Wogonin and NS-398, a COX-2 inhibitor, suppressed LPS-stimulated PGE<SUB>2</SUB> production in osteoblasts. NS-398 inhibited the effect of LPS on RANKL and OPG expression in osteoblasts. These results suggest that wogonin acts as an inhibitor of LPS-induced osteoclastogenesis through downregulation of RANKL and upregulation of OPG expression via blockage of PGE<SUB>2</SUB> production. Based on these results, wogonin has potential for use as a therapeutic agent in bacteria-induced bone resorption. Copyright © 2009 John Wiley & Sons, Ltd.</P>

Structure and Antiinflammatory Activity Relationships of Wogonin Derivatives

Jinhee Jang,김현표,Haeil Park 대한약학회 2005 Archives of Pharmacal Research Vol.28 No.8

A number of wogonin derivatives have been synthesized as congeners of wogonin and evaluated for their inhibitory activities of PGE2 production. Wogonin derivatives modified at the B ring of wogonin were obtained from 2,4-Dihydroxy-3,6-dimethoxyacetophenone (1) via several steps. Most wogonin derivatives exhibited much reduced inhibitory activities against COX-2 catalyzed PGE2 production compared to that of wogonin. Alkylation of 5,7-phenol groups and substitution at the B ring of wogonin generally caused reduction of inhibitory activity.

Structure and Antiinflammatory Activity Relationships of Wogonin Derivatives

Jang, Jin-Hee,Kim, Hyun-Pyo,Park, Hae-Il The Pharmaceutical Society of Korea 2005 Archives of Pharmacal Research Vol.28 No.8

A number of wogonin derivatives have been synthesized as congeners of wogonin and evaluated for their inhibitory activities of $PGE_2$ production. Wogonin derivatives modified at the B ring of wogonin were obtained from 2,4-Dihydroxy-3,6-dimethoxyacetophenone (1) via several steps. Most wogonin derivatives exhibited much reduced inhibitory activities against COX-2 catalyzed $PGE_2$ production compared to that of wogonin. Alkylation of 5,7-phenol groups and substitution at the B ring of wogonin generally caused reduction of inhibitory activity.

Synthetic Wogonin Derivatives Suppress Lipopolysaccharide-Induced Nitric Oxide Production and Hydrogen Peroxide-Induced Cytotoxicity

Chun Wanjoo,Lee Hee Jae,Kong Pil-Jae,Lee Gun Hee,Cheong Il-Young,Park Haeil,Kim Sung-Soo The Pharmaceutical Society of Korea 2005 Archives of Pharmacal Research Vol.28 No.2

Wogonin (5,7-dihydroxy-8-methoxyflavone) has been reported to exhibit a variety of biological properties including anti-inflammatory and neuroprotective functions. In this study, biological activities of diverse synthetic wogonin derivatives have been evaluated in two experimental cell culture models. Inhibitory activities of wogonin derivatives on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV2 microglial cells and on hydrogen peroxide ($H_{2}O_2$)-induced neuronal cell death in SH-SY5Y human neuroblastoma were examined. Wogonin derivatives such as WS2 and WS3 showed more potent suppressive activities on LPS-induced NO production and $H_{2}O_2$-induced cytotoxicity than wogonin itself. In addition, thiol substitution played a minor role in enhancing the activities of the derivatives. These findings may contribute to the development of novel anti-inflammatory and neuroprotective agents derived from wogonin.

황금(Scutellaria baicalensis) 조직배양에 의한 Baicalin, Baicalein 및 Wogonin 생산

황인택,이재진,이주영,백승우,김용호 한국자원식물학회 2015 한국자원식물학회지 Vol.28 No.4

Scutellaria baicalensis Georgi (SJ) is a perennial plant and its root has been used in oriental traditional medicine for treatment of fever, inflammation, diarrhea and anticancer effect, etc. In this study, plant tissue culture system for SJ was developed. Stem piece of younger plant was optimum explant for callus induction and growth on MS medium supplemented with NAA 1.0 ㎎/L plus BA 0.5 ㎎/L. Plantlet regeneration through callus culture was well on MS medium containing NAA 1.0 mg/L. SJ has been known biologically active substances such as baicalin, baiclein, and wogonin. This study was carried out to examine the effect of plant growth regulators for production of baicalin, baicalein, and wogonin through callus culture. The HPLC pattern of callus extract was identical to that of standard solution, it shows that the callus produced by tissue culture has the same flavonoids composition of SJ. Baicalin, baicalein, and wogonin production was 471.5~52.8 ㎍/g, 137.6~4.0 ㎍/g, and 16.6~1.3 ㎍/g, respectively, on MS media with nine different plant growth regulator combinations. This may indicate that plant tissue culture of SJ possible to produce the biologically active substances effectively. 황금의 식물조직배양 체계 확립 및 2차대사산물 생성 적정조건을 확인하고자 실험을 수행하였다. 황금 조직배양은 MS 배지에 여러 가지 explant를 치상한 결과 어린 식물의 줄기를 사용하여 PGRs인 NAA와 BA를 소량 혼합처리한 조건에서 callus의 분화가 수월하였으며, shoot는 explant에서 발생된 callus를 소량의 NAA가 첨가된 배지에 배양할 때 분화율이 높게 나타났다. Shoot는 PGRs가 포함되지 않은 MS배지에서 뿌리가 분화되었고 조직배양으로 재분화된 황금 식물개체는 토양으로 이식 후에도 왕성하게 생장함을 확인할 수 있었으며 따라서 본 연구에서는 황금의 식물조직배양 체계를 확립할 수 있었다. 한편, 식물조직배양을 통하여 2차대사 산물 축적이 가능한 지를 확인하고자 황금 callus를 대상으로 몇 가지 flavonids 함량을 HPLC 기기로 분석한 바 callus에서 baicalin, baicalein 및 wogonin을분석할 수 있었다. 이들 함량은 배지에 함유된 PGRs 농도에 따라 baicalin 471.5~52.8 ㎍/g, baicalein 137.6~4.0 ㎍/g, wogonin 16.6~1.3 ㎍/g의 함량 변이를 나타내었다. 따라서 식물조직배양을 통하여 황금에 함유된 baicalin, baicalein 및wogonin 등의 flavonoids를 생산할 수 있음을 확인할 수 있었다.

Neuroprotective Effect of Wogonin: Potential Roles of Inflammatory Cytokines

Piao, Hua-Zi,Jin, Shun-Ai,Chun, Hyang-Sook,Lee, Jae-Chul,Kim, Won-Ki The Pharmaceutical Society of Korea 2004 Archives of Pharmacal Research Vol.27 No.9

Wogonin (5,7-dihydroxy-8-methoxyflavone), an active component originated from the root of Scutellaria baicalensis Georgi, has been reported to possess antioxidant and anti-inflamma-tory properties. In this study, we investigated the neuroprotective effect of wogonin in a focal cerebral ischemia rat model. Wogonin markedly reduced the infarct volume after 2 h middle cerebral artery occlusion followed by 22 h reperfusion. Wogonin decreased the production of nitric oxide and inflammatory cytokines such as TNF-$\alpha$ and IL-6 in lipopolisaccharide-stimu-lated microglial cells. While wogonin reduced the activity of NF-$textsc{k}$B, it did not change the activ-ity of mitogen-activated protein kinases family members, p38, ERK and JNK. The lipopolisaccharide-stimulated production of NO and cytokines was significantly blocked by vari-ous kinds of NF-$textsc{k}$B inhibitors such as N-acetyl cysteine, pyrrolidinedithiocarbamate and MG-132. The data may indicate that wogonin has neuroprotective effect by preventing the over-activation of microglial cells, possibly by inactivating NF-$textsc{k}$B signaling pathway