아리스톨로크산 함유 생약제에 대한 안전성평가연구 : 생식·발생독성시험연구 Studies on the reproductive and developmental toxicity

손경희,이규식,김순선,김소희,곽승준,채수영,박철훈,김병호,최요우,이종필,제금련,강진석,장동덕,길광섭,최광식,박귀례 식품의약품안전청 2001 식품의약품안전청 연보 Vol.5 No.-

아리스톨로크산(ArA)은 Jlristolochia 속 식물에 많이 항유되어있는 성분으로 이 성분이 들어있는 약을 장기복용한 사람들에게서 신장암 등 비뇨기계 암의 발병를이 놀은 것으로 알려졌다. 본 연군에서는 아리스톨로크산 함유 생약제인 마두령의 안전성에 대한 촤학쩍 근거를 마련하기 위하여 마두령 단방과 마두령 복방에 대한 생식 ·살생독성시험을 실시하였다. 마두령 단방 건조추출물(0.j, 2, 8mg/kg; ArA 릉도) 및 복방 건조춘출물(0.05, 0.Smg/ifg; ArA 농도)을 젓드에 임신 전 14일부터 기간 항성기인 임신17일까지 경구 투여한 후 잉신 ?0일에 제왕절개하여 모체와 태자에 대한 검사를 실시하고 이를 아리스톨로크산(2, 8, 16mg/k끄 투여 실험과 비교하였다. 마두령 단방 8mg트 투여군의 체중증가량과 사접춰량이 대조군에 비해 감소하였으며, 아리스톨로르산은 8m을J'k핑 및 15m료토 투여시 임신 전 ·후의 체중증가량 및 사료섭취량이 모두 대조군에 비해 감소하옅다. 또한. 마두령 단방의 경우 8mgg으 투여시 잉신이 저해되는 것_근로 나타났으나. 아리스톨로크산은 임신율에 영향을 미치지 않았고 다민 16mgAg 투여군에서 태자 체중이 유의성있게 강소하였다. 마두령 단방 8m9,/kg 투여군곽 아리스톨크산 8mg/kg 및 16mgg 투여군에서 간장의 절대무게 강소, 진장의 장대무게 증가, 비장의 절대무게감소 등이 간찰되었으며, 조직병리학적검사에서는 마두령 단방 8m9./k9 투여관과 아리스톨로크 16mgg 투여군에서 신장의 손상이 나타났다. 전배자 배양 실험에서는, 마두령 단방은 Ir끄ml 이상에서 태자발달이 현저히 저해되었고, 마두령 복방의 겅우 0.Ofrg/ml 이상에서, 아리스톨로크산끈 경우 300r9/r리 이상의 농도에서 배자발달이 저해되는 것으로 나타났다. 따라4i 본 연구결과로 띠루어 마두령은 임상용량인 0.03mg (.f.rA)/kg/da)-에서는 생식 · 발생득성을 일으키지 않는 것으로 사료된다. Rapidly progressive interstitial renal fibrosis has recently been reported in young women who have been on a slimming regimen including chinese herbs. Aristolochic acid, suspected as the causal factor of this renal disease, is a well known carcinogen. It has been known that Madouling (Aristolochiae fructus) contains aristolochic acid. The objective of this study was to investigate the effects of Madouling, Madouling-tang, and aristolochic acid on reproductive and developmental toxicity. Female rats were administered orally with the extracts of Madouling (0.5, 2, 8mg/kg;ArA dose). madouling-tang (0.05, 0.5mg/kg), and aristolochic acid(2, 8, 16mg/kg) from 14 days before mating to day 17 of gestation. Madouling(8mg/kg) decreased fertility in the 8mg/kg group, but Madouling-tang and aristolochic acids did not. Significant decrease of mean fetal body weights were observed in the 16mg/kg group of aristolochic acids. External, visceral and skeletal malformation of fetuses were not observed with treatment. There were decrease in the absolute liver weights and the absolute spleen weight and increase in the relative kidney weights in the 8mg/kg group of Madouling and 8 and 16mg/kg groups of Aristolochic acid. Histopathological examination showed the discrete damage of kidney in the 8mg/kg group of Madouling and 16mg/kg groups of Aristolochic acid. In whole embryo culture, Madouling and Madouling-tang caused the retardation of growth and development of embryo in the dose of 1㎍/ml and 0.02㎍/kg, respectively while Aristolochic acids showed the similar effect in the dose of 300㎍/kg. These results indicate that Madouling, up to 0.05mg/kg (prescription dose to human) has no adverse effects on the fertility, reproduction and development of Sprague-Dawley rats.

Dual ionic liquid-immobilized silicas for multi-phase extraction of aristolochic acid from plants and herbal medicines

Fang, Luwei,Tian, Minglei,Yan, Xuemin,Xiao, Wei,Row, Kyung Ho Elsevier 2019 Journal of chromatography Vol.1592 No.-

<P><B>Abstract</B></P> <P>A convenient, and efficient multi-phase extraction method was used to isolate aristolochic acid from real samples. To increase the efficiency, a new dual ionic liquid-immobilized silica was used as a sorbent and the effects of the adsorption conditions were investigated. IM-BIM@Sil in a methanol/water (60:40, v/v) solution at 60 °C extracted the highest amount of aristolochic acid (16.69 mg/g) compared to the other sorbents examined. The sorbent was then applied to the multi-phase extraction of aristolochic acid. After extraction eight times, 2.4–70.9 × 10<SUP>−3</SUP> mg/g of aristolochic acid was isolated from nine real samples after several washing and elution steps. The recoveries of aristolochic acid ranged from 70.0% to 110.6% with relative standard deviations of 3.5%–9.1%, highlighting the accuracy of this method.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Dual ionic liquid-immobilized silicas were created. </LI> <LI> High efficiency multi-phase extraction was applied in isolation. </LI> <LI> High adsorption amount of aristolochic acid on new sorbent. </LI> </UL> </P>

Aristolochic Acid에 의한 Fanconi 증후군

신현필 ( Sin Hyeon Pil ),이태원 ( Lee Tae Won ),이영재 ( Lee Yeong Jae ),이상호 ( Lee Sang Ho ),임천규 ( Im Cheon Gyu ),김명재 ( Kim Myeong Jae ) 대한신장학회 2004 Kidney Research and Clinical Practice Vol.23 No.1

A unique type of rapidly progressive interstitial fibosis of the kidney designated Chinese herb nephropathy (CHN) has been reported in Belgian woman after intake of Chinese herbs. CHN contains variable clinical features from progressive renal failure with severe anemia to adult-onset Fanconi`s syndrome which have been reported mostly in Asian countries. We are reporting a case of 43 years-old female patient. The patient visited our hospital with symptoms of polyuria, polydipsia and generalized weakness and laboratory investigation showed hypokalemia, hyperchloremic metabolic acidosis, hypophosphatemia consitent with Fanconis`s syndrome. We found out her intake of Chinese herbal mixture by history taking. The Fanconi`s syndrome seemed to be revrersible but it progressed to renal failure after four months in spite of only ten days intake. Renal biopsy resulted typical findings of aristolochic acid induced nephrophathy. Aristolochic acids were also detected in herbal mixture by high performance chromtography. This case shows variable clinical course of aristolochic acid-induced nephrophathy. It is important to bear in mind that CHN could present variable clinical pattern and herbal mixture that include aristolochic acid should be avoided. (Korean J Nephrol 2004;23(1):147-151)

아리스톨로크산 함유 생약제에 대한 안전성평가연구 : 3개월간 반복투여독성시험을 통한 신장독성평가

황명실,박미선,문지영,이지선,염영나,이효민,신동환,강진석,윤은경,최미나,육미영,장동덕,길광섭,김승희,양기화 식품의약품안전청 2001 식품의약품안전청 연보 Vol.5 No.-

본 실험에서는 아리스톨로크산을 함운하고 있는 생약제중 하나인 마두령(.4risfoforfioe JTurruf)의 90일 반복투여독성시헌을 실f,」하였다. 마두령에서 아리스톨로크산을 정량분석한 결과 마두령 단일 건조븐말의 경운 2.112mg/g, 잉상에서 처방되고 있는 마든령복방 (마두령외 9가지 생약제 복합처방) 건조분말꼭 경무에는 0.066m9/5으로 각각 검출되었다. 본 실험에서 용량설정은 마두령 처리군 (저용량 군; 21.3m9/k9/day (임상용량), 중용량군; 2t3mgfg/day, 고용량군; 2430mg딘g b.w./day), 마두령복방 처리군 (427mg/kg b.w./da?; 임상용량) 및 positive control (아리스톨로크산) 처리군 (저용량군;0.05mgag/da17, 중용량군; 0.Smg/kg/day, 고용량군; 5mg./kg/dal·1으료 하여 랫드에게 경구투여하였다. 마두령단방 처궈근 및 마두령복방 처리군에서 저용량군과 중응량군에서는 잉상관찰 및 생화학적 분석에서 패조군과 유의적인 차이가 없었다. 그러나 마두령단방 고용량근에서는 간, 신장의 상대적인 무계가 증가되었고, 신장의 유두이행상피증식 및 암종이 관찰되었으며, 위에서는 편평세포암종이 관찰되었다. 하자만 간손상이나 간암은 유발하지는 않는 것으로 판단되었다. 결론끌으로 본 실험의 결과에서는 아리스톨로크산 함유 생약제인 마두령이 임상용량인 저응량 처리군에서는 독성을 나타내지 않았으나, 고용쏭으로 장기 복용시켰을 깅우 설치류의 전위부위 및 신장에서 독성이 있음을 알 수 있었다. Chines herbs nephropathy (CHN) has been described in young women who had taken a slimming pills containing some chines herbs. Aristolochic acid (AA), suspected substance as the causal factor of CHN, is known a carcinogen. The Aristolochiae fructus (fruit of Aristolochia contorta) was used in Korean Traditional Medicine consists of appropriate amounts of mixed natural products. Subchronic toxicity of A. furctus containing aristolochic acid was investigated in SD rats. The body weight and clinical signs were observed after orally administration of A. futctus at doses of 21.2, 213, and 2130 mg/kg/day; mixture (including 9 other herbs) at dose of 427 mg/kg/day; aristolochic aicd (Ⅰ+Ⅱ) at doses of 0.05, 0.5, and 5 mg/kg/day for 3 months. At the end of the treatment, high dose treated animals revealed a deficit in final body weight about 25% compared with that of control. Organ weights of kidney, liver, testis, or ovary were increased with dose dependent manner. No changes considered to be due to the administration of A. furctus, mixture or aristolochic acid were in hematological and clinical study. However, significant changes at histopathological study of kidney and stomach were boserved in high-dose treated groups. In conclusion, for human health safety it needs appropriate regulatory actions regarding the use of natural herbal medicines known or suspected of containing aristolochic acid.

유통 방기의 화학적 분석

허옥순,이진하,김세은,주인선,신동우,이정호,김지연,강숙경,이형규,백승화,문병우,김진수 식품의약품안전청 2000 식품의약품안전청 연보 Vol.4 No.-

방기는 대한약전 및 중국약전에 식물학적 및 화학적으로 다른 식물이 동일한 이름으로 수재되어 있어서 운통 및 사용에 혼란을 초래하고 있다. 현재 우리나라에서 유통되고 있는 방기는 패부분 중국 및 베트남에서 수입되고 있으며 방기 외에도 중국산 시장품인 분방기, 광방기가 다수 유통되고 았는 것으로 알려져 있다. 특히' 광방기는 함유성분인 ariStOBDChiC acid의 광범위한 발암성, 신독성이 알려지면서 각 국에서 사용을 금지하고 있으나 우리 나라예서는 유통 현황에 대한 실태조차 파악되지 않고 일는 실정이다. 따라서 방기에 대한 정확한 감별법과 품질기준을 마련하고 이에 따라 aristolochic acid를 함유하고 있는 광방기에 대하여는 수입 및 유통을 금지시킬 필요가 있다. 현재 방기는 외형, 현미경 검사 및 알카로이드 확인시험법으로 감별하게 되어 있으나 다양한 유사품의 정확한 식별을 왜해서는 알카로 이드 타입 및 함량 분포에 러한 연구에 기초한 화학적 감별의 필요성이 요구되고 있다. 이에 본 딴군에서는 방기류에 포함되꺼 있는 주요 알카로이드를 대상으로 확인 및 정량할 수 있는 동시분석법을 확립하였으며, 국내 유통방기 딘움목에 대하여 시험한 결과 유통품의 72.3%에서 독성물질인 aristolochic acid가 검출되어 rlris,!of ocho 속 광방기로 추정되었으며, a7.7%도 방기, 분방기, 목방기와는 다른 방기류 생약이 아닌 강츠로 추정되었다. 광방기 중의 aristolochic acid 함량은 평균 0.31 mg/g으로 측정되었다. 따라서, 유통방기의 대부분이. 약전품 방기가 아니어서 방기의 수입검사와 품질관레, 약효에 심각한 문제점이 있음이 딱혀졌다. 그러므로 광방기 및 유사품의 수입 및 사용을 금지시키고 방기류 생약에 대하여 화학적 성룬검사에 의한 엄격한 수입검사 기준을 설정하여 관리, 검사할 필요가 있다. ln Korean PhaγmaEopia(KP), Sinomeni stem is recorded to he originated from 51'norleriufl ucu#url(fvrenispennaceae 1. In other hand, the original plant of Sinomeni stem(Fangi)in Ehinese Pharmacopla(CP) is reEoFded as SHeffani'o #efFanf#c(Menispermaceae). Tn addition,)'aFious commercial froducis sucl) as fris#of ocfl'o JfHgj(AristolochiaEeae) and Eorufus4FiJoC us(b'lenisperrnaceae) , wh14H belong to different farriiIY and/or genus, are circulated becausethe!7 are all called as ending ,)ame Fangj. In Korea, most of Sinomenl stem are imported fromEi)ina and Vietnam, so it ii con3idered 41)at various mar14e1 products are imported andii#tula?ed. Recentl?-, it )vas Teponed ti)a4 aTlstoloct)ic acids contained in Aristolochla genusinduErd widespread renal 1o714it? and mutagenicity. Many countries prohibited import and useof plants containing aristolochIE acldi. Ho)Yever, present circulative status is not yet refoTted inKorea. T!)erefore, it no·fds to establish frofer discrimination method and quality standardization,to in)7estirate circul3ti4·e status and ?o eF☞Bliblt the use _fri'rfoforAr'o flnri It is kno)vn to have difficulty in discriminating various Sinomeni stem Products by only fts external form. Thus itneeds chemical analysis based on ma;or atkaloid type and content of each ptant.0 this study,we estabtished off#cient simultaneous analysis irlethod which identifeid and quantified majoralkaloids contained various Sinomeni stem products by HPLC. Thirty six samples werecollected and exantned by this method. As a resutt, aristolochic acids were detected in 72.3%of samples. Therefore, it was elucidated that frisfofochio Jhngi are circtllated in high mass.The samplcs of 27.7% were inferred to plants dif:erent from 51=Houleniuul ocufurT and ffepfaHiafefrnndro. These results demonstrate that the present status of circulated Sinomef stem hasserious problem. When the amount of aristolechic acids were determined by HPLC, the contentwas quite high in the range of 0.i2-1.6smg/g of dry plants. Therefore, it suggests thatAristolochia fangi should be proHbited by chemical analysis method due to the toxic effectattributed to the toxic effect of aristolochic acids.

Tissue-resident natural killer cells exacerbate tubulointerstitial fibrosis by activating transglutaminase 2 and syndecan-4 in a model of aristolochic acid-induced nephropathy

( Yu Mee Wee ),( Heounjeong Go ),( Monica Young Choi ),( Hey Rim Jung ),( Yong Mee Cho ),( Young Hoon Kim ),( Duck Jong Han ),( Sung Shin ) 생화학분자생물학회(구 한국생화학분자생물학회) 2019 BMB Reports Vol.52 No.9

Despite reports suggesting that tissue-resident natural killer (trNK) cells cause ischemic kidney injury, their contribution to the development of tubulointerstitial fibrosis has not been determined. This study hypothesized that the depletion of trNK cells may ameliorate renal fibrosis by affecting transglutaminase 2/syndecan-4 interactions. Aristolochic acid nephropathy (AAN) was induced in C57BL/6 mice as an experimental model of kidney fibrosis. The mice were treated with anti-asialo GM1 (ASGM1) or anti-NK1.1 antibodies to deplete NK cells. Although both ASGM1 and NK1.1 antibodies suppressed renal NKp46⁺DX5⁺ NK cells, renal NKp46⁺DX5^- cells were resistant to suppression by ASGM1 or NK1.1 antibodies during the development of tubulointerstitial fibrosis in the AAN-induced mouse model. Western blot analysis showed that both antibodies increased the expression of fibronectin, transglutaminase 2, and syndecan-4. These findings indicate that trNK cells played an exacerbating role in tubulointerstitial fibrosis by activating transglutaminase 2 and syndecan-4 in the AAN-induced mouse model. [BMB Reports 2019; 52(9): 554-559]

Melatonin Attenuates Mitochondrial Damage in Aristolochic Acid-Induced Acute Kidney Injury

Sun Jian,Pan Jinjin,Liu Qinlong,Cheng Jizhong,Tang Qing,Ji Yuke,Cheng Ke,wang Rui,Liu Liang,Wang Dingyou,Wu Na,Zheng Xu,Li Junxia,Zhang Xueyan,Zhu Zhilong,Ding Yanchun,Zheng Feng,Li Jia,Zhang Ying,Yua 한국응용약물학회 2023 Biomolecules & Therapeutics(구 응용약물학회지) Vol.31 No.1

Aristolochic acid (AA), extracted from Aristolochiaceae plants, plays an essential role in traditional herbal medicines and is used for different diseases. However, AA has been found to be nephrotoxic and is known to cause aristolochic acid nephropathy (AAN). AA-induced acute kidney injury (AKI) is a syndrome in AAN with a high morbidity that manifests mitochondrial damage as a key part of its pathological progression. Melatonin primarily serves as a mitochondria-targeted antioxidant. However, its mitochondrial protective role in AA-induced AKI is barely reported. In this study, mice were administrated 2.5 mg/kg AA to induce AKI. Melatonin reduced the increase in Upro and Scr and attenuated the necrosis and atrophy of renal proximal tubules in mice exposed to AA. Melatonin suppressed ROS generation, MDA levels and iNOS expression and increased SOD activities in vivo and in vitro. Intriguingly, the in vivo study revealed that melatonin decreased mitochondrial fragmentation in renal proximal tubular cells and increased ATP levels in kidney tissues in response to AA. In vitro, melatonin restored the mitochondrial membrane potential (MMP) in NRK-52E and HK-2 cells and led to an elevation in ATP levels. Confocal immunofluorescence data showed that puncta containing Mito-tracker and GFP-LC3A/B were reduced, thereby impeding the mitophagy of tubular epithelial cells. Furthermore, melatonin decreased LC3A/B-II expression and increased p62 expression. The apoptosis of tubular epithelial cells induced by AA was decreased. Therefore, our findings revealed that melatonin could prevent AA-induced AKI by attenuating mitochondrial damage, which may provide a potential therapeutic method for renal AA toxicity.

Electrochemical Behavior and Square Wave Voltammetric Determination of Aristolochic Acid-I

Mi Sun Kim,Younghee Hahn 대한약학회 2007 Archives of Pharmacal Research Vol.30 No.2

Electroanalytical procedure for the determination of nephrotoxic aristolochic acid-I in the medicinal plant has been developed in the presence of potential interferences of lead and cadmium by square wave voltametry (SWV). Among the phosphate buffers of pH values at 5.0, 6.1, 6.5 and 7.0, the phosphate buffers of pH 6.1 yielded the most accurate analysis of AA-I in the presence of Pb2+ and Cd2+; Pb2+ was precipitated as Pb(HPO4) and did not appear in the SW voltammogram, while Cd2+ appeared at -0.564 V which was well resolved from AA-I at -0.416 V. When the Ip of AA-I was plotted vs. concentrations between 1.67×10-8 M and 1.67×10-6 M in the presence of Pb2+ and Cd2+, a linear calibration curve was obtained with a slope of 6×108 nA/M and a correlation coefficient of 0.9999. The present method was applied to determine AA in the dried natural products of Aristolochia contorta Bunge; Total AA in the dried root and the ripe fructus of Aristolochia contorta Bunge were found as 25 ± 1 µg/g and 85 ± 3 µg/g, respectively.

증례 : 광방기에 의해 발생한 Chinese Herb Nephropathy 1예

이재욱 ( Jay Wook Lee ),손민정 ( Min Jung Sohn ),허남주 ( Nam Joo Heo ),주권욱 ( Kwon Wook Joo ),정윤철 ( Yoon Chul Jung ),이정상 ( Jung Sang Lee ),한진석 ( Jin Suk Han ) 대한내과학회 2006 대한내과학회지 Vol.71 No.2

본 증례의 환자는 근위부 신세뇨관 산증, 진행성 만성 신부전증, 세뇨관 위축 및 간질 섬유화 등 CHN에 잘 맞는 임상 증상 및 조직 소견을 보였고, 복용하던 한약재에서 AA가 검출되어 AA에 의한 CHN 또는 AAN으로 진단할 수 있었다. 이러한 환자의 신조직에서 AA-DNA adduct를 추가로 검출할 수 있다면 진단의 정확성을 더욱 높일 수 있을 것으로 보인다. Chinese herb nephropathy (CHN) is characterized by progressive tubulointerstitial nephritis and development of renal failure in a couple of years after diagnosis. Aristolochic acid (AA) is believed to be associated with the development of CHN. The authors report a case of CHN in which AA in the herb regimen was identified by high-performance liquid chromatography (HPLC). A 32-year-old female presented with nausea, vomiting and generalized weakness. She had been taking Chinese herbs for symptomatic care. Clinical and laboratory examinations revealed Fanconi syndrome, renal failure, and severe anemia. Renal biopsy showed severe tubulointerstitial nephritis with moderate tubular atrophy and interstitial fibrosis. She developed end-stage renal failure 4 months after diagnosis. The herb she had been taking was Aristolochia fangchi. HPLC technique was used to identify AA and to measure its concentration in the herb. From the clinical and laboratory data, the patient was diagnosed with CHN caused by aristolochic acid.(Korean J Med 71:224-228, 2006)

Chinese Herbs Nephropathy 1예

남기덕,이태원,노정현,양문호,조병수,홍성표,임천규,김명재 대한신장학회 2000 대한신장학회잡지 Vol.19 No.4