ORIGINAL RESEARCH : Silymarin-loaded solid nanoparticles provide excellent hepatic protection: physicochemical characterzation and in vivo evaluation

( Kwan Yeol Yang ),( Du Hyeong Hwang ),( Abid Mehmood Yousaf ),( Dong Wuk Kim ),( Young Jun Shin ),( Ok Nam Bae ),( Yong Ii Kim ),( Jong Oh Kim ),( Chul Soon Yong ),( Han Gon Choi ) 영남대학교 약품개발연구소 2013 영남대학교 약품개발연구소 연구업적집 Vol.23 No.0

BACKGROUND: The purpose of this study was to develop a novel silymarin-loaded solid nanoparticle system with enhanced oral bioavailability and an ability to provide excellent hepatic protection for poorly water-soluble drugs using Shirasu porous glass (SPG) membrane emulsification and a spray-drying technique. METHODS: A silymarin-loaded liquid nanoemulsion was formulated by applying the SPG membrane emulsification technique. This was further converted into solid state nanosized particles by the spray-drying technique. The physicochemicalcharacteristics of these nanoparticles were determined by scanning electron microscopy, differential scanning calorimetry, and powder X-ray diffraction. Their dissolution, bioavailability, and hepatoprotective activity in rats were assessed by comparison with a commercially available silymarin-loaded product. RESULTS: Formulation of a silymarin-loaded nanoemulsion, comprising silymarin, castor oil, polyvinylpyrrolidone, Transcutol HP, Tween 80, and water at a weight ratio of 5/3/3/1.25/1.25/100 was accomplished using an SPG membrane emulsification technique at an agitator speed of 700 rpm, a feed pressure of 15 kPa, and a continuous phase temperature of 25°C. This resulted in generation of comparatively uniform emulsion globules with a narrow size distribution. Moreover, the silymarin-loaded solid nanoparticles, containing silymarin/castor oil/polyvinylpyrrolidone/Transcutol HP/Tween 80 at a weight ratio of 5/3/3/1.25/1.25, improved about 1,300-fold drug solubility and retained a mean size of about 210 nm. Silymarin was located in unaltered crystalline form in the nanoparticles. The drug dissolved rapidly from the nanoparticles, reaching nearly 80% within 15 minutes, indicating three-fold better dissolution than that of the commercial product. Further, the nanoparticles showed a considerably shorter time to peak concentration, a greater area under the concentration-time curve, and a higher maximum concentration of silymarin compared with the commercial product (P < 0.05). In particular, the area under the concentration-time curve of the drug provided by the nanoparticles was approximately 1.3-fold greater than that of the commercial product. In addition, the silymarin-loaded nanoparticles significantly reduced carbon tetrachloride-induced hepatotoxicity, indicating improved bioactivity compared with silymarin powder and the commercial product. CONCLUSION: Silymarin-loaded nanoparticles developed using SPG membrane emulsification and spray-drying techniques could be a useful system for delivery of poorly water-soluble silymarin while affording excellent hepatic protection.

Improving the water solubility and antimicrobial activity of silymarin by nanoencapsulation

Lee, Ji-Soo,Hong, Da Young,Kim, Eun Suh,Lee, Hyeon Gyu Elsevier 2017 Colloids and surfaces Biointerfaces Vol.154 No.-

<P><B>Abstract</B></P> <P>The aims of this study were to improve the water solubility and antimicrobial activity of milk thistle silymarin by nanoencapsulation and to assess the functions of silymarin nanoparticle-containing film as an antimicrobial food-packaging agent. Silymarin nanoparticles were prepared using water-soluble chitosan (WCS) and poly-γ-glutamic acid (γ-PGA). As the WCS and silymarin concentrations increased, particle size and polydispersity index (PDI) significantly increased. Nanoencapsulation significantly improved the water solubility of silymarin 7.7-fold. Antimicrobial activity of silymarin was effectively improved when silymarin was entrapped within the nanocapsule compared to when it was not entrapped. Films incorporating silymarin nanoparticles had better antimicrobial activity than films incorporating free silymarin. The results suggest that silymarin nanoparticles have applications in antimicrobial food additives and food packing.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Silymarin was nanoencapsulated using chitosan and γ-poly (glutamic acid). </LI> <LI> The solubility of silymarin was significantly increased by nanoencapsulation. </LI> <LI> The antimicrobial activity of silymarin effectively improved by nanoencapsulation. </LI> <LI> Film containing silymarin nanoparticles could control growth of food microorganism. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P> <P>Nanoencapsulation with water-soluble chitosan and poly-γ-glutamic acid can be used to improve the solubility and antimicrobial activity of silymarin.</P>

간암세포주에서 상피간엽전환억제를 통한 Silymarin의 침윤 및 전이 억제 효과

김도훈 ( Do-hoon Kim ),박소정 ( So-jeong Park ),이승연 ( Seung-yeon Lee ),윤현서 ( Hyun-seo Yoon ),박충무 ( Chung Mu Park ) 대한임상검사과학회 2018 대한임상검사과학회지(KJCLS) Vol.50 No.3

발생하는 간암 중 가장 주요한 형태인 간세포암은 강한 전이 특성으로 인해 높은 재발율과 사망률을 보인다. Silymarin은 엉겅퀴에서 추출한 플라보노이드 성분으로 여러 암세포주에서 상피간엽전환(epithelial mesenchymal transition, EMT) 조절을 통해 항암효과를 보이는 것으로 보고되었다. 본 연구에서는 silymarin이 EMT의 조절을 통해 간세포암 세포주인 Huh7 cell의 침윤과 전이를 억제하는지를 분석하고자 하였다. Huh7 cell의 침윤과 전이 활성을 분석하기 위하여 wound healing assay와 in vitro invasion assay를 시행하였고 EMT 관련 유전자와 상위 신호전달물질의 발현 분석을 위해 Western blot assay를 실시하였다. 그 결과 silymarin은 농도 의존적으로 Huh7 cell의 침윤과 전이를 억제하였다. EMT 관련 유전자 중 세포 부착 단백질인 E-cadherin은 증가하였으나, 중간엽세포의 지표인 vimentin, 종양미세환경 조절에 관여하는 MMP-9의 발현은 억제되었고 이들의 활성에 관여하는 전사인자인 Snail과 nuclear factor (NF)-κB 또한 농도 의존적으로 활성이 감소하는 것을 확인할 수 있었다. 특히, 상위신호전달물질 중 silymarin은 phosphoinositide-3-kinase (PI3K)/Akt의 인산화 억제를 통해 EMT 관련 유전자들을 조절하는 것으로 나타났고 이것은 selective inhibitor인 LY294002의 처리 결과로 확인할 수 있었다. 결과적으로, silymarin은 PI3K/Akt 경로를 통해 EMT 관련 유전자의 발현을 조절함으로써 Huh7 cell의 침윤과 전이를 억제하는 것으로 생각된다. 이를 통해 silymarin이 간세포암의 전이 억제에 효과적인 항암물질의 후보가 될 수 있는 잠재력을 가진 후보물질이 될 수 있음을 보여주었다. Hepatocellular carcinoma (HCC), a major type of hepatoma, is associated with high recurrence and mortality because of its uncontrolled metastatic feature. Silymarin is a polyphenolic flavonoid from Silybum marianun (milk thistle) and exhibits anti-carcinogenic activity through modulation of the epithelial-mesenchymal transition (EMT) in several cancer cells. In this study, the inhibitory mechanism of silymarin against migration and invasion was investigated in the Huh7 HCC cell line. Wound healing and in vitro invasion assays were conducted to examine the effects of silymarin on migration and invasion. Western blot analysis was also applied to evaluate the inhibitory effects of silymarin on the EMT-related genes and their upstream signaling molecules. Silymarin inhibited the migratory and invasive activities of Huh7 cells. In addition, silymarin attenuated the protein expression levels of vimentin and matrix metalloproteinase (MMP)-9 as well as their transcription factors, Snail, and nuclear factor (NF)-κB, while the expression of E-cadherin was increased by the silymarin treatment. Among the upstream signaling molecules, the phosphorylation of Akt was inhibited by the silymarin treatment, which was confirmed by the selective inhibitor, LY294002. Consequently, silymarin inhibited the invasive and migratory activities in Huh7 cells through the modulation of EMT-related gene expression by the PI3K/Akt signaling pathway, which may have potential as a chemopreventive agent against HCC metastasis.

Enhanced bioavailability and hepatoprotective effect of silymarin by preparing silymarin-loaded solid dispersion formulation using freeze-drying method

Dong Yu Lim,Minyeong Pang,Jaehyeok Lee,Jihoon Lee,Ji-Hyeon Jeon,Jin-Hyang Park,Min-Koo Choi,Im-Sook Song 대한약학회 2022 Archives of Pharmacal Research Vol.45 No.10

This study aimed to develop a solid dispersionformulation of silymarin (Silymarin-SD) using freezedryingmethod to enhance its oral bioavailability (BA) byinhibiting the intestinal fi rst-pass eff ect and increasing itssolubility and permeability. Silymarin-SD formulation (i.e.,silymarin:tween 80:hydroxypropyl cellulose (HPC) = 1:1:3(w/w/w) signifi cantly increased silymarin permeability inthe duodenum, jejunum, and ileum by decreasing the effl uxratio of silymarin and by inhibiting silymarin-glucuronidationactivity, in which tween 80 played a crucial role. Asa result, orally administered Silymarin-SD formulationincreased plasma silymarin concentrations and decreasedsilymarin-glucuronide in rats compared with silymarin aloneand silymmarin: D -α-tocopherol polyethylene glycol 1000succinate (1:1, w/w) formulation. In addition to modulatingintestinal fi rst-pass eff ect, Silymarin-SD formulationshowed a signifi cantly higher cumulative dissolution for120 min compared with that of silymarin from the physicalmixture (PM) of the same composition as Silymarin-SD and silymarin alone; the relative BA of silymarin-SDincreased to 215% and 589% compared with silymarin-PM and silymarin alone, respectively. This could be attributedto the amorphous status of the Silymarin-SD formulationwithout chemical interaction with excipients, such as tween80 and HPC. Moreover, the hepatoprotective eff ect of Silymarin-SD in acetaminophen-induced acute hepatotoxicity,as estimated from the alanine aminotransferase and aspartateaminotransferase values, was superior to that of silymarin. Inconclusion, the increase in the dissolution rate and intestinalpermeability of silymarin, and the inhibition of silymaringlucuronidationby the Silymarin-SD formulation, preparedusing tween 80 and HPC, increased its plasma concentrationand resulted in a superior hepatoprotective eff ect comparedto silymarin.

Inhibition of Wnt Signaling by Silymarin in Human Colorectal Cancer Cells

( Hyun Ji Eo ),( Gwang Hun Park ),( Jin Boo Jeong ) 한국응용약물학회 2016 Biomolecules & Therapeutics(구 응용약물학회지) Vol.24 No.4

Silymarin from milk thistle (Silybum marianum) has been reported to show an anti-cancer activity. In previous study, we reported that silymarin induces cyclin D1 proteasomal degradation through NF-κB-mediated threonine-286 phosphorylation. However, mechanism for the inhibition of Wnt signaling by silymarin still remains unanswered. Thus, we investigated whether silymarin affects Wnt signaling in human colorectal cancer cells to elucidate the additional anti-cancer mechanism of silymarin. Transient transfection with a TOP and FOP FLASH luciferase construct indicated that silymarin suppressed the transcriptional activity of β-catenin/TCF. Silymarin treatment resulted in a decrease of intracellular β-catenin protein but not mRNA. The inhibition of proteasome by MG132 and GSK3β inhibition by SB216763 blocked silymarin-mediated downregulation of β-catenin. In addition, silymarin increased phosphorylation of β-catenin and a point mutation of S33Y attenuated silymarin-mediated β-catenin downregulation. In addition, silymarin decreased TCF4 and increased Axin expression in both protein and mRNA level. From these results, we suggest that silymarin-mediated downregulation of β-catenin and TCF4 may result in the inhibition of Wnt signaling in human colorectal cancer cells.

엉겅퀴 추출물 실리마린의 피부 미백효과

추수진 ( Soo Jin Choo ),류인자 ( In Ja Ryoo ),김영희 ( Young Hee Kim ),허광화 ( Guang Hua Xu ),김기호 ( Ki Ho Kim ),한창성 ( Chang Sung Han ),김수진 ( Su Jin Kim ),김진웅 ( Jin Woong Kim ),손의동 ( Eui Dong Son ),유익동 ( Ick Dong 대한화장품학회 2009 대한화장품학회지 Vol.35 No.2

천연물로부터 새로운 미백 소재를 개발하기 위하여 식물 추출물들의 미백활성을 조사한 결과, 엉겅퀴의 열매로부터 추출한 silymarin이 우수한 미백효과를 나타내는 것을 발견하였다. Silymarin으로부터 유효 성분을 분리하기 위하여 각종 컬럼 크로마토그래피 및 HPLC 등의 기법을 실시하여 silybin과 isosilybin을 분리한 후 이성질체인 silybin A와 B, 그리고 isosilybin A와 B를 각각 순수 분리하였다. Silymarin은 Mel-Ab melanocyte에서 세포독성에 영향을 주지 않는 동시에 멜라닌의 생성을 억제하였고 IC₅₀ 값은 28.2 μg/mL이었다. 또한 Silymarin은 cell-based tyrosinase의 활성을 저해하고, western blot 분석 결과 tyrosinase 단백질의 발현을 감소시키는 것을 확인하였다. Silymarin으로부터 분리한 활성 화합물인 silybin 및 isosilybin의 미백 효과를 측정한 결과, 각각 42.25 μM 및 16.32 μM의 IC₅₀ 값을 가지며 멜라닌의 생성을 억제하였으며 tyrosinase 단백질의 발현을 감소시켰다. Diastereoisomer 형태로 존재하는 silybin A 및 B, 그리고 isosilybin A 및 B의 멜라닌 저해활성을 측정한 결과, 네 가지 화합물 모두 농도 의존적으로 멜라닌의 생성을 억제하는 것으로 나타났다. 또한, 피부 미백 임상연구를 실시한 결과, silymarin 2 % 함유 크림을 사용할 경우 피부 미백 효과가 유효하게 나타남을 확인하였다. 이상의 결과로부터 본 활성물질 silymarin은 피부 미백 효과가 우수한 안전한 화장품 원료로서 사용할 수 있을 것으로 사료된다. We found that silymarin exhibited the inhibitory effect on melanogenesis in a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Silymarin is a standardized extract obtained from the dried seeds of milk thistle (Silybum marianum Gaertn.). Silymarin significantly prevented melanin production in a dose-dependent manner with an IC₅₀ value of 28.2 μg/mL without effects on cell viability. Also, silymarin inhibited tyrosinase activity in melanocyte, while it did not affect the catalytic activity of cell-free tyrosinase. Furthermore, Western blot analysis indicated that silymarin decreased the expression of tyrosinase protein. Silybin A/B and isosilybin A/B were also able to inhibit melanin production and tyrosinase expression in protein level. Double blind study on the clinical efficacy of a cream containing 2 % silymarin showed that silymarin have a significant skin whitening effect. Therefore, this study suggests that silymarin may be useful as a natural skin whitening agent.

엉겅퀴 유래 Silymarin의 단독 및 알코올 병용 시 혈압 조절 효과

제현동,민영실 대한산업경영학회 2022 산업융합연구 Vol.20 No.7

In the study, we endeavored to assess the convergence effect of Silybum marianum-derived silymarin and epidemiologically-correlated alcohol intake on vascular contractility and to determine the mechanism involved. There were few reports addressing the question whether thin or thick filament modulation is included in ethanol and silymarin-induced regulation. We hypothesized that ethanol at a low concentration and silymarin play a role in agonist-dependent regulation of vascular contractility. Denuded arterial muscles of Sprague-Dawley male rats were suspended in organ baths and isometric tensions were transduced and recorded using isometric transducers and an automatic data acquisition system. Interestingly, both silymarin and ethanol didn’t encourage silymarin alone-induced inhibition in agonists-induced contraction suggesting that endothelial nitric oxide synthesis might be involved in ethanol or silymarin-induced modulation of vascular contractility and additional pathways besides endothelial nitric oxide synthesis such as ROCK inactivation might be involved in the silymarin-induced modulation of vascular contractility. 역학 조사에서 알코올 섭취와 고혈압 증가 사이에 인과관계가 있어서 이번 연구에서 엉겅퀴 유래 silymarin의 단독 및 알코올 병용 투여에서 혈관수축 억제능을 관찰하였고 아직 불분명한 수축성 조절 기전에 대해 효능제 선택적 조절 가설을 수립하여 조사하였다. 내피가 손상된 혈관이 수조 내 현수되었고 혈관에 의한 기계적 신호가 등장력 변환기에서 전기적 신호로 변환되어 생리측정기에 표시되었다. 저농도의 ethanol과 silymarin은 혈관 내피에서 산화질소 생성 작용 외에 평활근에 대한 직접 작용으로 동맥의 수축성을 감소시킬 것으로 추측되었는데 인위적으로 내피가 손상된 동맥에서 ethanol과 병용된 silymarin이 silymarin 단독에 비해 굵은 미세섬유성 조절성 수축약 (fluoride, thromboxane mimetic)에 의한 혈관 수축 억제에 차이가 없었고 silymarin 단독에 비해 가는 미세섬유성 조절성 phorbol ester에 의한 혈관 수축 억제에 차이가 없었다. 따라서 silymarin 단독은 내피 의존성 산화질소 생성과 내피에 비의존적으로 평활근에서 주로 ROCK 활성 감소에 참여하여 결과적으로 평활근에서 악틴-미오신 상호작용을 억제하여 혈관을 이완시키고 ethanol은 내피 의존성 산화질소 생성 외에 평활근에 대한 작용이 없는 것으로 생각된다.

Inhibition of Wnt Signaling by Silymarin in Human Colorectal Cancer Cells

어현지,박광훈,정진부 한국응용약물학회 2016 Biomolecules & Therapeutics(구 응용약물학회지) Vol.24 No.4

Silymarin from milk thistle (Silybum marianum) has been reported to show an anti-cancer activity. In previous study, we reported that silymarin induces cyclin D1 proteasomal degradation through NF-κB-mediated threonine-286 phosphorylation. However, mechanism for the inhibition of Wnt signaling by silymarin still remains unanswered. Thus, we investigated whether silymarin affects Wnt signaling in human colorectal cancer cells to elucidate the additional anti-cancer mechanism of silymarin. Transient transfection with a TOP and FOP FLASH luciferase construct indicated that silymarin suppressed the transcriptional activity of β-catenin/TCF. Silymarin treatment resulted in a decrease of intracellular β-catenin protein but not mRNA. The inhibition of proteasome by MG132 and GSK3β inhibition by SB216763 blocked silymarin-mediated downregulation of β-catenin. In addition, silymarin increased phosphorylation of β-catenin and a point mutation of S33Y attenuated silymarin-mediated β-catenin downregulation. In addition, silymarin decreased TCF4 and increased Axin expression in both protein and mRNA level. From these results, we suggest that silymarin-mediated downregulation of β-catenin and TCF4 may result in the inhibition of Wnt signaling in human colorectal cancer cells.

Silymarin-Mediated Degradation of c-Myc Contributes to the Inhibition of Cell Proliferation in Human Colorectal Cancer Cells

Hyun Ji Eo,Jin Boo Jeong,Jin Suk Koo,Hyung Jin Jeong 한국자원식물학회 2017 한국자원식물학회지 Vol.30 No.3

In this study, we elucidated the molecular mechanism of silymarin by which silymarin may inhibits cell proliferation in human colorectal cancer cells in order to search the new potential anti-cancer target associated with the cell growth arrest. Silymarin reduced the level of c-Myc protein but not mRNA level indicating that silymarin-mediated downregulation of c-Myc may result from the proteasomal degradation. In the confirmation of silymarin-mediated c-Myc degradation, MG132 as a proteasome inhibitor attenuated c-Myc degradation by silymarin. In addition, silymarin phosphorylated the threonine-58 (Thr58) of c-Myc and the point mutation of Thr58 to alanine blocked its degradation by silymarin, which indicates that Thr58 phosphorylation may be an important modification for silymarin-mediated c-Myc degradation. We observed that the inhibition of ERK1/2, p38 and GSK3β blocked the Thr58 phosphorylation and subsequent c-Myc degradation by silymarin. Finally, the point mutation of Thr58 to alanine attenuated silymarin-mediated inhibition of the cell growth. The results suggest that silymarin induces the cell growth arrest through c-Myc proteasomal degradation via ERK1/2, p38 and GSK3β-dependent Thr58 phosphorylation.

Silymarin, a flavonoid antioxidant, protects streptozotocin-induced lipidperoxidation and β-Cell damage in rat pancreas

Manju Sharma,Tarique Anwer,K K Pillai,Syed Ehtaishamul Haque,A K Najmi,YasminSultana 경희대학교 융합한의과학연구소 2008 Oriental Pharmacy and Experimental Medicine Vol.8 No.2

The present study is aimed at finding the influence of silymarin (a flavonoid) (25 mg/kg & 50 mg/kg) in streptozotocin (STZ)-induced diabetic rats. Type 2 diabetes was induced by single intraperitoneal injection of STZ (100 mg/kg) to 3 days old rat pups. Silymarin was administered for 15 days after the animals were confirmed diabetic (75 days after STZ injection). Blood glucose, glycosylated hemoglobin (HbA1c), lipid peroxides (LPO) levels and reduced glutathione (GSH) contents in pancreas and liver were estimated following the established procedures. Biochemical observations were further substantiated with histological examination of pancreas. Blood glucose and HbA1c levels, which were elevated by STZ, were lowered to physiological levels by the administration of silymarin. The levels of LPO were significantly increased in STZ-induced diabetic rats. Silymarin reduced the LPO levels in both pancreas and liver. GSH contents which were reduced significantly in pancreas and liver of STZ-induced diabetic rats were brought back to near normal levels by silymarin treatment. Multifocal necrotic and degenerative changes of pancreas in STZ-diabetic rats were minimized to near normal morphology by administration of silymarin as evident by histopathological examination. Silymarin showed a dose dependent protective effect on STZ-induced β-cell damage. It could be attributed to the antioxidative and free radicals scavenging properties of the flavonoid. Thus, it may be considered as a natural antioxidant with potential therapeutic application in the treatment of type 2 diabetes. The present study is aimed at finding the influence of silymarin (a flavonoid) (25 mg/kg & 50 mg/kg) in streptozotocin (STZ)-induced diabetic rats. Type 2 diabetes was induced by single intraperitoneal injection of STZ (100 mg/kg) to 3 days old rat pups. Silymarin was administered for 15 days after the animals were confirmed diabetic (75 days after STZ injection). Blood glucose, glycosylated hemoglobin (HbA1c), lipid peroxides (LPO) levels and reduced glutathione (GSH) contents in pancreas and liver were estimated following the established procedures. Biochemical observations were further substantiated with histological examination of pancreas. Blood glucose and HbA1c levels, which were elevated by STZ, were lowered to physiological levels by the administration of silymarin. The levels of LPO were significantly increased in STZ-induced diabetic rats. Silymarin reduced the LPO levels in both pancreas and liver. GSH contents which were reduced significantly in pancreas and liver of STZ-induced diabetic rats were brought back to near normal levels by silymarin treatment. Multifocal necrotic and degenerative changes of pancreas in STZ-diabetic rats were minimized to near normal morphology by administration of silymarin as evident by histopathological examination. Silymarin showed a dose dependent protective effect on STZ-induced β-cell damage. It could be attributed to the antioxidative and free radicals scavenging properties of the flavonoid. Thus, it may be considered as a natural antioxidant with potential therapeutic application in the treatment of type 2 diabetes.