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Synthesis of Ginsenoside Polymer Conjugates and Novel Alpha Glucosyl Ginsenosides from Panax ginseng Meyer 원문보기

  • 저자

    Mathiyalagan Ramya

  • 학위수여기관

    Graduate school of Biotechnology, Kyung Hee University

  • 학위구분

    국내박사

  • 학과

    생명공학원

  • 지도교수

    Prof. Deok Chun Yang

  • 발행년도

    2014

  • 총페이지

  • 키워드

  • 언어

    eng

  • 원문 URL

    http://www.riss.kr/link?id=T13536715&outLink=K  

  • 초록

    암은 전세계적으로 가장 치료가 어렵고 현재까지 주요 사망 원인으로 알려져 있으며 천연물을 이용한 항암 치료가 많이 이뤄지고 있다. 인삼은 수 천년 동안 약용식물로 이용되어져 왔고, 그 효능과 약리 작용에 대한 연구가 많이 되어있다. 인삼 사포닌은 인삼의 triterpenoid로서 다양한 구조와 그에 따른 약리 효능에 대한 연구가 진행되고 있다. 섭취된 인삼 사포닌은 장내 세균에 의해 major ginsenoside 로부터 minor ginsenoside인 ginsenoside CK로 변환되어 장내 세포로부터 흡수, 혈액내로 이동 되어 다양한 약리 효과를 나타낸다. 하지만 이러한 ginsenodise CK와 PPD는 용해도가 낮고 암세포 뿐만 아니라 정상 세포에도 세포독성을 일으켜 부작용을 나타낸다고 보고된바 있다. Minor ginsenoside 인 ginsenoside CK와 PPD aglycon 의 용해도와 약물 전달 능력을 높이기 위하여 친수성 중합체인 Poly ethylene glycol (PEG) 또는 glycol chitosan (GC)을 이용하여 에스테르화 반응을 통한 공유결합체를 형성하여 이들 minor ginsenoside의 용해성과 약리 효능 및 약물 전달 효율을 극대화하고자 하였다. 이러한 중합체 (GC-CK, PEG-CK, PEG-PPD) 의 형성은 spherical형태를 띠고 있으며 입자직경은 296, 404,189nm로 나타남을 알 수 있었다. 또한 중성 pH7.0하에서는 중합체로부터 CK 또는 PPD가 해리되는 속도와 양이 매우 느리고 적은 반면 약산성, pH5.4에서는 CK와 PPD가 중합체로부터 빠르게 해리되어 나옴으로서 암세포 target 지향적, 선택적 약물 전달 가능성을 높일 수 있고 상대적으로 정상세포에서의 세포독성을 현저히 줄일 수 있다. 본 결합체의 구조적 특징을 조사하기위해 1H NMR, FTIR, FE-TEM 을 분석하였고, 용해도를 측정한 결과, CK보다 용해도가 월등히 향상되었음을 확인하였다. 암세포 특이적억제력을 조사하기위해 종양세포 HT29, HepG2, HT22, RAW264.7 등에 위 중합체를 농도별 처리하였을 때 CK보다 Chitosan-CK, PEG-CK가 항암활성이 높았고, 특히 GC-CK가 PEG-CK보다 항암활성이 더 높아 향후 인삼 사포닌 뿐만 아니라 개발된 약물의 선택적 전달 흡수를 위해 더 많은 연구가 요구되어지며 Chitosan GC개발이 요구된다. Sugar donor로서 maltose와 sugar acceptor 로서 ginsenoside Rg1을 함유하는 반응액에 rat small homogenate 첨가 후 50℃에서 반응시키면서 36 시간동안 주기적으로 반응액을 취하여 TLC로 분석한 결과, 효소액을 첨가하지 않은 control과 비교했을 때 효소액을 첨가한 반응액서는 ginsenoside Rg1보다 Rf값이 낮은 성분들이 생성되었으며 maltose가 가수분해되어 생성된 glucose도 검출되었다. 이러한 반응 생성물들은 반응 초기에 급격히 증가한 다음 반응시간이 결과함에 따라서 오히려 감소하는 경향을 보였다. 이러한 결과는 rat small intestine중에 함유된 α-glucosidase의 transglycosylation 반응에 의해 반응초기에 ginsenoside Rg1-α-glucosyl compounds가 생성된 다음 반응 후기에 α-glucosidase의 가수분해 작용에 의해 생성된 ginsenoside Rg1-α-glucosides가 다시 G-Rgl 가수분해되기 때문이라 판단된다. 또한 HPLC-MS, 1H-NMR and 13C-NMR 분석을 통해rat small homogenate 반응액으로부터 생성된 6개의 화합물은 다음과 같다. 6-O-[α-D-glcp-(1→4)-β-D-glcp]-20-O-(β-D-glcp)-20(S)-protopanaxatriol, 6-O-β-D-glcp-20-O-[α-D-glcp-(1→6)-(β-D-glcp)]-20(S)-protopanaxatriol, 6-O-β-D-glcp-20-O-[α-D-glcp-(1→4)-(β-D-glcp)]-20(S)-protopanaxatriol, 6-O-[α-D-glcp-(1→6)-β-D-glcp]-20-O-(β-glcp)-20(S)-protopanaxatriol, 6-O-[α-D-glcp-(1→3)-β-D-glcp]-20-O-(β-D-glcp)-20(S)-protopanaxatriol, 6-O-β-D-glcp-20-O-[α-D-glcp-(1→3)-(β-D-glcp)]-20(S)-protopanaxatriol. 인삼 사포닌에 당이 대부분 βform으로 결합한 반면 본 분리된 2종의 신규화합물은 α form으로 당이 결합된 것으로 NMR결과 본 신규 화합물은 6‐O‐β‐D‐glcp‐20‐O‐α‐D‐glcp‐(1→6)‐(β‐D‐glcp)‐20(S)‐protopanaxatriol 와6‐O‐α‐D‐glcp‐(1→6)‐β‐D‐glcp‐20‐O‐(β‐D‐glcp)‐20(S)‐ protopanaxatriol 로 확인되었으며, 향후 그 약리 효능의 차이를 검증할 필요가 있다고 사료된다. 본 연구는 유용하고 약리 효능이 뛰어난 신규 minor ginsenoside를 생성하기위해 polymer를 이용한 화학적방법과 α–glucosidase를 이용한 생물학적 효소방법을 실시하였다. Minor ginsenoside CK, PPD를 분리하여 보다 용해성이 높고, 세포독성이 낮아 선택적으로 암세포에 선택적으로 작용하여 항암효과가 뛰어난 GC-CK, PEG-CK, PEG-PPD등 다양한 중합체 형성을 시도, 결합체형성조건을 탐색하였으며, 항암효과와 암세포로의 특이적 약물 전달 가능성을 제시하였다. 2종의 신규 ginsenoside를 분리, 구조분석을 하였고, 본 연구를 통해 다양한 인삼 사포닌 생성 조건에 대한 기초자료를 제시하는 바이다.


    Cancer is one of the most incurable diseases, which is a major cause of death worldwide. Several compounds isolated from plant origin were demonstrated to possess anti-cancer effect. As a result, such compounds are being tested as anticancer agents in clinical trials and some of them were approved by Food and Drug Administration (FDA, USA). In this juncture, Korean ginseng (Panax ginseng Meyer) is considered one of the important medicinal plants in oriental medicine for more than thousands of years. Ginsenoside, a triterpenoid that is present only in ginseng is known to exhibit numerous pharmacological efficacies like increase immunization functions, anti-inflammatory effects, protective effects against Alzheimer's disease, anti-diabetic activity, improving liver functions including anti-cancer effects. Among the different ginsenosides, the Rg3, Rh2 and aglycone protopanaxadiol (PPD) were already in the clinical trials. However, after oral administration of crude and PPD type exacts, the major ginsenosides were mainly converted into minor ginsenosides such as Compound k (CK) and aglycone PPD due to hydrolysis of glucose molecules by intestinal microbiota. These both were the primary metabolite that reaches systemic circulations and higher uptake ratio than other ginsenosides such as Rg3 and Rh2 was reported. Moreover, these compounds have been reported for various pharmacological and potent cancer efficacies. However, the clinical application of ginsenoside is significantly hampered due to poor solubility. To improve the solubility, covalent conjugation of hydrophilic polymers to ginsenosides is one of the most promising approaches. In this study, biocompatible and biodegradable hydrophilic polymers such as poly ethylene glycol (PEG) and glycol chitosan (GC) were covalently conjugated to the surface of hydrophobic ginsenosides CK and aglycone PPD via hydrolyzable ester bonds. The resulting conjugates (GC-CK, PEG-CK and PEG-PPD) demonstrated increased solubility of ginsenosides in water. In aqueous condition, the polymeric conjugates formed self-assembled nanoparticles with spherical morphology and an average diameter of 296, 404 and 189 nm respectively. In vitro release studies demonstrated that CK and PPD were slowly released from the conjugates in a physiological buffer (pH 7.4), whereas the release rate of CK and PPD significantly increased under the acidic condition (pH 5.0). In vitro cytotoxicity assay in HT29 cancer cells revealed that CK released from GC-CK conjugates exhibited higher cytotoxicity compared to CK released from PEG-CK conjugates. Whereas, PEG-PPD conjugates exhibited lower cytotoxicity than PPD, presumably be due to slow release of PPD by the hydrolysis of ester bonds. Both GC-CK and PEG-CK conjugates maintained similar cytotoxicity in HepG2, and HT22 cancer cell lines. Moreover, both GC-CK and PEG-CK conjugates maintained good cell viability in pre-inflammation disease condition in RAW264.7 cells (murine macrophage cells) than CK alone. The inhibition of inflammation was measured by nitric oxide assay in the lipopolysaccharide activated RAW 264.7. The CK with GC inhibited NO production but CK from PEG was not much inhibited than free CK. The chemical conjugation of hydrophilic polymers to poorly insoluble drugs significantly improved their in vivo bioavailability. Accordingly, thus synthesized GC-CK conjugates exhibited a high anticancer activity in HT29 and HepG2 cancer cells. Interestingly the GC-CK conjugate was found to be non-toxic for RAW264.7 cells. Overall the results suggest that, GC-CK conjugates could be a useful carrier for intracellular release of CK in tumor and inflammation diseases. The chemical conjugation approach developed in this study could also be used to other ginsenosides to improve their solubility and in vivo anti-tumor activity. Finally, we attempted to conjugate glucose molecules to the ginsenosides by a transglycosylation reaction of the glycosyl hydrolase enzyme. For this, rat intestinal homogenates which exhibited a high alpha glucosidase activity was utilized as the catalyst, and maltose as the donor and ginsenoside Rg1 as a sugar acceptor. As a result, a serious of compounds was synthesized, isolated and purified. Based on the various physiochemical characterizations (HPLC-MS, 1H-NMR and 13C-NMR), the alpha glucosyl ginsenoside Rg1 conjugates was identified. The individual compounds named as 6-O-[α-D-glcp-(1→4)-β-D-glcp]-20-O-(β-D-glcp)-20(S)-protopanaxatriol, 6-O-β-D-glcp-20-O-[α-D-glcp-(1→6)-(β-D- glcp)]-20(S)-protopanaxatriol, 6-O-β-D-glcp-20-O-[α-D-glcp-(1→4)-(β-D-glcp)]-20(S)-protopanaxatriol, 6-O-[α-D-glcp-(1→6)-β-D-glcp]-20-O-(β-glcp)-20(S)-protopanaxatriol,6-O-[α-D-glcp-(1→3)-β-D-glcp]-20-O-(β-D-glcp)-20(S)-protopanaxatriol and 6-O-β-D-glcp-20-O-[α-D-glcp-(1→3)-(β-D-glcp)]-20(S)-protopanaxatriol. Among these six, 6‐O‐β‐D‐glcp‐20‐O‐α‐D‐glcp‐(1→6)‐(β‐D‐glcp)‐20(S)‐protopanaxatriol and 6‐O‐α‐D‐glcp‐(1→6)‐β‐D‐glcp‐20‐O‐(β‐D‐glcp)‐20(S)‐ protopanaxatriol are considered to be novel compounds of alpha-ginsenosidal saponins. To the best of our knowledge, for the first time in the literature, we have reported an unusual glycosylation in 6th and 20th positions of α‐D‐glucopyranosyl‐(1→6)‐β‐D‐glucopyranosyl sugar chain with 20(S)-protopanaxatriol saponins in Panax ginseng Meyer. Although β form are common, unusual α form ginsenosides may have a great impact to improve pharmacological efficacies for benefit of human kind.


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