플라티코딘 D와 효소에 의해 전환된 플라티코딘 D 강화분획물의 지방축척 억제효과

학위논문 (박사)-- 서울대학교 대학원 : 약학대학 약학과, 2019. 2. 김영식.길경 (Platycodon grandiflorum A. DC)의 유효성분이자 지표성분인 플라티코딘 D는 기존에 항암, 항산화, 항염증 등의 효능이 있는 것으로 보고가 되고 있다. 최근, 플라디코딘 D는 동물모델에서 지질대사 관련 개선능과 항비만 생리활성이 탁월한 것으로 많은 보고가 있었음에도 불구하고, 아직까지 세포수준에서 어떠한 기전으로 항비만 효과가 있는지 명확한 결과는 없고, 세포수준의 결과가 동물실험 상에서 동일하게 재현되지 않은 단점이 있다. 본 연구에서는 지방합성에 관여하는 peroxisome proliferator-activated receptor γ2 (PPARγ2)와 지방세포분화에 관여하는 mitogen activated protein kinase (MAPKs), 지방산화에 중추적 역할을 하는 AMP-activated protein kinaseα(AMPKα)의 활성을 측정하여, 지방세포에서 플라디코딘 D의 지방축척억제 효과를 확인하고 지방유도동물 모델에서 지방 억제 효과를 보고자 하였다. 플라디코딘 D가 탁월한 약리학적 가치를 가지고 있긴 하지만, 도라지 건조 중량의 미량으로 포함 되어 있고, 기존의 천연물분리 방법으로는 시간이 오래 걸리고, 수율이 낮은 단점이 있어서 플라디코딘 D를 상업적을 이용하는데 많은 어려움이 있었다. 본 실험실에서는 길경사포닌 중에 플라디코딘 D와 비슷한 화학 구조를 가진 플라디코딘 D유도체들을 효소처리를 하여 단시간 내에 플라디코딘 D로 전환, 플라디코딘 D의 함량을 높인 강화분획조성물을 만든 바 있다. 본 연구에서는 기존에 분리가 쉽지가 않던 플라디코딘 D를 대체하여 분리가 효율적이며 상업적 으로 이용가능성이 있는 플라티코딘 D 강화분획물 (PScell)의 지방축척 억제효과 를 확인하고자 하였다. 분화된 지방세포에 플라디코딘 D 5 μM을 처리하였을 경우 지방합성 마커인 PPARγ2와 C/EBPα가 뚜렸하게 억제되었고, 비만전사인자 (adipogenic transcription factor)인 AP2, FAS발현이 감소가 됨으로써 분화된 지방세포 내에 지방함량이 약 62.5% 정도 억제됨이 확인되었다. 또한, 지방세포 초기분화에 관여하는 것으로 알려진 유사분열 활성화 단백질 인산화효소 (MAPKkinase)는 플라디코딘 D 처리한 그룹에서 3 시간부터 24 시간까지 p-ERK를 꾸준히 억제 되었다. AMPK activator로 잘 알려진 aminoimidazole carboxamide ribonucleo- tide (AICAR)와 비교실험에서 플라디코딘 D 5 μM투여는 AICAR 1 mM 투여 보다 PPARγ2와 C/EBPα의 발현을 현저하게 억제되었고, p-AMPK와 p-ACC 발현은 유의성 있게 증가되었지만 AICAR보다는 약함이 확인되었다. 위 세포실험의 결과를 토대로, 본 실험자는 8주 동안 고지방 식이로 섭취한 C57BL/6 마우스에서 플라디코딘 D의 항비만효과를 수행하였다. 플라디코딘 D 투여그룹은 고지방식이그룹보다 경구투여 10일 이후 부터 체중이 유의성 있게 감소되었고, 실험 종료 후 고지방식이그룹보다 약 16 g정도 감소된 것으로 확인되었다. 섭취량은 투여 초기 4주 동안 유의성 있게 감소되었으나 이후 4주 투여에서는 복원됨으로써 다른 그룹간 섭취량에 차이가 없었다. 플라티코딘 D 투여 그룹에서 단위 면적당 지방의 부피와 지방세포 크기가 줄어들었고, 혈액내의 지질대사 개선 효과가 현저히 나타났다. 비만동물모델 지방조직에서 지방형성 (lipogenesis)의 표적 단백질인 PPARγ2와 C/EBPα의 발현정도가 억제되었고 AMPK 및 ACC의 발현이 증가되었으며, 간조직 또한 지방간의 핵심 마커인 sterol-regulatory element binding proteins 1 (SREBP-1)발현이 감소되었다. 뒤이어, 효소전환에 의해 플라디코딘 D가 강화된 분획물 (PScell)의 지방축척효과를 확인하였다. 플라티코딘 D 강화분획물을 지방세포분화 유도물질과 함께 농도별 (5, 7.5, 10 ㎍/㎖)로 처리한 결과, 분화된 지방세포에서 지방 축적이 농도별로 억제되었다. 8주간 진행된 동물실험에서 플라티코딘 D 강화분획물을 경구투여한 그룹에서는 고지방그룹보다 유의적인 체중감소와 복부지방량 감소 효과를 나타내었는데, 이것은 수치상 플라디코딘 D 와 약리활성수준이 비슷하거나 좀더 좋은 효능을 보일 것으로 예상되었다. 본 연구 결과들을 종합하여 볼 때, 플라디코딘 D와 플라디코딘 D 강화분획물은 비만 및 비만에 의해 유래된 지방간에 효과적인 약물일 것이라 기대된다. 또한, 플라디코딘 D 강화분획물은 그동안 상업화하기 어려웠던 플라디코딘 D의 대체물질이 될 수 있을 것이라 판단하지만, 산업화 과정에서는 단백질수준에서 기존 상품화 되어 있는 단일화합물이나 플라디코딘 D와 효능평가를 비교하는 후속실험이 필요할 것으로 사료된다.Platycodi Radix, the root of Platycodon grandiflorum (Jacq.) A. DC. (Campanulaceae), is traditionally used as a treatment for respiratory discomfort by practitioners of Traditional Chinese medicine, Japanese Kampo medicine and Korean medicine. Platycosides, the saponins found in the roots of Platycodon grandiflorum (Jacq.) A. DC. (Platycodi Radix), are typically composed of oleanane backbones with two side chains, one is a 3-O-glucose side chain linked by a glycosidic bond and the other being a 28-O-arabinose-rhamnose-xylose-apiose side chain joined by an ester bond. Platycodi Radix is known to have more than 20 saponins with similar structures. Among them, platycodin D is superior to other saponins and the distinctive compound found only in Platycodi Radix. It is known to be effective for those with a sore throat, bronchitis, cold, diabetes, inflammation and cancer. Although platycodin D has broad pharmacological value, because the total content of saponins is as low as 2% by dry weight of Platycodi Radix, it is difficult to isolate platycodin D from Platycodi Radix on a large scale. When attempting to overcome this disadvantage, we successfully isolated platycodin D in Platycodi Radix and several derivatives in a short period of time using a multi-step process which includes high-speed counter-current chromatography (HSCCC) and preparative reversed-phase high-performance liquid chromatography (HPLC) steps. We also successfully developed an enzyme-conversion technique through which platycodin D3 (PD3) and platycoside E (PE), which have two and three glucose units at C-3, respectively, are converted to platycodin D, consequently increasing the amount of platycodin D in saponin and thus making it an enriched fraction (henceforth PScell). In this study, we aim to determine the mechanism of how platycodin D, which is widely known to be effective on the bronchus, works on obesity. A second goal is to determine the effects of PScell on obesity. We initially examined platycodin D and platycodin D derivatives to determine how they inhibit lipid accumulation activity, finding that platycodin D is a more effective inhibitor than any of the platycodin D derivatives. To determine the optimal concentration of platycodin D, various doses were exposed to cells during MDI (isobutylmethylxanthine, dexamethasone and insulin)-induced differentiation. On day 8, the lipid contents in 3T3-L1 cytoplasm were measured by Oil Red O staining. The results showed that platycodin D (1, 2.5, 5 μM) blocked adipocyte differentiation in proportion to the platycodin D dose with platycodin D 5 μM inhibiting lipid accumulation in the cytoplasm by as much as 62.5% in a MDI-treated positive control without affecting cell viability. It was also found that platycodin D significantly inhibits fat accumulation of lipid droplets in the cytoplasm by inhibiting adipogenic-specific transcription factors PPARγ2 and C/EBPα in MDI-induced 3T3-L1 cells in a dose-dependent manner. Similarly, platycodin D inhibits adipocyte differentiation of 3T3-L1 cells through the ERK pathway within 24 hrs. Furthermore, we investigated the molecular mechanism of platycodin D, focusing on its ability to decrease the expression of adipogenic factors through AMP-activated protein kinase α (AMPKα) in adipocytes and to prevent abdominal fat accumulation in high-fat-diet-induced obesity among C57BL/6 mice. To verify the anti-obesity effect in vivo, a group of mice ate a normal diet while the others were fed a high-fat diet for eight weeks. The high-fat-diet mice were then divided into three subgroups: termed the aminoimidazole carboxamide ribonucleotide (AICAR) group, the vehicle group and the platycodin D group. It was found that platycodin D significantly reduced fat accumulation by inhibiting adipogenic signal transcriptional factors, in this case peroxisome proliferator-activated receptor γ2 (PPARγ2) and CCAAT/enhancer binding protein α (C/EBPα), through AMPK in vivo. Platycodin D also reduced both the body weight and fat mass and consequently improved lipid metabolism by increasing AMPK, as also seen in AICAR group, while also reducing PPARγ2 and C/EBPα expression levels in adipose tissue. In addition, glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels, which are used as indicators of hepatic disease in serum and the size of lipid drops during morphometric observations of fatty liver were significantly reduced in the platycodin D group. Moreover, protein expressions of AMP-activated protein kinase α (AMPKα) increased while sterol regulatory element binding protein-1 (SREBP-1) decreased in the platycodin D group compared to those of the high-fat group (HF). This outcome suggests that platycodin D can be used to inhibit lipid accumulation by reducing the expression levels of adipogenic factors related to the AMPK pathway and that it can be expected to improve lipid metabolism in obesity-associated hepatic lipogenesis. In addition, we attempted to investigate the fat accumulation effect of PScell, the fraction with an increased platycodin D content, by enzymatic transformation. Treatment of 3T3-L1 adipocytes with PScell (5, 7.5, 10 μg/mL) reduced lipid accumulation in a dose-dependent manner. In a mouse model, oral administration of PScell (70 mg/kg, 6 mL/kg) reduced high-fat-diet-induced body weight gain. Consistently, PScell alleviated total cholesterol, LDL cholesterol, triglyceride and glucose levels in mice serum. Liver and abdominal adipose tissues from the groups treated with PScell exhibited a decreased number of lipid droplets relative to the high-fat control group. PScell showed a stronger inhibitory effect on lipid accumulation at a slightly lower concentration than that of platycodin D numerically in in vitro and in vivo. From these results, we considered that PScell is a potential candidate as a treatment for obesity and fatty liver induced by a high-fat diet by replacing platycodin D with a low yield in Platycodi radix.CONTENTS ABSTRACT CONTENTS LIST OF FIGURES LIST OF TABLE I. INTRODUCTION 1. Platycodi Radix, Platicosides and Platycodin D 2. Adipocyte and adipogenesis 3. AMPK 4. Purpose of the study II. MATERIALS and METHODS 1. MATERIALS 1.1. Plant material 1.2. Chemicals and reagents 1.3. Cells culture 1.4. Animals 1.5. Apparatuses 2. METHODS 2.1. Preparation of crude sample 2.2. One-step separation of platycodin D by HSCCC 2.3. Sample preparation of enriched saponin fraction 2.4. Cell viability assay 2.5. Adipocyte differentiation and treatment 2.6. Oil Red O staining 2.7. Western blotting 2.8. Real time PCR 2.9. High fat diet induced obese mice 2.10. Serum analysis 2.11. Micro-computed tomography 2.12. Histological analysis 2.13. Data analysis III. RESULTS 3.1. Lipid accumulation inhibition activities of platycodin D and platycodin D derivatives isolated by HSCCC 3.2. Effects of platycodin D on lipid accumulation during adipocyte differentiation 3.3. Effects of platycodin D on the expression of p-ERK during adipocyte differentiation 3.4. Effects of platycodin D on the expression of a p-AMPKα pathway in 3T3-L1 cell 3.5. Effect of platycodin D on body weight, food intake and daily energy in high fat diet mice 3.6. Effect of platycodin D on white adipose tissue mass and serum profiles in high fat diet mice 3.7. Effect of platycodin D on adipogenic protein expression in high fat diet mice 3.8. Effects of platycodin D on lipid accumulation in liver of C57BL/6 model induced obesity 3.9. Effects of PScell on lipid accumulation inhibition activities during adipocyte differentiation 3.10. Effects of PScell on food intake, body weight gain in C57BL/6 model induced obesity 3.11. Effects of PScell on white adipose tissue and liver fatty droplet accumulation in C57BL/6 model-induced obesity 3.12. Effects of PScell on serum profiles in C57BL/6 model-induced obesity IV. DISCUSSION V. REFERENCES VI. ABSTRACT IN KOREAN VII. ACKNOWLEGEMENT LIST OF FIGURES Fig. 1. Structure of platycodin D Fig. 2. Physiological balance between hypertrophy and hyperplasia Fig. 3. Constitute of cells in white adipose tissue Fig. 4. Secretion adipokines of triglyceride overload hypertrophy adipocyte Fig. 5. Transcriptional factors of adipocyte differentiation PPARγ2, C/EBPα Fig. 6. MAPKs signal transduction pathways Fig. 7. Involvement of the MAPKs at the various steps of adipogenesis Fig. 8. Sampling-preparative isolation platycodin D from Platycodon grandiflorum A. DC. by HSCCC coupled with ELSD Fig. 9. Enzymetic biotransformation of the saponin-enriched fraction to platycodin D by cellulase Fig. 10. Large-scale modification of the saponin-enriched fraction for platycodin D Fig. 11. Adipocyte differentiation and treatment Fig. 12. High fat diet animal model experiments Fig. 13. Cytotoxicity and lipid accumulation inhibition activities of platycodin D and platycodin D derivatives Fig. 14. Effects of platycodin D on lipid accumulation during adipocyte differentiation Fig. 15. Effects of platycodin D on the expression of p-ERK during adipocyte differentiation Fig. 16. Effects of platycodin D on the activation of AMPKα during adipocyte differentiation Fig. 17. Effects of platycodin D on body weight gain, food intake and daily energy in C57BL/6 model-induced obesity Fig. 18. Effects of platycodin D on fat size, mass and serum profiles in C57BL/6 model-induced obesity Fig. 19. Effects of platycodin D on the expression of proteins related to lipid metabolism in white adipose tissue Fig. 20. Effects of platycodin D on lipid accumulation in liver of C57BL/6 model-induced obesity Fig. 21. Effects of PScell on lipid accumulation inhibition activities during adipocyte differentiation Fig. 22. Effects of PScell on food intake and body weight gain in C57BL/6 model-induced obesity Fig. 23. Effects of PScell on liver and white adipose tissue size fatty droplet accumulation in C57BL/6 model-induced obesity Fig. 24. Effects of PScell on serum profiles in C57BL/6 model-induced obesity LIST OF TABLE Table 1. The structures of various platycosidesDocto