患有肥胖及高血脂症二者之患者可暴露於心血管疾病之高風險，而且減少身體之白色脂肪組織(WAT)可最小化慢性心血管病症之風險。已報導許多減重藥物可升高心血管疾病之風險。此外，亦已注意到他汀類(statin，一種降膽固醇藥物)具有輕微至嚴重副作用。因此，研究者尋求針對抗肥胖及抗高血脂症施加之替代療法。 認為在肥胖之進展期間，WAT質量擴大取決於脂肪細胞增生及肥大二者。藉此，將脂肪細胞分化(自前脂肪細胞分化為脂肪細胞)表徵為與肥胖正相關之主要細胞過程。兩種關鍵轉錄因子，過氧化物酶體增殖物活化受體(PPAR)及CCAAT/增強子結合蛋白(C/EBP)，對調節與脂肪細胞分化相關之基因之轉錄活性起關鍵作用。增加之C/EBP-β可能產生PPAR-γ配體以觸發PPAR-γ介導之信號傳導路徑，使得脂肪細胞分化及脂肪生成。此外，5'AMP活化蛋白激酶(AMPK)可分別藉由抑制乙醯CoA羧化酶(ACC)來增加脂肪酸氧化以及藉由阻抑HMG-CoA還原酶(HMGCR)來減少膽固醇合成。先前報告揭露肥胖患者或小鼠中之脂聯素之血漿含量低於較瘦個體。脂聯素(由脂肪細胞分泌之脂肪因子中之一者)能夠促進脂肪細胞分化且藉由刺激AMPK活化來調節能量代謝。 已報導，YSY處方內之一些植物成份展現對於抗高血脂或抗動脈粥狀硬化效應之潛在活性(Aziz, Z 、 Wong, S. Y 與 Chong, N. J. (2013). Effects of Hibiscus sabdariffa L. on serum lipids: a systematic review and meta-analysis. J Ethnopharmacol, 150, 442-450 ； Bisen, P. S. 、 Baghel, R. K. 、 Sanodiya, B. S. 、 Thakur, G. S. 與 Prasad, G. B. (2010). Lentinus edodes: a macrofungus with pharmacological activities. Curr Med Chem, 17, 2419-2430 ； Fukushima, M. 、 Ohashi, T. 、 Fujiwara, Y 、 Sonoyama, K. 與 Nakano, M. (2001). Cholesterol-lowering effects of maitake (Grifola frondosa) fiber, shiitake (Lentinus edodes) fiber, and enokitake (Flammulina velutipes) fiber in rats. Exp Biol Med (Maywood), 226, 758-765 ； Tsi, D. 、 Das, N. P. 與 Tan, B. K. (1995). Effects of aqueous celery (Apium graveolens) extract on lipid parameters of rats fed a high fat diet. Planta Med, 61, 18-21 ； Zeng, F. 、 Zhao, C 、 Pang, J. 、 Lin, Z 、 Huang, Y 與 Liu, B. (2013). Chemical properties of a polysaccharide purified from solid-state fermentation of Auricularia auricular and its biological activity as a hypolipidemic agent. J Food Sci, 78, H1470-1475 ； Zhang, J. 、 Liang, R. 、 Wang, L. 、 Yan, R. 、 Hou, R. 、 Gao, S. 與 Yang, B. (2013). Effects of an aqueous extract of Crataegus pinnatifida Bge. var. major N.E.Br. fruit on experimental atherosclerosis in rats. J Ethnopharmacol, 148, 563-569)
。儘管單一植物成份可提供對於高血脂症之抑制效應，但多成份產物可能展現優良協同活性。Patients with both obesity and hyperlipidemia can be exposed to a high risk of cardiovascular disease, and reducing the body's white adipose tissue (WAT) can minimize the risk of chronic cardiovascular disease. Many weight loss drugs have been reported to increase the risk of cardiovascular disease. In addition, it has also been noted that statins (statin, a cholesterol-lowering drug) have mild to severe side effects. Therefore, researchers seek alternative treatments for anti-obesity and anti-hyperlipidemia. It is believed that during the progression of obesity, WAT mass expansion depends on both adipocyte hyperplasia and hypertrophy. In this way, adipocyte differentiation (differentiation from preadipocytes to adipocytes) is characterized as the main cellular process positively related to obesity. Two key transcription factors, peroxisome proliferator-activated receptor (PPAR) and CCAAT/enhancer binding protein (C/EBP), play a key role in regulating the transcriptional activity of genes involved in adipocyte differentiation. Increased C/EBP-β may produce PPAR-γ ligands to trigger PPAR-γ-mediated signal transduction pathways, resulting in adipocyte differentiation and adipogenesis. In addition, 5'AMP activated protein kinase (AMPK) can increase fatty acid oxidation by inhibiting acetyl CoA carboxylase (ACC) and reduce cholesterol synthesis by inhibiting HMG-CoA reductase (HMGCR), respectively. Previous reports have revealed that plasma levels of adiponectin in obese patients or mice are lower than leaner individuals. Adiponectin (one of the adipokines secreted by adipocytes) can promote adipocyte differentiation and regulate energy metabolism by stimulating AMPK activation. It has been reported that some of the plant ingredients in the YSY prescription exhibit potential activity against antihyperlipidemic or anti-atherosclerotic effects (Aziz, Z , Wong, S. Y and Chong, NJ (2013). Effects of Hibiscus sabdariffa L. on serum lipids: a systematic review and meta-analysis. J Ethnopharmacol, 150, 442-450 ; Bisen, PS , Baghel, RK , Sanodiya, BS , Thakur, GS and Prasad, GB (2010). Lentinus edodes: a macrofungus with pharmacological activities. Curr Med Chem, 17, 2419-2430 ; Fukushima, M. , Ohashi, T. , Fujiwara, Y , Sonoyama, K. and Nakano, M. (2001). Cholesterol-lowering effects of maitake (Grifola frondosa) fiber , shiitake (Lentinus edodes) fiber, and enokitake (Flammulina velutipes) fiber in rats. Exp Biol Med (Maywood), 226, 758-765 ; Tsi, D. , Das, NP and Tan, BK (1995). Effects of aqueous celery (Apium graveolens) extract on lipid parameters of rats fed a high fat diet. Planta Med, 61, 18-21 ; Zeng, F. , Zhao, C , Pang, J. , Lin, Z , Huang, Y and Liu, B. (2013). Chemical properties of a polysaccharide purified from solid-state fermentation of Auricularia auricular and its biological activity as a hyp olipidemic agent. J Food Sci, 78, H1470-1475 ; Zhang, J. , Liang, R. , Wang, L. , Yan, R. , Hou, R. , Gao, S. and Yang, B. (2013) . Effects of an aqueous extract of Crataegus pinnatifida Bge. var. major NEBr. fruit on experimental atherosclerosis in rats. J Ethnopharmacol, 148, 563-569) . Although a single plant component can provide an inhibitory effect on hyperlipidemia, a multi-component product may exhibit excellent synergistic activity.
本發明驚人地發現YSY之水萃取物可減輕體重、高血脂症、脂肪肝及動脈粥狀硬化。 除非另外定義，否則本文中所用之所有技術及科學術語均具有與熟習本發明所屬技術領域者通常所理解含義相同之含義。儘管類似於或等效於本文中所述之彼等方法及材料之任何方法及材料可用於本發明之實踐或測試中，但闡述較佳方法及材料。出於本發明之目的，以下術語定義於下文中。 術語「一(a及an)」係指一個或一個以上(即至少一個)冠詞之語法對象。 如本文所用，在申請專利範圍中，除非明確指示僅指替代物或除非替代物互相排斥，否則術語「或」係指「及/或」。 術語「改善(promote、promotion及promoting)」係指活性、反應、病況、疾病或其他生物學參數之增加。 術語「個體」包括活的生物體，例如人類、猴、母牛、綿羊、馬、豬、牛、山羊、狗、貓、小鼠、大鼠、培養細胞及其轉基因物種。在較佳實施例中，個體係人類。 術語「投與」包括容許本發明組合物實施其預期功能之投與途徑。 術語「治療(treat或treatment)」意指降低疾病或病況之效應之方法。治療亦可指降低疾病或病況自身之潛在病因之方法。治療亦可為自天然水準之任何降低且可為(但不限於)疾病、病況或疾病或病況之症狀之完全消除。 術語「預防(prevent、prevention或preventing)」意指與骨質疏鬆症相關症狀之抑制或防止。 術語「有效量」意指有效治療及/或預防疾病、病況或疾病或病況之症狀之本發明組合物之量。 在一態樣中，本發明提供包含木耳(wood ear,Auricularia auricular
)、香菇(Lentinus edodes
)、山楂(hawthorn fruit,Crataegus pinnatifida
)、洛神葵(roselle,Hibiscus sabdariffa
)、芹菜(Apium graveolens
)及李子(梅(Prunus mume
))之組合物之水萃取物，
其中該水萃取物係藉由以下獲得：將木耳、香菇、山楂、洛神葵、芹菜及李子混合以形成組合物，將該組合物浸泡於水中在約50℃至約80℃之溫度至少16小時及將所得組合物煮至沸騰以獲得該組合物之水萃取物。所得萃取物可藉由HPLC檢測。 在一個實施例中，組合物之浸泡時間係約18小時至約48小時、約18小時至約46小時、約18小時至約44小時、約18小時至約42小時、約18小時至約40小時、約18小時至約38小時、約18小時至約36小時、約18小時至約34小時、約18小時至約32小時、約18小時至約30小時、約18小時至約28小時、約18小時至約26小時、約20小時至約48小時或約22小時至約48小時。較佳地，組合物浸泡於水中約24小時。 在一個實施例中，浸泡溫度係約50℃至約75℃、約50℃至約70℃、約50℃至約65℃、約55℃至約75℃或約55℃至約70℃。較佳地，浸泡溫度係約60℃。 在一個實施例中，組合物中之木耳、香菇、山楂、洛神葵、芹菜及李子之比率以乾重計為約0.5至約1.5:約0.5至約1.5:約1.5至約2.5:約1.5至約2.5:約4.5至約5.5:約0.5至約1.5。較佳地，該比率以乾重計為約1:約1:約2:約2:約5:約1。 在另一態樣中，本發明提供包含木耳(wood ear,Auricularia auricular
)、香菇(Lentinus edodes
)、山楂(hawthorn fruit,Crataegus pinnatifida
)、洛神葵(roselle,Hibiscus sabdariffa
)、芹菜(Apium graveolens
)及李子(梅(Prunus mume
))之組合物之水萃取物，
其中該組合物包含具有滯留時間分別為約3.2分鐘至約4.5分鐘(較佳約3.978分鐘)及約5.4分鐘至約6.2分鐘(約5.943分鐘)之化合物，如藉由HPLC所量測。 在一個實施例中，水萃取物進一步包含滯留時間分別為約3.2分鐘至約4.5分鐘(較佳約3.978分鐘)、約2.7分鐘至3.2分鐘(較佳2.995分鐘)及約5.4分鐘至約6.2分鐘(約5.943分鐘)之化合物，如藉由HPLC所量測。在一實施例中，水萃取物進一步包含滯留時間為約3.2分鐘至約4.5分鐘(較佳約3.978分鐘)、約2.7分鐘至3.2分鐘(較佳2.995分鐘)及約5.4至約6.2分鐘(較佳約5.943分鐘)、約2.0分鐘至約2.5分鐘(較佳約2.307分鐘)、約9.0分鐘至約9.4分鐘(較佳約9.224分鐘)及約6.8分鐘至7.1分鐘(較佳約6.931分鐘)之化合物，如藉由HPLC所量測。除該等峰之外，水萃取物進一步包含滯留時間為約0.565分鐘、約0.751分鐘、約1.175分鐘、約7.996分鐘、約8.361分鐘、約10.879分鐘、約11.709分鐘、約12.303分鐘及約12.766分鐘之化合物，如藉由HPLC所量測。在一實施例中，本發明水萃取物具有如圖1中所示之HPLC概況。 根據本發明，本發明水萃取物具有如下表中所示之HPLC概況。
可採用業內已知之任何萃取技術以製備本發明萃取物。所得萃取物可進一步藉由層析分級分離。較佳層析係使用溶劑溶析之液相層析。較佳地，液相層析係高效液相層析(HPLC)或反相HPLC。在一實施例中，本文中所述HPLC係藉由使用移動相A為ddH2
O及移動相B為MeOH/甲酸(體積比率99.9:0.1)、C18管柱及流速為0.5 ml/min及UV檢測器來實施。 在一態樣中，本發明提供包含木耳(wood ear,Auricularia auricular
)、香菇(Lentinus edodes
)、山楂(hawthorn fruit,Crataegus pinnatifida
)、洛神葵(roselle,Hibiscus sabdariffa
)、芹菜(Apium graveolens
)及李子(梅(Prunus mume
))之組合物之水萃取物，其中該組合物包含分別具有以下滯留時間之化合物：約4.61分鐘至4.95分鐘(較佳約4.67分鐘)、約6.1分鐘至約6.5分鐘(較佳約6.29分鐘)、約6.8分鐘至約7.0分鐘(較佳約6.91分鐘)、約3.45分鐘至約3.62分鐘(較佳約3.53分鐘)及約2.96分鐘至3.16分鐘(較佳約3.06分鐘)，如藉由多反應監測(MRM)之萃取離子層析圖(XIC)所量測，其中MRM之XIC係在153.000/109.000 Da之HPLC-MS/MS下檢測且使用原兒茶酸作為標準品。 在另一態樣中，在一態樣中，本發明提供包含木耳(wood ear,Auricularia auricular
)、香菇(Lentinus edodes
)、山楂(hawthorn fruit,Crataegus pinnatifida
)、洛神葵(roselle,Hibiscus sabdariffa
)、芹菜(Apium graveolens
)及李子(梅(Prunus mume
))之組合物之水萃取物，
其中該組合物包含分別具有以下滯留時間之化合物：約4.35分鐘至約4.65分鐘(較佳約4.52分鐘)、約3.41分鐘至約3.62分鐘(較佳約3.55分鐘)、約4.0分鐘至約4.15分鐘(較佳約4.09分鐘)、約3.8分鐘至約4.0分鐘(較佳約3.95分鐘)及約5.18分鐘至約5.4分鐘(較佳約5.26分鐘)，如藉由多反應監測(MRM)之萃取離子層析圖(XIC)所量測，其中MRM之XIC係在153.000/109.000 Da之HPLC-MS/MS下檢測且使用綠原酸作為標準品。 在一態樣中，本發明提供包含如本文中所定義之水萃取物之製劑。 在另一態樣中，本發明提供用於治療及/或預防高血脂症、動脈粥狀硬化及/或肥胖之方法，其包含將有效量之本發明水萃取物投與個體。 本發明水萃取物可與醫藥上可接受之載劑、賦形劑及/或稀釋劑一起調配為用於投與之醫藥製劑或藥劑。 醫藥上可接受之載劑、稀釋劑及/或賦形劑意指載劑、稀釋劑及/或賦形劑必須與製劑之其他成份相容、不會不利地影響本發明水萃取物之治療益處且對其接受者無毒。 可藉由全身及/或局部遞送化合物之任何方法來投與水萃取物或其製劑以實踐本發明。該等方法包括經口途徑、非經腸途徑、十二指腸內途徑等。 在局部施加中，將水萃取物或其製劑局部地施加至需要其之位點。 對於局部施加，水萃取物或其製劑可調配於含有懸浮或溶解於一或多種載劑中之水萃取物或其製劑之適宜軟膏中。用於局部投與本發明水萃取物或其製劑之載劑包括(但不限於)：礦物油、液體石蠟脂、白石蠟脂、丙二醇、聚氧乙烯、聚氧丙烯化合物、乳化蠟、糖(例如乳糖)及水。另一選擇為，醫藥製劑可調配於含有懸浮或溶解於一或多種醫藥上可接受之載劑中之水萃取物或其製劑之適宜洗劑或乳霜中。適宜載劑包括(但不限於)礦物油、山梨醇酐單硬脂酸酯、聚山梨醇酯60、十六烷基酯蠟、鯨蠟硬脂醇、2-辛基十二烷醇、苯甲醇及水。 端視欲治療之具體病況、病症或疾病而定，可與水萃取物或其製劑一起施加額外治療劑。彼等額外藥劑可作為多劑量方案之部分自含水萃取物之組合物以任何順序依序投與(連續或間斷投與)。另一選擇為，彼等藥劑可為單一劑型之部分，與水萃取物或其製劑一起混合於單一組合物中(同時或并行投與)。 對於經口投與，可用於本發明中之醫藥組合物可採取以下形式：溶液、懸浮液、錠劑、丸劑、膠囊、粉劑、顆粒、半固體、持續釋放型調配物、酏劑、氣溶膠及諸如此類。含有各種賦形劑(例如檸檬酸鈉、碳酸鈣及磷酸鈣)之錠劑可連同各種崩解劑(例如澱粉(較佳馬鈴薯或木薯澱粉)及某些複合矽酸鹽)一起，連同黏合劑(例如聚乙烯吡咯啶酮、蔗糖、明膠及阿拉伯膠)一起採用。此外，潤滑劑(例如硬脂酸鎂、月桂基硫酸鈉及滑石粉)通常極為可用於壓錠目的。亦採用相似類型之固體組合物作為軟填充及硬填充明膠膠囊中之填充劑；就此而言，較佳材料亦包括乳糖或牛乳糖以及高分子量聚乙二醇。在期望經口投與水性懸浮液及/或酏劑時，可將本發明之水萃取物或其製劑與各種甜味劑、調味劑、著色劑、乳化劑及/或懸浮劑以及稀釋劑(例如水、乙醇、丙二醇、甘油及其各種相似組合)組合。 調配物之選擇取決於各種因素，例如藥物投與之模式(例如，對於經口投與，調配物呈錠劑、丸劑或膠囊之形式較佳)及原料藥之生物利用度。 本文所用術語「非經腸」係指包括靜脈內、肌內、腹膜內、胸骨內、皮下、髓內及關節內注射及輸注之投與模式。用於非經腸注射之醫藥組合物可包含醫藥上可接受之無菌水性或非水性溶液、分散液、懸浮液或乳液以及在即將使用前重構成無菌可注射溶液或分散液之無菌粉末。水性溶液尤其適用於靜脈內、肌內、皮下及腹膜內注射目的。就此而言，所採用之無菌水性介質均可易於藉由熟習此項技術者眾所周知之標準技術獲得。適宜水性及非水性載劑、稀釋劑、溶劑或媒劑之實例包括水、乙醇、多元醇(例如甘油、丙二醇、聚乙二醇及諸如此類)、羧甲基纖維素及其適宜混合物、植物油(例如橄欖油)及可注射之有機酯(例如油酸乙酯)。例如，可藉由使用諸如卵磷脂等塗覆材料、藉由維持所需粒徑(在分散液情形下)且藉由使用表面活性劑來維持適當流動性。 可用於本發明中之醫藥製劑亦可含有佐劑，例如(但不限於)防腐劑、潤濕劑、乳化劑及分散劑。微生物作用之防止可藉由納入各種抗細菌及抗真菌劑(例如，對羥基苯甲酸酯、氯丁醇、苯酚、山梨酸及諸如此類)來確保。包括諸如糖、氯化鈉及諸如此類之等滲劑亦可為合意的。可藉由納入延遲吸收之藥劑(例如單硬脂酸鋁及明膠)來造成可注射醫藥形式之延長吸收。 除水萃取物及其製劑外，懸浮液可含有懸浮劑，例如乙氧基化異硬脂醇、聚氧乙烯山梨醇及山梨醇酐酯、微晶纖維素、偏氫氧化鋁、膨潤土、瓊脂及黃蓍膠及其混合物。 出於經皮(例如局部)投與之目的，製備其他方面類似於上文非經腸溶液之稀釋無菌水性或部分水性溶液(濃度通常為約0.1%至5%)。 可用於本發明中之醫藥製劑亦可藉由經鼻氣溶膠或吸入投與。此等組合物係根據醫藥調配領域內熟知之技術製備，且可於鹽水中製備為溶液，使用苯甲醇或其他適宜防腐劑、吸收促進劑以增強生物利用度、氟碳化合物及/或其他習用增溶劑或分散劑。 用於直腸或陰道投與之組合物較佳為栓劑，其可藉由將本發明之水萃取物或其製劑與適宜無刺激性賦形劑或載劑(例如可可脂、聚乙二醇或栓劑蠟)混合製備，該等栓劑在室溫為固體但在體溫為液體，且因此可在直腸或陰道腔內融化並釋放藥物。 其他醫藥上可接受之載劑包括(但不限於)無毒固體、半固體或液體填充劑、稀釋劑、囊封材料，或任一類型之調配助劑，包括(但不限於)離子交換劑、氧化鋁、硬脂酸鋁、卵磷脂、血清蛋白(諸如人類血清白蛋白)、緩衝物質(諸如磷酸鹽、甘胺酸、山梨酸、山梨酸鉀)、飽和植物脂肪酸之偏甘油酯混合物、水、鹽或電解質(諸如硫酸魚精蛋白、磷酸氫二鈉、磷酸氫鉀、氯化鈉、鋅鹽)、膠體二氧化矽、三矽酸鎂、聚乙烯基吡咯啶酮、基於纖維素之物質、聚乙二醇、羧甲基纖維素鈉、聚丙烯酸酯、蠟、聚乙烯-聚氧丙烯-嵌段聚合物、聚乙二醇及羊毛脂。 固體醫藥賦形劑包括(但不限於)澱粉、纖維素、滑石粉、葡萄糖、乳糖、蔗糖、明膠、麥芽、稻、麵粉、白堊、矽凝膠、硬脂酸鎂、硬脂酸鈉、甘油單硬脂酸酯、氯化鈉、脫脂乳粉及諸如此類。液體及及半固體賦形劑可選自甘油、丙二醇、水、乙醇及各種油，包括石油、動物、植物或合成來源之彼等，例如，花生油、大豆油、礦物油、芝麻油等。較佳液體載劑(尤其用於可注射溶液)包括水、鹽水、水性右旋糖及二醇。 熟習此項技術者已知或將根據此揭示內容明瞭製備各種具有某一量之活性成份之醫藥組合物之方法。其他適宜醫藥賦形劑及其調配物闡述於Remington's Pharmaceutical Sciences, E. W. Martin編輯，Mack Publishing Company，第19版(1995)。 可用於本發明中之醫藥組合物可含有1%-100% (以重量計)之本發明之水萃取物或其製劑。在任何情形中，欲投與之組合物或調配物將含有一定量之本發明之水萃取物或其製劑，該量可有效治療所治療之個體之病況、病症或疾病。 熟習此項技術者將瞭解水萃取物或其製劑之醫藥上有效量可根據經驗確定且可以純形式或以醫藥上可接受之鹽、酯或前藥形式(若此等形式存在)採用。可將該等藥劑以與一或多種醫藥上可接受之賦形劑組合之醫藥組合物之形式投與患者。將理解，在投與(例如)人類患者時，本發明之藥劑或組合物之總日用量將在由主治醫師做出合理醫學判斷之範圍內決定。任一具體患者之特定治療有效劑量值將端視各種因素而定：欲達成之細胞反應之類型及程度；所採用特定藥劑或組合物之活性；所採用特定藥劑或組合物；患者之年齡、體重、一般健康狀況、性別及飲食；藥劑之投與時間、投與途徑及排泄速率；治療之持續時間；與特定藥劑組合或同時使用之藥物；及醫學領域中熟知之類似因素。在一實施例中，本發明水萃取物之有效量係約4 g/天至約18 g/天。在一些實施例中，有效量係約4 g/天至約16 g/天、約4 g/天至約14 g/天、約4 g/天至約12 g/天、約6 g/天至約18 g/天、約6 g/天至約14 g/天、約8 g/天至約18 g/天、約8 g/天至約16 g/天、約8 g/天至約14 g/天、約10 g/天至約18 g/天、約10 g/天至約16 g/天或約8 g/天至約14 g/天。較佳地，有效量係約12 g/天。亦可以患者特定之方式來配置劑量以在血液中提供預測定濃度之藥劑，如藉由業內接受之常規技術所測定。 另一選擇為，水萃取物或其製劑可與食品級載劑、賦形劑、稀釋劑及/或鹽一起調配為保健食品或膳食補充物。上文所提及之載劑、賦形劑、稀釋劑及/或鹽可用於本發明之保健食品或膳食補充物中。保健食品或膳食補充物可以各種形式存在，包括(但不限於)錠劑、膠囊、囊片、粉劑、飲品(包括奶昔)、固體食品類(包括零食棒)等。 熟習相關領域之技術者將易於明瞭可對本文中所述之方法及應用作出其他適宜修改及改寫，而不背離本發明或其任何實施例之範圍。提供以下實例以說明而非限制本發明。實例 材料及方法 1. 化學品及試劑
抗磷酸-ACC (乙醯CoA羧化酶；編號3661)、磷酸-AMPKα (5'-單磷酸腺苷活化蛋白激酶-α；編號2531s)及PPAR-γ (過氧化物酶體增殖物活化受體-γ；編號2435)之抗體係自Cell Signaling Technology, Inc. (Danvers, MA, USA)購得。識別β-肌動蛋白(編號ab6276)及C/EBP-β (CCAAT/增強子結合蛋白β；編號GTX61124)蛋白之抗體分別係自Abcam (Cambridge, MA, USA)及GeneTex, Inc. (USA)獲得。抗小鼠免疫球蛋白G (IgG) (編號sc2005)及兔IgG (編號sc2004)之辣根過氧化物標記之二級抗體係自Santa Cruz Biotechnology (Santa Cruz, CA, USA)獲得。所有其他試劑均係自Sigma-Aldrich (St Louis, MO, USA)購得。2. YSY 之製備
YSY由6種天然食物構成，包括木耳(wood ear,Auricularia auricular
)、香菇(Lentinus edodes
)、山楂(hawthorn fruit,Crataegus pinnatifida
)、洛神葵(roselle,Hibiscus sabdariffa
)、芹菜(Apium graveolens
)以及李子(梅(Prunus mume
))，
且其混合比率以乾重計為1:1:2:2:5:1。所有植物均係自標準合格之農場購得且其來源由中藥治療中心(Chinese herbal therapy center)、中國醫藥大學附設醫院(China Medical University Hospital ) (Taichung, Taiwan)驗證。將所有乾燥植物切成片，在60℃下浸泡於水中達24 h，且然後在100℃下煮以濃縮萃取物。3. YSY 之層析 概況及生物活性化合物之鑑別
藉由LC/ESI-MS/MS (Applied Biosystems Sciex, Foster City, CA, USA)實施YSY中前文所提及之諸如原兒茶酸(PCA)及綠原酸(CGA)等潛在生物活性化合物之鑑別以建立YSY萃取物之參照層析概況，以用於每一批之萃取程序之品質檢查。使用配備有電噴霧電離源且與Agilent 1200 HPLC (Agilent Technologies, Wilmington, DE, USA)連接之API 2000-三重四極質譜儀(Applied Biosystems/MDS Sciex, Foster City, CA, USA)來實施LC/ESI-MS/MS分析。使用Syncronis™ C18管柱(150 mm × 4.6 mm，5 μm；Thermo, Waltham, MA, USA)分離YSY (500 μg/mL)。移動相A係ddH2
O，而移動相B係MeOH/甲酸(體積比率99.9:0.1)，且流速設至0.5 ml/min。用於HPLC分析中之檢測器係UV檢測器。 使用電噴霧電離串聯式質譜術以負性多反應監測(MRM)模式來實施質譜分析。所有分析物均顯示[M-H]-
為最強前驅物離子，其可在負離子模式中檢測為[M-H]。對於每一分析物，使用100 ms作為留置時間檢測兩種MRM轉換，一種定量及一種定性；PCA：353/191、353/85；CGA：153/109、153/91。4. 高血脂動物模型
所有程序及動物護理均根據實驗動物服務中心(Laboratory Animal Service Center)、中國醫藥大學(China Medical University) (Taichung, Taiwan)之機構動物倫理指南批准且實施。將雄性ApoE-KO小鼠(4月齡)分為4組(n=8/組)，包括普通飲食組(正常飲食)、HFD (高脂飲食；正常飲食與60%脂肪混合)、HFD +低劑量YSY (0.4 g/kg/天)及HFD +高劑量YSY (2 g/kg/天)。將雄性倉鼠(8週齡)分為8組(n=6/組)，包括HFD、HFD + YSY (1.48 g/kg/天)、HFD +木耳(1.48 g/kg/天)、HFD +香菇(1.48 g/kg/天)、HFD +山楂(1.48 g/kg/天)、HFD +洛神葵(1.48 g/kg/天)、HFD +芹菜(1.48 g/kg/天)及HFD +梅(1.48 g/kg/天)。藉由HFD誘導降血脂動物持續4週且然後使用YSY萃取物或單一植物成份再治療干預持續8週。在研究結束時，藉由肌內注射Zoteil 50®
(0.1 ml) (Virbac Ltd, Carros, France)麻醉動物且然後收集包括血液、肝及內臟脂肪組織之生物樣本以評估組織病理學及血液生化檢查。5. 血液生化分析
在過夜饑餓後，在處死動物後藉由使用分離管收集全部血液，容許血液試樣在室溫下凝塊2 h，然後在4℃下以1000 ×g離心20 min。總膽固醇(TC)、三酸甘油酯(TG)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)、麩丙轉胺酶(GPT)及血脲氮(BUN)之血清含量之量測由Zhen- Xing Co., Ltd (Taichung, Taiwan)提供服務。此外，藉由商業ELISA套組(編號E90605Mu, USCN Life Science Inc., USA)根據製造商之說明書來量測血清脂聯素。6. 肝及內臟脂肪組織之冷凍切片
根據吾人之先前研究(Pan等人，2013)來實施肝組織中之脂質積累。肝組織使用生理鹽水灌注，在5%福馬林中和溶液(J.T. Baker, Inc., Philipsburg, NJ, USA)中固定24 h，且包埋於Tissue-Tek®
OCT化合物(編號4583, Sakura Finetek Inc., Torrance, CA, USA)中。將經包埋組織切成10 μm且使用蘇丹IV (對於肝組織)或油紅(對於脂肪組織)以及蘇木精/伊紅(Merck, Whitehouse Station, NJ, USA)染色。在400倍放大下獲得照片且於Alphalmager 2200®
文件系統(Alpha InnoTech, San Leandro, CA, USA)上定量。 7.脂質墊分析
在8週研究結束時收集脂質墊。解剖性腺周圍及腹脇部脂肪組織且然後使用1×磷酸緩衝鹽水(PBS)沖洗。 脂質墊比率係以如下等式(Eq. (1))計算： 脂質墊比率(%) = 性腺周圍及腹脇部脂肪組織(g)/體重(g)×100 (1)8. 西方墨點
使用PRO-PREP® 蛋白萃取溶液(500 μl/g)將肝組織均質化，且藉由使用Bio-Rad蛋白分析(Bio-Rad)來量測蛋白濃度。將等量(30 μg)之萃取之蛋白穿過10% SDS-PAGE電泳且然後轉移至聚偏二氟乙烯(PVDF)膜。藉由使用溶解於PBST (具有0.1% Tween 20之1×PBS)中之5%脫脂奶粉將膜封閉1 h且然後使用PBST洗滌。然後在室溫下將膜與特定一級抗體在適當稀釋下(p-ACC, 1:1000；p-AMPK, 1:1000；HMG-CoA, 1:500；PPAR-γ, 1:1000；C/EBP-β, 1:500；β-肌動蛋白，1:1000)一起培育2 h。然後，洗滌墨點且在室溫下與HRP-偶聯二級抗體在1:2000稀釋下一起培育1 h。使用化學發光試劑使發光信號顯影，藉由Fujifilm LAS-4000系統(San Leandro, CA, USA)獲取，且使用來自NIH (USA)之Image J軟體程式定量。9. 主動脈脂紋之 分析
在動物處死後收集主動脈且然後使用常用鹽水溫和沖洗。將所有組織試樣於4%多聚甲醛中培育10 min且然後在室溫下使用油紅溶液(於異丙醇中，5 mg/mL)染色15 min。將經染色主動脈使用若干不同濃度之異丙醇(90%、70%及50%)洗滌1 min，然後使用水沖洗。使用數位相機(編號D80；Nikon, Tokyo, Japan)獲得照片。10. 臨床研究
此人類研究係開放標記及平行先導性臨床試驗，其由中國醫藥大學附設醫院(Taichung, Taiwan)批准(編號DMR99-IRB-126)以研究YSY在健康及高血脂個體中之降脂效應。考慮登記由成人治療小組III (Adult Treatment Panel III, ATP III)界定患有高血脂症之年齡為22-65歲之成年人。排除懷孕或患有慢性代謝疾病之參與者。另外，所有個體均未正使用降脂藥物，例如他汀類、膽酸螯合劑、貝特類及菸鹼酸及可在試驗期間改變肝功能之藥品。根據ATP III指導，將合格參與者分為3組(正常、邊界及高組)。研究開始於4週基線期。研究前收集血樣作為空白對照。然後，所有個體接受12 g/天之YSY達2個月。在研究結束時，在YSY投與後24 h收集血樣。11. 統計學分析
數據表示為平均值± S.D。方差之分析用於評價差異之統計學顯著性，然後對於所有對之比較進行塔基測試(Tukey's test)。將p值＜0.05視為統計上顯著。使用Statistical Package for Social Sciences (SPSS X, Chicago, IL, USA)分析數據。實例 1 YSY 降低 HFD 飼養之小鼠之體重、脂肪組織 積累及血液脂質
吾人之實驗數據表明，相較於對照組，體重及內臟脂肪組織質量因YSY之高劑量處理而顯著降低(圖1A及1B)。對於確定YSY誘導之體重下降是否係由於食物攝取改變所致，在實驗時期期間記錄食物攝取。如圖1C中所示，在8週之研究後，在所有組之間在食物攝取量上不存在顯著差異。在本研究中，吾人亦探索YSY對HFD飼養之ApoE-KO小鼠之降血脂效應(表1)。實驗結果顯示，相較於對照組(單獨HFD)，在高劑量YSY處理後，若干生化參數(TC、TG及LDL)顯著降低。然而，HDL之血清含量藉由YSY未顯著改良(表1)。此外，觀察到在所有組之間GPT及BUN之血清含量無顯著差異，暗示發現在YSY處理後無明顯毒性。表 1
-經YSY處理之ApoE-KO小鼠中之血清生化概況. a
相較於對照組(單獨使用HFD處理)，p＜
0.05，b
相較於普通飲食組，p＜
0.05。實例 2 YSY 降低 HFD 飼養之小鼠中之脂肪生成
在8週之動物研究後，將內臟脂肪組織冷凍切片且經HE染色變色以評估脂肪細胞大小之改變，且組織病理學結果表明高劑量YSY處理減小脂肪細胞大小(圖2A)，此暗示YSY可調節脂肪細胞分化。因此，檢查參與脂肪細胞分化之3種關鍵蛋白(包括AMPK、PPAR-γ及C/EBP-β)，以闡明YSY之潛在調節。吾人之結果顯示，在經高劑量YSY處理之組中，YSY增加AMPK磷酸化且減少PPAR-γ及C/EBP-β表現(圖2B)。由於YSY降低WAT大小且抑制脂肪生成，故吾人進一步量測HFD飼養之ApoE-KO小鼠中之脂聯素之循環含量。發現YSY劑量依賴地增加脂聯素之血清含量(圖2C)。實例 3 YSY 改善 HFD 飼養之小鼠之脂肪肝及動脈粥狀硬化
YSY對內臟WTA之抑制效應揭露其預防肥胖之潛在效應。在此研究中，收集肝及主動脈用於組織病理學分析，且吾人之數據展示高劑量YSY處理可減輕脂肪肝及動脈粥狀硬化(圖3A及3B)。許多研究指示，AMPK路徑係脂質代謝之主要調節子之一，其影響脂肪肝及動脈粥狀硬化之發展(Beg、Allmann與Gibson, 1973； Carlson與Kim, 1973)。吾人之數據亦展示YSY藉助增加AMPK及ACC蛋白之磷酸化程度以及降低HMGCR之表現程度而促進脂質代謝(圖3C)。實例 4 YSY 改良人類個體之高血脂症
招募患有高血脂症之人類個體以確定YSY在動物模型中之降血脂效應。臨床結果指示，在投與2個月後，除在正常組中之外，YSY可顯著降低邊界及高組二者中之TC及TG之血清含量(表2)。表 2
-經YSY處理之人類個體中之血清生化概況.
邊界組(TC：200-239 mg/dL)及高組(TC：≥ 240 mg/dL)。使用威爾克森符號等級測試(Wilcoxon Signed Rank test)以測試3組之間之顯著差異。使用卡方測試(Chi square test)評估營養處理與患者組之間之關聯。a
相較於治療前相同組之基礎水準，p＜
0.05。實例 5 在 HFD 飼養之倉鼠中，相較於個別植物成份， YSY 展現最佳抗高血脂效應
實驗結果指示，僅梅子萃取物顯示在投與劑量(1.48 g/kg/天；圖4)下對血清LDL含量之降低效應。有趣地，在相同劑量下，YSY展現對膽固醇、TG及LDL之血清含量之多個及顯著抑制。實例 6 YSY 之層析 概況及生物活性化合物
在吾人之研究中，將標準化合物(PCA及CGA)製備為1 μg/mL之濃度。將YSY (500 μg)溶解於1 mL水中，且隨後藉由0.45 μm過濾器過濾用於HPLC。如圖5A及5B中所示，基於MRM之分析提及，相較於標準品(RT=4.68 min)，YSY中PCA之滯留時間(RT)係4.67 min；且相較於標準品(RT=4.59 min)，YSY中CGA之滯留時間係4.52 min。 此外，藉由HPLC分析YSY之水萃取物(圖6A)及作為比較之YSY之醇萃取物(圖6B)。HPLC概況顯示於圖6中且滯留時間、峰面積及峰之相對高度顯示於下表中。 The invention surprisingly found that the water extract of YSY can reduce body weight, hyperlipidemia, fatty liver and atherosclerosis. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below. The term "a" and "an" refer to the grammatical object of one or more articles (ie at least one). As used herein, in the scope of a patent application, the term "or" means "and/or" unless explicitly indicated to refer only to substitutes or unless substitutes are mutually exclusive. The term "promote, promotion, and promotion" refers to an increase in activity, response, condition, disease, or other biological parameters. The term "individual" includes living organisms such as humans, monkeys, cows, sheep, horses, pigs, cows, goats, dogs, cats, mice, rats, cultured cells, and transgenic species thereof. In the preferred embodiment, each system is human. The term "administration" includes administration routes that allow the composition of the invention to perform its intended function. The term "treat or treatment" means a method of reducing the effect of a disease or condition. Treatment can also refer to ways to reduce the underlying cause of the disease or condition itself. The treatment can also be any reduction from the natural level and can be, but not limited to, the complete elimination of the disease, condition or symptoms of the disease or condition. The term "prevent (prevent, prevention or preventing)" means the suppression or prevention of osteoporosis-related symptoms. The term "effective amount" means the amount of the composition of the present invention that is effective to treat and/or prevent a disease, condition or symptom of the disease or condition. In one aspect, the present invention provides wood ear ( Auricularia auricular ), mushrooms ( Lentinus edodes ), hawthorn (hawthorn fruit, Crataegus pinnatifida ), roselle, Hibiscus sabdariffa , celery ( Apium graveolens ) and plums ( Prunus mume ) water extract of the composition , wherein the water extract is obtained by mixing agaric, shiitake mushrooms, hawthorn, roselle, celery and plum to form a composition, and soaking the composition The resulting composition is boiled in water at a temperature of about 50°C to about 80°C for at least 16 hours to obtain a water extract of the composition. The resulting extract can be detected by HPLC. In one embodiment, the soaking time of the composition is about 18 hours to about 48 hours, about 18 hours to about 46 hours, about 18 hours to about 44 hours, about 18 hours to about 42 hours, about 18 hours to about 40 Hours, about 18 hours to about 38 hours, about 18 hours to about 36 hours, about 18 hours to about 34 hours, about 18 hours to about 32 hours, about 18 hours to about 30 hours, about 18 hours to about 28 hours, About 18 hours to about 26 hours, about 20 hours to about 48 hours, or about 22 hours to about 48 hours. Preferably, the composition is soaked in water for about 24 hours. In one embodiment, the soaking temperature is about 50°C to about 75°C, about 50°C to about 70°C, about 50°C to about 65°C, about 55°C to about 75°C, or about 55°C to about 70°C. Preferably, the soaking temperature is about 60°C. In one embodiment, the ratio of fungus, shiitake mushrooms, hawthorn, roselle, celery and plums in the composition is on a dry weight basis from about 0.5 to about 1.5: about 0.5 to about 1.5: about 1.5 to about 2.5: about 1.5 to About 2.5: about 4.5 to about 5.5: about 0.5 to about 1.5. Preferably, the ratio is about 1: about 1: about 2: about 2: about 5: about 1 on a dry weight basis. In another aspect, the invention provides wood ear ( Auricularia auricular ), mushrooms ( Lentinus edodes ), hawthorn fruit ( Crataegus pinnatifida ), roselle ( Hiscus sabdariffa ), celery ( Apium graveolens ) and A water extract of a composition of plum ( Prunus mume ) , wherein the composition includes a residence time of about 3.2 minutes to about 4.5 minutes (preferably about 3.978 minutes) and about 5.4 minutes to about 6.2 minutes (about 5.943 minutes) as measured by HPLC. In one embodiment, the water extract further comprises a residence time of about 3.2 minutes to about 4.5 minutes (preferably about 3.978 minutes), about 2.7 minutes to 3.2 minutes (preferably 2.995 minutes) and about 5.4 minutes to about 6.2 minutes The compound (about 5.943 minutes), as measured by HPLC. In one embodiment, the water extract further comprises a residence time of about 3.2 minutes to about 4.5 minutes (preferably about 3.978 minutes), about 2.7 minutes to 3.2 minutes (preferably 2.995 minutes) and about 5.4 to about 6.2 minutes (compared Preferably about 5.943 minutes), about 2.0 minutes to about 2.5 minutes (preferably about 2.307 minutes), about 9.0 minutes to about 9.4 minutes (preferably about 9.224 minutes) and about 6.8 minutes to 7.1 minutes (preferably about 6.931 minutes) Compounds, as measured by HPLC. In addition to these peaks, the water extract further includes retention times of about 0.565 minutes, about 0.751 minutes, about 1.175 minutes, about 7.996 minutes, about 8.361 minutes, about 10.879 minutes, about 11.709 minutes, about 12.303 minutes, and about 12.766 minutes. Compounds, as measured by HPLC. In one embodiment, the water extract of the present invention has an HPLC profile as shown in FIG. According to the present invention, the water extract of the present invention has an HPLC profile as shown in the following table. Any extraction technique known in the art can be used to prepare the extract of the present invention. The resulting extract can be further fractionated by chromatography. The preferred chromatography is liquid chromatography using solvent elution. Preferably, the liquid chromatography is high performance liquid chromatography (HPLC) or reverse phase HPLC. In one embodiment, the HPLC described herein is by using mobile phase A as ddH 2 O and mobile phase B as MeOH/formic acid (volume ratio 99.9:0.1), C18 column and flow rate of 0.5 ml/min and UV Detector to implement. In one aspect, the invention provides wood ear ( Auricularia auricular ), mushrooms ( Lentinus edodes ), hawthorn fruit ( Crataegus pinnatifida ), roselle, Hibiscus sabdariffa , celery ( Apium graveolens ) and plums ( Prunus mume ) water extract of the composition, wherein the composition includes compounds having the following residence times: about 4.61 minutes to 4.95 minutes (preferably about 4.67 minutes), about 6.1 minutes to about 6.5 minutes ( (Preferably about 6.29 minutes), about 6.8 minutes to about 7.0 minutes (preferably about 6.91 minutes), about 3.45 minutes to about 3.62 minutes (preferably about 3.53 minutes) and about 2.96 minutes to 3.16 minutes (preferably about 3.06 minutes) , As measured by extractive ion chromatogram (XIC) of multiple reaction monitoring (MRM), where XIC of MRM is detected under HPLC-MS/MS of 153.0000/109.000 Da and uses protocatechuic acid as standard . In another aspect, in one aspect, the present invention provides wood fungus (wood ear, Auricularia auricular ), shiitake mushroom ( Lentinus edodes ), hawthorn fruit (hawthorn fruit, Crataegus pinnatifida ), roselle, Hibiscus sabdariffa ), A water extract of a composition of celery ( Apium graveolens ) and plum ( Prunus mume ) , wherein the composition includes compounds each having the following residence time: about 4.35 minutes to about 4.65 minutes (preferably about 4.52 minutes), About 3.41 minutes to about 3.62 minutes (preferably about 3.55 minutes), about 4.0 minutes to about 4.15 minutes (preferably about 4.09 minutes), about 3.8 minutes to about 4.0 minutes (preferably about 3.95 minutes) and about 5.18 minutes to about 5.4 minutes (preferably about 5.26 minutes), as measured by extractive ion chromatogram (XIC) of multiple reaction monitoring (MRM), where XIC of MRM is detected under HPLC-MS/MS of 153.0000/109.000 Da And use chlorogenic acid as standard. In one aspect, the invention provides a formulation comprising an aqueous extract as defined herein. In another aspect, the present invention provides a method for treating and/or preventing hyperlipidemia, atherosclerosis, and/or obesity, which comprises administering an effective amount of the water extract of the present invention to an individual. The water extract of the present invention can be formulated into a pharmaceutical preparation or medicament for administration together with a pharmaceutically acceptable carrier, excipient and/or diluent. Pharmaceutically acceptable carriers, diluents and/or excipients mean that the carriers, diluents and/or excipients must be compatible with the other ingredients of the formulation and will not adversely affect the treatment of the water extract of the present invention Beneficial and non-toxic to its recipients. The invention can be practiced by administering the water extract or its preparation by any method of systemic and/or local delivery of the compound. Such methods include oral route, parenteral route, intraduodenal route and so on. In topical application, the water extract or its preparation is applied locally to the site where it is needed. For topical application, the water extract or its preparation can be formulated in a suitable ointment containing the water extract or its preparation suspended or dissolved in one or more carriers. Carriers for topical administration of the water extract of the present invention or its preparation include (but are not limited to): mineral oil, liquid paraffin, white paraffin, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax, sugar ( (Eg lactose) and water. Alternatively, the pharmaceutical preparation can be formulated in a suitable lotion or cream containing an aqueous extract or formulation thereof suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzene Methanol and water. Depending on the specific condition, disorder or disease to be treated, additional therapeutic agents may be applied together with the water extract or its preparation. These additional agents can be administered sequentially (intermittently or intermittently) in any order as part of a multi-dose regimen from the composition of the aqueous extract. Alternatively, they may be part of a single dosage form, mixed with the water extract or its preparation in a single composition (administered simultaneously or concurrently). For oral administration, the pharmaceutical compositions that can be used in the present invention can take the following forms: solutions, suspensions, lozenges, pills, capsules, powders, granules, semi-solids, sustained release formulations, elixirs, aerosols And so on. Lozenges containing various excipients (such as sodium citrate, calcium carbonate, and calcium phosphate) can be combined with various disintegrants (such as starch (preferably potato or tapioca starch) and certain composite silicates), together with binders (Eg polyvinylpyrrolidone, sucrose, gelatin and gum arabic) are used together. In addition, lubricants (such as magnesium stearate, sodium lauryl sulfate, and talc) are often extremely useful for tabletting purposes. Similar types of solid compositions are also used as fillers in soft-filled and hard-filled gelatin capsules; in this regard, preferred materials also include lactose or nougat and high molecular weight polyethylene glycols. When it is desired to administer the aqueous suspension and/or elixir orally, the water extract or its preparation of the present invention and various sweeteners, flavoring agents, colorants, emulsifiers and/or suspending agents and diluents ( For example, water, ethanol, propylene glycol, glycerin and various similar combinations). The choice of formulation depends on various factors, such as the mode of drug administration (eg, for oral administration, the formulation is preferably in the form of tablets, pills, or capsules) and the bioavailability of the drug substance. The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intramedullary and intraarticular injection and infusion. Pharmaceutical compositions for parenteral injection may contain pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders that are reconstituted into sterile injectable solutions or dispersions immediately before use. Aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. In this regard, the sterile aqueous medium used can be easily obtained by standard techniques well known to those skilled in the art. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerin, propylene glycol, polyethylene glycol and the like), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils ( (Eg olive oil) and injectable organic esters (eg ethyl oleate). For example, proper fluidity can be maintained by using a coating material such as lecithin, by maintaining a desired particle size (in the case of a dispersion), and by using a surfactant. The pharmaceutical preparations that can be used in the present invention may also contain adjuvants, such as (but not limited to) preservatives, wetting agents, emulsifying agents, and dispersing agents. The prevention of the action of microorganisms can be ensured by incorporating various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, sorbic acid, and the like). It may also be desirable to include isotonic agents such as sugar, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be caused by including agents that delay absorption (such as aluminum monostearate and gelatin). In addition to the water extract and its preparation, the suspension may contain suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar And tragacanth gum and its mixture. For the purpose of transdermal (eg, topical) administration, a diluted sterile aqueous or partially aqueous solution (concentration usually about 0.1% to 5%) similar to the parenteral solution above is prepared in other respects. The pharmaceutical preparations that can be used in the present invention can also be administered by nasal aerosol or inhalation. These compositions are prepared according to techniques well known in the field of pharmaceutical formulation, and can be prepared as a solution in saline, using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbon compounds, and/or other practices Solubilizer or dispersant. The composition for rectal or vaginal administration is preferably a suppository, which can be prepared by combining the water extract of the present invention or its preparation with a suitable non-irritating excipient or carrier (such as cocoa butter, polyethylene glycol or Suppositories (waxes) are prepared by mixing. These suppositories are solid at room temperature but liquid at body temperature, and therefore can melt in the rectum or vaginal cavity and release the drug. Other pharmaceutically acceptable carriers include (but are not limited to) non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials, or any type of formulation aids, including (but not limited to) ion exchangers, Alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphate, glycine, sorbic acid, potassium sorbate), partial glyceride mixture of saturated vegetable fatty acids, water , Salt or electrolyte (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt), colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances , Polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol and lanolin. Solid pharmaceutical excipients include (but are not limited to) starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, Glycerin monostearate, sodium chloride, skimmed milk powder and the like. The liquid and semi-solid excipients can be selected from glycerin, propylene glycol, water, ethanol, and various oils, including petroleum, animal, plant, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Preferred liquid carriers (especially for injectable solutions) include water, saline, aqueous dextrose, and glycols. Those skilled in the art know, or will know from the disclosure, methods for preparing various pharmaceutical compositions having a certain amount of active ingredients. Other suitable pharmaceutical excipients and their formulations are described in Remington's Pharmaceutical Sciences, edited by EW Martin, Mack Publishing Company, 19th Edition (1995). The pharmaceutical composition usable in the present invention may contain 1%-100% (by weight) of the water extract of the present invention or a preparation thereof. In any case, the composition or formulation to be administered will contain an amount of the water extract of the present invention or a preparation thereof, which amount is effective to treat the condition, disorder or disease of the individual being treated. Those skilled in the art will understand that the pharmaceutically effective amount of the water extract or its preparation can be determined empirically and can be used in pure form or in the form of a pharmaceutically acceptable salt, ester or prodrug (if these forms exist). The agents can be administered to a patient in the form of a pharmaceutical composition in combination with one or more pharmaceutically acceptable excipients. It will be understood that when administered to, for example, a human patient, the total daily dosage of the agent or composition of the present invention will be determined within the scope of reasonable medical judgment made by the attending physician. The specific therapeutically effective dose value of any particular patient will depend on various factors: the type and degree of cellular response to be achieved; the activity of the specific agent or composition used; the specific agent or composition used; the age of the patient, Body weight, general health status, gender, and diet; time of administration, route of administration, and excretion rate; duration of treatment; drugs used in combination or concurrent use with specific agents; and similar factors well known in the medical field. In one embodiment, the effective amount of the water extract of the present invention is about 4 g/day to about 18 g/day. In some embodiments, the effective amount is about 4 g/day to about 16 g/day, about 4 g/day to about 14 g/day, about 4 g/day to about 12 g/day, about 6 g/day To about 18 g/day, about 6 g/day to about 14 g/day, about 8 g/day to about 18 g/day, about 8 g/day to about 16 g/day, about 8 g/day to about 14 g/day, about 10 g/day to about 18 g/day, about 10 g/day to about 16 g/day, or about 8 g/day to about 14 g/day. Preferably, the effective amount is about 12 g/day. The dosage can also be configured in a patient-specific manner to provide a predicted concentration of the agent in the blood, as determined by conventional techniques accepted in the industry. Alternatively, the water extract or its preparation can be formulated as a health food or dietary supplement together with food-grade carriers, excipients, diluents and/or salts. The carriers, excipients, diluents and/or salts mentioned above can be used in the health food or dietary supplement of the present invention. Health foods or dietary supplements can exist in various forms, including (but not limited to) lozenges, capsules, caplets, powders, drinks (including milkshakes), solid foods (including snack bars), and the like. Those skilled in the relevant arts will readily understand that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of the invention or any of its embodiments. The following examples are provided to illustrate but not limit the invention. Example materials and methods 1. Chemicals and reagents anti-phospho-ACC (acetyl CoA carboxylase; No. 3661), phospho-AMPKα (5'-adenosine monophosphate activated protein kinase-α; No. 2531s) and PPAR-γ (Peroxisome Proliferator Activated Receptor-γ; No. 2435) The antibody system was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies recognizing β-actin (code ab6276) and C/EBP-β (CCAAT/enhancer binding protein β; code GTX61124) were from Abcam (Cambridge, MA, USA) and GeneTex, Inc. (USA) obtain. Horseradish peroxide-labeled secondary antibodies against mouse immunoglobulin G (IgG) (code sc2005) and rabbit IgG (code sc2004) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were purchased from Sigma-Aldrich (St Louis, MO, USA). 2. Preparation of YSY YSY is composed of 6 kinds of natural foods, including wood ear ( Auricularia auricular ), mushrooms ( Lentinus edodes ), hawthorn fruit ( Crataegus pinnatifida ), roselle, Hibiscus sabdariffa , celery ( Apium) graveolens ) and plums ( Prunus mume )) , and their mixing ratio is 1:1:2:2:5:1 on dry weight. All plants were purchased from standard qualified farms and their source was verified by Chinese herbal therapy center (Chinese herbal therapy center), China Medical University Hospital (Taichung, Taiwan). All dried plants were cut into pieces, soaked in water at 60°C for 24 h, and then boiled at 100°C to concentrate the extract. 3. The chromatographic profile of YSY and the identification of biologically active compounds are carried out by LC/ESI-MS/MS (Applied Biosystems Sciex, Foster City, CA, USA), such as protocatechuic acid (PCA) mentioned above in YSY And identification of potential biologically active compounds such as chlorogenic acid (CGA) to establish a reference chromatographic profile of YSY extracts for use in the quality inspection of each batch of extraction procedures. LC/ESI was implemented using an API 2000-triple quadrupole mass spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA, USA) equipped with an electrospray ionization source and connected to an Agilent 1200 HPLC (Agilent Technologies, Wilmington, DE, USA). -MS/MS analysis. A Synronis™ C18 column (150 mm × 4.6 mm, 5 μm; Thermo, Waltham, MA, USA) was used to separate YSY (500 μg/mL). Mobile phase A is ddH 2 O, and mobile phase B is MeOH/formic acid (volume ratio 99.9:0.1), and the flow rate is set to 0.5 ml/min. The detector used in HPLC analysis is a UV detector. Mass spectrometric analysis was performed using negative ion multiple reaction monitoring (MRM) mode using electrospray ionization tandem mass spectrometry. All analyzes were showed [MH] - strongest precursor ions, which can be detected as [MH] in negative ion mode. For each analyte, 100 ms was used as the retention time to detect two MRM transitions, one quantitative and one qualitative; PCA: 353/191, 353/85; CGA: 153/109, 153/91. 4. Hyperlipidemia animal model All procedures and animal care are approved and implemented in accordance with the institutional animal ethics guidelines of the Laboratory Animal Service Center and China Medical University (Taichung, Taiwan). Male ApoE-KO mice (4 months old) were divided into 4 groups (n=8/group), including normal diet group (normal diet), HFD (high fat diet; normal diet mixed with 60% fat), HFD + Low dose YSY (0.4 g/kg/day) and HFD + high dose YSY (2 g/kg/day). Male hamsters (8 weeks old) were divided into 8 groups (n=6/group), including HFD, HFD + YSY (1.48 g/kg/day), HFD + fungus (1.48 g/kg/day), HFD + Shiitake (1.48 g/kg/day), HFD + hawthorn (1.48 g/kg/day), HFD + Roselle (1.48 g/kg/day), HFD + celery (1.48 g/kg/day) and HFD + plum ( 1.48 g/kg/day). The hypolipidemic animals were induced by HFD for 4 weeks and then YSY extracts or single plant ingredients were used for re-treatment intervention for 8 weeks. At the end of the study, animals were anesthetized by intramuscular injection of Zoteil 50 ® (0.1 ml) (Virbac Ltd, Carros, France) and then biological samples including blood, liver and visceral adipose tissue were collected to evaluate histopathology and blood biochemical examination . 5. Blood biochemical analysis After starvation overnight, all blood was collected by using a separation tube after the animals were sacrificed, the blood sample was allowed to clot at room temperature for 2 h, and then centrifuged at 1000 × g for 20 min at 4°C. Amount of serum content of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), glutamate aminotransferase (GPT) and blood urea nitrogen (BUN) The test was provided by Zhen-Xing Co., Ltd (Taichung, Taiwan). In addition, serum adiponectin was measured by a commercial ELISA kit (No. E90605Mu, USCN Life Science Inc., USA) according to the manufacturer's instructions. 6. Frozen section of liver and visceral fat tissues According to my previous research (Pan et al., 2013), lipid accumulation in liver tissue was performed. Liver tissue was perfused with normal saline, fixed in 5% formalin neutralizing solution (JT Baker, Inc., Philipsburg, NJ, USA) for 24 h, and embedded in Tissue-Tek ® OCT compound (No. 4583, Sakura Finetek Inc ., Torrance, CA, USA). The embedded tissue was cut into 10 μm and stained with Sudan IV (for liver tissue) or oil red (for adipose tissue) and hematoxylin/eosin (Merck, Whitehouse Station, NJ, USA). Obtained at 400 times magnification photograph, and in Alphalmager 2200 ® file system (Alpha InnoTech, San Leandro, CA , USA) on quantitative. 7. Lipid pad analysis Lipid pads were collected at the end of the 8-week study. Adipose tissue around the gonads and ventral flank was dissected and then rinsed with 1× phosphate buffered saline (PBS). The lipid pad ratio is calculated by the following equation (Eq. (1)): Lipid pad ratio (%) = fat tissue around the gonads and abdominal flank (g)/body weight (g)×100 (1) 8. Western blot The liver tissue was homogenized using PRO-PREP® protein extraction solution (500 μl/g), and the protein concentration was measured by using Bio-Rad protein analysis (Bio-Rad). An equal amount (30 μg) of extracted protein was electrophoresed through 10% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked by using 5% skimmed milk powder dissolved in PBST (1×PBS with 0.1% Tween 20) for 1 h and then washed with PBST. Then at room temperature, the membrane and the specific primary antibody are appropriately diluted (p-ACC, 1:1000; p-AMPK, 1:1000; HMG-CoA, 1:500; PPAR-γ, 1:1000; C/ EBP-β, 1:500; β-actin, 1:1000) were incubated together for 2 h. Then, the ink dots were washed and incubated with HRP-conjugated secondary antibody at a dilution of 1:2000 at room temperature for 1 h. The luminescence signal was developed using chemiluminescence reagents, acquired by Fujifilm LAS-4000 system (San Leandro, CA, USA), and quantified using Image J software program from NIH (USA). 9. Analysis of aortic lipid streaks After the animals were sacrificed, the aorta was collected and then gently rinsed with common saline. All tissue samples were incubated in 4% paraformaldehyde for 10 min and then stained with oil red solution (in isopropanol, 5 mg/mL) for 15 min at room temperature. The stained aorta was washed with several different concentrations of isopropyl alcohol (90%, 70% and 50%) for 1 min, and then rinsed with water. A digital camera (No. D80; Nikon, Tokyo, Japan) was used to obtain the photos. 10. Clinical Research This human research department is an open-label and parallel pilot clinical trial approved by the Hospital Affiliated to China Medical University (Taichung, Taiwan) (No. DMR99-IRB-126) to study the decline of YSY in healthy and hyperlipidic individuals Lipid effect. Consider registering adults aged 22-65 years old with Hyperlipidemia as defined by Adult Treatment Panel III (ATP III). Exclude participants who are pregnant or have chronic metabolic diseases. In addition, all individuals were not using lipid-lowering drugs, such as statins, cholic acid chelators, fibrates and nicotinic acid, and drugs that could change liver function during the trial. According to the guidance of ATP III, the qualified participants will be divided into 3 groups (normal, border and high group). The study started at the 4-week baseline period. Blood samples were collected as blank controls before the study. Then, all individuals received 12 g/day of YSY for 2 months. At the end of the study, blood samples were collected 24 h after YSY administration. 11. Statistical analysis The data are expressed as mean ± SD. The analysis of variance was used to evaluate the statistical significance of the differences, and then Tukey's test was performed for all comparisons. The p value <0.05 was considered statistically significant. Statistical Package for Social Sciences (SPSS X, Chicago, IL, USA) was used to analyze the data. Example 1 YSY reduced body weight, adipose tissue accumulation, and blood lipid in mice fed with HFD . Our experimental data showed that body weight and visceral adipose tissue quality were significantly reduced by high-dose treatment of YSY compared to the control group (Figures 1A and 1B ). To determine whether the YSY-induced weight loss was due to changes in food intake, food intake was recorded during the experimental period. As shown in Figure 1C, after the 8-week study, there was no significant difference in food intake between all groups. In this study, we also explored the hypolipidemic effect of YSY on ApoE-KO mice raised in HFD (Table 1). The experimental results show that, compared with the control group (HFD alone), after high-dose YSY treatment, several biochemical parameters (TC, TG and LDL) were significantly reduced. However, the serum level of HDL was not significantly improved by YSY (Table 1). In addition, no significant differences in serum levels of GPT and BUN were observed between all groups, suggesting that no significant toxicity was found after YSY treatment. Table 1 -Serum biochemical profile in ApoE-KO mice treated with YSY. a compared to the control group (treated with HFD alone), p < 0.05, b compared to the ordinary diet group, p < 0.05. Example 2 YSY reduces fat production in HFD- fed mice. After an 8-week animal study, visceral adipose tissue was frozen sectioned and discolored by HE staining to assess changes in fat cell size, and histopathological results indicated high-dose YSY treatment Decreasing the size of adipocytes (Figure 2A) suggests that YSY can regulate adipocyte differentiation. Therefore, the three key proteins involved in adipocyte differentiation (including AMPK, PPAR-γ and C/EBP-β) were examined to clarify the potential regulation of YSY. Our results showed that in the group treated with high-dose YSY, YSY increased AMPK phosphorylation and decreased the performance of PPAR-γ and C/EBP-β (Figure 2B). Because YSY reduces the size of WAT and inhibits lipogenesis, we further measured the circulating content of adiponectin in ApoE-KO mice fed by HFD. It was found that YSY dose-dependently increased the serum content of adiponectin (Figure 2C). Example 3 YSY improves fatty liver and atherosclerosis in mice fed with HFD . The inhibitory effect of YSY on visceral WTA reveals its potential effect in preventing obesity. In this study, liver and aorta were collected for histopathological analysis, and our data showed that high-dose YSY treatment can reduce fatty liver and atherosclerosis (Figures 3A and 3B). Many studies indicate that the AMPK pathway is one of the major regulators of lipid metabolism, which affects the development of fatty liver and atherosclerosis (Beg, Allmann and Gibson, 1973; Carlson and Kim, 1973). Our data also shows that YSY promotes lipid metabolism by increasing the phosphorylation of AMPK and ACC proteins and reducing the expression of HMGCR (Figure 3C). Example 4 YSY improves hyperlipidemia in human subjects. Human subjects with hyperlipidemia are recruited to determine the hypolipidemic effect of YSY in animal models. Clinical results indicate that after 2 months of administration, YSY can significantly reduce the serum levels of TC and TG in both the border and high groups, except in the normal group (Table 2). Table 2 -Serum biochemical profile in human subjects treated with YSY. Boundary group (TC: 200-239 mg/dL) and high group (TC: ≥ 240 mg/dL). A Wilcoxon Signed Rank test was used to test the significant differences between the three groups. A Chi square test was used to assess the association between nutritional treatment and patient group. a Compared with the basic level of the same group before treatment, p< 0.05. Example 5 In the hamsters fed HFD, as compared to the individual components of the plant, showing the experimental result indicating the best YSY antihyperlipidemic effect, plum extract only displayed administered dose (1.48 g / kg / day; FIG. 4) of the The effect of reducing the level of serum LDL. Interestingly, at the same dose, YSY exhibited multiple and significant inhibition of the serum levels of cholesterol, TG and LDL. Example 6 Chromatographic profile of YSY and biologically active compounds In our research, standard compounds (PCA and CGA) were prepared at a concentration of 1 μg/mL. YSY (500 μg) was dissolved in 1 mL of water, and then filtered through a 0.45 μm filter for HPLC. As shown in Figures 5A and 5B, the MRM-based analysis mentions that the retention time (RT) of PCA in YSY is 4.67 min compared to the standard (RT=4.68 min); and compared to the standard (RT= 4.59 min), the retention time of CGA in YSY is 4.52 min. In addition, the water extract of YSY (FIG. 6A) and the alcohol extract of YSY as a comparison (FIG. 6B) were analyzed by HPLC. The HPLC profile is shown in Figure 6 and the retention time, peak area and peak relative height are shown in the table below.
