536405 A7 B7 五、發明說明（1 ) 發明背景 發明範圍 (請先閱讀背面之注咅？事項再填寫本頁) 本發明係關於抗骨質疏鬆症之藥劑’尤其是內含海藻 糖作爲有效成分的抗骨質疏鬆症藥劑。 先前技藝之描述 經濟部智慧財產局員工消費合作社印製 骨質疏鬆症（即Scalier1 s疾病）是一種骨骼病症’進 中骨骼的絕對量下降而無骨質上的改變。在生命體中’成 骨細胞不斷的進行骨生成，而破骨細胞不斷的進行骨質再 吸收。骨生成及骨質再吸收之速率失衡（某些降低負平衡 端的因素引起）可引發骨質疏鬆症。造成骨質疏鬆症的原 因主要可分成環境以及遺傳的因素；前者包括：老化及內 分泌疾病，例如：甲狀腺機能亢進、生殖腺功能不足、庫 辛氏症候群，等；而後者包括：動情激素受體基因異常、 骨生成遲滯、半胱胺酸尿症等。一旦引起骨質疏鬆症，則 會依續引發多孔的骨骼；骨骼皮質寬度變窄，骨髓腔擴 大，及降低網狀骨質的小樑骨。隨著骨質疏鬆症的發展， 骨骼的物理強度使病人常抱怨發生腰痛及關節痛，並使其 骨骼即使在輕微的震撼下亦易於骨折。 在治療骨質疏鬆症時’一般先對病人投予止痛藥以解 除疼痛，再投用與鈣代謝相關的藥劑（例如：活化的維生 素D製劑、降血鈣素、動情激素、及蛋白質同化荷爾 蒙）。此類常見的骨質疏鬆症藥劑爲荷爾蒙製劑，可引發 嚴重的副作用；某些病人可能在得到所要的療效之前被迫 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 536405 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（2 ) 停止投藥。 骨質疏鬆症隨著老化之加速而更形嚴重。已將老年人 逼入牆角，因此須要大力發展日常及易於服用之藥劑以有 效的治療/預防抗骨質疏鬆症，使老年人能健康的並無憂 無慮的享受退休生活。 發明摘要 如前述之觀點，依據本發明目的是提供一種抗骨質疏 鬆症之藥劑，其係以口服或非經腸投用具有效的治療/預 防性的效應。 本發明者篩選過各種物質，並意外的發現海藻糖（一 種雙醣類）可控制骨生成，降低負平衡端，在正常情況下 對哺乳動物及人類投用能對骨質疏鬆症產生治療性/預防 性效果而鮮少有副作用。本發明以提供一種口服的或非經 腸地投用藥劑（內含海藻糖作爲有效成分）對抗骨質疏鬆 症來達成上述目的。 海藻糖是一種雙醣類，由兩個葡萄糖分子在還原端結 合組成，其廣汎的分布在天然界中；出現在微生物、蘑 菇、昆蟲等。雖然對於用海藻糖作爲蔗糖之取代醣類之須 求在食品、化妝品、醫藥學上日益增加，海藻糖對哺乳動 物之生理作用除了在能量補充作用、皮膚之水分通透作 用、及血液中脂肪酸之控制作用之外則不淸楚。 海藻糖本身是一種已知的化合物。如上述所言，本發 明發現海藻糖在哺乳動物中具有優異的抗骨質疏鬆症功 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） --------------------訂---------線 (請先閱讀背面之注意事項再填寫本頁) -5- 536405 經濟部智慧財產局員工消費合作社印製 A7 ___B7______ 五、發明說明（3 ) 能。因此使用海藻糖作爲新穎的口服或非經腸投用藥劑對 抗骨質疏鬆症。 發明之詳細描述 本發明係關於以口服或非經腸投用內含海藻糖作爲有 效成分的抗骨質疏鬆症藥劑。據知，海藻糖有不同之鍵結 形式，故而有三種光學異構物，即α，α -、α，0 -及 /3，/3 -海藻糖。因爲此類化合物對於哺乳動物及人類呈 現相當地抗骨質疏鬆症作用，故可利於本發明。因此不論 此異構物之製備方法及性質爲何，依據本發明之藥劑的使 用總有效量中至少含有一種此類的光學異構物。 本發明中使用之海藻糖可用各種方法製備。因爲本發 明與生產海藻糖無關，所以不詳細說明其生產方法，若考 慮海藻糖之生產成本，其方法包括將非還原醣類形成酵素 及海藻糖釋放酵素與澱粉部分水解物接觸的步驟，可另人 滿意的使用如揭不於（本發明申請者申請之）Japanese Patent Kokai Nos. 1 43,876/95、2 1 3,283/95、322,883/95、 298,880/95 ^ 66,1 87/96 、 66,1 88/96 > 336,388/96 、及 84,5 8 6/96之方法。此類方法中，以相當低廉的澱粉作爲材 料來產生相當高產量的α，α -海藻糖。此方法得到之商 品實施例爲” TREHA0SE®” ，其爲可食用之海藻糖產物含 至少9 8 %海藻糖（乾燥固體爲準（d · s · b ·))， 以及” TREH AS TAR®” ，一種海藻糖糖漿含至少2 8 %海 藻糖（d.s.b·)，其係產自 Hayashibara Shoji，Inc.， 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） --------------------訂---------線 (請先閱讀背面之注意事項再填寫本頁) -6 - 536405 A7 B7 五、發明說明（4 )536405 A7 B7 V. Description of the invention (1) Background of the invention (please read the note on the back? Matters before filling out this page) The present invention is about an anti-osteoporosis agent 'especially containing trehalose as an active ingredient Anti-osteoporosis agent. Description of previous techniques Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economics Osteoporosis (ie Scalier 1 s disease) is a skeletal disorder ′ The absolute amount of bones has decreased without bone changes. In living organisms, osteoblasts continue to undergo osteogenesis, while osteoclasts continuously undergo bone resorption. Imbalances in the rate of osteogenesis and bone resorption (caused by factors that reduce the negative balance) can cause osteoporosis. The causes of osteoporosis can be divided into environmental and genetic factors; the former includes: aging and endocrine diseases, such as: hyperthyroidism, insufficient gonad function, Cushing's syndrome, etc .; and the latter includes: abnormal estrogen receptor genes , Delayed osteogenesis, cysteineuria and so on. Once osteoporosis is caused, porous bones are successively caused; the width of the skeletal cortex is narrowed, the bone marrow cavity is enlarged, and the trabecular bone of the reticular bone is reduced. With the development of osteoporosis, the physical strength of bones makes patients often complain of low back pain and joint pain, and makes their bones easy to fracture even under slight shock. When treating osteoporosis, 'the patient is usually given analgesics first to relieve pain, and then administered with calcium metabolism-related agents (such as activated vitamin D preparations, calcitonin, estrogens, and protein assimilation hormones) . Such common osteoporosis agents are hormonal preparations, which can cause serious side effects; some patients may be forced to apply Chinese National Standard (CNS) A4 (210 X 297 mm) before the desired effect is obtained. 536405 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 V. Description of Invention (2) Stop drug administration. Osteoporosis becomes more severe as aging accelerates. The elderly have been forced into the corner, so it is necessary to vigorously develop daily and easy-to-take medicines to effectively treat / prevent anti-osteoporosis, so that the elderly can enjoy their retirement life in a healthy and worry-free manner. SUMMARY OF THE INVENTION In view of the foregoing, it is an object of the present invention to provide an anti-osteoporosis agent which is effective for the therapeutic / preventive effect of oral or parenteral administration. The inventors have screened various substances and unexpectedly found that trehalose (a disaccharide) can control bone formation and reduce the negative balance end. Under normal circumstances, administration to mammals and humans can produce a therapeutic effect on osteoporosis / Preventive effect with few side effects. The present invention achieves the above object by providing an oral or parenteral agent (containing trehalose as an active ingredient) against osteoporosis. Trehalose is a disaccharide composed of two glucose molecules combined at the reducing end. It is widely distributed in the natural world; it appears in microorganisms, mushrooms, and insects. Although the demand for trehalose as a sugar substitute for sucrose is increasing in food, cosmetics, and medicine, the physiological effects of trehalose on mammals are in addition to energy supplementation, skin water permeability, and fatty acids in the blood. Beyond its controlling role, it is not terribly good. Trehalose itself is a known compound. As mentioned above, the present invention finds that trehalose has excellent resistance to osteoporosis in mammals. The paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) --------- ----------- Order --------- line (please read the precautions on the back before filling this page) -5- 536405 A7 printed by employee consumer cooperative of Intellectual Property Bureau of Ministry of Economic Affairs ___B7______ 5. Description of the invention (3) Yes. Therefore, the use of trehalose as a novel oral or parenteral agent is effective against osteoporosis. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an anti-osteoporosis agent for oral or parenteral administration containing trehalose as an effective ingredient. It is known that trehalose has different bonding forms, so there are three optical isomers, namely, α, α-, α, 0-and / 3, / 3-trehalose. Since such compounds exhibit considerable anti-osteoporosis effects on mammals and humans, they are advantageous for the present invention. Therefore, regardless of the preparation method and properties of this isomer, the total effective amount of the pharmaceutical agent according to the present invention contains at least one such optical isomer. The trehalose used in the present invention can be prepared by various methods. Because the present invention is not related to the production of trehalose, its production method is not described in detail. If the production cost of trehalose is considered, the method includes the steps of contacting a non-reducing sugar-forming enzyme and a trehalose-releasing enzyme with a starch partial hydrolysate. Another satisfactory use is not disclosed (applied by the applicant of the present invention) Japanese Patent Kokai Nos. 1 43,876 / 95, 2 1 3,283 / 95, 322,883 / 95, 298,880 / 95 ^ 66,1 87/96, 66, 1 88/96 > 336,388 / 96 and 84,5 8 6/96. In such methods, relatively inexpensive starch is used as a material to produce a relatively high yield of α, α-trehalose. The commercial examples obtained by this method are "TREHA0SE®", which is an edible trehalose product containing at least 98% trehalose (based on dry solids (d · s · b ·)), and "TREH AS TAR®" , A trehalose syrup containing at least 28% trehalose (dsb ·), which is produced by Hayashibara Shoji, Inc., this paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ---- ---------------- Order --------- line (please read the precautions on the back before filling this page) -6-536405 A7 B7 V. Description of the invention (4)
Okayama，Japan ;以及試劑級的海藻糖其純度至少爲9 9 % (d · s · b ·)，其係產自 Hayashibara Biochemical Laboratories，Inc.，Okayama, Japan ; α，α —海藻糖之製備 可用麥芽糖/海藻糖轉換酵素，如揭示於Japanese Patent Kokai NOS. 1 70,977/95、263/96、及 1 49,980/96 ;或常見的 麥芽糖及海藻糖磷酸化酶組合。 產生具有αΘ -光學異構物之海藻糖，可將環麥牙 糖葡聚糖葡聚糖轉移酶及Α -半乳糖苷酶與澱粉部分水解 物及乳糖之混合物依據揭示於（本發明申請者申請之） Japanese Patent Kokai-Nos. 144,694/92 及 179,490/92 之方法 反應。/3，Θ -海藻糖可用常見的化學合成得之。本發明 中，在口服投用時此類海藻糖異構物不須經高度純化，並 且不須分離其他的雜質。此類光學異構物可爲未經純化的 形式或包括生產過程中產生之其他醣類副產物的組合物形 式，或包括其他不會明顯防礙海藻糖於哺乳動物及人類中 對抗骨質疏鬆症作用的適當成分之混合物形式。在注射本 藥劑對抗人類及動物的骨質疏鬆症時，較佳者在使用前應 將海藻糖中之熱原用移除，其係使用一種或多種去鹽的方 法（例如離子交換器、巨多孔的樹脂、活化的活性碳及濾 膜）；吸收的方法；及過濾的方法。 當投用至哺乳動物及人類時，海藻糖之作用可將骨生 成及骨吸收控制在正常的狀況；海藻糖在此方法的作用下 可維持正常的平衡狀況，以及在負平衡時使骨生成及骨吸 收速率失調之輕度骨質疏鬆症（如老年人骨質疏鬆症其骨 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I丨丨丨--------% (請先閱讀背面之注意事項再填寫本頁) 訂---------線· 經濟部智慧財產局員工消費合作社印製 經濟部智慧財產局員工消費合作社印製 536405 A7 B7 五、發明說明（5 ) 生成速率降低至正常水準以下因此骨骼含量下降）及高度 骨質疏鬆症（如副甲狀腺分泌過多症其骨吸收速率遠超過 正常水準而使骨骼之含量降低）恢復至理想的狀況。由於 此類作用，故在對骨骼狀況良好之哺乳動物及人類投用海 藻糖時可維持/改進骨骼之健康狀況，對於患有骨質疏鬆 症之人類病人則可減輕其腰痛及關節痛、增加骨骼之含 量、且強化骨骼使之不易骨折。因此本發明中”抗骨質疏 鬆症之藥劑”指醫藥學的產品及食品（包括：飮料），內 含海藻糖作爲有效成分並可治療及/或預防骨質疏鬆症。 本藥劑對抗骨質疏鬆症之特殊效用可用以下描述的測試作 爲指數，例如卵巢摘除之老鼠其股骨的重量加以證實。 依據本發明之抗骨質疏鬆症藥劑可僅含海藻糖或內含 海藻糖及其他有助於以便海藻糖投藥之成分的組合物。當 組合物以口服方式投用時，其一般的形式爲食品或口服投 用的醫藥組合物，包括：食品及管灌的液體，尤其是液 體、懸浮液、乳狀液、乳膏、漿糊、粉末、顆粒形式、及 其他須要形狀的固體食品及醫藥學的產品形式。本藥劑之 食品形式可爲組合製劑，例如：使用水、醇類、戊基類物 質、蛋白質、纖維、醣類、脂質、維生素、礦物、風味 劑、著色劑、甜劑、調味劑、風味劑、穩定劑、抗氧化 劑、及抗菌劑、以及健康食品材料，例如：促進雙叉乳酸 桿菌生長之醣類、奶粉、牛奶蛋白質水解物，包括：酪蛋 白鈣肽類及酪蛋白磷酸肽類、乳醯肝褐質、大豆異黃酮、 血粉、骨粉、及粉末狀的珊瑚，全可作爲食品之材料及/ 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） --------------------訂---------線 (請先閱讀背面之注意事項再填寫本頁) -8- 536405 A7 B7 五、發明說明（6 ) (請先閱讀背面之注意事項再填寫本頁) 或成分。此類組合物可爲食物的形式或管灌的液體形式。 爲了製備本藥劑之醫藥學形式，海藻糖可製作成組合物， 其係使用一種或多種載體、賦形劑/輔助劑、稀釋劑、及 穩定劑，以及視須要之鈣製劑，例如：乳酸鈣、甘油磷酸 鈣、氫磷酸鈣、及L -天門冬胺酸鈣；以及其他藥劑，例 如：止痛藥、消炎劑、活化的維生素D製劑、維生素K製 劑、降血鈣素製劑、動情激素製劑、及蛋白質組成代謝激 素製劑。視用途而定，此抗骨質疏鬆症之藥劑通常含有至 少0 · 1 w / w %之海藻糖，口服時較佳者至少爲1 w / w % 〇 經濟部智慧財產局員工消費合作社印制衣 以口服方式使用本藥劑對抗人類之骨質疏鬆症時’本 藥劑對人類投用後具有中度抗骨質疏鬆症之作用。視用途 而定，在維持/改進骨骼狀況及預防骨質疏鬆症時，本藥 劑是以食品的形式對人類口服投用。治療骨質疏鬆症、預 防骨折、及解除骨質疏鬆症之腰痛及關節痛時’本藥劑一 般是以食品的形式或口服投用的醫藥學產品’例如：液 體、糖漿、粉末、顆粒、藥片、或膠囊對人類投用。本藥 劑之每日劑量通常約〇 · 5至約1 〇 〇公克/成年人/ 天，視海藻糖含量較佳者約1至約5 0公克/成年人/ 天。本藥劑之投用劑量爲1 一 5次/週（隔天投用）。 製備抗骨質疏鬆症之本藥劑的注射形式一般係以有效 量的海藻糖爲底質與一種或多種非海藻糖之成分溶解於 水。并入本藥劑之成分包括：醣類，例如：葡萄糖、麥芽 糖、果醣、山梨糖醇、木糖醇；電解質，例如：氫氧化 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公β ~Γ_ 536405 Α7 Β7 五、發明説明（7 ) (請先閱讀背面之注意事項再填寫本頁} 鈉、氯化鈉、碘化鈉、乙酸鈉、乳酸鈉、檸檬酸鈉、磷酸 氫鈉、磷酸二氫鉀、乙酸鈣、乳酸鈣、甘油磷酸鈣、葡萄 糖酸鈣、硫酸鎂、硫酸鋅、氯化鋅、硫酸鐵、氯化亞鐵、 硫酸銅、及硫酸錳；胺基酸，例如：L 一異白胺酸、L〜 白胺酸、L 一離胺酸、L 一甲硫胺酸、L 一苯基丙胺酸、 L 一蘇胺酸、L 一色胺酸、L 一纈胺酸、L 一精胺酸、L 一組織胺酸、甘胺酸、L 一丙胺酸、L 一半胱胺酸、L — 天門冬胺酸、L 一麩胺酸、L 一脯胺酸、L 一絲胺酸、及 -甲狀腺胺酸；維生素，例如：硫胺素氯化物、核黃素單 磷酸鹽、吡哆醇氯化氫、氰基氰鈷胺素、泛酸鈣、菸鹼醯 胺、葉酸、生物素、二酒石酸膽鹼、L 一抗壞血酸、乙酸 視黃酯、生育酚乙酸鹽、及維他命K 1 ;植物及動物脂 質，例如：大豆油、紅花油、亞麻子油、椰子油、鯨油、 及海洋生物之油；鈣製劑，例如：乳酸鈣、甘油磷酸鈣、 氫磷酸鈣、及L -天門冬胺酸鈣；及藥劑，例如：活化的 維生素D製劑、維生素K、降血鈣素、動情激素、蛋白質 組成代謝激素、止痛藥、及消炎劑。 經濟部智慧財產局員工消費合作社印製 製備上述依據本發明之抗骨質疏鬆症的注射藥劑時， 可將海藻糖與一種或多種上述之非海藻糖成分溶於水，使 海藻糖含量爲0 · lw/w%或更多，更佳者爲1 一 1〇 w / W %。 所形成的混合物用濾膜過濾滅菌，將無菌濾液注入適 當的容器（視須要可調整滲透壓）中，並將容器直接密封 或在冷凍乾燥內容物後密封。溶液之P Η以調至大約中性 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） -10- 536405 Α7 ___ Β7 五、發明說明（8 ) P Η較佳，更佳者之ρ η約爲6 · 0至約7 · 5。加入適 量之酸及/或鹼或醫藥學上可接受的緩衝溶液，例如：磷 酸鹽及碳酸鹽緩衝溶液，可將溶液保存在此ρ Η之下。 注射本藥劑對抗人類骨質疏鬆症時，本藥劑有優異之 抗骨質疏鬆症作用而無嚴重之不良副作用。爲了維持/改 進骨絡之健康狀況以及預防骨質疏鬆症，可將本注射藥劑 溶解於少量之溶液（不多於1 〇 0毫升）後在1小時內一 次注射、滴注等方法對人類投用。要減輕腰痛及關節痛以 及預防骨折時，可將本注射藥劑溶解於少量之溶液（不多 於5 0 0毫升）後用一次注射、滴注等方法對人類投用。 在各例子中，投藥速度視海藻糖之重量而定，較佳者應不 高於0 · 5公克/公斤體重/小時，且投藥時間較佳者應 介於二小時之內。本注射藥劑對抗骨質疏鬆症可以（例 如：）皮下、肌肉內、腹腔內、或靜脈內注射的途徑對人 類投用；以靜脈內投藥最佳。視用途而定，一般對人類投 用之藥劑劑量約爲0 · 5至約1 0 0公克/成年人/天， 且視海藻糖之劑量而定以約1至約5 0公克/成年人/天 較佳。 以下之實驗1 - 3係說明依據本發明抗骨質疏鬆症之 口服藥劑的效應及滿意度，實驗4 -5係說明依據本發明 抗骨質疏鬆症之注射藥劑的效應及滿意度： 實驗1 動物實驗 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) . 線· 經濟部智慧財產局員工消費合作社印製 -11 - 536405 A7 B7 五、發明說明（9 ) (請先閱讀背面之注意事項再填寫本頁) 依據常見的方法，將雌性d d Y老鼠（四週大、依體 重分成3 4隻/群）用戊巴氏妥納麻醉並摘除其卵巢。第 二天，以溶於蒸餾水之” TREHAOSE®” （結晶之海藻糖粉 末含至少9 8%海藻糖（d · s · b ·)產自Hayashibara Shoji，Inc·. Okayama, Japan )對老鼠口服投用，其劑量爲 〇 . 01 、0 · 1或1公克/公斤體重/次（每天一 次），每週五次。四週後完成投藥，將老鼠用一般方法稱 重、犧牲，摘除兩側之股骨，在1 1 0 °C下加熱過夜乾燥 股骨並稱其乾重。飼料爲低鈣飼料以便易於區別骨骼之含 量。 經濟部智慧財產局員工消費合作社印製 同時，並用與海藻糖組相同的方法進行以下之控制組 實驗：控制組1 ，以等量蒸餾水替代水溶性海藻糖溶液讓 老鼠服用；控制組2 ，對老鼠投用雌二醇作爲骨質疏鬆症 之治療劑，其係溶於以等量之蒸餾水替代水溶性海藻糖溶 液，劑量爲1毫克/公斤體重/次（每週二次）；控制組 3，老鼠進行剖腹手術之僞手術，不摘除卵巢，以等量蒸 餾水替代水溶性海藻糖溶液投用。海藻糖及控制組1至3 之老鼠股骨乾重列於表1。將某些經海藻糖劑量爲0 . 1 公克/公斤體重/次投用之老鼠、及摘除控制組1至3 (不服用海藻糖）的老鼠置於顯微鏡下觀察其脛骨。顯微 鏡下觀察之檢體用一般的方法製備，用福馬林溶液固定摘 除之脛骨，用水淸洗、去除石灰、切開延長軸、用蛋白酶 K處理以去除骨髓細胞。脛骨檢體在低真空電子顯微鏡下 放大2 4倍觀察並照相。結果示於圖1至3。圖中白色的 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -12- 536405 A7 _____B7_____ 五、發明說明（10) 部分是網狀骨質的小樑。老鼠摘除卵巢後之骨質疏鬆症稱 爲卵巢切除的骨質疏鬆症，是後更年期骨質疏鬆症之老年 式 模 越 卓 的 症 鬆 疏 質 骨 人 711鼇 (請先閱讀背面之注意事項再填寫本頁) 訂---------線· 經濟部智慧財產局員工消費合作社印製 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -13- 536405 A7 B7 發 經濟部智慧財產局員工消費合作社印製 明 I嗽 11ΠΠ tj m — 朝 餾 讲 餾 讲 餾 傅 5g 朝 ^ - S职 _ W 國_ 轻靡 <NI Ο (ΝΙ Ο οο VO ι—< ο ν/Ί 〇\ \ 4 Ο cn \〇 r—H 〇 CS1 卜 r—Η Ο O 寸 CNl o 十1 +1 +1 +1 +1 十1 \ i csi Csl CN \o CNl cn 寸 CN cn cs O' CNl CN r- CN o CNl un CO _ S JrP 圍 -齡 οο \ο oo un Ο CNl 寸 υη 卜 +1 +1 +1 +1 +1 +1 r—Η ΐ i σ\ ! 崎 CO oo νο cn οο o oo 寸 oo oo a\ a\ H \ i _ m Η ιφπ 职 國w 33 ο CN o CSI CSI CN 寸 CSI t—H CSI 〇 CNl +1 十1 +1 +1 +1 十1 C<I CSI cn 卜 J—i cn \ i cn cn r—H CO o csi cn 00 1 i cn ng Μ Μ 爷 m <imii 櫞 m /S Omil 啊 m igirn <4 ^ 機 0 避S _ m 檄一 避S tlmi] Pt| m 避C 11 1 1 1 喔 --------------------訂---------線 i^w.(請先閱讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -14 - 536405 經濟部智慧財產局員工消費合作社印製 Α7 Β7 五、發明說明（12) 由表1之數據顯示’哺乳動物口服海藻糖後能顯著的 抑制降低骨質含量之骨質疏鬆症，雖然其效果不如雌二 醇。此抑制降質骨質含量效應之劑量約爲0 · 1公克/公 斤體重/次（海藻糖），且劑量在1公克/公斤體重/次 時，效應更爲增加。海藻糖之作用可從圖1至3中加以了 解。比較圖1及圖2之脛骨（圖3爲正常之脛骨圖），控 制組1之脛骨（如圖2二中間至較低之部分）可淸楚的發 現其網狀骨質小樑減少且髓腔擴大，係爲典型的骨質疏鬆 症，此骨骼結構上非常脆弱。海藻糖處理組之脛骨（如圖 1 )，其網狀骨質小樑的減少及髓腔擴大明顯的受到抑 制，且此骨骼在結構上較強。實驗經小心觀察後，海藻糖 之處理沒有引發異常的現象。未經發表的數據顯示，老鼠 經以蔗糖替代海藻糖投用後依海藻糖組老鼠的相同方法進 行測試。結果與控制組老鼠（摘除卵巢並投用蒸餾水）沒 有顯著的差別。 與上述實驗相同，從第一組及第三組海藻糖處理 (0 · 1公克/公斤體重/次）之某些老鼠（上述已被摘 除脛骨的老鼠之外）身上摘取脛骨。從各脛骨中用一般的 方法收集骨髓細胞懸浮於α - Μ E Μ培養液（補充1 0 V / ν胎牛血淸）以生成細胞密度爲1 · 5 X 1 0 6細胞/毫 升。將產生的細胞懸浮液置每孔於體積爲〇 · 5毫升之2 4孔培養盤。各孔中加入〇 · 2 5毫升/孔之細胞懸浮液 (細胞密度爲4 X 1 0 4細胞/毫升之成骨細胞）（類似上 述懸浮細胞之新鮮製備的培養液）。將溶於上述新鮮製備 --------訂---------線 (請先閱讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -15- 536405 A7 B7 五、發明說明（13) 培養液之1 α，2 5 —二羥基維生素D3之4X 1 〇— 9M 溶液加至各孔，體積爲0 · 2 5毫升/孔。產生之細胞懸 浮混合物在5 v / v % C〇2培養箱中、3 7 °C下反應六 天，其間並更換新鮮製備含1 α，2 5 -二羥基維生素D 3 (1 X 1 〇— 9Μ)及1 0 ν/ν%胎牛血淸培養液。 完成培養後，移除上淸液各孔之細胞用福馬林固定’ 以一般之方法用” NAPHTHOL AS-MS PHOSPHATE"及"RED VIOLET LB SALT"(購自 Sigma Chemicaal Company，ST. Louis， M〇，US A)對細胞（具有抗酒石酸之酸性磷酸酯酶活性）染 色。分化的破骨細胞具有抗酒石酸之酸性磷酸酯酶活性， 並有多核之特徵。染色後各孔於顯微鏡下觀察並計算多核 細胞以測定破骨細胞之數目。結果顯示，控制組1之破骨 細胞數目至少是控制組2的3倍，海藻糖組之破骨細胞數 目約爲控制組2的2倍。此結果顯示海藻糖具有抑制破骨 細胞分化活性。從上述控制組1的實驗動物中發現骨質疏 鬆症相隨之骨骼含量降低與引發破骨細胞分化有良好的相 關性，且於海藻糖組的實驗動物中發現骨骼含量降低之抑 制與抑制引發破骨細胞分化有良好的相關性。實驗1之數 據顯示海藻糖能控制骨生成’從負平衡端傾向正常狀並具 中度之抗骨質疏鬆症作用，海藻糖之作用係關於上述抑制 破骨細胞分化之作用。 實驗2 臨床測試 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 丨丨！--------€ (請先閱讀背面之注意事項再填寫本頁) 訂---------線· 經濟部智慧財產局員工消費合作社印製 -16- 經濟部智慧財產局員工消費合作社印製 536405 A7 B7 五、發明說明（14) 將三十四位志願者，在6 0至7 2歲之間、患有老年 人骨質疏鬆症以及外傷後骨質疏鬆症伴隨之腰痛及關節痛 男女各1 7位，分成性別數目相近的兩組，並服用以描述 於實施例5之方法製備的抗骨質疏鬆症之本藥劑或與實施 例5相似的安慰劑（用蔗糖替代海藻糖），劑量爲每次飯 後五藥片/次，爲期一個月。實驗由醫技人員進行並於服 藥時詢問志願者之狀況。完成實驗後，依據調查結果將測 試樣品之效應分成四級；”非常有效”、”稍微有 效”、”沒有變化”、及”變差”。效率（％ )以各組中 回答”非常有效”、”稍微有效”之志願者所占志願者總 數之百分比表示。結果示於表2。 表2 測試樣品 判斷 依據本發明之抗骨質疏鬆症藥劑 安慰劑 非常有效 2 0 輕微有效 5 1 未改變 1〇 15 效差 〇 1 效率 4 1 6 表2的數據顯示，本藥劑在抗骨質疏鬆症上能有效的 消除骨質疏鬆症伴隨之腰痛及關節痛。安慰劑組之效應低 於1 0 %，而本藥劑組則高過4 0 %。無志願者抱怨使用 ^氏張尺錢财@ ®家鮮（CNS)A4絲（21G X 297公釐）— " .!! 羲------- —訂---------線 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 536405 A7 B7 五、發明說明（15) 藥劑本後他的或她的心智及身體狀況，而大多數的志願者 睡眠良好，舒服的鬆驰，規則的排便，且降低前述工作及 走路時之缺點。結果顯示本藥劑在抗骨質疏鬆症上有中度 之抗骨質疏鬆症 作用。 實驗3 急性毒性測試 將適量之” TREHAOSES ”結晶海藻糖粉末（具有9 8 % 之海澡糖（d · s · b ·)，購自 HayashibaraShoji， Inc.，Okayama，Japan )溶於含5 w/w%阿拉伯膠之生理 食鹽水，溶液用一般的方法滅菌。將滅菌之溶液用胃管腹 腔內地或口服投用至一組十隻的Μ Y老鼠（2 0 — 2 5公 克重量），然後觀察七天。結果顯示與投藥路徑無關，即 使投用最大劑量（1 5公克/公斤體重）亦無老鼠死亡。 數據顯示本可藥劑每日投用至哺乳動物及人類對抗骨質疏 鬆症而少有副作用。 實驗4 動物實驗 將含1 0 w / w %海藻糖（得自描述於實施例7 )之 注射液用磷酸鹽緩衝的生理食鹽水（以下簡稱爲” P B S ” ）稀釋，用孔隙度爲0 · 2 2 p m之濾膜過濾， 成爲0·lw/w%或lw/w%之海藻糖溶液。用此溶 液及上述之注射方法測試樣品。依據〜般的方、法，將雌性 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） --------------------訂---------線 (請先閱讀背面之注意事項再填寫本頁) -18- 536405 A7 B7 五、發明說明（16 ) (請先閱讀背面之注意事項再填寫本頁) d d Y老鼠（4週大）依據其體重每3 4隻分成一組，用 戊巴氏妥鈉麻醉，並摘除其卵巢。第二天，從老鼠的尾靜 脈注射各測試樣品（劑量爲5毫升/公斤體重/次），每 天一次每週五次。四週後完成投藥，將老鼠稱重，用一般 的方法犧牲，並摘取其兩側之股骨，1 1 0 °C下加熱過夜 乾燥股骨並稱乾重。用低鈣飼料餵食老鼠去除骨骼含量間 之差異。 經濟部智慧財產局員工消費合作社印製 平行控制組用與海藻糖組相同的方法處理：於控制組 1 ，老鼠投用等量之P B S替代測試樣品；控制組2 ，老 鼠施用假剖腹手術而不摘除卵巢，投用等量之P B S替代 測試樣品。測試樣品及控制組1及2之老鼠股骨乾重列於 表3。將某些投用海藻糖（1 w / w %之海藻糖溶液）之 老鼠以及控制組1及2非海藻糖投樂組老鼠摘除脛骨’在 顯微鏡下觀察脛骨。顯微鏡觀察的樣品係依一般的方法製 備，用福馬林溶液固定摘除之脛骨，用水淸洗’去除石 灰，延長軸切開，用蛋白酶K處理去除骨髓細胞。脛骨樣 品在相對低真空電子顯微鏡，2 4倍放大下照相。數據分 別爲圖4至6 。此類圖中，白色部份爲網狀骨質的小樑 骨。老鼠摘除卵巢之後的骨質疏鬆症稱爲卵巢切除的骨質 疏鬆症，是老年人骨質疏鬆症中後更年期骨質疏鬆症的卓 越模式。 •19- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 536405 A7 B7 五、發明說明（彳7) 經濟部智慧財產局員工消費合作社印製 C<i 鹚 \ < 5g 勒 餾 讲 餾 餾 讲 _ φπ 國 轻 _ m igac 〇 CN 〇 ο oo \ i 〇 m CSI o Ί—Η \ < 〇 〇 r-H 〇 +1 寸 1 < 〇〇 CSI +1 cn csi CN +1 a\ CO VO csi +1 1 \ 卜 CS +1 O' 卜 csi _ 國 轻 职 O' r- un ON ί—Η un v〇 十1 ο 〇\ +1 〇\ CN CO +1 C^I 寸 oo +1 CTN VO 〇〇 十1 卜 σ> 〇〇 _ m η 盤 _ 圍 33 _ r—Η csi +1 cn C^l CO 〇\ t—H +1 r—H CO o C<I +1 σ> \ < cn CN CSI +1 〇 C<1 cn τ—Η CN +1 cn CN m ng Μ oo PQ oo PQ Oh /-*"s 聽 檄 Π§ > _ "I Μ — ^ S ng檄 _娣 ilitn 承 寂 — 聽 πα機 n赠 *w.v 懸 1111Π U w --------------------訂---------線 IAW.(請先閱讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -20- 536405 Α7 Β7 五、發明說明（18) (請先閱讀背面之注音？事項再填寫本頁) 表3的數據顯示，當哺乳動物靜脈內投用海藻糖能顯 著的抑制骨質疏鬆症伴隨之骨質含量之下降。此抑制骨質 含量下降效應之劑量爲大約5毫升/公斤體重/次之1w / w %海藻糖，且此效應在相同劑量之1 〇 w / w %海藻 糖下更形增加。海藻糖之作用可參見圖4至6。比較圖4 及5之脛骨而圖6爲正常之脛骨，控制組1之脛骨參見圖 5之中間至下半段部分顯示，網狀骨質小樑的減少及骨髓 腔擴大（此爲典型的骨質疏鬆症），此骨骼在物理上較脆 弱。海藻糖組之脛骨如圖4，網狀骨質小樑的減少及骨髓 腔擴大受到明顯的抑制，此骨骼在物理上較強硬。小心觀 察後發現海藻糖投藥後沒有引發異常。未顯示數據的實驗 中用葡萄糖、麥芽糖、山梨糖醇或木糖醇替代海藻糖投用 至老鼠，並用相同於海藻糖組之方法對老鼠進行測試。其 結果與控制組（老鼠摘除卵巢並於腹腔內投用P B S )並 無顯著的差別。 經濟部智慧財產局員工消費合作社印製 與上述實驗相同，從第一組及第三組用1 0 %海藻糖 溶液處理（劑量約爲5毫升/公斤體重/次）之某些老鼠 (上述已被摘除脛骨的老鼠之外）身上摘取脛骨。從各脛 骨中用一般的方法收集骨髓細胞懸浮於^ 一 Μ E Μ培養液 (補充1 Ο ν / ν胎牛血淸）以生成細胞密度爲1 . 5 X 1〇6細胞/毫升。將產生的細胞懸浮液置每孔於體積爲 0 . 5毫升之2 4孔培養盤。各孔中加入0 · 2 5毫升/ 孔之細胞懸浮液（細胞密度爲4 X 1 0 4細胞/毫升之成骨 細胞）（類似上述懸浮細胞之新鮮製備的培養液）。將溶 -21 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 536405 A7 B7 五、發明說明（19 ) (請先閱讀背面之注咅？事項再填寫本頁) 於上述新鮮製備培養液之1 ^ ’ 2 5 -二羥基維生素D 3之 4xi〇- 9M溶液加至各孔，體積爲0 · 25毫升孔。產 生之細胞懸浮混合物在5 v / v % C〇2培養箱中、3 7 °C下反應六天，其間並更換新鮮製備含1 α ’ 2 5 -二羥 基維生素D3 (1x10 — 9Μ)及ΙΟν/ν%胎牛血淸 培養液。 經濟部智慧財產局員工消費合作社印製 完成培養後，移除上淸液各孔之細胞用福馬林固定， 以一般之方法用” NAPHTHOL AS-MS PHOSPHATE”及"RED VIOLET LB "(購自 Sigma Chemicaal Company, ST. Louis，Μ〇， US A)對細胞（具有抗酒石酸之酸性磷酸酯酶活性）染色。 分化的破骨細胞具有抗酒石酸之酸性磷酸酯酶活性並有多 核之特徵。染色後各孔於顯微鏡下觀察並計算多核細胞以 測定破骨細胞之數目。結果顯示，控制組1之破骨細胞數 目至少是控制組2的3倍，海藻糖組之破骨細胞數目約爲 控制組2的2倍。此結果顯示海藻糖具有抑制破骨細胞分 化活性。從上述控制組1的實驗動物中發現骨質疏鬆症相 隨之骨骼含量降低與引發破骨細胞分化有良好的相關性， 且於海藻糖組的實驗動物中發現骨骼含量降低之抑制與抑 制引發破骨細胞分化有良好的相關性。實驗1之數據顯示 海藻糖能控制骨生成，從負平衡端傾向正常狀並具中度之 抗骨質疏鬆症作用’海藻糖之作用係關於上述抑制破骨細 胞分化之作用。 實驗5 本紙張尺度適用中國國家標準（CNS)A4規格（21〇 X 297公釐） -22- 536405 A7 B7 五、發明說明（2〇) 急性毒性測試 (請先閱讀背面之注意事項再填寫本頁) 依據常見的方法，將學名爲Oryctolagus cuniculus ( — 種日本的白兔）品系的雄性兔子，各重約三公斤，每五隻 一組安置於籠中，從心耳靜脈連續投用注射實施例7及8 之方法得到之（完整的或溶解於適量的蒸餾水後）注射 液，投藥速率爲5毫升/公斤體重/小時，各組爲不同之 投藥期間至多爲六小時，然後觀察其狀況七天。結果顯示 即使使用最大可能劑量（3 g藻糖/公斤體重）亦無老鼠 死亡。數據顯示對人類及動物投用本藥劑可抗骨質疏鬆症 而少有副作用。 以下實施例1 - 6描述依據本發明口服施用地藥劑較 佳的具體實施例，且以下實施例7 - 8描述依據本發明注 射藥劑較佳的具體實施例： 實施例 1 健康食品 經濟部智慧財產局員工消費合作社印製 依據常見的方法，將馬鈴薯內含之還原糖在2 0 °C儲 存狀況以及相對溼度爲8 5 %下自動水解兩週，馬鈴薯用 水淸洗、剝皮、分級，並用離心切片機切成厚度 1 · 5 m m之薄片。薄片用水淸洗去除表面的澱粉粒，並 在1 7 0 °C下油炸約5分鐘，接著去除炸油。使用鹽劑 (含七份重量之鹽，三份之重量” TREHAOSE®” （至少含 9 8 %海藻糖（d · s · b ·)，之結晶海藻糖粉末，購 自 Hayashibara Shoji，Inc.，Okayama，Japan )粉末調味，並 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -23- 536405 A7 B7 五、發明說明（21 ) 在炸馬鈴薯片上灑上適量的風味劑，然後移至機器稱重， 裝塡，及包裝以得到含海藻糖之荼點。 (請先閱讀背面之注意事項再填寫本頁) 產品具有令人滿意的風味及滋味，可作爲健康食品以 維持/改進骨豁之健康狀態。 實施例 2 健康食品 將2 5份重量之攪拌好的奶油依續加入1 8份重量 之” T R E H A〇S E ® ” （至少含9 8 %海藻糖，d · 經濟部智慧財產局員工消費合作社印製 s · b ·，之結晶海藻糖粉末，購自Hayashibara Shoji， Inc.，Okayama, Japan )及1 〇份重量之蛋，並將混合物攬 拌成乳膏。將四十七份重量之麵粉加至乳膏，混合物揉好 後，用布捲起，並放置2 0分鐘。將產生之麵團作成棒 狀，直徑3公分，用鱲紙捲好，並在4 °C下放置二小時。 將麵團切成環狀，5 m m厚，置於含油之平底盤，在1 7 0 °C下烤1 0分鐘，塗覆上” TREHASTARE®” （至少含2 8 %海藻糖，d · s · b ·，之海藻糖糖漿，購自 Hayashibara Shoji，Inc·, Okayama, Japan )，並再烤 1 〇 分 鐘後得到冰盒餅乾。 產品具有令人滿意的風味及滋味，可作爲健康食品以 維持/改進骨骼之健康狀態。 實施例 3 健康食品 24 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 536405 A7 B7 五、發明說明（22 ) (請先閱讀背面之注意事項再填寫本頁) 將七份重量之冷凍乾燥茶萃取物及三份重量之” T R EHASTA®” （至少含 28% 海藻糖，d · s · b ·， 之海藻糖糖獎，購自 Hayashibara Shoji，Inc·，Okayama， Japan )溶於適量的水。產生的溶液可用一般的方法發酵並 噴灑9 0份重量之乾燥茶葉。依據常見的方法筛選茶葉’ 切割、乾燥，並用分離器去除雜質’每兩公克之含量用曰 本紙（Japanese paper )包裝成含海藻糖之茶包。 飮用前，將產品浸於1 8 0毫升之冷水約1 0分鐘或 於1 8 0毫升水中加熱至9 0 — 1 0 0 °C約兩分鐘。產品 具有令人滿意的風味及滋味，可作爲健康食品以維持/改 進骨骼之健康狀態。 實施例 4 健康食品 將2.7份重量之”丁£丁尺！；®” （含至少50% 經濟部智慧財產局員工消費合作社印製 麥芽四糖（d · s · b ·)之麥芽四糖糖漿，購自 Hayashibara Shoji，Inc, Okayama, Japan )、七份重量之” T REHASTARS®” （至少含 28% 海藻糖，d.s. b ·，之海藻糖糖漿，購自 H ayashibara Sh〇jl，Inc.， Okayama, Japan )、五份重量之咖啡萃取物、2 · 2份重量 之全脂奶粉、一份重量之脫脂奶粉、0 · 0 4份重量之蔗 糖脂肪酸酯類、0 · 0 6份重量之碳酸氫鈉、及8 2份重 量之水用一般的方法混合以得到含海藻糖之咖啡飮料。 產品具有令人滿意的風味及滋味，可作爲健康食品以 -25- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 536405 A7 B7 五、發明說明（23) 維持/改進骨骼之健康狀態。 (請先閱讀背面之注意事項再填寫本頁) 實施例5 健康食品添加物 將五十五份重量之” TREHAOSE®” （至少含 9 8 %海藻糖，d · s · b ·，之結晶海藻糖粉末，購自 Hayashibara Shoji, Inc., Okayama, Japan ) 、4 0 · 5 份重 量之玉米澱粉、及2 · 5份重量之結晶纖維素混合。混合 物中噴灑加入適量的水並用一般的方法揉製，經流床造 粒、硏磨、並篩選得到製作藥片的粉末。此後將粉末與兩 份重量之蔗糖脂肪酸酯類（作爲亮光劑）混合並用製作藥 片的機器（穿孔之直徑爲1 1 m m )製作成藥片，得到之 藥片約3 0 0毫克/藥片。產品具有令人滿意的吞嚥性且 在腸道中易於崩解，可作爲健康食品的添加物以維持/改 進骨骼之健康狀態。 經濟部智慧財產局員工消費合作社印製 實施例 6 健康食品添加物 將三十九份重量之” TREHAOSE®” （至少含9 8 %食 品級之海藻糖，購自 Hayashibara Shoji，Inc.，Okayama, Japan ) 、2 5份重量之粉未狀的天然珊瑚、九份重量之牛 骨粉、1 2份重量之粉未狀的酸乳酪、1 〇份重量之關華 豆膠、1 · 9份重量之維生素C、及0 · 1份重量之糖基 維生素P之混合物中噴灑加入適量的水揉製。產生之混合 -26- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 536405 A7 _ B7 五、發明說明（24 ) (請先閱讀背面之注意事項再填寫本頁) 物用流床造粒、硏磨，並篩選得到粉末以製作藥片。將粉 末與三份重量之糖脂肪酸酯類（作爲光澤劑）均勻混合， 且用製作藥片的機器（穿孔之直徑爲6 mm)製作成藥 片’得到之藥片每片約含2 0 0毫克重量之海藻糖。 產品添加鈣後呑嚥容易並可作爲健康食品添加物以維 持/改進健康。 實施例7 注射液 將用離子交換器及活化的活性碳以一般的方法移除熱 原的” TREHAOSE® ” （至少含9 8 %食品級之海藻糖，購 經濟部智慧財產局員工消費合作社印製 自 Hayashibara Shoji，Inc., Okayama，Japan )，加熱溶於蒸 餾水作爲注射液，其中用適量的0 · 5 M磷酸鹽緩衝溶液 (ρΗ7 · 0)加以平衡，以生成l〇w/w%之海藻糖 溶液。此溶液用孔隙度0 · 2 2 p m之濾膜過濾，將2〇 〇毫升量之Μ液分裝至玻璃瓶。用氮氣取代瓶內之氣體、 密封、及用高壓蒸氣滅菌得到無色的不透明溶液，在波長 430 nm下穿透度爲99.9%。 產品有令人滿意的穩定性可有效的作爲注射液治療/ 預防骨質疏鬆症。 實施例 8 乾燥的注射液 將用離子交換器及活化的活性碳以一般的方法移除熱 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -27- 536405 A7 B7 五、發明說明（25) (請先閱讀背面之注意事項再填寫本頁) 原的” TREHAOSE®” （至少含9 8 %食品級之海藻糖，購 自 Hayashibara Shoji，Inc.，Okayama，Japan )，加熱溶於蒸 餾水作爲注射液，其中用適量的0 . 5 M磷酸鹽緩衝溶液 (Ρ Η 7 · 0 )加以平衡，以生成1 0 w / w %之海藻糖 溶液。此溶液用孔隙度0 · 2 2 p m之濾膜過濾，將2 0 ◦ -毫升量之爐液分裝至玻璃瓶。玻璃瓶內此無色的不透 明溶液，在波長430nm下穿透度爲99 · 9%，經冷 凍乾燥後瓶內之氣體用惰性氣體取代並將玻璃瓶用壓蒸氣 滅菌得到乾燥的注射液。 產品在注射使用前可溶於適量之蒸餾水。產品具有令 人滿意的穩定性可有效的作爲注射液治療/預防骨質疏鬆 症。 經濟部智慧財產局員工消費合作社印製 如上述所言，本發明是基於發現海藻糖可控制骨生 成，降低負平衡，使成理想的狀況，故有優良的抗骨質疏 鬆症之作用。因此作用，本藥劑（內含海藻糖爲有效成 分）可維持並改進骨骼的健康狀況並避免骨質疏鬆症。當 投用至患有老年人骨質疏鬆症（包括後更年期的骨質疏鬆 症）；甲狀腺機能亢進、生殖腺功能不足、庫辛氏症候 群、骨生成遲滯、及高半胱胺酸尿症；外傷之副作用及過 度投用類固醇荷爾蒙因此骨骼易骨折；動情激素受體基因 異常的病人時本藥劑能消除骨質疏鬆症之腰痛及關節痛並 增加骨骼含量避免骨折。海藻糖是廣汎分布之天然醣類； 可每日服用而少有副作用。因爲本藥劑抑制人類及哺乳動 物低密度脂蛋白（L D L )含量之增加，對後更年期的女 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -28- 536405 A7 B7 五、發明說明（26) (請先閱讀背面之注意事項再填寫本頁) 性投用海藻糖在治療/預防循環疾病（包括：血液動力學 的病症、動脈硬化、血栓形成、心肌梗塞、及過量L D L 引起的大腦栓塞）上有令人滿意的效應。 依據本文對本發明較佳具體實施例的描述，據了解亦 可作出各種修飾，此類修飾包含於本發明之精神與範圍之 內故屬於本發明之申請專利範圍之內。 圖形簡述 圖1是中等光度影像圖（x24)，該圖爲卵巢經移 除之老鼠，經海藻糖處理後老鼠脛骨的低真空掃描式電子 顯微鏡影像圖。 圖2是中等光度影像圖（x24)，該圖爲卵巢經移 除之老鼠，未經海藻糖處理老鼠脛骨的低真空掃描式電子 顯微鏡影像圖。 圖3是中等光度影像圖（x2 4)，該圖爲卵巢未經 移除之老鼠，未經海藻糖處理老鼠脛骨的低真空掃描式電 子顯微鏡影像圖。 經濟部智慧財產局員工消費合作社印製 圖4是中等光度影像圖（X 2 4)，該圖爲卵巢經移 除之老鼠，經海藻糖處理後老鼠脛骨的低真空掃描式電子 顯微鏡影像圖。 圖5是中等光度影像圖（X 2 4 )，該圖爲卵巢經移 除之老鼠，未經海藻糖處理老鼠脛骨的低真空掃描式電子 顯微鏡影像圖。 圖6是中等光度影像圖（X 2 4 )，該圖爲未經卵巢 -29- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 536405 A7 __B7 五、發明說明（27 ) 式 描 掃. 空 真 低 的 骨 脛 鼠 老 彐二 理 處 糖 藻 海 經。 未圖 ， 像 鼠影 老鏡 之微 除顯 移子 經電 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -30-Okayama, Japan; and reagent-grade trehalose with a purity of at least 99% (d · s · b ·), which is produced by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan; α, α-trehalose can be prepared Maltose / trehalose converting enzymes, as disclosed in Japanese Patent Kokai NOS. 1 70,977 / 95, 263/96, and 1 49,980 / 96; or a common combination of maltose and trehalose phosphorylase. To produce trehalose with αΘ-optical isomers, the mixture of cyclomestosan dextran glucan transferase and A-galactosidase with starch partial hydrolysate and lactose can be disclosed based on (Applicant of the present invention Application) Japanese Patent Kokai-Nos. 144,694 / 92 and 179,490 / 92. / 3, Θ-trehalose can be obtained by common chemical synthesis. In the present invention, such trehalose isomers need not be highly purified and need not be separated from other impurities when administered orally. Such optical isomers may be in an unpurified form or a composition including other carbohydrate by-products produced during the production process, or include other trehalose that does not significantly prevent trehalose from combating osteoporosis in mammals and humans. Acting as a mixture of appropriate ingredients. When injecting this agent against osteoporosis in humans and animals, it is better to remove the pyrogen from trehalose before use, which is one or more methods of desalting (such as ion exchanger, macroporous Resin, activated carbon and filter membrane); methods of absorption; and methods of filtration. When administered to mammals and humans, the effect of trehalose can control bone formation and bone resorption in a normal state; trehalose can maintain a normal balance state under the action of this method, and make bone formation under negative balance And mild osteoporosis of bone resorption rate disorders (such as osteoporosis in the elderly, the bone paper size of the paper applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) I 丨 丨 丨 ------- -% (Please read the precautions on the back before filling out this page) Order --------- Line Printed by the Employee Consumption Cooperative of the Intellectual Property Bureau of the Ministry of Economy Printed by the Employee Consumption Cooperative of the Intellectual Property Bureau of the Ministry of Economy V. Description of the invention (5) The generation rate is reduced to below the normal level and the bone content is reduced) and high osteoporosis (such as parathyroid secretion whose bone resorption rate is far higher than the normal level and the bone content is reduced) is restored to the ideal situation. Due to such effects, it can maintain / improve bone health when trehalose is administered to mammals and humans with good bone conditions, and it can reduce back pain and joint pain and increase bones in human patients with osteoporosis Content, and strengthen the bones to make it difficult to fracture. Therefore, the "anti-osteoporosis agent" in the present invention refers to pharmaceutical products and foods (including: condiments) that contain trehalose as an active ingredient and can treat and / or prevent osteoporosis. The special effect of this agent against osteoporosis can be confirmed by the test described below, for example, the weight of the femur of a mouse with ovaries removed. The anti-osteoporosis agent according to the present invention may contain only trehalose or a composition containing trehalose and other ingredients that facilitate the administration of trehalose. When the composition is administered orally, its general form is a food or a pharmaceutical composition for oral administration, including: food and tube-filled liquids, especially liquids, suspensions, emulsions, creams, pastes , Powder, granular form, and other solid food and pharmaceutical product forms that require shapes. The food form of this medicine can be a combination preparation, for example: using water, alcohols, pentyls, protein, fiber, sugars, lipids, vitamins, minerals, flavors, colorants, sweeteners, flavoring agents, flavoring agents , Stabilizers, antioxidants, and antibacterial agents, and health food materials, such as sugars, milk powder, and milk protein hydrolysates that promote the growth of Lactobacillus bifidus, including: casein calcium peptides and casein phosphopeptide醯 Liver brown, soy isoflavones, blood meal, bone meal, and powdered coral, all can be used as food materials and / This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) ---- ---------------- Order --------- line (please read the precautions on the back before filling this page) -8-536405 A7 B7 V. Description of the invention (6) (Please read the notes on the back before filling out this page) or ingredients. Such compositions may be in the form of food or liquid forms for tube filling. To prepare the pharmaceutical form of the medicament, trehalose can be made into a composition which uses one or more carriers, excipients / auxiliaries, diluents, and stabilizers, and calcium preparations as needed, such as calcium lactate , Calcium glycerophosphate, calcium hydrophosphate, and L-aspartate; and other agents, such as: analgesics, anti-inflammatory agents, activated vitamin D preparations, vitamin K preparations, calcitonin preparations, estrogens, And protein constitutes a metabolic hormone preparation. Depending on the application, this osteoporosis-resistant medicine usually contains at least 0.1 w / w% of trehalose, preferably at least 1 w / w% when taken orally. When this medicine is used orally to combat osteoporosis in humans, 'This medicine has a moderate anti-osteoporosis effect in humans after administration. Depending on the application, this medicine is administered orally to humans in the form of food when maintaining / improving bone conditions and preventing osteoporosis. For the treatment of osteoporosis, prevention of fractures, and relief of osteoporosis from low back pain and joint pain 'This medicine is generally in the form of food or orally administered pharmaceutical products' For example: liquid, syrup, powder, granules, tablets, or Capsules are intended for human use. The daily dose of this medicine is usually about 0.5 to about 1000 g / adult / day, and depending on the trehalose content, about 1 to about 50 g / adult / day is preferred. The dosage of this medicine is 1-5 times / week (administered every other day). The injection form for the preparation of the anti-osteoporosis agent is generally an effective amount of trehalose as a substrate and one or more non-trehalose components are dissolved in water. Ingredients incorporated into this agent include: sugars, such as: glucose, maltose, fructose, sorbitol, xylitol; electrolytes, such as: hydroxide This paper is sized to the Chinese National Standard (CNS) A4 (210 X 297) β ~ Γ_ 536405 Α7 Β7 V. Description of the invention (7) (Please read the notes on the back before filling out this page} Sodium, sodium chloride, sodium iodide, sodium acetate, sodium lactate, sodium citrate, sodium hydrogen phosphate, phosphoric acid Potassium dihydrogen, calcium acetate, calcium lactate, calcium glycerophosphate, calcium gluconate, magnesium sulfate, zinc sulfate, zinc chloride, iron sulfate, ferrous chloride, copper sulfate, and manganese sulfate; amino acids, such as: L Monoisoleucine, L ~ leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-valine, L Monoarginine, L-histidine, glycine, L-alanine, L-cysteine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, And -thyroxine; vitamins such as: thiamine chloride, riboflavin monophosphate, pyridoxine chloride Hydrogen, cyanocobalamin, calcium pantothenate, nicotinamide, folic acid, biotin, choline ditartrate, L-ascorbic acid, retinyl acetate, tocopheryl acetate, and vitamin K1; plant and animal lipids , Such as: soybean oil, safflower oil, linseed oil, coconut oil, whale oil, and oils from marine life; calcium preparations, such as: calcium lactate, calcium glycerol phosphate, calcium hydrogen phosphate, and L-aspartate; and Pharmaceuticals, such as: activated vitamin D preparations, vitamin K, calcitonin, estrogens, protein-forming metabolic hormones, analgesics, and anti-inflammatory agents. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs to prepare the above-mentioned antibiotics When injecting osteoporosis, trehalose and one or more of the above non-trehalose components can be dissolved in water, so that the trehalose content is 0 · lw / w% or more, more preferably 1 to 10w / W%. The resulting mixture is filtered and sterilized with a filter membrane, and the sterile filtrate is poured into a suitable container (adjustable osmotic pressure if necessary), and the container is directly sealed or sealed after freeze-drying the content. The liquid P Η is adjusted to approximately neutral. The paper size applies the Chinese National Standard (CNS) A4 specification (210 × 297 mm) -10- 536405 Α7 ___ B7 V. Description of the invention (8) P Η is better, and the better ρ η is about 6 · 0 to about 7 · 5. Add appropriate amount of acid and / or base or pharmaceutically acceptable buffer solution, such as phosphate and carbonate buffer solution, the solution can be stored in ρ Η When injecting this medicine against human osteoporosis, this medicine has excellent anti-osteoporosis effect without serious adverse side effects. In order to maintain / improve the health of bones and networks and prevent osteoporosis, this injection can be dissolved After a small amount of solution (not more than 100 ml), it is administered to humans by one injection, drip, etc. within 1 hour. To reduce back pain and joint pain and prevent fractures, this injection can be dissolved in a small amount of solution (not more than 500 ml) and administered to humans by one injection, drip, etc. In each case, the dosing rate depends on the weight of the trehalose. The better one should be no more than 0.5 g / kg body weight / hour, and the better one should be within two hours. This injectable agent can be used to treat osteoporosis (for example :) subcutaneously, intramuscularly, intraperitoneally, or intravenously. It is best administered intravenously. Depending on the application, the dosage of pharmaceuticals generally administered to humans is about 0.5 to about 100 g / adult / day, and depending on the dose of trehalose, about 1 to about 50 g / adult / It's better. The following Experiments 1-3 illustrate the effects and satisfaction of oral agents against osteoporosis according to the present invention, and Experiments 4-5 describe the effects and satisfaction of injections with anti osteoporosis according to the present invention: Experiment 1 Animal Experiment This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back before filling out this page). Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs -11-536405 A7 B7 V. Description of the invention (9) (Please read the precautions on the back before filling out this page) According to common methods, female dd Y mice (4 weeks old, divided into 3 4 per group according to weight) are given pentostoleran Anesthetize and remove their ovaries. The next day, rats were orally administered with "TREHAOSE®" (crystallized trehalose powder containing at least 98% trehalose (d · s · b ·) in distilled water from Hayashibara Shoji, Inc .. Okayama, Japan) dissolved in distilled water. For use, the dosage is 0.01, 0.1 or 1 g / kg body weight / time (once a day), once a week. After four weeks of administration, the rats were weighed and sacrificed in the usual way, the femurs on both sides were removed, and the femurs were dried overnight by heating at 110 ° C and weighed dry. The feed is low-calcium for easy identification of bone content. At the same time, it was printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, and the following control group experiments were performed using the same method as the trehalose group: the control group 1 replaced the water-soluble trehalose solution with the same amount of distilled water for rats to take; the control group 2 Mice administered estradiol as a therapeutic agent for osteoporosis, which was dissolved in an equivalent amount of distilled water instead of a water-soluble trehalose solution at a dose of 1 mg / kg body weight / time (twice a week); control group 3, The rats were subjected to a pseudo-laparotomy operation without removing the ovaries, and the equivalent amount of distilled water was used instead of the water-soluble trehalose solution. The femoral stem weights of mice in the trehalose and control groups 1 to 3 are listed in Table 1. Certain mice that received trehalose at a dose of 0.1 g / kg body weight / time, and mice from the control group 1 to 3 (without taking trehalose) were placed under a microscope to observe their tibia. Specimens for observation under a microscope were prepared by ordinary methods. The removed tibia was fixed with formalin solution, washed with water, removed lime, cut the extension shaft, and treated with proteinase K to remove bone marrow cells. Tibial specimens were observed and photographed at 24x magnification under a low vacuum electron microscope. The results are shown in Figs. The white paper size in the picture is in accordance with Chinese National Standard (CNS) A4 specification (210 X 297 mm) -12- 536405 A7 _____B7_____ 5. Description of the invention (10) Part is a trabecular bone. Osteoporosis after ovarian removal in mice is called osteoporosis of ovariectomy, and it is a senile model of postmenopausal osteoporosis. Osteoporosis 711 (Please read the precautions on the back before filling out this page ) Order --------- Line · Printed by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, the paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) -13- 536405 A7 B7 Economic Development Ministry of Intellectual Property Bureau's Employee Cooperative Cooperative Prints I1111ΠΠ tj m — North Korea Distillation and Distillation Process 5g North Korea-S Position _ W 国 _ 轻 迷 < NI Ο (ΝΙ Ο οο VO ι— < ο ν / Ί 〇 \ \ 4 〇 cn \ 〇r—H 〇CS1 卜 r—Η Ο O inch CNl o ten 1 +1 +1 +1 +1 ten 1 \ i csi Csl CN \ o CNl cn inch CN cn cs O 'CNl CN r- CN o CNl un CO _ S JrP Wai-age οο \ ο oo un Ο CNl inch υη bu +1 +1 +1 +1 +1 +1 r—Η ΐ i σ \! Qi CO oo νο cn οο o oo inch oo oo a \ a \ H \ i _ m Η ιφπ working country w 33 ο CN o CSI CSI CN inch CSI t—H CSI 〇CNl +1 ten 1 +1 +1 +1 Ten 1 c < I CSI cn J-i cn \ i cn cn r-H CO o csi cn 00 1 i cn ng Μ Μ e m < imii 橼 m / S Omil ah m igirn < 4 ^ machine 0 avoid S _ m 檄 one avoid S tlmi] Pt | m avoid C 11 1 1 1 oh -------------------- order --- ------ line i ^ w. (Please read the precautions on the back before filling this page) This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) -14-536405 Ministry of Economic Affairs Wisdom Printed by the Consumer Affairs Cooperative of the Property Bureau A7 B7 V. Description of the invention (12) According to the data in Table 1, 'Mammalian oral trehalose can significantly inhibit osteoporosis that reduces bone content, although its effect is not as good as that of estradiol. The dose for suppressing the effect of degrading bone content is about 0.1 g / kg body weight / time (trehalose), and the effect is further increased when the dose is 1 g / kg body weight / time. The effect of trehalose can be understood from Figures 1 to 3. Comparing the tibia of Figures 1 and 2 (Figure 3 is the normal tibia), the tibia of control group 1 (the middle to the lower part of Figure 2) can be clearly found to have a reduced reticular trabeculae and a medullary cavity Enlargement is a typical osteoporosis, which is very fragile in structure. In the trehalose-treated tibia (see Figure 1), the reduction of reticular trabeculae and the enlargement of the medullary cavity were significantly suppressed, and the bone was stronger in structure. After careful observation in the experiment, the trehalose treatment did not cause any abnormalities. Unpublished data show that mice were tested in the same way as mice in the trehalose group after administration of sucrose instead of trehalose. The results were not significantly different from those in the control group (with ovaries removed and administered with distilled water). As in the experiment described above, the tibia was removed from certain mice (except the mouse from which the tibia had been removed) treated with trehalose (0.1 g / kg body weight / time) in the first and third groups. Bone marrow cells were collected from each tibia by a general method and suspended in α-M E M culture medium (supplemented with 10 V / v fetal bovine blood pupa) to generate a cell density of 1.5 x 106 cells / mL. The resulting cell suspension was placed in a 24-well culture plate having a volume of 0.5 ml per well. 0.5 ml / well of cell suspension (osteoblasts with a cell density of 4 x 104 cells / ml) was added to each well (similar to the freshly prepared culture medium of the suspended cells described above). Will be dissolved in the freshly prepared -------- order --------- line (please read the precautions on the back before filling this page) This paper size applies to China National Standard (CNS) A4 specifications (210 X 297 mm) -15- 536405 A7 B7 V. Description of the invention (13) 1 α, 2 5-dihydroxyvitamin D3 4X 1 0-9M solution was added to each well with a volume of 0 · 2 5 ml / well. The resulting cell suspension mixture was reacted in a 5 v / v% CO 2 incubator at 37 ° C. for six days, during which time it was replaced and freshly prepared to contain 1 α, 2 5 -dihydroxyvitamin D 3 (1 X 1 0— 9M) and 10 ν / ν% fetal bovine blood maggot culture broth. After the completion of the culture, the cells in each well of the supernatant liquid were removed and fixed with formalin. "NAPHTHOL AS-MS PHOSPHATE" and "RED VIOLET LB SALT" (purchased from Sigma Chemicaal Company, ST. Louis, M. 〇, US A) stain cells (with tartrate-resistant acid phosphatase activity). Differentiated osteoclasts have tartrate-resistant acid phosphatase activity and have multinucleate characteristics. After staining, each well is observed under a microscope and observed Calculate multinucleated cells to determine the number of osteoclasts. The results show that the number of osteoclasts in control group 1 is at least three times that of control group 2, and the number of osteoclasts in trehalose group is about twice that of control group 2. This result It shows that trehalose has the ability to inhibit osteoclast differentiation. From the experimental animals of the control group 1 above, it was found that the osteoporosis phase and the decrease in bone content have a good correlation with the initiation of osteoclast differentiation. A good correlation was found between the inhibition of reduced bone content and the inhibition of osteoclast differentiation. The data from Experiment 1 show that trehalose can control bone formation. The end tends to be normal and has a moderate anti-osteoporosis effect. The effect of trehalose is on the above-mentioned effect of inhibiting osteoclast differentiation. Experiment 2 Clinical test This paper is in accordance with Chinese National Standard (CNS) A4 (210 X 297) (Mm) 丨 丨! -------- € (Please read the notes on the back before filling out this page) Order --------- Line Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs -16- Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 536405 A7 B7 V. Description of the Invention (14) Thirty-four volunteers, aged 60 to 72, will suffer from osteoporosis and trauma in the elderly Back osteoporosis associated with low back pain and joint pain, 17 males and 1 females, divided into two groups of similar genders, and took the anti-osteoporosis drug prepared by the method described in Example 5 or similar to Example 5 Placebo (sucrose is used instead of trehalose) at a dose of five pills / time for one month after each meal. The experiment was performed by medical technicians and asked the volunteers' status when taking the medicine. After the experiment was completed, Effect of test sample Level 4; "Very effective", "Slightly effective", "No change", and "Worse". The efficiency (%) of the volunteers who answered "very effective" and "slightly effective" in each group accounted for volunteers The percentage of the total is expressed. The results are shown in Table 2. Table 2 Test samples judged that the anti-osteoporosis agent according to the present invention is very effective in placebo 2 0 slightly effective 5 1 unchanged 1015 poor efficacy 0 1 efficiency Table 1 The data show that this agent can effectively eliminate low back pain and joint pain associated with osteoporosis in anti-osteoporosis. The effect in the placebo group was less than 10%, while the effect in this drug group was more than 40%. No volunteers complained about the use of ^ Zhang Zhangqiancai @ ® 家 鲜 (CNS) A4 丝 (21G X 297mm) — ". !! 羲 ------- —Order ------- --Line (Please read the notes on the back before filling this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 536405 A7 B7 V. Description of the invention (15) His or her mental and physical condition after the pharmacy, and Most volunteers sleep well, relax comfortably, regular bowel movements, and reduce the disadvantages of the aforementioned work and walking. The results show that this agent has a moderate anti-osteoporosis effect on anti-osteoporosis. Experiment 3 Acute toxicity test A suitable amount of "TREHAOSES" crystalline trehalose powder (with 98% sea bath sugar (d · s · b ·), purchased from HayashibaraShoji, Inc., Okayama, Japan) was dissolved in a solution containing 5 w / w% Gum Arabic physiological saline, the solution is sterilized by ordinary methods. The sterilized solution was intraperitoneally or orally administered to a group of ten MY mice (20 to 25 g in weight) by a gastric tube, and then observed for seven days. The results showed no relation to the route of administration, and no mouse died even at the maximum dose (15 g / kg body weight). The data show that the agent can be administered daily to mammals and humans to combat osteoporosis with few side effects. Experiment 4 Animal experiments: An injection containing 10 w / w% of trehalose (obtained from Example 7) was diluted with a phosphate buffered saline solution (hereinafter referred to as "PBS"), with a porosity of 0 · The filter was filtered at 2 2 pm and became a trehalose solution of 0.1 lw / w% or lw / w%. Samples were tested using this solution and the injection method described above. According to the general method and method, the paper size of the female is applied to the Chinese National Standard (CNS) A4 (210 X 297 mm) -------------------- Order --------- Line (Please read the precautions on the back before filling this page) -18- 536405 A7 B7 V. Invention Description (16) (Please read the precautions on the back before filling out this page) dd Y mice (4 weeks old) were divided into groups of 34 each according to their body weight, anesthetized with sodium pentosate, and their ovaries were removed. The next day, each test sample (dose 5 ml / kg body weight / time) was injected from the tail vein of the mouse, once every day and every five days. After four weeks of administration, the rats were weighed, sacrificed by ordinary methods, and the femurs on both sides were removed. The femurs were heated at 110 ° C overnight to dry the femur and weighed dry. The mice were fed low calcium diets to remove differences in bone content. Printed by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, the parallel control group was treated in the same way as the trehalose group: in control group 1, rats were given the same amount of PBS as the test sample; in control group 2, the rats were given a pseudo-laparotomy without The ovary was removed and the test sample was replaced with an equal amount of PBS. The femoral stem weights of the test samples and control groups 1 and 2 are shown in Table 3. The tibia was removed from certain mice administered with trehalose (1 w / w% trehalose solution) and mice from the control groups 1 and 2 non-trehalose-administered groups, and the tibia was observed under a microscope. The sample for microscopic observation was prepared according to a general method, and the removed tibia was fixed with formalin solution, washed with water 'to remove ashes, extended axotomy, and treated with proteinase K to remove bone marrow cells. The tibial specimens were photographed under a relatively low vacuum electron microscope at a magnification of 24x. The data are shown in Figures 4 to 6, respectively. In such figures, the white part is the trabecular bone of the reticular bone. Osteoporosis after ovarian removal in mice is called osteoporosis with ovariectomy, and it is an excellent model of postmenopausal osteoporosis in elderly osteoporosis. • 19- This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) 536405 A7 B7 V. Description of invention (彳 7) Printed by the Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs C < i 鹚 \ < 5g lenticulation and distillation _ φπ National Light _ m igac 〇 CN ο oo \ i 〇 m CSI o Ί—Η \ < 〇 〇 r-H 〇 +1 inch 1 < 〇〇CSI +1 cn csi CN +1 a \ CO VO csi +1 1 \ 卜 CS +1 O '卜 csi _ National light duty O' r- un ON ί—Η un v〇 十 1 ο 〇 \ +1 〇 \ CN CO +1 C ^ I inch oo +1 CTN VO 〇〇 十一 卜 σ > 〇〇_ m η disk_ circumference 33 _ r—Η csi +1 cn C ^ l CO 〇 \ t-H +1 r—H CO o C < I +1 σ > \ < cn CN CSI +1 〇 C < 1 cn τ—Η CN +1 cn CN m ng Μ oo PQ oo PQ Oh /-* " s listen 檄 Π§ > _ " I Μ — ^ S ng 檄 _ 娣 ilitn 承 寂 — listen to πα Machine n gift * wv hanging 1111Π U w -------------------- order --------- line IAW. (Please read the precautions on the back first (Fill in this page again.) This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) -20- 536405 Α7 Β7 5. Invention Description (18) (Please read the note on the back? Matters before filling this page The data in Table 3 shows that when mammals are administered intravenously with trehalose, it can significantly inhibit the decrease in bone content associated with osteoporosis. The dose of the effect of inhibiting the decrease of bone content is about 5 ml / kg body weight / second 1 w / w% trehalose, and the effect is even more increased at the same dose of 10 w / w% trehalose. The effect of trehalose can be seen in Figures 4 to 6. Comparing the tibia of Figures 4 and 5 and Figure 6 is the normal tibia. The tibia of control group 1 is shown in the middle to lower half of Figure 5. It shows the reduction of reticular trabeculae and the enlargement of the bone marrow cavity (this is a typical osteoporosis Disease), this bone is physically weak. The tibia in the trehalose group is shown in Figure 4. The decrease in reticular trabeculae and the enlargement of the bone marrow cavity were significantly suppressed. This bone was physically stronger. After careful observation, no abnormalities were found after trehalose administration. In experiments where no data was shown, glucose, maltose, sorbitol or xylitol was used instead of trehalose in mice, and mice were tested in the same way as the trehalose group. The results were not significantly different from those in the control group (when the ovaries were removed and the PB was administered intraperitoneally). Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, the same as the above experiment, some mice treated with 10% trehalose solution (dose about 5 ml / kg body weight / time) from the first group and the third group (the above has been The tibia was removed from the mouse except the tibia. Bone marrow cells were collected from each tibia by a general method and suspended in 1 μM EM culture solution (supplemented with 100 ν / ν fetal bovine blood limulus) to generate a cell density of 1.5 X 106 cells / ml. The resulting cell suspension was placed in a 24-well culture plate having a volume of 0.5 ml per well. Add 0.25 ml / well of cell suspension (osteoblasts with a cell density of 4 X 104 cells / ml) to each well (similar to the freshly prepared culture medium of the suspension cells). Will dissolve-21-This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 536405 A7 B7 V. Description of the invention (19) (Please read the note on the back? Matters before filling out this page) The above-mentioned freshly prepared culture solution of 1 ^ '2 5 -dihydroxyvitamin D 3 in 4xi-9M was added to each well with a volume of 0. 25 ml. The resulting cell suspension mixture was reacted in a 5 v / v% CO2 incubator at 37 ° C for six days, during which time it was replaced and freshly prepared to contain 1 α '2 5 -dihydroxyvitamin D3 (1x10 — 9M) and ΙΟν / ν% fetal bovine blood maggot culture solution. After printing by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, the cells were removed from each well of the supernatant solution and fixed with formalin, and "NAPHTHOL AS-MS PHOSPHATE" and "RED VIOLET LB " (purchase Cells (with tartrate-resistant acid phosphatase activity) were stained from Sigma Chemicaal Company, ST. Louis, MO, US A). Differentiated osteoclasts have tartrate-resistant acid phosphatase activity and are multinucleated. After staining, each well was observed under a microscope and multinucleated cells were counted to determine the number of osteoclasts. The results showed that the number of osteoclasts in control group 1 was at least three times that of control group 2, and the number of osteoclasts in trehalose group was about twice that of control group 2. This result shows that trehalose has the ability to inhibit osteoclast differentiation. It was found from the experimental animals of the control group 1 that the osteoporosis phase followed by a decrease in bone content has a good correlation with the initiation of osteoclast differentiation, and in the experimental animals of the trehalose group, it was found that the inhibition of the decrease in bone content and the inhibition of osteoclasts Osteoblast differentiation has a good correlation. The data of Experiment 1 show that trehalose can control osteogenesis, tend to be normal from the negative balance end, and has a moderate anti-osteoporosis effect. The effect of trehalose is on the above-mentioned effect of inhibiting osteoclast differentiation. Experiment 5 This paper size is in accordance with Chinese National Standard (CNS) A4 (21〇X 297mm) -22- 536405 A7 B7 V. Description of the invention (20) Acute toxicity test (Please read the precautions on the back before filling in this Page) According to a common method, male rabbits of the scientific name Oryctolagus cuniculus (— a Japanese white rabbit) strain, each weighing about three kilograms, are placed in groups of five in a cage, and are continuously injected from the heart ear vein. Example The injection solution obtained by the methods 7 and 8 (complete or dissolved in a suitable amount of distilled water) injection solution, the injection rate is 5 ml / kg body weight / hour, each group for a different administration period up to six hours, and then observe its condition for seven days. The results showed that even with the maximum possible dose (3 g fucose / kg body weight), no mice died. The data show that administration of this agent to humans and animals is resistant to osteoporosis with few side effects. The following Examples 1 to 6 describe preferred specific examples of the medicament for oral administration according to the present invention, and the following Examples 7 to 8 describe preferred specific examples of the medicament for injection according to the present invention: Example 1 Intellectual Property of the Ministry of Health Food Economy Printed by the Consumer Cooperative of the Bureau of the People's Republic of China, the reducing sugar contained in potatoes was automatically hydrolyzed for two weeks at a storage condition of 20 ° C and a relative humidity of 85%, and the potatoes were washed with water, peeled, classified, and centrifuged. The microtome cuts into thin slices of 1 · 5 mm. The flakes were rinsed with water to remove the starch granules on the surface, and fried at 170 ° C for about 5 minutes, and then the frying oil was removed. Salt crystals (containing seven parts by weight of salt, three parts by weight of "TREHAOSE®" (at least 98% trehalose (d · s · b ·), crystalline trehalose powder, purchased from Hayashibara Shoji, Inc., Okayama, Japan) powder seasoning, and this paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) -23- 536405 A7 B7 V. Description of the invention (21) Sprinkle an appropriate amount of flavor on potato chips , Then move to the machine to weigh, decorate, and package to get the trehalose-containing point. (Please read the precautions on the back before filling this page) The product has satisfactory flavor and taste, and can be used as a healthy food. Maintain / improve the healthy state of bones. Example 2 Healthy Food Add 25 parts by weight of stirred cream to 18 parts by weight of "TREHA〇SE ®" (containing at least 98% trehalose, d. Economical The Ministry of Intellectual Property Bureau employee consumer cooperative printed s · b ·, crystalline trehalose powder, purchased from Hayashibara Shoji, Inc., Okayama, Japan) and 10 parts by weight of eggs, and mixed the mixture into a cream. Forty-seven parts by weight Add the powder to the cream. After kneading the mixture, roll it up with a cloth and let it stand for 20 minutes. The resulting dough is made into a stick shape with a diameter of 3 cm, rolled with a paper towel, and left at 4 ° C for two hours. Cut the dough into rings, 5 mm thick, place on a flat pan with oil, bake at 170 ° C for 10 minutes, and coat with "TREHASTARE®" (with at least 28% trehalose, d · s · b., trehalose syrup, purchased from Hayashibara Shoji, Inc., Okayama, Japan), and baked for 10 minutes to obtain ice box biscuits. The product has satisfactory flavor and taste, and can be used as a healthy food to maintain / Improve the health status of bones. Example 3 Healthy food 24-This paper size applies the Chinese National Standard (CNS) A4 (210 X 297 mm) 536405 A7 B7 V. Description of the invention (22) (Please read the precautions on the back first (Fill in this page again.) Seven parts of freeze-dried tea extract and three parts of "TR EHASTA®" (at least 28% trehalose, d · s · b ·, trehalose sugar award, purchased from Hayashibara Shoji , Inc., Okayama, Japan) is dissolved in an appropriate amount of water. The solution can be fermented by ordinary methods and sprayed with 90 parts by weight of dry tea leaves. According to common methods, the tea leaves are 'cut, dried, and impurities are removed by a separator', and the content of every two grams is packed in Japanese paper. Tea bag with trehalose.飮 Before use, immerse the product in 180 ml of cold water for about 10 minutes or heat it in 180 ml of water to 90-100 ° C for about two minutes. The product has satisfactory flavor and taste, and can be used as a healthy food to maintain / improve the health of bones. Example 4 Healthy food will be 2.7 parts by weight of "Ding £ ding !!" (contains at least 50% of malt tetraose (d · s · b ·) printed by maltose tetrasaccharide (d · s · b ·) of the consumer cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Sugar syrup, purchased from Hayashibara Shoji, Inc, Okayama, Japan), seven parts by weight of "T REHASTARS®" (containing at least 28% trehalose, ds b ·, trehalose syrup, purchased from Hayashibara Shoji, Inc. ., Okayama, Japan), five parts by weight of coffee extract, 2.2 parts by weight of whole milk powder, one part by weight of skim milk powder, 0.4 parts by weight of sucrose fatty acid esters, and 0.6 parts by weight Sodium bicarbonate and 82 parts by weight of water were mixed in a usual manner to obtain a trehalose-containing coffee sauce. The product has satisfactory flavor and taste, and can be used as a healthy food. -25- This paper size applies Chinese National Standard (CNS) A4 (210 X 297 mm) 536405 A7 B7 V. Description of the invention (23) Maintenance / improvement Bone health. (Please read the precautions on the back before filling this page) Example 5 Healthy food additives will be 55 parts by weight of "TREHAOSE®" (containing at least 98% trehalose, d · s · b ·, crystalline seaweed) Sugar powder, commercially available from Hayashibara Shoji, Inc., Okayama, Japan), 40. 5 parts by weight of corn starch, and 2.5 parts by weight of crystalline cellulose. The mixture is sprayed with an appropriate amount of water and kneaded by a general method, granulated, honed, and sieved by a fluid bed to obtain a powder for making tablets. Thereafter, the powder was mixed with two parts by weight of sucrose fatty acid esters (as a brightener) and made into tablets using a machine for making tablets (perforated diameter of 11 mm) to obtain tablets of about 300 mg / tablet. The product has satisfactory swallowability and is easy to disintegrate in the intestine. It can be used as an additive to health foods to maintain / improve the health of bones. Example 6 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. Healthy food additives will be 39 parts by weight of "TREHAOSE®" (containing at least 98% of food grade trehalose, purchased from Hayashibara Shoji, Inc., Okayama, Japan), 25 parts by weight of powdered natural coral, 9 parts by weight of beef bone meal, 12 parts by weight of powdered yogurt, 10 parts by weight of Guanhua bean gum, and 1.9 parts by weight Spray a mixture of vitamin C and 0.1 parts by weight of sugar-based vitamin P by kneading with an appropriate amount of water. The resulting mixture-26- This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 536405 A7 _ B7 V. Description of the invention (24) (Please read the precautions on the back before filling this page) Granulation, honing in a fluid bed, and screening to obtain powders to make tablets. Mix the powder with three parts by weight of sugar fatty acid esters (as a gloss agent), and use a tablet making machine (the diameter of the perforation is 6 mm) to make tablets. Each tablet contains about 200 mg by weight. Trehalose. The product is easy to choke after adding calcium and can be used as a health food supplement to maintain / improve health. Example 7 The injection solution will remove the pyrogen "TREHAOSE®" (containing at least 98% food-grade trehalose by ordinary means using an ion exchanger and activated activated carbon, purchased from the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs). Made from Hayashibara Shoji, Inc., Okayama, Japan), heated and dissolved in distilled water as an injection solution, in which an appropriate amount of 0.5 M phosphate buffer solution (ρΗ7 · 0) was used to balance to produce 10 w / w% Trehalose solution. This solution was filtered through a membrane filter with a porosity of 0.22 pm, and 200 ml of M liquid was dispensed into glass bottles. Replace the gas in the bottle with nitrogen, seal, and sterilize with high pressure steam to obtain a colorless opaque solution with a transmission of 99.9% at a wavelength of 430 nm. The product has satisfactory stability and can be effectively used as an injection to treat / prevent osteoporosis. Example 8 The dry injection solution will be removed with an ion exchanger and activated activated carbon in a general way. The paper size applies the Chinese National Standard (CNS) A4 (210 X 297 mm) -27- 536405 A7 B7 5 2. Description of the invention (25) (Please read the notes on the back before filling out this page) The original "TREHAOSE®" (containing at least 98% food-grade trehalose, purchased from Hayashibara Shoji, Inc., Okayama, Japan), Heated and dissolved in distilled water as an injection, which was equilibrated with an appropriate amount of 0.5 M phosphate buffer solution (P 7 · 0) to generate a 10 w / w% trehalose solution. This solution was filtered through a filter membrane with a porosity of 0.22 pm, and the furnace liquid in a volume of 20 ◦-ml was dispensed into a glass bottle. This colorless, opaque solution in a glass bottle has a transmission of 99 · 9% at a wavelength of 430 nm. After freeze-drying, the gas in the bottle is replaced with an inert gas and the glass bottle is autoclaved to obtain a dry injection solution. The product is soluble in distilled water before injection. The product has satisfactory stability and is effective as an injection to treat / prevent osteoporosis. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs As mentioned above, the present invention is based on the discovery that trehalose can control bone formation, reduce negative balance, and make it an ideal state, so it has an excellent anti-osteoporosis effect. Therefore, this medicine (containing trehalose as an effective ingredient) can maintain and improve bone health and avoid osteoporosis. When administered to patients with osteoporosis in the elderly (including postmenopausal osteoporosis); hyperthyroidism, hypogonadism, Cushing's syndrome, delayed bone formation, and homocysteineuria; side effects of trauma And excessive use of steroid hormones makes bones easy to fracture; in patients with abnormal estrogen receptor genes, this agent can eliminate back pain and joint pain of osteoporosis and increase bone content to avoid fractures. Trehalose is a widely distributed natural sugar; it can be taken daily with few side effects. Because this agent inhibits the increase of low density lipoprotein (LDL) content in humans and mammals, the Chinese National Standard (CNS) A4 size (210 X 297 mm) is applicable to the post-menopausal female paper size. -28- 536405 A7 B7 5 、 Explanation (26) (Please read the precautions on the back before filling this page) Sexual use of trehalose in the treatment / prevention of circulatory diseases (including: hemodynamic disorders, arteriosclerosis, thrombosis, myocardial infarction, and overdose LDL-induced cerebral embolism) has a satisfactory effect. According to the description of the preferred embodiments of the present invention, it is understood that various modifications can also be made. Such modifications are included in the spirit and scope of the present invention and therefore fall within the scope of the patent application of the present invention. Brief Description of the Figures Figure 1 is a medium-photographic image (x24) of a low-vacuum scanning electron microscope image of the tibia of a mouse with ovaries removed and trehalose-treated mice. Figure 2 is a mid-photographic image (x24) of a low-vacuum scanning electron microscope image of the tibia of a mouse with ovaries removed and without trehalose treatment. Figure 3 is a medium photometric image (x2 4), which is a low-vacuum scanning electron microscope image of the tibia of a mouse without an ovary removed and a trehalose-treated mouse. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Figure 4 is a mid-photographic image (X 2 4). This image is a low-vacuum scanning electron microscope image of the tibia of a mouse with ovary removed and trehalose treated. Figure 5 is a mid-photographic image (X 2 4), which is a low-vacuum scanning electron microscope image of the tibia of a mouse with ovaries removed and without trehalose treatment. Figure 6 is a mid-photometric image (X 2 4), this figure is without ovaries-29- This paper size applies Chinese National Standard (CNS) A4 specifications (210 X 297 mm) 536405 A7 __B7 V. Description of the invention (27 ) Scanning style. The emptiness of the lower bone tibia and the old man's sacrifice at the second place of the seaweed. Not shown, the micro-movement of a telescope like a mouse shadow old mirror (please read the precautions on the back before filling out this page) Printed on paper scales applicable to the Chinese National Standard (CNS) A4 Specifications (210 X 297 mm) -30-