褐藻膠包覆柚皮苷與透鈣磷石作為骨質疏鬆症治療之研究

Abstract

骨質疏鬆症是一種罹患機率隨著年齡增加而增加的老年疾病，隨著骨質的流失、骨組織的強度下降，骨折的風險便會持續增加，據統計有1/5的男性與1/3的女性終其一生會罹患此症。 目前對於骨質疏鬆症的治療手段多有其缺陷。因此，本研究欲開發一種新型態的口服藥劑，將透鈣磷石與柚皮苷包覆在褐藻膠中形成微粒以方便吞食，透鈣磷石提供穩定且易於吸收的鈣質來源，柚皮苷則是已被證實能夠提升骨質密度的中藥萃取物。 本研究使用XRD、SEM與EDS來確認透鈣磷石的合成與褐藻膠包覆透鈣磷石和柚皮苷的微粒性質，並且確認其在胃酸的環境(pH = 2)中能夠大量電解並釋放出鈣離子，以增加鈣質在體內的吸收率。體外與體內測試皆分為生物相容性與有效性的測試。在體外測試階段，使用L929細胞確認了本研究使用的材料有著良好的生物相容性，並使用人類骨肉瘤細胞MG-63來進行成骨細胞的成骨活性試驗，發現本研究之褐藻膠微粒萃取液對於促進成骨作用的效果較一般培養液與已知能增加成骨作用活性之成骨培養液佳。因此進一步以大鼠之體內測試加以驗證之。 體內測試以卵巢切除誘發骨質流失並再飼養兩個月以使大鼠骨質疏鬆，並分成卵巢未切除組、切除不處理組與切除給藥組。本研究將微粒磨碎並且以管餵的方式每日餵食大鼠，餵食六周後以X光測量其股骨之骨質密度並且在下一周犧牲。血清生化學與組織切片的結果皆顯示本實驗材料無明顯生物毒性，在有效性測試方面，骨密度測試、無機物比例評估與骨組織切片之結果皆顯示本實驗材料相較於切除不處理組能略為提升骨質；而在血清生化學分析中，可以發現實驗材料組的成骨指標相較於其他組別有大幅的增加，顯示本實驗材料能有效增加成骨作用之速率，以降低骨折風險。

Osteoporosis, one of the most prevalent geriatric syndromes, is a disease caused by the loss of bone mass and causing the decreasing of bone strength and the increasing of bone fracture risk. The prevalence rate and fatality rate are proportional to age, which imposes an onerous burden on both society and economic system. Statistically, one-fifth of men and one-third of women will suffer this disease during their lifetime, so it is important to have an effective solution to this “silent killer.” Current treatments for osteoporosis all have some disadvantages such as adverse events, aggravating medication restrictions, and limited efficacy. To find a better solution for osteoporosis, we are going to develop a new oral formula. Two molecules, brushite and naringin, were chosen to increase bone mass. Brushite can stably and considerably release calcium and serves as calcium supplement in this study. Naringin, an extract of traditional Chinese medicine called Gu-Sui-Bu (骨碎補), has a proved effect on reducing the bone fracture risk. Furthermore, the two molecules were encapsulated in alginate hydrogel for patients to easily swallow. XRD, SEM, and EDS analyses indicated the syntheses of brushite and alginate sphere were successful. The solubility of brushite at pH = 2 was ideal for calcium releasing and absorbing. In vitro studies and in vivo studies were both divided into biocompatibility and validity test. For in vitro studies, we followed ISO 10993 to conduct biocompatibility and cytotoxicity test with L929 cells. For validity test, human osteosarcoma cell line MG-63 served as osteoblast to evaluate the osteogenic activity. The outcome indicated that the extract of ANB could significantly increase the osteogenic activity compared with normal medium and osteogenic medium. The animal model of in vivo study was SD rat and osteoporosis was induced by ovariectomy. The rats were divided into three groups, control group, ovariectomy group, and ANB group. After rats were fed by oral gavage for 6 weeks, we measured BMD of rats and sacrificed them on the next week. Serum biochemistry and histological examination indicated that the materials used in this study had ideal biocompatibility and biotoxicity and suitable for lives to intake. BMD test, inorganic matrix assessment, and histological examination indicated that bone mass of ANB group would slightly increase compared with ovariectomy group. Serum biochemistry showed that the concentration of bone formation marker of ANB group was much higher than that in other groups, which indicated that ANB could obviously increase the rate of bone formation. If the body is treated for longer time, ANB can furtherly decrease the risk of fragility fracture.