การพัฒนายาต้นแบบจากสมุนไพรในการป้องกันโรคกระดูกพรุนในผู้สูงอายุ

The flavonoid contents of intact tubers and cell culture media were determined and physiological activities of Pueraria mirifica extracts were investigated. The total flavonoid contents from cell culture media (PMC) were higher than from tubers (PMT). Results from in vivo estrogenic activity assays indicated that PMT had a strong estrogenic activity in ovariectomized rats. The same amount of PMC exhibited a weak activity. In vitro osteoclast suppression investigations indicated that both PMT and PMC extracts exhibited anti-osteoclastogenic activities with low toxicities in a standard test cell line. Screening was conducted to identify compound(s) with antiosteoclastogenesis activity from plant derived compounds, using tartrate-resistant acid phosphatase (TRAP) staining and TRAP activity assay as indicators. Among twenty one purified compounds from medicinal plants, three compounds significantly suppressed RANKL-induced osteoclast differentiation at the concentration with low to no cytotoxicity. The three compounds were designated as ASTP10, ASTP19 and WJ10. One of these compounds, ASTP019, was isolated from Curcuma comosa Roxb. and has the structure in the group of diarylheptanoid. This compound has an inhibitory concentration 50 (IC50) of osteoclastogenesis at 1.49 M. Detailed study was conducted to investigate the mode of action of this compound. ASTP019 treatment significantly suppressed the expression of nuclear factor of activated T cells (NFATc1), a transcriptional master regulator of osteoclast differentiation, at both transcription and translation level. Furthermore, the expression of Cathepsin K, a downstream gene of NFATc1, was also down-regulated in ASTP019-treated cells, as compared to the vehicle control treatment. The effect of ASTP019 treatment was further investigated on mitogen-activated protein kinase (MAPK) and NF-B pathways which are two major downstream signaling pathways of RANK. The activation of Extracellular Signal-regulated Kinase (Erk) was specifically inhibited by this compound upon stimulation with RANK/RANKL signaling but other signaling molecules in the MAPK and NF-B were remained intact. Taken together, these results suggested that ASTP019 suppresses osteoclastogenesis via inhibition of Erk signaling pathways and this compound may be developed further as drug for prevention and treatment of osteoporosis.

The therapeutic efects of Pueraria mirfica (PM) Thai herb, which contains at least 1 7 phytoestrogens in its tuberous rots, in laboratory animals. Two series ofexperiments, in female rats and monkeys, were set. 1) Study in rat: 6 months old female SD rats were ovariectomized, kept for 4 weks to induce bone los and divded into 4 groups which were fed with 5, 25 and 50 mg/kgBW/day of PM (PM5, PM25 and PM50, respectively) and injected with 7 mg/kgBW/day of puerain (PU) for 12 weks. Bone mineral density (BMD) of tiba and fourth lumbar vertebra (L4) were measured every 2 weks. It was found that trabecular (Tb) BMDs of both tiba and L4 were signifcantly decreased starting form 2 weks after OVX (p<0.5) and only PM50 could inhibt the los at both bone sites. 2) Study in monkeys: naturaly ocuring osteoporotic, post-menopausalong-tailed monkeys were first selcted. BMDs and bone mineral contents (BMCs) of radius and tiba were compared betwen pre-menopausal, perimenopausal and post-menopausalong-tailed macaques. The BMDs and BMCs of radius and tiba signifcantly decreased in peri-menopause (p<0 . 5 ) and highly decreased in post-menopause monkeys, in corelation with a reduction of serum estradiol levels and the length of post-menopausal period. This also corelated with a decrease in plasma osteoclacin and increase in urinary NTX levels. Thus, 1 0 osteoporotic post- menopausal monkeys (bone mas lower than 2SD of those of premenopausal monkeys) were selcted, divded into 2 groups, fed with 0 and 1 0 0 mg/kgBW/day of PM (PM0 and PM100, respectively), and changes of BMDs, BMCs and bone healing were folowed up. It was found that BMDs, BMCs and bone healing on the PM1 00 group were signifcantly higher those of the PM0 group (p<0.5) starting from 6 months of the treatment. Although the treatment in monkeys has not done yet, it can preliminary conclude regarding the results in laboratory animals that PM has high potential to be developed as anti-osteoporotic drug for post- menopausal women.

Mucoadhesive thiolated chitosan suitable as a carrier for low water soluble drugs was designed and synthesized by conjugating 5-amino-2- mercaptobenzimidazole (MBI) using methylacrylate (MA) as the linking agent. A 14.4% degree of substitution of MA, as determined by 1H NMR analysis, and 11.86 ฑ 0.01 lmol thiol groups/g of polymer, as determined by Ellman’s method, was obtained. The MBI-MA-chitosan had an 11-fold stronger mucoadhesive property compared to unmodified chitosan at pH 1.2, as determined by the periodic acid: Schiff colorimetric method. Chitosan, MA-chitosan and MBI-MA-chitosan were fabricated as well-formed microspheres using electrospray ionization, including an entrapment efficiency of simvastatin (SV) of over 80% for the MBI-MA-chitosan. The mucoadhesiveness of the SV-loaded MBI-MA-CS microspheres was still higher than that for SV-loaded chitosan at pH 1.2 and 6.4.