Thesis (M.Sc. (Pharmacology))--Mahidol University, 1986.

Thesis (Ph. D.)--Harvard University, 1975. / "Annex: 'Renaissance humanism and botany, ' Annals of science 33 (1976), 519-542 [and] 'Publishing scholarly books in the sixteenth century, ' Scholarly publishing, April 1983, 259-274." Includes bibliographical references (p. 261-283) and index.

Thesis (Ph. D.)--University of California, San Diego, 2009. / Title from first page of PDF file (viewed July 9, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 369-389).

Thesis (Ph. D.)--Harvard University, 1975. / "Annex: 'Renaissance humanism and botany, ' Annals of science 33 (1976), 519-542 [and] 'Publishing scholarly books in the sixteenth century, ' Scholarly publishing, April 1983, 259-274." Includes bibliographical references (p. 261-283) and index.

Made available in DSpace on 2014-06-11T19:27:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-03-12Bitstream added on 2014-06-13T20:36:15Z : No. of bitstreams: 1 machado_ac_me_araca.pdf: 1826186 bytes, checksum: 81765d2ec1f653c7404b19dc2370a8ad (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo do presente estudo foi avaliar a compatibilidade biológica do extrato aquoso e hidroalcoólico de folhas da planta Aroeira do Sertão (Astronium urundeuva) empregando-se o teste edemogênico e implantes em subcutâneo de rato. Utilizou-se 48 ratos machos wistar com peso aproximado de 250g. Para quantificação do edema, 18 animais anestesiados, receberam injeção intravenosa de Azul de Evans a 1% (0,2 mg/kg). Após 30 minutos, foram injetados 0,1 mL do extrato e solução fisiológico na região dorsal do animal. Os ratos foram sacrificados após 3 e 6 horas. As peças obtidas foram colocadas em formamida por 72 horas em estufa a 45ºC. A leitura foi realizada em espectrofotômetro com comprimento de onda de 630 nm. Para a implantação no subcutâneo do rato (reação tardia), 30 ratos receberam implantes de tubos de polietileno contendo os extratos na região dorsal sendo posteriormente sacrificados após 7 e 28 dias para remoção das peças. As peças foram processadas, cortadas e coradas com Hematoxilina e Eosina. Os resultados foram obtidos pela leitura dos espécimes em microscópio óptico, em aumentos de 10 e 40x, para avaliação da espessura da cápsula fibrosa e quantificação do infiltrado inflamatório. Não foi observada diferença significante (p>0,05) na quantificação de edema dos diferentes grupos nos tempos operatórios, mas verificou-se haver diferença significante (p<0,05) entre as soluções analisadas, independente do tempo de estudo. A solução hidroalcoólica apresentou maior edema que a solução de aroeira aquosa e solução fisiológica. Foi apontado resultados semelhantes no período de 7 dias para os três grupos experimentais. Para o período de 28 dias, houve redução acentuada do número de células inflamatórias para a solução fisiológica e extrato aquoso / The aim of the present study was to evaluate submucous tissue response to the extract of Aroeira’s leaf employing the endemogenic analysis and implants in rats. The test groups consisted of aqueous and hydroalcoholic Aroeira’s extracts and the control group consisted of physiological saline. Forty eight male Wistar rats weighing 250g were selected. For the edema quantification, 18 animals under anesthesia received intravenous injection of 1% Evans Blue (0,2mg/Kg). After 30 minutes, the extracts (0,1ml) and the physiological saline were injected on the rats’ dorsum. The animals were killed after 3 and 6 hours. The samples were put in formamide for 72h in heater at 45°. The readings were realized in spectrophotometer with 630nm wavelength. For the submucous implantation, 30 rats received a polyethylene tube containing the extracts on their dorsum. The animals were killed after 7 and 28 days. The samples were processed for histological analysis and evaluated in optical microscope (10x and 40x original augmentation). The fiber capsule thickness was measured and the inflammatory infiltrate was quantified. The Two-way ANOVA, Mann-Whitney and Kruskall- Wallis tests were used for statistical analysis with a 95% confidence interval. There were no statistical significant differences (P>0.05) between groups in relation to edema quantification in the different periods. There were significant differences (P<0.05) between the solutions analyzed independent of the time period. The hydroalcoholic solution resulted in more edema than the aqueous and saline solutions. Similar results were found on the 7-day period for the 3 groups. On the 28-day period there was a notable reduction on inflammatory cell number for the saline and aqueous extract groups

Introdução: O Phyllanthus niruri (P. niruri) ou quebra pedra é uma planta com ação antilitogênica. No entanto, estudos clínicos nesta área ainda são escassos na literatura. O objetivo principal deste estudo foi avaliar prospectivamente os efeitos do P. niruri nos parâmetros metabólicos de pacientes com litíase urinária e, secundariamente, avaliar o impacto da ingestão do chá da planta na eliminação de cálculos urinários. Material e Métodos: Foram estudados 56 pacientes portadores de cálculos renais <10 mm. Avaliação clínica, metabólica e ultrassonográfica do trato urinário foram realizadas antes do uso do P. niruri (infusão de 500 ml/dia com 9 g do extrato seco da planta), após a administração deste por 15 semanas e, finalmente após 12 semanas sem o uso (período de \"Wash out\"). Utilizamos o teste ANOVA e o teste de Tukey para comparação entre os períodos do estudo. O nível de significância considerado foi de 5%. Resultados: Trinta e seis pacientes (64%) eram mulheres. A média de idade dos 56 pacientes foi 44,1±9,16 anos. O IMC médio foi 27,2±4,4 Kg/m2. Não se observou alteração nos parâmetros antropométricos, séricos, no volume urinário ou efeitos adversos significativos durante todo o período de estudo. Houve redução da pressão arterial diastólica de 76±10,5 para 72,5±10,5 mmHg (p=0,02), quando comparado o período de uso do chá e o período de \"Wash out\". Aumento significativo dos valores do potássio urinário de 50,5±20,4 para 56,2±21,8 mEq/vol.24h (p=0,017); da relação magnésio/creatinina de 58±22,5 para 69,1±28,6 mEq/gCr.24h (p=0,013) e da relação potássio/creatinina, de 39,3±15,1 para 51,3±34,7 mEq/gCr.24h (p=0,008) foi observado ao final do período de uso do chá quando comparado com a avaliação inicial. O número de cálculos renais por paciente reduziu de 3,21±2,02 para 2,02±2,07 cálculos (p < 0,001) após o consumo do P. niruri quando comparado com o momento inicial. Na avaliação inicial, 24 pacientes apresentaram hipercalciúria e hipocitratúria (42,8%), seis apresentaram hiperuricosúria (10,7%) e cinco hiperoxalúria (8,9%). Após o uso do P. niruri, nos pacientes portadores de hiperuricosúria houve redução de 0,77±0,22 para 0,54±0,07 mg/vol.24h (p=0,0057) no valor do ácido úrico urinário. Nos portadores de hipocitratúria, o citrato urinário aumentou de 211,8±123,7 para 322,3±145,8 mg/vol.24h (p=0,0282) e nos pacientes com hiperoxalúria houve a redução no oxalato urinário de 59,0±11,7 para 28,8±16,0 mg/vol.24h (p=0,0002). Conclusão: O consumo do P. niruri se mostrou seguro e não provocou efeitos adversos ou alterações séricas relevantes e elevou a excreção urinária de magnésio e potássio. Algumas alterações metabólicas urinárias predisponentes a formação de cálculos normalizaram em subgrupos de pacientes estudados. O consumo do P. niruri contribuiu na eliminação de cálculos urinários / Introduction: Phyllanthus niruri (P. niruri) or breake-stone is a plant commonly used to reduce stone risk, however, clinical studies on this issue are lacking. The aim of this study was to prospectively evaluate the effects of P. niruri in metabolic parameters of patients with kidney stones and secondarily to evaluate the impact of the plant intake in the elimination of urinary calculi. Material and Methods: We studied 56 patients with kidney stones < 10 mm. Clinical, metabolic, and imaging studies were performed prior to P. niruri (tea infusion of 500 ml/day with 9 g of the dried plant extract), after 15 weeks of tea administration and finally after 12 weeks without the intake plant (wash out). ANOVA test for repeated measures and Tukey test and McNemars test for categorial variables. The significance level was set at 5%. Results: Thirty-six patients (64%) were female and mean age was 44.1±9.16 years-old. The mean BMI was 27.2±4.4 Kg/m2. There was no change in anthropometric and serum parameters or urinary volume throughout the study period. There was a reduction in diastolic blood pressure from 76±10.5 during the tea use to 72.5±10.5 mmHg after the wash out (p=0.02). When the tea use period was compared to the baseline assessment, there was a significant increase in urinary potassium from 50.5±20.4 to 56.2±21.8 mg/24-hour (p=0.017), magnesium/creatinine ratio from 58±22.5 to 69.1±28.6 mg/gCr24-hour (p=0.013) and potassium/creatinine ratio from 39.3±15.1 to 51.3±34.7 mg/gCr24-hour (p=0.008). The number of kidney stones per patient decreased from 3.21±2.02 to 2.02±2.07 calculi (p < 0.001) after consumption of tea compared with the initial stage. Initial evaluation showed hypercalciuria and hypocitraturia in 24 patients (42.8%), hyperuricosuria in six (10.7%) and hyperoxaluria in five cases (8.9%). In patients with hyperuricosuria there was a decrease in the amount of urinary uric acid from 0.77±0.22 to 0.54±0.07 mg/24-hour (p=0.0057). After the use of P. niruri, in patients with hypocitraturia, urinary citrate increased from 211.8±123.7 to 322.3±145.8 mg/24-hour (p=0.0282). In patients with hyperoxaluria there was a reduction in urinary oxalate from 59.0±11.7 to 28.8±16.0 mg/24-hour (p=0.0002). Conclusion: P. niruri intake is safe and does not cause significant adverse effects or significative serum metabolic changes. The use of the tea plant increases urinary excretion of magnesium and potassium. Some urinary metabolic changes predisposing to the formation of calculi normalized in subgroups of patients studied. The consumption of P. niruri contributed to the elimination of urinary calculi

丹参是一种著名的传统中药，富含丹酚酸和丹参酮。其中，丹参酮的潜在抗肿瘤作用近年来引起众多关注。本研究评价了主要的丹参酮及其衍生物对结肠癌细胞的细胞毒性。结果显示DHTS具有最强的抗结肠癌活性和显著的肿瘤特异性细胞毒性，其细胞毒性主要由于凋亡诱导而不是引起坏死。初步的构效关系分析提示丹参酮母环结构中的A环和B环增加的离域性有助于提高其对结肠癌细胞的细胞毒性，而非二维结构及较小的D环也是进行结构改造的可能方向。 / 基于以上发现，本研究进一步探讨了DHTS的体内外抗肿瘤活性及内在机制。本研究发现DHTS的促凋亡活性并不依赖于p53的表达，而依赖于caspase活性及线粒体介导的细胞质中氧自由基 ROS及钙离子的聚集。DHTS可引起浓度及时间依赖caspase-9/-3/-7的活化而并未显著引起caspase-8的活化，这一现象发生于同样以浓度及时间依赖方式进行的线粒体中cytochrome c及AIF转位之后。在DHTS诱导的结肠癌细胞凋亡中，cytochrome c及caspase介导的凋亡通路及AIF介导的凋亡通路均被激活并显示出两条通路之间的交叉调控。 / 此外，线粒体在DHTS的促凋亡活性中的作用在本研究中被深入探讨。本研究发现线粒体可能是DHTS的一个直接靶点, 而氧化磷酸化复合体III则更可能是其作用位点。DHTS可以引起迅速而明显的线粒体功能障碍，随之引起细胞质中大量的氧自由基及钙离子聚集，诱导凋亡的产生。 / 与体外结果一致，本研究证实了DHTS对免疫缺陷小鼠中的结肠癌移植廇也具有明显的抗肿瘤作用。与溶媒对照组比较，DHTS治疗组中移植廇的增长显著被减缓，在治疗终点时的廇体积与重量也显著被降低。TUNEL检测确认DHTS诱导移植廇中癌细胞的显著凋亡。免疫荧光检测也发现DHTS诱导caspase-3及caspase-7在移植廇中癌细胞的明显活化。 / 综上所述，本研究提供了丹参酮抗结肠癌活性的一些初步构效关系的信息，为提高丹参酮抗结肠癌活性的结构改造提供一定的参考。更重要的是，本研究证明了DHTS的体内外抗结肠癌活性并探讨了其作用机制及可能靶点，为DHTS作为新的应用于抗结肠癌药物或辅助治疗用药提供了临床前研究证据。 / Salvia miltiorrhiza Bunge, also known as Danshen, rich in phenolic acid and tanshinones, has been widely used to treat various kinds of diseases including heart diseases and hepatitis in China with minimal side effects. Among the tanshinones, tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone I are the major bioactive constituents in this herb. In this study, the anti-colon cancer potential of five tanshinones and six derivatives of tanshinone IIA were evaluated in several colon cancer cell lines. It was found that apoptosis but not necrosis contributed significantly to the cytotoxicity of the tanshinones. Dihydrotanshinone I (DHTS) was confirmed to be the most potent and selective anti-cancer compound among the tanshinones tested in this study. Preliminary SAR (structure activity relationship) of tanshinones reveals that the increase of delocalizability of A and B rings in the chemical structure of the tanshinones enhances their cytotoxicity on cancer cells, while compounds with a non-planar and small sized D ring region are better choices for anti-cancer effect. / The underlying mechanisms of the anti-colon cancer activity of DHTS were further studied. It was found that apoptosis induced by DHTS was p53 independent but caspase dependent, which was closely related to intracellular accumulation of ROS (reactive oxidant stress) and calcium mediated by mitochondria. A concentration- and time-dependent activation of caspase-9,-3,-7 but not caspase-8 by DHTS in HCT116 cells was detected after the translocation of cytochrome c and AIF (apoptosis inducing factor) from mitochondria. In this process, the crosstalk between the caspase-dependent and caspase-independent pathways was firstly shown in the apoptotic mechanism of DHTS. To this end, the release of cytochrome c happened first and the translocation of apoptosis inducing factor (AIF) was prevented by a pan caspase inhibitor. In the meantime, the release of cytochrome c and activation of caspase-9 and PARP (poly-ADP-ribose polymerase) cleavage were decreased after AIF knockdown. Especially, mitochondrion was suggested to be the direct target of DHTS and OXPHOS complex III but not OXPHOS complex I was probably the acting site of DHTS. / In accordance with the results obtained in vitro, the potential anti-colon cancer activity of DHTS was also observed in nude mice with xenograft tumors and the compound did not produce any observable systemic toxicity. DHTS efficiently delayed tumor growth by decreasing the tumor size and weight through the induction of apoptosis in cancer cells but not by inhibition of cell proliferation. In the same tissues, a distinct activation of caspase-3 and caspase-7 in tumor cells was also detected by immunofluorescence assay. / Collectively, the present study provides preliminary information about the SAR of the anti-colon cancer activity for tanshinones. It also confirms that DHTS is a promising compound for anti-cancer action both in vitro and in vivo. In addition, this study gives us a better understanding regarding the mechanisms of how DHTS induces apoptosis in cancer cells. All these findings could provide solid pre-clinical evidence to propel the development and application of DHTS and perhaps its derivatives as novel therapeutic or adjuvant agents for the treatment of colon cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Lin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 122-132). / Abstracts also in Chinese. / Wang, Lin.

A method is presented for the simultaneous identification of nine compounds in samples of A. membranaceus and A. membranaceus var. mongholicus. Compounds identified in the extracts of the two plants included glycosides, saponins and flavonoids. They are identified as Calycosin-7-O-beta-D-glucoside (C1), Ononin (C2), (6aR,11aR)-3-hydroxy- 9,10-dimethoxypterocarpan-3-O-beta-D-glucoside (C3), (3R)-7,2'-dihydr- oxy-3',4'-dimethoxyisoflavan-7-O-beta-D-glucoside (C4), Calycosin (C6), Astragaloside IV (C5 ), Formononetin (C7), (6aR,11a R)-3-hydroxy-9,10-di-methoxypterocarpan (C8), and Isomucronulatol (C9). / An HPLC-DAD-MS method is proposed for the differentiation of Rhizoma Coptidis and Cortex Phellodendri samples. This method can also be used to identify two common species of Rhizome Coptidis, i.e., C. chinensis and C. deltoidea, and two species of Cortex Phellodendri, i.e., P. chinensis and P. amurense. From the experiment results, there are thirteen, twelve and seven common components found in samples Rhizoma Coptidis, P. amurense and P. chinensis, respectively. Nine compounds in Rhizoma Coptidis were identified to be alkaloids. The common components in Cortex Phellodendri included four alkaloids and two lactones, i.e., obaculacotone and obacunone, present in all samples of P. amurense. / High Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass-Spectrometry has been applied to the analysis and standardization of Chinese Herbal Medicines. The applications included quantitative study of Astragaloside in Radix Astragali, investigation on the chromatographic fingerprint of Radix Astragali, differentiation of Cortex Phellodendri and Rhizoma Coptidis, and identification of constituents in Sanhuang Xiexin Decoction. / In the quantitative study of Astragaloside, an Multiple Reaction Monitoring scan mode was used. The linearity between 2 and 500 mg/L is 0.9996. The precision of injection and reproducibility of method is 1.72% and 3.27% respectively. A total of 20 samples from local market and mainland China were analyzed and the results are comparable to those obtained from HPLC-ELSD analysis. / The present study also proposed a HPLC separation and online identification for the 15 constituents in a composite Chinese herbal formula, the Sanhuang Xiexin Decoction. It provided a possible starting point to evaluate related herbal preparations containing Rhizoma Coptidis, Radix Scutellariae and Rhizoma Rhei. Thirteen constituents in the decoction were identified, including five major alkaloids from Rhizoma Coptidis, five anthraquinones from Rhizoma Rhei and two favonoids and one glycoside from Radix Scutellariae. / "November 2007." / Adviser: Chi Chun Tao. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4726. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 177-200). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

病原體感染是包括植物的高等生物的主要健康危害之一。為抵禦入侵者，大多數植物會製造防禦蛋白，包括凝集素、蛋白酶抑製劑、抗真菌蛋白、核糖核酸酶和核糖體失活蛋白，並分佈在不同的器官，如葉、根、種子和塊莖。一些植物防禦蛋白被發現能表現出多種生物活性，如抗腫瘤活性、抗細菌活性和抗病毒活性，能抵抗多種植物病原菌和人類病原體。因此，一些植物防禦蛋白可能有潛力用於治療人類疾病，或保護農作物免受感染。 / 我們在研究中從不同的植物來源成功純化出各種防禦蛋白，包括：小芋頭塊莖中的血凝素、日本長芋中的凝集素、東北紅豆中的血凝素和抗真菌多肽、棕色芸豆中的凝集素、抗真菌多肽和胰蛋白酶抑製劑，玉豆一號中的凝集素以及小斑豆中的胰蛋白酶抑製劑。小芋頭血凝素被發現能誘導脾細胞的有絲分裂反應。日本長芋凝集素和東北紅豆血凝素被發現能對一些腫瘤細胞株（如乳腺癌MCF7細胞及鼻咽癌CNE2細胞）發揮抗增殖的作用。棕色芸豆凝集素能誘導脾臟細胞的有絲分裂反應以及抑制腫瘤細胞株（如乳腺癌MCF7細胞、肝癌HepG2及鼻咽癌CNE1和 CNE2細胞）的生長，而棕色芸豆抗真菌蛋白能抑制數種病原真菌物種的生長。研究這些防禦蛋白的生物活性有助找出其潛在應用價值，如藥用前景。 / Infection from pathogens is one of the major health hazards in higher organisms including plants. To defend against harmful invaders, most plants produce a variety of defense proteins including lectins, protease inhibitors, antifungal proteins, ribonucleases and ribosome-inactivating proteins. They may be present in different organs of the plants, such as leaves, roots, seeds and tubers. Some of the plant defense proteins were found to exhibit a variety of biological activities such as anti-tumor activity, anti-bacterial activity and anti-viral activity that act against various plant pathogens and also some human pathogens. Therefore, some plant defense proteins may have potential for therapeutic applications in human diseases, or protecting the crops from infections. / This study involved purification of defense proteins from different plant sources. The proteins that were successfully isolated included a hemagglutinin from small taro tubers, a lectin from Japanese yam tubers, a lectin and an antifungal peptide from northeast red beans, a lectin, an antifungal peptide and a trypsin inhibitor from brown kidney beans, a lectin from French bean cultivar no. 1 and a trypsin inhibitor from mini pinto beans. The small taro hemagglutinin was found to induce mitogenic response in splenocytes. The Japanese yam lectin and northeast red bean hemagglutinin were found to exert anti-proliferative activity toward some tumor cell lines including MCF7 and CNE2 cells. The brown kidney bean lectin induced a mitogenic response from murine splenocytes as well as inhibited the growth of tumor cell lines including MCF7, HepG2, CNE1 and CNE2 cells, while the brown kidney bean antifungal protein inhibited the growth of several pathogenic fungal species including M. arachidicola, S. turcica and B. maydis. Studying the biological activities of these defense proteins helps to find out their potential applications like therapeutic uses. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chan, Yau Sang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves i-xvii). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i-ii / 論文摘要 --- p.iii / Acknowledgements --- p.iv / List of Publications --- p.v / Table of Contents --- p.vi-vii / List of Figures --- p.viii-ix / List of Tables --- p.x / List of Abbreviations --- p.xi / Chapter Chapter 1 --- Introduction on plant defense proteins / Chapter 1.1 --- General introduction to plant defense proteins --- p.1-2 / Chapter 1.2 --- An overview on lectins --- p.3-18 / Chapter 1.2.1 --- History of lectins --- p.3-6 / Chapter 1.2.2 --- Classification of lectins --- p.7-11 / Chapter 1.2.3 --- Biological activities of lectins --- p.12-16 / Chapter 1.2.4 --- Applications of plant lectins --- p.16-18 / Chapter 1.3 --- An overview on defensins --- p.18-25 / Chapter 1.3.1 --- Types of defensins --- p.18-21 / Chapter 1.3.2 --- Mechanism of anti-microbial activity of defensins --- p.22-23 / Chapter 1.3.3 --- Application of defensins --- p.23-25 / Chapter 1.4 --- An overview on trypsin inhibitors --- p.25-38 / Chapter 1.4.1 --- Serpins --- p.26-28 / Chapter 1.4.2 --- Kunitz-type protease inhibitors --- p.29-31 / Chapter 1.4.3 --- Bowman-Birk protease inhibitors --- p.32-34 / Chapter 1.4.4 --- Physiological functions of protease inhibitors --- p.35-38 / Chapter 1.5 --- Aim of study --- p.38-41 / Chapter Chapter 2 --- Isolation and characterization of a hemagglutinin from small taros and a lectin from yam tubers / Chapter 2.1 --- Introduction --- p.42-45 / Chapter 2.2 --- Materials and Methods --- p.46-55 / Chapter 2.3 --- Results --- p.56-78 / Chapter 2.4 --- Discussion --- p.79-84 / Chapter Chapter 3 --- Isolation and characterization of two defense proteins from seeds of Phaseolus vulgaris cv. “northeast red bean“ / Chapter 3.1 --- Introduction --- p.85-86 / Chapter 3.2 --- Materials and Methods --- p.87-93 / Chapter 3.3 --- Results --- p.93-119 / Chapter 3.4 --- Discussion --- p.120-129 / Chapter Chapter 4 --- Isolation and characterization of three defense proteins from seeds of Phaseolus vulgaris cv. “brown kidney bean“ / Chapter 4.1 --- Introduction --- p.130-131 / Chapter 4.2 --- Materials and Methods --- p.131-136 / Chapter 4.3 --- Results --- p.136-175 / Chapter 4.4 --- Discussion --- p.176-189 / Chapter Chapter 5 --- Isolation and characterization of a lectin from French bean cultivar no. 1 beans and a trypsin inhibitor from mini pinto beans / Chapter 5.1 --- Introduction --- p.190-191 / Chapter 5.2 --- Materials and Methods --- p.191-194 / Chapter 5.3 --- Results --- p.195-212 / Chapter 5.4 --- Discussion --- p.213-221 / Chapter Chapter 6 --- General discussion / Chapter 6.1 --- Summary on purification protocols of the defense proteins in the study --- p.222-228 / Chapter 6.2 --- Chemical properties of the defense proteins in the study --- p.228-232 / Chapter 6.3 --- Biological activities of the defense proteins in the study --- p.232-238 / Chapter 6.4 --- Potential application of these defense proteins and future perspectives --- p.238-242 / References --- p.i-xvi

傳統中醫中藥理論遵從"腎主骨"概念。因此，中醫在治療與骨有關的疾病時一般都處方"補腎"類中藥。 / ELP是一例中藥草本 "補腎" 複方。其包含三種中藥，包括淫羊藿（E）、女貞子（L）和補骨脂（P）。動物體內實驗和臨床研究已證明ELP有效治療絶經後骨質疏鬆症。可是，經口服吸收後的血清中的ELP有效物質對細胞的成骨影響從未進行過相關研究。ELP對預防在缺乏體力活動下所引起的骨質疏鬆症的療效也屬未知。此外，基於其"補腎"的特性，ELP可能潛在著能促進骨折癒合的功能。本研究的目的包括研究血清中ELP的有效物質在細胞和分子水平上的護骨能力，並測試其對預防於失重狀態下引起的骨質疏鬆症（慢性骨紊亂）的效能。本研究還旨在考察 ELP在促進骨癒合 （急性骨紊亂）上的作用。本研究分為三部分。 / 第一部分 -- 骨代謝的體外研究:健康大鼠分別口服草本配方ELP、EL、及單味中草藥提取物E或L、並以蒸餾水作為對照（H2O），口服給藥二小時後收集其血清作體外血清藥理學研究。分別考察含藥血清對各細胞系包括UMR106、RAW264.7、和從大鼠骨中分離出的骨髓間充質幹細胞（MSC）的增殖和分化屬性的影響，並以液質聯用技術（LC-MS）來分析血清內所含中藥的化學成份。 / 第二部分 -- 骨質疏鬆症的體內研究:以尾吊雄性大鼠作為卸荷狀態骨質疏鬆症的動物模型。在不同的給藥組中，大鼠口服高中低三種劑量的ELP（ELP-H、ELP-M和ELP-L），或三個不同抗骨質疏鬆藥物，包括雷洛昔芬（Ral），阿侖膦酸鈉（Aln）和雷奈酸鍶（Strn）作為陽性對照組，並以蒸餾水為安慰劑對照（TS）。另一組大鼠則沒有尾吊，作為正常對照（Non-TS）。本部分分析在吊尾期間大鼠體內生化指標和骨密度（BMD）的變化，及其後各組在骨小梁微結構和骨骼生物力學上的差異。 / 第三部分 -- 骨缺損癒合的體內研究:兩個鑽孔性骨缺損模型分別建立於老年雌性大鼠的左股骨骨幹和右脛骨近端骺端。其後動物分成4組：（1）ELP 口服給藥（ELP）；（2）CDNR外敷治療（CDNR為另一中藥複方，包含紅花（C）、續斷（D）、三七（N）和大黃（R））；（3）ELP口服給藥結合CDNR外敷治療（ELP+CDNR）；（4）和蒸餾水餵養（Control）。通過監測骨缺損癒合的過程、檢測大鼠血液中生化標誌物的變化、骨骼生物力學測試和形態計量學分析，考察ELP及其與CDNR在骨缺損癒合上的協同作用。 / 第一部分的結果顯示，口服給藥二小時後，大鼠血清中淫羊藿的標記化合物淫羊藿苷（icariin）無被檢出。在EL或E的給藥大鼠血清中，檢出淫羊藿苷的其中一個代謝產物icariside I；而其另一個代謝產物icariside II，則在ELP的給藥大鼠血清中檢測到。L和P的常見標記化合物則能從相應餵飼L和P的大鼠血清中檢出。體外血清藥理學研究結果表明含藥（ELP）大鼠血清對細胞無毒性作用，且能促進 UMR106 細胞增殖和上調其Runx2 基因表達。然而，含藥血清無增加UMR106細胞的鹼性磷酸酶活性和鈣沉積。它抑制 RAW264.7細胞的分化及其基質金屬蛋白酶9（MMP-9）和組織蛋白酶 K的基因表達。它亦能促進MSC細胞的增殖，增強其鹼性磷酸酶活性和Runx2與ALP基因的表達。 / 第二部分的結果指出ELP-H能減少吊尾大鼠股骨遠端及腰椎骨密度的百分比損失，抵抗股骨遠端骨小梁微結構惡化和加強股骨骨幹骨缺損部位的生物力學特性。此外，ELP-H還能降低血液骨鈣素和抗酒石酸酸性磷酸酶5b（TRAP5b）的濃度。研究亦發現ELP對骨密度、結構參數和生化指標的影響存在劑量依賴性。整體上而言，ELP在預防卸荷骨質疏鬆症的影響類似於Ral和Aln，而非Strn。 / 第三部分的結果表明，從顯微電腦掃描或形態計量學上分析，所有實驗組跟對照組間均沒有顯著性差異。但值得注意的是，ELP+CDNR大大提高了股骨骨幹骨缺損在癒合過程中的歸一化生物力學屬性。而ELP單獨用藥則減少了TRAP5b的濃度。 / 總之，這項研究結論出血清藥理學研究加上LC-MS的應用能作為找出中藥中有效成分的有效途徑。本研究還展示ELP的含藥血清對骨細胞有護骨作用。ELP可防預在卸荷狀態下形成的骨質疏鬆症，它還有助於提升外敷中藥複方CDNR在骨缺損癒合過程中的療效。從這項研究的三個部分中歸納出的共同點說明，儘管ELP擁有刺激成骨的能力，它的護骨作用主要是透過它的抗骨吸收效果。ELP在慢性（防止骨質疏鬆症）和急性（促進骨癒合）骨紊亂上均有療效。 / Traditional Chinese Medicine (TCM) claims that bone health lies in the functioning of the "Kidneys". When the "Kidney" is strong, our body can stimulate growth and transformation of the bone marrow, which nourishes and strengthens the skeleton. Therefore, "Kindey-tonifying" herbs are usually used to cure bone diseases. / ELP is a "Kidney-tonifying" Chinese herbal formula containing three Chinese herbs including Herba Epimedii (E), Fructus Ligustri Lucidi (L) and Fructus Psoraleae (P). It has been proven effective to treat postmenopausal osteoporosis through in vivo and clinical studies. However, ELP is for oral administration. The osteogenic properties of its post-absorption metabolites have never been studied. The efficacy of ELP on prevention of osteoporosis development due to physical inactivity is also unknown. With its "Kindey-tonifying" property, ELP is also considered as a potential agent to facilitate fracture healing. / The aims of this study included to investigate the osteoprotective effects of ELP metabolites at cellular and molecular levels and to prove the efficacy of ELP on prevention of osteoporosis development in unloading condition - a chronic bone disorder. It also aimed to study the effect of ELP on promotion of bone defect healing - an acute bone disorder. This study was divided into three parts. / Part 1 - in vitro study of bone metabolism: Healthy rats were fed with herbal formula ELP or EL, single herbal extracts of E or L or distilled water as control (H₂O). Sera were then collected for in vitro seropharmacological study. Cell lines including UMR106 and RAW264.7, as well as mesenchymal stem cell (MSC) isolated from rats, were cultured with the sera. Their proliferation and differentiation properties of the cells were analyzed. In addition, the chemical profiles of the herbal extracts within the sera were analyzed using liquid chromatography-mass spectrometry (LC-MS). / Part 2 - in vivo study of osteoporosis: Tail-suspension male rats were used as the unloading osteoporotic animal model. The rats in different groups were fed with three different doses of ELP (ELP-H, ELP-M and ELP-L), or three different anti-osteoporosis drugs including raloxifene (Ral), alendronate (Aln) and strontium ranelate (Strn) as positive controls or distilled water as placebo control (TS). One group of rats was non-tail-suspended as normal control (Non-TS). Changes in bone mineral density (BMD), microarchitecture of trabeculae and biomechanical properties of the bone of the rats were analyzed. Changes in biochemical markers within the tail-suspension period were also studied. / Part 3 - in vivo study of bone defect healing: two drilled-hole bone defects were created in the diaphysis of left femur and proximal metaphysis of right tibia, respectively, of aged female rats. Animals were divided into 4 groups: (1) administered with ELP orally (ELP); (2) treated with another herbal formula CDNR containing Carthami Flos (C), Dipsaci Radix (D), Notoginseng Rhizoma (N) and Rhei Rhizoma (R) topically (CDNR); (3) treated with oral ELP and topical CDNR at the same time (ELP+CDNR); and (4) fed with distilled water (Control). The effects of ELP and the synergistic effects of ELP+CDNR on facilitation of the bone defect healing were monitored in vivo using viva-CT and through measurement of biochemical markers biweekly. After euthanasia of the rats, the bones were harvested for biomechanical test and histomorphometrical analysis. / Results: Part 1 revealed that the common marker compound, icariin, had not been detected in the sera of all the rats. Instead, one of the metabolites of E, icariside I, was found in the sera of the rats fed with EL or E, while another metabolite, icariside II, was detected in the serum of the rats fed with ELP. Common marker compounds of L and P were observed in the sera of the rats fed with the herbal items accordingly. The in vitro studies in this Part showed that there was no cytotoxic effect of the rat sera on the cells. The post-absorbed ELP metabolites in rat serum promoted UMR106 proliferation by 25.7%, (p < 0.05) and upregulated the Runx2 gene expression by 1.18 fold (p < 0.05) after cultured for 2 and 3 days, respectively. However, they could not increase the ALP activity and calcium deposition of UMR106. They also inhibited RAW264.7 differentiation by 29.2 % (p < 0.05) and downregulated the MMP9 and Cathepsin K gene expression of RAW264.7 by 0.46 (p < 0.05) and 0.36 (p < 0.01) fold, respectively. The ELP metabolites promoted the proliferation of MSC by 14.4 % (p < 0.001) and resulted in 42.6 % higher ALP activity than the control serum (p < 0.05). They also upregulated the Runx2 and ALP gene expression at both Day 4 and Day 7 of culture significantly. / Part 2 showed that compared with the tail-suspension control (TS), ELP in high dose (ELP-H) reduced the percentage loss of total and trabecular BMD by 5.46 and 8.52 %, respectively (p < 0.05 both) in distal femur, and by 4.67 % (p < 0.05) in trabecular region of lumbar spine of the tail-suspended rats. Analysis from micro-CT showed that microarchitectural parameters BV/TV, Tb.Th and TV density of the distal femur of ELP-H were 17.62, 11.90 and 8.09 % higher than those of the TS (p < 0.05, for all). 3-point bending test on mid-shaft femur of the rats revealed that the yield load, ultimate load and stiffness of the drill-defect of ELP-H were higher than those of TS significantly. All of the biochemical markers decreased significantly from baseline (Day 0) to Day 28 in ELP-H. In addition, osteocalcin and TRAP5b concentrations of ELP-H were lower than those of TS significantly at Day 28. The effect of ELP on BMD, microarchitectural parameters and biochemical markers were in dose-dependent manner. In general, the osteoprotective effect of ELP-H on unloading bone was similar to Ral and Aln, but not Strn. / Part 3 indicated no significant difference in BV/TV and BMD among all groups at each time point. Histomorphometrical analysis from fluorescent labeling and Goldner’s trichrome staining showed no statistical difference in new bone formation between the Control and other treatment groups. Notably, the normalized yield load, ultimate load and failure of ELP+CDNR were significantly higher than those of Control by 20.38 % (p < 0.05), 23.17 % (p< 0.001) and 25.55 % (p< 0.001), respectively. Analysis on the change of biochemical markers showed that the bone formation marker BALP increased while bone resorption markers Dpd and TRAP5b decreased within the 42-day monitoring period. BALP activity of both Control and ELP increased significantly but only ELP reduced the TRAP5b concentrations starting from Day 14 post-op. There was no statistical difference when the concentrations of the biochemical markers were compared horizontally among the 4 groups at the same time point. / In conclusion, the current study demonstrated that seropharmacological study incorporating with the application of LC-MS can be a potential efficient approach to find out active ingredients of medicine herbs. Post-absorbed metabolites of ELP also showed their osteoprotective effects on bone cells. Aqueous extract of ELP could prevent the development of osteoporosis in unloading condition and such effect was dose-dependent. It also helped elevating the efficacy of a topical applied herbal formula CDNR on improving the bone strength of healing bone defects. A common finding from the 3 parts of this study illustrated that the osteoprotective effect of ELP was mainly achieved by its anti-resorptive efficacy on bone, although it possess an ability to stimulate osteoblastogenesis. ELP was found effective for both chronic (prevent osteoporosis development) and acute (facilitate bone healing) bone disorders. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Siu, Wing Sum. / "November 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 201-227). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.vi / ACKNOWLEDGEMENTS --- p.ix / TABLE OF CONTENTS --- p.xi / LIST OF FIGURES --- p.xvii / LIST OF TABLES --- p.xxiii / PUBLICATIONS --- p.xxiv / ABBREVIATION --- p.xxv / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter 1.1 --- TRADITIONAL CHINESE MEDICINE (TCM) AND BONE DISEASES --- p.1 / Chapter 1.2 --- CELLULAR AND MOLECULAR MECHANISMS ON BONE METABOLISM --- p.2 / Chapter 1.2.1 --- Bone formation by osteoblast --- p.3 / Chapter 1.2.2 --- Bone resorption by osteoclasts --- p.4 / Chapter 1.3 --- OSTEOPOROSIS --- p.5 / Chapter 1.3.1 --- Postmenopausal osteoporosis --- p.6 / Chapter 1.3.2 --- Disuse osteoporosis --- p.8 / Chapter 1.3.3 --- Basic principle of TCM on osteoporosis --- p.10 / Chapter 1.3.4 --- Common Chinese herbal medicine reported to have anti-osteoporotic effects --- p.11 / Chapter 1.4 --- BONE FRACTURE --- p.11 / Chapter 1.4.1 --- Biology and repair of bone fracture --- p.12 / Chapter 1.4.2 --- TCM on promotion of fracture healing --- p.13 / Chapter 1.4.3 --- Theories of TCM on fracture healing --- p.15 / Chapter CHAPTER 2: --- OSTEOPOROSIS AND HERBS --- p.16 / Chapter 2.1 --- CHINESE HERBAL MEDICINE SELECTED IN THIS PART --- p.16 / Chapter 2.2 --- DESIGN OF STUDY --- p.19 / Chapter 2.3 --- HYPOTHESES AND OBJECTIVES --- p.19 / Chapter 2.4 --- BACKGROUND OF THE STUDY --- p.23 / Chapter 2.4.1 --- In vitro study of ELP on bone cells --- p.23 / Chapter 2.4.2 --- In vivo study of ELP on postmenopausal osteoporosis --- p.23 / Chapter 2.4.3 --- Clinical study of ELP on postmenopausal osteoporosis --- p.24 / Chapter CHAPTER 3: --- PART 1 IN VITRO SEROPHARMACOLOGICAL STUDY ON OSTEOPOROSIS --- p.26 / Chapter 3.1 --- OBJECTIVES --- p.26 / Chapter 3.2 --- SEROPHARMACOLOGICAL APPROACH TO STUDY ELP --- p.26 / Chapter 3.3 --- TYPES OF CELLS INVOLVED IN THE CURRENT STUDY --- p.27 / Chapter 3.3.1 --- UMR106 --- p.28 / Chapter 3.3.2 --- RAW264.7 --- p.28 / Chapter 3.3.3 --- Mesenchymal stem cell (MSC) --- p.28 / Chapter 3.4 --- IN VITRO ASSESSMENTS ON BONE METABOLISM --- p.29 / Chapter 3.4.1 --- Bone formation --- p.29 / Chapter 3.4.1.1 --- 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay --- p.29 / Chapter 3.4.1.2 --- Bromodeoxyuridine (BrdU) assay --- p.30 / Chapter 3.4.1.3 --- Total alkaline phosphatase (ALP) activity measurement --- p.30 / Chapter 3.4.1.4 --- Calcium deposition analysis --- p.30 / Chapter 3.4.2 --- Bone degradation --- p.31 / Chapter 3.4.2.1 --- Tartrate-resistant acid phosphatase (TRAP) staining --- p.31 / Chapter 3.4.3 --- Phenotypic markers of cells involved in bone remodeling using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) --- p.31 / Chapter 3.5 --- MATERIAL AND METHODS --- p.37 / Chapter 3.5.1 --- Preparation of herbal extracts --- p.37 / Chapter 3.5.2 --- Serum preparation for seropharmacological study --- p.38 / Chapter 3.5.2.1 --- Administration of herbal extracts and blood collection --- p.38 / Chapter 3.5.2.2 --- Serum preparation --- p.38 / Chapter 3.5.3 --- Analysis of marker compounds in serum using liquid chromatographymass spectrometry (LC-MS) --- p.39 / Chapter 3.5.3.1 --- Serum preparation --- p.39 / Chapter 3.5.3.2 --- Operation of LC-MS --- p.39 / Chapter 3.5.4 --- Isolation and characterization of MSC from bone marrow --- p.40 / Chapter 3.5.5 --- Cell culture --- p.42 / Chapter 3.5.5.1 --- General materials --- p.42 / Chapter 3.5.5.2 --- UMR106 --- p.43 / Chapter 3.5.5.3 --- RAW264.7 --- p.44 / Chapter 3.5.5.4 --- Bone Marrow MSC --- p.45 / Chapter 3.5.6 --- Assays analyzing the responses of cells on the effect of metabolites of herbs in serum --- p.46 / Chapter 3.5.6.1 --- General materials --- p.46 / Chapter 3.5.6.2 --- Assays for bone formation --- p.50 / Chapter 3.5.6.3 --- Assays for bone degradation --- p.55 / Chapter 3.5.7 --- Statistical analysis --- p.56 / Chapter 3.6 --- RESULTS --- p.57 / Chapter 3.6.1 --- Chemical characterization of ELP extract --- p.57 / Chapter 3.6.2 --- Marker compounds found in rat serum using LC-MS --- p.58 / Chapter 3.6.3 --- Effects of herbal metabolites on UMR106 --- p.61 / Chapter 3.6.3.1 --- Effect on cell viability --- p.61 / Chapter 3.6.3.2 --- Effects on cell proliferation and differentiation --- p.61 / Chapter 3.6.3.3 --- Regulation on osteogenesis through gene expression --- p.63 / Chapter 3.6.4 --- Effects of herbal metabolites on RAW264.7 --- p.67 / Chapter 3.6.4.1 --- Effect on cell viability --- p.67 / Chapter 3.6.4.2 --- Inhibitory effect on RAW264.7 --- p.67 / Chapter 3.6.4.3 --- Regulation on osteoclastogenesis through gene expression --- p.67 / Chapter 3.6.5 --- Effects of herbal metabolites on bone marrow mesenchyma stem cell (MSC) --- p.70 / Chapter 3.6.5.1 --- Confirmation of MSC isolated from bone marrow of rat using flow cytometry --- p.70 / Chapter 3.6.5.2 --- Effect on cell viability --- p.70 / Chapter 3.6.5.3 --- Effects on cell proliferation and differentiation --- p.71 / Chapter 3.6.5.4 --- Regulation on osteogenesis through gene expression --- p.71 / Chapter 3.7 --- DISCUSSION --- p.75 / Chapter CHAPTER 4: --- PART 2 IN VIVO STUDY ON DISUSE OSTEOPOROSIS . --- p.83 / Chapter 4.1 --- OBJECTIVES --- p.83 / Chapter 4.2 --- POTENTIAL EFFECT OF ELP ON DISUSE OSTEOPOROSIS --- p.83 / Chapter 4.3 --- ANIMAL MODELS FOR OSTEOPOROSIS STUDY --- p.84 / Chapter 4.3.1 --- Conventional ovariectomized animal model for the studies of osteoporosis --- p.85 / Chapter 4.3.2 --- Animal models for study of disuse osteoporosis --- p.85 / Chapter 4.3.2.1 --- Bandaging or casting --- p.86 / Chapter 4.3.2.2 --- Tail-suspension (TS) --- p.86 / Chapter 4.4 --- ASSESSMENTS ON DISUSE OSTEOPOROSIS DEVELOPMENT --- p.87 / Chapter 4.4.1 --- Bone mineral density (BMD) measurement --- p.87 / Chapter 4.4.2 --- Micro-architecture analysis --- p.87 / Chapter 4.4.3 --- Bone strength assessment --- p.88 / Chapter 4.4.4 --- Bone turnover monitoring by measuring biochemical markers --- p.89 / Chapter 4.4.4.1 --- Bone formation markers --- p.89 / Chapter 4.4.4.2 --- Bone resorption markers --- p.91 / Chapter 4.5 --- MATERIAL AND METHODS --- p.95 / Chapter 4.5.1 --- Preparation of herbal extracts --- p.95 / Chapter 4.5.2 --- Tail-suspension rat model --- p.95 / Chapter 4.5.3 --- Animal arrangement and grouping --- p.97 / Chapter 4.5.4 --- Administration of herbal extracts and drugs --- p.97 / Chapter 4.5.5 --- Assessments on disuse osteoporosis development --- p.98 / Chapter 4.5.5.1 --- Bone mineral density measurement using Peripheral Quantitative Computed Tomography (pQCT) --- p.98 / Chapter 4.5.5.2 --- Bone micro-architecture analysis using Micro-computed Tomography (μCT) --- p.99 / Chapter 4.5.5.3 --- Bone strength assessment through biomechanical bending test --- p.100 / Chapter 4.5.5.4 --- Bone turnover monitoring by measuring biochemical markers --- p.100 / Chapter 4.5.5.4.1 --- Serum collection --- p.100 / Chapter 4.5.5.4.2 --- Measurements of biochemical markers --- p.101 / Chapter 4.5.6 --- Statistical analysis --- p.105 / Chapter 4.6 --- RESULTS --- p.106 / Chapter 4.6.1 --- Effects of ELP on bone mineral density (BMD) --- p.106 / Chapter 4.6.2 --- Effects of ELP on bone micro-architecture --- p.118 / Chapter 4.6.3 --- Effects of ELP on biomechanics of bone --- p.122 / Chapter 4.6.4 --- Effects of ELP on bone turnover --- p.125 / Chapter 4.7 --- DISCUSSION --- p.132 / Chapter CHAPTER 5: --- PART 3 IN VIVO STUDY ON BONE DEFECT HEALING --- p.140 / Chapter 5.1 --- HERBAL ITEMS SELECTED IN THIS PART --- p.140 / Chapter 5.2 --- DESIGN OF STUDY --- p.143 / Chapter 5.3 --- HYPOTHESES AND OBJECTIVES --- p.144 / Chapter 5.4 --- SPECIFIC STRATEGY ON PROMOTION OF FRACTURE HEALING OF TCM --- p.144 / Chapter 5.5 --- POTENTIAL EFFECT OF ELP ON BONE HEALING --- p.144 / Chapter 5.6 --- ANIMAL MODELS --- p.146 / Chapter 5.6.1 --- Bone fracture model --- p.147 / Chapter 5.6.2 --- Drill-hole bone defect model --- p.147 / Chapter 5.7 --- ASSESSMENTS ON BONE HEALING --- p.149 / Chapter 5.7.1 --- Micro-architecture analysis --- p.149 / Chapter 5.7.2 --- Bone strength assessment --- p.150 / Chapter 5.7.3 --- Bone turnover monitoring by measuring biochemical markers --- p.151 / Chapter 5.7.4 --- Histomorphometry --- p.151 / Chapter 5.8 --- MATERIALS AND METHODS --- p.153 / Chapter 5.8.1 --- Preparation of herbal extracts --- p.153 / Chapter 5.8.1.1 --- ELP --- p.153 / Chapter 5.8.1.2 --- CDNR --- p.153 / Chapter 5.8.2 --- Production of drill-hole bone defect --- p.154 / Chapter 5.8.2.1 --- Femur --- p.155 / Chapter 5.8.2.2 --- Tibia --- p.155 / Chapter 5.8.2.3 --- Animal arrangement and grouping --- p.157 / Chapter 5.8.3 --- Herbal formulae administration and application --- p.157 / Chapter 5.8.3.1 --- Oral administration --- p.157 / Chapter 5.8.3.2 --- Topical application --- p.157 / Chapter 5.8.4 --- Assessments on bone healing --- p.158 / Chapter 5.8.4.1 --- Bone micro-architecture and bone density measurement using in vivo micro-computed tomography (vivaCT) --- p.158 / Chapter 5.8.4.2 --- Bone strength assessment through biomechanical bending test --- p.159 / Chapter 5.8.4.3 --- Bone turnover monitoring by measuring biochemical markers --- p.160 / Chapter 5.8.4.4 --- Histomorphometry --- p.160 / Chapter 5.8.4.4.1 --- Fluorochrome double labeling --- p.160 / Chapter 5.8.4.4.2 --- Tissue processing and sectioning --- p.161 / Chapter 5.8.4.4.3 --- Staining of sections --- p.162 / Chapter 5.8.4.4.4 --- Image analysis --- p.164 / Chapter 5.8.5 --- Statistical analysis --- p.165 / Chapter 5.9 --- RESULTS --- p.166 / Chapter 5.9.1 --- Effect of ELP and CDNR on bone micro-architecture --- p.and / Chapter bone --- density at the bone defect site --- p.166 / Chapter 5.9.2 --- Histomorphometrical findings in treatment of bone healing --- p.172 / Chapter 5.9.3 --- Effect of ELP and CDNR on biomechanics of bone --- p.175 / Chapter 5.9.4 --- Effect of ELP and CDNR on bone turnover --- p.178 / Chapter 5.10 --- DISCUSSION --- p.184 / Chapter CHAPTER 6: --- GENERAL DISCUSSION AND CONCLUSION --- p.193 / Chapter 6.1 --- UNKNOWN AREAS FOR THE STUDY OF ELP --- p.193 / Chapter 6.2 --- SUMMARY OF CRUCIAL FINDINGS OF THE OSTEOGENIC EFFECTS OF ELP IN EACH PART OF THIS STUDY --- p.194 / Chapter 6.2.1 --- Part 1: in vitro seropharmacological study on osteoporosis --- p.194 / Chapter 6.2.2 --- Part 2: in vivo study on disuse osteoporosis --- p.195 / Chapter 6.2.3 --- Part 3: in vivo study on bone healing --- p.196 / Chapter 6.3 --- COMMON OSTEOGENIC EFFECT OF ELP IN THE THREE PARTS OF THE WHOLE STUDY --- p.197 / Chapter 6.4 --- LIMITATIONS OF THE PRESENT STUDY --- p.197 / Chapter 6.5 --- SIGNIFICANCES OF THIS STUDY --- p.199 / Chapter 6.6 --- FUTURE STUDIES --- p.199 / BIBLIOGRAPHY --- p.201