| This dissertation was divided into seven chapters.In Chapter 1, a detailed review was given about the latest research progress of resource chemistry of Chinese medicinal materials of animal horns origined from family Cervidae.Furthermore, the medicinal historical of Elaphuri Dividiani Cornu (EDC) and its modern researches were also summarized.In Chapter 2, the resource chemistry evaluation on the EDC was performed, and the results were summarized in four sections.Section 1: resource chemistry evaluation of the nucleosides and nucleobases.To explore the profiles of the nucleosides and nucleobases in EDC, A rapid and sensitive hydrophilic interaction liquid chromatography method was developed and validated for the identification and quantitative determination of 17 nucleosides and nucleobases by using ultra-highperformance liquid chromatography separation system coupled to triple quadrupole mass spec-trometer. Furthermore, Multivariate statisticalanalysis was also used to investigate the differences in the nucleosides and nucleobases profiles among the samples. The results showed that all the target compounds of 17 nucleosidesand nucleobases were presented in almost all of the samples. The total contents of nucleosides and nucleobases in EDC ranged from 5.971 to 49.70μg/g,and varied obviously in samples from various ages or different sections. The average total content of the three sections was the highest in the EDC of 2 -year-old, which was close to that in CC, followed by 5, 6-8, 3- year-old, and that in 4- year-old was the lowest. Interestingly, the total contents in different sections of 2, 6-8-year-old EDC and Cervi Cornu (CC) decreased downward from the upper to the base, respectively. In most EDC samples, xanthine, hypoxanthine, uracil,thymine, gua-nine and guanosine accounted for more than 70% of the weight oftotal nucleosides and nucleobases. The PC A results showed that CC could be preliminarily discriminated from EDC and Anter button by the profiles of nucleobases andnucleosides.Section 2: resources chemistry evaluation of the free amino acids.With the application of HILIC-UPLC-TQ-MS, a rapid, sensitive and non-derivative method for analysis of amino acids has been established and validated. The results showed that almost all of the samples were rich in 24 free amino acids, although their contents were varied obviously in samples from various ages or different sections. The average total content of the three sections was the highest in the 6-8-year-old EDC, which was close to that in 2- year-old, followed by 5, 3, 4-year-old. Interestingly, the total contents indifferent sections of 3 and 4-year-old EDC and Cervi Cornu (CC) decreased downward from the base to the upper, respectively. In most EDC samples,phenylalanine, leucine, valine, alanine, glycine, glutamic acid, aspartic acid, arginine and lysine accounted for more than 89% of the weight of total nucleosides and nucleobases. Moreover, the non-protein amino acids such as y-aminobutyric acid, hydroxyproline, citrulline, taurine and ornithine were also detected in the samples.Section 3: resources chemistry evaluation of the inorganic elementsInorganic elements in EDC and CC were analyzed by ICP-AES using microwave digestion combined with multivariate statistical analysis. Total of 21 inorganic elements including five essential macro elements of calcium (Ca), phosphorus (P), magnesium (Mg), potassium(K),sodium (Na), and ten essential trace elements of iron (Fe), copper (Cu), zinc (Zn), manganese (Mn),chromium (Cr), selenium (Se), nickel (Ni), molybdenum (Mo), silicon (Si), strontium (Sr) were found in all the samples, but stannum (Sn), arsenic (As),cobalt (Co), boron (B), mercury (Hg),vanadium (V), titanium (Ti) and zirconium (Zr) were not detected. The results showed that the content of five macro elements was close to that in CC, and Ca and P content reached 24.36% and 10.85%,respectively. The content of Zn, Fe, Si and Sr were higher relatively in the ten essential trace elements. There were no differences in kinds of elements in three sections of EDC except Fe,Si, Mn and aluminum (Al). The correlation analysis showed that there were 36 elements in pair were significantly positively correlated.Section 4: resource chemistry evaluation of the saccharides.The total contents of hexosamine, neutral and acidic polysaccharides in EDC and CC were determined using UV-Vis spectrophotometry. The results showed that the contents of the three polysaccharides varied obviously in samples from various ages or different sections. The total content of the three polysaccharides were the highest in 2-year-old EDC, followed by 5, 6-8,3 and 4. The average content of three polysaccharides in the three sections were all the highest in 2-year-old EDC, and the neutral polysaccharides contents were close to that in CC, but the acidic polysaccharides content was lower than CC. The neutral polysaccharides were the highest in all the EDC samples from various ages, followed by hexosamine and acidic polysaccharides.In Chapter 3, the evaluation of biological activities on nourishing yin effect of EDC was studied.Section 1: a yin-deficiency model induced by thyroxine and reserpine in mice was established to study the nourishing yin effect of EDC. The powder of EDC, the water extract of EDC and the resulting residue were tested. The content of interleukin-2 (IL-2) and tumor necrosis factor a(TNF-a) in plasma, the activities of superoxide dismutase (SOD) and glutathione peroxidase(GSH-Px), the malondialdehyde (MDA) content in liver were evaluated. In addition, the viscera index of spleen, kidney and thymus were also measured. Compared to the model group, both of the powder of EDC and the residue could significantly increase the SOD and GSH-Px activity, and the viscera index of spleen. And they could also reduce the conten of MDA and TNF-a, and the viscera index of kidney. The water extract of EDC could increase the SOD and GSH-Px activities,reduce the MDA and the viscera index of kidney. The mechanism of noumishing yin effect of EDC might be related to the regulation of the antioxidant capacity, endocrine and immune systems of the organism.Section 2: a yin-deficiency model induced by thyroxine and reserpine in rats was established to study the nourishing yin effect of EDC. The water extract of EDC and the resulting residue were tested. The viscera index of spleen and thymus, and the contents of corticosterone (Cort),testosterone (T), estradiol (E2), adrenocorti cotrophic hormone (ATCH), thyroid stimulating hormone (TSH), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate(cGMP),triiodothyronine (T3),thyroid hormone (T4), immunoglobulin G (IgG) and immunoglobulin M (IgM) were determined. The results showed that the water extract of EDC and its resulting residue could significantly increase the viscera index of spleen, the content of cGMP and IgG,and significantly decrease the content of E2 and cAMP/cGMP. And the water extract of EDC could also significantly increase the T and ATCH content.In chapter 4, a blood-deficiency model induced by acetylphenyhydrazine and cyclophosphamide in mice was established to study the supplementing essence and blood effect of EDC. The powder of EDC, the water extract of EDC and the resulting residue were tested. The white blood cell (WBC), red blood cell (RBC), errohemoglobin (HGB), and hematocrit (HCT)count in peripheral blood of mice were determined. The cell cycle in the bone marrow was measured, and the spleen pathology was also boversed. The results showed that the powder and the residue could significantly increase the WBC count, and the water extract also have a tendency to increase the RBC count. The water extract and the residue could promote the transformation of the bone marrow cells from G0/G1 phase to S phase. And the water extract could also inprove the spleen pathological conditions. These results indicated that EDC have a protective effect on the blood-deficiency model.In chapter 5, the evaluation of biological activities on anti-aging effect of EDC was studied.Section 1: an aging was established to study the anti-aging effect of EDC. The powder of EDC, the water extract of EDC and the resulting residue were tested. The content of interleukin-2(IL-2) and tumor necrosis factor a (TNF-a) in plasma, the activities of superoxide dismutase (SOD)and glutathione peroxidase (GSH-Px), the malondialdehyde (MDA) content in liver, kidney and brain, the activity of MAO in brain, and the hydroxyproline content in the back skin of mice were evaluated. In addition, the viscera index of spleen and thymus were also measured. Compared to the model group, both of the powder of EDC and the water extract of EDC could significantly increase the SOD activity in liver, kidney and brain, the GSH-Px activity in kidney and brain, the viscera index of spleen and thymus, and the hydroxyproline content in the back skin of mice. And they could also decrease the MDA content in liver and brain, and the MAO in brain. The resulting residue could significantly increase the SOD and GSH-Px activities in kidney, the hydroxyproline content in the back skin, and the viscera index of thymus. In addition, it could also decrease the MDA and MAO content in brain. The mechanism of anti-aging of EDC might be related to the elevation of activities of antioxidant enzymes, suppression activities of MAO in brain,enhancement of immune function, slowing down skin aging.Section 2: The effect of EDC on the life-span in Drosophila melanogaster was explored. The water extract of EDC was tested and the life-span of Drosophila melanogaster was obversed. The results showed that the water extract of EDC could significantly extend the half-death time,maximum life-span and mean life-span of Drosophila melanogaster, which indicated that EDC could prolong the life-span in Drosophila melanogaster.In chapter 6, the evaluation of biological activities on strengthening bones and tendons effect of EDC was studied.Section 1: an osteoporosis model in ovariectomized rats was established to study the strengthening bones and tendons effect of EDC. The powder of EDC, the water extract of EDC and the resulting residue were tested. The contents of calcium (Ca),phosphorus (P),magnesium(Mg) and alkaline phosphatase (AKP) in serum were determined. And the bone minaral density(BMD) and bone minaral content (BMC) in the femur were also measured. Compared to the model group, the powder of EDC, the water extract of EDC and the residue could significantly increase the content of P. Both of the powder of EDC and the residue could significantly increase the BMD,BMC, and the AKP level, the water extract of EDC also could significantly increase the AKP level and BMD. These results indicated that EDC have an anti-osteoporosis effect on the ovariectomized rats.Section 2: A model of osteoporosis induced by prednisolone in zebrafish was established to study the anti-osteoporosis effect of EDC. The different extracts of EDC including total water extract of EDC (ML 1) and its different sections of nominal molecular weight cutoff MW> 1 OOkDa(ML2), lOkDa <MW <100kDa (ML3), 3kDa <MW <10kDa (ML4),MW <3kDa (ML5) and the 95% ethanol extract of EDC (ML6) were tested. After the zebrafish larvae were stained with alizarin red, the quantitative analysis of the head skeleton mineral area (MA) and integrated optical density (IOD) in the digital imaging was performed by a professional image analysis software to reflect the amount of zebrafish head skeleton mineral area. Compared to the model group, the MA and IOD in the low dose group of ML1 and ML3 and in the high dose group of ML5 were significantly increased; the MA in the low dose group of ML2 was also significantly increased;the MA and IOD in the group of ML4 increased as the dose was increased, and they obviously increased at a high dose. In addition,there were no significant differences in the MA and IOD between the group of ML6 and the model. The results showed that EDC could prevent osteoporosis induced by prednisolone in zebrafish, and the water-soluble components in the EDC might be the material foundation of anti-osteoporosis.Section 3: Estrogenic activity of EDC was detected on the MG-63 cells which were transfected with an estrogen receptor element (ERE). The different extracts of EDC including total water extract of EDC (ML1) and its different sections of nominal molecular weight cutoff MW>100kDa (ML2), lOkDa <MW <100kDa (ML3), 3kDa <MW <10kDa (ML4), MW <3kDa(ML5) and the 95% ethanol extract of EDC (ML6) were screened by testing the luciferase intensity on the MG-63-ERE cell model to estimate the estrogenic activity, and estradiol was selected as the positive control. The results showed that ML3, ML4, ML5 and ML6 could significantly increase the luciferase intensity, which indicated that they have obvious estrogenic activitives, and a clear dose-effect relationship was obversed in the samples of ML4 and ML6.In chapter 7, the COI sequence of several animal horns derived from TCM were analyzed in an attempt to acquire DNA barcoding markers for species identification and lay a foundation for adulterants identification, and the genetic relationship among the original animals of various horns wre also dissected. Eleven samples of seven kinds of animal horns origined from family Cervidae,Bovidae and Rhinocerotidae were sequenced, the intergeneric, interspecific, intraspecific genetic distances were calculated, and the phylogenetic tree of all the accessions tested was built based on the distance data obtained. The COI sequences of several animal horns were acquired successfully through PCR amplification using universal primers LCO1490/HCO2198. In the dendrogram constructed, the samples of various horns clustered in three families and formed a relatively independent branch respectively with high support level, and could separate from each other.Pantholops hodgsoni Abel clustered together with family Cervidae, Ceratotherium simum Cottoni with Bubalus bubalis Linnaeus and Bos grunniens Linnaeus were also clustered together, which showed a close genetic relationship. The COI sequence of several animal horns derived from TCM laid a foundation for the accurate identification of each animal horn and its adulterants, the close genetic relationship between Ceratotherium simum Cottoni and Bubalus bubalis Linnaeus showed in the dendrogram constructed confirmed the potentiality of water buffalo horn as the alternative resource for rhino horn, and it also provided an effective method for searching substitution of precious horns. |
PDF Full Text Request