| Deer bones are the bones of the deer family(Cervusnippon Temminck)or red deer(Cervuselaphus Linnaeus).Ancient books record that deer bones are sweet,slightly hot,non-toxic,have the effect of tonifying fatigue,strengthening muscles and bones,and can be used to treat osteoporosis,rheumatic limb pain and so on.Osteoporosis is a systemic disorder of bone metabolism that usually manifests as decreased bone density and decreased bone mass.In this experiment,we used the raw material of deer bones of the sika deer to extract the active substance with the effect of promoting osteoblast proliferation by ultra-high pressure extraction technology.The extraction yield and total protein content were used as the indexes,and the extraction conditions were optimized by single-factor and orthogonal experiments to determine the best extraction process with the extraction temperature of45℃,material-to-liquid ratio of 7,holding time of 25 min and pressure of 400 Mpa.The sika deer bone samples and bovine bone samples at 5 different time points,June(the initial formation stage of dikang antler),July(the rapid growth stage of dikang antler),August(the initial formation stage of trident antler),September(the beginning of ossification stage of trident antler)and November(the ossification stage of trident antler and shedding of antler skin),were extracted using the preferred process and acted on mouse osteoblasts MC3T3-E1,respectively,and it was concluded that bovine bone extract could not promote osteoblast proliferation,and the sika deer bone in the 1.25 mg/m L concentration group at 5different samples at 1.25 mg/m L concentration group had the function of promoting MC3T3-E1 cell proliferation,which were significantly different from the blank control group and positive control group of bovine bone samples(P < 0.05 or P < 0.01),comparing the samples at 5 time points of June,July,August,September and November,the proliferation effect of the samples in June,September and November was significantly better than the proliferation effect of the samples in July and August,all of which were significant(P < 0.01).The above results showed that sika deer bone at different time points could significantly promote the proliferation of osteoblasts,and the deer bone samples collected at different time points had certain differences in promoting the proliferation of osteoblasts.There was a positive correlation between the change curve of sika deer bone density and the curve of promoting the proliferation of osteoblasts.Therefore,when developing health food or drug raw materials with bone density increasing function with sika deer bones as raw materials,we should pay attention to the differences in drug efficacy caused by different harvest dates.According to the results of this experiment and comprehensively considering the economic value of sika deer before and after antler production,this paper believes that the male sika deer bones collected before October to the next year are more in line with the requirements for genuine collection of deer bones.This study provides a reference for the precise selection of deer bone raw materials in medicine and health food.With male sika deer bone as the main raw material after antler production,with compatibility of Panax notoginseng,Drynariae rhizoma,calcium carbonate and calcium gluconate,the health food of Panax notoginseng deer bone capsule with enhanced bone density function was developed.Based on the Chinese Pharmacopoeia,the daily dosage of compatible raw materials was determined: 1 g of deer bone,1 g of Panax notoginseng,2.4g of Drynariae rhizoma,0.9 g of calcium carbonate,and 0.6 g of calcium gluconate.The optimal extraction process of Panax notoginseng and Drynariae rhizoma was screened by orthogonal test to investigate the material-liquid ratio,extraction time and number of times of extraction,which was 10 times of water addition for the first time,8 times for the second and third time,and 3 times of extraction for 1.5 h.The preparation forming process was investigated and three batches of pilot scale experiments were conducted.Panax notoginseng and Drynariae rhizoma were extracted according to the optimal extraction parameters to obtain the filtrate,concentrated 1.23-1.27 under reduced pressure(50-60°C),dried(-0.06-0.08 MPa,70-75°C),crushed and sieved(80 mesh).Add sika deer bone powder(Co60 irradiation),calcium carbonate and dextrin to mix well,make soft material(70% ethanol),granulate at 20 mesh,dry at 70°C,whole at 18 mesh,and capsule to obtain Panax notoginseng deer bone capsule with a loading capacity of 0.45 g in per capsule.A high performance liquid phase assay for total saponin of Panax notoginseng,the signature ingredient of Panax notoginseng in the formulation,and methodological investigation was carried out.The results showed that the method for the determination of Panax notoginseng total saponin was reasonable and stable,and could be used as an effective method to detect the content of Panax notoginseng in this product.The safety of Panax notoginseng deer bone capsule was evaluated by the investigation of acute toxicity test,genotoxicity test and 30 days frequent poison feeding test.Through the investigation of rat acute poisoning experiments,it can be seen that Panax notoginseng deer bone capsules were gastrically observed for 14 days,and no death occurred.The tolerable amount of acute toxicity through oral passage is greater than 15 g/kg.bw,and this product is non-toxic.The results of genotoxicity tests show that this product has no genotoxicity.After 30 days of feeding tests in rats,no abnormal changes in important indicators such as overall health status,physiological and biochemical function and organ histomorphology of rats were caused,which proved that Panax notoginseng deer bone capsules were safe and non-toxic side effects.SD rats fed with low-calcium diet were used for functional evaluation.After long-term administration of Panax notoginseng deer bone capsule,it was found that there was no adverse effect on the growth and development of rats,the calcium absorption yield was higher,and it could significantly increase the bone density and bone calcium content,and it was determined that Panax notoginseng deer bone capsule has good function of increasing bone density. |
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