| Vitamin D2(VD2) was the important one of the vitamin D family. VD2 can regulate calcium and phosphorus metabolism, and promote bones more healthy. Edible fungus contains abundant VD2 precursors-ergosterol, which can be converted into VD2 under UV irradiation. However, different conversion conditions show significant difference on the VD2 yield. this paper carried out the process about optimizing the transformation of ergosterol to VD2 in Flammulina velutiper powder, on which as the basis to develop natural health food source of vitamin D,and research about anti-osteoporosis activity of the developed products.The main research contents including determination of ergosterol and VD2 by HPLC and the extraction process optimizing of ergosterol and VD2 in edible fungi powder,optimization of ergosterol transform to VD2, the protective effect of three natural antioxidants on the stability of VD2 anti-oxidants, development of natural source VD2 health food and preliminary exploration on the effect of manufactured natural VD2 health food on protection of osteoporosis in SD rat with ovaries removed. Those research were established aimming to provide the scientific basis and lay the theoretical foundation for research and development of natural source vitamin D health food products. The main research results were as follows:1. Determination of ergosterol and VD2 by HPLC and the extraction process optimizing of ergosterol and VD2 in F. velutiper powderThe extraction conditions of ergosterol was optimized as: the samples were ultrasonic extracted by alcohol at 40? for 15 min for 2 times, and the ratio of liquid to material was 40:1 (mL: g). The extraction conditions of VD2 was optimized as: the samples were ultrasonic extracted by alcohol at 60? for 25 min for 2 times, and the ratio of liquid to material was 15:1 (mL: g). The HPLC system was performed on an RP C18 column(4.6 mm×100 mm, 3.5?m), with the mobile phase was acetonitrile-0.1 % formic acid (95:5) at a flow rate of 1.0 mL·min-1, the column temperature was 30? , the injection volume was 10?L, and the detection wavelength of VD2 and ergosterol were 264 nm and 282 nm, respectively. Result:The liner ranges calibration curves of ergosterol and VD2 were 1.00?60.00 ?g·mL-1(R2=0.9994) and 1.00?40.00 ?g·mL-1(R2=0.9997). The average recoveries(n=9) were 100.31 % and 98.69 %.The above method can be used to simultaneously determine the content of ergosterol and VD2 in edible fungus powder. The average determined VD2 content of 4 kinds of VD2 drugs mentioned as multiple elements capsule, vitamin AD soft capsule,vitamin AD drops, VD2 calcium hydrophosphate tablet and VD2 formulated health care foods by ourselves were 101.44 %, 112.56 %, 115.38 %, 105.42 %, and 86.43 %, respectively, to the labeled content by using the method in this paper. It showed that the method above was sensitive, reliable, simply, rapid and accurate. It can be used to determine the content of VD2 in the formulated health care foods and drugs.2. Optimization of ergosterol transform to VD2The F. velutiper powder was exposured under ultraviolet irradiation coupled with stirred simultaneously, and the conditions of ergosterol transforming to VD2 were optimized by single factor test and orthogonal test in several factors, including material status, irradiation temperature, irradiation time, single wavelength ultraviolet, combination of multiple wavelength ultraviolet, sequenced combination of multiple wavelength ultraviolet and material-liquid ratios. The orthogonal test showed that the factors affecting the VD2 UV conversion was irradiation time, ultraviolet wavelengths, solid-liquid ratio, and irradiation temperature in sequence. Ultimately the optimal transformation conditions in F. velutiper powder was determined as the material status of turbid liquid, material-liquid ratios 1:20 (g:mL), exposured under UV light of 254 nm wavelength and with magnetic stirring simultaneously for 2 h under 40?. In this optimized condition the VD2 content in F. velutiper powder was high about 592.31 ?g·g-1.3. The protective effect of three natural antioxidants on the stability of VD2Resveratrol, tea polyphenols, and soy isoflavones were mixed with VD2 in 1:1 ,1:10, and 10:1 mass ratio respectively, and changes of VD2 stability was determined by ultra high performance liquid chromatography after the mixtures were placed for 10 days, 26 days, and 36 days. The results showed that three test natural antioxidants added with VD2 in 1:1, 1:10 and 10:1 quality proportion has good protective effect on the stability of VD2, and their protective effect are more significant when longer duration of place. The protective effect showed the best when VD2 and antioxidant mixed in 1:10 mass ratio, the retention rate of Tea polyphenols, soybean isoflavones and resveratrol was over 84% after placed for 36 days, and increased by 72.72 %, 68.08 % and 75.32 % compared with the control respectively.4. Development of natural source VD2 health foodThe preliminarily formula of natural source VD2 health foodpreliminarily ewas:F.velutiper powder with UV irradiated for 10?30 %, soy isoflavone for 0.5-2 %, maltodextrin for 5?20 %,magnesium stearate for 1 ?3 %,essence for 0?5 %,milk powder for 5?10 %, corn starch for 10?30 %, and croscarmellose sodium for 5?10 %; The productive process was preliminarily determined followed as the preparation of preparation of transformed F.velutiper powder, hybrid, granulating, dry, particle uniformizing, hybrid secondary,tableting, screening and sterilization. The average VD2 content in natural source VD2 health food was 361.15 IU per tablet. The weight difference test, content uniformity test, stability test and tablet disintegration time limit test were carried out. The above experimental results conformed to the stipulations of the state pharmacopoeia for the requirements of pharmaceutical tablets. And the result provided a certain theoretical basis and data support to the production process of natural source VD2 health food.5. Preliminary exploration on the effect of manufactured VD2 Health Food on protection of osteoporosis in SD rat with ovaries removedThe osteoporosis model was established by removing the ovaries of the rats. The rats were divided into Sham group (control), OVX group (model group), OVX+VD2 group, and OVX+manufactured VD2 health food group (OVX+MVHP) randomly. After continuously administrate drug by drenching for 12 weeks every other day, the weight and concentrations of serum calcium and serum phosphorus were determined. The results showed that the difference of weight between OVX+VD2 group and OVX group was significant(p<0.1), and the difference of serum calcium concentration between the OVX+ MVHP group and the OVX group was significant (p<0.05), and there was also significant difference between OVX+MVHP group and OVX group of the serum phosphorus concentration (p<0.1).The results preliminary showed that the natural source VD2 health food developed by ourselves exhibit some effect in prevention and treatment of osteoporosis SD rats. |
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