| Background : Anoectochilus roxburghii(Wall.)Lindl.)is a perennial herb of the genus Anoectochilus,family Orchidaceae,and is a cherished medicinal and food plant in China,with the effects of nourishing the skin,harmonizing the five organs,and prolonging life.Anoectochilus roxburghii is widely consumed in the southeastern coastal region and is in great demand.However,its seeds are tiny and difficult to sprout,its growth conditions are demanding,and it has been over-harvested over the years,resulting in a great depletion of Anoectochilus roxburghii’s wild resources.In addition,the current Anoectochilus roxburghii industry has many problems such as low level of development,single product form,and narrow audience.Therefore,there is an urgent need for research on the development of plant resources,breakthrough innovation in artificial rapid breeding technology,and the development of its deep processing products.Objective: This project aims to establish a more optimal system for the rapid propagation of Anoectochilus roxburghii in group culture to provide technical support for the conservation of Anoectochilus roxburghii wild germplasm resources and the realization of Anoectochilus roxburghii factory nursery.At the same time,through its traditional use of the test,the proposed use of Fagopyrum tataricum(L.)Gaertn into the compound,the preparation of a low-sugar,potent compound gel gummy for the elderly population.The research of this project can provide a good theoretical basis and application technology support for the conservation of Anoectochilus roxburghii resources and the upgrading of its industry.Research content:1.In this study,we selected nodal stem segments of Anoectochilus roxburghii as explants,and optimized the system for fast-growth of Anoectochilus roxburghii in three aspects: primary shoot induction medium,secondary proliferation medium,and strong rooting medium.The ratio of phytohormone naphthalene acetic acid(NAA)to 6-benzyladenine(6-BA)(1:3,1:5,1:10)and the types of natural additives(mashed banana,mashed potato,mashed tomato)and their concentrations(20~80 g/L)were optimized using the induction rate and net plant height rate of nodal stem segments of Anoectochilus roxburghii as evaluation criteria.Then,we optimized the ratio of phytohormone naphthalene acetic acid(NAA)to 6-benzyladenine(6-BA)(1:3,1:5,1:10),the types of natural additives(mashed banana,mashed potato,mashed tomato)and their concentrations(20~80 g/L),and the concentration of nano-carbon(0.125~0.500 mg/L).Finally,we optimized the different concentrations of nanocarbon(0.125~0.500 mg/L)with the number of roots,number of leaf emergence and net plant height as the evaluation indexes of the rootless seedlings growing more than 2.5 cm,and establisheda new fast breeding system for Anoectochilus roxburghii to realize the rapid breeding of Anoectochilus roxburghii.2.By combining Fagopyrum tataricum with Anoectochilus roxburghii,the content of the main antioxidant components(total flavonoids and total polyphenols)contained in both was used as an evaluation criterion to optimize the combination ratio and the first extraction concentration,time and temperature;DPPH· and ABTS+ free radical scavenging ability was used as an evaluation index and isoradiometric analysis was performed to determine the best in vitro antioxidant synergistic effect by its combination(Anoectochilus roxburghii-Fagopyrum tataricum Mixed Extracts,AFME)in vitro antioxidant synergistic effects were used to determine the optimal compounding ratio.Meanwhile,KM mice(5weeks old,male,18 g±2 g)were used to construct a D-galactose(D-gal)induced aging mice group,and the optimal compounding ratio of AFME(1:1)was selected.and low(AFME-L)dose groups and vitamin C group(Vitamin C,VC),and the general signs,body mass,learning and memory ability,organ index,liver index,serum MDA and SOD content,liver GSH-PX activity,GSH,MDA and AGEs content,and liver pathological changes of mice were used as evaluation indexes to evaluate its anti-aging effects.3.The fudge formulation was determined by using the sensory scores of the gels obtained from the compounding of four edible gums with AFME(1:1)as the main ingredient,and the screening of single gums and flavor correcting agents by using the textural properties and sensory scores,and the analysis of the sensory scores of the gels obtained from the compounding of four edible gums by using the response surface design experiment.The fondant was prepared and its active ingredient content,textural properties and hygienic physical and chemical indexes were measured,and its shelf life was determined by accelerated shelf life test(ASLT).Result:1.The results of shoot induction rate showed that the ratio of NAA to 6-BA concentration for induction of adventitious shoots was 1:10,the best addition was banana mud 80 g/L,the medium formulation was MS+NAA 0.2 mg/L+6-BA 2 mg/L+banana mud 80 g/L,the shoot induction rate increased to 82% after 30 d,and the net plant height rate was 21.4%;the best hormone ratio in the successional proliferation culture was The optimal hormone ratio in the successional proliferation culture was 1:10 between NAA and 6-BA,the optimal addition was banana mud 80 g/L,and the optimal nanocarbon concentration was 0.125 g/L.The optimal proliferation formula was MS + NAA 0.3 mg/L + 6-BA 3 mg/L + banana mud 80 g/L +nanocarbon 0.125 g/L,and the proliferation multiplicity reached 5.76 after 40 d,and the shoots were stout and growing well;the optimal nanocarbon concentration in the rooting culture of strong seedlings was 0.125 g/L.The optimal nanocarbon concentration in culturewas 0.375 g/L.The optimal medium formulation: 1/2 MS + NAA 0.3 mg/L + banana mash 80g/L + nanocarbon 0.375 g/L,the rooting rate was 96.53% at 40 d,the number of leaves emerged was 2.20%,the net plant height rate was 1.79%,the rooting was early,the fibrous roots were many and long,the stems were sturdy and dark green in color.2.The results showed that the optimal compounding ratio of AFME was 1:1,the first extraction concentration was 85%,the extraction time was 30 min,and the extraction temperature was 60°C.The total flavonoid content of AFME obtained was 23.64 mg/g and the total polyphenol content was 28.52 mg/g.Isoradiometric analysis showed that the γ values of DPPH and ABTS+ radicals scavenged by AFME(1:1)were 0.918 and 0.930,respectively.0.918 and 0.930;γ values of AFME(1:2)were 0.954 and 0.999;γ values of AFME(2:1)were0.985 and 0.973,and the in vitro antioxidant synergistic effects were AFME(1:1)> AFME(1:2)> AFME(2:1),indicating a synergistic effect of Anoectochilus roxburghii and Fagopyrum tataricum.Gavage of AFME(1:1)(2800 mg/kg,calculated as raw drug concentration)for 9 w,can significantly improve the aging mice appear yellow hair loss,slow mobility,body mass,brain and thymus index decreased,and significantly improve the liver index,learning and memory capacity,serum SOD activity,liver GSH-PX activity,significantly reduce the MDA content and GSH content and improve hepatocyte degeneration and necrosis and sinusoidal dilatation in liver tissue.3.The best gum base formula was obtained by screening the types and concentrations of single gums,and the best gum base formula was obtained by combining gelatin,gellan gum,xanthan gum and acacia bean gum,and the best response surface optimization was obtained as follows: gelatin 8.30%,gellan gum 0.86%,xanthan gum 1.20%,acacia bean gum 0.90%;the best correction agent formula was obtained as follows: erythritol 15%,citric acid 0.25%,sodium citrate 0.2%.The results of orthogonal experiments showed that the best fondant production process was: boiling time of 20 min,boiling temperature of 80℃,and 20% of sesame and walnut powder were added together.According to the best recipe and process of making the goldenrod buckwheat compound fondant,its total flavonoid content 2.17%,total polyphenols 1.81%;fondant hardness 55 gf,elasticity 0.8649,chewiness 83.57 gf,mucoadhesive 0.9662,reversibility 1.0072;reducing sugar 35.2%,dry weight loss 19.18%,water activity 0.795;E.coli,colony The shelf life at room temperature was 17 d.The total number of coliform bacteria was not detected.Conclusions:In this experiment,the bioengineering technology was used to rapidly breed Anoectochilus roxburghii seedlings and to establish a rapid culture system for Anoectochilus roxburghii plants.The antioxidant effect of the compound in vitro and in vivo was alsoinvestigated to provide an experimental basis for developing the medicinal value of cultivated Anoectochilus roxburghii and thus conserving wild Anoectochilus roxburghii resources;and a Anoectochilus roxburghii health food for the elderly population was prepared,which is moderately sweet and sour with good taste,and evaluated for quality in view of the current accelerated aging and the health problems it brings;It further improves the market recognition of Anoectochilus roxburghii products and expands the market application and economic value of Anoectochilus roxburghii. |
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