Study On Hypoglycemic Activities Of Callus Before And After Chromosome Doubling Of Zhongsang 5801

Long-term high blood sugar in the human body can lead to complications such as diabetes,cardiovascular disease,nerve damage,kidney damage,and eye diseases.The twigs,leaves and roots of Morus alba are often used as traditional hypoglycemic Chinese medicinal materials to control blood sugar levels.Due to their rich content of polyphenols,flavonoids,alkaloids,terpenes,and other bioactive components,they have significant hypoglycemic effects.However,the complete basis and mechanism of their hypoglycemic activity have not been fully elucidated.In response to the national“Healthy China 2030”strategic plan and the transformation and upgrading needs of the mulberry industry,the breeding objectives of mulberry trees are gradually inclined to obtain varieties that can produce important secondary metabolites.Therefore,it is of great significance to clarify the variation rules of hypoglycemic active components from mulberry with chromosome doubling,hybridization,and other breeding modes.The artificially homologous tetraploid mulberry variety Jialing30（2n=4X=56）,widely cultivated in the western region of China,was selected by chromosome doubling from the excellent diploid mulberry Zhongsang 5801（2n=2X=28）.As a dioecious cross-pollinating woody plant cultivated in the wild,mulberry trees have a long growth cycle,and the content of secondary metabolites is greatly affected by various factors of the ecological environment.In order to accurately explore the changes in the types and content of hypoglycemic active substances with chromosome doubling,this study obtained mulberry Zhongsang 5801and Jialing 30 callus in the same growth state through plant tissue culture and used ligand fishing combined with mass spectrometry technology to quickly screen and identify the hypoglycemic active components in mulberry callus.Then,using molecular docking to further explore its possible mechanisms.The main research content and results are as follows:1.Establishment of a mulberry callus culture systemUsing the tender stem segments of Zhongsang 5801 and Jialing 30 as explants,sterile seedlings（induction rate 39.23%,59.59%）and callus（induction rate 89.38%,96.67%）were successfully induced.The callus were proliferated（proliferation coefficient 19.42,23.88）,and a mulberry wounded tissue culture system was established,obtaining highly dedifferentiated,well-grown,and non-browning callus materials of Zhongsang 5801 and Jialing 30.2.Determination of the main substance content in mulberry twigs and callusContinuous reflux extraction was used to extract dried mulberry twigs and callus from two varieties using petroleum ether,ethyl acetate,anhydrous ethanol,and water sequentially.The contents of total polyphenols,total flavonoids,total polysaccharides,total reducing sugars,total alkaloids,and 1-deoxynojirimycin,which is closely related to hypoglycemic activity were determined in each extract.The results showed that the content distribution of substances varied among different varieties,growth stages,and solvent fractions.The overall content of each substance in the mulberry twigs of Zhongsang 5801 was higher than that in Jialing 30,while the overall content of each substance in Jialing 30’s callus was higher than that of Zhongsang 5801.The ethyl acetate fraction of mulberry callus had the highest content of total polyphenols and total flavonoids,with Jialing 30（125.18 mg/g,119.82 mg/g）being significantly higher than Zhongsang 5801（67.79 mg/g,42.20 mg/g）.The anhydrous ethanol fraction had the highest content of total reducing sugars and total polysaccharides,with Jialing 30（345.09 mg/g,712.22 mg/g）being significantly higher than Zhongsang 5801（175.79 mg/g,404.78 mg/g）.Total alkaloids and 1-deoxynojirimycin were nearly not detected in all extracts of mulberry wound healing tissues except for the water fraction of Jialing 30.3.Antioxidant and hypoglycemic activity of mulberry callusUsing ascorbic acid（VC）as a positive control drug and 4 personal oxidative stress models of hydroxyl freedom（·OH）,nitric oxide radical（NO·）,superoxide anion radical（O₂^-·）,and lipid peroxidation（LPO）as evaluation indicators.Each segmented antioxidant activity of the mulberry callus was investigated.In·OH scavenging activity test,the ethyl acetate and water fractions of Jialing 30(IC₅₀=0.109 mg/mL,0.104 mg/mL)were significantly better than those of Zhongsang 5801(IC₅₀=0.128 mg/mL,0.193 mg/mL)and comparable to the positive control VC(IC₅₀=0.108 mg/mL).In NO·scavenging activity test,except for the petroleum ether fraction,both varieties exhibited good activity,with Jialing 30 water fraction(IC₅₀=0.070 mg/mL)showing the best activity,comparable to the positive control VC(IC₅₀=0.058 mg/mL).In O₂^-·scavenging experiment,the petroleum ether and ethyl acetate fractions of Jialing 30(IC₅₀=0.190 mg/mL,0.154 mg/mL)exhibited good activity.In the lipid peroxidation（LPO）inhibition test,except for the water fraction and Zhongsang 5801 anhydrous ethanol fraction,all extracts showed activity better than the positive control VC(IC₅₀=0.024 mg/mL),with Zhongsang 5801(IC₅₀=0.006mg/mL)and Jialing 30(IC₅₀=0.003 mg/mL)ethyl acetate fractions showing the best activity.Using acarbose（Acarbose）and sodium orthovanadate（NaVO₃）as positive control drugs,and three key targets of blood glucose regulation ofα-glucosidase（AG）,α-amylase（AA）and protein tyrosine 1B（PTP-1B）as evaluation indicators.Each segmented hypoglycemic activity of mulberry callus was investigated.In AG inhibition activity test,the ethyl acetate fractions of Zhongsang 5801 and Jialing 30(IC₅₀=1.296μg/mL,0.426μg/mL)and the anhydrous ethanol fraction of Jialing 30(IC₅₀=0.899μg/mL)showed significant inhibitory effects.In AA inhibition activity test,only the ethyl acetate fraction of Jialing 30(IC₅₀=0.010 mg/mL)showed significant inhibitory effect,and the activity was better than the positive control acarbose(IC₅₀=0.019 mg/mL).In PTP-1B inhibition activity test,both varieties exhibited good inhibitory activity in the petroleum ether and ethyl acetate fractions,with the petroleum ether fraction of Jialing 30(IC₅₀=0.378μg/mL)showing a significant inhibitory effect.Combining the results of the main content and activity measurements,the ethyl acetate extracts of the mulberry callus were determined to be the main effective parts for hypoglycemic activity.4.Rapid screening and identification of hypoglycemic active componentsBased on the ligand fishing and mass spectrometry techniques,α-glucosidase andα-amylase were immobilized on aminated iron oxide magnetic nanoparticles.Fourteen potential blood sugar-lowering active components（Moracin C,Moracin N,Isorhamnetin-3’-O-（6’’-malonylglucoside）,Apiin,Chalcomoracin,Mongolicin F,Warfarin,2,4,2’,4’-Tetrahydroxy-3-（3-methyl-2-butenyl）-chalcone,Kuwanon C,Kuwanon H,Morusin,Kuwanon V,Mulberrofuran B,Ursolic acid,Oleanolic acid）were successfully screened and identified from the ethyl acetate extract of mulberry callus.5.Validation and preliminary mechanisms exploration of potential hypoglycemic active componentsSix representative compounds with strong responses,including Kuwanon C,Kuwanon H,Morusin,Mulberrofuran B,Ursolic acid and Oleanolic Acid,were selected for qualitative,quantitative analysis,and activity validation.Molecular docking was used to predict the enzyme binding sites of the potential blood sugar-lowering active components identified from the screening.The results showed that all six compounds were detected in the ethyl acetate fraction of mulberry callus.Among them,Mulberrofuran B(IC₅₀=3.270μg/mL)showed the bestα-glucosidase inhibitory activity,and Kuwanon H(IC₅₀=3.256μg/mL)exhibited the bestα-amylase inhibitory activity.Ursolic acid and Oleanolic Acid did not showα-amylase inhibitory activity within a certain concentration range.Molecular docking results showed that the potential blood sugar-lowering active components exhibited good binding ability toα-glucosidase andα-amylase.Amino acid residues Asp307,Asp215,Arg315,Lys156,Ser311,Asp242,Asp352,and Ser304 inα-glucosidase,and Thr6,Gly9,and Gln7 inα-amylase were the possible active binding sites.In summary,this study comprehensively elucidated the main material basis for the hypoglycemic activity of mulberry callus in Zhongsang 5801 and Jialing 30,laying the foundation for the study of the changes of hypoglycemic active components in polyploid breeding of mulberry.This research has certain reference and guiding significance for development of natural plants hypoglycemic drugs and directional breeding of mulberry based on specific biological active components.