| Medicinal mulberry is a black mulberry（M.nigra）species growing in Xinjiang,China,with the highest chromosome ploidy.The medicinal mulberry is rich in anthocyanins,flavonoids and other nutrients.It is a traditional Uygur medicine for the treatment of tonsillitis,sore throat,and other diseases of the Uygur people.It has good pharmacological activities such as anti-oxidation,anti-inflammation,anti-tumor and anti-bacterial activities.Inflammation is a complicated immune defense process caused by organ damage,bacterial or viral infection,toxic drugs and other damage factors,and multi-cellular and multi-factor involvement.Its basic symptoms include redness,heat,swelling and pain.It is one of the most common basic pathological processes in human diseases.Inflammation can be divided into acute inflammation and chronic inflammation.On the one hand,inflammation is a defensive and protective mechanism of the body,usually acute inflammation,which can effectively prevent the malignant development of infection and injury.On the other hand,the persistence of inflammation or chronic long-term inflammation can cause heavy or light damage to the body.Such as pain caused by pharyngitis,life-threatening inflammation caused by serious drug allergy,increased cholesterol and thrombosis,rheumatoid arthritis pain and so on.The currently used steroids and non-steroidal anti-inflammatory drugs on the market have some side effects,every year millions of people are affected by its side effects.Therefore,the development of natural non-toxic side effects of anti-inflammatory drugs to fight chronic or persistent inflammation,is of great significance.In this study,orthogonal test and response surface test were used to optimize the extraction process of total flavonoids of Morus nigra（MnTF）.At the same time,we developed the ultra-performance liquid chromatography–tunable ultraviolet/mass single-quadrupole detection（UPLC-TUV/QDa）method to identify and analyze the total flavonoids（TF）,anthocyanins and flavonols of 26 mulberry germplasms.The changes of anthocyanin and flavonol content in different maturity were analyzed.The main types of mulberry were classified according to the distribution of the components.Furthermore,we compared and analyzed the differences of the flavonoids between black mulberry and non-black mulberry.Through the analysis of toxicity and analgesia,the detection of cytokines,the analysis of the protein expression of inflammation,the analysis of antioxidant and antibacterial activities of mulberry,to explore its main anti-inflammatory and analgesic active ingredients and anti-inflammatory pathways.The main results are as follows:1.Optimization of MnTF extraction processThe three main factors that screened the extraction rate of MnTF were ethanol volume fraction,temperature and extraction times by orthogonal test of 6 factors and 2levels.The optimum extraction conditions of MnTF obtained by single factor test and three-factor-three response surface test were as follows:48%ethanol,80℃,repeat extraction 4 times,MnTF yield was predicted to be 2.32%,measured 2.31%.After comparing the extraction efficiency,antioxidant activity（including reducing power,hydroxyl radical scavenging ability and superoxide anion radical scavenging ability）,and the actual efficiency and benefit of different extraction schemes,the optimal extraction scheme for MnTF was determined as follows:38%Ethanol,extracted twice at 60℃for 15 min/time,the ratio of solid to liquid was 1:10,the ultrasonic power was500 W and the yield of MnTF was 2.22%.2.Analysis of TF,Anthocyanins and Flavonols of 26 Mulberry GermplasmsTF content reflects the antioxidant activity and nutritional value of fruits.Through established UPLC-TUV/QDa method,the main flavonoids in mulberry,including 3kinds of anthocyanins and 5 kinds of flavonols,can be rapidly analyzed.The survey of26 mulberry germplasms revealed that the black mulberry is rich in flavonoids,and has the highest levels of TF（35.39 mg/g）,cyanidin-3-O-glucoside（C3G,8.29 mg/g）,cyanidin-3-O-rutinoside（C3R,2.89 mg/g）and quercetin（74.44μg/g）.Analysis of the composition of different color or maturity of black mulberry fruits showed that the content of anthocyanins increased rapidly during the red black fruit period,and then the content of anthocyanins decreased after the majority color of fruits became black.At the whole mature stage,the content of flavonols is low,but the content of rutin gradually increased during this period.Anthocyanidin and flavonol content of mulberry fruit with different fruit color were significantly different.Through statistical analysis,the higher the color of mulberry fruit was,the higher the content of anthocyanins and flavonols.These mulberry fruits could be divided into three categories according to their compositions and color:Black mulberry fruits,their anthocyanins and flavonols content are high,and the color are dark purple,such as M.nigra and Hongguo2;Red or black mixed mulberry fruits,mulberry fruits contain a small amount of anthocyanins and flavonols,such as Morus mongolica and Morus alba‘Pendula’;White mulberry fruits,these mulberries do not contain anthocyanins and have low flavonols,such as Morus alba cv.Zhenzhubai and Morus alba‘Baiyuwang’.3.Antinociceptive and Antibacterial Properties of Black and Non-Black MulberriesThe antinociceptive and antibacterial activities of TF from black mulberry（MnTF,TF of Morus nigra）and non-black mulberry（MmTF,TF of Morus mongolica;and MazTF,TF of Morus alba cv.Zhenzhubai）fruits were studied.MnTF was rich in anthocyanins（11.3 mg/g）and flavonols（0.7 mg/g）.Comparatively,MmTF and MazTF had low flavonol contents and MazTF had no anthocyanins.Through the monitoring of mice body weight and viability test of RAW264.7 cell showed that TF of all the three kinds of mulberry（TFs）have no side effects.Through formalin-induced pain-like behavior test,xylene-induced ear edema test,and carrageenan-induced paw edema test in mice showed that MnTF had better antinociceptive and antibacterial effects than MmTF and MazTF.This activity of MnTF increased in a dose-dependent manner.The levels of pro-inflammatory cytokines IL-1β,TNF-α,IFN-γ,and NO in serum of mice were detected by ELISA.MnTF（50,100mg/kg）significantly inhibited or eliminated these cytokines.At inflammation-related protein levels,MnTF significantly inhibited the expression of iNOS,COX2,phospho-p65（p-p65）and phospho-IκBα（p-IκBα）,and increased the expression of anti-inflammatory cytokine IL-10.Mice analgesic tests with monomeric compounds showed that C3G,rutin（Ru）,and isoquercitrin（IQ）are the major active ingredients in the analgesic process.MnTF shows good reducing power and free radical scavenging activity（OH-,O2-,DPPH,and ABTS）,which may be the basis of its good anti-inflammatory analgesic activity.MnTF showed significantly higher antinociceptive and antibacterial activities toward Escherichia coli,Pseudomonas aeruginosa and Staphylococcus aureus than MmTF and MazTF.The minimum bactericidal concentrations（MBC）of MnTF for these three bacteria were 2,2,1.8 mg/mL,respectively.In this study,the optimal extraction process of MnTF was obtained,and the main three anthocyanins and five flavonols in mulberry were identified by UPLC-TUV/QDa.The distribution of total flavonoids,anthocyanin monomers and flavonol monomers in26 germplasm resources was further analyzed.According to the composition distribution,the main types of mulberry can be divided into black,red and white mulberry.The anti-inflammatory and analgesic activities of black mulberry and non-black mulberry were further analyzed.The results show that MnTF has higher analgesic,detumescence,and antibacterial activity than MmTF and MazTF.These characteristics are related to its rich anthocyanins（C3G and C3R）and flavonols（Ru）,and to inflammatory cytokines,iNOS.The inhibition of COX2 and nuclear factor-kappa B（NF-kB）pathway-related proteins is closely related. |
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