| Mulberry, locally named as mulberry jujube or mulberry fruit, is the fruit of Morus alba with deep purple or black oval and can be used as food and medicine. It has long been recorded in many ancient Chinese classic medical literatures, for example, “Tang Materia Medica”(AD 657-659) recorded that eating mulberry alone can cure diabetes and “Compendium of Materia Medica”(AD 1518-1593) reported that mulberry juice can be good for a hangover and mulberry wine can eliminate edema. The 2015 edition of “Chinese Pharmacopoeia” records: mulfruit is the dry fruit of mulberry of moraceae; the fruits turn red in 4 – 6 month and can be harvested, then be dried; mulberry is sweet, acid and cold and belongs to the meridian of heart, liver and kidney; also has the effects of blood ziyin and fluid dryness; in addition, mulberry is used to cure the symptom of dizziness tinnitus, palpitations insomnia, weigh, early white, tianjin thirst, internal heat diabetes and blood deficiency constipation. Modern pharmacology has demonstrated that mulberry has various kinds of biological activities, including hepatoprotective, antioxidative, antibacterial, anti-inflammatory, hypolipidemic, hypoglycemic, immunomodulating activities and so on.According to many herbal medicine literatures, mulberry is recorded as cold-natured, hence not suitable for those people having cold-nature spleen and stomach. Recently, some clinical reports have been published about hemorrhagic proctitis of children after eating mulberry. In addition, some literatures stressed that children and adults with defective gastrointestinal function should eat few mulberries because of Trypsin inhibitor. However, till now, there is no research in our knowledge that focuses on investigating TI contained in mulberry. Herein, we systematically explored the inhibition effect on trypsin of mulberry.(1) The preparation of mulberry extract(ME) and the content determination of active ingredients in ME. Mulberry extract was prepared with ethanol combining with ultrasonic, then the content of anthocyanins, polyphenols, flavonoids, polysaccharides and proteins in ME were determined by pH-differential method, Folin-Ciocalteu method, aluminum nitrate method, phenol-sulfuric acid colorimetric method and coomassie brilliant blue method, respectively.(2) Trypsin inhibitory effect of mulberry and the enzyme inhibition kinetics curve. The activity of trypsin inhibitor in mulberry and the enzyme inhibition kinetics were determined by BAPNA method. The results showed that mulberry can inhibit the activity of trypsin and inhibition belonged to a type of a combined non-competitive and anti-competitive inhibition.(3) The research that type of trypsin inhibitor in mulberry and its inactivation. At the research beginning, we hypothesized that trypsin inhibitor in mulberry may be tannin belonging to polyphenol which was abundant in mulberry. In order to check the speculation,an enzymatic oxidation experiment, in which a kind of polyphenol oxidase(PPO) extracted from snow pear was mixed with the ME solution under oxidative condition, was performed. However, our findings indicated that the inhibitory effect of ME after the treatment with the polyphenol oxidase on trypsin showed no considerable drop, it was thus concluded that polyphenols were not the main TI in mulberry. Reminded by soybean trypsin inhibitor studies, we speculated that TI in mulberry might be proteinaceous material. It has been demonstrated that trypsin inhibitors in most plants are mostly Bowan-Birk and / or Kunitz protease inhibitor, among them, Bowan-Birk protease inhibitor has lower molecular weight(about 8 kDa) and can only be precipitated with acetone, while Kunitz protease inhibitor has relatively higher molecular weight(about 20 kDa) and can be precipitated with ammonium sulfate. So, in order to get the possible protease inhibitor in mulberry, ME was precipitated with acetone and ammonium sulfate, successively. Then, the inhibitory effects of the resultant precipitates on trypsin were evaluated. The results showed that both the protein precipitates had inhibitory effect on trypsin, the acetone precipitate had far more strong inhibitory effect on trypsin than that of ammonium sulfate precipitate, implying that the main trypsin inhibitor in mulberry should probably be Bowman-Birk protease inhibitor. A fine thermal stability is found in trypsin inhibitor in mulberry, so we inactivated trypsin inhibitor in mulberry with alkaline protease, we found that the activity of trypsin inhibitor in mulberry which deactivated by alkaline protease(T-ME) greatly reduced, it concluded that the inhibitor can be deactivated by alkaline protease.(4) The research that the injury on pancreatic tissue and the effect on immunity of ME and T-ME in mice. The Kunming mice(10 ~ 12g) were used as the experimental objects, animals were weighed and then random Ly divided into the following five groups: normal control group(NC), low-dose ME group(Low ME, 1.8mg·g-1·d-1), high-dose ME group(High ME, 5.4mg·g-1·d-1), low-dose T-ME group(Low T-ME, 1.8 mg·g-1·d-1) and high-dose T-ME group(High T-ME, 5.4mg·g-1·d-1). The mice were dosed 28 days continuously, the mice were sacrificed at the 14 th day and 28 th day respectively, then the pancreatic tissue was taken out. At the 28 th day, after the mice were sacrificed, immune organ index and cellular immunity indicators were determined. The results indicated that mulberry can injure pancreatic tissue in immature mice, furthermore, mulberry has the immunity-enhancing function, but the TI contained in mulberry can weaken this immunological enhancement in mice.(5) The influence on immunity of ME and T-ME in mice with leukopenia induced by twice cyclophosphamide injection. Mice were twice injected cyclophosphamide to induce leukopenia, the mice were divided into the following six groups: normal control group(NC), negative control(CY), low-dose ME group(Low ME, 1.8mg·g-1·d-1), high-dose ME group(High ME, 5.4mg·g-1·d-1), low-dose T-ME group(Low T-ME, 1.8 mg·g-1·d-1) and high-dose T-ME group(High T-ME, 5.4mg·g-1·d-1). Changes in peripheral hemogram, immune organ index and related immunity indexes were observed at 10 th day and 17 th day. The results showed that mulberry had leukogenic effect, improved hemopoietic function and enhanced immunity. In addition, the effect on leukogenic, hemopoietic and immunity of mulberry reduced after the second injection of cyclophosphamide, it may be associated with that the immunity or body recovery ability of mice were weaken after the second injection of cyclophosphamide. |
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