The results showed that on day 7 after the first vaccination, the levels of sIgA in the trachea wash fluids from the MLP-2H,MLP-2M,and MLP-2Lgroups were higher than those from the VC and BC groups. abilities of these polysaccharides to stimulate spleen lymphocyte proliferation in micein vitrowere compared by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. MLP-2 was more effective than MLP-1; therefore, MLP-2 was chosen for the study of its immune-enhancing effectsin vivo. For thein vivoexperiments, 14-day-old chickens immunized with Newcastle disease (ND) vaccine were orally administered MLP-2, andAstragaluspolysaccharide (APS) was used as the control. Each chicken was orally administered 4 mg or 8 mg of MLP-2 for seven consecutive days starting three days before ND vaccine immunization. MLP-2 significantly improved the ND serum antibody titer and interleukin-2 (IL-2), interferon- (IFN-) and immunoglobulin A (sIgA) concentrations in tracheal and jejunal wash fluids, and increasing numbers of immune globulin A-positive (IgA+) cells in cecal tonsils and increased body weight. These results indicated that MLP-2 could significantly enhance immune activity and could therefore be utilized as an immunopotentiator drug candidate. == Introduction == The modulation of the immune response plays an important role in preventing diseases, and increasing research attention has been paid to the immunomodulation and immunostimulation induced by active substances [1]. Current immunomodulators include immune adjuvants such as aluminum hydroxide, Freund’s adjuvant (FA) and albumen adjuvants. However, neither aluminum hydroxide nor FA can induce strong cellular immunity, and FIA can cause local stimulation, tissue damage, and even carcinogenesis [2]. Albumen adjuvants were developed recently but were too expensive to be commercialized. Therefore, there is an urgent need to research and develop a new-type immune adjuvant with high efficiency, low toxicity and extensive resources [3]. Many Chinese herbal polysaccharides have obvious advantages in improving humoral immunity and cellular immunity, such asCyrtomium macrophyllum[4],Cassia obtusifolia[5],Atractylodes macrocephalaKoidz (RAMPS) [6], RAMPStp and RAMPS60c [7]. Numerous Chinese herbal polysaccharides can also enhance the mucosal immunity of animals. For example, oral administration of Si Jun Zi Tang polysaccharide can increase the number of IgA+cells in the small intestine in mice and enhance VU 0240551 mucosal immunity [8]. Therefore, Chinese herbal polysaccharides have obvious benefits in improving humoral immunity, cellular immunity and mucosal immunity and could become promising compounds in the development of immunomodulators. Mulberry (Morus alba) can grow very well under various climatic conditions ranging from temperate to tropical. Mulberry leaves can be used to feed silkworms, and it contains many active ingredients with a wide range of pharmacological activities. For example, rutin and quercetin in mulberry leaves can enhance antioxidative activity and treat hyperglycemia [9]. Deoxynojirimycin has -glucosidase inhibitory activity, allowing it to reduce blood glucose levels [10]. Most of these active ingredients are found in the ethanol extracts of mulberry leaves. Mulberry leaf polysaccharide (MLP), another major active component, can promote insulin expression and modulate hepatic glucose metabolism in alloxan-induced diabetic mice [11]. To some extent, MLP can also act VU 0240551 as an antioxidant and antibacterial agent [12]. In veterinary clinics, adding MLP to pig feed during early VU 0240551 weaning can improve intestinal microbial ecology, reduce the occurrence of diarrhea, and improve overall growth [13]. In addition to the above effects, MLP can significantly improve the carbon clearance capacity and spleen lymphocyte transformation capability induced by ConA and can notably increase the thymus index [14]. Furthermore, previous studies have shown that Mouse monoclonal antibody to Hsp27. The protein encoded by this gene is induced by environmental stress and developmentalchanges. The encoded protein is involved in stress resistance and actin organization andtranslocates from the cytoplasm to the nucleus upon stress induction. Defects in this gene are acause of Charcot-Marie-Tooth disease type 2F (CMT2F) and distal hereditary motor neuropathy(dHMN) this porcine reproductive and respiratory syndrome (PRRS)-specific antibody titer could be significantly improved when pigs immunized with a PRRS vaccine were orally administered MLP [15], which indicates that MLP has some immunomodulatory properties. In this study, MLP was extracted by the ethanol precipitation method and further purified by DEAE-cellulose-52 and Sephadex G-100 column chromatography to afford two major purified polysaccharides (MLP-1 and MLP-2). The purified polysaccharides were characterized, and their immune properties were investigated. The purpose of this study was to investigate the effects of purified MLP-1 and MLP-2 on immune cellsin vitroand on humoral immunity and respiratory and intestinal mucosal immunity in chickensin vivo. This study provides a theoretical basis for the development of new animal immunopotentiators. == Materials and methods == == Drug preparation == The mulberry leaves were purchased from a mulberry plantation in Bozhou (Anhui, China), washed with tap water, and air dried at room temperature. MLP was extracted as follows: temperature of 100C, time of 2 hours, and ratio of water to raw material of 25 mL/g. The resulting solution was filtered, and the supernatant was mixed with ethanol in a 1:3 (v/v) ratio and stored overnight. The precipitates were.