Phytochemical Constituents Propolis Flavonoid, Immunological Enhancement, Anti-porcine Parvovirus Activities of isolated from the Propolis

Propolis was widely used in health preservation and disease healing, it contains many ingredients. The previous study had been revealed that the propolis has a wide range of efficacy, such as antiviral, immune enhancement, anti-inflammatory and so on, but its antiviral components and underlying mechanism of action remain unknown. In this study, we investigated the chemical composition, and anti-PPV and immunological enhancement of Propolis Flavonoid(PF). Chemical composition of PF was distinguished by UPLC-Q/TOF-MS/MS analysis.The presence and characterized of 26 major components was distinguished in negative ionization modes.To evaluate the effects of PF used as adjuvant on the immune response porcine parvovirus (PPV). Thirty Landrace-Yorkshire hybrid sows were randomly assigned to 3 groups, and the sows in adjuvant groups were intramuscular injected PPV vaccine with 2.0 mL PF adjuvant (PA), oilemulsion adjuvant (OA), respectively. After that, serum hemagglutination inhibition antibody titers, IgM and IgG subclasses, eripheral lymphocyte proliferation activity, and concentrations of cytokines were measured. Results indicated an enhancing effect of PA on IgM, IL-2, IL-4, IFN-γ and the IgG subclass responses. These findings suggested that PA could significantly enhance the immune responses. Furthermore, we screened the chemical components the effective of anti-PPV, Ferulic acid have an excellently anti-PPV effective.

,as well as anti-PPV effect in vitro.
Porcine parvovirus (PPV) is one of main pathogens that cause swine reproductive disorder and increasing economic losses in the world. [9][10][11][12] Several studies have pointed out that vaccination is an effective method for controlling this disease. [13][14] The successful vaccination depends on their association with potent adjuvant which can increase the immunogenicity of vaccine. A better adjuvant can activate specific effectors of the immune system, such as cytotoxic or auxiliary T cells (Th1/Th2) and strengthen the humoral and/or cellular immune responses against that antigen. 15-16 On the other hand, suitable adjuvant should have lower toxicity and side effects. 17 But there are some shortages in commonly used adjuvants, for example, oilemulsion adjuvant (OA) can cause inflammation, induration or necrosis in the local, and disseminated granulomas in lungs, liver, kidneys, heart, lymph nodes and skeletal muscles in rabbits or rats. 18 Aluminum salt adjuvant (AA) is a widely used adjuvant in human vaccines licensed by the US FDA, 19 but it is a poor inducer of Th1 cellular immune response and easy to induce immunoglobulin E antibody response associated with some allergic reactions. [20][21] Thus,nature product Propolis as a immunologic adjuvant has attracted more and more attention due to its minor side effects and more pharmacological efficacy.
Most of the studies in the literature have investigated the anti-PPV and immunological enhancement effective of the propolis flavonoid isolated from the Propolis. 22 Propolis flavonoid (PF), a kind of ingredient extracted from propolis, as a harmless natural adjuvant has been used in chickens vaccinated with activated vaccine, and the results showed that PF could improve the immune-enhancing activity in the humoral and cellular immune response. [23][24] Since the major effective of propolis are derived from active ingredient, the anti-PPV and immunological enhancement effective is highly dependent on its extraction method and content of active ingredients of propolis flavonoid. Ethanol extracted propolis is still the main method for propolis flavonoids.However，still little is known regarding the anti-ppv effective of propolis flavonoids compounds,such as Ferulic acid,Quercetin,Chrysin and so on. 4 Thus,this study a ultra-performance liquid chromatography-quadrupole/time-offlight mass (UPLC-Q/TOF-MS) spectrometry was used to distinguish and exhibit the Standard chemical map of propolis flavonoid.In addition,this study was to evaluate the adjuvant effect and characteristic of PF on the humoral and cellular immune response of immunized pigs and screen out good anti-PPV propolis flavonoids compounds.    The crude total propolis flavonoids was about 80% in the alcohol extraction.

Materials and
According to the analytical results for the chemical constituents(as shown in Figure   1A),the different components in the Supernatant and Substratum of propolis extract are mainly small polar components.We analyzed the chemical fingerprint (UHPLC-Q/TOF-MS) of PF ( Figure 1B).The analysis in negative ionization modes revealed the presence and characterized of 26 major components (Table 1).  to obtain m/z 178, which was speculated to be Ferulic Acid by comparing the mass spectrogram information with the reference substance.    vaccination, IgM level of PA group only was numerically higher than the other adjuvant groups, and higher than that of BC group.In addition,IgA serotype is monomer, and the immune function is weak.The IgA level of all groups have not significant difference, due to the sample was the serum( Figure 3B). IgG1 levels in three adjuvant groups were higher than that of BC group when the samples were collected after immunization. On days 7, 14, and 35 after vaccination, IgG1 levels of PA and OA groups were higher than that of BC group; on day 21 after vaccination, IgG1 level of PA group was higher than that of BC group( Figure 3C).PA triggered stronger IgG2 level than the OA and BC groups on days 14, 21 and 35, respectively( Figure 3D).The IgG3 level was higher in the pigs of PA group than those of the pigs immunized with other adjuvant groups. However, no significant difference was observed for the responses of IgG3 between OA and BC groups ( Figure 3E). As  Table 2. In all adjuvant groups, the peak values of lymphocyte proliferation activity were on day 7 after vaccination.

Identification and characterized of propolis flavonoids
The A570 values in PA group were significant highest and significantly higher than those of the other two adjuvant groups from days 7 to 21 . On day 35, the A570 values in three adjuvant groups were significant higher than that of BC group.  a -c Data within a column without the same superscripts differ significantly (p<0.05).

Serum antibody titer
The serum HI antibody titers of each group are shown in Table 3. As compared with the BC group, the antibody titers in all vaccination groups increased (p<0.05) from days 7 to 35. On days 7 and 14 after vaccination, the antibody titers in PA and OA groups were significant higher than that of AA groups. On days 21, 28, 35 and 42, the antibody titers in PA group were significant lower than those of OA group, while significant higher than those of AA group.

Serum cytokine level detected by Elisa assays
Serum cytokine levels were listed as Figure 4.The serum IL-2,IL-4 and IFN-γ levels in three adjuvant groups were significant higher when compared with the BC group( Figure 4A,4B and 4F).Interestingly, the serum IL-6,IL-10,IL-12 levels in three groups have no significant higher when compared with the BC group( Figure 4C,4D and 4E).IL-6 and IL-12 could promote the express level of TNF-α.In addition.In spire of this found,we think PF could immune enhancement in vivo. increased cellular immune responses. [26][27] In this study, PF was various in regards to their adjuvant effects on the immune responses stimulated the immune system cells to produce cytokines. As shown in Fig 4,  Th1 activation contributes to cell-mediated immunity whereas Th2 activation favours the humoral immune response. 29 Th1/Th2 balance is a prerequisite for the functionality of immune system against infections. PF immunomodulatory action has been widely investigated lately, both in vitro and in vivo. [30][31] PF has been suggested to be a promising adjuvant substance in duck inactivated vaccines. 32 Important functional properties of immune cells are their capability to synthesize and secrete soluble polypeptide factors referred to as cytokines. Most cytokines are secreted and then bind to specific receptors on the surface of target cells. Upon binding they act to regulate growth and/or differentiation and to optimize the immune response. Using a Polish sample, Ansorge 33 found that propolis has immunoregulatory effects that may be mediated by Erk2 MAP kinase signals that promote cellular growth. Oršoli´c suggested that propolis stimulated macrophages thus influencing specific and non-specific immune defence mechanism 34 . Activation of macrophages is important for the modulating property of tested compounds, since it leads to the production of