Purification And Structural Characterization Of Flavonoids And Polysaccharides From Platycladus Orientalis(L.) Franco Leaves And Their Biological Activities

The leaves of Platycladus Orientalis(L.) Franco is used as medicinal materials for a long history and could be used as functional food materials approved by China Food and Drug Administration(CFDA). In this study, the purification, structural characterization of flavonoids and polysaccharides from Platycladus orientalis(L.) Franco leaves and their biological activities such as anti- inflammatory effect of flavonoids, immuno-stimulating activity and antiviral activity of polysaccharides were investigated.The purification of flavonoids from he crude water extract of the Platycladus orientalis(L.) Franco leaves using macroporous adsorption resins. For purification of flavonoids, six resins(NKA-9, ADS-F8, ADS-17, AB-8, D101 and ADS-5) were screened. Among them, AB-8 resin showed very good adsorption and desorption capacities. The static adsorption and desorption kinetics on AB-8 resin for enriching flavonoids were investigated. The adsorption isotherms results showed that low temperature was good for the adsoption of flavonoids when the equilibrium concentration was higher than 0.10 mg/mL. Optimal conditions for the dynamic experiments with AB-8 can be summarized as follows: total flavonoids content in loading solution: 0.60 mg/mL, processing volume: 2 BV, flow rate: 1.0 mL/min, temperature: 25 °C, desorption solvent: 40% ethanol solution(v/v). The POF had three thermally decomposed temperatures which were 347.6 °C, 437.5 °C and 494.8 °C according to the results of simultaneous thermogravimetry-differential scanning calorimetry(STA/TG-DSC) analyses. The ultraviolet- visible(UV-Vis) spectroscopy and FT-IR spectroscopy were applied to structural characterization. Identifications were performed using liquid chromatography-mass spectrometry(LC-MS) by comparing the mass spectra of POF with earlier publications. The main flavonoids were identified as boeravinone B, esculin, amentoflavone, glabridin, afromosin, sophoracoumestan B, licoflavanone and rivularin 2′-O-glucuronide. Meanwhile, POF in the dosages range of 25 to 400 μg/mL showed a significant anti- inflammatory effect on lipopolysaccharide(LPS)- induced RAW 264.7 mouse macrophage cells for inhibition on NO, IL-6 and TNF-α secretion in a dose-dependent maner in RAW 264.7 cells. These results indicated the potential application of POF in medicine and food fields to reduce the risk of chronic inflammatory diseases.A new polysaccharide fraction, here named POP1, was obtained from the leaves of Platycladus Orientalis(L.) Franco by water extraction followed with alcohol precipitation. Structure characterization revealed that POP1 had an average molecular weight of 8.10 × 103 Da and consisted of glucose(64.96%), arabinose(12.58%), mannose(10.97%), galactose(6.55%) and rhamnose(5.74%). The main linkages type of POP1 were comprised of(1â†'5)-linked α-L-Ara,(1â†'3)-linked α-L-Man,(1â†'6)-linked β-L-Rha,(1â†'4)-linked α-D-Glc,(1â†'6)-linked α-D-Glc,(1â†'6)-linked β-D-Gal,(1â†'3,6)-linked β-D-Gal and terminated with α-L-Man and α-D-Glc residues basing on the periodate oxidation-Simith degradation, methylation and NMR analyses. Simultaneous thermogravimetry-differential scanning calorimetry was applied to analyze the stability of POP1. The results showed that POP1 had good thermal stability and the thermally decomposed temperature of POP1 was around 320.9 °C. Meanwhile, POP1 exhibited excellent immuno-stimulating activity by enhancing NO, TNF-α, IL-6 and IL-12 secretion in mouse macrophage cell lines RAW 264.7 via activating the related mRNA expression. Besides, POP1 showed significant anti-HBV activity through inhibiting the expression of HBs Ag(IC50=1.33 ± 0.12 mg/mL) and HBe Ag(IC50=1.67 ± 0.13 mg/mL) and interfering the HBV DNA replication(IC50=0.80 ± 0.03 mg/mL). These results suggested that POP1 could be developed as an immunoregulatory ingredient for functional foods or vaccine adjuvant for HBV infection treatment or prevention.Another new polysaccharide fraction, here named POP2, was extracted and purified from the leaves of Platycladus Orientalis(L.) Franco. Structure analyses revealed that POP2 had an average molecular weight of 9.69 × 103 Da and consisted of glucose(63.95%), arabinose(14.39%), galactose(11.42%) and mannose(10.24%). The main linkage types of POP2 were comprised of(1â†'4)- linked α-D-Glc,(1â†'6)- linked α-D-Glc,(1â†'6)-linked β-D-Gal,(1â†'5)-linked α-L-Ara, and terminated with α-L-Man and α-D-Glc residues basing on the monosaccharide composition analysis, linkage analysis by methylation and NMR analysis. RAW 264.7 mouse macrophage cells could be polarized to M1 subtype by 1.0 μg/mL LPS sitimulating for 8 h using the IL-6, TNF-α and iNOS mRNA expression as screening indexes. Meanwhile, RAW 264.7 cells could be polarized to M2 subtype by 20 ng/mL IL-4 stimulating for 8 h according to the mRNA expression of ARG1 and MCP-1. POP2 in the dosages range of 31.25 to 500 μg/mL could not only enhance the secretion of inflammatory cytokines such as IL-6 and TNF-α, but also increase the release of anti- inflammatory cytokines such as IL-10 in LPS- induced macrophage cells. Besides, the secretion of NO was significantly inhibited by POP2 in M1 subtype model. POP2 could enhance the release of inflammatory cytokines such as NO, IL-6 and TNF-α, while the secretion of the anti- inflammatory cytokine TGF-β was significantly suppressed in IL-4 induced macrophage cells. These results suggested that POP2 had multiple functions on macrophage polarization and could regulate the balance of the production of inflammatory and anti- inflammatory cytokines. So POP2 had the potential to be developed as a bioactive ingredient for functional foods and dietary supplements.