Determination And Purification Of Flavonoids From Citrus Reticulata Cv. Suavissima And Citrus Changshanensis Fruit And Their Bioactivity Evaluation

Flavonoids profile and their bioactivities (antioxidant, anti-cancer, and anti-diabetic activity) were investigated in different parts of Citrus reticulata cv. Suavissima (Ougan) and Citrus changshanensis (Huyou) fruit. Separation and purification of main flavonoids compounds from Ougan and Huyou fruits was established and the contribution of individual flavonoid for different bioactivities were analyzed and discussed as well. The main results are as follows:1. Eight flavonoid compounds, including four flavanone glycosides, i.e. naringin, hesperidin, neohesperidin, poncirin, and four polymethoxylated flavones (PMFs), i.e. sinensetin, nobiletin, tangeretin,5-hydroxy-6,7,8,3’,4’pentamethoxyflavone (5HPMF) were identified from Ougan fruit. Ten flavonoid compounds, including six flavanone glycosides, i.e. eriocitrin, neoeriocitrin, naringin, narirutin, hesperidin, neohesperidin and four PMFs, i.e. HexaMF, nobiletin, HeptaMF, tangeretin were identified from Huyou fruit. In both cultivars, flavanones were detected in all fruit parts, i.e. flavedo, albedo, segment membrane (SM), and juice sacs (JS), while PMFs were only present in the flavedo. In addition, albedo contained the highest contents of flavonoids in both cultivars. Neohesperidin represented as the majority of flavonoids in Ougan fruit, while naringin and neohesperidin were predominant flavonoids in Huyou fruit. All the fruit extracts exhibited DPPH redical scavenging activity where flavedo showed the highest scavenging rate. Low temperature storage at6℃for120d maintained the majority of flavonoids in both Ougan and Huyou fruit, though the content of individual flavonoids changed to different extents.2. Efficient separation and purification of neohesperidin and naringin from the albedo of Ougan and Huyou were established by the combination of macroporous resin column chromatography and high-speed countercurrent chromatography (HSCCC). After the first cleaning-up with D101resin, the purity of neohesperidin and naringin increased from4.70%in the crude extract to32.38%in the resin-refined sample, and from3.92%to38.07%, respectively. Further HSCCC purification with a two-phase solvent system composed of chloroform-methanol-n-butanol-water (4:3:1:2, v/v) resulted in final purity of more than97%for neohesperidin and naringin, respectively. Further isolation and purification of three flavanone glycosides (naringin, hesperidin, neohesperidin) and four PMFs (sinensetin, nobiletin, tangeretin,5HPMF) from Ougan flavedo was carried out by solvent extractions, Sephadex LH-20gel filtration, and HSCCC. All the purified compounds were analyzed and confirmed by HPLC and LC-MS.3. The anti-cancer activities of all the four parts of Ougan and Huyou fruit were investigated. Results showed that only Ougan flavedo extract exhibited significant inhibitory effect on the growth of HepG2liver, HL-60leukemia, and MDA-MB-231breast cancer cell lines. Results of antiproliferative activity of individual flavonoids showed that four PMFs (sinensetin, nobiletin, tangeretin,5HPMF) exhibited significant inhibition effects on the proliferation of the three cancer cells tested, indicating that the antiproliferative activity exerted from the Ougan flavedo may partly attribute to PMFs. Two flavanone aglycones (naringenin, hesperitin) also exhibited significant antiproliferative activity, indicating that the primary metabolites of corresponding flavanone glycosides (naringin, hesperidin, neohesperidin) may act on the cancer cells in vivo. In addition, nobiletin and tangeretin showed significant inhibitory effects on the migration of SKOV3ovarian cancer cells in the wound healing assay.4. The anti-diabetic activities of all the four parts of Ougan and Huyou fruit were investigated. Four parts of Huyou fruit all showed increased effects on glucose consumption in HepG2cells while Ougan extracts showed low activity. Results of individual flavonoid showed that cells treated with naringin and neohesperidin showed increased consumption of glucose, which was similar to metformin. In addition, combination treatment of naringin and neohesperidin resulted in synergistic effect. Western blot showed that the enhancement of glucose consumption was associated with increased phosphorylation of AMP-activated protein kinase (AMPK). In addition, naringenin and hesperetin exhibited increased glucose comsumption and a-glucosidase inhibitory activities, indicating that the primary metabolites of naringin and neohesperedin may have hypoglycemic effect in vivo.Our results showed that Ougan and Huyou fruits are rich sources of flavonoids. Ougan fruit extracts exhibited antiproliferation effect on cancer cells. Huyou fruit extract exhibited anti-diabetic activity through inducation of glucose consumption.