Studies On The Functional Components Of Black Pigment From Vaccinium Bracteatum Thunb Leaf (VBTL)

Vaccinium Bracteatum Thunb, a resource of Chinese traditional medicine, belongs to the genus Rhododendron. It is grown massively in China but can be found worldwide.In this work, systematic studies were carried out on quantitative detection, extraction technology, isolation, purification, identification, antioxidant activity, free radical scavenging activity, and retina light damage prevention activity determination of Vaccinium Bracteatum Thunb leaf (VBTL) black pigment. The relationship was then discussed between the molecular structures and the physiological functions of VBTL black pigment.Based on the UV spectra of VBTL extracts and probe into the reaction mechanisms by three spectrophotometric methods and comparison of the relevant results with HPLC method, a relatively simpler, more accurate method, i.e., a colorimetric method (390nm) using AlCl₃ as the reagent was established for quantitative analysis of VBTL black pigment.A two step extraction process of VBTL black pigment was established, with each step using following conditions: extraction solvent 60%(v/v) ethanol, temperature 60℃, duration 1 hour, and material /ethanol ratio 1:35. By this optimized process 34.71mg flavones could be obtained from one gram VBTL, which accounts for 97% of total flavones.Eleven kinds of macroporous resins were screened with regard to absorption and desorption capacity for VBTL black pigment flavones. The AB-8 resin was determined to be the best one for previous purification of VBTL black pigment due to its relatively higher absorption and desorption capacity. Dynamic absorption capacity tests reveal that AB-8 presents the highest absorption capacity at pH 5.05, sample concentration 0.68¹.33mg/mL, and sample loading rate 3mL/min. When 95% ethanol was used as the eluent, the desorption rate reached 95%, and the purity of the flavones was increased from 14.5% to 50.32%. It was found that AB-8 should be regenerated after three times usage.The functional components in black pigment from VBTL were chromatographically purified with AB-8, polyamide, silica gel, and TSK gel Toyopearl HW-40. Five components, respectively coded B, C, D, E, and F, were thus purified. And by structural analyses with UV spectroscopy, HPLC-MS and NMR spectroscopy, these components were determined to be quercetin, chrysin, apigenin, kaempferol, and luteolinidin, respectively.The antioxidant properties of the five purified components were investigated by the ferric thiocyanate method and liposome phospholipid peroxidation method. Besides chrysin showing slightly higher antioxidant activities than rutin, all the other four components showed antioxidant activities significantly higher than rutin. The antioxidant activities of all five components increased significantly with the increase of their concentration. The antioxidant activities of samples tested follows the order: quercetin≈luteolinidin>apigenin>kaempferol≈Vc>chrysin>rutin>BHT> raw extracts. The half-inhibition concentration (IC₅₀) of the five samples against ferric thiocyanateperoxidation were determined as following: 0.0006188, 304.8, 2.9127, 4.0040 and 2.5450 u g/mL respectively for quercetin, chrysin, apigenin, kaempferol, and luteolinidin. All the samples can inhibit the generation of conjugated diene hydroperoxide during liposome oxidation following the order in capacity: quercetins?luteolinidin>apigenin>kaempferol>Vc>chrysin??rutinR=5BHT> raw extracts. The half-inhibition concentration (IC50) of the five samples were respectively 0.03986, 3.4417^ 0.1730, 0.5184, 0.2004ug/mL.The superoxide anion [O2 ? ] and hydroxyl radical [ ? OH] scavenging capacities of these componnents were investigated respectively in the pyrogallol-lumino and o-phenanthroline hydrate-CuSO4-Ascorbic acid-H2O2 luminescence system. The IC50 of [O2 ? ] scavenging of five components were determined: 5.48, 17.44, 10.73, 12.74, and 5.91 V mol/L. The IC50 of [ ? OH] scavenging of the five components were also determined: 5.63, 22.45, 11.58, 16.61, and 5.99 u mol/L.The electroretinogram (ERG) indicated that VBTL black pigment could effectively prevent rabbit retina from being light damaged. The total superoxide (T-SOD) levels and the maleic dialdehyde (MDA) levels in the rabbit retina were measured. It was found that T-SOD decreased and MDA increased in the control group (rabbit retina light damaged), while T-SOD and MDA levels of the test group (rabbit retina light damaged, and VBTL black pigment treated) remained similar to that of natural rabbit retina. Based on the mechanism of retina light damaging and experimental observations as above, the retina protection effects of VBTL black pigment was probable to be ascribed to their antioxidant and free radical scavenging activities.The relationship between the molecular structure and the antioxidant and free radical scavenging functions of the components were discussed. The activities of these components were not only associated with the conjugate double bond system, but with the hydroxyl substitutes in their molecular structures.