| Natural colored cotton is a kind of environmental-friendly textile fiber with many excellent properties, such as antioxidant, antibacterial, and ultraviolet proofing properties, thus it has good market application value and development prospect. However, the development and utilization of naturally colored cotton is still limited by its own shortcomings, i.e. incomple chromatography, color unstability and not enough brightness. Therefore, a clear structure of natural colored cotton pigment is of vital significance to development and utilization of natural colored cotton. In this paper naturally green cotton was studied. Firstly, the pigment of green cotton was segmented and enriched by normal phase silica gel chromatography column and physicochemical properties of purified pigment was analyzed. Then, the purified pigment was further purified by polyamide chromatography column and a relatively pure substance of pigment was obtained after semi-prepared HPLC separated again. UV spectrum, ESI-MS and NMR was used to analyze its structure. At last, the antioxidant stability of green cotton pigment was studied.The crude extracted pigment of green cotton was divided into three sections of purification by normal phase silica gel chromatography column eluted with different polarity of dichloromethane and methanol system and the purified pigment sections was analyzed by HPLC and UV spectrum. The HPLC chromatograms showed that green cotton pigment has multiple components and the components with green color portion in pigment was proved to be lipophilic substances. 10 % dichloromethane eluted pigment contained more substances, which need further purify. The UV spectrum indicated that green cotton pigment consisted of many different types of pigment substance, then, the high polarity of the fraction of pigment was identified as flavonoids, and pigment eluted by 40 % dichloromethane had the physicochemical properties of 3-hydroxy substituted dihydroflavone containing two phenolic hydroxyl groups. Optimization of the separation of pigment eluted by 100 % dichloromethane with semi-prepared HPLC found that the structure of two main components with green light in pigment were similar with the same UV absorption peaks, of which wavelength were at 241 nm, 286 nm, 313 nm.Polyamide chromatography column was used to further purify the pigment eluted by 10 % dichloromethane in silica gel chromatography column with smaller gradient of dichloromethane and methanol system and four components were obtained, which separated by semi-preparation again. The ESI-MS showed that the four components had the same molecular weight of 340, indicating that they were isomeride, in addition, they had same molecular debris with mass charge ratio of 163. The comprehensive inference from UV spectrum, ESI-MS, NMR showed that a higher content of four components was esters with long aliphatic chain and a probable structure was 1, 2, 3-three hydroxy caffeic acid ester, but the specific structure needs further study.Scavenging DPPH radical method was used to study on the stability of antioxidant activity of green cotton pigment. The result shows that in acidic and alkalescent conditions, the pigment of natural green cotton has high and stable ability of scavenging DPPH free radical, but its antioxidant capacity decreased rapidly under strong alkaline conditions. The DPPH radical scavenging activity of green cotton pigment fell to 2.97 %, when the pigment was treated in the alkali solution with the pH value of 13.When adjusted to neutral, green cotton pigment recovered to 81.38 % of its original radical scavenging ability. The main structure of pigment was unchanged when in the alkaline condition, but the structure of some components of the pigment had changed irreversibly. It was found that no significant change in radical scavenging ability of green cotton pigment was caused, when treating the pigment at 150 ℃ for 1 h. Light irradiation had significant effect on antioxidant activity of the pigment of natural green cotton. The radical scavenging ability of green cotton pigment dropped to 57.10 % when the pigment was exposed to the sun light for 5 days. |
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