Authors: Lin Shuying¹, Gao Jianhua¹, Guo Qingquan², Ning Zhengxiang¹
(1. School of Food and Biological Engineering, South China University of Technology, Guangzhou, Guangdong 510640; 2. Institute of Chemical Engineering, South China University of Technology, Guangzhou, Guangdong 510640)
Abstract
Dihydromyricetin (DMY or DHM) is an important flavonoid with significant antioxidant activity. The solubility changes were determined using the dissolution and weighing method, and the chemical structure changes were studied using UV-visible spectroscopy. The results show that Dihydromyricetin solution is prone to oxidation, making it relatively unstable. It remains stable under conditions not exceeding 100°C, with heating time less than 30 minutes, and in acidic to neutral pH conditions. However, transition metal ions such as Al³⁺, Fe³⁺, and Cu²⁺ catalyze the oxidation of Dihydromyricetin.
Keywords: Dihydromyricetin; Spectroscopy; pH value; Chemical structure; Stability
1.Introduction
Dihydromyricetin (3,5,7,3’,4’,5’-hexahydroxy-2,3-dihydroflavonol, Dihydromyricetin) is a polyphenolic flavonoid found extensively in Vitaceae plants, especially in the genus Ampelopsis. In young stems and leaves of Ampelopsis plants, the content can reach 20% to 28% of dry weight, with even higher concentrations in young leaves. Products made from these leaves, such as vine tea, have been shown to have anti-inflammatory, cough suppressant, expectorant, analgesic, antibacterial, antihypertensive, lipid-lowering, anti-cancer, and hepatoprotective effects. Additionally, Dihydromyricetin exhibits excellent preservative and antioxidant properties, showing potential as a new natural preservative and antioxidant with broad development prospects.
Given the unique effects of Ampelopsis, research on the extraction and physiological activity of Dihydromyricetin has become increasingly active. Current research focuses mainly on two aspects: solvent extraction methods, including compound organic solvent and water extraction, and chromatographic methods, primarily macroporous adsorption resin extraction.
Flavonoid compounds generally exhibit UV-visible absorption spectra, and UV-visible spectroscopy can accurately and rapidly detect changes in their chemical structure. Therefore, this study primarily used UV-visible spectroscopy to examine the effects of pH, temperature, and metal ions on the chemical structure stability, solubility, and color of Dihydromyricetin to provide a basis for its application in the food, pharmaceutical, and cosmetic industries.
2.Materials and Methods
2.1Experimental Materials, Instruments, and Reagents
· Materials: Young leaves of Ampelopsis, collected from Baiyun Mountain, Guangdong, identified according to literature.
· Instruments: UV-visible spectrophotometer, electronic balance, thermostatic instrument.
· Reagents: All chemical reagents used were of analytical grade.
2.2Experimental Methods
2.2.1Extraction and Preparation of Dihydromyricetin
Dried young leaves of Ampelopsis were boiled with water, concentrated, and filtered. The solution was left to stand until pale yellow crystalline particles precipitated, which were then vacuum filtered and dried to obtain a crude product. This crude product was extracted using a Soxhlet extractor, and the lower layer solution was concentrated. The concentrated solution was diluted with distilled water (1:100 by mass), filtered, and left to stand, leading to the precipitation of white crystals. The crystals were recrystallized several times to obtain fine white needle-like crystals, identified as Dihydromyricetin with a purity of 95% as determined by high-performance liquid chromatography (HPLC).
2.2.2Detection of Thermal Stability of Dihydromyricetin in Aqueous Solution
0.5 g of Dihydromyricetin (purity > 95%) was dissolved in 900 mL of water and boiled. Samples were taken every 5 minutes up to 600 minutes, diluted to 25 mL, and scanned in the wavelength range of 200-800 nm.
2.2.3Detection of pH Effects on Dihydromyricetin Stability
1.0-1.5 g of Dihydromyricetin (purity > 95%) was dissolved in water solutions with different pH values, heated to 100°C for 5 minutes to dissolve, then cooled to room temperature. After 72 hours, the crystal state, color, and color of the supernatant were observed. The samples were vacuum filtered, and the mass of the residue was measured to calculate the solubility of Dihydromyricetin under different pH conditions. The supernatant was scanned in the wavelength range of 220-800 nm to compare stability at different pH levels.
2.2.4Detection of Metal Ion Effects on Dihydromyricetin Stability
Approximately 0.05 g of Dihydromyricetin (purity > 95%) was dissolved in 2000 mL of distilled water. Samples of 50 mL were taken, and 1 mL of 0.1 mol/L metal ion solution was added. The mixture was allowed to stand for 6 hours and then scanned in the wavelength range of 200-800 nm.
3. Results and Discussion
3.1 Thermal Stability of Dihydromyricetin in Aqueous Solution
The thermal stability of Dihydromyricetin is closely related to time. As shown in Figure 1a, after heating a Dihydromyricetin solution at 100°C for 30 minutes, no changes were observed in the UV-visible spectrum. However, after 35 minutes of heating, significant changes occurred. The absorption peak at 294 nm and the shoulder around 324 nm remained unchanged, but the peak heights decreased, and an additional absorption in the 350-400 nm range began to appear. The color of the solution visibly darkened, indicating that the Dihydromyricetin configuration was changing, possibly due to oxidation.
Further research found that Dihydromyricetin’s melting point is around 245°C. Beyond this temperature, decomposition occurs. However, under dry conditions and below this temperature, Dihydromyricetin demonstrates good thermal stability.
3.2 Effect of pH on Dihydromyricetin Stability
As shown in Figures 2-4, the UV-visible spectrum of Dihydromyricetin changes significantly with increasing pH. Under acidic conditions (pH 2.0-3.5), Dihydromyricetin shows a characteristic absorption peak at 294 nm with a shoulder around 324 nm. No significant absorption is observed in the visible light region, indicating that Dihydromyricetin’s configuration is stable in acidic conditions. Under neutral conditions, the absorption peak at 294 nm decreases, while the shoulder at 324 nm increases, indicating partial changes in Dihydromyricetin’s configuration. In alkaline conditions, the absorption peak at 294 nm disappears, the shoulder at 324 nm increases, and a new absorption peak appears around 498 nm, indicating a significant change in Dihydromyricetin's configuration.
Based on these findings, it is recommended to use and store Dihydromyricetin under acidic to neutral conditions (pH < 7.0) to maintain its antioxidant properties.
3.3 Effect of Metal Ions on Dihydromyricetin Stability
Metal ions in food raw materials and processing may affect Dihydromyricetin stability. The study found that Dihydromyricetin reacts differently to various metal ions. Transition metal ions such as Al³⁺, Fe³⁺, and Cu²⁺ significantly alter Dihydromyricetin’s absorption spectrum, leading to changes in its structure due to oxidative reactions.
4. Conclusion
1.DMY or DHM becomes unstable in aqueous solutions with increasing time and temperature, undergoing irreversible oxidation. It is recommended to avoid excessive heating during extraction and processing, keeping boiling time under 30 minutes.
2.pH is a crucial factor affecting Dihydromyricetin stability. Acidic to neutral conditions (pH < 7.0) are suitable for Dihydromyricetin application and preservation.
3.Metal ions like Al³⁺, Fe³⁺, and Cu²⁺ should be avoided during Dihydromyricetin extraction and application due to their catalytic effects on Dihydromyricetin oxidation.
References:
1.Zhang Yousheng, Yang Weili, Xiong Haoping. "Determination of Myricetin in Ampelopsis Plants by RP-HPLC." Chinese Herbal Medicine, 2001.
2.He Guixia, Pei Gang, Zhou Tianda, et al. "Determination of Total Flavonoids and Dihydromyricetin Content in Ampelopsis." China Journal of Chinese Materia Medica, 2000.
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