Comparison and product analysis of alkaline hydrolysis and acid hydrolysis extraction processes for Lycium barbarum combined with polyphenols
Phenolic substances are widely distributed in the plant kingdom, including flavonoids, flavonoids, anthocyanins, catechins, etc., and are one of the most abundant secondary metabolites in plants. Due to its powerful antioxidant properties and significant effects in preventing various oxidative stress-related diseases such as antioxidant, anti-inflammatory, anti-tumor, hypotensive, hypoglycemic, and lipid-lowering effects, phenolic substances in food and natural/traditional Chinese medicine are receiving increasing attention. In plants, phenols exist in both free and bound forms, namely soluble phenolic substances that can be extracted by solvents and non extractable phenolic compounds (NEPC) that cannot be directly extracted by solvents. The latter binds to macromolecules such as sugars, hemicellulose, pectin, and proteins through covalent bonds such as ester bonds and glycosidic bonds, and is the main form of phenolic components in plants. Research on phenolic substances in food has found that NEPC exhibits better biological activity than free phenolic substances. For example, compared to free phenols, NEPC in grains shows higher peroxide radical scavenging ability and cellular antioxidant activity. It is generally believed that the biological activity of phenolic substances in plants is closely related to their bioavailability. Research on drug metabolism has found that free phenols may be degraded by digestive juices, while NEPC is not digested due to its association with large molecules. After entering the intestine, it is hydrolyzed by gut microbiota to exert biological activity and even exhibit effects similar to sustained-release formulations. In addition, some NEPC can be converted into other compounds that affect the human body. Therefore, in-depth analysis of NEPC in food and natural/traditional Chinese medicine can help comprehensively evaluate the biological activity of phenolic substances.
Lycium barbarum L. is a dried and mature fruit of Lycium barbarum L., a plant in the Solanaceae family. Modern pharmacological studies have shown that Lycium barbarum has multiple potential biological activities, most of which are related to antioxidant activity, which is the synergistic effect of Lycium barbarum polysaccharides and phenolic compounds. Our research group found in previous studies that gallic acid and ferulic acid can be detected in the alkaline hydrolysis products of Lycium barbarum polysaccharides, indicating the presence of NEPC. Relevant analysis results suggest that NEPC in Lycium barbarum plays an important role in its antioxidant activity. At present, research on phenolic compounds in goji berries mostly focuses on soluble phenols. Neglecting NEPC may underestimate the total content, antioxidant capacity, and positive impact of phenolic compounds in goji berries on human health.
To analyze NEPC, appropriate hydrolysis methods should be used first to break ester and glycosidic bonds and release phenolic components. At present, the most commonly used hydrolysis methods in literature are acid hydrolysis and alkaline hydrolysis. About 30% of studies use acid (usually hydrochloric acid or sulfuric acid) to hydrolyze the covalent bonds between NEPC and macromolecules. Compared to alkaline hydrolysis, acid hydrolysis can release more phenolic components, but the high temperature, high acidity conditions, and prolonged hydrolysis required for acid hydrolysis may often lead to the degradation of phenolic components. Common phenolic compounds in plants, such as ferulic acid, often bind to cell walls through ester bonds. Most studies use alkaline hydrolysis to analyze NEPC in plants. When hydrolyzing NEPC with inorganic bases such as sodium hydroxide, high temperatures are not required, but the reaction often needs to be carried out in the absence of light under nitrogen protection to prevent the degradation of phenolic components.
In order to comprehensively evaluate the phenolic components in goji berries, this study used acid hydrolysis and alkali hydrolysis to remove free phenolic components from goji berries. The yield of phenolic components was used as the evaluation index, and single factor experiments and response surface methodology were used to evaluate the effects of acid-base concentration, hydrolysis time, and solid-liquid ratio on the evaluation index. The process conditions for alkali hydrolysis and acid hydrolysis were optimized; And UPLC-Q-TOF-MS was used to compare the phenolic components in samples obtained by different hydrolysis methods, providing reference for selecting appropriate hydrolysis methods for different phenolic components.

 

Phenolic compounds are the most abundant secondary metabolites in plants and also one of the most concerned bioactive substances. Phenolic compounds in plants exist in both free and bound forms, with NEPC being the main component of phenolic substances in plants. Most previous studies have focused on the composition and biological activity of free phenols, with less attention paid to NEPC. Currently, an increasing number of studies suggest that NEPC may be an important active substance in food and natural/traditional Chinese medicine. Similarly, research on the free phenolic components in goji berries has been very in-depth, but there have been no reports of NEPC. The preliminary research of the research team found that Lycium barbarum polysaccharides can be hydrolyzed with weak sodium hydroxide (0.2mol/L) to obtain phenolic substances, indicating the possible presence of phenolic substances linked by ester bonds. It is generally believed that NEPC interacts with macromolecules such as cellulose, proteins, and lignin in a covalent bond form, or binds to plant fibers or embedded in cells through ionic bonds or physical interactions. The NEPC in goji berries is linked to polysaccharides or plant substrates, and further confirmation is needed.
NEPC cannot be directly extracted using water or alcohol solvents. To analyze NEPC in goji berries, alkaline hydrolysis or acid hydrolysis methods must be used to prepare NEPC. Alkali can break the ester bonds between phenolic components and substances such as sugars, proteins, pectin, fibers, etc., thereby releasing phenols and being detected. Sodium hydroxide is the most commonly used alkaline hydrolysis compound. During the alkaline hydrolysis process, phenolic components often undergo degradation under conditions such as oxygen, high temperature, and light. Therefore, they are often hydrolyzed at low temperatures in the absence of light under nitrogen protection. In this study, low-temperature hydrolysis was also carried out under nitrogen protection using sodium hydroxide as the hydrolysis catalyst. Acid hydrolysis usually uses strong acids such as hydrochloric acid. Hydrochloric acid can not only hydrolyze ester bonds, but also hydrolyze glycosidic bonds, effectively releasing monomers or oligomers of NEPC. In this study, the NEPC in goji berries measured by alkaline hydrolysis was 0.65 ± 0.05mg/100g, while it was 0.52 ± 0.03mg/100g in acid hydrolysis products. The difference between the two may be due to the different mechanisms of acid hydrolysis and alkaline hydrolysis. Both single factor experiments and response surface experiments showed that the optimal time required for alkaline hydrolysis was the same as that for acid hydrolysis. However, the yield of NEPC prepared by alkaline hydrolysis was higher, which may be due to the fact that alkaline hydrolysis more interrupted the binding of NEPC with structural proteins, dietary fiber, etc. Acid treatment breaks glycosidic bonds and dissolves sugars. Under acidic conditions, hydroxyl groups in polyphenol molecules are easily protonated, causing polyphenol molecules to break and release monomers or oligomers. Acidic hydrolysis at high temperatures can result in the loss of some phenolic substances; Under alkaline conditions, the hydroxyl groups in polyphenol molecules are more easily deprotonated than under acid hydrolysis conditions, resulting in slightly higher alkaline hydrolysis yields and reduced losses.
Previous studies have found differences in NEPC obtained through alkaline hydrolysis and acid hydrolysis. Caffeic acid, catechins, and gallic acid derivatives can be detected in the acid hydrolysis products of flour, while ferulic acid only exists in alkaline hydrolysis products, indicating that there may be more types of phenolic substances obtained from acid hydrolysis. This study used UPLC-Q-TOF-MS to analyze the composition of substances in the NEPC hydrolysis products of goji berries. The total ion chromatogram (see Figure 6) showed that there was no difference in the types of compounds between alkaline hydrolysis and acid hydrolysis products, but the number of acid hydrolysis products was significantly higher than that of alkaline hydrolysis products. This phenomenon may be due to the easy deprotonation and destruction of phenolic hydroxyl groups in strong alkaline environments, which makes them undetectable by mass spectrometry, while phenolic substances are more stable and easier to detect in acidic environments; On the other hand, alkaline hydrolysis can only break the ester bonds between phenols and macromolecules, while acids can hydrolyze not only ester bonds but also glycosidic bonds, so there are more types of phenolic substances that can be detected. From the perspective of products, the main ones are phenylpropanoid derivatives. Among the 11 identified compounds, there are 8 phenylpropanoids, 1 coumarin, 1 phenylpropanoid derivative, and 1 benzophenone. In the alkaline hydrolysis product, only four compounds can be identified, with ferulic acid being the main substance; And all 11 compounds identified can be detected in acid hydrolysis products. It is worth noting that among the 8 phenolic acids, 2 compounds may be the products of coumarin lactone ring opening during the hydrolysis of NEPC, rather than the native substances in goji berries. This may be related to the high concentration of acids used in the hydrolysis process. In the later stage, the concentration of acids or bases can be reduced to obtain a more complete molecular structure. Moreover, the NEPC contained in goji berries mainly consists of phenylpropanoid acid and coumarin components. Whether there are other components still needs further analysis of the hydrolysis products. In order to conduct in-depth analysis of NEPC in goji berries, mass spectrometry detection should be used as the guide in the later stage, and the concentration of acid/alkali during hydrolysis should be adjusted to obtain the true native NEPC belonging to goji berries.