A novel method for real-time adulteration identification of heated-honey processed Astragali radix using REIMS-iKnife and its regulatory effects on gut microbiota, SCFAs, and immune function in spleen-deficient rats.

BACKGROUND

Astragali radix (AR) is a well-known traditional Chinese medicine (TCM). AR processed with heated honey (HAR) can enhance the effect in treating spleen deficiency. However, some people have used heated sugar as a substitute for heated honey in processing AR. The mechanism underlying the effect of HAR in ameliorating spleen deficiency has not been fully elucidated so far. Therefore, this study aimed to determine whether AR has been processed with heated honey or heated sugar. Subsequently, we compared the differential effects of AR and HAR on the composition of intestinal microbiota, short-chain fatty acid (SCFA) metabolism, and immune responses in spleen-deficient rats.

METHODS

Rapid evaporative ionization mass spectrometry (REIMS) was employed to detect AR processed with different types of heated honey, different types of heated sugar, and heated honey mixed with heated soft sugar in various proportions. 16S rRNA high-throughput sequencing was conducted to analyze the effects of AR and HAR on intestinal flora diversity in spleen-deficient rats. The regulatory effects of AR and HAR on SCFA metabolism in spleen-deficient rats were measured using headspace gas chromatography-mass spectrometry (HS-GC-MS). The effects of AR and HAR on specific and non-specific immunoreactivity in spleen-deficient rats were evaluated using enzyme-linked immunosorbent assay (ELISA). Furthermore, correlations were detected between different intestinal flora, short-chain fatty acids, and immune response factors.

RESULTS

We successfully identified AR processed with heated potpourri honey, linden honey, and locust flower honey as genuine heated honey processed products. Additionally, we achieved accurate identification of AR processed with heated soft sugar or granulated white sugar and determined the proportion of adulterated sugar in samples. Both AR and HAR increased the abundance of beneficial gut bacteria, reduced the abundance of pathogenic bacteria, upregulated the synthesis of propionic acid and butyric acid, and modulated the metabolism of isovaleric acid. Notably, the positive effect of HAR on propionic acid expressinon was stronger than that of AR. Meanwhile, both AR and HAR significantly modulated the expression levels of immune factors in serum and colon samples, with HAR exhibiting superior regulatory effects compared to AR.

CONCLUSION

The procedure developed in this study can help rapidly and accurately determine whether sugar has been added during the heated honey processing of AR. The enhanced effect of HAR on spleen deficiency may be associated with its regulatory effect on the abundance of Lactobacillus and Turicibacter genera. Through this effect, it promotes the synthesis of propionic acid, upregulates anti-inflammatory factors, downregulates pro-inflammatory factors, and enhances the immunomodulatory effect.