Optimization of Aqueous and Lipid-Soluble Extraction Methods for Alfalfa (Medicago sativa)

Beneficial effects of alfalfa on the intestinal microbiome and immune system have been noted in murine, swine, and poultry literature; however, a disparity exists based on consistency in cutting used, extraction method and analysis, and outcomes measured in animal models. The present objective was to optimize the preparation of aqueous and lipid-soluble extracts of first and fifth cutting alfalfa to be used in a future animal study. Based on literature reviews, aqueous components were extracted in a 1:5 alfalfa: water ratio at room temperature or heated (60°C) with continuous stirring for 24 hours and a solvent change once at 12 hours. Chloroform extracts were completed in 1:4 or 1:5 alfalfa: chloroform ratios at room temperature or heated (40°C) with continuous stirring for a total of 72 hours, and extract collection and solvent change every 24 hours. Aqueous extracts were lyophilized and analyzed for CP, crude fat (CF), and GE. Chloroform extracts were evaporated to dryness and analyzed for CP and CF. Statistical differences between temperatures, ratios, and alfalfa cutting were analyzed on JMP using a Welch’s t-test with significance reported at P<0.05. Room temperature aqueous extracts contained on average 25.64% CP (n=8, all analyses), 1.87% CF, and 3588.71 cal/g GE versus 25.99% CP, 1.38% CF, and 3660.61 cal/g GE when heated. Increasing extraction temperature resulted in 0.35% more CP, 0.49% less CF, and 1.96% more GE. Extraction temperature and cutting did not significantly affect CP (P=0.71 and 0.59, respectively), CF (P=0.22 and 0.17), or GE (P=0.06 and 0.36). Alfalfa extracted with chloroform at the 1:4 ratio resulted in 14.06% CP (n=14) and 39.28% CF (n=12) compared to 15.68% CP and 34.59% CF at the 1:5 ratio. Room temperature extracts contained 15.48% CP and 34.83% CF, whereas heated extracts contained 14.27% CP and 39.04% CF. The 1:5 ratio resulted in a 1.62% increase in CP (P=0.25) and a 4.69% decrease in CF (P=0.20). Heating decreased CP by 1.21% (P=0.39) and increased CF by 4.21% (P=0.25). Chloroform extracts of fifth cutting alfalfa contained 20.64% less CP (n=16; P=0.009) and 25.5% more CF (n=12; P=0.001) than first cutting extracts. The results from these trials led to the selection of room temperature aqueous extracts and 1:4 chloroform extracts with gentle heating for use in animal feeding trials to determine the bioactive effects of whole and extracted first and fifth cutting alfalfa on the immune system and intestinal microbiome.