Extraction, purification, and quantification of hesperidin from the immature Citrus grandis/maxima fruit Nepal cultivar

Abstract

Abstract. Manikyam HK, Tripathi P, Patil SB, Lamichhane J, Chaitanya MVNL, Patil AR. 2022. Extraction, purification, and quantification of hesperidin from the immature Citrus grandis/maxima fruit Nepal cultivar. Asian J Nat Prod Biochem 20: 21-26. Hesperidin, a flavanone group of flavonoids and a 7-O-rutinoside of hesperitin, is abundant in citrus fruits. Pomelo or Citrus grandis (L.) Osbeck/ C. maxima (Burm.) Merr. is grown and cultivated in Nepal as seasonal edible fruit. Pomelo has flavanone and other chemical constituents. Flavanones like naringin, hesperidin, neohesperidin, and naringenin are present in the fruits of C. grandis. Many extraction techniques have been reported in the extraction and purification of hesperidin citrus fruits like sweet lemons, grapefruits, mandarins, etc. However, little information has been provided on hesperidin content in pomelo fruits. In order to estimate the quantity of hesperidin in the pomelo fruits of Nepal, we have first made a study. The immature pomelo fruits of size 2-7 cm were collected from the cultivation field and dried completely. The pulverized fruit powder with moisture content less than 15% was subjected to 5% methanolic acetic extraction and Dimethylformamide (DMF) solvent extraction in Soxhlet apparatus by refluxing at 90°C in 1:15 ratio in three subsequent extractions of each 1 hour. The concentrates were crystalized at freezing temperatures in methanol and further purified by repeating the crystallization process. The white amorphous powder was subjected to RP-HPLC analysis method, chromatographic conditions 285 nm wavelength, C18 column 4.6 mm x 15 cm x 5 µm, flow rate 1.2 mL/minute with injection volume 10 µL run time of 30 minutes, column temperature 40°C. Hesperidin yields were 0.12 g/100 g with acetic acid methanol with a purity of 87% and 0.17 g/100 g in DMF extraction with a purity of 90% with recoveries of 87% in acetic acid methanol extraction, and 90% in DMF extraction when compared with standard 93%. Further extraction does not yield any hesperidin content indicating a total of 0.15-0.17 g/100 g of hesperidin content in the whole immature fruit of C. grandis/maxima. Thus we found DMF and 5% methanolic acetic solvents as preferable solvents to extract hesperidin.