Hesperidin (hesperidin) is mainly extracted from secondary metabolites of rutaceae citrus plants, and citrus peel as well as the white intima is the main enrichment site. Peels of navel orange (Citrus sinensis) consist of Hesperidin up to 3.5% (dry weight), while in grapefruit (Citrus maxima), there is only 0.6%-1.2%. The Hesperidin content in the peel of late-maturing variety “Voolingxia Orange” was 42% more than that of the early-maturing variety, and the sample retention rate of natural air drying within 7 days after harvesting was 98.3%, as reported in the Journal of Agricultural and Food Chemistry in 2023. In industrial extraction, 12-15 kg crude extract can be obtained by extracting 1 ton fresh citrus peel with ethanol-water mixed solvent (60% v/v). Hesperidin purity is increased from 35% to above 90% and the yield is sacrificed by approximately 22% after macroporous resin purification.
The supply chain of Hesperidin across the globe depends greatly on geographical locations. China’s Hunan Province Citrus aurantium, planting area 680,000 mu, provided 53% of the world’s raw material, and the standard content of Hesperidin in dried young fruits was ≥7.0% (Chinese Pharmacopoeia 2020 edition). The citrus processing factories in Brazil’s Sao Paulo state extract 2,300 tons of Hesperidin annually from orange peel waste, accounting for 31 percent of the global total output, but the 2022 frost calamity reduced the supply of raw materials by 17 percent, and the market price rose from $45 / kg to $78. The University of Catania in Italy found through research that the potassium content (1.8%-2.3%) of soil in the citrus orchard on Mount Etna enhanced the biosynthesis efficiency of Hesperidin by 15%-18%, and the unit price of the peel extract of the local specialty variety “blood orange” can reach 125 euros/kg.
Innovation in extraction technology has significantly contributed to the production efficiency of Hesperidin. Supercritical CO₂ extraction process (40MPa, 55 ° C) reduces the single extraction cycle from 24 hours to 4.5 hours with traditional solvents, and the solvent residue is reduced from 500ppm to less than 8ppm. A biotech company in Jiangsu Province utilized membrane separation and compound crystallization technology to reduce the energy consumption of the 300 tons/year Hesperidin production line by 37%, and the product particle size D50 was maintained at 15μm±2μm, meeting injectable grade API standards. The immobilized enzymatic hydrolysis process invented by Nori in 2021 raised the conversion rate of citrus flavone glycosides from 72% to 94%, lowering the production cost of Hesperidin by $19 / kg. The KIST Institute in South Korea engineered citrus cell lines through CRISPR-Cas9 technology to enhance the biosynthesis flux of Hesperidin by 2.3 times.
The manufacturing of medicinal grade Hesperidin is under strict quality requirements. The European Pharmacopoeia (EP 10.0) necessitates that the total impurity of the concerned substances in apis should not exceed 0.5% and residue on ignition is < 0.1%. The USP 43 standard stipulates that the deviation of the retention time of the main peak should be not more than ±0.3 minutes and RSD of the content determination should be < 1.0%. In 2023, the FDA disclosed in a warning letter to an Indian supplier that the level of the heavy metal lead in its Hesperidin raw material was 3.7 times the standard (detection value 0.37ppm vs standard 0.1ppm), which caused the recall of 12 batches of drugs. The International Organization for Standardization (ISO 20457:2018) specifies that the microbial specifications of food additive grade products will meet: total aerobic bacteria count < 1000 CFU/g, and yeast mold < 100 CFU/g.
Emerging biotechnology is expanding the sources of Hesperidin. By utilizing hairy root culture technology, Meiji University in Japan heterogeneously expressed the citrus flavone synthetase gene in ginseng cells, and Hesperidin yield was 1.2mg/g dry weight, 80-fold that from natural sources. In 2022, Amyris, a synthetic biology company, used engineered yeast strains for large-scale production of Hesperidin in a 120m³ fermenter with fermentation titer 8g/L and purity 99.5%, yet the production cost was still 42% higher compared to plant extraction. The Chinese Academy of Agricultural Sciences established a solid-state fermentation process of citrus residue by Aspergillus niger CICC 2487, which increased the degradation rate of pectin to 92% and the bioavailability of Hesperidin from 13% to 67%.