Determination And Correlative Content And Bioactivity Of Limonin, Nomilin, Acridone From Citrus

In the present study, we modified and established the method for determination and extraction of limonin, nomilin and acridone in different organs (fruit, root, stem and leaf) and tissues of C. changshanensis, C. grandis, C. unshiu, and C. reticulate. In addition, the contents and bioactivities such as antioxidant and antibacterial activity were evaluated for limonin and nomilin from citrus fruit or root extracts of different cultivars, developmental stages, tissues and organs, and those in the raw crystal of limonin and nomilin from segment membrane (SM) of C. grandis. The main results were as follows:The developed method for determination and extraction of limonin and nomilin from citrus were simple, easy-to-do, and high efficient. Both TLC and HPLC were combined for the quantification and qualification of limonin and nomilin from different citrus samples. The HPLC conditions were: detection wavelength were 210 nm; C₁₈ column, methanol: acetonitrile: phosphate buffer (pH=3.5) =10:40:39 were used as the mobile phase; the relative standard deviation (RSD) for limonin and nomilin were 6.48 and 2.90% (n=5), respectively; the limit of detection (LOD) for both compounds were 0.03μg/g (S/N=3). For extraction of limonin and nomilin from citrus, methylene chloride were used as the extraction solution, after 60 min of water bath extraction at 50℃, the recoveries for limonin and nomilin were 90.45 and 91.09%, respectively.The HPLC detection system, which were established based on the fluorescent characteristics of 9(10H)acridone structure, included: an excitation wavelength of 395 nm and an emission wavelength of 435 nm, a gradient combination of deionized water (A) and methanol (B) were used as the elute solution. Such detection system were proved to be high sensitivity and stability, and the limit of detection (LOD) were 0.67 ng/g (S/N=3), the relative standard deviation (RSD) for the contents between 0.25-4.00μg/mL were 1.29-2.85% (n=5). The developed method for extraction and determination of acridone used ethanol as the extraction solution, after 60 min ultrasonic extraction at a frequency of 26 Hz and a power of 500 W at 30℃, the recovery of 9 (10H) acridone were between 97.12-101.56%.During the fruit development, there was an increase followed by a decrease for the contents of limonin and nomilin in the citrus fruit of all the four tissues studied, i.e. flavedo, albedo, SM, and juice vesicle (JV). Highest limonin contents were found in the flavedo of C. reticulat and segment membrane (SM) of C. changshanensis, which were 1.53 and 3.52 mg/g, respectively. Highest nomilin contents were found in the flavedo of C. grandis and SM of C. changshanensis, which were 2.82 and 2.92 mg/g, respectively. Both the limonin and nomilin cotents in the albedo of C. Grandis kept at low level throughout the developmental stages, which were lower than 0.09 and 0.08 mg/g, respectively. In the SM of mature C. Grandis fruit, both the limonin and nomilin increased from the 0.0001 and 0.06 mg/g at 3w before harvest to 0.42 and 0.51 mg/g at harvest. The results of study of different citrus organs showed an aboundant accumulation of both the limonin and nomilin in the ctirus root, the total amount of which were higher than their counterparts in the fruit, leaf and stem. The total amount of limonin and nomilin contents in the fibre of C. reticulate were 2.56 mg/g, while the corresponding amount in the fruit, leaf and stem of the same developmental stage were 1.35, 0.64, and 0.36 mg/g, respectively.Study of antioxidant activity of the raw crystal of limonin and nomilin from SM of C. grandis showed: the antioxidant capacity of limonoid raw crystal of citrus fruit was 10.34 times that of Vc; the antioxidant capacity of crude extract of citrus root was 12.59-17.21 times that of Vc. Different inhibition effect were found for the limonin, nomilin and acridone extract from citrus fruit or root against the growth of two bacteria and two fungus. The diameter of inhibition circle of limonin and nomilin extract against Penicillium digitatum and Aspergillus niger were 2.09 and 2.25 cm, respectively (the control were 1.26 and 1.25 cm, respectively), however, it showed no effect on the growth of Escherichia coil and Staphylococcus aureus, the diameter of inhibition circle were 1.27 and 1.28 cm, respectively (the control were 1.27 and 1.26 cm, respectively).The present study showed a genetic, developmental and tissue diversity of both the content and bioactivity of limonin and nomilin in citrus. The developed detection and extraction system for 9 (10H) acridone were easy-to-do and stable, which could be used for further study of content and bioactivity of acridone alkaloid in citrus.