Limonoid Aglycones Research Articles

Content changes of bitter compounds in ‘Guoqing No.1’ Satsuma mandarin (Citrus unshiu Marc.) during fruit development of consecutive 3 seasons

The main bitter compounds (nomilin, limonin and naringin) in the fruit tissues of ‘Guoqing No.1’ Satsuma mandarin (Citrus unshiu Marc.) were determined throughout the fruit development of 3 consecutive growing seasons. Although fluctuating largely at the corresponding developing stages of the 3years, the contents of these compounds in fruit tissues mostly displayed a declining trend, which implied that the rhythm of the metabolism of these bitter compounds was not consistent among years and was largely growing season dependent. Regarding their distribution, fruit flavedo might be a weak sink that contained the lowest level of naringin, while the segment membrane accumulated large amount of limonin and nomilin, which indicated a possible tissue bias pattern for biosynthesis or accumulation of those compounds. Partial correlation coefficient analysis revealed a synergistic accumulation of naringin and the two limonoid aglycones in fruit tissues during fruit development, indicating an integrated metabolism of flavonoids and limonoids.

Secondary Metabolites of Ponderosa Lemon (Citrus pyriformis) and their Antioxidant, Anti-Inflammatory, and Cytotoxic Activities

Column chromatography of the dichloromethane fraction from an aqueous methanolic extract of fruit peel of Citrus pyriformis Hassk. (Rutaceae) resulted in the isolation of seven compounds including one coumarin (citropten), two limonoids (limonin and deacetylnomilin), and four sterols (stigmasterol, ergosterol, sitosteryl-3-beta-D-glucoside, and sitosteryl-6'-O-acyl-3-beta-D-glucoside). From the ethyl acetate fraction naringin, hesperidin, and neohesperidin were isolated. The dichloromethane extract of the defatted seeds contained three additional compounds, nomilin, ichangin, and cholesterol. The isolated compounds were identified by MS (EI, CI, and ESI), 1H, 13C, and 2D-NMR spectral data. The limonoids were determined qualitatively by LC-ESI/MS resulting in the identification of 11 limonoid aglycones. The total methanolic extract of the peel and the petroleum ether, dichloromethane, and ethyl acetate fractions were screened for their antioxidant and anti-inflammatory activities. The ethyl acetate fraction exhibited a significant scavenging activity for DPPH free radicals (IC50 = 132.3 microg/mL). The petroleum ether fraction inhibited 5-lipoxygenase with IC50 = 30.6 microg/mL indicating potential anti-inflammatory properties. Limonin has a potent cytotoxic effect against COS7 cells [IC50 = (35.0 +/- 6.1) microM] compared with acteoside as a positive control [IC50 = (144.5 +/- 10.96) microM].

Limonoid content of sour orange varieties

Modern Citrus cultivars are thought to have arisen from three parents: the pummelo, the mandarin, and citron. Taxological and genetic data support that sweet and sour oranges share a common parentage. However, as their name suggests, the organoleptic properties of the fruit from these two families is distinctly different. Analysis of the limonoid content of sour orange varieties has been limited. Juice samples prepared from a selection of sour orange cultivars were evaluated for their limonoid A-ring lactone, aglycone, and glucoside contents. Limonoate A-ring lactone concentrations ranged from 11.1 to 44. 9 mg L⁻¹, whereas nomilinoate A-ring lactone levels were found not to exceed 1.2 mg L⁻¹. Total limonoid aglycone and total limonoid glucoside concentrations varied from 2.4 to 18.4 mg L⁻¹ and from 149.0 to 612.3 mg L⁻¹, respectively. Limonoid glucoside profiling by liquid chromatography-mass spectrometry suggest that the sour oranges are distinctly different from sweet oranges and other citrus species. Limonoid aglycone and A-ring contents across sweet and sour oranges are similar, whereas limonoid glucoside profiles are distinctly different. Juice prepared from Citrus maderaspatana had the highest limonoid concentrations among the samples tested and could potentially be used for the isolation of limonoid A-ring lactones and glucosides.

Chemical composition and biological activity of Citrus jambhiri Lush

The fresh peel of Citrus jambhiri was extracted with aqueous methanol and the residue was fractionated using light petroleum, chloroform and ethyl acetate. The constituents of the extracts were separated by column chromatography employing solvents of different polarity. The chemical structure of the isolated compounds was then identified by MS and NMR. Column chromatography of the petroleum fraction resulted in the isolation of nobiletin, 5-O-demethylnobiletin, tangeretin, 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone, 3,5,6,7,8,3′,4′-heptamethoxyflavone, and a mixture of β-sitosterol and stigmasterol. The chloroform fraction afforded 6-demethoxynobiletin, 5,4′-dihydroxy-6,7,8,3′-tetramethoxyflavone, limonin and nomilin. The flavonoid glycosides naringin, hesperidin and neohesperidin were isolated from the ethyl acetate fraction. The chemical structure of the isolated compounds was established by MS and NMR (APT, COSY, HSQC, HMBC, and NOESY). LC–ESI-MS analysis of the ethyl acetate fraction afforded eight flavonoid glycosides, while the dichloromethane fraction of the defatted seeds contained seven limonoid aglycones. The chloroform fraction exerted the strongest DPPH∗ free radical scavenging activity in comparison to other fractions. The petroleum fraction showed a significant inhibition of lipoxygenase indicating an anti-inflammatory action (IC50 29±1μg/mL). Some of the isolated polymethoxyflavones exhibited strong cytotoxicity against COS7, HeLa and Caco-2 cell lines.

Simultaneous Extraction of Bioactive Limonoid Aglycones and Glucoside from Citrus aurantium L. Using Hydrotropy

Citrus limonoids were demonstrated to possess potential biological activities in reducing the risk of certain diseases. Limonoids are present in citrus fruits in the form of aglycones and glucosides. At present, limonoid aglycones and limonoid glucosides are extracted in multiple steps using different solvents. In order to understand their potential bioactivity, it may be beneficial to isolate and purify these compounds using environment friendly methods. A new method of extraction and purification of limonoids was established using a hydrotrope polystyrene adsorbent resin. Extraction of aglycones and glucosides was achieved in a single step, using an aqueous solution of sodium cumene sulphonate (Na-CuS). Sour orange (Citrus aurantium L.) seed powder was extracted with 2 M Na-CuS solution at 45 degrees C for 6 h. The filtered extract was diluted with water and loaded on an SP 700 adsorbent column. The column was washed with distilled water to remove the hydrotrope and then eluted using water and methanol in different compositions to obtain three compounds. The structures of the isolated compounds were confirmed by NMR spectroscopy as deacetyl nomilinic acid glucoside (DNAG), deacetyl nomilin (DAN) and limonin (LIM).

Hydrotropic extraction of bioactive limonin from sour orange ( Citrusaurantium L.) seeds

Limonoids are potential bioactive compounds present only in citrus among fruits and vegetables. A new process for extraction of limonoid aglycones from sour orange ( Citrus aurantium L.) seeds was investigated using aqueous hydrotropic solutions. The extraction efficiency was dependent on hydrotrope concentration, extraction temperature and percent of raw material loaded. Two hydrotropes such as sodium salicylate (Na-Sal) and sodium cumene sulphonate (Na-CuS) were studied using Box-Behnken experiment design. Response surface analysis (RSA) of data was performed to study the effect of parameters on extraction efficiency. Prominent limonoid aglycone such as limonin was extracted and quantified for process optimization. Both hydrotropes gave maximum limonin yield at 2 M concentration, extraction temperature of 45 °C and 10% solid loading. A maximum limonin yield of 0.65 mg/g seeds was obtained using Na-CuS whereas only 0.46 mg/g seed was obtained using Na-Sal. Using this process, the use of organic solvents can be reduced dramatically to keep the process environmental friendly for the extraction of bioactive compounds.

Colorimetric Method for the Estimation of Total Limonoid Aglycones and Glucoside Contents in Citrus Juices

A method for estimating the total limonoid aglycone and glucoside concentrations in Citrus samples in terms of limonin and limonin glucoside equivalents is presented. The method consists of extraction followed by colorimetric quantification. The colorimetric quantification was based on the formation of red to orange colored derivatives resulting from the treatment of limonin, limonin glucoside, or a fruit extract with 4-(dimethylamino)benzaldehyde (DMAB) in the presence of perchloric and acetic acids. Absorbance maxima for the limonin and limonin glucoside derivatives were found to be 470 and 503 nm, respectively. The influence of DMAB concentration, reaction time, and solvent composition on color development and sensitivity were investigated and optimal assay conditions established. With a microplate format under these conditions, the limits of detection and quantification were determined to be 0.25 and 0.50 microg/mL for limonin and 0.50 and 1.0 microg/mL for limonin glucoside.

Purification of citrus limonoids and their differential inhibitory effects on human cytochrome P450 enzymes

AbstractRecent studies demonstrated that citrus limonoids and flavonoids possess numerous health promoting properties. In the present study, glucosides of limonoids and flavonoids were purified from citrus molasses and limonoid aglycones from citrus seeds. Glucosides were separated on styrene (divinylbenzene), Q‐sepharose resins with increasing concentration of sodium chloride. A pH‐dependent cold precipitation was carried out for the isolation of naringin in large quantity. Major aglycones such as limonin and nomilin were isolated from seeds by direct crystallization and minor limonoids were purified by vacuum liquid chromatography. The structures of the isolated compounds were confirmed by NMR spectra. Individual limonoids were tested for O‐dealkylase and hydroxylase activities of human cytochrome P450 (CYP) isoenzymes such as CYP1A2, CYP1B1, CYP3A4 and CYP19, using ethoxyresorufin, methoxyresorufin and dibenzylfluorescein as substrates. Partial to high inhibition of CYPs was observed in dose‐dependent assays. Significant (P < 0.001) reductions in enzyme activities were observed with purified compounds above 2 µmol. Kinetic analyses indicated that limonin glucoside inhibited CYP19 competitively (IC50, 7.1 µ mol L−1), whereas Nomilinic acid glucoside inhibited it noncompetitively (IC50, 9.4 µ mol−1). Nomilinic acid glucoside was the most potent limonoid, with an overall IC50 of < 10 µ mol, for all the enzymes tested. The differential inhibition of CYPs can be ascribed to structural variations of the limonoid nucleus. Limonoid inhibition of key CYPs involved in carcinogenesis supports growing evidence that citrus limonoids act as anticancer agents. Copyright © 2007 Society of Chemical Industry

Simultaneous determination of citrus limonoid aglycones and glucosides by high performance liquid chromatography

High performance liquid chromatography (HPLC) method has been developed for simultaneous quantification of limonoid aglycones and glucosides on a reversed phase C 18 column using a binary solvent system, coupled with diode array detector. Seven limonoids such as limonin, nomilin, isolimonic acid, ichangin, isoobacunoic acid, limonin 17-β- d glucopyranoside and deacetyl nomilinic acid 17-β- d glucopyranoside were separated and detected at 210 nm. Furthermore, limonoids were separated, identified and quantified in four varieties of citrus fruits and seeds using developed method. Limonin and limonin glucoside were found to be the predominant limonoid aglycone and glucoside, respectively, in all tested samples. The sensitivity of the method was found to be 0.25–0.50 μg for tested limonoids.

Citrus Bioactive Limonoid Extraction using Environment-friendly Hydrotropy

Citrus consumption has been shown to promote human health due to presence of several bioactive compounds. In the process of understanding the health benefits of citrus, we need to isolate and characterize these compounds. Limonoids are one of such prominent, but lesser-known phytonutrients that have been shown to prevent cancers of the mouth, skin, lung, breast, and colon. With the growing interest in the health-promoting properties of citrus limonoids, the demand for these bioactives has significantly increased. It has been critical to explore environment-friendly extraction methods rather than using hazardous organic solvents. A water-based hydrotropic extraction of limonoid aglycones from sour orange (Citrus aurantium L.) seeds was developed. Two hydrotropes, sodium salicylate (Na-Sal) and sodium cumene sulfonate (Na-CuS), were studied for extraction efficiency using the Box Behnken experiment design method. The extraction efficiency of prominent aglycone limonin was observed depending on hydrotrope concentration, extraction temperature, and percentage of raw material loading. Response Surface Analysis (RSA) of data predicted the optimum conditions for maximum yield. Recovery of aglycones from filtered extract is also easily achieved by mere dilution using water at pH 3 or 7 or by partitioning the extract with dichloromethane. At optimum conditions, limonin yield of 0.46 mg/g seeds in the case of Na-Sal extraction and 0.65 mg/g seeds in the case of Na-CuS extraction was achieved. The results demonstrated that the hydrotropic extraction process of limonoid aglycones has practical commercial importance. This project is based upon work supported by the USDA–CSREES IFAFS #2001 52102 02294 and USDA–CSREES #2005-34402-14401 “Designing Foods for Health” through the Vegetable and Fruit Improvement Center.

Identifying citrus limonoid aglycones by HPLC‐EI/MS and HPLC‐APCI/MS techniques

HPLC coupled with normal phase electron ionisation (EI) and atmospheric pressure chemical ionisation (APCI)/ mass spectrometry methods has been applied to identify 17 known neutral limonoid aglycones from Citrus sources. The HPLC-MS data from the known limonoids provided chromatographic characteristics, APCI-derived molecular weight data and EI fragmentation data for each limonoid. EI fragmentation patterns for the limonoids were correlated with structural characteristics. The EI fragmentation patterns coupled with APCI-derived molecular weights were utilised as a potential method by which to discern the structural character of unknown citrus limonoids.

Chromatographic Techniques to Purify Individual Limonoids from Seeds and Molasses of Citrus Fruits

Limonoids, chemically related triterpinoids predominantly found in citrus and neem relatives, are known to play a pivotal role in the prevention of different types of cancer and cardiovascular diseases. Since the concentrations of these compounds are low in the plant tissues, the isolation of pure compounds is the limiting factor for the individual activity studies in animal models. In this study, combinations of chromatographic techniques were used to isolate limonoid aglycones and limonoid glucosides from citrus byproducts such as seeds and molasses. The compounds were initially extracted with different polar solvents and the concentrated extracts were passed through a series of adsorbent resin (SP-70) and ion-exchange resins (WA-30, Dowex-50, Q-sepharose) to remove further impurities. The use of increasing ionic strength of NaCl from 0 to 800 mm to release the exchanged compounds from the ion exchange columns further separated the limonoids from flavonoids, which was confirmed through TLC, UV, and analytical HPLC methods. Individual compounds were further purified using flash chromatography and preparative HPLC methods and identified by using LC-MS analysis. Direct crystallization of limonin resulted in a 17% increase in the yield as compared to the previously reported methods. The results suggest that application of these purification methods are useful for the bulk purification of compounds in order to further investigate their biological activity.

Electron ionization mass spectrometry of citrus limonoids.

Electron ionization (EI) mass spectrometry was used to differentiate four structurally closely related citrus limonoid aglycones, including limonin, nomilin, obacunone, and deacetylnomilin. The limonoids were isolated and purified from citrus seeds. Structures of major fragment ions were elucidated by high-resolution mass spectrometry (HRMS) and fragmentation pathways were proposed. The fragmentation patterns observed in the EI spectra can be used as important references for the positive characterization of limonoid aglycones.

Mass spectrometry and tandem mass spectrometry of citrus limonoids.

Methods for atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) of citrus limonoid aglycones and electrospray ionization tandem mass spectrometry (ESI-MS/MS) of limonoid glucosides are reported. The fragmentation patterns of four citrus limonoid aglycones (limonin, nomilin, obacunone, and deacetylnomilin) and six limonoid glucosides, that is, limonin 17-beta-D-glucopyranoside (LG), nomilin 17-beta-D-glucopyranoside (NG), nomilinic acid 17-beta-D-glucopyranoside (NAG), deacetyl nomilinic acid 17-beta-D-glucopyranoside (DNAG), obacunone 17-beta-D-glucopyranoside (OG), and obacunoic acid 17-beta-D-glucopyranoside (OAG) were investigated using a quadruple mass spectrometer in low-energy collisionally activated dissociation (CAD). The four limonoid aglycones and four limonoid glucosides (LG, OG, NAG, and DNAG) were purified from citrus seeds; the other two limonoid glucosides (NG and OAG) were tentatively identified in the crude extract of grapefruit seeds by ESI mass spectrometry in both positive and negative ion analysis. Ammonium hydroxide or acetic acid was added to the mobile phase to facilitate ionization. During positive ion APCI analysis of limonoid aglycones, protonated molecular ion, [M + H]+, or adduct ion, [M + NH3 + H]-, was formed as base peaks when ammonium hydroxide was added to the mobile phase. Molecular anions or adduct ions with acetic acid ([M + HOAc - H] and [M + HOAc]-) or a deprotonated molecular ion were produced during negative ion APCI analysis of limonoid aglycones, depending on the mobile-phase modifier used. Positive ion ESI-MS of limonoid glucosides produced adduct ions of [M + H + NH3]+, [M + Na]+, and [M + K]+ when ammonium hydroxide was added to the mobile phase. After collisionally activated dissociation (CAD) of the limonoid aglycone molecular ions in negative ion APCI analysis, fragment ions indicated structural information of the precursor ions, showing the presence of methyl, carboxyl, and oxygenated ring structure. CAD of the adduct ion [M + H + NH3]+ of limonoid glucosides produced the aglycone moiety corresponding to each glucoside. The combination of mass spectrometry and tandem mass spectrometry provides a powerful technique for identification and characterization of citrus limonoids.

Limonoids in Seeds of Iyo Tangor (Citrus iyo hort. ex Tanaka)

Iyo tangor (Citrus iyo hort. ex Tanaka) seeds contained limonin, nomilin, obacunone and deacetylnomilin, in order of decreasing concentration. They also contained the 17-β-D-glucopyranosides of nomilin, obacunone, limonin, deacetynomilin and nomilinic acid. Total limonoid aglycone concentration in the seeds was 873 mg per 100 g on a dry weight basis and total limonoid glucoside concentration was 446 mg. The composition and relative concentration of each limonoid aglycone and glucoside in Iyo tangor seeds were very similar to those of other citrus species distributed widely in western Japan such as Valencia orange (C. sinensis Osbeck), Sanbokan (C. sulcata hort. ex Tanaka) and Hyuganatsu (C. tamurana hort. ex Tanaka). These data reveal that Iyo tangor taxonomically belongs to the same group as those citrus species.

Molecular cloning and characterization of a novel gene encoding limonoid UDP-glucosyltransferase in Citrus

We isolated a cDNA clone encoding limonoid UDP-glucosyltransferase (limonoid GTase) from the albedo of Satsuma mandarin ( Citrus unshiu Marc.) and investigated the contribution to limonoid glucoside accumulation in fruit. The isolated cDNA clone (CitLGT) was 1732 bp in length encoding 511 deduced amino acids with a predicted molecular mass of 57.5 kDa. The products of in vitro translation from an expression vector had the limonoid GTase activity. Southern blot analysis of genomic DNA indicated that CitLGT was present as a single copy gene in the Citrus genome. The amount of transcript corresponding to CitLGT mRNA changed the same way as the fluctuation of limonin glucoside content during fruit development of navel orange ( Citrus sinensis Osb.). This indicates that the transcription of CitLGT regulates the conversion of limonoid aglycones to glucosides in citrus fruit.

Antifeedant activity of citrus limonoids against Colorado potato beetle: comparison of aglycones and glucosides

The importance of plant allelochemicals in plantherbivore interactions is well known. Although some of these phytochemicals act as phagostimulants for specific herbivores, the majority of allelochemicals examined appear to function primarily in plant defense, acting as insect antifeedants, growth regulators, and/or toxins (Jermy, 1966; Bernays & Chapman, 1977). As such, these chemicals have great potential for use in pest management, either directly, as natural plant protectants, or as models for the development of synthetic products. Citrus limonoids, a group of terpenoid allelochemicals found in the plant family Rutaceae have previously been shown to act as insect antifeedants (Klocke & Kubo, 1982; Alford et al., 1987; Mendel et al., 1991) as well as mammalian anticarcinogens (Lam & Hasegawa, 1989; Miller et al., 1989). They are also useful as chemotaxonomic markers (Hasegawa & Miyake, 1996). Thus, citrus limonoids are important functional chemicals in agriculture and medicine. Limonoid aglycones, which are present in young and growing citrus tissues, are converted to their respective 17β-D-glucoside derivatives during late stages of fruit growth and maturation (Hasegawa et al., 1991; Fong et al., 1992). Limonoid glucosides, such as limonin 17β-D-glucopyranoside, are accumulated in mature fruit tissues and seeds as major secondary metabolites (Fong et al., 1989; Hasegawa et al., 1991). Thirty-six limonoid aglycones and 17 limonoid glucosides have been isolated from Citrus and its hybrids, yet few have been investigated for their biological activities. None of the limonoid glucosides have previously been examined for antifeedant activity against insects. In this study we compared the antifeedant activity of limonoid aglycones and glucosides against Colorado potato beetle, Leptinotarsa decemlineata (Say), larvae.

Limonoids and Flavonoids in Juices of Oroblanco and Melogold Grapefruit Hybrids

ABSTRACTOroblanco and Melogold are hybrids obtained from pummelo and grapefruit. Limonoids and flavonoids in both juices were analyzed. Oroblanco and Melogold juices contained low concentrations of limonoid glucosides, an average of 99 and 59 ppm, respectively. However, they contained relatively high concentrations of bitter limonoid aglycones, limonin and nomilin, at levels above the limonin bitterness threshold. For comparison, limonoid glucosides in juices of grapefruit, another pummelo hybrid, were also analyzed. Limonin glucoside was the major limonoid glucoside in all juices analyzed. Nomilin glucoside and nomilinic acid glucosides were also present. Oroblanco and Melogold juices contained bitter flavonoids normally found in grapefruit and pummelo including naringin, neohesperidin and poncerin in total amount of 440 ppm in Oroblanco juice and 495 ppm in Melogold juice. They also contained several other nonbitter flavonoids found in grapefruit.

Biochemistry and biological functions of citrus limonoids

Abstract Limonoids are one of bitter principles in citrus juices. Excessive bitterness lowers the quality and value of citrus juices. Nomilin is considered to be the precursor of all other limonoids accumulated in Citrus and related species. It is biosynthesized from acetate via the terpenoid biosynthetic pathway in the phloem region of stems and then translocated to the leaves, fruit tissues, peels, and seeds where it is further metabolized to other limonoids. The citrus limonoid aglycones are then glucosidated by limonoid UDP‐D‐glucose transferase in maturing fruit tissues and seeds. These limonoid glucosides are accumulated in such high concentrations that they are one of major secondary metabolites in citrus fruit tissues, and they play an important role in fruit quality and possibly in human health. Research on creation of transgenic citrus trees that produce fruits free of the limonoid bitterness problem is in progress. Limonoids have been shown to induce glutathione S‐transferase activity and inhib...

Limonoids in Pummelos [Citrus grandis (L.) Osbeck

ABSTRACTJuice and seeds from 16 cultivars of mature pummelo fruits were analyzed for limonoid content. Pummelo juice contained an average of 18 ppm limonin and 29 ppm total limonoid glucosides. Compared to other juices, ptmnnelo contained very high concentrations of limonin and very low concentrations of limonoid glucosides. Limonin, nomilin, obacunone and trace amounts of deacetyhromilin were found in pummelo seeds. The 17–β‐D‐glucopyranoside derivatives (glucosides) of nomilin, nomilinic acid and obacunone were also present. Total limonoid aglycone concentration in the seeds ranged from 773 ppm to 9,900 ppm and total limonoid glucosides ranged from 130 ppm to 1,912 ppm.