Citrus Limonoids Research Articles

Effect of Citrus Limonoids on Hepatic Gene Expression Involved in Lipid Metabolism in Rats

Effect of Citrus Limonoids on Hepatic Gene Expression Involved in Lipid Metabolism in Rats

Suppression of intestinal carcinogenesis in APC-mutant mice by the citrus limonoid limonin

Suppression of intestinal carcinogenesis in APC-mutant mice by the citrus limonoid limonin

Limonin – A citrus limonoid, establish anticancer potential by stabilizing lipid peroxidation and antioxidant status against N-nitrosodiethylamine induced experimental hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common category of liver cancer, representing 83% of all cases. The intention of the present investigation was designed to explore the defensive efficiency of limonin against N-nitrosodiethylamine (DEN, 200mg/kg) induced phenobarbital promoted experimental hepatocellular carcinoma in male Wistar albino rats. Hepato carcinonogen DEN-treated experimental rats showed an elevated level of HCC marker enzyme alpha-fetoprotein, enhanced lipid peroxidation due to oxidative stress and free radical formation. In parallel, there was a drastic decline in the level of antioxidant enzymes due to the induction of DEN in Group II animals. Treatment with limonin, due to its pharmacological and antioxidant potential enhanced marker enzyme (AFP), lipid peroxidation and diminished antioxidant status was brought back near standard level. In addition, histopathological analysis was also carried out to mark the pathological alterations. In Group II animals, severe lesions and cirrhosis of the liver cells were noted. Limonin administration establishes its anticancer activity by totally refurbishing the smashed hepatocytes in Group III animals. Our study strongly put forward that limonin has excellent antioxidant and therapeutic property against DEN-induced hepatocarcinogenesis by suppressing LPO and oxidative stress mediated free radicals generation and through modulating antioxidants defense mechanism.

Limonin, A Citrus Limonoid, had no Apparent Effect on Cognitive Dysfunction in Mice with Chronic Cerebral Hypoperfusion

Background: Limonoids are highly oxygenated triterpenoid compounds abundantly contained in citrus fruits. It has been reported that limonoids have several pharmacological activities such as anti-cancer, anti-malarial, and antimicrobial activities. Moreover, recent reports have indicated that limonoids may have a preventive effect on cardiovascular disease and metabolic disorders. However, very little information is available about the effects of limonoids on cognitive impairment due to Alzheimer disease and vascular dementia. In the present study, we investigated whether limonin, one of the major limonoids, has a preventive effect on cognitive dysfunction in a mouse vascular dementia model induced by chronic cerebral hypoperfusion. Materials and Methods: Ten-week-old C57BL/6J male mice were subjected to bilateral common carotid artery stenosis (BCAS) using external microcoils. Mice were administered limonin (10 mg/kg/day) from 8 to 16 weeks of age. Spatial working memory was evaluated by the Morris water maze test at 16 weeks of age. Blood pressure was measured by tailcuff method. Cerebral blood flow was assessed by 2D laser speckle flowmetry. Results: Chronic cerebral hypoperfusion induced by BCAS significantly impaired cognitive function compared with that of the control group. However, administration of limonin did not ameliorate cognitive dysfunction induced by BCAS. Moreover, cerebral blood flow was reduced by BCAS, but this reduction was not improved by limonin treatment. There was no significant change in blood pressure in each mouse. Conclusion: Limonin had no apparent effect on cognitive impairment attributed to chronic cerebral hypoperfusion. Keywords: Blood pressure, cerebral blood flow, cognitive function, common carotid artery stenosis, hypoperfusion, limonoid, limonin, Morris water maze, mouse model, spatial learning, vascular dementia.

Limonoids and their anti-proliferative and anti-aromatase properties in human breast cancer cells

Lemons are a widely used citrus crop and have shown several potential health benefits. In the present study, the mechanism and effectiveness of the anti-cancer and anti-aromatase properties of limonoids were investigated for the first time. Defatted lemon (Citrus lemon L. Burm) seed powder was extracted with ethyl acetate (EtOAc) and methanol (MeOH) for 16 h each, successively. These extracts were fractionated using 1D (silica) and 2D (ion exchange and SP-70 columns) column chromatography to obtain nine limonoids. The compounds were identified by TLC, HPLC, and LC-MS techniques. A panel of 9 purified limonoids, including limonin, nomilin, obacunone, limonexic acid (LNA), isolimonexic acid (ILNA), nomilinic acid glucoside (NAG), deacetyl nomilinic acid glucoside (DNAG), limonin glucoside (LG) and obacunone glucoside (OG) as well as 4 modified compounds such as limonin methoxime (LM), limonin oxime (LO), defuran limonin (DL), and defuran nomilin (DN), were screened for their cytotoxicity on estrogen receptor (ER)-positive (MCF-7) or ER-negative (MDA-MB-231) human breast cancer cells. We further tested the mechanism of the anti-proliferative activity of limonoids using an in vitro aromatase enzyme assay and western blot with anti-caspase-7. Among the tested limonoids, 11 limonoids exhibited cytotoxicity on MCF-7 whereas 8 limonoids showed cytotoxicity against the MDA-MB-231 cell lines. Although most of the limonoids showed anti-aromatase activity, the inhibition of proliferation was not related to the anti-aromatase activity. On the other hand, the anti-proliferative activity was significantly correlated with caspase-7 activation by limonoids. Our findings indicated that the citrus limonoids may have potential for the prevention of estrogen-responsive breast cancer (MCF-7) via caspase-7 dependent pathways.

Citrus limonoids and curcumin additively inhibit human colon cancer cells

In the current study, we examined the ability of limonoids, including limonin, limonin glucoside (LG) and curcumin, to inhibit proliferation of human colon cancer (SW480) cells. Additionally, we studied the effect of combining these two classes of natural compounds on inhibition of proliferation and the possible mode of cytotoxicity. The SW480 cells were treated with compounds individually and in combination to understand the effect on cell death, DNA fragmentation, caspase-3 activity and the expression of Bax, Bcl-2 and caspase-3 proteins. Results of cell proliferation assays suggest that combinations of limonoids with curcumin at three different ratios (1 : 3, 1 : 1 and 3 : 1) to a final concentration of 50 ppm demonstrated up to 96% inhibition of cell proliferation. The MTT assay results were also confirmed by counting viable cells. Further, incubation of cells with combinations of limonoids and curcumin resulted in elevation of total cellular caspase-3 activity by 3.5-4.0 fold along with a 2- to 4-fold increase in the Bax/Bcl-2 ratio. The expression of pro-caspase-3 and its cleaved products in cells treated with curcumin (individually or combination) indicates higher potency of the combination to induce apoptosis. For the first time, this study provides compelling evidence of the pharmacodynamic additive effect of limonoids and curcumin in inhibiting human colon cancer cells. The above results were also confirmed by fluorescence microscopy of SW480 cells treated with limonoids, curcumin and combination, after tagging with fluorescent probes. These results suggest that consumption of curcumin and limonoids together may offer greater protection against colon cancer.

Isolimonic acid interferes with Escherichia coli O157:H7 biofilm and TTSS in QseBC and QseA dependent fashion

BackgroundE. coli O157:H7 (EHEC) is an important human pathogen. The antibiotic treatment of EHEC reportedly results in release of Shiga toxin and is therefore discouraged. Consequently, alternative preventive or therapeutic strategies for EHEC are required. The objective of the current study was to investigate the effect of citrus limonoids on cell-cell signaling, biofilm formation and type III secretion system in EHEC.ResultsIsolimonic acid and ichangin were the most potent inhibitors of EHEC biofilm (IC25=19.7 and 28.3 μM, respectively) and adhesion to Caco-2 cells. The qPCR analysis revealed that isolimonic acid and ichangin repressed LEE encoded genes by ≈3 to 12 fold. In addition, flhDC was repressed by the two limonoids (≈3 to 7 fold). Further studies suggested that isolimonic acid interferes with AI-3/epinephrine activated cell-cell signaling pathway. Loss of biofilm inhibitory activity of isolimonic acid in ΔqseBC mutant, which could be restored upon complementation, suggested a dependence on functional QseBC. Additionally, overexpression of qseBC in wild type EHEC abated the inhibitory effect of isolimonic acid. Furthermore, the isolimonic acid failed to differentially regulate ler in ΔqseA mutant, while plasmid borne expression of qseA in ΔqseA background restored the repressive effect of isolimonic acid.ConclusionsAltogether, results of study seem to suggest that isolimonic acid and ichangin are potent inhibitors of EHEC biofilm and TTSS. Furthermore, isolimonic acid appears to interfere with AI-3/epinephrine pathway in QseBC and QseA dependent fashion.

Limonin 7-methoxime interferes with Escherichia coli biofilm formation and attachment in type 1 pili and antigen 43 dependent manner

Citrus limonoids were reported to inhibit autoinducer-mediated bacterial cell–cell signaling, Escherichia coli O157:H7 biofilms and the Type III secretion system. The goal of the present study was to understand the effect of specific modifications in limonin molecule on cell–cell signaling inhibitory activity. The limonin was modified into de-furan limonin, limonin 7-oxime, limonin 7-methoxime. The modified limonoids were examined for their cell–cell signaling and biofilm inhibitory properties. The results of the study suggest that the furan ring and C-7 substitutions were important structural features in the limonin molecule to inhibit cell–cell signaling and biofilm formation in Vibrio harveyi. The C-7 modifications of limonin showed increased potency to inhibit the E. coli biofilm. Furthermore, the data indicates that limonin 7-methoxime reduced the attachment of E. coli to plastic surface by interacting with type 1 pili and Ag43. In addition, limonin 7-methoxime seems to reduce the adhesion of E. coli to spinach leaves and colon epithelial cells.

Inhibitory effects of limonin on six human cytochrome P450 enzymes and P-glycoprotein in vitro

Among the various possible causes for drug interactions, pharmacokinetic factors such as inhibition of drug-metabolizing enzymes and transporters, especially cytochrome P450 (CYP) isoenzymes and P-glycoprotein (P-gp), are regarded as the most frequent and clinically important. Limonin is a widely used dietary supplement and one of the most prevalent citrus limonoids, which are known to have inhibitory effects on CYPs and P-gp. In this study, the in vitro inhibitory effects of limonin on the major human CYP isoenzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) activities in human liver microsomes were examined using liquid chromatography–tandem mass spectrometry. The inhibitory effects of limonin on P-gp activity in a human metastatic malignant melanoma cell line WM-266-4 were examined using a calcein-AM fluorometry screening assay. It demonstrates that limonin has negligible inhibitory effects on human CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and P-gp. However, potent inhibition of CYP3A4 by limonin is observed with IC50 values of 6.20μM (CYP3A4/testosterone) and 19.10μM (CYP3A4/midazolam). This finding has important implications with regard to food–drug interactions between limonin and several narrow therapeutic index drugs that are metabolized by CYP3A4.

Structure–function relationships of citrus limonoids on p38 MAP kinase activity in human aortic smooth muscle cells

Limonoids, abundantly present in citrus fruits, have potential role in reducing risk of different type of cancer. In the present study, we hypothesized that seven structurally different limonoids would involve in inflammatory pathway via modulating p38 MAP kinase activity at various extent in vascular smooth muscle cells. Results demonstrated that the different functional groups containing limonoids had differential effects on the p38 MAP kinase activity in human aortic smooth muscle cells. Among seven limonoids, nomilin exhibited the highest (38%) inhibition of p38 MAP kinase activity, followed by limonin (19%), deacetyl nomilin (19%), and defuran nomilin (17%). While defuran limonin and methyl nomilinate showed no significant decrease in p38 MAP kinase activity, obacunone significantly increased the p38 MAP kinase activity by 38%. Furthermore, TNF-α induced p38 MAP kinase activity in the smooth muscle cells was completely inhibited by nomilin. Thus our data provide the first evidence that nomilin is the potent natural inhibitor for p38 MAP kinase activity in human aortic smooth muscle cells. These data also suggest that a seven-membered A ring with acetoxy group, present in nomilin, seems to be essential for its inhibitory activity on p38 MAP kinase.

Anti-obesity and anti-hyperglycemic effects of the dietary citrus limonoid nomilin in mice fed a high-fat diet

TGR5 is a member of the G protein-coupled receptor family and is activated by bile acids (BAs). TGR5 is thought to be a promising drug target for metabolic diseases because the activation of TGR5 prevents obesity and hyperglycemia in mice fed a high-fat diet (HFD). In the present study, we identified a naturally occurring limonoid, nomilin, as an activator of TGR5. Unlike BAs, nomilin did not exhibit the farnesoid X receptor ligand activity. Although the nomilin derivative obacunone was capable of activating TGR5, limonin (the most abundant limonoid in citrus seeds) was not a TGR5 activator. When male C57BL/6J mice fed a HFD for 9 weeks were further fed a HFD either alone or supplemented with 0.2% w/w nomilin for 77 days, nomilin-treated mice had lower body weight, serum glucose, serum insulin, and enhanced glucose tolerance. Our results suggest a novel biological function of nomilin as an agent having anti-obesity and anti-hyperglycemic effects that are likely to be mediated through the activation of TGR5.

Citrus Flavonoids Luteolin, Apigenin, and Quercetin Inhibit Glycogen Synthase Kinase-3β Enzymatic Activity by Lowering the Interaction Energy Within the Binding Cavity

Pancreatic cancer studies have shown that inhibition of glycogen synthase kinase-3β (GSK-3β) leads to decreased cancer cell proliferation and survival by abrogating nuclear factor κB (NFκB) activity. In this investigation, various citrus compounds, including flavonoids, phenolic acids, and limonoids, were individually investigated for their inhibitory effects on GSK-3β by using a luminescence assay. Of the 22 citrus compounds tested, the flavonoids luteolin, apigenin, and quercetin had the highest inhibitory effects on GSK-3β, with 50% inhibitory values of 1.5, 1.9, and 2.0 μM, respectively. Molecular dockings were then performed to determine the potential interactions of each citrus flavonoid with GSK-3β. Luteolin, apigenin, and quercetin were predicted to fit within the binding pocket of GSK-3β with low interaction energies (-76.4, -76.1, and -84.6 kcal·mol(-1), respectively) and low complex energies (-718.1, -688.1, and -719.7 kcal·mol(-1), respectively). Our results indicate that several citrus flavonoids inhibit GSK-3β activity and suggest that these have potential to suppress the growth of pancreatic tumors.

Mosquito larvicidal activity of citrus limonoids against Aedes albopictus

Citrus limonoids, nomilin and limonin, were used for larvicidal assay against Aedes albopictus utilizing WHO methodology. LC(50s) were 305.83, 176.08, and 136.07 μM for nomilin and 850.09, 600.72, and 407.09 μM for limonin after 24, 48, and 72 h, respectively. LT(50) assays exhibited that Savage citrange oil was the best at all concentrations (400, 500, 600, and 700 ppm) while Fairchild and Cassa grande were the weakest oils at 400 ppm, but at 500, 600, and 700 ppm, Carrizo citrange remained at the bottom with highest LT(50) values. Results exhibited that nomilin was more toxic than limonin and therefore provided a clear indication that limonoids in sample oils influenced the potential of respective oil. Out of the 10 tested citrus seed oils, Savage citrange (Citrus sinensis) comprised the maximum amount of limonin (2823.59 μg/ml) followed by grapefruit, Sacaton citrumelo, and Jaffa. When this oil (Savage citrange) was evaluated for bioassay against larvae of Ae. albopictus, it reflected complete dominance (LC(50) and LT(50)) as compared to rest of the oils. Although Jaffa (Citrus paradisi) was found to contain nomilin and limonin, it was found less effective as compared to Savage citrange. The oils from Minneola and Chinese lime did not contain limonin and nomilin, and were therefore weak in terms of LC(50) values. Presence of limonin and nomilin in plant products is therefore a significant indicator of the pest control that needs to be exploited in other plants as well.

Citrus limonoids interfere with Vibrio harveyi cell–cell signalling and biofilm formation by modulating the response regulator LuxO

Citrus limonoids are unique secondary metabolites, characterized by a triterpenoid skeleton with a furan ring. Studies have demonstrated beneficial health properties of limonoids. In addition, certain citrus limonoids play a role in plant defence against insect pests. In the present study, five limonoids were purified from sour orange and evaluated for their ability to inhibit cell-cell signalling. The purified limonoids were tested for their ability to interfere with cell-cell signalling and biofilm formation in Vibrio harveyi. Isolimonic acid, deacetylnomilinic acid glucoside and ichangin demonstrated significant inhibition of autoinducer-mediated cell-cell signalling and biofilm formation. Furthermore, isolimonic acid and ichangin treatment resulted in induced expression of the response regulator gene luxO. In addition, luxR promoter activity was not affected by isolimonic acid or ichangin. Therefore, the ability of isolimonic acid and ichangin to interfere with cell-cell signalling and biofilm formation seems to stem from the modulation of luxO expression. The results suggest that isolimonic acid and ichangin are potent modulators of bacterial cell-cell signalling.

Simultaneous separation and identification of limonoids from citrus using liquid chromatography‐collision‐induced dissociation mass spectra

Limonoids are considered as potential cancer chemopreventive agents and are widely distributed in the Citrus genus as aglycones and glucosides. In the present study, reversed-phase HPLC coupled with CID mass spectra was developed for the simultaneous separation and identification of aglycones and glucosides of limonoids from citrus. Five aglycones such as limonin, deacetyl nomilin, ichangin, isolimonoic acid and nomilin were identified by positive ion CID MS/MS, whereas five glucosides, viz. limonin glucoside, isoobacunoic acid glucoside, obacunone glucoside, deacetyl nomilinic acid glucoside and nomilinic acid glucoside were analyzed by negative ion CID mass spectra. The developed method was successfully applied to complex citrus samples for the separation and identification of aglycones and glucosides. Citrus seeds were extracted with methanol and partially purified and analyzed by LC-CID mass spectra. The separation was achieved by C-18 column; eight limonoids were identified by comparing the retention times and mass spectral fragmentation. To the best of our knowledge, this is the first report on the identification of citrus limonoids using CID technique.

In vivo induction of phase II detoxifying enzymes, glutathione transferase and quinone reductase by citrus triterpenoids

BackgroundSeveral cell culture and animal studies demonstrated that citrus bioactive compounds have protective effects against certain types of cancer. Among several classes of citrus bioactive compounds, limonoids were reported to prevent different types of cancer. Furthermore, the structures of citrus limonoids were reported to influence the activity of phase II detoxifying enzymes. The purpose of the study was to evaluate how variations in the structures of citrus limonoids (namely nomilin, deacetyl nomilin, and isoobacunoic acid) and a mixture of limonoids would influence phase II enzyme activity in excised tissues from a mouse model.MethodsIn the current study, defatted sour orange seed powder was extracted with ethyl acetate and subjected to silica gel chromatography. The HPLC, NMR and mass spectra were used to elucidate the purity and structure of compounds. Female A/J mice were treated with three limonoids and a mixture in order to evaluate their effect on phase II enzymes in four different tissues. Assays for glutathione S-transferase and NAD(P)H: quinone reductase (QR) were used to evaluate induction of phase II enzymatic activity.ResultsThe highest induction of GST against 1-chloro-2,4-dinitrobenzene (CDNB) was observed in stomach (whole), 58% by nomilin, followed by 25% isoobacunoic acid and 19% deacetyl nomilin. Deacetyl nomilin in intestine (small) as well as liver significantly reduced GST activity against CDNB. Additionally isoobacunoic acid and the limonoid mixture in liver demonstrated a significant reduction of GST activity against CDNB. Nomilin significantly induced GST activity against 4-nitroquinoline 1-oxide (4NQO), intestine (280%) and stomach (75%) while deacetyl nomilin showed significant induction only in intestine (73%). Induction of GST activity was also observed in intestine (93%) and stomach (45%) treated with the limonoid mixture. Finally, a significant induction of NAD(P)H: quinone reductase (QR) activity was observed by the limonoid mixture in stomach (200%). In addition, the deacetyl nomilin treatment group displayed an increase in QR activity in liver (183%) and intestine (22%).ConclusionThe results of the present study suggests that, dietary intake of citrus limonoids may provide a protective effect against the onset of various cancers by inducing the activity of certain phase II detoxifying enzymes in specific organs.

Grapefruit bioactive limonoids modulate E. coli O157:H7 TTSS and biofilm

Limonoids are important constituents of the grapefruit and other citrus fruits. Research on health benefits suggests that citrus limonoids may act as anti-cancer, cholesterol lowering, anti-HIV and anti-feedant compounds. However, antimicrobial activities of citrus limonoids are not reported. In the present investigation, limonoids were purified from grapefruit seed and evaluated for their potential to antagonize cell-to-cell communication, biofilm formation and expression of Enterohemorrhagic Escherichia coli (EHEC) type three secretion system (TTSS). The results of the present study suggest that, certain limonoids are inhibitory to the cell-to-cell communication, biofilm formation and EHEC TTSS. Specifically, obacunone demonstrated strong inhibition of EHEC biofilm formation and TTSS. Furthermore, obacunone and other limonoids seem to inhibit the biofilm formation and TTSS in quorum sensing dependent fashion. The results indicate that certain grapefruit limonoids may possibly help in antagonizing the EHEC infection process, and may serve as lead compound in development of new antipathogenic molecules.

Bioactive Compounds: Historical Perspectives, Opportunities, and Challenges

Mom's conventional wisdom of eating fruits and vegetables to lead a healthy life has evolved with scientific, fact-finding research during the past four decades due to advances in science of "Foods for Health". Epidemiological and prospective studies have demonstrated the vital role of fruits, vegetables, and nuts in reducing the risk of cancer and cardiovascular diseases. In recent years, several meta-analyses strongly suggested that by adding one serving of fruits and vegetables to daily diet, the risk of cardiovascular diseases will be decreased up to 7%. The multidisciplinary and partnership efforts of agriculture and medical scientists across the globe stimulated interest in establishing certain interdisciplinary centers and institutes focusing on "Foods for Health". While the consumption of various healthy foods continues, several questions about toxicity, bioavailability, and food-drug interactions of bioactive compounds are yet to be fully understood on the basis of scientific evidence. Recent research on elucidation of the molecular mechanisms to understand the "proof of the concept" will provide the perfect answer when consumers are ready for a "consumer-to-farm" rather than the current "farm-to-consumer" approach. The multidisciplinary research and educational efforts will address the role of healthy foods to improve eye, brain, and heart health while reducing the risk of cancer. Through this connection, this review is an attempt to provide insight and historical perspectives on some of the bioactive compounds from the day of discovery to their current status. The bioactive compounds discussed in this review are flavonoids, carotenoids, curcumin, ascorbic acid, and citrus limonoids.

Limonin Methoxylation Influences the Induction of Glutathione S-Transferase and Quinone Reductase

Previous studies have indicated the chemopreventive potential of citrus limonoids due to the induction of phase II detoxifying enzymes. In the present study, three citrus limonoids were purified and identified from sour orange seeds as limonin, limonin glucoside (LG), and deacetylnomilinic acid glucoside (DNAG). In addition, limonin was modified to defuran limonin and limonin 7-methoxime. The structures of these compounds were confirmed by NMR studies. These five compounds were used to investigate the influence of phase II enzymes in female A/J mice. Our results indicated the highest induction of glutathione S-transferase (GST) activity against 1-chloro-2,4-dinitrobenzene (CDNB) by DNAG (67%) in lung homogenates followed by limonin-7-methoxime (32%) in treated liver homogenates. Interestingly, limonin-7-methoxime showed the highest GST activity (270%) in liver against 4-nitroquinoline 1-oxide (4NQO), while the same compound in the stomach induced GST by 51% compared to the control. The DNAG treated group induced 55% in stomach homogenates. Another phase II enzyme, quinone reductase (QR), was significantly induced by limonin-7-methoxime by 65 and 32% in liver and lung homogenates, respectively. Defuran limonin induced QR in lung homogenates by 45%. Our results indicated that modification of limonin has differential induction of phase II enzymes. These findings are indicative of a possible mechanism for the prevention of cancer by aiding in the detoxification of xenobiotics.

Liquid chromatography–electrospray ionisation mass spectrometry method for the rapid identification of citrus limonoid glucosides in citrus juices and extracts

A rapid and selective liquid chromatography–electrospray ionisation mass spectrometry (LC–ESI-MS) method to screen citrus samples for limonoid glucosides and estimate their relative concentrations has been developed. This method utilises a phenyl stationary phase, whereas previous methods have relied on C-18. Samples may be analysed directly without treatment other than dilution. Peak areas from the extracted deprotonated molecular ion mass signals for individual limonoid glucosides were normalised against the sum of the areas to establish their relative concentrations. The method was successfully applied to the analysis of various juice, extracts, and liquid samples of partially purified limonoid glucosides.