Citrus Limonoids Research Articles

Bile acids acting as a feeding signal and functional foods mimicking bile acid function.

To elucidate the function of the bile acid-binding receptor TGR5 in skeletal muscle, we developed transgenic mice expressing human TGR5 in the skeletal muscle tissue. A significant increase in muscle mass was observed in these transgenic mice, whereas a decrease in muscle mass was observed in the TGR5-deficient mice. Following treadmill exercise, TGR5 gene expression increased in response to ER stress induced in skeletal muscle via an ER stress response motif present in its promoter region. Exercise and rapid postprandial elevation in blood bile acid concentrations can be considered the primary stimuli for the TGR5-mediated increase in skeletal muscle mass. We developed a scoring system to identify food ingredients with TGR5 agonist activity, and identified the citrus limonoid nomilin. Similar effects were observed for other triterpenoids in addition to nomilin. Cell culture and in vivo experiments demonstrated that these food factors increase protein synthesis and muscle mass.

Nomilin Reversed Cardiotoxicity Caused by Co-exposure to Zearalenone and Deoxynivalenol via the Keap1/Nrf2 Signaling Pathway in Zebrafish.

The contamination of food and feed by mycotoxins, particularly zearalenone (ZEA) and deoxynivalenol (DON), is a global issue. Prenatal exposure to ZEA and DON can result in congenital cardiac malformations in fetuses. Addressing the prevention and mitigation of embryonic cardiotoxicity caused by these toxins is crucial. Citrus limonoid nomilin (NOM) is an extract known for its pathological properties in various diseases. This study investigated the potential mechanism of NOM in mitigating cardiotoxicity caused by ZEA and DON co-exposure in a zebrafish model. The findings indicated that NOM pretreatment alleviated cardiac developmental toxicity induced by ZEA and DON and normalized the expression of key genes involved in heart development, including gata4, vmhc, nkx2.5, and sox9b. Co-exposure to NOM, ZEA, and DON enhanced SOD and catalase activity, increased glutathione levels, and reduced ROS and malondialdehyde production. Furthermore, NOM reduced cardiac oxidative damage by activating the Keap1/Nrf2 signaling pathway. In summary, this study offers new insights for preventive interventions against congenital heart disease caused by mycotoxin exposure.

Identifying citrus limonoids as a potential fusion inhibitor of DENV-2 virus through its in silico study and FTIR analysis.

The online version contains supplementary material available at 10.1007/s40203-024-00207-2.

Metabolomic navigated Citrus waste repurposing to restore amino acids disorder in neural lesion

The fruit juice food industry produces huge waste annually, mainly Citrus peel and seeds. We investigated their chemical composition using hydrophilic interaction chromatography (HILIC-) and reverse phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS), revealing 277 compounds, mainly containing flavonoids and limonoids. As the primary representative component in Citrus waste, limonin was selected to be explored new bio-functions. We applied Zebrafish larvae to study the metabolomic response invoked by limonin. The differential metabolites (DMs) varied depending on the exposing concentration of limonin. Enrichment analysis indicated that the identified DMs related to inflammation and neurologic disorders, including epilepsy which were newly discovered for limonoids and Citrus waste. Limonin was found to restore amino acids disorder to take neuroprotection against epilepsy. Our findings provided a new bio-function and purpose for Citrus waste and limonoids. Also, we demonstrated a concise case to repurpose food waste for new applications under metabolome investigation.

Antioxidant Activity of Citrus Limonoids and Investigation of Their Virucidal Potential against SARS-CoV-2 in Cellular Models.

The COVID-19 pandemic represents an unprecedented global emergency. Despite all efforts, COVID-19 remains a threat to public health, due to the complexity of mass vaccination programs, the lack of effective drugs, and the emergence of new variants. A link has recently been found between the risk of developing a severe COVID-19 infection and a high level of oxidative stress. In this context, we have focused our attention on natural compounds with the aim of finding molecules capable of acting through a dual virucidal–antioxidant mechanism. In particular, we studied the potential of grapefruit seed extracts (GSE) and their main components, belonging to the class of limonoids. Using chemical and biological approaches including isolation and purification of GSE, antioxidant and virucidal assays, we have shown that grapefruit seed constituents, belonging to the class of limonoids, are endowed with remarkable virucidal, antioxidant and mitoprotective activity.

Effect Of Thymus Vulgaris, Mentha Piperita And Pelargonium Citrosum Leaf, Syzygium Aromaticum Buds And Citrus Limonoids Peels Extracts As Mosquito Larvicidal And Pupicidal Agent

Mosquito control depends on application of synthetic insecticides. This has been limited due to its harmful effect on human health. This increased needs for botanical origin alternatives as a sustainable and more safe method of mosquito control. The current study aimed to evaluate larvicidal and pupicidal effect of thyme ( Thymus vulgaris ), peppermint ( Mentha piperita ) and citronella leaf ( Pelargonium citrosum ), clove buds ( Syzygium aromaticum ) and lemon peels ( Citrus limonoids) methanolic extracts. Tested plants were extracted and three concentrations of these extracts were prepared (25, 50 and 100 mg/ml) then their larvicidal and pupicidal activities were evaluated against 20 larvae and 20 pupas for each concentration. Mortality rate were calculated after 24 hrs. Adult mosquitoes were identified to genus level. Clove showed the highest larvae mortality rate (100%) for the three tested concentrations, followed by thyme, lemon and peppermint. Citronella had no effect as larvicidal agent. On the other hand, pupicidal activity test of thyme showed the highest results followed by clove. While lemon didn’t show any activity. Mosquito was identified as Culex. The result showed that buds extract of S. aromaticum and leaf extracts of T. vulgaris and peels extract of C. limonoids could serve as potential larvicidal extracts against the Culex mosquito.

Limonoids as an effective Anticancer Agent: A Review

Citrus limonoids (CLs) are a group of highly oxygenated terpenoid found mostly in the seeds, fruits and peel tissues of citrus fruits such as lemons, limes, pumellos, oranges, grapefruits, bergamots, and mandarins. [10] The term limonoids was derived from limonin, which is the first tetranortriterpenoid obtained from bitter principles of citrus [6]. CLs are found as both free aglycones and corresponding -D glucosides, the former mostly occurring in seeds while the latter are formed during fruit maturation. Conversion of agycon to glycon is carried out by two enzymes, uridine diphosphoglucose- limonoid glycosyl t ransferase and limonoid D-ring lactone hydrolase. [8,9]. Glucosides have an open D ring to accommodate an attached glucose moiety; in aglycones the D-ring is closed [9] Citrus fruits are particularly high in a class of phytochemicals known as the limonoids1and recent research suggests that limonoids appearto possess substantial anticancer activity. [7] The present review focused on pharmacological studies of citrus limonoids as an anticancer agent.

Citrus limonoids induce apoptosis and inhibit the proliferation of pancreatic cancer cells.

In our recent study, we demonstrated that certain limonoids isolated from citrus seeds induced apoptosis in human pancreatic (Panc-28) cells. In this study, limonin, nomilin and limonexic acid (LNA) were investigated for understanding the possible mode of cytotoxicity in cultured pancreatic cancer (Panc-28) cells. All three limonoids inhibited Panc-28 cell proliferation, with IC50 values less than 50 μM after 72 h of incubation. The induction of apoptosis was confirmed through the cleavage of caspase-3, decreased mitochondrial membrane potential, and expression of apoptosis-related proteins. The Bax/Bcl2 expression ratio was increased up to 11-fold in cells pre-treated with 60 μM limonoids for 48 h. Apart from this, the limonoids also induced the expression of p21, and exhibited anti-inflammatory activity through decreasing the expression of cox-2, NF-κB and IL-6. Based on these results, we were interested in understanding the possible mode of inhibition by LNA, which exhibited the highest activity. The treatment of Panc-28 cells resulted in dose- and time-dependent induction of apoptosis-inducible proteins. In addition, treatment with 60 μM LNA resulted in the activation of Akt-associated signals to induce apoptosis, and the same was confirmed by the effects of the compounds on pAkt, p53, VEGF and caspase proteins. The results of this study demonstrated the cytotoxicity of limonoids to human pancreatic cancer cells through the modulation of genes involved in proliferation and survival.

Limonoids from Citrus: Chemistry, anti-tumor potential, and other bioactivities

• This review describes the recent progress in Chemical structures and bioactivities of limonoids. • This review summarizes the origin of Citrus limonoids from different species. • The possibility of Citrus limonoids as drug lead was discussed. • The recent progress in the metabolic study of Citrus limonoids was discussed. Citrus limonoids are tetranortriterpenoids compounds mainly found in oranges, lemons, grapefruits, and other fruits of Citrus . They are proved to be the leading cause of bitterness in Citrus fruits and are mainly consumed for therapeutic purposes and as food. Numerous studies have focused on Citrus limonoids and intend to develop new chemotherapeutic or complementary medicine in recent years. Citrus limonoids showed various bioactivities such as anti-tumor, antioxidative, anti-inflammatory, anti-neurological diseases, immunomodulatory, anti-insect, anti-bacteria, antiviral activities, etc. This review summarized limonoids from Citrus to date, along with their chemical structures and biological activities with a particular focus on their anti-tumor potential.

Role of Citrus Limonoid as a Possible Bioavailability Enhancer

The oral delivery is the most practical route to deliver drugs into the body, however drug-metabolizing enzymes and drug transporters can play important roles in modulating drug absorption. This study intended to find a natural bioenhancer for improving drug bioavailability. Two limonoids, including limonin deepoxy and nomilin, isolated from pomelo pulp were studied and the inhibition effects on human CYP3A4 and P-gp were investigated. Testosterone 6β-hydroxylation was performed in recombinant human CYP3A4 to discover the effects on CYP activity. Daunorubicin transport in Caco-2 and calcein-AM uptake in LLC-PK1and LLC-GA5-COL300 were conducted to evaluate the effects on P-gp function. The results show that both limonin deepoxy and nomilin could inhibit CYP3A4 and only nomilin exhibited mechanism-based inhibition. Nomilin was able to inhibit human P-gp in the concentration-dependent manner. Taken together, nomilin demonstrated strong activities on both CYP3A4 and P-gp, indicating that nomilin could possibly be used as a bioavailability enhancer.

Elucidation of osteoporosis prevention mechanism by citrus limonoid

Elucidation of osteoporosis prevention mechanism by citrus limonoid

Effect of Post-Harvest LED and UV Light Irradiation on the Accumulation of Flavonoids and Limonoids in the Segments of Newhall Navel Oranges (Citrus sinensis Osbeck).

To investigate the effect of post-harvest light irradiation on the accumulation of flavonoids and limonoids, harvested Newhall navel oranges were continuously exposed to light-emitting diode (LED) and ultraviolet (UV) light irradiation for 6 days, and the composition and content of flavonoids and limonoids in the segments were determined using UPLC-qTOF-MS at 0, 6, and 15 days after harvest. In total, six polymethoxylated flavonoids (PMFs), five flavone-O/C-glycosides, seven flavanone-O-glycosides, and three limonoids were identified in the segments. The accumulation of these components was altered by light irradiation. Red and blue light resulted in higher levels of PMFs during exposure periods. The accumulation of PMFs was also significantly induced after white light, UVB and UVC irradiation were removed. Red and UVC irradiation induced the accumulation of flavone and flavanone glycosides throughout the entire experimental period. Single light induced limonoid accumulation during exposure periods, but limonoid levels decreased significantly when irradiation was removed. Principal component analysis showed a clear correlation between PMFs and white light, between flavonoid glycosides and red light and UVC, and between limonoids and UVC. These results suggest that the accumulation of flavonoids and limonoids in citrus is regulated by light irradiation. White light, red light and UVC irradiation might be a good potential method for improving the nutrition and flavor quality of post-harvest citrus.

Variation in limonin and nomilin content in citrus fruits of eight varieties determined by modified HPLC.

The nomilin and limonin content in citrus fruits of different varieties was determined at fruit growth and maturation stages by HPLC. The results showed that the two limonoids can be separated, identified, and quantified in citrus fruits within 10min by the developed method. The method exhibited good precision, repeatability, stability, and recovery rate. The content of limonin and nomilin in most citrus fruits presented an increasing trend initially, and then decreased during fruit growth and maturation; a peak was observed at the young fruit or fruit expansion stage. The dropped fruits also contained some amount of limonoids, suggesting their industrial application. The variation and cluster analyses results revealed that the orange varieties contained the highest amount of limonoids at the mature stage. The results of this study will enable better use of citrus limonoids.

Citrus limonoids: mechanism, function and its metabolic engineering for human health

Citrus limonoids: mechanism, function and its metabolic engineering for human health

Citrus Limonoid Glucosyltransferase: AKey Player For Natural Debittering And Anticancerous Potential

Citrus fruits and juices are rich source of health benefitting phytochemicals which play a vital role in balanced diet and disease prevention. Citrus limonoids and flavonoids are the major phytochemicals which are of great interest in pharmaceutical industries because of their demonstrated anticancerous, antioxidant, anti-inflammatory, hormonal stimulation, antibacterial and antiviral actions. Citrus limonoid biosynthetic pathway contains an important regulatory limonoid glucosyltransferase enzyme (LGT). LGT is the natural debittering enzyme encoded by a single copy gene which has been isolated from different Citrus spp. This enzyme is mainly responsible for conversion of all limonoid aglycones (mostly bitter) to their corresponding glucosides (mostly nonbitter) but only during late fruit developmental stage of citrus. Citrus LGT belongs to glycosyltransferase super family whose members are the wide managers to catalyze the transfer of sugar molecules to their acceptor molecules to play several key modifications in plant secondary metabolites. These reveal great significance value in plant cell metabolism especially in detoxification of xenobiotics, production and storage of natural products. Despite to the fact that over expression of LGT in citrus will lead to reduce the delayed bitterness caused by limonin (an aglycone) but in addition will enhance the accumulation of limonoid glucosides in fruits. Further, recent studies suggest that citrus limonoids especially glucosides have shown importance against brain, pancreas, colon, and breast cancers. Thus, future studies should be focused on utilizing the potential of LGT present in citrus plants in terms of anticancerous properties as well as reducing the delayed bitterness problem important for citrus juice industry

Identification of key amino acid residues in the hTGR5-nomilin interaction and construction of its binding model.

TGR5, a member of the G protein-coupled receptor (GPCR) family, is activated by bile acids. Because TGR5 promotes energy expenditure and improves glucose homeostasis, it is recognized as a key target in treating metabolic diseases. We previously showed that nomilin, a citrus limonoid, activates TGR5 and confers anti-obesity and anti-hyperglycemic effects in mice. Information on the TGR5–nomilin interaction regarding molecular structure, however, has not been reported. In the present study, we found that human TGR5 (hTGR5) shows higher nomilin responsiveness than does mouse TGR5 (mTGR5). Using mouse–human chimeric TGR5, we also found that three amino acid residues (Q77ECL1, R80ECL1, and Y893.29) are important in the hTGR5–nomilin interaction. Based on these results, an hTGR5–nomilin binding model was constructed using in silico docking simulation, demonstrating that four hydrophilic hydrogen-bonding interactions occur between nomilin and hTGR5. The binding mode of hTGR5–nomilin is vastly different from those of other TGR5 agonists previously reported, suggesting that TGR5 forms various binding patterns depending on the type of agonist. Our study promotes a better understanding of the structure of TGR5, and it may be useful in developing and screening new TGR5 agonists.

A Limonoid Kihadanin B from Immature Citrus unshiu Peels Suppresses Adipogenesis through Repression of the Akt-FOXO1-PPARγ Axis in Adipocytes.

Citrus limonoids are secondary metabolites and exhibit a variety of biological activities. In this study, we elucidated the suppression of adipogenesis by a Citrus limonoid kihadanin B and determined its molecular mechanism in mouse 3T3-L1 adipocytes. Kihadanin B was purified from the peels of immature Citrus unshiu by HPLC, and its chemical structure was determined by NMR and mass spectrometry. Kihadanin B reduced the lipid accumulation with the reduction of the expression levels of the adipogenic and lipogenic genes, but did not affect lipolysis in adipocytes. Phosphorylation levels of Akt and a forkhead transcriptional factor, FOXO1, a repressor of PPARγ, were lowered by kihadanin B. Furthermore, kihadanin B increased the binding level of FOXO1 to the PPARγ gene promoter in adipocytes. These results indicate that a Citrus limonoid kihadanin B repressed the adipogenesis by decreasing lipid accumulation through the suppression of the Akt-FOXO1-PPARγ axis in 3T3-L1 adipocytes.

The Chemistry and Pharmacology of Citrus Limonoids.

Citrus limonoids (CLs) are a group of highly oxygenated terpenoid secondary metabolites found mostly in the seeds, fruits and peel tissues of citrus fruits such as lemons, limes, oranges, pumellos, grapefruits, bergamots, and mandarins. Represented by limonin, the aglycones and glycosides of CLs have shown to display numerous pharmacological activities including anticancer, antimicrobial, antioxidant, antidiabetic and insecticidal among others. In this review, the chemistry and pharmacology of CLs are systematically scrutinised through the use of medicinal chemistry tools and structure-activity relationship approach. Synthetic derivatives and other structurally-related limonoids from other sources are include in the analysis. With the focus on literature in the past decade, the chemical classification of CLs, their physico-chemical properties as drugs, their biosynthesis and enzymatic modifications, possible ways of enhancing their biological activities through structural modifications, their ligand efficiency metrics and systematic graphical radar plot analysis to assess their developability as drugs are among those discussed in detail.

Citrus limonoid nomilin inhibits osteoclastogenesis in vitro by suppression of NFATc1 and MAPK signaling pathways

BackgroundAnimal experiment studies have revealed a positive association between intake of citrus fruits and bone health. Nomilin, a limonoid present in citrus fruits, is reported to have many biological activities in mammalian systems, but the mechanism of nomilin on bone metabolism regulation is currently unclear. PurposeTo reveal the mechanism of nomilin on osteoclastic differentiation of mouse primary bone marrow-derived macrophages (BMMs) and the mouse RAW 264.7 macrophage cell line into osteoclasts. Study designControlled laboratory study. Effects of nomilin on osteoclastic differentiation were studied in in vitro cell cultures. MethodsCell viability of RAW 264.7 cells and BMMs was measured with the Cell Counting Kit. TRAP-positive multinucleated cells were counted as osteoclast cell numbers. The number and area of resorption pits were measured as bone-resorbing activity. Osteoclast-specific genes expression was evaluated by quantitative real-time PCR; and proteins expression was evaluated by western blot. ResultsNomilin significantly decreased TRAP-positive multinucleated cell numbers compared with the control, and exhibited no cytotoxicity. Nomilin decreased bone resorption activity. Nomilin downregulated osteoclast-specific genes, NFATc1 and TRAP mRNA levels. Furthermore, nomilin suppressed MAPK signaling pathways. ConclusionThis study demonstrates clearly that nomilin has inhibitory effects on osteoclastic differentiation in vitro. These findings indicate that nomilin-containing herbal preparations have potential utility for the prevention of bone metabolic diseases.

Determination of Citrus Limonoid Glucosides by High Performance Liquid Chromatography Coupled to Post-Column Reaction with Ehrlich’s Reagent

A method for the identification and quantification of citrus limonoid glucosides in juices, based upon high performance liquid chromatography (HPLC) separation coupled to post-column reaction with Ehrlich’s reagent, has been developed. This method utilizes a phenyl stationary phase and an isocratic mobile phase. Limit of detection for the method was determined to be 50 ng of limonin glucoside. Limonin glucoside and the other limonoid glucosides tested responded equivalently to the post-column reaction with Ehrlich’s reagent suggesting that limonin glucoside may be used as a surrogate for the quantification of other limonoid glucosides, including nomilin, deacetyl nomilin, nomilinic acid, deacetyl nomilinic acid, and obacunone glucosides. For most juice samples, no sample treatment is required to determine limonin glucoside content. Concentration by solid phase extraction is recommended for juices with low levels of limonin glucoside and to facilitate the detection and quantification of other limonoid glucosides present at low concentrations. With the exception of blood oranges, the method was successfully applied to the analysis of juices from a variety of citrus varieties.