Naringin Levels in Citrus Tissues : II. Quantitative Distribution of Naringin in Citrus paradisi MacFad

The quantitative distribution of the flavanone-7-neohesperidoside, naringin, in seeds, seedlings, young plants, branches, flowers, and fruit of Citrus paradisi Macfad., cv `Duncan' was analyzed by radioimmunoassay. High levels of naringin were associated with very young tissue and lower levels were found in older tissues. Seed coats of ungerminated seeds and young shoots had high naringin concentrations whereas cotyledons and roots had very low concentrations. Light-grown seedlings contained nearly twice as much naringin as etiolated seedlings and, in young plants and branches, the naringin content was highest in developing leaves and stem tissue. In flowers, the ovary had the highest levels of naringin, accounting for nearly 11% of the fresh weight. There was a net increase in the total naringin content of fruits during growth. However, due to the large increase in fruit size, there was a concomitant decrease in the naringin concentration as the fruit matured.     

Naringin dietary supplementation at 0.15% rates does not provide protection against sub-clinical acidosis and does not affect the responses of fattening lambs to road transportation

Forty Assaf fattening lambs (initial age 13 to 15 weeks) offered a diet of barley straw and a commercial concentrate were used to assess the effect of naringin (a type of citrus flavonoid with proven antioxidant, antimicrobial and anti-inflammatory properties in monogastric animals) at a dose of 1.5 g/kg per dry matteron plasma lipid peroxidation thiobarbituric acid reactive substances (TBARS), immune response, ruminal bacterial community and protection provided by the ruminal wall against subclinical acidosis. After 49 days of the experimental diets, lambs were subjected to a 4-h transportation stress period. As expected, TBARS values were significantly increased in all the lambs just after the transportation period, but no effect of naringin was observed. Although naringin lowered red blood cell count, neither the total white blood cells counts nor the production of IFN-γ were affected by naringin. No anti-inflammation activity preventing rumenitis was detected, but a clear effect on ruminal bacterial community was observed in lambs consuming naringin. Further experiments, using different doses of naringin might show health benefits of naringin supplementation in lambs, but a clear beneficial effect on health was not readily apparent in this study.     

Naringin Alleviates Diabetic Kidney Disease through Inhibiting Oxidative Stress and Inflammatory Reaction

Naringin, a flavanone glycoside extracted from Citrus grandis Osbeck, has a wide range of pharmacological effects. In the present study we aimed at demonstrating the protective effect of naringin against diabetic kidney disease (DKD) and elucidating its possible molecular mechanism underlying. The beneficial effect of naringin was assessed in rats with streptozotocin (STZ)-induced diabetes and high glucose-induced HBZY-1 cells. According to our results, first we found that naringin relieved kidney injury, improved renal function and inhibited collagen formation and renal interstitial fibrosis. Second, we confirmed that naringin restrained oxidative stress by activating Nrf2 antioxidant pathway. Moreover, the results suggested that naringin significantly resisted inflammatory reaction by inhibiting NF- κ B signaling pathway. Taken together, our results demonstrate that naringin effectively alleviates DKD, which provide theoretical basis for naringin clinically used to treatment of DKD.     

Direct enzymatic glucosylation of naringin in grapefruit juice by alpha-D-glucosidase from the marine mollusc Aplysia fasciata

The enzymatic glucosylations of naringin, performed using alpha-D-glucosidase, identified in the Mediterranean mollusc Aplysia fasciata is reported. The enzyme actively operates on maltose and has an interesting transglycosylation potential using this donor. We also investigated the use of this marine alpha-glucosidase for a food-compatible glucosylation of naringin to produce new enzymatically modified carbohydrate possessing naringin derivatives. The regioselective formations of the beta-gluco-C6 alpha-glucosyl derivative and of the corresponding isomaltosyl diglucoside of naringin were obtained in high yield and efficiency of reaction. Suspensions of naringin can be used up to approximately 90 mg/mL initial acceptor concentration. In different experiments it was demonstrated that the enzyme was still active after 48 h in presence of this high amount of acceptor and that one of the diasteromers of the naringin is preferred by the enzyme from A. fasciata during glucosylation/deglucosylation enzymatic steps. Finally, the feasibility of efficient naringin glucosylation in grapefruit juice was also demonstrated at optimal pH of the enzyme and low maltose concentrations.     

Alleviation of iron induced oxidative stress by the grape fruit flavanone naringin in vitro

Iron is an essential element that participates in several metabolic activities of cells; however, excess iron is a major cause of iron-induced oxidative stress and several human diseases. The protective effect of naringin, a grape fruit flavanone, was studied in iron overloaded isolated mouse liver mitochondria, where the isolated mitochondrial fraction was incubated with various concentrations of naringin before ferric ion loading. Iron overloading of mitochondrial fraction resulted in an increase in lipid peroxidation, protein oxidation, and DNA damage, whereas iron overload reduced the glutathione (GSH) concentration, glutathione-S-transferase (GST), glutathione peroxidase (GSHPx), catalase and superoxide dismutase (SOD) activities. Pretreatment of mitochondrial fraction with naringin inhibited iron-induced lipid peroxidation, protein oxidation, and DNA damage. Conversely, naringin supplementation arrested iron-induced depletion in the GSH contents, GSHPx, GST, SOD and catalase activities significantly. Ferric iron reduction assay revealed that naringin could not reduce ferric iron into ferrous iron indicating that it did not exhibit prooxidant activity. Iron free coordination site assay indicated that naringin was unable to occupy all the active sites of iron indicating that naringin did not completely chelate iron. Our study demonstrates that naringin was able to share the burden of endogenous oxidants by inhibiting the iron-induced depletion of all important antioxidant enzymes as well as GSH and may act as a good antioxidant.     

Juvenile-Specific Localization and Accumulation of a Rhamnosyltransferase and Its Bitter Flavonoid in Foliage,Flowers, and Young Citrus Fruits

1-2-Rhamnosyltransferase catalyzes the production of disaccharide-flavonoids that accumulate to 75% of dry weight. Vast energy is expended in a short time span to produce these flavonoids. The highest rhamnosyltransferase activities and immunodetected concentrations were observed in early development of Citrus grandis (pummelo), coinciding with up to 13% of fresh weight as naringin. The concentration of naringin in leaves, petals, receptacles, filaments, albedo, and flavedo drops drastically during development and correlates directly with a decrease in the activity and amounts of 1-2-rhamnosyltransferase. Anthers had minute rhamnosyltransferase activities and low concentrations of naringin. Conversely, high 1-2-rhamnosyltransferase activity and naringin concentrations appeared in both young and mature ovaries, as well as in young fruits. The total amounts of naringin in mature leaves decreased without detectable in vitro degradation of naringin in leaves. There was still a net accumulation of naringin in the albedo and flavedo of older fruit even though these tissues had only traces of 1-2-rhamnosyltransferase. Traces of enzyme synthesis in fruits, or import of the product from leaves, may explain the net accumulation of naringin in growing fruits. Unlike the late-expressed genes for glycosyltransferases in anthocyanin biosynthesis, the rhamnosyltransferases from Citrus are active only in juvenile stages of development.     

Naringinase-catalyzed hydrolysis of naringin adsorbed on macroporous resin

Naringenin, a natural plant flavonoid found in citrus fruits, has been reported to exhibit a wide range of pharmacological functions, including anticancer, antioxidant, antiatherogenic, antithrombotic, and vasodilator activities. Naringenin can be produced from the naringinase (NGase)-catalyzed enzymatic hydrolysis of naringin. However, the poor solubility of naringin in aqueous systems considerably limits the efficiency of naringenin biocatalysis. In this work, a novel substrate adsorption system was proposed for naringin adsorption to increase the efficiency of naringin hydrolysis and naringenin production. Three Amberlite macroporous resins, namely, XAD-4, XAD-7HP and XAD-16, were investigated for their naringin adsorption capacities and effects on NGase hydrolysis. Results indicated that the physical properties of the resins played a critical role in naringin adsorption and naringenin enzymatic synthesis. Naringin hydrolysis was carried out using free and adsorbed substrates. The substrate adsorption strategy could increase the catalytic efficiency at a high naringin concentration. In addition, the reaction conditions for enzymatic naringenin synthesis were optimized, and naringenin was prepared at a liter scale with a high substrate concentration. These results suggested that substrate adsorption is a promising strategy to increase the enzymatic hydrolysis efficiency of naringenin in aqueous systems.     

Determination of naringin and naringenin in human urine by high-performance liquid chromatography utilizing solid-phase extraction

An HPLC method for determining a flavonoid naringin and its metabolite, naringenin, in human urine is presented for application to the pharmacokinetic study of naringin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using hesperidin for naringin or hesperetin for naringenin as internal standard and solid-phase extraction using a strong anion exchanger, Sep-Pak Accell QMA cartridge. The HPLC assay was carried out using an Inertsil ODS-2 column (250×4.6 mm I.D., 5 μm particle size). The mobile phases were acetonitrile–0.1 M ammonium acetate–acetic acid (18:81:1, v/v; pH 4.7) for naringin and acetonitrile–0.1 M ammonium acetate–triethylamine (25:75:0.05; v/v; pH 8.0) for naringenin. The flow-rate was 1.0 ml min⁻¹. The analyses were performed by monitoring the wavelength of maximum UV absorbance at 282 nm for naringin and at 324 nm for naringenin. The lower limits of quantification were ca. 25 ng/ml for naringin and naringenin with R.S.D. less than 10%. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 5 ng for naringin and 1 ng for naringenin. A preliminary experiment to investigate the urinary excretion of naringin, naringenin and naringenin glucuronides after oral administration of 500 mg of naringin to a healthy volunteer demonstrated that the present method was suitable for determining naringin and naringenin in human urine.     

Regioselective acylation of flavonoids catalyzed by immobilized Candida antarctica lipase under reduced pressure

A single-step acylation of rutin and naringin, catalyzed by immobilized Candida antarctica lipase B in 2-methyl-2-butanol, occurred preferentially on the primary hydroxyl group. Using palmitic methyl ester as acyl donor, the acylation rate of naringin was 10-fold higher than that of rutin. Under optimal conditions, i.e. a molar ratio acyl donor/naringin of 7:1 and 200 mbar, 92% naringin was acylated.     

Functional characteristics of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. BL-31 highly specific for intermolecular transglycosylation of bioflavonoids

The functional characteristics of a beta-cyclodextrin glucanotransferase (CGTase) excreted from alkalophilic Bacillus sp. BL-31 that is highly specific for the intermolecular transglycosylation of bioflavonoids were investigated. The new beta-CGTase showed high specificities for glycosyl acceptor bioflavonoids, including naringin, rutin, and hesperidin, and especially naringin. The transglycosylation of naringin into glycosyl naringin was then carried out under the conditions of 80 units of CGTase per gram of maltodextrin, 5 g/l of naringin, 25 g/l of maltodextrin, and 1 mM Mn2+ ion at 40 degrees C for 6 h, resulting in a high conversion yield of 92.1%.     

Investigation of the Interaction of Naringin Palmitate with Bovine Serum Albumin: Spectroscopic Analysis and Molecular Docking

Background

Bovine serum albumin (BSA) contains high affinity binding sites for several endogenous and exogenous compounds and has been used to replace human serum albumin (HSA), as these two compounds share a similar structure. Naringin palmitate is a modified product of naringin that is produced by an acylation reaction with palmitic acid, which is considered to be an effective substance for enhancing naringin lipophilicity. In this study, the interaction of naringin palmitate with BSA was characterised by spectroscopic and molecular docking techniques.

Methodology/Principal Findings

The goal of this study was to investigate the interactions between naringin palmitate and BSA under physiological conditions, and differences in naringin and naringin palmitate affinities for BSA were further compared and analysed. The formation of naringin palmitate-BSA was revealed by fluorescence quenching, and the Stern-Volmer quenching constant (K_SV) was found to decrease with increasing temperature, suggesting that a static quenching mechanism was involved. The changes in enthalpy (ΔH) and entropy (ΔS) for the interaction were detected at −4.11±0.18 kJ·mol⁻¹ and −76.59±0.32 J·mol⁻¹·K⁻¹, respectively, which indicated that the naringin palmitate-BSA interaction occurred mainly through van der Waals forces and hydrogen bond formation. The negative free energy change (ΔG) values of naringin palmitate at different temperatures suggested a spontaneous interaction. Circular dichroism studies revealed that the α-helical content of BSA decreased after interacting with naringin palmitate. Displacement studies suggested that naringin palmitate was partially bound to site I (subdomain IIA) of the BSA, which was also substantiated by the molecular docking studies.

Conclusions/Significance

In conclusion, naringin palmitate was transported by BSA and was easily removed afterwards. As a consequence, an extension of naringin applications for use in food, cosmetic and medicinal preparations may be clinically and practically significant, especially in the design of new naringin palmitate-inspired drugs.     

Naringin prevents ovariectomy-induced osteoporosis and promotes osteoclasts apoptosis through the mitochondria-mediated apoptosis pathway

Naringin, the primary active compound of the traditional Chinese medicine Rhizoma drynariae, possesses many pharmacological activities. The present study is an effort to explore the anti-osteoporosis potential of naringin in vivo and in vitro. In vivo, we used ovariectomized rats to clarify the mechanisms by which naringin anti-osteoporosis. In vitro, we used osteoclasts to investigate naringin promotes osteoclasts apoptosis. Naringin was effective at enhancing BMD, trabecular thickness, bone mineralization, and mechanical strength in a dose-dependent manner. The result of RT-PCR analysis revealed that naringin down-regulated the mRNA expression levels of BCL-2 and up-regulated BAX, caspase-3 and cytochrome C. In addition, naringin significantly reduced the bone resorption area in vitro. These findings suggest that naringin promotes the apoptosis of osteoclasts by regulating the activity of the mitochondrial apoptosis pathway and prevents OVX-induced osteoporosis in rats.     

C-2 stereochemistry of naringin and its relation to taste and biosynthesis in maturing grapefruit

Circular dichroism and proton magnetic resonance spectroscopy can be used to determine the C-2 chirality of naringin, the main bitter principle of grapefruit. Thus, the aglycone chirality of naringin has been studied as a function of grapefruit maturity. The amount of (2S) isomer is 85–92% in immature grapefruit but only 55–60% in mature grapefruit. These results are discussed in relation to the current postulates relating to flavanone biosynthesis. A naringin sample predominant in the (2R) isomer has been prepared and found by some tasters to be more bitter than the (2S) naringin. It is concluded that the naringin isomerization in ripening grapefruit is not responsible for debittering.     

柚皮苷及其与吴茱萸碱联合用药对APP~（swe）/PS~（ΔE9）转基因痴呆模型小鼠学习记忆能力的影响

目的探讨柚皮苷及柚皮苷与吴茱萸碱联合用药对APPswe/PSΔE9转基因痴呆模型小鼠学习记忆能力的影响,研究柚皮苷与吴茱萸碱联合使用是否具有协同或叠加作用。方法 APPswe/PSΔE9转基因痴呆模型小鼠随机分组,每组12只,雌雄各半,分别选择在3月龄和5月龄两个时间段开始进行治疗,各给药组按设定剂量分别单独或联合加入鼠粮中。3月龄给药16周、5月龄给药4周后进行水迷宫实验,以安理申作为阳性药对照,并与安慰剂组小鼠进行比较。Thioflavin-S荧光染色检测单独使用柚皮苷治疗转基因小鼠脑组织老年斑的形成。结果单独使用柚皮苷4周和16周,缩短寻找平台的潜伏期分别为22%、32%;单独使用吴茱萸碱4周和16周,缩短寻找平台的潜伏期分别为33%、10%。柚皮苷治疗16周减少海马老年斑26%。柚皮苷与吴茱萸碱联合应用能够显著缩短寻找平台的潜伏期47%。结论单独使用柚皮苷或吴茱萸碱均能改善痴呆模型小鼠的学习记忆能力,柚皮苷长期的使用优于短期的治疗效果,但是吴茱萸碱短期有效,长期作用反而下降;柚皮苷与吴茱萸碱联合应用能够显著改善APPswe/PSΔE9痴呆模型小鼠的学习记忆能力,但联合用药组并未表现出强于单独给药组的协同保护效应。     

Naringin alters the cholesterol biosynthesis and antioxidant enzyme activities in LDL receptor-knockout mice under cholesterol fed condition

The purpose of the current study was to evaluate the lipid lowering and antioxidant capacity of naringin in LDL receptor knockout (LDLR-KO) mice fed a cholesterol (0.1 g/100 g) diet. As such, naringin or lovastatin (0.02 g/100 g) was supplemented in a cholesterol diet for 6 weeks. The naringin and lovastatin supplementation significantly lowered the plasma total cholesterol level compared to the control group. The plasma and hepatic triglyceride level was only lowered by the lovastatin supplement, while the hepatic cholesterol content was lowered by both the naringin and lovastatin supplements compared to the control group. The hepatic HMG-CoA reductase activity was significantly lower in the naringin and lovastatin supplemented groups than in the control group, whereas the ACAT activity was unaffected. The excretion of total sterol was significantly higher in the naringin and lovastatin groups compared to the control group due to significant changes in the acidic and neutral sterol, respectively. When comparing the hepatic antioxidant enzyme activities, the superoxide dismutase, catalase, and glutathione reductase activities were all significantly higher in the naringin-supplemented group than in the control group, while only the lovastatin supplement increased the glutathione reductase activity. Accordingly, the current results confirmed that naringin lowers the plasma cholesterol level via the inhibition of hepatic HMG-CoA reductase activity and increases the excretion of fecal sterol. Naringin was also found to improve the activities of hepatic antioxidant enzymes against oxidative stress in a hypercholesterolemic animal model, i.e. cholesterol-fed LDLR-KO mice.     

Molecular characterization and enzymatic hydrolysis of naringin extracted from kinnow peel waste

Kinnow peel, a waste rich in glycosylated phenolic substances, is the principal by-product of the citrus fruit processing industry and its disposal is becoming a major problem. This peel is rich in naringin and may be used for rhamnose production by utilizing α-L-rhamnosidase (EC 3.2.1.40), an enzyme that catalyzes the cleavage of terminal rhamnosyl groups from naringin to yield prunin and rhamnose. In this work, infrared (IR) spectroscopy confirmed molecular characteristics of naringin extracted from kinnow peel waste. Further, recombinant α-L-rhamnosidase purified from Escherichia coli cells using immobilized metal-chelate affinity chromatography (IMAC) was used for naringin hydrolysis. The purified enzyme was inhibited by Hg2+ (1 mM), 4-hydroxymercuribenzoate (0.1 mM) and cyanamide (0.1 mM). The purified enzyme established hydrolysis of naringin extracted from kinnow peel and thus endorses its industrial applicability for producing rhamnose.     

An enzymatic colorimetric method for measuring naringin using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) in the presence of peroxidase

A sensitive colorimetric method for naringin estimation using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as peroxidase substrate is described. The method is based on the coupling reaction of an ABTS radical cation with an oxidation product of naringin formed by peroxidase. This coupling reaction leads to the formation of a purple-colored compound with a maximum absorption at 560 nm. A molar absorption coefficient at this wavelength was calculated to be 13,286 +/- 200 M-1 cm-1. The lowest amount of naringin that can be detected is 1 nmol. The application of this method to the quantification of naringin in grapefruit tissues is presented.     

Comparison of metabolic pharmacokinetics of naringin and naringenin in rabbits

Naringin and naringenin are antioxidant constituents of many Citrus fruits. Naringenin is the aglycone and a metabolite of naringin. In order to characterize and compare the metabolic pharmacokinetics of naringenin and naringin, naringenin was administered intravenously and orally to rabbits, and naringin was administered orally. The concentration of naringenin in serum prior to and after enzymatic hydrolysis was determined by HPLC method. The pharmacokinetic parameters were calculated by using WINNONLIN. The results showed that the absolute bioavailability of oral naringenin was only 4%, whereas after taking the conjugated naringenin into account, it increased to 8%. When naringin was administered orally, only little naringenin and predominantly its glucuronides/sulfates were circulating in the plasma. The ratio of AUC of naringenin conjugates to the total naringenin absorbed into the systemic circulation after oral naringenin was much higher when compared to that after i.v. bolus of naringenin, indicating that extensive glucuronidation/sulfation of naringenin occurred during the first pass at gut wall. Oral dosing of naringin resulted in even higher ratio of AUC of naringenin conjugates to the total naringenin than that after oral naringenin. Our results also showed that there were great differences in pharmacokinetics of naringin and naringenin. Oral naringin resulted in latter Tmax, lower Cmax and longer MRT (mean residence time) for both naringenin and its conjugated metabolites than those after oral naringenin.     

Naringin in Ganshuang Granule suppresses activation of hepatic stellate cells for anti‐fibrosis effect by inhibition of mammalian target of rapamycin

A previous study has demonstrated that Ganshuang granule (GSG) plays an anti‐fibrotic role partially by deactivation of hepatic stellate cells (HSCs). In HSCs activation, mammalian target of rapamycin (mTOR)‐autophagy plays an important role. We attempted to investigate the role of mTOR‐autophagy in anti‐fibrotic effect of GSG. The cirrhotic mouse model was prepared to demonstrate the anti‐fibrosis effect of GSG. High performance liquid chromatography (HPLC) analyses were used to identify the active component of GSG. The primary mouse HSCs were isolated and naringin was added into activated HSCs to observe its anti‐fibrotic effect. 3‐methyladenine (3‐MA) and Insulin‐like growth factor‐1 (IGF‐1) was added, respectively, into fully activated HSCs to explore the role of autophagy and mTOR. GSG played an anti‐fibrotic role through deactivation of HSCs in cirrhotic mouse model. The concentration of naringin was highest in GSG by HPLC analyses and naringin markedly suppressed HSCs activation in vitro, which suggested that naringin was the main active component of GSG. The deactivation of HSCs caused by naringin was not because of the autophagic activation but mTOR inhibition, which was supported by the following evidence: first, naringin induced autophagic activation, but when autophagy was blocked by 3‐MA, deactivation of HSCs was not attenuated or reversed. Second, naringin inhibited mTOR pathway, meanwhile when mTOR was activated by IGF‐1, deactivation of HSCs was reversed. In conclusion, we have demonstrated naringin in GSG suppressed activation of HSCs for anti‐fibrosis effect by inhibition of mTOR, indicating a potential therapeutic application for liver cirrhosis.     

Naringin abrogates osteoclastogenesis and bone resorption via the inhibition of RANKL-induced NF-κB and ERK activation

Osteolytic bone diseases including osteoporosis are commonly accompanied with enhanced osteoclast formation and bone resorption. Naringin, a natural occurring flavonoid has been found to protect against retinoic acid-induced osteoporosis and improve bone quality in rats. Here, we showed that naringin perturbs osteoclast formation and bone resorption by inhibiting RANK-mediated NF-κB and ERK signaling. Naringin suppressed gene expression of key osteoclast marker genes. Naringin was found to inhibit RANKL-induced activation of NF-κB by suppressing RANKL-mediated IκB-α degradation. In addition, naringin inhibited RANKL-induced phosphorylation of ERK. This study identifies naringin as an inhibitor for osteoclast formation and bone resorption, and provides evidence that natural compounds such as naringin might be beneficial as an alternative medicine for the prevention and treatment of osteolysis.