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 inhibit the formation of certain chemically induced carcinogenesis in laboratory animals. Some of the limonoids possess antifeedant activity against insects, suggesting that one of the biological functions of limonoids in plants is pest control. The citrus limonoids are unique for many species and varieties, and make excellent taxonomic markers. Thus, limonoids are important citrus constituents to citrus growers, processors, breeders, and the consumer.     

Limonoid glucosides in Orange Molasses

The concentrations of limonoid glucosides in orange molasses, a byproduct of citrus juice processing, were measured. The 17-β-D-gluco-pyranoside derivatives (glucosides) of limonin, nomilin, nomilinic acid and obacunone were present and quantified. Orange molasses contained an average of 568 ppm of total limonoid glucosides at 11.8°Brix. Orange molasses (3.8L) contained up to 12.5g of total limonoid glucosides. The molasses could be used as a source for commercial isolation of citrus limonoid glucosides.     

Limonoids and Flavonoids in Juices of Oroblanco and Melogold Grapefruit Hybrids

Oroblanco 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.     

Limonoids in Pummelos [Citrus grandis (L.) Osbeck]

Juice 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.     

Recovery of Limonoid Glucosides from Citrus Molasses

ABSTRACT: A method to recover multi-gram quantities of limonoid glucosides from citrus molasses using ion-exchange and styrene/divinylbenzene resins has been developed. A cation exchange resin is used to decolorize the molasses, while an anion exchanger is used to separate limonoid glucosides from other negatively charged compounds. Styrene/divinylbenzene resins are used to bind limonoid glucosides and remove water-soluble materials. The method affords approximately 5 grams of a water-soluble powder containing over 60% by weight limonoid glucosides per liter of molasses. An analysis of the efficiency of the method shows that less than 10% of the total limonoid glucosides are lost during recovery.     

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.     

Limonoid debittering of citrus juices using immobilized bacterial cell systems

Limonoids such as limonin and nomilin cause bitterness in a variety of commercial citrus juices. This is a major problem of the world‐wide citrus product industry and also has significant negative economic impact on processors and growers also. This article reviews research on immobilized bacterial cell systems that can be used for the reduction of limonoid bitterness in citrus juices.     

Limonoid Glucosides in Fruit, Juice and Processing by-products of Satsuma Mandarin (Chus unshiu Marcov.)

In a study on improving utilization of waste from processing of Satsuma mandarin (Citrus unshiu Marcov.) the limonoid glucosides of fruit, juice, and by-products were measured using HPLC and TLC. All materials had the 17-β-D-glucopyranoside derivatives of limonoids reported in other commercial citrus fruit. Citrus molasses was a good source for industrial scale extraction of limonoid glucosides. An extraction system using polystyrene divinylbenzene resins, was developed which could be expanded to industrial scale.     

柑橘中β-葡萄糖苷酶的研究进展

β-葡萄糖苷酶广泛存在于自然界中,在柑橘果皮、果汁以及果肉中都能被检测到。柑橘中β-葡萄糖苷酶对柑橘香气形成、柑橘果实加工具有重要作用。本文总结了柑橘中β-葡萄糖苷酶的酶活测定和分离纯化方法,阐述了柑橘中β-葡萄糖苷酶对柑橘果实香气的影响,及其在柑橘柠檬苦素类物质及花色苷降解中的作用,并且指出了柑橘β-葡萄糖苷酶研究中存在的问题以及今后研究的方向。      

甜橙种子中柠檬苦素类似物配糖体的测定

利用液质联用 (LC ESI MS)技术对甜橙种子的乙醇提取物进行了检测 ,从中发现了两种柠檬苦素类似物的配糖体———奥巴叩酮配糖体和诺米林配糖体 .采用13C和1H核磁共振谱法对化合物的结构进行了进一步证实 .     

Limonoid content of sour orange varieties

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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. Published 2011 by John Wiley & Sons, Ltd.     

Biosynthesis of limonoid glucosides in lemon (Citrus limon)

TLC analyses of lemon (Citrus limon (L) Burm f) fruit at various stages of maturity showed that limonoid glucosides were synthesised during the late stages of fruit growth. No limonoid glucosides were found in the stem and leaf. Use of radioactive tracers showed that ¹⁴C-nomilin was converted to ¹⁴C-nomilin 17-β-D -glucopyranoside in fruit tissues and seeds. Glucosides are indeed the major end products of limonoid metabolism.     

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

Recent 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     

柑桔中类柠檬苦素含量及分布研究

对几种柑桔品种果皮、种子、果汁中类柠檬苦素的含量进行了测定,结果表明,柑桔果实中不同部位类柠檬苦素含量不同,以种子中含量最高,果皮次之,而果汁中含量最低。不同品种间类柠檬苦素含量依次为:邓肯葡萄柚>琯溪蜜柚>南充实生甜橙>锦橙>大红袍红桔。     

柑橘类果汁脱苦技术研究进展

柚皮苷和类柠檬苦素是柑橘类果汁的主要苦味物质,其存在明显影响了柑橘类果汁的品质。介绍了脱除柑橘类果汁苦味的一些技术：代谢脱苦、吸附脱苦、酶法脱苦、屏蔽法脱苦、分离技术脱苦和基因脱苦等。     

冷冻对柑橘汁过滤性能的影响

过滤是果汁生产中固液分离的重要手段。冷冻至 - 2 2℃的柑橘汁的粘度从原来的 16 .5 m· Pa/s降低至 2 .80 m·Pa/s,室温下自然解冻后出现固液分离现象 ,经离心分离后 ,平均过滤速度是未冷冻过的柑橘汁过滤速度的 3倍 ,但冷冻温度对可溶性固形物含量基本没有影响     

柑桔汁中的果胶酯酶及其钝化特性

果胶酯酶（ＰＥ或ＰＭＥ）的活性是导致柑桔汁混浊态的丧失的主要原因,因此研究其理化性质,钝化其活性对提高柑桔汁质量意义重大。介绍了柑桔汁中果胶酯酶的性质及钝化其活性的一些措施。     

Kinetic Models for Thermal Inactivation of Multiple Pectinesterases in Citrus Juices

First-order kinetic model of a two-component system was applied to investigate heat inactivation of multiple pectinesterases in citrus juices. The conventional log-linear model was applied to determine D- and z-values of thermolabile and thermostable pectinesterases in citrus juices, respectively, for use in the two-component model. A parameter for concentration of enzyme populations in the juice was included in the model. The two-component model predicted results were in better agreement with the observed heat pasteurization curves than those predicted by the log-linear kinetic model. The two-component model is especially useful for optimizing temperature-time conditions for pasteurizing citrus juices.     

A review of pectin methylesterase inactivation in citrus juice during pasteurization

BackgroundPectin, naturally found in citrus, plays a key role in the quality of the obtained juices. Pectin methylesterase enzyme (PME) influences the cloud stability, viscosity, color, mouth feeling and flavor of the juices by de-esterification of pectin. Iinactivation of PME is introduced as a pasteurization index in citrus juices, due to its higher thermal resistance than the spoilage microorganisms.Scope and approachInactivation of PME using different thermal (conventional, microwave and ohmic heating) and non-thermal (pulsed electric field, high pressure processing and high pressure carbon dioxide) processes is important in juice production. The aim of this study was to review the effect of these processing methods on the PME inactivation in different citrus juices.Key finding and conclusionUsing non-thermal methods in combination with moderate thermal methods can be more effective in PME inactivation with minimum loss in citrus juice quality.     

橙汁饮料中橙与橘成分双重实时荧光定量PCR检测技术

目的：建立双重实时荧光定量PCR检测橙汁饮料中橙与橘成分的方法。方法：通过设计和筛选橙、橘成分的引物和探针,检测水果寻找橙和橘的荧光扩增规律,利用橙和橘在FAM/VIC两个荧光通道上的比值差异来判定橙、橘成分。采用模拟橙汁掺杂柑橘汁试验验证方法检测的最低掺杂比例,对市售橙汁饮料进行检验来验证方法可行性。结果：纯橙水果在FAM和VIC两通道均有荧光扩增曲线,FAM/VIC通道荧光比值在0.5±0.2的范围内,而纯橘水果仅在FAM通道有很强的荧光扩增曲线。模拟掺杂试验中,可以检测出橙汁中掺有10%及以上的柑橘汁。结论：该方法能够检测橙汁饮料中的橙和橘成分,可用于市售橙汁饮料的鉴伪。