Radioimmunoassay for the determination of limonin in Citrus

An ¹²⁵I-radioimmunoassay technique has been developed for the triterpenoid bitter principle, limonin. Synthesis of the iodinated tracer and the limonin—bovine serum albumin conjugate are described. The antibody has a high affinity (K_a 1.1 x 10⁹l/mol) and specificity for limonin and the detection limit of the assay is 0.07 ng or 0.7 ppb. Standard curves are linear over a range of 0.5–100 ng limonin, assays can be performed in crude extracts, and several hundred samples can be processed per day. The distribution of limonin in fruits and vegetative parts of Citrus paradisi has been determined, highest values (0.92%) being found in the seeds, lowest (0.0007%) in the juice vesicles of ripe fruits. The potential of this assay method in citrus research is discussed.     

Effect of compound sequence on bitterness enhancement

The nature and occurrence of carry-over effects, i.e. the response to a stimulus is influenced by previous samples, were examined for selected bitter compounds. A time-intensity procedure was used to rate the bitterness of six compounds (caffeine, denatonium benzoate, limonin, naringin, quinine and sucrose octa-acetate). For each subject concentrations of these compounds were determined that were approximately equal in intensity to 1.18 x 10(-5) M limonin. To test carry-over effects of each compound the 36 paired sequences (pairs) were evaluated. Within a session three pairs were tested, between which two-stage rinses were used to remove any effects of the previous pairs. Within a pair only water rinses were used between stimuli. For all compounds carry-over or sensitization effects were observed in which values for maximum intensity, rate of onset and total area under the time-intensity curve were higher for a compound when tested in the second position than in the first. In addition, the degree of sensitization and susceptibility to sensitization were compound-specific. Caffeine increased the bitterness by the largest amount for all other compounds, while it was least affected. Regardless of the compound in the first position, the bitterness of quinine and denatonium were most enhanced.     

Structure and Bitterness in Flavanone Glycosides

Naringenin-7-β-kojibioside, -7-β-sophoroside, -7-[α-d-galactosyl(l→2)β-d-glucoside], -7-[β-d-glucosyl(l→2)β-d-galactoside], and also hesperetin-7-β-kojibioside and -7-β-sophoroside were prepared by the coupling of naringenin or hesperetin with the α-acetobromo derivatives of the appropriate disaccharides, followed by saponification.

Their relative bitterness values were discussed in comparison with naringin and neo-hesperidin.     

Citrus limonoids obacunone and limonin inhibit azoxymethane-induced colon carcinogenesis in rats

Obacunone and limonin are bitter limonoids in citrus. Their modifying effects on the development of aberrant crypt foci (ACF), the activity of detoxification enzymes, glutathione S-transferase (GST) and quinone reductase (QR), and cell proliferation activity were investigated in male F344 rats treated with azoxymethane (AOM). Obacunone and limonin were administered in the diet, during the initiation (for 4 weeks) or postinitiation phase (for 4 weeks) of AOM-induced tumorigenesis. Feeding of obacunone and limonin (0.02% or 0.05%) caused significant reduction (55-65% by "initiation" feeding and 28-42% by "postinitiation" feeding) in the yield of ACF. The ability to reduce the proliferating cell nuclear antigen-labeling index in crypts and correlated well with the prevention of ACF. In a subsequent long-term experiment (38 weeks), in which rats were initiated with AOM and fed 0.05% obacunone or 0.05% limonin during the initiation or post-initiation phase, both compounds in diet caused significant reduction (65%-92% inhibition) in the incidence of colonic adenocarcinoma. Thus, citrus bitter limonoids obacunone and limonin possess chemopreventive effects on chemically induced rat colon carcinogenesis.     

pH influence on the consumption of limonin species by Rhodococcus fascians cells

Summary The ability of Rhodococcus fascians cells to degrade limonin and limonin species (limonoate, limonoate-D-ring lactone and limonoate-A-ring lactone) was checked against pH. These studies showed a marked effect of pH on cell growth mainly due to substrate availability (limonin species). Evolution of limonin and its species within the medium were followed at different pH values. The best substrate for Rhodococcus fascians at pH 7.0 was limonoate whereas at pH 4.0 to 5.5 it appeared to be limonin. Results suggest that the citrus juice debittering process start only once the natural precursor of limonin (limonoate A ring lactone) has been transformed into limonin, the equilibrium displacement being governed by the citrus juice pH.     

Glycosides and Oligosaccharides in the l-Rhamnose Series

In order to investigate the substrate-specificity of α-l-rhamnosidase induced in Aspergillus species, the titled compounds were synthesized employing the various kinds of methods.

One is the Helferich reaction by using the sirupy l-rhamnose teteraacetate and appropriate phenols in the presence of p-toluenesulphonic acid or zinc chloride etc. The other method is Königs-Knorr reaction by using triacetyl α-l-rhamnosyl bromide and appropriate phenols in the presence of mercuric salts. The excellent result was obtained by the former method than the latter one. Deacetylation of the resulting esters (MeOH-NaOMe) gave the titled compounds.

Naturally occurring flavanone-7-neohesperidoside, naringin and neohesperidin contained in citrus peels were synthesized. β-Neohesperidose heptaacetate was treated with hydrogen bromide in acetic acid, giving hexaacetyl α-neohesperidosyl bromide. The latter compound coupled with phloroacetophenone in the presence of silver carbonate in quinoline yielded phloroacetophenone-4-neohesperidoside after deacetylation.

Condensation of phloroacetophenone-4-neohesperidoside with p-hydroxybenzaldehyde and isovanilline respectively in the presence of strong alkali afforded the corresponding chalcone-neohesperidosides, which were converted by ring closure to naringin and neohesperidin respectively.

Furthermore, the reactivity among phloroacetophenone-4-glycosides, namely β-d-gluco-side, β-d-xyloside, and β-neohesperidoside and fifteen kinds of substituted benzaldehydes was investigated. Phloroacetophenone-4-β-d-glucoside reacted with p-hydroxybenzaldehyde, p-anisaldehyde isovanillin and protocatechualdehyde. In the case of phloroacetophenone-4-β-d-xyloside the same result was obtained except the case of protocatechualdehyde.

In the case of phloroacetophenone-4-neohesperidoside reacted only with p-hydroxybenzaldehyde and isovanillin.     

Characterization of a naringinase from Aspergillus oryzae 11250 and its application in the debitterization of orange juice

Naringinase plays a rather important role in reducing the bitterness of juice by hydrolyzing naringin. A novel extracellular naringinase was purified from Aspergillus oryzae 11250 cultured in the presence of orange peel. A 26.78-fold purification rate was achieved by salt-induced precipitation, followed by anion-exchange and gel filtration chromatography with 32% recovery and specific activity of 2194.62 units per mg protein (U/mg). The optimum pH and temperature for naringinase activity were 5.0 and 45 °C, respectively. This enzyme was stable at 30 °C for 5 h. The K_m and V_max of naringinase toward naringin determined by Lineweaver-Burk method were 1.60 ± 0.13 mM and 126.21 ± 5.52 μmol/(min mg), respectively. The enzyme activity was inhibited completely by Ag⁺ at 10 mM. Naringinase is capable of hydrolyzing naringin, neohesperidin, and some other glycosides. A supplement of 6 U/mL of this naringinase in citrus juice sufficiently removed naringin to relieve the bitterness of citrus juice. These properties make the enzyme an ideal candidate for commercial application in the debitterization of orange juice.     

Marked Increase in PROP Taste Responsiveness Following Oral Supplementation with Selected Salivary Proteins or Their Related Free Amino Acids

The genetic predisposition to taste 6-n-propylthiouracil (PROP) varies among individuals and is associated with salivary levels of Ps-1 and II-2 peptides, belonging to the basic proline-rich protein family (bPRP). We evaluated the role of these proteins and free amino acids that selectively interact with the PROP molecule, in modulating bitter taste responsiveness. Subjects were classified by their PROP taster status based on ratings of perceived taste intensity for PROP and NaCl solutions. Quantitative and qualitative determinations of Ps-1 and II-2 proteins in unstimulated saliva were performed by HPLC-ESI-MS analysis. Subjects rated PROP bitterness after supplementation with Ps-1 and II-2, and two amino acids (L-Arg and L-Lys) whose interaction with PROP was demonstrated by ¹H-NMR spectroscopy. ANOVA showed that salivary levels of II-2 and Ps-1 proteins were higher in unstimulated saliva of PROP super-tasters and medium tasters than in non-tasters. Supplementation of Ps-1 protein in individuals lacking it in saliva enhanced their PROP bitter taste responsiveness, and this effect was specific to the non-taster group.¹H-NMR results showed that the interaction between PROP and L-Arg is stronger than that involving L-Lys, and taste experiments confirmed that oral supplementation with these two amino acids increased PROP bitterness intensity, more for L-Arg than for L-Lys. These data suggest that Ps-1 protein facilitates PROP bitter taste perception and identifies a role for free L-Arg and L-Lys in PROP tasting.     

Naringin levels in citrus tissues : I. Comparison of different antibodies and tracers for the radioimmunossay of naringin

The preparation of a tritiated radiotracer that was used in the radioimmunoassay of naringin (naringenin-7-O-α-rhamnosyl- (1-2)-β-d-glucopyranoside) and which was synthesized by reduction of the carbonyl group of the flavanone is reported. The resulting assay has a detection limit of 0.5 picomole per 0.1 milliliter, is specific for the 7-neohesperidoside substitution on flavanones, and can measure naringin in crude extracts of plant tissues. This radioimmunoassay is compared with three other naringin immunoassays which use antibodies raised against two different haptens and different tracers labeled with ¹²⁵I or ³H. The applicability of the methods to the quantification of naringin and other flavanone neohesperidosides in citrus tissue is discussed.     

A New Proteolytic Enzyme from Achromobacter lyticus M497-1

Potent antioxidative hydroxyflavanones were produced with Aspergillus saitoi from hesperidin or naringin, which are flavanone glycosides in citrus fruit with weak antioxidative activity. The hydroxyflavanone produced from hesperidin was identified as 8-hydroxyhesperetin (8-HHE), a novel substance, and those from naringin were identified as carthamidin (6-hydroxynaringenin) and isocarthamidin (8-hydroxynaringenin) by FAB-MS, ¹H-NMR and ¹³C-NMR analyses. The antioxidative activity of these hydroxyflavanones was examined by using the free radical-scavenging system of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the methyl linoleate oxidation system. The hydroxyflavanones (8-HHE, carthamidin, and isocarthamidin) exhibited stronger activity than the flavanone glycosides (hesperidin or naringin) and their aglycones (hesperetin or naringenin). The activity of 8-HHE and isocarthamidin was comparable to that of α-tocopherol, and that of carthamidin was weaker than that of isocarthamidin. The hydroxyflavanones, which were hydroxylated on A ring of flavanone by Aspergillus saitoi, were obtained as potent antioxidants.     

Suppression of Bitterness by Sodium: Variation Among Bitter Taste Stimuli

Taste interactions between salts (NaCl, LiCl, KCl, L-arginine:L-asparticacid, Na-acetate and Na-gluconate) and bittertasting compounds(urea, quinine HCI, magnesium sulphate, KCI, amiloride HCI andcaffeine) were investigated. In each study binary combinationsof three or four concentrations of one bitter compound withfour concentrations (0, 0.1, 0.3 and 0.5 M) of one salt wererated for bitterness and saltiness using the method of magnitudeestimation. In most cases, perceived bitterness was suppressedby salts, although the degree of suppression varied. In general,bitterness suppression was not accompanied by an equivalentreciprocal suppression of saltiness. Only MgSO₄ and amiloridehad suppressing effects on the saltiness of NaCl at the intermediateconcentrations and no bitter compound affected the saltinessat the high concentrations of NaCl. Since salt suppressed thebitterness of urea effectively, a detailed analysis of suppressionof the bitterness of urea by different salts was conducted.Those studies indicated that the key component in this effectwas the sodium or lithium ion for two reasons: first, all threesodium salts and the lithium salt had a suppressive effect onbitterness, whereas KCl did not; secondly, the effect of a salton suppression of the bitterness of urea was independent ofits perceived saltiness; that is, NaCl, Na-acetate (which isperceived as less salty than NaCl), and Na-gluconate (whichis perceived as less salty than Na-acetate) reduced bitternesscomparably. These results suggest that there is a major peripheralcomponent to the suppression of the bitterness of urea, andperhaps other bitter tasting compounds, by sodium. Chem. Senses20: 609–623, 1995.     

Flavonoids in Citrus Hybrids

The occurrence and distribution of flavanone glycosides in the leaves and fruits of many kinds of artificial citrus hybrid plants were investigated by polyamide thin-layer chromatography. The citrus hybrids can be divided into two broad categories, a) those containing rutinosyl glycosides, b) those containing neohesperidosyl glycosides in accordance with the case of natural citrus species. The fiavonoid patterns of rutinosyl glycosides are classified into the following groups, a) hesperidin, b) narirutin, c) hesperidin and narirutin, d) didymin and narirutin, e) hesperidin, narirutin and eriocitrin and f) hesperidin and eriocitrin, while the pattern of neohesperidosyl glycosides fall into six groups, a) naringin, b) neohesperidin and naringin, c) neohesperidin, naringin and neoeriocitrin, d) neohesperidin and neoeriocitrin, e) naringin and neoeriocitrin, and f) poncirin, neohesperidin, naringin and neoeriocitrin. It is worthy of note that a hybrid (accession number 1088) between C. unshiu and C. hassaku contains only narirutin. Among the ninty-four hybrids examined, fifty-three varieties were obviously different from female parents in their flavonoid pattern and could be judged as true hybrids by fiavonoids but the others could not.

Additionally, a survey of fiavonoids in newly found natural pummelo- and Daidai hybrids were carried out in connection with their origin.     

Limonin biosynthesis from obacunone via obacunoate in Citrus limon

Radioactive tracer work showed that [¹⁴C]obacunone was converted to at least four metabolites in Citrus limon. Two were identified as obacunoate and limonin. When [¹⁴C]methyl obacunoate was fed, limonin was found to be one of the metabolites. Based on these results and data accumulated thus far, biosynthetic pathways of limonoids in citrus are proposed.     

Dose-Dependent Effects of L-Arginine on PROP Bitterness Intensity and Latency and Characteristics of the Chemical Interaction between PROP and L-Arginine

Genetic variation in the ability to taste the bitterness of 6-n-propylthiouracil (PROP) is a complex trait that has been used to predict food preferences and eating habits. PROP tasting is primarily controlled by polymorphisms in the TAS2R38 gene. However, a variety of factors are known to modify the phenotype. Principle among them is the salivary protein Ps-1 belonging to the basic proline-rich protein family (bPRP). Recently, we showed that oral supplementation with Ps-1 as well as its related free amino acids (L-Arg and L-Lys) enhances PROP bitterness perception, especially for PROP non-tasters who have low salivary levels of Ps-1. Here, we show that salivary L-Arg levels are higher in PROP super-tasters compared to medium tasters and non-tasters, and that oral supplementation with free L-Arg enhances PROP bitterness intensity as well as reduces bitterness latency in a dose-dependent manner, particularly in individuals with low salivary levels of both free L-Arg and Ps-1 protein. Supplementation with L-Arg also enhanced the bitterness of caffeine. We also used ¹H-NMR spectroscopy and quantum-mechanical calculations carried out by Density Functional Theory (DFT) to characterize the chemical interaction between free L-Arg and the PROP molecule. Results showed that the –NH₂ terminal group of the L-ArgH⁺ side chain interacts with the carbonyl or thiocarbonyl groups of PROP by forming two hydrogen bonds with the resulting charged adduct. The formation of this PROP•ArgH+ hydrogen-bonded adduct could enhance bitterness intensity by increasing the solubility of PROP in saliva and its availability to receptor sites. Our data suggest that L-Arg could act as a ‘carrier’ of various bitter molecules in saliva.     

Citrus limonoid by-products as insect control agents

Limonoids are a group of chemically related bitter tetranortriterpene derivatives found predominantly in Rutaceae and Meliaceae plants (Ourison et al., 1964). Interest in the Rutaceae limonoids has centered around limonoid removal from consumable citrus products. For example, bitterness in citrus juices (as well as in other citrus products) due to limonoids has become an increasingly serious economic problem (Wilson & Crutchfield, 1968; Sinclair, 1972). Interest in the Meliaceae limonoids, on the other hand, has centered on their efficacy as pest control and/or antitumor agents (Kubo & Klocke, 1981, 1982; Nakanishi, 1977, 1980). For example, azadirachtin, isolated from several Meliaceae trees, has proven to be a potent natural product against a myriad of insect and nematode pests (Warthen, 1979). In fact, we have isolated azadirachtin from the fresh fruit of Azadirachta indica as a potent insect ecdysis inhibitor against four agricultural pest insects with artificial diet feeding assay (Kubo & Klocke, in litt).     

Riboflavin-binding protein is a novel bitter inhibitor

Riboflavin-binding protein (RBP) from chicken egg, which was recently reported to be a selective sweet inhibitor for protein sweeteners, was also found to be a bitter inhibitor. RBP elicited broadly tuned inhibition of various bitter substances including quinine-HCl, naringin, theobromine, caffeine, glycyl-L-phenylalanine (Gly-Phe), and denatonium benzoate, whereas several other proteins, such as ovalbumin (OVA) and beta-lactoglobulin, were ineffective in reducing bitterness of these same compounds. Both the bitter tastes of quinine and caffeine were reduced following an oral prerinse with RBP. It was found that RBP binds to quinine but not to caffeine, theobromine, naringin, and Gly-Phe. However, the binding of RBP to quinine was probably not responsible for the bitter inhibition because OVA bound to quinine as well as RBP. Based on these results, it is suggested that the bitter inhibitory effect of RBP is the consequence of its ability to interact with taste receptors rather than because it interacts with the bitter tastants themselves. RBP may have practical uses in reducing bitterness of foods and pharmaceuticals. It may also prove a useful tool in studies of mechanisms of bitter taste.     

Bitter Peptides in Cow Milk Casein Digests with Bacterial Proteinase

Isolation and determination of amino acid sequence of the bitter peptides formed in the digestion of cow milk casein with alkaline proteinase of Bacillus subtilis were investigated. The casein digest with the enzyme was extracted with butanol and the extracted bitter peptides were fractionally purified by treating with several other organic solvents followed by subjecting to chromatography and gel-filtration. The amino acid sequence of one of the bitter peptides was determined as follows: Arg-Gly-Pro-Pro-Phe-Ileu-Val. Liberation of N-terminal Arg with trypsin or bacterial aminopeptidase did not affect the bitterness. Also, splitting off of Val and Ileu or Ileu-Val at the C-terminus by carboxypeptidase, or a bacterial neutral proteinase gave no influence on the bitterness. However, liberation of Arg and Gly from the peptide with bacterial aminopeptidase gave rise to a non bitter peptide.     

The molecular and enzymatic basis of bitter/non‐bitter flavor of citrus fruit: evolution of branch‐forming rhamnosyltransferases under domestication

Domestication and breeding of citrus species/varieties for flavor and other characteristics, based on the ancestral species pummelo, mandarin and citron, has been an ongoing process for thousands of years. Bitterness, a desirable flavor characteristic in the fruit of some citrus species (pummelo and grapefruit) and undesirable in others (oranges and mandarins), has been under positive or negative selection during the breeding process of new species/varieties. Bitterness in citrus fruit is determined by the composition of branched‐chain flavanone glycosides, the predominant flavonoids in citrus. The flavor‐determining biosynthetic step is catalyzed by two branch‐forming rhamnosyltransferases that utilize flavanone‐7‐O‐glucose as substrate. The 1,2‐rhamnosytransferase (encoded by Cm1,2RhaT) leads to the bitter flavanone‐7‐O‐neohesperidosides whereas the 1,6‐rhamnosytransferase leads to the tastelessflavanone‐7‐O‐rutinosides. Here, we describe the functional characterization of Cs1,6RhaT, a 1,6‐rhamnosyltransferase‐encoding gene directing biosynthesis of the tasteless flavanone rutinosides common to the non‐bitter citrus species. Cs1,6RhaT was found to be a substrate‐promiscuous enzyme catalyzing branched‐chain rhamnosylation of flavonoids glucosylated at positions 3 or 7. In vivo substrates include flavanones, flavones, flavonols and anthocyanins. Cs1,6RhaT enzyme levels were shown to peak in young fruit and leaves, and gradually subside during development. Phylogenetic analysis of Cm1,2RhaT and Cs1,6RhaT demonstrated that they both belong to the branch‐forming glycosyltransferase cluster, but are distantly related and probably originated separately before speciation of the citrus genome. Genomic data from citrus, supported by a study of Cs1,6RhaT protein levels in various citrus species, suggest that inheritance, expression levels and mutations of branch‐forming rhamnosyltransferases underlie the development of bitter or non‐bitter species/varieties under domestication.     

Meloxicam Taste-Masked Oral Disintegrating Tablet with Dissolution Enhanced by Ion Exchange Resins and Cyclodextrin

The purpose of this study was to develop taste-masked oral disintegrating tablets (ODTs) using the combination of ion exchange resin and cyclodextrin, to mask the bitter taste and enhance drug dissolution. Meloxicam (MX) was selected as a model drug with poor water solubility and a bitter taste. Formulations containing various forms of MX (free drug, MX-loaded resin or resinate, complexes of MX and 2-hydroxypropyl-β-cyclodextrin (HPβCD) or MX/HPβCD complexes, and a mixture of resinate and MX/HPβCD complexes) were made and tablets were prepared by direct compression. The ODTs were evaluated for weight variation, thickness, diameter, hardness, friability, disintegration time, wetting time, MX content, MX release, degree of bitter taste, and stability. The results showed that thickness, diameter, weight, and friability did not differ significantly for all of these formulations. The tablet hardness was approximately 3 kg/in.², and the friability was less than 1%. Tablets formulated with resinate and the mixture of resinate and MX/HPβCD complexes disintegrated rapidly within 60 s, which is the acceptable limit for ODTs. These results corresponded to the in vivo disintegration and wetting times. However, only tablets containing the mixture of resinate and MX/HPβCD complexes provided complete MX dissolution and successfully masked the bitter taste of MX. In addition, this tablet was stable at least 6 months. The results from this study suggest that the appropriate combination of ion exchange resin and cyclodextrin could be used in ODTs to mask the bitter taste of drug and enhance the dissolution of drugs that are weakly soluble in water.