Features of citrus terpenoid production as revealed by carotenoid, limonoid and aroma profiles of two pummelos (Citrus maxima) with different flesh color.

BACKGROUND: Terpenoids are major components of carotenoids, limonoids and aromas in citrus fruits, resulting in fruit coloration, bitterness and aroma. In this study the carotenoid, limonoid and volatile profiles of red-flesh Chuhong pummelo (CH) and pale green-flesh Feicui pummelo (FC) were investigated by HPLC and GC/MS. RESULTS: Large differences were found in constituents of carotenoids and limonoids in juice sacs and flavedo and of aromas in flavedo of the two pummelos. For carotenoids in juice sacs, CH contained 57 times the amount in FC, mainly all-trans-lycopene and phytoene, whereas in flavedo it contained only 25% of that in FC, the latter showing a high proportion of ß-carotene and other chloroplastic carotenoids. In comparison with FC, limonin and nomilin aglycone production was boosted in juice sacs of CH while being almost absent in flavedo. For volatiles in flavedo, the total amount was significantly higher in CH. PCA suggested that germacrene-type sesquiterpenoids, etc. were principal in distinguishing volatile profiles of the two pummelos. CONCLUSION: The data showed a different tissue-biased pattern of carotenoid and limonoid aglycone synthesis in pummelos with different flesh color, and the possible independently regulated synthesis of those metabolites in different fruit tissues. Furthermore, decreased carotenoid and limonoid aglycone production accompanied by increased accumulation of volatile terpenoids in flavedo of red-flesh CH was identified, indicating that a total capacity or a balance of production of various terpenoids might exist in pummelo fruit tissues. It was also suggested that substrate concentration is not the key factor affecting product concentrations during the synthesis of monoterpene derivatives.

Species-related difference between limonin and obacunone among five liver microsomes and zebrafish using ultra-high-performance liquid chromatography coupled with a LTQ-Orbitrap mass spectrometer.

RATIONALE: Limonin and obacunone are two major limonoids distributed in the Rutaceae and Meliaceae families. Their defined anti-tumor activity is closely connected with the furan ring and the multi-carbonyls in their structures. In vivo and in vitro biotransformations may influence their structures and further change their effects. The metabolic profiles of limonin and obacunone have not been studied previously. In order to clarify their in vivo and in vitro metabolism, a comparative investigation of their metabolic pathways in five different species of liver microsomes and zebrafish was carried out. METHODS: In the present study, ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC/HRMS) and related electrospray ionization (ESI) tandem mass spectrometric (MS/MS) dissociation of limonin and obacunone were applied for the analysis. Each metabolite was identified by its accurate mass data. Human liver microsomes (HLMs), monkey liver microsomes (MLMs), dog liver microsomes (DLMs), rat liver microsomes (RLMs), mice liver microsomes (XLMs) and zebrafish were included in the biotransformations. RESULTS: One phase I metabolite of limonin (M1-1) and two phase I metabolites of obacunone (M2-1, M2-2) were identified by accurate mass measurement and MS/MS fragmentation behaviors. A reduction reaction was regarded as the major metabolic pathway of limonoids in liver microsomes. The reduction reaction site of M1-1 and M2-1 was at the C-16 carbonyl, while for M2-2 it was at C-7. M1-1 was the major and unique metabolite of limonin and the metabolic rate of limonin varied from 11.5% to 17.8% in liver microsomes (LMs). M2-2 was the main metabolite of obacunone in LMs and zebrafish. M1-1 and M2-1 were only detected in LMs while M2-2 was found in both LMs and zebrafish incubation systems. The metabolic rate of obacunone varied from 2.5% to 19.1% and the content of M2-2 was about five times higher than that of M2-1. CONCLUSIONS: The ESI-HR-MS/MS fragmentation behaviors of limonin and obacunone were investigated for the first time. A qualitative and semi-quantitative method was developed for the in vivo and in vitro metabolic analysis of limonin and obacunone. The results demonstrated that the metabolic processes of limonin and obacunone were different between LMs and zebrafish. However, both of these two parent compounds presented similar metabolic processes in five species of LMs. This was caused by the metabolic difference between mammals and fish or because limonin probably cannot be absorbed in zebrafish.

[Determination of obacunone and obaculactone in different processing products of Phellodendri amurensis cortex].

OBJECTIVE: To study the impact of different processing methods on the content of limonin compounds in Phellodendri Amurensis Cortex. METHODS: Used RP-HPLC method to determine the content of obacunone and obaculactone in different processing products of Phellodendri Amurensis Cortex. The chromatographic separation was carried out on Kromasil C18 (250 mm x 4.6 mm,5 micro m), with mobile phase of acetonitrile and water (50: 50) at a flow rate of 1.0 mL/min. The column temperature was 25 degrees C and the detection wavelength was 205 nm. RESULTS: The content of obacunone and obaculactone had significant differences in different processing products. The sequence of the content changes of obacunone was as follows: raw products > fried carbon products > wine fried products salt fried products. The content of obaculactone was fried carbon products approximately wine fried products approximately salt fried products > raw products. CONCLUSION: The loss of obacunone in fried carbon products is much more than that of wine fried products or salt fried products. The content of obaculactone have similar degree of increase, increasing rate is 18.15%, 15.62% and 15.84%, respectively.

[Simultaneous determination of jatrorrhizine, palmatine, berberine, and obacunone in Phellodendri Amurensis Cortex by RP-HPLC].

OBJECTIVE: To develop a HPLC method to determine the content of jatrorrhizine, palmatine, berberine, and obacunone in Phellodendri Amurensis Cortex simultaneously. MEHTOD: The separations were carried out at 25 degrees C on a Phenomenex Gemini C18 column (4.6 mm x 250 mm, 5 microm) eluted with acetonitril and water containing 0.1% phosphoric acid in gradient mode. The flow rate was 1.0 mL x min(-1), detection wavelengthes were 345 nm for jatrorrhizine , palmatine, berberine and 210 nm for obacunone. RESULT: The average recoveries of jatrorrhizine, palmatine, berberine, and obacunone were 98.94%, 101.17%, 96.22% and 98.90%, respectively. CONCLUSION: The method is simple, accurate and repeatable, and can be used in content determination of jatrorrhizine, palmatine, berberine, and obacunone in Phellodendri Amurensis Cortex.

Developing an activity and absorption-based quality control platform for Chinese traditional medicine: Application to Zeng-Sheng-Ping(Antitumor B).

ETHNOPHARMACOLOGICAL RELEVANCE: Zeng-Sheng-Ping (ZSP), also called antitumor B, is a marketed Chinese traditional medicine used for cancer prevention. AIM OF THE STUDY: Currently, for the quality control of Chinese traditional medicines, marker compounds are not selected based on bioactivities and pharmaceutical behaviors in most of the cases. Therefore, even if the "quality" of the medicine is controlled, the pharmacological effect could still be inconsistent. The aim of this study is to establish an activity and absorption-based platform to select marker compound(s) for the quality control of Chinese traditional medicines. MATERIALS AND METHODS: We used ZSP as a reference Chinese traditional medicine to establish the platform. Activity guided fractionation approach was used to purify the major components from ZSP. NMR and MS spectra were used to elucidate the structure of the isolated compounds. MTT assay against oral carcinoma cell line (SCC2095) was performed to evaluate the activities. UPLC-MS/MS was used to quantify the pure compounds in ZSP and the active fraction. The permeabilities of the identified compounds were evaluated in the Caco-2 cell culture model. The intracellular accumulation of the isolated compounds was evaluated in the SCC2095 cells. RESULTS: The major compounds were identified from ZSP. The contents, anti-proliferation activities, permeabilities, and intracellular accumulations of these compounds were also evaluated. The structure of these purified compounds were identified by comparing the NMR and MS data with those of references as rutaevine (1), limonin (2), evodol (3), obacunone (4), fraxinellone (5), dictamnine (6), maackiain (7), trifolirhizin (8), and matrine (9). The IC50 of compounds 5, 6, and 7 against SCC2095 cells were significantly lower than that of ZSP. The uptake permeability of compounds 5, 6, and 7 were 2.58 ± 0.3 × 10(-5), 4.33 ± 0.5 × 10(-5), and 4.27 ± 0.8 × 10(-5) respectively in the Caco-2 cell culture model. The intracellular concentrations of these compounds showed that compounds 5, 6, and 7 were significantly accumulated inside the cells. CONCLUSION: Based on the activity against oral carcinoma cell line as well as the absorption permeability, compound 5, 6, and 7 are selected as quality control markers for ZSP. An activity and absorption-based platform was established and successfully used for the quality control of ZSP.

Content changes of bitter compounds in 'Guoqing No.1' Satsuma mandarin (Citrus unshiu Marc.) during fruit development of consecutive 3 seasons.

The main bitter compounds (nomilin, limonin and naringin) in the fruit tissues of 'Guoqing No.1' Satsuma mandarin (Citrus unshiu Marc.) were determined throughout the fruit development of 3 consecutive growing seasons. Although fluctuating largely at the corresponding developing stages of the 3 years, the contents of these compounds in fruit tissues mostly displayed a declining trend, which implied that the rhythm of the metabolism of these bitter compounds was not consistent among years and was largely growing season dependent. Regarding their distribution, fruit flavedo might be a weak sink that contained the lowest level of naringin, while the segment membrane accumulated large amount of limonin and nomilin, which indicated a possible tissue bias pattern for biosynthesis or accumulation of those compounds. Partial correlation coefficient analysis revealed a synergistic accumulation of naringin and the two limonoid aglycones in fruit tissues during fruit development, indicating an integrated metabolism of flavonoids and limonoids.

Effect of Liberibacter infection (huanglongbing or "greening" disease) of citrus on orange juice flavor quality by sensory evaluation.

UNLABELLED: Some anecdotal reports suggest that infection of citrus trees with Candidatus Liberibacter asiaticus (Las), the suspected causal agent of huanglongbing (HLB) disease, imparts off flavor to orange juice. It is of interest to the industry to know how Las infection affects juice quality with respect to cultivar, maturity, or processing method. Hamlin, Midsweet, and Valencia oranges were harvested over 2 y from trees that tested negative (Las-) or positive (Las+) for Las from different groves and included normal looking (nonsymptomatic) and symptomatic fruit (small, green, and lopsided) from Las+ trees. In the 1st year, fruit were manually juiced, while in the 2nd year, a commercial process was used. Juice from Las+ trees was compared to juice from Las- trees in difference-from-control tests, and by descriptive analysis. Results showed large variability due to tree, harvest date, and cultivar. Juice from Hamlin Las+ trees tended to be more bitter and sour than its Las- counterpart. In contrast, hand processed Valencia juice from Las+ trees was perceived to have some off-flavor and bitterness compared to control, but the following year, commercially processed Valencia juice from Las+ trees was perceived to be only slightly more sour than juice from Las- trees for the April harvest, and to be sweeter for the June harvest. When juice from individual replicates was pooled to be more representative of a commercial situation, there was no difference between Las+ and Las- juice in Valencia. Trained panel differences were noted for juice from Hamlin Las+ fruit, especially for symptomatic fruit. PRACTICAL APPLICATION: Assumptions that juice made from oranges harvested from Huanglongbing (from infection with Liberibacter sp.) affected trees is off-flavored appeared to be generally more true for Hamlin juice than for Midsweet or Valencia, especially for Hamlin juice made from symptomatic fruit. For Midsweet and Valencia, flavor differences between juice made from fruit harvested from diseased or healthy trees varied greatly between trees, season, and even processing method. Under a commercial processing situation, where juice is blended from several varieties, seasons, and multiple locations, it is expected that off-flavor will not be a major problem.

Mass spectrometry and tandem mass spectrometry of citrus limonoids.

Methods for atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) of citrus limonoid aglycones and electrospray ionization tandem mass spectrometry (ESI-MS/MS) of limonoid glucosides are reported. The fragmentation patterns of four citrus limonoid aglycones (limonin, nomilin, obacunone, and deacetylnomilin) and six limonoid glucosides, that is, limonin 17-beta-D-glucopyranoside (LG), nomilin 17-beta-D-glucopyranoside (NG), nomilinic acid 17-beta-D-glucopyranoside (NAG), deacetyl nomilinic acid 17-beta-D-glucopyranoside (DNAG), obacunone 17-beta-D-glucopyranoside (OG), and obacunoic acid 17-beta-D-glucopyranoside (OAG) were investigated using a quadruple mass spectrometer in low-energy collisionally activated dissociation (CAD). The four limonoid aglycones and four limonoid glucosides (LG, OG, NAG, and DNAG) were purified from citrus seeds; the other two limonoid glucosides (NG and OAG) were tentatively identified in the crude extract of grapefruit seeds by ESI mass spectrometry in both positive and negative ion analysis. Ammonium hydroxide or acetic acid was added to the mobile phase to facilitate ionization. During positive ion APCI analysis of limonoid aglycones, protonated molecular ion, [M + H]+, or adduct ion, [M + NH3 + H]-, was formed as base peaks when ammonium hydroxide was added to the mobile phase. Molecular anions or adduct ions with acetic acid ([M + HOAc - H] and [M + HOAc]-) or a deprotonated molecular ion were produced during negative ion APCI analysis of limonoid aglycones, depending on the mobile-phase modifier used. Positive ion ESI-MS of limonoid glucosides produced adduct ions of [M + H + NH3]+, [M + Na]+, and [M + K]+ when ammonium hydroxide was added to the mobile phase. After collisionally activated dissociation (CAD) of the limonoid aglycone molecular ions in negative ion APCI analysis, fragment ions indicated structural information of the precursor ions, showing the presence of methyl, carboxyl, and oxygenated ring structure. CAD of the adduct ion [M + H + NH3]+ of limonoid glucosides produced the aglycone moiety corresponding to each glucoside. The combination of mass spectrometry and tandem mass spectrometry provides a powerful technique for identification and characterization of citrus limonoids.

Sensitive analytical method for the novel H1-receptor antagonist HSR-609 in human plasma and urine by high-performance liquid chromatography with pre-column fluorescent derivatization.

A simple and sensitive method for quantitation of HSR-609 (I) in human plasma and urine was developed using HPLC with the fluorescence labelling reagent 4-(N,N-dimethylaminosulfonyl)-7-N-piperazino-2,1,3-benzox adi azole (DBD-PZ). Compound I was extracted from human plasma and urine, and derivatized by reaction with DBD-PZ in the presence of Mukaiyama reagent A, an equimolar solution of 2,2'-dipyridyl disulfide (DPDS) and triphenylphosphine (TPP) in acetonitrile. The reaction mixture was cleaned up by liquid liquid extraction following the derivatization. The conjugate was analyzed by ion-pair-HPLC with fluorometric detection. The quantitation limits for I were 0.5 ng/ml in plasma and 5 ng/ml in urine. Using this method, plasma concentration and urinary excretion of I were studied after oral administration of I to human volunteers.