Oxygen Heterocyclic Compounds In Citrus Juices And Essential Oils: Analytical Methodology, Chemical Characterization, And Blood Pressure Lowering Activity Studies

Oxygenated heterocyclic compounds?OHCs?are a large class of secondary metabolites found primarily in citrus.OHCs are heterocyclic aromatic compounds containing oxygen in the central ring.They mainly include three chemical families:polymethoxyflavones?PMFs?,coumarins and furanocoumarins?FCs?which have a wide range of biological activities that have human health implications.These potent compounds are found at trace levels in citrus juices,the main product from fruit processing,and different juices contain different OHCs at vastly different levels.Due to their low concentrations and structural similarities,they are difficult to accurately measure and little information on juice OHCs can be found in the literature.The amounts and kinds of OHCs in many citrus juices as well as compositional changes during thermal processing is largely unknown.OHCs content in citrus peel essential oils which are one of the most important by-products in processing,need more comprehensive study too.Few methods exist could quantify these complicated components in all three classes in a single analysis.Furthermore,these existing methods are time consuming and require the use of highly expensive LC-MS/MS instruments.For these reasons,we developed a set of procedures for the analysis of citrus OHCs in citrus juices and oils that was rapid,accurate and sensitive using relatively inexpensive instrumentation;we also developed a matched set of procedures for the pretreatment of citrus juice samples that was able to completely extract juice OHCs,concentrate them from natural low levels and remove interfering compounds.Using these methods,concentrations of 38 OHCs in 138 juice samples and 24 cold-pressed oil?CPO?samples were analyzed.Changes in coumarin and furanocoumarin side chain structures and their reaction kinetics in juices of different pH and temperatures were examined.Sweet orange CPO which contains abundant OHCs,was employed to study the alleviating bioactivity on hypertensive rats.?1?Development of analytical and sample preparation methods and methodological verification:A high-performance liquid chromatographic method to separate all 38 OHCs in all three chemical classes from a single injection was developed and optimized.This method employed a quaternary mobile phase and used photo diode array-fluorescence detection?HPLC-PDA-FL?.Firstly,various binary solvent compositions of methanol/acetonitrile/tetrahydrofuran?THF?with water were employed to determine the relative elution order of the 38 OHCs under reverse phase isocratic conditions;then the quaternary mobile phase gradient was designed and optimized by using the log capacity factor plots of each binary system to individually resolve all 38 target OHCs.The optimized chromatographic conditions consisted of a C8 column employing four solvent reservoirs containing:A0.025%phosphoric acid?aq.?,B methanol,C acetonitrile,D water/acetonitrile/THF?55:20:25?were mixed to produce the desired solvent gradient composition.The mixed solvent gradient completely separated all target OHCs within 50 min.Chromatographic resolution was no less than 1.5,selectivity 1.01-1.20,theoretical plates 18,000-1,500,000.The UV absorbance?210-400 nm?and fluorescence emission spectra?340-560 nm?of all 38 OHCs were determined to create a library of spectral properties which was used to identify unknown peaks in samples.Based on the spectral library,330 nm UV wavelength was used to monitor all OHCs;both 400 and 450 nm emissions were monitored since not all OHCs fluoresced at the same wavelength.These different response ratios of peak area from standards at FL 450 nm/FL 400 nm,UV 330 nm/FL 450nm and UV 330 nm/FL 400 nm,were used to establish a response ratio data base whose collective values were highly individual.Therefore,we developed a unique method by combination of retention time with UV and fluorescence spectral properties as well as response ratios;it was a highly effective means of identifying peaks in a wide range of citrus samples.The external standard calibration method with an internal standard?psoralen?correction was used for quantitation.To optimize sensitivity,quantitation wavelengths were set to UV 330,270,250 nm and FL 400,450,500 nm.The results showed excellent linearity?R²>0.9990?with the UV limits of detection?LOD?ranging from 0.01-0.31mg/L,limits of quantitation?LOQ?0.04-1.05 mg/L,with fluorescence LOD ranging from0.0001-10.5 mg/L,and LOQ 0.0004-35.0 mg/L.Analysis of a picogram level of coumarin substances such as scopoletin could be achieved by using FL signal.Studies of recovery,repeatability and precision showed good results.To completely extract citrus juice OHCs,juice samples were firstly centrifuged to separate liquid serum from solids?pulp cloud?.The serum passed through a C-18 solid phase extraction?SPE?cartridge;interfering substances were washed out by 27%aqueous acetonitrile solution?pH 4.0?;OHCs eluted from the cartridge with ethyl acetate.Key parameters such as the flow rate,sample load and polarity of washing solvent were determined.Pulp cloud sediment was extracted using liquid-solid extraction;extraction efficiency of five organic solvents was studied and pure acetonitrile was optimal.The SPE eluent and sediment extract were dried under nitrogen and re-dissolved with methanol before HPLC injection.?2?Composition and distribution characteristics of OHCs in citrus CPOs and juices:Twenty-four CPO samples and 138 juice samples collected mainly from the Chongqing region were analyzed using the newly developed methods.Economic species including sweet orange,mandarin,grapefruit,pummelo,lemon,citron and kumquat were examined and their OHCs pattern characterized.The resulting composition and concentration data were evaluated using principal component analysis?PCA?to show the specific clustering in the data.Application of OHCs pattern to authenticate citrus juice was discussed.CPO samples contained 34 of the target OHCs.Most of the OHCs in sweet orange oil and mandarin oil consisted of PMFs,accounting for 92.2%⁹9.9%of total OHCs.Heptamethoxyflavone was the major component in sweet orange oil,followed by nobiletin and tangeretin.In mandarin oil tangeretin was the major component,followed by 5-demethylnobiletin and nobiletin.OHCs in CPOs of pummelo,grapefruit,lemon,citron and kumquat were primarily composed of coumarins and FCs,which account for more than 93.8%of total OHCs.Highest amounts of total OHCs were found in pummelo and grapefruit CPOs.Total concentration of OHCs in Guanxi pummelo oil was 74.0 g/L compared to sweet orange oils which were about 1.0 g/L.Thirty-three OHC variable values in CPOs were used to form seven principal components?PCs?for each sample.Two-dimensional score plots showed that the CPO samples were divided into three large groups,i.e.pummelo and grapefruit,mandarin and sweet orange,and citron and lemon.The characteristic OHCs from CPOs in each group that determined by 3-D loading graph were as follows:in lemon and citron,are 5-geranyloxy-7-methoxycoumarin and 8-geranyloxypsoralen;in pummelo and grapefruit are auraptene and meranzin;in mandarin and sweet orange are primarily five PMFs.Citrus juice samples contained 36 target OHCs.The major difference between juice and CPO data is that coumarins and FCs which contain the substituted epoxy group,such like meranzin,6',7'-epoxybergamottin,oxypeucedanin,byakangelicol and heraclenin,were present in the essential oil and a few low acid juices,whereas their corresponding hydration products,i.e.meranzin hydrate,6',7'-dihydroxybergamottin,oxypeucedanin hydrate,byakangelicin and heraclenol,were only found in the acidic juices.In addition,juice PMFs included isosinensetin which was not detected in CPO.The total content of OHCs in citrus juice was 2-5 orders lower than that of CPO.The average total OHCs concentration of each species of juice from highest to lowest was:grapefruit 39.44 mg/L,pummelo 13.10 mg/L,mandarin 8.28 mg/L,citron and lemon 2.32 mg/L,sweet orange2.15 mg/L and kumquat<1.00 mg/L.Thirty-five OHCs in the juice were used as variables to form seven PCs.Using these PCs to classify 138 juice samples,the results showed that juices were more tightly clustered and better separated by species than CPO.The six common species formed six distinct eigenvectors that were well separated from each other.The characteristic OHCs components of each species were as determined from PCA loading values.In lemon juice was primarily oxypeucedanin hydrate and byakangelicin;in pummelo and grapefruit juice was 6',7'-DHB.Using the citrus juice OHCs data set,it was possible to discriminate mixed?adulterated?citrus juice like orange juice with 30%or higher amount of mandarin juice so that help to verify juice authenticity.?3?Alteration characteristics and reaction kinetics of OHCs epoxides during juicing process:The reaction products and rate of meranzin and 6',7'-epoxybergamottin?6',7'-EB?were studied at 25°C in model solutions at three acidity levels?pH 2.0,3.5 and 5.0?and also those in pH 3.5 juice serum.The effects of thermal pasteurization/pectinesterase inactivation on the degrading of meranzin and 6',7'-EB in pH 5.0 and 3.5 juice serum were also examined.In general,the epoxy moiety of coumarins and FCs reacted with water following an acid catalyzed nucleophilic addition mechanism,producing the corresponding o-diols such as meranzin hydrate?MH?and 6',7'-dihydroxybergamottin?6',7'-DHB?.These reactions were fastest at pH 2.0 and hardly occurred at pH 5.0.Meranzin reacted in two consecutive steps in model solution,at pH 2.0 and 25°C.The first step was the side chain hydration reaction,during which the 8-?3,3-dimethyl-2-epoxyethyl?group was added with water to form an 8-?2,3-dihydroxy-3-methyl butyl?group.This reaction followed a pseudo-first-order with the rate constant k₁=0.0697 min^-1,and the half-life t_{1/2 1}=9.94 min.The second step reaction?s?consumed the first step product,MH,following an apparent first order rule,but the final products were unknown.The decomposition rate was slower,with t_{1/2 2}=34.2-42.6 min.In the pH 3.5 model solution at the same temperature,only the first reaction occurred following a pseudo-first order with k=0.00726 min^-1 and t_1/2=95.47 min.6',7'-EB firstly underwent parallel reaction in the pH 2.0 model solution,25°C.The main reaction was side chain hydration,a pseudo-first order reaction,during which 5-?6',7'-epoxy-geranyloxy?group reacted with water to form 5-?6',7'-dihydroxy-geranyloxy? group.The rate constant k₁=0.0597 min^-1 and half-life t_{1/2 1}=11.6 min.The side reaction that shortened the side-chain from C10 to C5 produced isoimperatorin?IIR?.This reaction also followed a pseudo-first-order with a half-life t_{1/2 2}=84.9 min.There were consecutive reactions which diminished 6',7'-DHB and IIR following apparent first-order rules,though the corresponding products have not been identified.In the pH 3.5 model solution at the same temperature,only the first step two reaction occurred with the main reaction k₁=0.00532 min^-1,t_{1/2 1}=130.3 min.The meranzin and 6',7'-EB epoxide hydration rates in pH 3.5 citrus juice serum,25°C,were four-times faster of those in model solution of the same condition,with a k_mer=0.0305±0.0025 min^-1,t_{1/2 mer}=22.7±2.0 min,and a k_EB=0.0206±0.0019 min^-1,t_1/2EB=33.9±3.5 min.The reason causing acceleration requires further investigation.Meranzin and 6',7'-EB were added to juice serum of pH 5.0 and pH 3.5,and the solutions were heated at 95°C for 30 s,similar to commercial juice pasteurization.Concentrations of the epoxides in the pH 5.0 serum were unchanged after heating.When the epoxides were similarly heated in the pH 3.5 juice serum,they were reduced by 98.9%and 96.3%respectively thus indicating that the rate of hydration was significantly pH and temperature dependent.?4?The prophylactic and relief effects of PMFs-rich long-leaf orange CPO on L-N^?-nitroarginine?L-NNA?induced hypertensive SD rats:Seven-week-old male SD rats were divided into six groups:normal group,hypertension control group,orange peel oil-low dose?OPO-L?group,orange peel oil-high dose?OPO-H?group,D-limonene blank control group and captopril treatment group,with 10 rats in each group.Except the normal group was not given any treatment,all the other groups were given L-NNA by gavage with 700 mg/kg bodyweight?bw?per day for61 days.Rats in the OPO-L and OPO-H groups were respectively given orange CPO by gavage at 5 and 10 mL/kg bw per day.Rats in the limonene group were given D-limonene at 10 mL/kg bw per day.For the treatment group,rats were given captopril at 15.6 mg/kg bw daily.Systolic and diastolic pressure were measured by tail-cuff method at the caudal artery by every 3 days.After the last measurement completed on the 61st day,all rats were executed by CO₂ asphyxia.After dissection,blood and tissues such as heart,liver and kidney were collected and the content of five bioactive factors related to vasodilation or vasoconstriction were determined.Serum levels of endothelin-1?ET-1?,calcitonin gene-related peptide?CGRP?,vascular endothelial growth factor?VEGF?and E-selectin were determined by ELISA kits and the contents of NO and malondialdehyde?MDA?in serum,heart,liver and kidney were determined by biochemical kits.The mRNA transcriptional levels of six genes,i.e.heme oxygenase-1?HO-1?,adrenomedullin?ADM?,receptor activity modifying protein 2?RAMP2?,and the neuronal,endothelial and inducible isoforms of nitric oxide synthases?nNOS,eNOS and iNOS?,all of which are related to blood pressure modulation were measured by q-PCR.The content of PMFs in orange CPO was determined by a fast HPLC-PDA method and D-limonene determined by GC-MS.After 61 consecutive days of intragastric administration of L-NNA,the systolic and diastolic blood pressure in the hypertensive control group reached 174±6 and 138±4mmHg respectively,nearly 85/75 mmHg higher than that of the normal group.Ingestion of orange CPO by gavage of 10 mL/kg bw?containing 1.64 mg PMFs/kg bw,OPO-H group?significantly?p<0.05?decreased systolic and diastolic blood pressure by 54/43mmHg,respectively.Gavage with 5 mL/kg bw?containing 0.82 mg PMFs/kg body weight,OPO-L group?also showed significant but weaker hypotensive effect?p<0.05?.Sweet orange CPO significantly increased the level of NO and CGRP and reduced the level of MDA,ET-1,VEGF and E-selectin in blood serum,heart,liver and kidney tissues of hypertensive rats,thereby relaxing blood vessels and preventing oxidative damage and angiogenesis in vivo.CPO up-regulated the mRNA expression of HO-1,nNOS and eNOS in myocardium,and meanwhile down-regulated the myocardial expression of ADM,RAMP2 and iNOS mRNA,demonstrating an improvement of cardiovascular relaxation and cardiomyocytes protection.All the results were positively dose-dependent within the selected dosage range.Results also showed that the effect of OPO-H group was slightly lower than that of captopril group;the contribution of D-limonene,the most abundant chemical accounted for 95.8%of the sweet orange CPO,was excluded as little positive effect presented by the limonene control group.As HPLC analysis showed that almost all non-volatiles in orange CPO composed of PMFs,the hypotensive effect of CPO was attributed to the activity of PMFs.