Isolation , Structure Elucidation of Ferulic and Coumaric acids from Fortunella japonica Swingle leaves and their Structure Antioxidant activity relationship

Objective: The present study deals with isolation, structure elucidation of ferulic and coumaric acids from Fortunella japonica Swingle leaves and their structure antioxidant activity relationship. Structural analysis was conducted using UV, IR, mass and Nuclear Magentic Resonance (NMR) spectroscopy. Methodology: In particular, completely assigned 1 H and 13 C-NMR data are presented. Also, the present study was extended to investigate the hepa- toprotective activity of isolated compounds against paracetamol-induced toxicity in freshly isolated rat hepatocytes model. Freshly isolated rat hepa- tocytes were exposed to paracetamol (5 mM) along with/without various concentrations of the isolated compounds (40-80µg/ml). Results and Dis- cussion: Exposure of isolated hepatocytes to paracetmol resulted in lipid hydroperoxides formation, depletion in protein thiols, superoxide dismutase (SOD), succinate dehydrogenase (SDH), catalase (CAT), glutathione per- oxidase (GPx) and glutathione reductase (GR) levels as well as elevation of alanine transaminase (ALT), aspartate transaminase (AST), lactate de- hydrogenase (LDH) and alkaline phosphatase (ALP). The isolated coumaric and ferulic acids have been found to efficiently inhibit paracetamol-induced biochemical alterations, namely oxidative stress biomarkers and protein oxidation. It also significantly prevented paracetamol-induced loss in the activity of antioxidant enzyme and the important endogenous antioxidant glutathione. Conclusion: The study suggests that coumaric and ferulic ac- ids can act as an antioxidant and hepatoprotective in physiological systems.


INTRODUCTION
Leaves of Fortunella japonica Swingle or kumquat are slow-growing evergreen shrubs, sometimes bearing small thorns, they belong to family Rutaceae, either forming the genus Fortunella or placed within genus Citrus.The leaves are dark glossy green, the tree producing edible golden yellow colored fruits, the plant is native to south Asia, they have long been cultivated in Japan and Taiwan, they were introduced to Europe in 1846 by Robert Fortune. 1 The leaves and fruits of the Fortunella species have been used in folk medicine in China and recently more attempts have been made to study the pharmacological activities of some bioactive compounds isolated from kumquat, the peel of the fruit has been extensively studied. 2 Phenolic composition and antioxidant characteristics of kumquat were only confined to the peel content of the fruits. 2he most widely distributed polyphenolic compounds in plant tissues are hydroxycinnamic acids.Some of the most common naturally occurring HCAs are p-coumaric acid, ferulic acid, sinapic acid, and caffeic acid.Their biological effects are strongly dependent on the number and position of hydroxyl groups. 3 free radical can be defined as any molecular species capable of independent existence that contains an unpaired electron in an atomic orbital. 4Free radicals and other reactive oxygen species are derived either from normal essential metabolic processes in the human body or from external sources such as exposure to X-rays, ozone, cigarette smoking, air pollutants, and industrial chemicals. 5A role of oxidative stress has been postulated in many conditions, including atherosclerosis, inflammatory condition, certain cancers, and cardiovascular diseases. 6erulic and coumaric acids (Figure 1) are an important biological and structural component of the plant cell wall. 7Due to their ability to stop radical chain reactions by resonance followed by polymerization, ferulic acid offers protection against UV-radiation and is responsible for crosslinking polysaccharides and other cell wall polymers. 8The antioxidant activity of ferulic and coumaric acids has been reported for scavenging NO, O 2 -and -OH. 9 Ferulic acid also has a hepatoprotective effect against toxicity induced in vivo by carbon tetrachloride. 10aracetamol is a widely used analgesic and antipyretic medication. 11aracetamol causes acute hepatic necrosis in rats and other animal species. 12However, when given in large single-dose ingestions, paracetamol can induce liver, kidney, and other organ damages in both humans and animals. 13At therapeutic doses, paracetamol mainly undergoes glucuroni dation and sulfation in the liver. 14On the other hand, at over dosages, paracetamol is metabolized by the cytochrome P450 (CYP450) oxidative system that is mainly localized in the liver endoplasmic reticulum, and generates N-acetyl-p-benzoquinone-imine (NAPQI). 15This metabolite depletes hepatic glutathione and then binds covalently to intracellular proteins, including mitochondrial proteins. 15This situation leads to the formation of reactive oxygen and nitrogen species, and initiates lipid peroxidation that eventually results in destruction, necrosis, or apoptosis of the liver cells. 14n continuation of our interested research program in the isolation and therapeutic evaluation of natural products, 16,17 the present study report here in, a facile route to isolate ferulic and coumaric acids from Fortunella japonica Swingle leaves and investigate their in-vitro hepatoprotective effects against paracetamol-induced oxidative damages in isolated hepatocytes.

Plant extraction
The leaves (1.5 Kg) of Fortunella japonica Swingle were shade dried then mechanically powdered and subjected to extraction by percolator, using methanol , the extract was concentrated to dryness under reduced pressure and controlled temperature by distillation in vacuum to yield a dark green colored mass, which was dissolved in 500 ml of water and extracted eight times with 500 ml of n-hexane, then extracted five times with 500 ml of ethyl acetate and finally three times with 500 ml of n-butanol, after removing the solvents, the n-butanol fraction afforded 22 g.

Investigation of the n-butanol fraction
The n-butanol fraction (12 g) was loaded on a column chromatography (100 L x 4.5 ID cm), packed with ion exchange resin, Diaion HP-20 as a stationary phase.Elution started with distilled water followed by mixtures of (water/ methanol) by stepwise addition of 10% of methanol till methanol 100%.Twenty ml fractions were collected and checked for purity by TLC (with developing system composed of methylene chloride and methanol of different proportions) and paper chromatography using solvent system S1-S2 Compounds were identified based on chromatographic properties and confirmed by UV, IR, mass, 1 H-and 13 C-NMR spectral data.

Biological testing Preparation of Suspensions
Isolated ferulic and coumaric acids were dissolved in DMSO and the volume was made up to 10 ml with distilled water to obtain a stock solution of 1 mg/ml concentration and stored at -20°C prior to use.El-gizawy and Hussein.: Hepatoprotective Activity Relationship of Ferulic and Coumaric Acids Further dilutions were made to obtain different concentrations ranging from 40 to 80 µg/ml with respective media and used for in vitro investigations.

Animals used
Adult albino rats (about 3-4 months old, 230-250 g) housed in temperature and humidity controlled room (24°C ± 2°C) with a 12-hr light/ dark cycle and fed the standard laboratory diet and water ad-libitum were used.The guidelines issued by Animal Ethics Committee of October 6 th University.

Hepatoprotective Effect of ferulic and coumaric acids in freshly isolated rat hepatocytes
The healthy rats were sacrificed by cervical decapitation and the healthy hepatocytes were performed using the collagenase perfusion method. 18he obtained hepatocytes were suspended in Krebs-Henseleit buffer at a concentration of 5×10 6 cells/ml.

Paracetamol-induced in vitro hepatocytes injury
Paracetamol-induced hepatocytes injury was carried out.After an incubation of 24 h, the hepatocytes were exposed to paracetamol (5 mM) along with/without various concentrations (40, 50, 60, 70 and 80 µg/ml) of ferulic and/or coumaric acids or DMSO alone (as normal).After 60 min of paracetamol challenge, each sample was divided into two parts (0.5 ml each), one aliquot was used for the determination of TBARS level and the other one was centrifuged the clear supernatant was used for the determination of AST, ALT, ALP and LDH activities, while the residue was use for the estimation of GSH, SOD, CAT, GPx, GR, succinate dehydrogenase, protein thiol and total protein.

Measurement of thiobarbaturic acid reactive substances (TBARS) level of hepatocytes
A thiobarbituric acid reactive substances assay kit (ZeptoMetrix) was used to measure the lipid peroxidation products, TBARS equivalents. 31In brief, hepatocytes (0.5 ml) homogenized with 0.1 M sodium phosphate buffer (pH 7.4).One hundred microliters of homogenate was mixed with  El-gizawy and Hussein.: Hepatoprotective Activity Relationship of Ferulic and Coumaric Acids 2.5 mL reaction buffer (provided by the kit) and heated at 95 o C for 60 min.After the mixture had cooled, the absorbance of the supernatant was measured at 532 nm using a spectrophotometer.The lipid peroxidation products are expressed in terms of TBARS equivalents.

Measurement of superoxide dismutase (SOD) activity of hepatocytes
The epinephrine method was used for superoxide dismutase (SOD) measurement.One mL of reaction mixture contained 50 mM sodium carbonate buffer (pH 10.0), 25 µL of 20 Mm epinephrine in 0.1 N HCl, and about 20 µg of protein in the enzyme sample.In the blank cuvette, the same amount of enzyme and buffer were taken, except epinephrine.
Absorbance was recorded at 320 nm for 6 min.The activity was calculated using the difference between absorbance of standard and absorbance of enzyme and is expressed as units/mg protein. 24asurement of Catalase (CAT) activity of hepatocytes Catalase (CAT) activity of hepatocytes was determined at 25℃ with spectrophotometer according to the previous study. 25

Measurement of Glutathione peroxidase (GPx) activity of hepatocytes
GPx activity of hepatocytes was determined with a commercial kit (Randox Laboratories, UK) according to the method by Paglia and Valentine,. 26Twenty microliters of the diluted sample was added to 1 mL of mixed substrate (4 mmol/L GSH, 0.5 U/L GR and 0.34 mmol/L NADPH dissolved in 50 mmol/L phosphate buffer, pH 7.2, 4.3 mmol/L EDTA).Forty microliters of cumene hydroperoxide (diluted in deionized water) was added to the mixture and GPx activity was measured at 37°C on a Hitachi U-2000 Spectrophotometer at 340 nm for 3 min.The activity was expressed as mU/mg protein in hepatocytes.

Measurement of Glutathione reductase (GR) activity of hepatocytes
GR activity of hepatocytes was measured with a commercial kit (biodiagnostic, USA) according to the method by Staal et al.,. 27Two hundred microliters of the diluted sample was added to 400 μL of 2.4 mmol/L GSSG buffer (dissolved in 125 mmol/L potassium phosphate buffer, pH 7.5, 2.5 mmol/L EDTA).Four hundred microliters of 0.55 mmol/L NADPH (dissolved in deionized water) was added to the mixture and GR activity was measured at 340 nm for 5 min on a spectrophotometer.The activity was expressed as mU/mg protein in hepatocytes.

Measurement of succinate dehydrogenase activity of hepatocytes
Succinate dehydrogenase possesses dye reductase properties and is the only membrane bound enzyme in citric acid cycle.Phenazine methosulphate and 2, 6-dichlorophenol indophenol (DCIP) are electron acceptor dyes in the assay. 28In this assay, the reaction mixture containing hepatocytes residue is mixed with 0.1 M potassium phosphate buffer (pH 7.4) and 0.1 M sodium cyanide and incubated at 37°C for 10 min.Sodium cyanide completely inhibits oxygen consumption in respiring cells and thus fairly prevents the loss of succinate by oxidation.The contents were split into two cuvettes followed by the addition of 2 mM phenazine methosulphate and 50 µM 2,6-dichlorophenolindophenol.Addition of 50 µL of 0.4 M succinate to the experimental cuvette marks the start of the reaction, whereas 50 µL of deionized water was added in the blank cuvette.Absorbance was recorded at 600 nm for 6 min.

Measurement of protein thiols level of hepatocytes
In this assay, hepatocytes residue was suspended in 14% perchloric acid, centrifuged at 4,500× g for 5 min, and the pellet was suspended in 7% perchloric acid and again centrifuged at 4,500× g for 5 min.To the pellet were added, 10% Triton X-100, 0.2 M potassium phosphate buffer (pH 7.4).Subsequently, addition of 2 mM 5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB in the buffer was followed by measurement of absorbance at 412 nm after incubation for 5 min in the dark. 27

Measurement of total reduced glutathione level of hepatocytes
About 60 µL of O-phosphoric acid was added to hepatocytes residue and centrifuged at 3000 rpm.The supernatant was then mixed with 0.1 M sodium phosphate buffer, 0.05 M EDTA (pH 8.0), and 100 µL O-phthaldehyde (1 mg/mL) and incubated at room temperature for 15 min to measure GSH levels.The absorbance was read at 410 nm against a reagent blank. 23

STATISTICAL ANALYSIS
All data were expressed as mean of 6 replicates ± SD.All analyses utilized SPSS, 13.0 statistical package for Windows (SPSS, 13.0 software, Inc.,).
A one-way analysis of variance(ANOVA) was employed for comparisons of means of the different groups.A p-value 0.05 was accepted as statistically significant. 32Control positive (paracetamol group) was compared with control negative one.Ferulic and coumaric acids groups were compared with paracetamol treated group.

Structure elucidation of the isolated compounds
Buff powder and yellow crystals, obtained from C1 and C2 fractions.The structure of isolated compounds with melting point of 214 and 168°C is presented in Figure 1. 1 HNMR spectrum of ferulic acid displayed the characteristic signal for a methoxy group at δ 3.84 (s).Also, methoxy group signal was disappeared in 1 HNMR spectrum of coumaric acid (Table 1 and 2).The compounds spectrum also showed three aromatic proton at δ (6.85 & 6.8), (6.83 & 7.5) and ( 7.51 & 7.49) characteristics for the H-6, H-5 and H-3 of aromatic part of coumaric and ferulic acids, respectively.Also, The presence of further two protons doublets with J=15 Hz at δ (6.37 & 7.64) and (6.27 & 7.64) indicated the presence of H-2'and H-1' in the side chain of compounds, respectively.The 13 CNMR spectrum showed the presence of specific signals (aromatic carbon, aliphatic chain and methoxy group) in agreement with the proposed structure of ferulic acid (4-hydroxy-3-methoxycinnamic acid).Also, the 13 CNMR spectrum showed the absence of OCH 3 signal in agreement with the proposed structure of coumaric acid (4-hydroxycinnamic acid) (Table 1 &  (1539, 1620) cm -1 (aromatic C=C) confirms the skeleton of coumaric and ferulic acids, respectively.As it can be concluded, the IR spectrum of isolated compound is completely in agreement with the proposed structure of coumaric and ferulic acid.Based on the above data and available literature, this is the first report to isolate coumaric acid and ferulic acid from the leaves of Fortunella japonica Swingle.

Biological activity
The effects of the isolated ferulic and coumaric acids on freshly isolated rat hepatocytes intoxicated with paracetamol are recorded in Table 3.A significant decrease in the levels of protein thiols and total protein levels (P<0.05) and a significant elevation in the levels of thiobarbituric acid reactive substances (TBARS) (P<0.05) were observed in hepatocytes exposed to paracetamol when compared to normal group.
El-gizawy and Hussein.: Hepatoprotective Activity Relationship of Ferulic and Coumaric Acids with ferulic and coumaric acids showed a significant restoration of SDH, SOD, CAT, GPx, GR and GSH toward the normal (P<0.05,when compared with paracetamol treated group) and is dose dependent.

DISCUSSION
The isolated compounds were elucidated by, 1 HNMR, 13 CNMR, IR and MS as well as comparison of the data with those reported in the literature. 33aracetamol is a commonly used, mild analgesic drug.It is considered safe in normal dosage, ingestion of large quantities of paracetamol can result in hepatic necrosis and acute renal failure in man 33 .This toxicity has been attributed to the formation of a highly reactive metabolic species, the N-acetyl-p-benzoquinone imine. 34bles 4-6 showed that the paracetamol (5 mM) produced a marked increase in the leakage of ALT, AST, ALP and LDH within 60 min. of incubation period.Also, elevation in leaked of ALT, AST, ALP and LDH to 3 folds as compared to the respective negative control (p<0.05).Pre-incubation of the hepatocytes with ferulic and coumaric acids in combination (40, 50, 60, 70 and 80 µg) offered a significant protection against paracetamol-induced ALT, AST, ALP and LDH leakage as early as 60 min.as compared to the control positive group (p<0.05).The most effective dose for ALT, AST, ALP and LDH is 80 µg.
The results in of the present study illustrated that the paracetamol (5 mM) produced a marked depletion of SDH, SOD, CAT, GPx, GR and GSH within 60 min of incubation period as compared to the respective negative control group (p<0.01).Pre-incubation of the hepatocytes El-gizawy and Hussein.: Hepatoprotective Activity Relationship of Ferulic and Coumaric Acids phenolics are now widely accepted as physiologic antioxidants that have a significant potential to protect against many degenerative diseases linked to free radical-related tissue damage.
The current study deal with the isolation of ferulic and coumaric acids from the leaves of Fortunella japonica Swingle and investigation of their structure antioxidant activity relationship in order to determine their possible roles in a paracetamol-induced liver toxicity.These cells, when treated with the ferulic and coumaric acids showed a significant restoration of the altered biochemical parameters toward the normal when compared with paracetamol treated group and is dose dependent.
Lipids are known to react with oxidizing hydroxyl radical ( .OH) to produce carbon-centered radical by: 1) hydrogen atom abstraction, and 2) addition to unsaturation.These lipid radicals (L .) react with oxygen to produce lipid peroxyl radical (LO 2. ), which initiates chain reaction causing lipid damage.The presence of coumaric and ferulic acids during paracetamol toxicity significantly inhibited the formation of these lipid oxidation products.Coumaric and ferulic acids are known to react with oxidizing radicals induced by paracetamol and could serve as a strong free radical inhibitor or scavenger. 9,10any attempts at explaining the structure activity relationships of some phenolic compounds have been reported in the literature. 35It has been reported that the antioxidant activity of phenolic compounds may result from the neutralization of free radicals initiating oxidation processes, or from the termination of radical chain reactions, due to their hydrogen donating ability. 36It is also known that the antioxidant activity of phenolic compounds is closely associated with their structures, such as substitutions on the aromatic ring and side chain structure. 37It is also proposed that the higher antioxidant activity of compounds 2 and 3 is related to the presence of hydroxyl groups. 35The structural requirements considered essential for effective radical scavenging by coumaric and ferulic acids is the presence of p-hydroxyl groups in aromatic ring and conjugated double bond.The presence of double bond between aromatic ring and carbonyl group in the isolated acids makes the electrons more delocalized to form quinone structure which possesses electron donating properties and is a radical target 37 (Figure 2).These positive results suggest that coumaric and ferulic acids are a hepatoprotective agent against paracetamol induced hepatotoxicity.Therefore it may be considered as a potential molecules for alternative treatments of hepatocytes damage.It was further evaluated by a study done by Shanmugarajan et al., 38 which confirmed the protective effects of ferulic acid against D-galactosamine; a hepatotoxin employed in studies involving liver disease, because it causes damage (necrosis) similar to the injury resultant of viral hepatitis in humans. 39erulic acid also has a hepatoprotective effect against toxicity induced in vivo by carbon tetrachloride, as reported by Srinivasan et al.,. 40Treatment with the acid significantly decreased the index of lipid peroxidation in the liver and significantly increased the activities of superoxide dismutase, catalase and glutathione peroxidase. 41oumaric and ferulic acids also imparted protection to the antioxidant enzyme SOD, CAT, GPx and GR from paracetamol-induced oxidative damage, which has been measured in terms of enzyme activity.The hepatocytes protective action of isolated acids toward SOD, CAT, GPx and GR has been found to be significant, even at 40 µg/ml, and increased from 50 to 80 µg/ml.The presence of isolated phenolic acids also inhibited the paracetamol-induced loss of activity of the hepatocytes marker enzyme SDH significantly from 40 to 80 µg/ml.The protection exerted by coumaric and ferulic acids against paracetamol-induced loss of endogenous antioxidant GSH has been found even at 40 µg/ml, and increased from 50 to 80 µg/ml concentration range.Isolation and structure elucidation of coumaric and ferulic acids from Fortunella japonica Swingle leaves as well as their structure antioxidant activity relationship has not been reported earlier to our knowledge, and this study is perhaps the first observation of its kind.
Treatment with coumaric and ferulic acids significantly increased the activities of SOD, CAT, GPx and GR in isolated hepatocytes.Phenolic acids are shown to preserve physiological integrity of the cells exposed to various stress.This can be attributed to the effective antioxidant property of coumaric and ferulic acids.Normally phenolic compounds act by scavenging free radicals and quenching the lipid peroxidative side chain.Phenolic compounds can act as free radical scavengers by virtue of their hydrogen donating ability and forming aryloxyl radicals. 42It has been proposed that hydroxyl and hydroperoxyl radicals initiate H + abstraction from a free phenolic substrate to form phenoxyl radical that can rearrange to quinonemethide radical intermediate, 43 which is excreted via bile.Srinivasan et al., 40 have also been reported phenolic acids as a natural protector against carbon tetrachloride induced toxicity.

CONCLUSION
In conclusion, the present study indicates that coumaric and ferulic acids isolated from the leaves of Fortunella japonica Swingle are an efficient antioxidant against paracetamol-induced hepatocytes toxicity in isolated hepatocytes model, even at 40 µg/ml.the isolated phenolic acids are able to protect major biochemical components of the cells (lipids and proteins) from stress (paracetamol)-induced oxidative damage, and almost complete protection of lipids and proteins has been observed from 40 to 80 µg/ml.Other features like, natural occurrence and dietary isolated acids makes it attractive and suitable candidate as an antioxidant both in vitro and in vivo.Further, the ongoing studies with cell system may reveal the extent of protection exerted by coumaric and ferulic acids in vivo systems.
Diluted sample was added to 59 mmol/L H 2 O 2 (dissolved in 50 mmol/L potassium phosphate buffer, pH 7.0) and CAT activity was measured at 240 nm for 3 min.One unit of CAT activity was defined as the mmol of H 2 O 2 degraded/min/mg protein.The activity was expressed as U/mg protein in hepatocytes.

Table 3 : Effects of different concentrations of ferulic and coumaric acids treatment on protein thiols and total protein level as well as thiobarbituric acid reactive substances (TBARS) formation induced by paracetamol using isolated suspended rat hepatocytes
Values are given as mean ± SD for six replicates.Values are statistically significant at *P<0.05.Paracetamol group was compared with normal control.Ferulic and coumaric acids groups were compared with paracetamol group.

Table 4 : Effects of different concentrations of ferulic and coumaric acids treatment on alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) activity of paracetamol intoxicated freshly isolated rat hepatocytes
Values are given as mean ± SD for six replicates.Values are statistically significant at *P<0.05.Paracetamol group was compared with normal control.Ferulic and coumaric acids groups were compared with paracetamol group.

Table 5 : Effects of different concentrations of ferulic and coumaric acids treatment on superoxide dismutase (SOD), succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) activity as well as level of reduced glutathione (GSH) of paracetamol intoxicated freshly isolated rat hepatocytes
Proposal mechanism of coumaric and ferulic acids antioxidant activity