ChemiCal Constituents of Clove

Chemical Constituents of Clove (Syzygium aromaticum, Fam. Myrtaceae) and their Antioxidant Activity Mahmoud I. Nassar1*, Ahmed H. Gaara1 Ahmed H. El-Ghorab2, Abdel-Razik H. Farrag3, Hui Shen4, Enamul Huq4 and Tom J. Mabry4

Abstract Sixteen volatile compounds were identified from the n-hexane extract of the buds of Syzygium aromaticum by using gas chromatography-mass spectroscopy (GC-MS). The major components were eugenol (71.56 %) and eugenol acetate (8.99 %). The dichloromethane extract of the buds yielded limonin and ferulic aldehyde, along with eugenol. The flavonoids tamarixetin 3-O-b-D-glucopyranoside, ombuin 3-O-b-D-glucopyranoside and quercetin were isolated from the ethanol extract; identifications of all these compounds were established by chemical and spectroscopic methods including 1D and 2D NMR. This is the first report of limonin, ferulic aldehyde and these flavonoids from this plant. All extracts and the isolated flavonoids showed strong antioxidant activity against 1, 2-diphenyl picrylhydrazyl (DPPH). Among the tested extracts, the ethanol extract of the clove buds showed remarkable scavenging activity, as compared with synthetic antioxidants such as butylated hydroxyl toluene (BHT). The ethanol extract of clove showed remarkable hepatoprotective activity against paracetamol-induced liver injury in female rats. Keywords: Syzygium aromaticum, Myrtaceae, volatile compounds, limonin, ferulic aldehyde, flavonoids, antioxidant activity, hepatoprotective activity.

2002). In addition to fruits and vegetables that are recommended at present as optimal sources of such components, the supplementation of human diet with spices or herbs, containing especially high amounts of compounds capable of deactivating free radicals (Madsen and Bertelsen, 1995). The benefits resulting from the use of natural

INTRODUCTION Chemical constituents with antioxidant activity found in high concentrations in plants (Velioglu et al, 1998) determine their considerable role in the prevention of various degenerative diseases (Challa et al, 1997; Diplock et al, 1998; Willett and Willett,

Natural Compounds Chemistry Department, National Research Centre, 12311 Dokki, Cairo, Egypt. Flavors and Aromatic Department, National Research Centre,12311 Dokki,Cairo, Egypt. 3 Pathology Department, National Research Centre,12311 Dokki, Cairo, Egypt. 4 Molecular Cell and Developmental Biology, University of Texas at Austin, Austin 78712, USA. *Corresponding author: Tel: 202-22337651, Fax: 202-33370931, Email: [email protected] 1 2

Mahmoud I. Nassar,

products rich in bioactive substances has promoted the growing interest of pharmaceutical, food and cosmetic industries. Syzygium species (Fam. Myrtaceae) have been reported to possess antibacterial (Shafi et al, 2002) and anti-inflammatory activity (Muruganadan et al, 2001). It was reported that the buds of Syzygium aromaticum (L.) Merr. & Perry (clove) were used in folk medicine as diuretic, odontalgic, stomachic, tonicardiac, aromatic condiment properties and condiment with carminative and stimulant activity (Boulos, 1983). The antimicrobial activity of the essential oils from clove and rosemary (Rosmarinus officinalis L.) has been tested alone and in combination (Fu et al, 2007). In addition, the antimicrobial activity of clove essentials oil have been studied against a large number of multi-resistant Staphylococcus epidermidis as well as the composition of the oil by GC/MS analysis (Chaieb et al, 2007). The antioxidant activity of commercial clove leaf essential oil (Eugenia carophyllus) and the main constituent eugenol was tested (Jirovetz et al, 2007). Cytotoxicity of clove oil and its major components has been investigated (Prashar et al, 2006). Several compounds from S. aromaticum have been found to possess growth inhibitory activity against oral pathogens, namely 5, 7-dihydroxy-2-methylchromone-8-C-βD-glucopyranoside, biflorin, kaempferol, rhamnocitrin, myricetin, gallic acid, ellagic acid and oleanolic acid (Cai and Wu, 1996). Also, an orsellinic acid glucoside has been isolated from S. aromaticum (Charles et al, 1998). Recently, flavonoide triglycosides have been isolated (Nassar, 2006). The evaluation of antioxidant properties of the raw material allows the determination of its suitability as high quality food beneficial for human health and therefore is of considerable importance. The aim of this study was to isolate and identify the volatile components and nonvolatile compounds (limonin and ferulic aldehyde and several flavonoids) via solvent extraction,

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from S. aromaticum as well as determine the antioxidant activity of the extracts and isolated flavonoids by using 2, 2-diphenyl2-picrylhydrazyl (DPPH) and the hepatoprotective activity. RESULTS AND DISCUSSION The n-hexane extract of the buds of S. aromaticum gave an orange oil with a characteristic clove odor. The volatile compounds of the hexane extract were determined by GC/MS analyses (Table 1). Because the authentic compounds of the most of these components were available, the quantitative calculations were based upon the relative areas of the corresponding GC signals. GC/ MS analyses also established the percentage composition of the 16 volatiles detected in the n-hexane extract of the buds (Table 1); eugenol (71.56%) and eugenol acetate (8.99%) were the major components. Fractionation of the methylene chloride extract of the buds on silica gel and Sephadex LH20 columns afforded a tetrahentriterpene, limonin (1) and an aromatic aldehyde, ferulic aldehyde (2), along with eugenol. The ethanol extract was subjected to polyamide column chromatography eluted with water/methanol step gradient. The obtained fractions were further purified on Sephadex LH-20 columns to give the flavonoids tamarixetin 3-O-β-D-glucopyranoside (3), ombutin 3-O-β-D-glucopyranoside (4) and quercetin (5) (Fig. 1). The CI/MS spectrum of 1 showed a molecular ion peak [M+1]+ at m/z 471, C26H30O8. HRCI showed an ion peak at m/z 471.20113 (calc. 471.20189). 1HNMR and 13C-NMR spectral data of 1 were identical to that previously reported for limonin (Patra et al, 1988). The 13C-NMR spectrum showed 26 nonequivalent carbon resonances, three of which appeared at δ 206, 169 and 166.6 ppm and represented three carbonyl groups. A DEPT experiment showed the presence of four methyl groups,

Chemical constituents of clove (Syzygium aromaticum, Fam. Myrtaceae)

Rev. Latinoamer. Quím. 36/1 (2007)

Table 1: The volatile compounds identified in the n-hexane extract of Syzygium aromaticum buds by using GC-MS No. Compound Conc. (%) Type Identification method 1 p-Cymene 0.9 M MS & KI 2 5-Hexene-2-one 0.67 LOC MS & KI 3 Thymol 0.87 LOC MS & KI 4 Eugenol 71.56 LOC MS & KI & AU 5 Eugenyl acetate 8.99 LOC MS & KI & AU 6 Caryophyllene oxide 1.67 LOC MS & KI 7 Guaiol 0.90 HOC MS & KI 8 Benzene-1-butylheptyl 0.55 LOC MS 9 Nootkatin 1.05 S MS & KI 10 Isolongifolanone (trans) 0.86 S MS & KI 11 Hexadecanoic acid 0.50 LOC MS 12 9,17-Octadeca-dienal 0.24 HOC MS 13 Octadecanoic acid butyl ester 0.33 HOC MS 14 Phenol-4-(2,3-dihydro-7 0.98 HOC MS -methoxy-3-methyl-5 1-propenyl)-2 -benzofurane 15 (Dodecatrienoic acid-3,7, 0.38 HOC MS 11- trimethylethyl ester 16 Vitamin E acetate 0.43 HOC MS M, monoterpene; S, sesquiterpene; LOC, lightly oxygenated compound; HOC, Heavily oxygentaed compound; MS, confirmed by comparison with mass spectrum; KI, confirmed by comparison with Kovat’s index on a DB5 column (Adams 1995); AU: authentic compound; Conc. (%) based on peak area integration

Fig. 1. Chemical structures of isolated compounds.

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five methylenes and nine methine groups. Limonin is reported here from S. aromaticum for the first time. The CI/MS of 2 showed a molecular ion peak [M+1]+ at m/z 179, C10H10O3. 1H-NMR spectral data of 2 were identical to that previously reported for ferulic aldehyde (Grande et al, 1985). 13C-NMR of compound 2 showed 10 nonequivalent carbon resonances, one of which appeared at δ 193.5 and was attributed to an aldehydic group and another one appeared at δ 56.01 assigned to a methoxyl group. The spectrum also showed in the downfield region, six peaks of a trisubstituted benzene ring together with two resonances for two olefinic carbons. The ESI/MS of 3 showed a molecular ion peak [M+1]+ at m/z 479, C22H22O12. It showed UV spectral data with diagnostic reagent identical to those of 4’- substituted flavonol glycoside. Acid hydrolysis of 3 afforded glucose and tamarixetin. The 1 H-NMR spectrum of 3 showed signals pattern of a quercetin moiety in the down field region, a methoxyl singlet appeared at δ 3.83 and a doublet at δ 5.55 (J = 7.6 Hz) was assigned to an anomeric proton of glucopyranosyl moiety. The 13C-NMR of 3 showed 22 nonequivalent carbon resonances, one of which was for a carbonyl at δ 177.5 and another was for a methoxyl at δ 55.75, in addition to six peaks were attributed to the glucose moiety (Agrawal and Banzal, 1989). The protonated carbons were assigned using HMQC. In HMBC, the anomeric proton showed cross peak with C-3 at δ 133.0, whereas the methoxyl group showed correlation with C-4’ at δ 146.9. These data confirmed that compound 3 was tamarixetin 3-O-b-D-glucopyranoside (Harborne, 1999), which was also isolated for the first time from S. aromaticum. The ESI/MS of 4 showed a molecular ion peak [M+1]+ at m/z 493, C23H25O12. It showed UV spectral data with diagnostic reagent identical to those of 7, 4’- disubstituted flavonol glycoside. Acid hydrolysis of 4 afforded glucose and ombutin. The 1H-

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NMR spectrum of 4 showed a signal pattern very similar to that of 3 with an extra methoxyl singlet at δ 3.85. Also, the 13C-NMR of 4 was similar to that of 3 with an extra methoxyl carbon resonance at δ 56.1. The protonated carbons were assigned using HMQC. Similarly, the locations of the two methoxyl groups at C-7 and at C-4’ as well as the location of glucose moiety at C-3 were all established by HMBC correlations. These data confirmed that compound 4 is ombutin 3-O-b-D-glucopyranoside, also isolated for the first time from S. aromaticum, previously reported only from Gynostemma yixingense (Fam. Cucurbitaceae) (Si et al., 1994). Antioxidant activity Many aromatic plants and spices especially clove buds and their essential oils have been known to support various biological activities such as antimicrobial and antioxidant properties (Fu et al, 2007). The radical scavenging effects (percentage of quenched radicals) were determined for clove buds extracts and their constituents. The clove buds extracts or their constituents when mixed with DPPH decolorized it due to hydrogen donating ability. All the tested samples (n-hexane, methylene chloride and ethanol extracts as well as quercetin, compound 3 and 4) revealed scavenging effects on DPPH (10 to 93 %) as shown in Fig. (2). Antioxidants are believed to neutralize the free radicals in lipid chains by contributing a hydrogen atom usually from a phenolic hydroxyl group, which in turn converts phenolic groups into stable free radicals that do not intiate or propagate further oxidation of lipids. It was observed that the scavenging activity of volatile extract of clove buds at all concentrations from 50 to 400 mg/ml is rather strong (42 -83 %). The remarkable antioxidant activity of hexane extract might


be due to the higher concentration of phenolic compounds such as eugenol (71.56%), eugenol acetate (8.99 %) and thymol (0.87 %). These results are in accord with previous literature (Lee and Shibamoto, 2001; El-Ghorab and El-Massry, 2003). The dichloromethane and ethanol extracts as well as of the isolated flavonoids of S. aromaticum buds were found to act as strong free radical scavengers in comparison with commercial antioxidants BHT as indicated by DPPH assays (Fig. 2). All the extracts and the isolated flavonoids exhibited potential antioxidant activity against DPPH radicals at different concentrations (50 to 400 mg/mL). All extracts of clove buds at higher concentration (400 mg/ml) have remarkable inhibition of DPPH radical scavenging activity (45 to 93 %) in comparison with 400 mg/ml BHT (95 %) (Fig. 2). It is well known that free radicals play an important role in autoxidation of unsaturated lipids in food stuffs (Kaur et al, 1991). Quercetin has a moderate antioxidant activity (46 %) at 400 mg/mL in comparison with BHT (70 %) at 50 mg/mL.


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Our results are consistence with Miller (1996), who found that quercetin scavenges oxygen-free radicals, and inhibits the enzyme xanthine oxidase. Among the tested extracts, the ethanol extract of the clove buds showed remarkable scavenging activity (93%), as compared with synthetic antioxidants such as BHT (95%). These results demonstrated that the extracts of S. aromaticum buds and the isolated flavonoids have effective activity as hydrogen donors and as primary antioxidants by reacting with lipid radicals. Hepatoprotective study: As the ethanol extract of clove showed the high antioxidant activity, this study evaluates the hepatoprotective activity of it on the paracetamol- induced liver injury. The serum biochemical analysis indicated that paracetamol treatment resulted in a significant increase (P < 0.05) of ALT (41.1 ± 0.8 u/L), AST (44 ± 2.7 u/L), and alkaline phosphatase levels (199.8 ± 21 u/

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L) as compared with those of control (29.5 ± 2.3, 36 ± 1.6 and 130 ± 8 u/L respectively). On the other hand, pretreatment the ethanol extract of clove succeeded in restoring all the biochemical parameters towards the normal values of the controls (ALT (26.1 ± 1.3 u/L), AST (33± 3.1 u/L), and alkaline phosphatase levels (164.6 ± 4.6 u/L). The activities of enzymes AST, ALT and ALP in serum are used routinely to assess the functional status of the liver in both clinical and experimental settings. They are used as serum markers of hepatic damage. Elevated levels of these enzymes in serum in paracetamol-treated group point to liver dysfunction. These findings were confirmed by histological observations of liver. Liver sections from control rats showed normal lobular architecture and normal hepatic cells with a well-preserved cytoplasm and well-defined nucleus and nucleoli (Fig. 3a). Histopathological examination of the livers of animals given only the ethanol extract of clove showed no significant morphological changes, as compared to animals in the control group (Fig. 3b). Liver sections from animals administered with paracetamol showed marked hepatocytes necrosis especially in the centrolobular, sinusoidal congestion, broad infiltration of the lymphocytes loss of cell boundaries and hepatic architecture, and ballooning degeneration. Some cells showed loss of nucleus and nucleoli. Also, areas of edema were found (Fig. 3c-e). Pretreatment with the ethanol extract of clove showed normal lobular structure with hardly ascertainable regenerative activity in paracetamol-treated animals (Fig. 3-f). The rise in serum levels of ALP, AST and ALT has been attributed to the damaged structural integrity of the liver (Chenoweth and Hake, 1962); because these are cytoplasmic in location and are released into circulation after cellular damage (Sallie et al., 1991). The extent of hepatic damage is assessed by the level of increased cytoplasmic enzymes (AST and ALT) in circulation

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(Sallie et al., 1991). Zimmerman and Seeff, (1970) reported that due to liver injury, the transport function of the hepatocytes gets disturbed, resulting in the leakage of plasma membrane, thereby causing an increased enzyme level in the serum. The reversal of increased serum enzymes in paracetamol-induced liver damage by the ethanol extract of clove may be due to the prevention of leakage of the intracellular enzymes by its membrane stabilizing activity, which in agreement with the commonly accepted view that serum levels of transaminases return to normal with healing of hepatic parenchyma and the regeneration of hepatocytes (Thabrew et al, 1987). From the foregoing findings it can be speculated that the observed increasing effect of ALT, AST and ALP levels in serum in rats treated with paracetamol alone were due to hepatocellular damage and the ethanol extract of clove afforded protection from such paracetamol-induced liver damage. A possible mechanism for protection by clove against paracetamol-induced liver damage could involve clove components acting as free radical scavengers intercepting those radicals involved in paracetamol metabolism by microsomal enzymes. Thus, by trapping oxygen related free radicals clove extract could hinder their interaction with polyunsaturated fatty acids and would abolish the enhancement of lipid peroxidative processes leading to MDA formation. Therefore, clove extract may be a useful agent for the normalization of paracetamol induced impaired membrane function. Thus, from the foregoing findings, it was observed that the ethanol extract of clove is a promising hepatoprotective agent and this hepatoprotective activity may be due to its antioxidant and normalization of impaired membrane function activity.



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Fig. 3: Photomicrographs of liver of rats show A) control with normal structure, b) rats given the extract of clove (500 mg/kg b. wt.) with normal structure, c, d, e) rats treated with paracetamol (650 mg/kg b.wt) showing different lesions, loss of cell boundaries and hepatic architecture and marked hepatocytes necrosis, ballooning degeneration and loss of nucleus and nucleoli (c) broad infiltration of the lymphocytes (d) sinusoidal congestion and area of edema (E) and f) rats given the extract and paracetamol with normal structure (H & E X 300).

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EXPERIMENTAL

Ferulic aldehyde (2):

Plant material

CI/MS m/z (rel. int.), 179(100) [M+1]+, 13C-NMR (CDCl3, 125 MHz): 146.9 (C-1), 148.9 (C-2), 109.4 (C-3), 126.7 (C-4), 124.0 (C-5), 114.9 (C-6), 152.8 (C-7), 126.5 (C-8), 193.5 (C-9), 56.0 (OMe).

The buds of S. aromaticum were obtained from a local market (Harraz Company for Medicinal Plants, Cairo, Egypt). A voucher specimen has been deposited in the National Research Centre Herbarium, Cairo, Egypt.

Tamarixetin 3-O-β-D-glucopyranoside (3): Extraction and isolation The dry powdered buds of S. aromaticum (500 g) were subjected to successive extractions, using n-hexane, dichloromethane and ethanol to give 14, 27 and 30 gm of extracts, respectively. The n-hexane extract was analyzed for its volatile components using GC/MS. The dichloromethane extract was subjected to Si-gel CC eluted by CH2Cl2/ethyl acetate step gradient. The eluted fractions were subjected to repetitive separation and purification on Sephadex LH-20 columns eluting with CH2Cl2-MeOH (1: 1) to give 12 mg of limonin, 8 mg of ferulic aldehyde and 10 mg eugenol. The ethanol extract was subjected to polyamide column chromatography eluted with water/methanol step gradient. The obtained fractions were further purified on Sephadex LH-20 columns to give 22 mg of tamarixetin 3-O-b-D-glucopyranoside (3),36 mg of ombutin 3-O-b-D-glucopyranoside (4) and 14 mg of quercetin (4).

ESI/MS m/z (rel. int.), 479 (60), 13C-NMR (DMSO-d6, 125 MHz): 156.4 (C-2), 133.0 (C-3), 177.5 (C-4), 161.3 (C-5), 98.8 (C-6), 164.2 (C-7), 93.8 (C-8), 156.5 (C-9), 104.1 (C-10), 121.1 (C-1`), 113.5 (C-2`), 149.5 (C3`), 146.9 (C-4`), 115.2 (C-5`), 122.1 (C-6`), 55.75 (3`-OMe),100.8 (C-1``), 74.4 (C-2``), 76.5 (C-3``), 70.0 (C-4``), 77.5 (C-5``), 60.7 (C-6``). Ombuin 3-O-β-D-glucopyranoside (4): ESI/MS m/z (rel. int.), 493 (45) 13C-NMR (DMSO-d6, 125 MHz): 156.3 (C2), 133.2 (C-3), 177.5 (C-4), 160.9 (C-5), 97.9 (C-6), 165.1 (C-7), 92.3 (C-8), 156.6 (C-9), 105.0 (C-10), 120.9 (C-1`),113.5 (C2`), 149.5 (C-3`), 146.9 (C-4`), 115.2 (C-5`), 122.2 (C-6`), 56.1 (7-OMe), 55.7 (4`-OMe), 100.7 (C-1``), 74.3 (C-2``), 76.4 (C-3``), 69.8 (C-4``), 77.5 (C-5``), 60.6 (C-6```). DPPH radical scavenging assay:

Limonin (1): CIMS m/z (rel. int.), 471 (100), 13C-NMR (CDCl3, 125 MHz): 79.2 (C-1), 35.6 (C-2), 169.0 (C-3), 80.3 (C-4), 60.6 (C-5), 36.4 (C6), 206.0 (C-7), 51.3 (C-8), 48.1 (C-9), 45.9 (C-10), 18.9 (C-11), 30.8 (C-12), 37.9 (C-13), 65.7 (C-14), 53.9 (C-15), 166.6 (C-16), 77.8 (C-17), 65.3 (C-18), 119.9 (C-19), 141.1 (C20), 109.7 (C-21), 143.2 (C-22), 20.7 (C-23), 17.6 (C-24), 21.4 (C-25), 30.2 (C-26).

Radical scavenging activity of clove buds extracts and their constituents against stable DPPH (2, 2-diphenyl-2-picrylhydrazyl hydrate was determined spectrophotometrically. When DPPH reacts with an antioxidant compound, which can donate hydrogen, it is reduced. The changes in color (from deep violet to light yellow) were measured at 517 nm on a UV/visible light spectrophotometer. Radical scavenging activity of extracts was measured accord-


ing to Miliauskas et al, 2004, as described below. Extract solutions (volatile oils, dichloromethane and ethanol extracts) were prepared by dissolving 0.1 g of dry extract in 10 ml of methanol. The solution of DPPH in methanol (6x10-5 M) was prepared daily, before UV measurements. Various concentrations of each extract (50, 100, 200 and 400 mg/mL) were added to solutions (1 ml) of DPPH in methanol. The mixtures were shaken vigorously and left to stand at room temperature for 30 min; the absorbances of the resulting solutions were measured spectrophotometrically at 517 nm. In this assay, the percentage of DPPH reduction by different extracts of clove was compared to that of BHT. The experiment was carried out in triplicate. Radical scavenging activity was calculated by the following formula:

% Inhibition = [(AB-AA)/AB]X 100, AB absorption of blank sample (t=0 min); A absorption of tested extract solution (t=30 min). Instrumentation: An HP model 6890 GC interfaced to an HP 5791A mass selective detector (GC/MS) was used for mass spectral identification of the GC components at MS ionization voltage of 70 eV. A 30 m x 0.25 mm i.d. (df = 0.25 lm) DB-5 bonded-phase fused-silica capillary column (J&W Scientific) was used for GC. The linear velocity of the helium carrier gas was 30 cm/s. The injector and the detector temperatures were 250 ºC. The oven temperature was programmed from 35 to 220 ºC at 3 ºC /min and held for 40 min. Kovat’s indices were determined by co-injection of the sample with a solution containing homologous series of n-hydrocarbons (C8-C26) under the same conditions as described above.


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The separated components were identified by matching with NIST mass – spectral library data, and by comparison of Kovat’s indices with those of authentic components and with published data (Adams, 1995). The quantitative determination was carried out based on peak area integration. Hepatoprotective assay Paracetamol hepatotoxicity was induced in female albino rats of Sprage Dewally strain weighing between 170 and 200 g. Animals bred and maintained in the Lab Animal House, National Research Centre, Cairo. Four groups of animals (6 rats each) were used in this study. Group 1, control, group 2 treated with the ethanol extract of clove (500 mg/kg b.wt.) for seven days, group 3, treated with paracetamol (i.p. at a dose of 650 mg/kg b.wt according to Parmar et al, (1995) and group 4, treated with the extract for seven days and at the eighth day injected with paracetamol as in group 3. After 24 h of the last treatment blood samples were withdrawn from ratino bulber venous plexus with under light anaesthesia and were kept at room temperature to coagulate. The blood samples were centrifuged and the separated serum was used for the estimation of AST, ALT, and ALP. The activity of AST and ALT were measured according to the method described by Reitman and Frankel (1957). The estimation of ALP was carried out by the methods of King and Armstrong (1980). Animals were then sacrificed and dissected. Their livers were taken out, washed with water, dried gently with filter paper and preserved in 10% formalin. Sections (4–5 mm thick) were prepared and then stained with hematoxylin and eosin dye for microscopic examination. All data were expressed as means ± standard errors, and analyzed with one-way analysis of variance (ANOVA).

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Student’s t-test was used to calculate statistical signiﬁcance by SPSS software. P < 0.05 and