Extraction of Eugenol in Clove Extract and Evaluation of Its Antioxidant Activity
Extraction of Eugenol in Clove Extract and Evaluation of Its Antioxidant Activity
Received: 02 Mar 2024; Received in revised form: 11 Apr 2024; Accepted: 22 Apr 2024; Available online: 30 Apr 2024
©2024 The Author(s). Published by Infogain Publication. This is an open access article under the CC BY license
(https://creativecommons.org/licenses/by/4.0/).
Abstract— Higher total phenolic content was observed in steam-distilled clove extract compared to its
oleoresin. Ensuring antioxidant activity of steam distilled and clove oleoresin using β-carotene–linoleic
acid model system was found to be 85.51±0.19% and 77.88±0.15%, respectively, at a level of 200 ppm. The
radical-scavenging activity of steam-distilled extract and oleoresin of clove were 88.93±0.23% and
80.84±0.36%, respectively, at the same level of 200ppm by using the DPPH method. Eugenol content in the
steam-distilled extract of clove (0.518±0.005mg/ml) was significantly higher than that of clove oleoresin
(0.433±0.007mg/ml). Recovery of eugenol content affected by the period of refluxing and clove extract
refluxed for 4 hr found to be highest recovery in the steam distilled clove extract (0.763±0.007) than its
clove oleoresin (0.635±0.020) with 13.904 min retention time using RP-HPLC. The steam-distilled clove
extract was found to have the highest antioxidant activity than its oleoresin counterparts.
Keywords— Antioxidant, β-carotene–linoleic acid, DPPH, eugenol, phenolic content.
clove oleoresin antioxidant activity. We also examined the observations, the tubes were submerged in a water bath set
antioxidant activity of the major eugenol in steam-distilled at 50°C. After that, it was independently added to a model
clove extract and clove oleoresin using RP-HPLC. system of β-carotene linoleic acid, and the activity was
measured spectrophotometrically 700 (Specord) at 470
nm. The following expression was used to assess the
II. MATERIALS AND METHODS
extracts antioxidant activity (AA) in terms of the photo-
2.1 Clove Extract: oxidation of beta-carotene. AA is represented by the
Clove oleoresin was obtained from Synthite Industry Ltd. antioxidant activity, Ao is the initial absorbance of sample,
in Kerala, India, while the steam-distilled extract was At is the absorbance of sample after time t, A0o is the
provided by Katyani Exports in Delhi, India. initial absorbance of control, and A0t is the absorbance of
control after time t respectively.
2.2 Determination of antioxidant capacity: To assess
antioxidant potential, total phenolic content, β carotene
linoleic acid model system, and radical scavenging assays
were used (Prior et al., 2005).
2.5 Radical-scavenging activity by DPPH model system
2.3 Total phenolic content
According to the method described by Blois (1958), the
Folin-Ciocalteu's technique was used to determine the total extracts of steam-distilled and oleoresin clove were taken
phenolic content of clove oleoresin and steam distilled at a rate of 200 ppm, dissolved in ethanol, and tested for
clove extract (Kahkonen et al., 1999). In a test tube, 400 μl their capacity to scavenge free radicals in the DPPH
of adequately diluted sample/gallic acid standard was system. Test tubes containing 200 ppm of sample extract
placed. It was combined using a vortex mixer after adding diluted in ethanol (1 ml) were then filled with 4 ml of a 0.1
2000 μl of diluted Folin-Ciocalteu's reagent. After 3 mM ethyl acetate solution of DPPH and shaken
minutes, 1600 μl of sodium carbonate solution was added vigorously. Tubes were left at 27°C for 20 min. Without
and incubated at room temperature for 30 minutes in the any additional extract, the control was made as described
dark. Instead of sample, 400 μl of distilled water was used above, and ethanol was used to adjust the baseline. Optical
for blank preparation. Using a Spectrophotometer densities (OD) of the samples were measured
(Specord 700), the absorbance of the samples was spectrophotometrically (Specord 700) at 517 nm. Radical-
measured against a blank at 765nm. The concentration of scavenging activity was expressed as % inhibition
gallic acid (400 μl of 10-100 g/ml) was collected in place percentage.
of the sample for standard curve preparation and quantified
with regard to the standard curve. The outcomes were
expressed as gallic acid equivalents (GAE), milligrams per
gm of spice extract. 2.6 Extraction and quantification of eugenol from clove
2.4 β-carotene–linoleic acid model system extract
The antioxidant activity of solvent extracts was evaluated The preparation of the sample is the first and most
using the procedure described by Marco (1968), with important step. It is necessary to extract the appropriate
slight modifications. β-carotene (0.2 mg) in chloroform chemical components from the spice materials in order to
(0.5 ml) was mixed with 20 mg linoleic acid and 200 mg proceed with further separation and characterization. 30
Tween 40. Nitrogen gas was used to evaporate the mg of clove extracts (steam distilled and oleoresin clove)
chloroform at 40°C. The resultant solution was was taken in a round-bottom flask, 30 ml of ethanol was
immediately diluted with 10 ml of double distilled water, added to the flask and allowed to reflux. Using a water
and the emulsion was thoroughly stirred for one minute reflux condenser, the sample was allowed to reflux for
using a magnetic stirrer. The emulsion was diluted further varying amounts of time ranging from 2 to 5 hours.
with 40 mL of distilled water. Aliquots (4 mL) of this Following the process of refluxing, the mixture was
reagent were placed into separate stopper test tubes evaporated on a water bath until it reached the desired
holding 1 mL of the necessary amount of ethanol-based level of dryness. After re-dissolving the resulting solution
sample extracts. At a rate of 200 ppm, the steam-distilled in 30 mL of ethanol, it was filtered using a PTFE
and oleoresin of clove extract dissolved in ethanol. A (Polytetrafluoroethylene) syringe filter with a 0.45-m pore
control was created using 1 ml of ethanol and 4 ml of size and then immediately injected into an HPLC system
emulsion. Optical densities of all samples were tested in a volume of 20 μL. Quantification was performed based
immediately (t = 0), again after 15 minutes, then every 30 on the standard curve of eugenol concentration in ethanol,
minutes for the next 3 hours (t = 180). In between which ranged from 0.25 to 1.5 mg/10 ml. At a wavelength
of 280 nm, the analysis was carried out using a C18 Data are presented as means ± SEM (n=3).
column (5 μm, 4.5 250mm, 100 A0). The mobile phase a-bMeans with different lowercase superscripts letters are
consisted of methanol and water at a volumetric ratio of significantly different (P < 0.05) from each other.
60:40. The temperature of the column was maintained at
300C and the flow rate was held constant at 0.8ml/min.
3.2 Antioxidant activity by β-carotene–linoleic acid
model system
III. RESULTS AND DISCUSSION Table 2 shows the antioxidant activity of clove extract
3.1 Total Phenolic Content by Folin-Ciocalteu method (steam distilled and oleoresin) at 200 ppm using β-
carotene-linoleic acid linked oxidation model system
The Folin–Ciocalteu method (Kahkone et al., 1999) was
(Marco, 1968). At a concentration of 200 ppm, the
used to measure the total amount of phenolic substance. A
antioxidant activity of steam distilled clove extract
standard curve of gallic acid (ranging from 10 to 100
μg/ml) was prepared to evaluate the overall phenolic (84.950±0.23%) was substantially (P>0.05) higher than
content of oleoresin and steam-distilled clove extracts and that of oleoresin (77.886±0.31%). This difference can be
attributed to the difference in total phenolic content of
results were expressed as mg gallic acid equivalents
steam distilled clove extract and clove oleoresin, which is
(GAE) per gram of spice extract. Table 1 displayed the
dependent on extraction method and solubility of oil
results that oleoresin had a GAE of 177.039±0.35mg and
the steam-distilled clove extract had a GAE of components. This is consistent with the findings of
256.506±0.45mg/gm respectively. The reason for this Mostafa et al., (2004), who found that super critical fluid
(SFE) extraction increases the yield of phenolic
difference was probably because different extracts
compounds due to an increase in the solubility of oil
contained different antioxidant chemicals that bind to
components.
water and fat (flavonoids, terpenoids, carotenoids,
phytoestrogens). Zhou et al. (2011) looked at the Table 2. Antioxidant activity of steam distilled and clove
hyrophillic and lipophillic antioxidant activity of loquat oleoresin
fruits and found that the overall antioxidant activity and Samples Antioxidant activities (%) at
phenolic content were linked to hydrophilic antioxidant 200ppm
compounds in a good way. Vicas et al. (2009) found
similar results by using FRAP (ferric tripyridyltriazine Clove oleoresin 77.886±0.15b
complex) of mistletoe (Visum album) had about 100 times Steam distilled clove 85.510±0.19a
less lipophillic antioxidant activity (LAA) than hydrophilic extract
antioxidant activity (HAA). Also, HAA was linked to Data are presented as means ± SEM (n=3).
higher levels of total phenolics in the leaves (R2=0.9363)
and stems (R2=7337) of mistletoe (Visum album). In a-bMeans with different lowercase superscripts letters are
2005, Shan et al. said that clove bud's main phenolic significantly different (P < 0.05) from each other.
substances are phenolic acids (gallic acid),
flavonolglucosides, tannin, and phenolic volatile oils 3.3 Radical-scavenging activity by DPPH assay
(eugenol and acetyl eugenol).The phenolic chemical
The radical-scavenging activity of clove extracts (steam
content of clove extract that was made by steam
distilled and oleoresin) was tested in the DPPH system at
distillation was higher. It also depends on the method used
200 ppm and the findings are shown in Table 3. Steam
to separate the oil and how well the different parts of the
distilled clove extract and its oleoresin were shown to have
oil dissolve (Reverchon, 1997; Mostafa et al., 2004). A
radical-scavenging activities of 88.935±0.23% and
study by Ozka et al. (2012) found that a steam-distilled
80.841±0.36%, respectively. The radical-scavenging
extract of the Rosa damascena flower had a high phenolic
potential of steam distilled clove extract was found to be
content.
considerably (P>0.05) higher than that of its oleoresin
Table 1. Total phenolic content of steam distilled and counterpart. This disparity in radical-scavenging
clove oleoresin (antioxidant) activity could be attributed to differing
hydrophilic and lipophillic antioxidant chemicals. Another
Samples Total phenolic content (mg of reason could be that the total phenolic content of steam
GAE/gm) distilled clove extract and clove oleoresin differs. The
Clove Oleoresin 177.039±0.35b results of the radical-scavenging (antioxidant) assay were
similar with the data obtained from the total phenolic
Steam distilled 256.506±0.45a
content and the β-carotene-linoleic acid model system. Our including antioxidant action. Eugenol was measured using
findings suggested that there was a positive relationship a modified HPLC method established by Yun et al.,
between phenolic content and antioxidant activity, since (2010). The extraction process was optimized for
the higher activity of the steam distilled clove extract may quantitative analysis to improve the sensitivity and
be related to its higher phenolic content. selectivity for separation of eugenol content.
Table 3: Radical-scavenging activities of clove extracts 3.4.1 Optimization of eugenol extraction using two
(steam distilled and oleoresin) as expressed in % different solvent
inhibition The extraction technique of eugenol content was
Samples % Inhibition standardized, and the efficiency of several extraction
solvents from clove extracts (steam distilled and oleoresin)
Clove oleoresin 80.841±0.36b
were assessed in order to get chromatograms with superior
Steam distilled clove extract 88.935±0.23a resolution of neighboring peaks in a short period. The
Data are presented as means ± SEM (n=3). eugenol content of steam distilled clove extract and clove
oleoresin was extracted using two different solvents,
a-bMeans with different lowercase superscripts letters are
methanol (absolute) and ethanol (95%). The results
significantly different (P < 0.05) from each other.
showed that ethanol extraction yielded more eugenol than
methanol extraction. As a result, ethanol was chosen as the
3.4 Quantitative analysis of eugenol content in clove extraction solvent for determining eugenol. Also, for
extracts (oleoresin and steam distilled) by RP-HPLC. industrial uses, ethanol is probably preferable to methanol
since the resulting solvent residues are less hazardous. The
Eugenol (4-allyl-2-methoxyphenol), a well-known clove
results are shown in Fig 1.
phenolic component, has numerous therapeutic effects,
Fig.1: Two different solvent used to estimate eugenol content from clove extracts (oleoresin and steam distilled) as expressed
in (mg/ml)
The mean changes between the samples were analyzed by one-way ANOVA.a-b Means with different lowercase superscripts
letters are significantly different (P < 0.05) from each other. Error bars show the variations of three determinations in terms
of standard error of mean.
3.4.2 Optimization of eugenol extraction using two minutes. It was discovered that raising the refluxing time
different solvent different time interval of refluxing period from 2 hours to 4 hours increased the eugenol content,
To establish the ideal extraction time for eugenol content, whereas increasing the refluxing time to 5 hours lowered
several refluxing times at different intervals such as 2 hr, 4 the eugenol content. As shown in fig 2, the quantitative
hr, and 5 hr were tried in terms of eugenol content HPLC results demonstrate that the 4 hr extraction time has
extraction. The steam distilled clove extract and oleoresin the highest value of eugenol concentration in the steam
were refluxed with each solvent for 2 to 5 hours using a distilled clove extract (0.763±0.007) than its clove
water reflux condenser. Following the refluxing, the oleoresin (0.635±0.020). Aside from the solvent and
samples were evaporated on a water bath until dry. The method of extraction, environmental factors and different
resulting solution was redissolved with the solvent and species may also account for the variance in extract
filtered through a 0.45m PTFE syringe filter before being activity. Fig 2 shows that the eugenol concentration of
injected directly into the HPLC apparatus. The analysis steam distilled clove extract was found to be higher than
was carried out at a flow rate of 0.8ml/min at a column that of its oleoresin counterparts. Selected solvents, such
temperature of 30°C. The eugenol concentration of as 95% ethanol, were employed because they were stable
oleoresin and steam distilled clove extract was determined and compatible with reversed-phase HPLC separation of
using a eugenol standard curve (range from 0.25 to 1.5 eugenol content.
ppm). Eugenol had a retention time of 13.904 ± 0.5
Fig.2. Eugenol content obtained at different time interval of refluxing (mg/ml) of clove extracts (steam distilled and
oleoresin)
The mean changes between the samples were analyzed by one-way ANOVA. a-b Means with different lowercase
superscripts letters are significantly different (P < 0.05) from each other. A-B Means with different uppercase superscripts
letters are significantly different (P < 0.05) from each other. Error bars show the variations of three determinations in terms
of standard error of mean
trout and its effects on swimming performance. North capacity of some Thai indigenous plants. Food Chemistry,
American Journal of Fisheries Management, 17(2):301– 100:1409-1418.
307. [18] Prior, X. and Wu, K. (2005). Standardized method for the
[3] Arnao, M. B.; Cano, A. and Acosta, M. (2001). The determination of antioxidant capacity and phenolics in foods
hydrophilic and lipophilic contribution to total antioxidant and dietary supplements. Journal of Agriculture Food
activity. Food Chemistry, 73:239-244. Chemistry, 53:4290-4302
[4] Bhuiyan, I. M.; Begum, J. and Akter, F. (2010).Constituents [19] Pokorny, J. and Korczak, J. (2001). Preparation of natural
of the essential oil from leaves and buds of clove antioxidants. In J. Pokorny, N. Yanishlieva, & M. H.
(Syzigiumcaryophyllatum(L.) Alston).African Journal of Gordon (Eds.), Antioxidants in food – practical application,
Plant Science, 4(11): 451-454. Cambridge: Woodhead Publishing Ltd. pp. 311–330.
[5] Blois, M. S. (2001). Antioxidant determination by the use of [20] Rajakumar, D.V. and Rao, M. N. A.
stable free radical. Nature, 181:1199–1200. (1993).Dehydrozingerone and isoeugenol as inhibitors of
[6] Jayaprakasha, G. K.; Singh, R. P. and Sakariah, K. K. lipid peroxidation and as free radical scavengers.
(2001). Antioxidant activity of grape seed (Vitisvinefera) Biochemistry Pharmacology, 46:2067–2072.
extracts on peroxidation models in vitro.Food Chemistry, [21] Reverchon, E. (1997). Supercritical fluid extraction and
73:285–290. fractionation of essential oilsandrelated products. Journal of
[7] Kahkonen, M. P.; Hopia, A. I.; Vuorela, H. J.; Rauha, J.; Supercritical fluids, 10:1–37.
Pihlaja, K.; Kujala, S. T. and Heinonen, M. (1999). [22] Rice-Evans, C. A.; Miller, N. J.; Bolwell, P. M.; and
Antioxidant activity of plant extracts containing Pridham, J. B. (1995). The relative antioxidant activities of
phenolic compounds. Journal of Agriculture and Food plant-derived polyphenolic flavonoids. FreeRadical
Chemistry,47:3954-3962. Research, 22:375-383.
[8] Kildeaa, M. A.; Allanb, G. L. and Kearney, R. E. (2004). [23] Shan, B.;Yizhong. Z.; Sun, M.and Corke, H.
Accumulation and clearance of the anaesthetics clove oil (2005).Antioxidant capacity of 26 spice extracts and
and AQUI-S_ from the edible tissue of silver perch characterization oftheir phenolic constituents. Department of
(Bidyanusbidyanus). Aquaculture,232:265–277. Botany and Department of Zoology, The University of
[9] Lee, K. G. and Shibamoto, T. (2001). Antioxidant property Hong Kong, Pokfulam Road, Hong Kong.Journal of
of aroma extract isolated from clove buds. Food Chemistry, Agriculture and Food Chemistry,53(20):7749–7759.
74(4):443-448. [24] Shelef, L.A. (1983). Antimicrobial effects of spices. Journal
[10] Liu, H. and White, P. J. (1992). Oxidative stability of of Food Safety, 6:29– 44.
soybean oils with altered fatty acid compositions. Journal of [25] Singleton, V.; Orthofer, R. and Lamuela-Raventos, R.
American Oil Chemists Societ,69:528–532 (1999). Analysis of total phenols and other oxidation
[11] Marco, G. J. (1968). A Rapid method for Evaluation of substrates and antioxidants by means of FolinCiocalteu
Antioxidants.Journal of American Oil Chemists reagent. In L. Packer (Ed.). Oxidants and antioxidants, part
Society,45:594-596. A, methods in enzymology, New York; Academic press,
[12] Mostafa, K.; Yadollah, Y.; Fatemeh, S. and Naader, B. 299:152-178.
(2004). Comparison of essential oil composition of [26] Soares, J. R.; Dinis, T. C. P.; Cunha, A. P. and Almeida, L.
Carumcopticum obtained by supercritical carbon dioxide M. (1997) Antioxidant activities of some extracts of Thymus
extraction and hydrodistillation methods. Food zygis. Free Radical Research26:469-478.
Chemistry,86:587–591. [27] Soto, C.G. and Burhanuddin, C.G. (1995) Clove oil as a fish
[13] Mylonasa, C. C.; Cardinalettia, T. G.; Sigelakia, I. and anaesthetic for measuring length and weight of rabbit fish
Polzonetti-Magni, A. (2005). Comparative efficacy of clove (Siganuslineatus). Aquaculture136:149–152.
oil and 2-phenoxyethanol as anesthetics in the aquaculture [28] Vicas, S.; Prokisch, J.; Rugina, D. and Socaciu, C. (2009).
of European sea bass (Dicentrarchuslabrax) and gilthead Hydrophilic and lipophilic antioxidant activity of Mistletoe
sea bream (Sparusaurata) at different temperatures. (Viscum album) as determined by FRAP method.
Aquaculture, 246:467–481. NotulaeBotanicaeHortiAgrobotanici Cluj-Napoca,
[14] Nagababu, E. and Lakshmaiah, N. (1992). Inhibitory effect 37(2):112-116.
of eugenol on non-enzymatic lipid peroxidation in rat liver [29] Yun,J. (2010). Quantitative analysis of eugenol in clove
mitochondria. Biochemical Pharmacology,43:2393–2400. extract by a validated HPLC method.Journal of AOAC
[15] Ozcelik, B.; Lee, J. H. and Min, D. B. (2003). Effects of international,93(6):1806-1810
light, oxygen, and pH on the absorbance of 2, 2-diphenyl-1- [30] Zhou, C.; Li, X.; Xu, C.; Sun, C. and Chen, K. (2011).
picrylhydrazyl (DPPH). Journal of Food Science, 68:487- Hydrophilic and lipophilic antioxidant activity of loquat
490. fruits. Journal of Food Biochemistry, DOI: 10.1111/j.1745-
[16] Ozkan, G.; Sagdic, O.; Baydar, N. G. and Badyar, H. 4514.2011.00574.x
(2004). Antioxidant and Antibacterial Activities of Rosa
Damascena Flower Extracts. Food Science and Technology
International, 10(4):277-281
[17] Pitchaon, M.; Suttajit, M. and Pongsawatmani, R. (2007).
Assessment of phenolic content and free radical scavenging