Comparative Profiling of Clove Extract and Its Component Antioxidant Activities Against Five Reactive Oxygen Species Using Multiple Free Radical Scavenging

Abstract

Clove (Syzygium aromaticum L.) is recognized to have strong antioxidant activity, as revealed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, β-carotene bleaching, and ferric reducing power assays. The present study was undertaken to determine the radical scavenging profiles of clove extract against five reactive oxygen species (ROS: hydroxyl radical, superoxide, alkoxyl radical, peroxyl radical, and singlet oxygen) using the multiple free-radical scavenging (MULTIS) method. The clove extract was shown to be effective in scavenging singlet oxygen. Further, ROS scavenging measurements for three antioxidant components of cloves (eugenol, β-caryophyllene, and eugenol acetate) were carried out. The MULTIS measurements suggested that eugenol plays the major role in the overall scavenging activity of clove as a food. Based on the results, the singlet oxygen scavenging mechanism of eugenol is discussed. This paper presents a quantitative analysis of the comparative antioxidant capacities of foods against various types of ROS.

Introduction

The free radicals produced by oxidation processes are disease-promoting agents that impair human health (Man and Jaswir, 2000). Addition of antioxidants is a useful way to mitigate the formation of such free radicals. In previous studies, cloves (Syzygium aromaticum L.) were demonstrated to show high antioxidant activity, as revealed by various assays such as free radical scavenging, β-carotene bleaching, ferric reducing power, and total phenol contents (Perez-Roses et al., 2016; Ghadermazi et al., 2017; He et al., 2018).

Cloves are the aromatic buds of evergreen trees belonging to the family Myrtaceae, which are native to the Maluku Islands of Indonesia. Clove buds are deep brown in color and have a strong pungent odor. The dried harvested buds have been used as aromatic stomachic and anti-inflammatory agents, and as a cooking spice since ancient times. Recently, Kasai et al. confirmed the antioxidant activity of clove buds against superoxide and hydroxyl radicals by using the spin trapping method (Kasai et al., 2016). So far, the quenching ability against 2,2-diphenyl-1-picrylhydrazyl (DPPH) has been used as one of the techniques for quantifying the antioxidant capacity of foods (Perez-Roses et al., 2016; Ghadermazi et al., 2017; He et al., 2018). However, it is reasonable to assume that the reactivity of antioxidant compounds with stable free radical DPPH may be different from that of actual biological ROS. Iuga et al. suggested possible mechanisms for antioxidant reactions depending on the nature of the active species: 1) hydrogen atom transfer (HAT), 2) proton-coupled electron transfer (PCET), 3) sequential electron proton transfer (SEPT), and 4) radical adduct formation (RAF) (Iuga et al., 2012). The direct and quantitative determination of scavenging abilities against various ROS has been hampered by experimental difficulties. Oowada et al. proposed an electron spin resonance (ESR) spin-trapping method based on multiple free-radical scavenging (MULTIS) to quantify the antioxidant abilities against multiple ROS (Oowada et al., 2012). Recently, we extended this method to the antioxidant analysis of food samples such as rosemary (herb) and ginger root (Sueishi et al., 2018; Sueishi et al., 2019). Antioxidants have scavenging abilities against multiple ROS, and the scavenging rate depends on the kind of ROS. The antioxidant abilities of clove against various ROS have not been well established. Further, scavenging data for pure antioxidant compounds against various ROS are still lacking.

In this study, the scavenging abilities of clove extract and its components (eugenol, β-caryophyllene, and eugenol acetate) were quantified against five ROS (hydroxyl radical (HO·), superoxide (O₂⁻·), alkoxyl radical (RO·), peroxyl radical (t-BuOO·), and singlet oxygen (¹O₂)) using the MULTIS method and compared with those of rosemary and ginger, so-called antioxidant foods. The MULTIS measurements showed that clove has effective antioxidant activity for ¹O₂ scavenging. Based on the MULTIS values, the ¹O₂ scavenging mechanism for the clove-related antioxidant component eugenol is discussed in detail and the antioxidant profile of eugenol against five biological ROS was compared with those of structural analogues.

Materials and Methods

Chemicals    The spin trap (ST) 5,5-dimethyl-1-pyrroline N-oxide (DMPO: Tokyo Chemical Ind.) was used to detect HO· and RO· radicals. 5-(2,2-Dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO) used for the detection of O₂⁻· and t-BuOO· was purchased from RRINC. 4-Hydroxy-2,2,6,6-tetramethyl-piperidine (4-HO-TEMP) was obtained from Tokyo Chemical Ind. to quantify ¹O₂. The antioxidant components of clove were obtained commercially and used as received (purity > 98%): eugenol (Tokyo Chemical Ind.), eugenol acetate (Tokyo Chemical Ind.), and β-caryophyllene (Sigma-Aldrich Chemical Co.). Other antioxidants (p-coumaric acid, caffeic acid, ferulic acid, kaempferol, rutin, myricetin, apigenin, and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (purity > 98%)) and precursors/sensitizers of the ROS used in this study were purchased from Sigma-Aldrich Chemical Co. and Tokyo Chemical Ind. Acetonitrile (Wako Pure Chemical Ind.) and water, purified by distillation, were used as a mixture solvent (1:1, v/v) in the ESR spin-trapping analysis.

Sample procedure and ESR spin-trapping measurements    Clove bud powder was purchased from Japanese markets: company A (harvested in Malaysia (n = 3)), company B (origin unknown (n = 3)), and company C (harvested in Indonesia (n = 3)). The clove powder (1 g) was agitated in 10 mL of acetonitrile at 353 K for 2 h. After filtration, the extract was obtained at 298 K.

The concentrations of the five ROS were quantified with and without antioxidants using the ESR spin-trapping method. The clove extract sample ([clove extract] = 0.03 - 0.1% (clove extract concentration of total volume)) was added to the spin-trap solution ([DMPO] = 3 - 10 mM (M = mol dm⁻³) for HO· and RO·, [CYPMPO] = 5 mM for O₂⁻· and t-BuOO·, and [4-HO-TEMP] = 60 mM for ¹O₂). The five ROS were independently generated by UV/Vis light illumination (RUV203S, Radical Research Inc.). The detailed procedures for ESR measurements have been described elsewhere (Sueishi et al., 2018; Sueishi et al., 2019). The HO·, RO·, and t-BuOO· radicals were generated by UV irradiation of H₂O₂ (10 mM), 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) (3 mM), and t-butylhydroperoxide (40 mM), respectively. ¹O₂ and O₂⁻· were formed from the photosensitizers pterin-6-carboxylic acid (40 µM) and riboflavin (50 µM), respectively. The ESR signal intensities of the ROS adducts were recorded on a JEOL FA-200 X-band ESR spectrometer.

Determination of ROS scavenging activities    From the ESR signal intensities of the ROS adducts, the scavenging rate constants and rates (antioxidant activities) were determined. Based on the competitive relationship between the ST and antioxidant compound (AOx), a simple formula for calculating the ROS scavenging capacity (relative rate constant (k_AOx/k_ST)) can be derived, as follows (Oowada et al., 2012; Kohri et al., 2009):   

where k_AOx and k_ST denote the rate constants of the ROS scavenging reaction of the AOx and ST, respectively. I and I₀ are the ESR signal intensities in the presence and absence of the antioxidants, respectively: [antioxidant (eugenol, eugenol acetate, β-caryophyllene, p-coumaric acid, caffeic acid, ferulic acid, kaempferol, rutin, myricetin, apigenin, and trolox)] = 0.040 – 0.12 mM for RO·, 0.004 – 0.080 mM for HO·, 0.002 – 0.040 mM for ¹O₂, 0.020 – 0.060 mM for O₂⁻·, and 0.020 – 0.40 mM for t-BuOO·. The measurement was repeated five times for three independent samples, and errors were shown as probable errors. The k_AOx/k_ST value was determined from the slope of the plot of (I₀ -I)/I against [AOx]₀/[ST]₀. The antioxidant trolox is conventionally used as a standard, and the relative scavenging rate constants are expressed as antioxidant capacity in trolox equivalent units (TEU) (Prior et al., 2003).

The ROS scavenging activities of foods are due to the activities of multiple antioxidants. The total scavenging abilities of a food can be expressed as the sum of the scavenging rates of all its antioxidant components, depending on the rate constants (antioxidant ability) and concentrations of antioxidant components (Sueishi et al., 2018; Sueishi et al., 2019):   

where ν_clove and ν_ST are the scavenging rates of clove and the ST solution, respectively. [AOx(i)%] denotes the concentration of the antioxidant component (i) in volume %.

Results and Discussion

Antioxidant activity of clove extracts    The relative ROS scavenging rates (antioxidant activities) of the clove extracts were determined according to Eq. (2). The relative scavenging rates (ν_{clove extract (100%)}/ν_{ST (1 mM)} (ν_{clove extract (100%)} denotes the scavenging rate of clove extract solution)) of three cloves (companies A, B, and C) are listed in Table 1. As a standard scavenger, trolox has been customarily used. In Table 1, the MULTIS values relative to that of 100 mM trolox solution are expressed as trolox equivalent unit 100 (TEU100).

Table 1. Relative scavenging abilities of clove extracts against five ROS in a mixture (1:1, v/v) of acetonitrile and water at room temperature

Antioxidants	TEU100 (νclove extract (100%)/νST (1 mM)b in parentheses)
	RO·	HO·c	1O2	O2−·	t-BuOO·
Company A	0.27	1	6.69	0.92	0.61
	(3150±80)	(3190±360)	(1580000±8310)	(7680±280)	(1730±150)
Company B	0.18	0.89	5.51	0.82	0.51
	(2080±30)	(2840±240)	(130000±55000)	(6850±610)	(1430±150)
Company C	0.22	1	7.03	1.03	0.57
	(2580±60)	(3190±350)	(1660000±50000)	(8670±180)	(1620±150)
Grated gingerd	0.06	1.48	1.27	0.23	0.23
(Sample B)	(645±32)	(4720±110)	(299000±12000)	(1930±90)	(662±29)
Rosemary extractd	0.26	0.56	1.07	0.9	1.77
(Jan. 2017)	(3010±50)	(1780±170)	(251500±18400)	(7570±830)	(5000±250)
Trolox (100 mM)	1	1	1	1	1
	(11500±300)	(3190±330)	(236000±7000)	(8390±230)	(2830±130)

^a  TEU100 = ν_{clove extract (100%)}/ν_{trolox (100 mM)}

^b  ST = DMPO for HO· and RO·, CYPMPO for O₂⁻· and t-BuOO·, and 4-HO-TEMP for ¹O₂

^c  The HO· scavenging ability in an acetonitrile-water mixture (3:7, v/v)

^d  Cited from Sueishi et al., 2019.

Table 1 shows that there are no significant differences among the antioxidant activities of the three cloves, judging from the experimental errors. It is useful to compare the antioxidant activities of the clove extract with those of other antioxidant food samples. In previous studies, the ROS antioxidant profiles of rosemary extract and grated ginger root were determined using the MULTIS method (Sueishi et al., 2018; Sueishi et al., 2019). In order to visualize the results shown in Table 1, the MULTIS values were illustrated in a radar chart, Figure 1a. This radar chart indicates that each antioxidant food has a specific ROS-scavenging profile. Rosemary is an effective scavenger of the peroxyl radical, and ginger is a good scavenger of the hydroxyl radical. Clove is an effective scavenger of singlet oxygen species as compared with ginger and rosemary.

Fig. 1.

(a) Radar chart comparison of antioxidant capacity (TEU100) of clove extract (company A), rosemary extract, and grated ginger. (b) Radar chart illustration of antioxidant profile of clove extract for the TEU100 values of eugenol against various ROS. The HO· scavenging ability of eugenol is adjusted to 1.0.

ROS scavenging ability of antioxidant components    Eugenol, β-caryophyllene, and eugenol acetate are the major antioxidant components in clove oil (Dorman et al., 2000; Chaieb et al., 2007); therefore, three antioxidants are assumed to be contained in the clove extract. The relative ROS-scavenging rate constants of the three clove components were determined by the MULTIS method according to Eq. (1). The results are listed in Table 2, together with the TEU. The TEU values reveal that eugenol and eugenol acetate are effective scavengers of singlet oxygen and that β-caryophyllene shows effective scavenging ability for hydroxyl radicals.

Table 2. TEU values of clove's antioxidant components and relative scavenging rate constants k_AOx/k_ST in a mixture (1:1, v/v) of acetonitrile and water at room temperature

Antioxidant	TEU valuesa (kAOx / kST in parentheses)
	RO· [vs DMPO]	HO·b [vs DMPO]	1O2 [vs 4-HO-TEMP]	O2−· [vs CYPMPO]	t-BuOO· [vs CYPMPO]
Eugenol	0.13	0.56	4.05	0.56	0.39
	(15.4 ± 0.2)	(17.9 ± 1.0)	(9560 ± 90)	(47.2 ± 2.0)	(10.9 ± 0.3)
b-Caryophyllene	0.22	1.03	0.38	∼0	∼0
	(25.5 ± 0.9)	(32.8 ± 1.2)	(899 ± 49)	(∼0)	(∼0)
Eugenol acetate	0.082	0.57	1.58	∼0	0.14
	(9.43 ± 0.48)	(18.1 ± 0.6)	(3740 ± 110)	(∼0)	(3.94 ± 0.23)
Troloxc	1	1	1	1	1
	(115 ± 3)	(31.9 ± 3.3)	(2360 ± 70)	(83.9 ± 2.3)	(28.3 ± 1.3)

^a  The relative scavenging rate constant to that of trolox (TEU = k_AOx/k_trolox).

^b  The HO· scavenging ability in an acetonitrile-water mixture (3:7, v/v).

^c  Cited from Sueishi et al., 2019.

Figure 1b shows a radar chart for the antioxidant abilities of clove extract (company A), together with the MULTIS values of eugenol. In this chart, the HO· scavenging ability of eugenol is adjusted to 1.0 (= HO· scavenging ability of clove extract). It is noteworthy that the scavenging ability profile of eugenol is similar to that of the clove extract sample. This suggests that the antioxidant capacity of clove as a food is comparable to the eugenol-like antioxidant profile, although some other antioxidant components have been reported in the food analysis of clove (Dorman et al., 2000; Chaieb et al., 2007).

Mechanism of singlet oxygen scavenging    Aromatic antioxidant compounds such as flavonoids are known to have effective antioxidant activities for ¹O₂ scavenging. The ¹O₂-scavenging TEU values determined for various aromatic antioxidants containing eugenol in a mixture of acetonitrile and water are listed in Table 3, together with the available oxidation potential (Eox) values. As shown in Figure 2, the antioxidants having smaller Eox values showed higher TEU, suggesting that the antioxidants in Table 3 act as electron donors. This finding is in agreement with the results reported by Mukai et al. (Mukai et al., 2005). They suggested that the transition state in the ¹O₂ quenching reaction of aromatic antioxidants has the property of a charge-transfer intermediate and showed that the ¹O₂ quenching reaction proceeds by energy transfer (physical quenching).

Table 3. TEU and Eox values for ¹O₂ scavenging of various antioxidant compounds in a mixture (1:1, v/v) of acetonitrile and water at room temperature

Antioxidant	TEUa	Eox/Vb vs Ag/AgCl
Eugenol	4.05	0.52
p-Coumaric acid	3.13	0.58
Caffeic acid	7.03	0.21
Ferulic acid	3.81	0.43
Kaempferol	4.07	0.24
Rutin	4.87	0.36
Myricetin	7.25	0.12
Apigenin	1.79	0.66

^a  The relative ¹O₂ scavenging rate constant to that of trolox (TEU = k_AOx/k_trolox).

^b  Cited from Refs. Bortolomeazzi et al., 2007; Zhang et al., 2011; Hotta et al., 2002.

Fig. 2.

Relationship between ¹O₂ scavenging abilities (TEU) and the oxidation potential (Eox) of each antioxidant.

Carotenoids such as β-carotene are important biological compounds that can inactive electronically excited ¹O₂ molecules (Mascio et al. 1991). The TEU value of β-carotene for ¹O₂ scavenging can be calculated to be TEU = 29.8 using the ¹O₂ quenching rate constants reported by Mascio et al. (Mascio et al. 1991). The ¹O₂ scavenging ability of β-carotene is 7.4 times larger than that of eugenol (TEU = 4.05), suggesting that β-carotene has high antioxidant activity for ¹O₂ scavenging.

Comparison of antioxidant profiles of various antioxidants    It is of interest to determine how structural alteration of eugenol analogues influences the antioxidant ability. Antioxidant activities of phenyl-type antioxidants depend on OH groups in the phenyl ring (Sueishi et al., 2014). It is useful to compare the antioxidant profile of the clove component eugenol with those of various aromatic antioxidants having a similar structure (substituent effect of ortho-position for 4-OH group in phenyl ring). The TEU values of caffeic acid, ferulic acid, and apigenin against RO·, HO·, t-BuOO·, and O₂⁻· in a mixture of acetonitrile and water were quantified as follows: TEU (caffeic acid, ferulic acid, and apigenin) = (0.24, 0.10, and 0.64 for RO·), (1.94, 1.24, and 0.64 for HO·), (1.06, 0.15, and 0.83 for t-BuOO·), and (3.57, 0.47, and 0.24 for O₂⁻·), respectively. The TEU values of caffeic acid in a mixture of acetonitrile and water showed large variations to previous scavenging data in aqueous solution (Sueishi et al., 2018). Although it is not easy to explain the kinetic scavenging data, we consider that the difference in ROS scavenging mechanisms under experimental conditions is an important factor. Using the above TEU values for various ROS and the ¹O₂ scavenging data (Table 3) observed in a mixture of acetonitrile and water, the radar chart of the antioxidant profiles of eugenol and similar aromatic antioxidants against five ROS was obtained (Figure 3), together with the structures of four antioxidants. Although eugenol shows a similar antioxidant profile for ROS scavenging activity as those of ferulic acid and apigenin, caffeic acid has the more effective antioxidant abilities for HO·, O₂⁻·, and ¹O₂ scavenging. As suggested above, the small Eox of caffeic acid is responsible for more effective ¹O₂ scavenging ability. In the case of HO· and O₂⁻· scavenging by caffeic acid, stabilization of the phenolic radicals produced by the ortho-dihydroxyl groups leads to enhanced antioxidant activities in the antioxidant reactions, which is consistent with the antioxidant activities of resveratrol analogs (Rossi et al., 2008). The antioxidant ability was interpreted based on the effects of a substituent that modifies the original eugenol structure. The MULTIS value is useful information for the development of functional foods.

Fig. 3.

Radar chart showing the antioxidant profiles of antioxidant compound eugenol and three other aromatic antioxidants, together with the structures of four antioxidants.

Conclusion

The ROS scavenging abilities of clove extract and its antioxidant components were determined by the ESR spin-trapping (MULTIS) method. The MULTIS measurements showed that the antioxidant capacity of clove extract is comparable to the eugenol-like antioxidant profile, and eugenol, an antioxidant component in clove, has effective scavenging ability against singlet oxygen. The ¹O₂ scavenging mechanism of eugenol was revealed on the basis of the relationship between the scavenging ability (TEU) and Eox of each antioxidant. The MULTIS method is shown to be useful for studying the comparative antioxidant activities of foods.

Acknowledgements    We thank Dr. Yashige Kotake (RRINC-USA) for the helpful discussion.

References

•  Bortolomeazzi,  R.,  Sebastianutto,  N.,  Toniolo,  R., and  Pizzariello,  A. (2007). Comparative evaluation of the antioxidant capacity of smoke flavouring phenols by crocin bleaching inhibition, DPPH radical scavenging and oxidation potential. Food Chem., 100, 1481-1489.
•  Chaieb,  K.,  Hajlaoui,  H.,  Zmantar,  T.,  Kahla-Nakbi,  A.B.,  Rouabhia,  M.,  Mahdouani,  K., and  Bakhrouf,  A. (2007). The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): a short review. Phytother. Res., 21, 501-506.
•  Dorman,  H.J.D.,  Figueredo,  A.C.,  Barroso,  J.G., and  Deans,  S.G. (2000). In vitro evaluation of antioxidant activity of essential oils and their components. Flavour Fragr. J., 15, 12-16.
•  Ghadermazi,  R.,  Keramat,  J., and  Goli,  S.A.H. (2017). Antioxidant activity of clove (Eugenia caryophyllata Thunb), oregano (Oringanum vulgare L) and sage (Salvia officinalis L) essential oils in various model systems. Int. Food Res. J., 24, 1628-1635.
•  He,  F.,  Chen,  J.,  Dong,  K.,  Leng,  Y.,  Xu,  J.,  Hu,  P.,  Yao,  Y.,  Xiong,  J., and  Pei,  X. (2018). Multi-technical analysis on the antioxidative capacity and total phenol contents of 94 traditional Chinese dietary medicinal herbs. Food Sci. Nutr., 6, 1358-1369.
•  Hotta,  H.,  Nagano,  S.,  Ueda,  M.,  Tsujino,  Y.,  Koyama,  J., and  Osakai,  T. (2002). Higher radical scavenging activities of polyphenolic antioxidants can be ascribed to chemical reactions following their oxidation. Biochim. Biophys. Acta, 1572, 123-132.
•  Iuga,  C.,  Alvarez-Idaboy,  J.R., and  Russo,  N. (2012). Antioxidant activity of trans-resveratrol toward hydroxyl and hydroperoxyl radicals: a quantum chemical and computational kinetics study. J. Org. Chem., 77, 3868-3877.
•  Kasai,  H.,  Shirao,  M., and  Ikegami-Kawai,  M. (2016). Analysis of volatile compounds of clove (syzygium aromaticum) buds as influenced by growth phase and investigation of antioxidant activity of clove extracts. Flavour Fragr. J., 31, 178-184.
•  Kohri,  S.,  Fujii,  H.,  Oowada,  S.,  Endo,  N.,  Sueishi,  Y.,  Kusakabe,  M.,  Shimmei,  M., and  Kotake,  Y. (2009). An oxygen radical absorbance capacity-like assay that directly quantifies the antioxidant's scavenging capacity against AAPH-derived free radicals. Anal. Biochem., 386, 167-171.
•  Man,  Y.B.C. and  Jaswir,  I. (2000). Effect of rosemary and sage extracts on frying performance of refined, bleached and deodorized (RBD) palm olein during deep-fat frying. Food Chem., 69, 301-307.
•  Mascio,  P.D.,  Murphy,  M.E., and  Sies,  H. (1991). Antioxidant defense systems: the role of carotenoids, tocopherols, and thiols. Am. J. Clin. Nutr., 53, 194S-200S
•  Mukai,  K.,  Nagai,  S., and  Ohara,  K. (2005). Kinetic study of the quenching reaction of singlet oxygen by tea catechins in ethanol solution. Free Rad. Biol. Med., 39, 752-761.
•  Oowada,  S.,  Endo,  N.,  Kameya,  H.,  Shimmei,  M., and  Kotake,  Y. (2012). Multiple free-radical scavenging capacity in serum. J. Clin. Biochem. Nutr., 51, 117-121.
•  Perez-Roses,  R.,  Risco,  E.,  Vila,  R.,  Penalver,  P., and  Canigueral,  S. (2016). Biological and nonbiological antioxidant activity of some essential oils. J. Agric. Food Chem., 64, 4716-4724.
•  Prior,  R.L.,  Hoang,  H.,  Gu,  L.,  Wu,  X.,  Bacchiocca,  M.,  Howard,  L.,  Hampsh-Woodill,  M.,  Huang,  D.  Ou,  B., and  Jacob,  R. (2003). Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORACFL)) of plasma and other biological and food samples. J. Agric. Food Chem., 51, 3273-3279.
•  Rossi,  M.,  Caruso,  F.,  Opazo,  C., and  Salciccioli,  J. (2008). Crystal and molecular structure of piceatannol; scavenging features of resveratrol and piceatannol on hydroxyl and peroxyl radicals and docking with transthyretin. J. Agric. Food Chem., 56, 10557-10566.
•  Sueishi,  Y.,  Hori,  M.,  Ishikawa.  M.,  Matsu-ura,  K.,  Kamogawa,  E.,  Honda,  Y.,  Kita,  M., and  Ohara,  K. (2014). Scavenging rate constants of hydrophilic antioxidants against multiple reactive oxygen species. J. Clin. Biochem. Nutr., 54, 67-74.
•  Sueishi,  Y.,  Sue,  M., and  Masamoto,  H. (2018). Seasonal variations of oxygen radical scavenging ability in rosemary leaf extract. Food Chem., 245, 270-274.
•  Sueishi,  Y.,  Masamoto,  H., and  Kotake,  Y. (2019). Heat treatments of ginger root modify but not diminish its antioxidant activity as measured with multiple free radical scavenging (MULTIS) method. J. Clin. Biochem. Nutr., 64, 143-147.
•  Zhang,  D.,  Xie,  L.,  Jia,  G.,  Cai,  S.,  Ji,  B.,  Liu,  Y.,  Wu,  W.,  Zhou,  F.,  Wang,  A.,  Chu,  L.,  Wei,  Y.,  Liu,  J., and  Gao,  F. (2011). Comparative study on antioxidant capacity of flavonoids and their inhibitory effects on oleic acid-induced hepatic steatosis in vitro. Euro. J. Med. Chem., 46, 4548-4558.