Components, Antioxidant and Antibacterial Activity of Tomato Seed Oil

Abstract

In this study, chemical components, antioxidant and antibacterial activity of tomato seed oil were investigated. GC-MS analysis of tomato seed oil showed 87 constituents representing 95.20% of the oil, and the major components cycloeucalenol (25.67%), oleic acid (16.70%), linoleic acid (7.85%), palmitic acid (6.08%) and octadecanoic acid (5.78%) constituted 62.08% of it. Antioxidant activity of the tomato seed oil was characterized by Hydroxyl Radical Scavenging Activity (HRSA) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH). In the HRSA test, tomato seed oil (EC₅₀ = 2.76 ± 0.16 µg/mL) showed better antioxidant activity than that of butylated hydroxytolune (BHT) (EC₅₀ = 436.70 ± 0.02 µg/mL). But in DPPH assay, the tomato seed oil (IC₅₀ = 1590.01 ± 0.01 µg/mL) exhibited relatively weaker antioxidant activity than that of BHT (IC₅₀ = 69.01 ± 0.02 µg/mL). Finally, the tomato oil exhibited moderate to potent, broad-spectrum antibacterial activities against seven clinically significant bacterial strains.

Introduction

Recently, there is an increased interest in the natural sourced matter, such as polyphenols, flavonoids, vitamin C and vitamin E, ubiquitous in fruits, teas, vegetables, cereals, and medicinal plants, for antioxidant and antibacterial compounds use (Hseu et al., 2008; Hussain et al., 2008; Ozturk and Ercisli, 2007; Zhou and Elias, 2013). In general, such natural matter is highly effective free radical scavenger and they are way less toxic than synthetic antioxidants (Barana et al., 2012; Pokorný, 2007; Zhu et al., 2011; Zou et al., 2004), such as BHA, BHT, propyl gallate and citric acid used in the food industry. These additives can cause nasty side effects in the food processing. (Kapoor et al., 2009). Based on these facts, the plant kingdom with a variety of natural compounds has attracted a lot of attention. Finding natural plant materials with both antioxidation and antibacterial effects becomes an ideal goal in the research field of food additives research (Ebrahimabadi et al., 2010).

In recent decades, the consumption of tomatoes and tomato products has been suggested to be able to reduce the risk of certain chronic diseases by preventing doxorubicin-induced cardiac myocyte oxidative DNA damage, reduce the levels of serum prostatespecific antigen, and positively modulating other disease (Agarwal and Rao, 1998; Ferreira et al., 2007; Van Breemen et al., 2011; Walfisch et al., 2003). These are primary due to the existence of a variety of antioxidants in tomoato (García-Alonso et al., 2009), including fatty acid, alcohol, tocopherol and phenolic compounds, (Martínez-Valverde et al., 2002).

Tomato seed oil is considered a good source of an edible oil and one of the major food ingredients across the world. It can be extracted from tomato seed, which is the major by-product of the tomato paste manufacturing industry. It is considered to be a good source of edible oil and one of the major food ingredients across the world (Evangelos et al., 1998). In order to make full use of tomato seed oil and to improve its economic value, it is very important to perform a full analysis of the oil. Giannelos et al. (2005) determined linoleic acid, oleic acid and other 16 organic acids using GC-FID by direct injection. Eller et al. (2010) determined 14 phytosterols using GC-MS after Trimethylsilyl (TMS) derivatization. However, their analysis only determined partial components of the oil since most of the volatile compounds could not be analyzed directly usig GC or GC-MS. To overcome this problem, a suitable derivatization reagent should be chosen to increase the number of compounds that can be analyzed by GC. In recent years, N,O-bistrimethylsilyl- fluoroacetamide (BSTFA) with 1% of trimethylchlorosilane (TMCS) has been widely applied for its good thermal stability and volatility, easy preparation, and a wide range of derived objects (Angerosa et al., 1995; Schummer et al., 2009).

In this study, BSTFA was used as derivative to investigate the components of the tomato seed oil with GC-MS. The antioxidant and antibacterial activities of the tomato seed oil were also investigated. The obtained data will allow the future application of the most promising tomato seed oil in food systems associating two activities that will contribute enormously to improve food safety and quality.

Materials and Methods

Materials    N,O-bistrimethylsilyl-fluoroacetamide (BSTFA) was obtained from Aladdin Reagent Co., Ltd (Shanghai, China). Trimethylchlorosilane (TMCS) and methoxyammonium chloride were purchased from Alfa Aesar Chemical Co. (Zhengzhou, China). 2,2-diphenyl-1-picrylhydrazyl (DPPH) and BHT were obtained from J&K Chemical Ltd. (Beijing, China). Folin-Ciocalteu's phenol reagent was purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd. (Tianjin, China). Other chemicals such as ethanol, acetone, H₂O₂, disodium hydrogen phosphate, sodium dihydrogen phosphate and so on were all purchased from Beijing Chemical Reagent Co. (Beijing, China). All reagents were used as received without further treatment.

Tomato seed oil was gifted from Xinjiang Tomato Technology & Development Co., Ltd. (Xinjiang, China).

Chromatographic analysis    The chemical composition of tomato seed oil was determined according Eller's report with slight modification (Eller et al., 2010). TMCS derivatives of the tomato seed oil was made as following: 20 mg the oil, 100 µL each pyridine and BSTFA with 1% TMCS were mixed, and reacted at 37°C for 30 min, then the reaction temperature increased at 5°C/min to 65°C to guarantee the reaction completely. After cooling to room temperature, 20 µL the oil were diluted to 2 mL with acetone, a 1 µL derivatized solution was injected into GC-MS (QP2010 Ultra, Shimadzu, Japan) equipped with an Rxi-5Sil MS fused silica capillary column (30 m × 0.25 mm × 0.25 µm). Splitless mode was used. The injector and ion source temperature were both 300°C. Electron energy was 70 ev. Column initial temperature was set at 80°C for 3 min, then increased at 5°C/min to 255°C, held for 5 min, finally ramped at 2°C/min to 320°C, where it was held for another 15 min. Helium was used as the carrier gas with a flow rate of 1.0 mL/min. Data was collected in the full scan mode (m/z 50 - 800). The identity of the components was assigned by matching their spectral data with those detailed in the Wiley 229.L, Wiley 7.L and NIST 08.L libraries. The relative percentage of the oil constituents was expressed as percentage by peak area normalization, without using correction factors.

Antioxidant activity    Antioxidant activity of tomato seed oil was screened using both DPPH free radical scavenging and hydroxyl radical (OH·) scavenging methods..

(1) DPPH radical scavenging assay

The DPPH assay usually involves hydrogen atom transfer reaction, but, based on kinetic data, an electron transfer mechanism has also been suggested for this assay (Li et al., 2008). Briefly, the tomato seed oil (concentrations ranging from 0.1 to 8 mg/mL) and the synthetic standard antioxidantBHT, ranging from 10⁻⁴ to 1 mg/mL, were prepared in ethanol. Each concentration level of tomato oil (or BHT) was mixed with 0.05 mg/mL DPPH (2 mL each) ethanol solution. Mixtures were shaken vigorously and incubated under room temperature for 30 min in the dark. Absorbance measurements were read at 517 nm under room temperature. A blank sample containing the same amount of ethanol and DPPH solution was used as negative control. All tests were performed in triplicates and average of the values used to calculate I%. Inhibition of free radical DPPH in percent (I%) was calculated as follow:

  

Where A_blank is the absorbance of the control reaction, and A_sample is the absorbance of the test compound. The sample concentration providing 50% inhibition (IC₅₀) was calculated by plotting inhibition percentages against concentrations of the sample.

(2) Hydroxyl radical scavenging assay

The scavenging activity of tomato seed oil on OH· was measured using a Fenton reaction with a few modifications (Jin et al., 1996). OH· could oxidize Fe²⁺ into Fe³⁺, and only Fe²⁺ could combine with 1,10-phenanthroline to form a red colored complex with the maximum absorbance at 536 nm. The concentration of hydroxyl radical was determined by the degree of decolourization of the reaction solution. The reaction mixture contained 1 mL of 0.75 M 1,10-phenanthroline, 2 mL of 0.2 M phosphate buffer (pH = 7.4), 1 mL of 0.75 mM FeSO₄·7H₂O, 1 mL H₂O₂ (0.01% v/v), and 1 mL tomato oil sample (concentrations ranging from 0.01 to 200 µg/mL) or BHT (as a positive control, ranging from 1 to 500 µg/mL), and incubated at 37°C for 60 min in a water bath, the absorbance of reaction mixture was measured at 536 nm against reagent blank.

  

Where A_s, A_n, and A_b were the absorbance values determined at 536 nm of the sample or BHT, the negative control, and the blank after reaction, respectively.

Antibacterial activity (disc diffusion assay)    For the disc diffusion assay (Bubonja-Sonje et al., 2011), the tomato seed oil was dissolved in acetone (concentrations ranging from 100 to 5000 µg/mL). A filter paper disk (Whatman No. 1, 6 mm diameter) containing 20 µL of each concentration of the oil and acetone (as negative control) were placed on the agar surface. Disks of roxithromycin (30 µg/mL) and chloramphenicol ophthalmic solution (6 µL/disck) were used as positive control. The plates were incubated overnight at 37°C, and the diameter of any resulting zones of growth inhibition was measured. Results were expressed as the percentage of inhibition growth compared with the roxithromycin (30 µg/mL) and chloramphenicol ophthalmic solution (6 µL/disc).

Statistical Analysis    he obtained antioxidant and antibacterial results were stated in mean ± standard deviation. Analysis of variance for individual parameters was performed on the basis of mean values to determine the significance at p < 0.05.

Results and Discussion

Chemical components of the tomato seed oil    GC-MS analyses of the tomato oil led to the identification of 87 compounds (see Fig. 1), representing 95.20% of the total oil. The identified compounds (peak area ≥ 0.10%) are listed in Table 1 in the order of their elution from an Rxi-5Sil MS column. As shown in Table 1, the major compounds detected in the oil were cycloeucalenol (25.67%), oleic acid (16.70%), linoleic acid (7.85%), palmitic acid (6.08%), and octadecanoic acid (5.78%), which mainly belong to alcohol, acid, phenol and some other essential phytochemicals.

Fig. 1.

Typical chromatogram of a GC-MS analysis of tomato seed oil.

Table 1. Main components (%) of the tomato seed oil

No.	Compounda	Composition (%)	SIb	No.	Compounda	Composition (%)	SIb
1	cycloeucalenol	25.67	75	27	myristic	0.30	81
2	oleic acid	16.70	96	28	squalene	0.30	96
3	linoleic acid	7.85	89	29	stearaldehyde	0.20	94
4	5.alpha-cholest-8-en-3.beta-ol, 14-methyl-, acetate	6.20	69	30	9-octadecenoic acid methyl ester	0.20	82
5	palmitic acid	6.08	93	31	4,15-octadecadienyl acetate	0.20	84
6	octadecanoic acid	5.78	93	32	cycloartenol	0.20	79
7	octadecanoic acid, 2,3-hydroxy propyl ester	3.50	91	33	octadecyl aldehyde	0.20	87
8	2-methyl-prop-2-enyloxy	2.40	73	34	oleic acid methyl ester	0.18	92
9	olealdehyde	2.29	94	35	9,17-octadecadienal	0.17	94
10	1-monopalmitin	2.20	80	36	phenol	0.16	94
11	1,3-dipalmitin	2.10	75	37	heneicosane	0.15	92
12	triarachidin	1.30	75	38	2-hydroxycyclopentadecanone	0.14	76
13	cis-9-hexadecenal	1.20	89	39	ambrettolid	0.14	75
14	trilinolein	1.20	88	40	erucyl amide	0.13	86
15	pseudoephedrine	1.16	83	41	oleamide	0.13	86
16	linoleoyl chloride	0.75	87	42	9-octadecenoic acid	0.12	82
17	glycerol	0.70	63	43	hexadecanoic acid, methyl ester	0.11	95
18	sitosterol	0.60	83	44	1,11,13-octadecatriene	0.11	82
19	glyceryl linoleate	0.54	90	45	heptadecanoic acid	0.11	83
20	pentadecanoic acid	0.51	88	46	pentatriacontane	0.11	71
21	cycloartanyl acetate	0.50	78	47	9,12-octadecadienoic acid, methyl ester	0.10	94
22	tocopherol	0.50	81	48	stigmasterol	0.10	92
23	tetrapentacontane	0.40	93	49	tetradecane	0.10	75
24	tetradecanoyl chloride	0.40	81	50	cholestadiene	0.10	90
25	oleoyl chloride	0.31	91	51	heptadecane	0.10	89
26	9,12-octadecadienyloxyethanol	0.30	87	52	lanosteryl tosylate	0.10	62

^aCompounds listed in order of elution from Rxi-5Sil MS column; ^bSI, Similarity;

Percentages are the means of three runs and were obtained from electronic integration measurements using selective mass detector.

Among 87 identified compounds, only 8 of them have been reported, which are oleic acid, palmitic acid, heptadecanoic acid, octadecanoic acid, linoleic acid, 9-octadecenoic acid (Giannelos et al., 2005), stigmasterol and cycloartenol (Eller et al., 2010). The presences of phenolic acids, polyphenols and flavonoids were firstly discovered, these compounds play very important in their antioxidant activities and antibacterial properties.

Antioxidant activity    Due to the complex reactive facets of phytochemicals in general and tomato seed oil in particular, it has been recommended that the antioxidant activities of the oil must be tested by at least two systems to establish authenticity (Tenore et al., 2011). For this reason, the antioxidant activity of tomato seed oil was demonstrated by two spectrophotometric methods: DPPH and HRSA. In this study, the ability of tomato oil sample to scavenge DPPH radical and OH· radical were determined on the base of its concentration providing 50% inhibition (IC₅₀ and EC₅₀). The IC₅₀ or EC₅₀ value was defined as the concentration of the sample necessary to cause 50% inhibition, which was obtained by interpolation from linear regression analysis (Yang et al., 2010). A lower IC₅₀ or EC₅₀ value is associated with a higher radical scavenging activity.

DPPH radical scavenging activity    DPPH radical scavenging assay is widely used to test the ability of compounds to act as free radical scavengers or hydrogen donors, and to evaluate antioxidant activity of foods (Bubonja-Sonje et al., 2011). DPPH is a stable organic free radical with an absorption wavelength at 517 nm, which can readily experience reduction in the presence of an antioxidant. It loses this absorption on accepting an electron or a free radical species, which results in a visually noticeable discolouration from purple to yellow. It can accommodate many samples in a short period and is sensitive enough to detect active ingredients at low a concentrations (Hseu et al., 2008; Zhu et al., 2011).

Here, DPPH radical was firstly chosen to investigate the scavenging activity of tomato seed oil, and the results were illustrated in Table 2. The inhibition value (IC₅₀) observed for the oil was significantly higher than that of BHT due to a low amount of phenolic components in the oil (Salleh et al., 2011). Earlier study has shown that kenaf seed oil might actually possess better antiradical activity if compared with some commercial edible oils (canola oil, corn oil and so on) and it can potentially serve as high antioxidative edible oil. Compared with this study, the IC₅₀ value for tomato seed oil was 1590 µg/mL, only about 13% of that of kenaf seed oil, 12270 µg/mL (Chan and Ismail, 2009). This result showed that the tomato seed oil might actually possess better antiradical activity if compared with kenaf seed oil and it can potentially serve as a high antioxidative edible oil.

Table 2. Antioxidant activity of the tomato seed oil and BHT in DPPH free radical scavenging activity and HRSA assay methods.

Sample	DPPH IC50 (µg/mL)	HRST EC50 (µg/mL)
Tomato seed oil	1590.01 ± 0.01	2.76 ± 0.16
BHT	69.01 ± 0.02	436.70 ± 0.02

Data represent mean ± standard deviation of three independent experiments.

Hydroxyl radical scavenging activity    HRSA method was also applied to determine antioxidant effect of the oil. The hydroxyl radical is one of representative reactive oxygen species generated in the body. In the HRSA assay, the ability of sample to scavenge hydroxyl radical was determined on the base of its concentration providing 50% inhibition (EC₅₀). EC₅₀ values of the oil and positive control (BHT) are listed in Table 2. The inhibition value (EC₅₀) observed for the oil was significantly lower than that of BHT (p < 0.05). The tomato seed oil had the low DPPH radical scavenging activity (IC₅₀ = 1590.01 ± 0.10 µg/mL) while showed the strongest HRSA activity (EC₅₀ = 2.76 ± 0.16 µg/mL). The difference in the HRSA and DPPH assays for sample might be due to the difference in the size of radicals or in the accessibility of antioxidants to the radical Centre (Joubert et al., 2004).

Based on above experimental results, it can be concluded that the tomato seed oil may possess highest antiradical activity among commercial edible oils, which is probably due to the higher level of contained unsaturated fatty acids (such as linoleic acid) and other antioxidant contents, such as cycloeucalenol (25.67%), tocopherol (0.48%) and sitosterol (0.60%)(Fang et al., 2005).

Antibacterial activity    Disc diffusion test may be used as a preliminary screen for susceptibility testing, and this method is generally more sensitive and allows quantitative determination of oil antibacterial efficiencies. The tomato seed oil (20 µL per disc) was tested for its putative antibacterial activity against seven model bacteria, including Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Acinetobacter baumannii (A. baumannii), Staphylococcus epidermidis (S. epidermidis), Enterococcus faecalis (E. faecalis), Shigella flexneri (S. flexneri), and Proteus mirabilis (P. mirabilis) at a various concentration ranging from 0.1 to 5 mg/mL. The results are summarized in Table 3, it can be seen that the oil had no considerable antibacterial activity against A. baumannii, S. epidermidis and E. faecalis at tested concentrations; but showed medium antibacterial activities to S. aureus, E. coli, S. flexneri and P. mirabilis, the higher concentration, the more antibacterial efficiencies attained.

Table 3. Antibacterial activity of the tomato seed oil.

	Inhibition zone (mm)						Chloramphenicol	Roxithromycin
organisms	acetone	2 µg/disc	10 µg/disc	20 µg/disc	50 µg/disc	100 µg/disc	6 µL/disc	30 µg/disc
S. aureus	10.1 ± 1.5	10.6 ± 0.5	11.2 ± 1.9	12.2 ± 2.2	12.5 ± 2.7	10.8 ± 1.7	21.1 ± 1.0	12.2 ± 1.5
E. coli	10.2 ± 0.8	10.2 ± 1.4	12.2 ± 1.6	15.7 ± 0.5	10.5 ± 0.0	11.4 ± 1.4	15.4 ± 0.5	12.1 ± 0.1
A baumannii	12.8 ± 1.0	12.8 ± 1.0	12.8 ± 1.0	12.8 ± 1.0	12.8 ± 1.0	12.8 ± 1.0	12.8 ± 1.0	12.8 ± 1.0
S. epidermidis	9.9 ± 0.3	9.9 ± 0.3	9.9 ± 0.3	9.9 ± 0.3	9.9 ± 0.3	9.9 ± 0.3	9.9 ± 0.3	9.9 ± 0.3
E. faecalis	9.7 ± 0.9	9.7 ± 0.9	9.7 ± 0.9	9.7 ± 0.9	9.7 ± 0.9	9.7 ± 0.9	17.4 ± 0.9	9.7 ± 0.9
S. flexneri	10.0 ± 0.7	12.9 ± 1.3	13.1 ± 4.8	13.5 ± 2.2	15.8 ± 0.9	17.0 ± 2.0	20.7 ± 4.3	17.3 ± 7.3
P. mirabilis	6.0 ± 0.0	9.6 ± 0.3	9.8 ± 0.0	10.4 ± 0.4	11.0 ± 0.0	12.0 ± 0.0	6.0 ± 0.0	7.1 ± 0.0

Data represent mean ± standard deviation of three independent experiments.

Standard antibiotic gentamycins, Chloramph- enicol and Roxithromycin, were used as control samples. In tested condition, just like the tomato seed oil, both standard antibiotic gentamycins showed no antibacterial activities to A. baumannii, S. epidermidis. Compared with no antibacterial activities using the oil and Roxithromycin, Chloramphenicol showed antibacterial activities to E. faecalis. Both standard antibiotic gentamycins showed little antibacterial activities to P. mirabilis, in contrast, the tomato seed oil showed a moderate antibacterial activity. To the other four bacteria, the antibacterial activities of the oil were less than those of control samples. Taken together, the tomato seed oil showed varied levels of antibacterial activity against four of seven tested bacteria. These may be caused by complex components in the oil, as some of which, i.e. polyphenols, flavonoids, and vitamin E, possess antibacterial activity (Hussain et al., 2008).

Conclusions

In conclusion, GC-MS analyses of the tomato seed oil led to the identification of 87 compounds, representing 95.20% of the total oil. The major components were found to be cycloeucalenol (25.67%), oleic acid (16.70%), linoleic acid (7.85%), palmitic acid (6.08%) and octadecanoic acid (5.78%). The tomato seed oil showed high antioxidant activities with a of EC₅₀ of 2.76 ± 0.16 µg/mL and IC₅₀ of 1590.01 ± 0.01 µg/mL. Compared with Chloramphenicol and Roxithromycin, the tomato seed oil showed moderately antibacterial activity, which suggested that the tomato seed oil exhibited a potent broad spectrum antimicrobial activity. Based on above results, tomato seed oils display remarkable biological activities such as antioxidant and antibacterial properties which are useful for preserving foods from decay and contamination and/or preventing living tissues from various diseases. These results encourage complementary and more in-depth studies on the antioxidant and antibacterial properties of chemical composition in the tomato seed oil.

Acknowledgements    This work was funded under the Technology Support Project of Xinjiang (201191170).

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