Oxidative Stability of Cooking Oil Blend Stabilized with Leaf Extract of Eucalyptus citriodora

Abstract

The present study was conducted to assess the effects of Eucalyptus citriodora (E. citriodora) leaf extract on the oxidative stability of blend of canola, rapeseed, and sunflower oils (45:20:35 v/v, respectively) under accelerated storage. The blended oil was stabilized with 300 mg/L ethanolic extract (source of total phenolic content and total flavonoid content [5.23 ± 0.19 and 1.18 ± 0.04 g/100 g d.wt. of extract]) of E. citriodora leaves. The oxidative stability was measured on the basis of parameters such as free fatty acid contents, peroxide value, sponification value, iodine value, color, cloud point, and refractive index. After a 100 day incubation period, the increase in refractive index, free fatty acid, and peroxide, and sponification values in stabilized and non-stabilized oil blends were 0.0028 and 0.0047, 0.20 and 2.37% as oleic acid, 12.54 and 21.12 meq/kg of oil and 10.04 and 17.01 meq of KOH/g of oil, respectively, as compared with initial values. However, a decrease of 10.0 and 16.9 g of iodine/100 g of oil was recorded in oil iodine values of both stabilized and non-stabilized blended vegetable oils. Results showed that E. citriodora leaf extract was found effective to maintain the oxidative stability of blended vegetable oils for long duration (6 months) as compared with control oil samples. Therefore, it can be concluded that E. citriodora leaf extract is a cheap rich source of natural antioxidants that can be easily used for the stabilization of vegetable oils in the food processing industries.

Keywords:

• Cooking oil
• Oxidative stability
• E. citriodora
• Peroxide value
• Free fatty acids

Introduction

Vegetable oils (VOs) are recognized as an important source of essential fatty acids and the precursors of important hormones, such as prostaglandins that control many physiological factors like blood pressure, cholesterol level, and the reproductive system.^[1,2] However, the high temperature and prolonged storage processing of oils and fats lead to rancidity and defective nutrition due to lipid peroxidation (LP).^[3] LP in fats and fatty foods not only deteriorates their quality and brings about chemical spoilage, but also generates free radicals and reactive oxygen species (ROS), implicated in carcinogenesis, mutagenesis, aging, inflammation, and cardiovascular diseases and also are the exacerbating factors in cellular injury and the aging process.^[3,4] Oxidative reactions limit the shelf life of fresh and processed lipids, which is a serious concern in the VO and fat industry.^[1] A variety of antioxidants occur naturally in foods including VOs that are not only essential for our health, but also important for maintaining the quality of food under long-term storage.^[5–7] Normally the concentration of these antioxidative compounds in VOs is not enough for their long-term storage especially during food manufacturing. So they require the addition of antioxidants externally to prolong their shelf life, make them look better, and stop them from spoiling. In this regard synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and ter-butyl hydroquinone (TBHQ) are commonly used to improve the shelf life and the oxidative stability of lipids and lipid containing foods. However, in most food products it is discouraged because of their toxicity and carcinogenicity.^[8,9] Natural antioxidants, including plant extracts, have attracted great attention from consumers all over the world as an alternate source of synthetic antioxidants^[10–15] and a lot of research performed on such plants has lead to the development of some commercial products containing natural antioxidants derived from various plant parts.^[16–18] In some earlier studies, efforts have been made to reduce the oxidation of VOs during storage by using products obtained from different plants.^[19,20]

E. citriodora has been traditionally used in medicines and insecticides in Africa and some other countries. Chemically it is rich in oxygenated compounds (mono terpenoids and phenolic compounds) including α-Pinene, 6-Methyl-5-hepten-2-one, Sabinene, β-Pinene, Myrcene, and p-Cymene. E. citriodora has citronellal (40.0%), isopulegol (14.6%), and citronellol (13%) as major components.^[21] E. citriodora plants are present on large scale in different agro-climatic regions of the world, as well as in Pakistan, and their leaves serve as huge amount of agricultural waste having no potential use. The high polyphenolic (natural antioxidants) content of E. citriodora encouraged us to use it for the stabilization of VO blends of canola, rapeseed, and sunflower oils. These VOs are rich source of essential fatty acids with high unsaturation and are commonly used in the oil industries for their commercial use. The blend of these VOs is also preferentially used by the consumers due to their easy availability in the market and better nutritional quality as compared with others. To the best of our knowledge, not a single report is available in literature on the oxidative stability of cooking oil blend of canola, rapeseed and sunflower oils by the leaf extract of E. citriodora. Therefore, the present study was conducted to study the oxidative stability of blended VO stabilized with E. citriodora leaf extract.

Materials and methods

Preparation of Leaf Extract and Oil Pre-Treatment

The leaves of E. citriodora were collected from University of Agriculture Faisalabad, Pakistan. The leaves were washed thoroughly with tap water to remove any wastes and dust particles. The leaves were then dried in the open air under shade for 4 weeks until the constant weight was achieved. The leaf extract was prepared following the method reported by Iqbal et al.^[22] The extracts were filtered (using pure ethanol) and concentrated using vacuum rotary evaporator (Heidloph, Unimax 2010, Germany) at reduced pressure and element temperature (40°C). The dried extract was weighed and stored in a refrigerator in air tight vials. The samples of VOs were obtained from the United Industries Limited, Kashmir Road, Faisalabad, Pakistan and stored in 1.5 L translucent polyethylene terephthalate (PET) bottles at 4°C after purging with 99.99% pure nitrogen. The blend of canola, rapeseed, and sunflower oils was prepared with 45:20:35 ratios, respectively. The reason for the use of the previously mentioned oil blend is due to its easy availability in the local market and wide use by the consumers as cooking oil with acceptable unsaturation.

Evaluation of Antioxidant Activity of the Extract of E. citriodora Leaves

The antioxidant activity of solvent extracted E. citriodora leaves extract was evaluated by some in vitro assays like total phenolic content (TPC) and total flavonoid content (TFC). TPC was determined following the method described by Chaovanalikit and Wrolstad^[23] and the TFC was estimated by the procedure reported by Dewanto et al.^[24] The scavenging of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical and induced percent inhibition of linoleic acid peroxidation was performed following the methods described by Bozin et al.^[25] and Iqbal and Bhanger,^[26] respectively.

Stabilization of Blended VOs

The contracted ethanolic extract of E. citriodora leaves was added (300 mg/L) to pre-heated cooking oil blend. The mixture was stirred for 30 min at 100°C for uniform dispersion. A control (without addition of extract) was also prepared for the same oil blend under same conditions.

Heat Treatment of VO Samples

All oil samples (stabilized and non-stabilized) were separately filled in 60 mL amber-colored glass vials, closed immediately with rubber seals and aluminium stoppers, and marked accordingly. All vials were heated in an oven at 65°C for 100 days continuously for a period of 8 h/d following Sultana et al.^[27] The rest of the period (16 h) the samples were kept in dark under ambient environmental conditions (30 ± 2ºC). The oxidative stability parameters were studied after every 10 days periodically. This kind of treatment will be helpful for industrialists and whole sale dealers, who intentionally keep their oils and fats (packed in tin containers, PET bottles, and PET bags, etc.) outside their shops, thus directly exposing them to sunlight, for the purposes of advertising or due to insufficient space in their shops.

Evaluation of Oxidative Stability Parameter of Oil Samples

Determination of color, refractive index (RI), and cloud point (CP)

The color of the oil samples (treated and non-treated) was measured according to the method reported by Anwar et al.^[19] using a Livibond Tintometer (Livibond Ltd., Salisbury, Wiltshire, UK). The RI of the oil samples was estimated by using refractometer (70,000 CX, ATAGO, and Japan). A sample of 0.2 mL was loaded to refractometer stage and the reading was noted after 5 s on a digital scale. Before loading the sample, the stage was wiped out with a piece of tissue soaked in methanol for an accurate measurement of RI. The CP of oil samples was measured below 0°C by taking 25–50 mL of oil samples in 50 mL beaker with a thermometer. The beaker containing oil sample was allowed to cool down slowly and note the temperature at which the whitish appearance started

Determination of free fatty acids (FFA), peroxide value (PV), iodine value (IV), and sponification value (SV)

Determination of FFA, PV, and IV for both treated and non-treated oil samples was made following the American Oil Chemist’s Society (AOCS) official methods, F 9a-44, Cd 8-53, and Cd 1-25, respectively.^[28] The SV of oil samples was determined following International Union of Pure and Applied Chemistry (IUPAC) method.^[29]

Statistical Analysis

All the experiments were conducted in completely randomized design (CRD) with three replications of each treatment. The data were presented as mean ± standard error. Furthermore, the data obtained was analyzed using analysis of variance (ANOVA) and Duncan’s Multiple Range test (DMR) and correlation statistical techniques on statistical software, Costat computer package (version 6.303, PMB 302, Monterey CA, 93940 USA).

Results and discussion

In the present study, the results regarding the percent yield, total phenolics, total flavonoids, and DPPH radical scavenging activity of ethanolic extracts from E. citriodora leaves are presented in Table 1. It was found that the overall yield of E. citriodora leaves extract obtained with absolute ethanol was 10.88 ± 0.45 g/100 g of dry weight. The obtained extract was found a good source of polyphenols such as TPC (5.23 ± 0.19 g/100 g d. wt. measured as gallic acid equivalents) and TFC (1.18 ± 0.04 g/100 g d. wt. measured as catechin equivalents) that shows its high oxidative stability capacity. The results of present study regarding polyphonic content in leaves extracts are comparable with earlier studies of Rubilar et al.^[30] on murta (Ugni molinae Turcz) leaves. They also reported that murta leaves extract (rich in polyphenoles) found effective to increase the soybean oil oxidative stability and the oxidative stability potential of the extract was measured in terms of DPPH radical scavenging activity and inhibition of linoleic acid peroxidation that were ranged in 70.56 ± 1.56 (% inhibition of DPPH) and 66.66 ± 1.99 (% as linoleic acid), respectively. So, the composition and antioxidative properties of the extract determines its potential use in the oil industry for the long duration storage/stabilization of oil without oxidative deterioration

TABLE 1 E. citriodora leaf extract antioxidative properties and changes in color of vegetable oil stabilized with the leaves extract (mean ± SD)

Leaf extract properties
Yield of leaf extracts (g/100 g d. wt.)	TPC (g/100 g d. wt. as GAE)	TFC (g/100 g d. wt. as CE)	Inhibition of linoleic acid peroxidation (%)	DPPH scavenging activity (%)
10.88 ± 0.45	5.23 ± 0.186	1.18 ± 0.035	66.66 ± 1.99	70.56 ± 1.56
Induction period (days)	Color
	Sample		Control
	R-unit	Y-unit	R-unit	Y-unit
00	1.00 ± 0.01^c	10.00 ± 0.02^a	1.00 ± 0.01^c	10.00 ± 0.01^a
10	1.00 ± 0.02^c	10.00 ± 0.02^a	1.00 ± 0.02^c	10.00 ± 0.01^a
20	1.00 ± 0.01^c	10.00 ± 0.01^a	1.00 ± 0.01^c	10.00 ± 0.02^a
30	1.00 ± 0.01^c	10.00 ± 0.01^a	1.00 ± 0.02^c	10.00 ± 0.01^a
40	1.00 ± 0.01^c	10.00 ± 0.02^a	1.00 ± 0.01^c	10.00 ± 0.01^a
50	0.90 ± 0.02^a	9.00 ± 0.01^ab	0.90 ± 0.02^a	9.00 ± 0.01^ab
60	0.90 ± 0.02^a	9.00 ± 0.02^ab	0.90 ± 0.01^a	9.00 ± 0.01^ab
70	0.80 ± 0.01^a	8.00 ± 0.02^bc	0.80 ± 0.02^a	8.00 ± 0.01^bc
80	0.70 ± 0.02^ab	7.00 ± 0.01^cd	0.70 ± 0.01^ab	7.00 ± 0.02^cd
90	0.70 ± 0.01^ab	7.00 ± 0.01^cd	0.70 ± 0.02^ab	7.00 ± 0.01^cd
100	0.60 ± 0.01^b	6.00 ± 0.02^d	0.60 ± 0.01^b	6.00 ± 0.02^d
Decrease from day 0	0.4	4.0	0.4	4.0

Tables 1 and 2 shows the changes in the color, RI, and CP of VOs stabilized with E. citriodora leaves extract subjected to daily oven treatment (65°C) for 100 days. The color (red + yellow) values of the oil samples subjected to ambient storage (30 ± 2ºC in dark) decreased slowly with the passage of time. The statistical analysis of the data did not show any significant (p ≤ 0.05) differences in discoloration of blended oil samples during the preliminary period of storage (up to 40 days). However, with an increase in incubation period, the color values of the blended oil samples became significantly (p ≤ 0.05) different in comparison with fresh VOs in both stabilized and non-stabilized samples. The maximum discoloration (0.4) was recorded after 100 days incubation period. However, the difference between values of non-stabilized and stabilized oil samples was non-significant at the end of the experimental period of 100 days. No previously reported data was found for the color of blended VO; however, literature revealed that oxidative deterioration lead to the discoloration of VOs.^[31]

TABLE 2 Different physical and oxidative stability attributes of vegetable oils stabilized with E. citriodora leaves extract (mean ± SD)

	CP (°C)		RI		FFA (percent as oleic acid)		PV (meq/kg of oil)		SV (mg of KOH/g of oil)		IV (g of iodine/100 g of oil)
Induction period (days)	Sample	Control	Sample	Sample	Control	Sample	Control	Control	Sample	Control	Sample	Control
00	7.6 ± 0.3^a	7.6 ± 0.3^b	1.4655 ± 0.002^a	1.4655 ± 0.002^a	0.15 ± 0.03^db	0.15 ± 0.03^hi	0.13 ± 0.00^k	0.13 ± 0.00^k	191.11 ± 0.61^a	191.11 ± 0.12^a	116.3 ± 1.1^a	116.3 ± 1.1^a
10	7.5 ± 0.2^b	7.6 ± 0.4^b	1.4655 ± 0.001^a	1.4656 ± 0.001^a	0.15 ± 0.03^db	0.19 ± 0.02^hi	1.45 ± 0.01^j	2.47 ± 0.06^j	191.18 ± 0.63^a	191.44 ± 0.11^a	114.1 ± 0.5^b	113.5 ± 0.8^b
20	7.6 ± 0.3^a	7.6 ± 0.2^b	1.4656 ± 0.002^a	1.4657 ± 0.001^a	0.16 ± 0.03^dcb	0.24 ± 0.04^gh	5.93 ± 0.03^i	7.73 ± 0.19^i	191.83 ± 0.45^a	192.79 ± 0.25^a	113.1 ± 0.7^bc	112.5 ± 0.4^bc
30	7.6 ± 0.2^a	7.7 ± 0.4^a	1.4656 ± 0.001^a	1.4658 ± 0.002^a	0.16 ± 0.02^dcb	0.38 ± 0.03^g	7.63 ± 0.28^h	8.94 ± 0.40^h	192.94 ± 0.37^a	192.62 ± 0.35^a	112.3 ± 0.5^cd	111.2 ± 0.8^c
40	7.6 ± 0.1^a	7.5 ± 0.2^c	1.4658 ± 0.001^a	1.4660 ± 0.002^a	0.17 ± 0.02^dcb	0.59 ± 0.01^fg	8.15 ± 0.32^g	9.32 ± 0.04^g	193.01 ± 0.91^a	194.73 ± 0.41^a	111.7 ± 1.1^d	109.5 ± 0.6^d
50	7.6 ± 0.2^a	7.7 ± 0.3^a	1.4661 ± 0.002^a	1.4664 ± 0.002^a	0.22 ± 0.03^da	0.74 ± 0.04^f	8.63 ± 0.05^f	9.65 ± 0.05^f	195.05 ± 0.67^a	196.61 ± 0.48^a	110.9 ± 0.9^de	107.3 ± 0.7^e
60	7.5 ± 0.2^b	7.6 ± 0.5^b	1.4665 ± 0.005^a	1.4669 ± 0.003^a	0.23 ± 0.04^da	1.03 ± 0.02^e	9.15 ± 0.07^e	11.06 ± 0.11^e	195.86 ± 0.37^a	198.34 ± 0.51^a	110.5 ± 0.3^de	106.1 ± 0.4^ef
70	7.4 ± 0.3^a	7.5 ± 0.3^c	1.4669 ± 0.002^a	1.4675 ± 0.002^a	0.25 ± 0.05^da	1.29 ± 0.03^d	10.24 ± 0.14^d	14.03 ± 0.52^d	197.03 ± 0.58^a	200.42 ± 0.46^a	109.4 ± 0.4^e	105.4 ± 0.7^f
80	7.6 ± 0.4^a	7.6 ± 0.7^b	1.4673 ± 0.003^a	1.4683 ± 0.004^a	0.27 ± 0.04^da	1.83 ± 0.09^c	11.31 ± 0.34^c	16.52 ± 0.38^c	197.90 ± 0.82^a	201.84 ± 0.57^a	107.5 ± 0.8^f	104.3 ± 0.9^f
90	7.5 ± 0.3^b	7.7 ± 0.4^a	1.4677 ± 0.005^a	1.4690 ± 0.002^a	0.31 ± 0.09^ca	2.19 ± 0.05^b	11.96 ± 0.24^b	18.73 ± 0.13^b	199.74 ± 0.56^a	204.76 ± 0.34^a	107.1 ± 0.6^f	102.5 ± 1.2^g
100	7.6 ± 0.1^a	7.5 ± 0.3^c	1.4683 ± 0.003^a	1.4702 ± 0.002^a	0.35 ± 0.08^a	2.52 ± 0.04^a	12.67 ± 0.19^a	21.25 ± 0.27^a	201.15 ± 0.48^a	208.12 ± 0.27^a	106.3 ± 0.2^f	99.4 ± 0.7^h
Decrease/ increase	0	–0.1	0.0028	0.0047	0.20	2.37	12.54	21.12	10.04	17.01	–10.0	–16.9

Values in columns with same alphabets in superscript do not differ significantly.

CP is considered as an important parameter in blended VOs and affected by blend ratio, as presented in Table 2. CP of all oil samples (stabilized and non-stabilized) was found between 7.5–7.7°C, it means that heat treatment (65°C) has no significant effect on CP. The statistical analysis also showed no significant variation in CP of both non-stabilized and stabilized VO samples. No previously reported data was found for CP of blended VO.

RI values for blended VOs (stabilized and non-stabilized) were determined at 40°C. The data presented in Table 2 shows that the RI values were in range 1.4655–1.4702 and 1.4655–1.4683 for non-stabilized and stabilized oil samples, respectively. These results revealed that the RI values increased slowly throughout the incubation period and this increase in RI was slightly greater for the control samples than the oil samples stabilized with E. citriodora leaf extract. However, both the stabilized and non-stabilized oil samples stored at same conditions exhibit a non-significant change in the RI values. In the present study the increase in RI was 0.0028 and 0.0047 for stabilized and non-stabilized oil samples after 100 day incubation period at 65°C. These values are comparable to the results reported for canola and sunflower oil^[32] and very close to the results presented earlier for sunflower oil.^[33]

FFAs formed by the hydrolytic rancidity of oil during its storage usually reported as percentage of oleic acid of oil.^[26,31] It determines the oxidative damage of oils during storage, as well as under varied temperature conditions. The oils with less increase in FFA value during storage are more stable and show less oxidative damage. In the present study, the FFA (percentage as oleic acid) values for both stabilized and non-stabilized oils shows that FFA values increased significantly in both stabilized and non-stabilized oil samples during storage (Table 2). Before storage the FFA value for both oil samples was 0.150 ± 0.03. During the induction period a significant increase in FFA values was recorded in both types of oil samples. This increase in FFA during the induction period was less in oil samples stabilized with E. citriodora leaf extract as compared with non-stabilized VO samples. The values for both stabilized and non-stabilized oil samples were between 0.167–2.52 and 0.159–0.350, respectively, from the first to the last analysis. However, this increase in FFA was more pronounced in the case of the control samples which show that the E. citriodora leaves extract was efficient to slow down the rate of formation of FFA. The statistical analysis showed significant (p ≤ 0.05) variations in FFA contents of both non-stabilized and stabilized oil samples throughout the incubation period. An increase of 0.20 and 2.37 (percentage as oleic acid) in FFA values was recorded both in stabilized and non-stabilized oil samples, respectively, after 100 days of the incubation period showing a significant role of E. citriodora leaf extract in slowing down the oil oxidation process. These results are comparable with the previous report on sunflower oil stabilized with olive waste cake extract^[11] and greater than the values reported in some important VOs.^[33]

Oil PV is the weight of active oxygen in milligrams in 1 g of the oil and referred as the formation of primary oxidation products in oils during storage. It is directly related to storage time and found an important parameter to access the oxidative stability of oil.^[33,34] Results presented in Table 2 shows that the PV of both stabilized and non-stabilized blended VO samples at the initial time (0 days) of storage was 0.139 meq/kg of oil. A significant increase (from 2.471–21.25 and 1.458–12.67 for non-stabilized and stabilized oil samples, respectively) in PV value was recorded throughout the incubation period. The results showed that an increase of 12.54 and 21.12 meq/kg of oil in PV value of stabilized and non-stabilized VOs, respectively, after a 100 day induction period at 65°C. This shows that the PV of the non-stabilized oil samples increased at a higher rate as compared with those of stabilized samples. So, it can be found from PV of oil samples that E. citriodora leaf extract is effective in slowing down the oil deterioration during storage. The lesser amount of deterioration of oils due to E. citriodora leaf extract might be due its high levels of polyphenols (TPC and TFC) as reported previously,^[30] effective in scavenging the produced free oxygen, leading to reduced oxidative deterioration of VOs. This less increase in PV in stabilized oil samples is positively related with the reduced FFA values. Results for PV in the present study are comparable with the values reported by Sultana et al.^[20] in stabilized corn oil.

SV is also a very important tool to assess the degree of saturation and un-saturation of oil and it is directly related to mean molecular weight of fatty acids present in the VOs. High SV shows the presence of a greater number of the saturated fatty acids while its low value means the high degree of unsaturation of oil.^[35] In the present study, the SV of both blended oil samples (stabilized and non-stabilized) increased on storage with the passage of time. However, this increase in SV was more in non-stabilized oil samples. It shows that the increase in oil saturation, in term of SV, was less in oil samples stabilized with E. citriodora leaf extract as compared with non-stabilized oil samples. In other words, E. citriodora leaf extract found effective in maintaining the oil unsaturation for a longer duration. As shown in Table 2 the initial SV was 191.11 ± 0.61 mg of KOH/g of oil for both oil samples that increased to 208.12 and 201.15 in non-stabilized and stabilized blended VOs, respectively. Overall, an increase of 10.04 and 17.01 mg of KOH/g of oil was recorded in SV of both stabilized and non-stabilized VO samples, respectively, after the 100 day induction period. It means a smaller increase in oil saturation was recorded in stabilized oil samples. These SV of oil samples are comparable with the values for sunflower and canola oil ^[33] and greater than the values for rapeseed oil.^[36] However, these values were found greater than the values reported in Moringa seed oil.^[31]

The oil IV indicates the degree of unsaturation of an oil or fat. It is defined as the number of grams of iodine absorbed by 100 g of oil or fat.^[36] A significant decrease in IV of both stabilized and non-stabilized oil samples was recorded during the induction period and the maximum decrease was recorded after a 100 day induction period. However, this decrease in oil unsaturation was more in non-stabilized blended oil samples. The values presented in Table 2 indicate that a decrease of 10.0 and 16.9 in oil IV of both stabilized and non-stabilized blended VOs, respectively, was recorded in the present study. This indicates that a relatively small decrease in oil unsaturation was recorded in blended VOs that were stabilized with E. citriodora leaf extract. These IVs for VOs in the present study were found less than the values reported in sunflower oil blended with olive waste cake extract^[11] and in the limits reported for rapeseed oil.^[36] However, a big difference was found between the values of present study and the values reported in Moringa oleifera.^[31] Furthermore, in the present study, oil IV and PV were negatively related with oil SV. The values of correlation coefficients among different oil physico-chemical attributes are given in Table 3. Of the different oil oxidative stability measuring parameters, a significant positive correlation (r_s = 0.799***) was recorded in FFA and PV, and PV and SV (r_s = 0.399*), while a negative correlation was found in oil IV and FFA (r_s = –0.801***) and PV (r_s = –0.921***).

TABLE 3 Correlation coefficients (R²) among physio-chemical properties of cooking oil stabilized with E. citriodora leaves extract (mean ± SD)

	FFA	PV	SV	IV	CP	RI	R	Y
FFA	1
PV	0.799***	1
SV	0.252^ns	0.399*	1
IV	–0.801***	–0.921***	–0.370^ns	1
CP	0.179^ns	0.079^ns	–0.030^ns	–0.111^ns	1
RI	0.087^ns	0.044^ns	0.025^ns	–0.092^ns	0.046^ns	1
Color (R)	–0.598***	–0.829***	–0.295^ns	0.823***	–0.043^ns	0.024^ns	1
Color (Y)	–0.598***	–0.823***	–0.295^ns	0.823***	–0.043^ns	0.024^ns	1.00***	1

*significant at 0.05 levels;

**significant at 0.01 levels;

***significant at 0.001 levels.

Conclusions

First, from the results of the present study, it was concluded that E. citridora leaf ethanolic extract is a rich source of natural polyphenols (phenolics and flavonoids) that can be used as natural antioxidant for oil stabilization and its storage for a long duration in the food industry. It was found that the antioxidative function of E. citridora leaf extract increases the shelf life of blended VOs, is associated with less decrease in oil unsaturation in terms of oil IV, after storage for a long period under accelerated conditions. Though some other studies are also available about the stabilization of VOs by extracts of different plant parts, those studies are on pure VOs and not on blended oils. Second, in other studies the extract used for oil stabilization is taken from plants that are not easily available in other geographical areas but the E. citridora plants have the ability to grow under varied environmental conditions and is a rich source of antioxidative compounds. So, the E. citridora leaf extract could be easily used in the oil and fat industry to reduce the oxidative damage of VOs under long-term storage as an alternative to synthetic antioxidants.

Acknowledgments

The authors are very thankful to Muhammad Pervaiz Ahmed Awan (General Manager Works), Hafiz Muhammad Waqar Azeem (Quality Control Manager), Zulfeqar Ahmed (Chemist), and the team of the quality control lab of United Industries (Pvt.) Ltd. Faisalabad for their cooperation for this research.