Regular paper

Assessing the pharmacological and biochemical effects of Salvia hispanica (Chia seed) against oxidized Helianthus annuus (sunflower) oil in selected animals

Tariq Aziz†1✉, Fawad Ihsan†2, Ayaz Ali Khan2✉, Shafiq ur Rahman3, Ghazala Yasmin Zamani4, Metab Alharbi5, Abdulrahman Alshammari5 and Abdullah F. Alasmari5

1Department of Agriculture, University of Ioannina, Arta 47100, Ioannina, Greece; 2Department of Biotechnology, University of Malakand, Chakdara, 18800, Pakistan; 3Department of Environmental Sciences, Shaheed Benazir Bhutto University, Sheringal Dir upper, Pakistan; 4Department of Biotechnology, Bacha Khan University, Charsadda, Pakistan; 5Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia

Oil oxidation is important in terms of taste, nutritive component quality and toxic effect of the oil. In this study, the oxidized sunflower oil was used along with chia seed in rabbits for the determination of its effects on various hematological and serum biochemical parameters as well as on liver histopathology. Three rabbits were fed with oxidized oil (obtained by heating) at the dose rate of 2 ml/kg body weight by mixing it with green fodder. The other rabbit groups were fed with Chia seed at dose rate of 1, 2 and 3 g/kg along with oxidized sunflower oil. Chia seed was fed alone to three rabbits at the dose rate of 2 g/kg body weight. All rabbits were fed regularly for twenty-one days. For the determination of hematological and biochemical parameters, whole blood and serum samples were collected on different days during feeding period. For histopathology, liver samples were used. Significant changes (p<0.05) were noted in the hematology and biochemical indices in the rabbits that were fed with oxidized sunflower oil alone, and along with different doses of Chia seed. In a dose-dependent manner, all these parameters were significantly improved (p<0.05), when the amount of Chia seed was increased. The biochemical and hematological indices were in normal range in the group fed only with Chia seed. In oxidized oil fed group, liver histopathological analysis showed that cholestasis was present at both sides (bile pigment secretion) and zone 3 necrosis with mild inflammatory cells. Mild vacuolization of hepatocytes was also observed. In Chia seed fed group, hepatocyte vacuolization and mild necrosis was noted. It was concluded that oxidized sunflower oil alters the biochemical and hematological parameters and causes liver abnormalities. Chia seeds act as an antioxidant and retrieve those alterations.

Keywords: Chia seed, hematological parameters, oxidized sunflower oil, rabbits

Received: 17 January, 2023; revised: 09 February, 2023; accepted:
17 February, 2023; available on-line: 27 February, 2023

✉e-mail: iwockd@gmail.com (TA), ayazkhan@uom.edu.pk (AAK)

Acknowledgments of Financial Support: The authors greatly acknowledge and express their gratitude to the Researchers Supporting Project number (RSP2023R462), King Saud University,
Riyadh, Saudi Arabia

†Equal contribution

Abbreviations: ALT, alanine transaminase; B, basophils; C, total cholesterol; E, eosinophils; H, hemoglobin; HDL, high density lipoproteins; L, lymphocytes; LDL, low density lipoproteins; M, monocytes; N, neutrophils; P, platelets; S, blood glucose; TG, triglycerides; WBCs, white blood cells

Introduction

Vegetable oils and fats are important constituents of foods and are essential components of our daily diet (Brahmi et al., 2020). Vegetable oils are obtained by mechanical expelling or solvent extraction of oleaginous seeds (soybeans, rapeseed, sunflower, etc.) or oleaginous fruit like palm and olive (Vidrih et al., 2010). Vegetable oils generally contain triglycerides (about 98 g/100 g) (Qian et al., 2020). Triglyceride is formed from a reaction between glycerol and fatty acids and other substances in a small proportion (Gnanaprakasam et al., 2021). Some of them such as diglycerides, vitamins, phytosterols, tocopherols and polyphenols have important health benefits in humans (Gharby et al., 2021; Chew et al., 2016), and therefore, they should not be removed during processing. Other compounds known for their negative effect on the quality and stability of oils include free fatty acids, unsaponifiable matters, waxes, pigments, solid impurities (mainly fibers), oxidation products (peroxides, aldehydes, ketones, alcohols, and oxidized fatty acids) (Gharby et al., 2016; Aliyar-Zanjani et al., 2019; Said et al., 2022). Several plants contain different chemicals which can be used for treatment of various diseases if they are consumed entirely or in parts with lower cost and less side effects (Sana et al., 2022; Bisht et al., 2021). Sunflower oil is one of the most widely grown essential oils in the world. The total world’s oilseed production is forecasted at nearly 647 million tons (United States Department of Agriculture Foreign Agricultural Service Oilseeds: World Markets and Trade. 2022). In 1998, the world’s seed oil was about 28.5 million tons and, as a vegetable oil, only soybean (Glycine max) and Brassica species (Brassica napus and Brassica campestris) produced more oil (Flagella et al., 2002). Sunflower (Helianthus annuus) is used in food, for oil, as a dye, for medicinal purposes, and as an ornamental plant species. Sunflower oil has been used since ancient times as a food and as a medicinal plant to cure many ailments. From a dietary point of view, a diet enriched in monounsaturated fatty acids has been recommended to reduce cholesterol in blood plasma (Dimitrijevic and Horn 2018). Sunflower is often used to produce oils from seeds but is also used as a protein source for human consumption as well as food (Choe and Min 2006). Oil oxidation is important in terms of palatability, nutritional quality and acidity of edible oils. Various chemical compounds, autoxidation and photosensitized oxidation are responsible for the degradation of edible oils during production and storage with respect to oxygen (Khan et al., 2022; Knez et al., 2019).

Salvia hispanica, also called Chia, is an annual herbaceous plant native to southern Mexico and northern Guatemala. It belongs to Lamiales, family Labiatae, subfamily Nepetoideae, and genus Salvia (Segura-Campos et al., 2014). The Salvia genus comprises of about 900 species, which have been extensively distributed for thousands of years in many regions of the world, including South Africa, North, Central and South America, and Southeast Asia (Grancieri et al., 2019; Ullah et al., 2016; Campos et al., 2016; Das et al., 2018; Mohd Ali et al., 2012).

Many studies have reported that Chia today is grown not only in Mexico and Guatemala, but also in Australia, Bolivia, Columbia, Peru, Argentina, America, and Europe. Today, Mexico is known as the largest producer of Chia in the world (Silva et al., 2016). Chia is the dietary seed of Salvia hispanica, a flowering plant, known for its antioxidants that is often used in food production (Coorey et al 2016). Recently, Chia seeds have been given more consideration and have become one of the most popular foods in the world based on their medicinal values and nutritional properties (Ullah et al., 2016; Das et al., 2018; Mohd Ali et al., 2012; Silva et al., 2016). Coorey et al. (2016) demonstrated that Chia is an excellent food ingredient as it contains a huge amount of α-linolenic acid and is easily added to commercial foods. In addition to that it has also been reported that Chia seeds contain high percentage of fatty acids, which make it crucial for health, antioxidant, and antimicrobial property (Ullah et al., 2016; Mohd Ali et al., 2012; Ixtaina et al., 2008; Reyes et al., 2008). Furthermore, several other studies (Grancieri et al., 2019; Silva et al., 2016; Ixtaina et al., 2008; Reyes et al., 2008; Ayerza et al., 2016; Muñoz et al., 2012) demonstrated that the Chia is an oil seed composed of fats, carbohydrates, dietary fiber, proteins, vitamins (A, B, K, E, D), minerals and antioxidants and its benefits of using as a nutritional supplement are numerous, such as supporting digestive system, helping the intestinal mucosa, stronger bones, reducing the risk of constipation, irritable bowel disease, heart diseases, diabetes, and many more (Silva et al., 2016; Correy et al., 2016; Ixtaina et al., 2008; Reyes et al., 2008; Ayerza et al., 2016; Muñoz et al., 2012; de Falco et al., 2018). In the region of Malakand, Khyber Pakhtunkhwa, Pakistan, different food items are fried and cooked using either ghee or oils. Persistent heating causes ghee and oil oxidation, hence, making it toxic. Therefore, the present study was aimed to check the toxic effects of oxidized sunflower (Helianthus annus) oil on hematological and biochemical parameters in rabbits. As Chia seeds (Salvia hispanica) are sources of one of the potent antioxidants, they were used to check its curing potential against the toxic effects of oxidized sunflower (Helianthus annuus) oil.

Materials and Methods

Materials

Sunflower oil was purchased from local market of Matta, Swat, Khyber Pakhtunkhwa, Pakistan. The sunflower oil was selected based on its high linoleic acid and oleic acid content. Chia seeds and rabbits (n=60) were purchased in the local market of Chakdara, Lower Dir, Khyber Pakhtunkhwa. EDTA containing tubes and gel tubes were purchased from the local market for whole blood collection and serum isolation. Formaldehyde and chloroform were provided by organic chemistry laboratory, Department of Chemistry, University of Malakand.

Thermal oxidation of oil

Sunflower oil samples were subjected to a five-hour long regular heating on hot plates at 100°C. These samples were then kept at –20°C to prevent them from further chemical changes.

Experimental animal clustering and feeding

Rabbits were reared in Bio-park of University of Malakand. Food and water were freely available to all the animals. They had an average weight of 1200 grams at the start of the experiment, and among the 60 rabbits, 21 individuals were selected for the experiment based on body weight and health status. The study was started after the approval of the ethical committee, Department of Biotechnology, University of Malakand. Rabbits were divided into seven groups, each having three rabbits (n=3). Groups were labelled as NC, NO, OO, C, CO1, CO2 and CO3 representing negative control, normal sunflower oil, oxidized oil, Chia seed only, Chia seed with oxidized oil (low dose), Chia seed with oxidized oil (medium dose) and Chia seed with oxidized oil (high dose), respectively. The negative control group was fed with green fodder and water. NO was fed with normal sunflower oil at a dose of 2 ml/kg with fodder and water. Group OO was fed with oxidized sunflower oil at a dose of 2 ml/kg. Group C was given Chia seed at a dose of 2 g/kg. Group CO1 was fed with Chia seed at a dose of 1g/kg and oxidized sunflower oil at a dose of 2 ml/kg. Group CO2 was fed with Chia seed at a dose of 2 g/kg and oxidized sunflower oil at a dose of 2 ml/kg. Group CO3 was fed with Chia seed at a dose of 3 g/kg and oxidized sunflower oil at a dose of 2 ml/kg. The feeding was continued for 21 consecutive days and blood samples were collected at day 0, 11 and 21 for hematological parameters and serum biochemical parameters.

Hematological and serum biochemical parameters

The whole blood was used for the analyses of hemoglobin (Hb), platelets (P), white blood cells (WBCs), neutrophils (N), lymphocytes (L), monocytes (M), eosinophils (E) and basophils (B) count by using a fully automated blood hematology analyzer (ERBA – XL 1000). About 5 ml of blood was collected and transferred to gel tubes to isolate serum. The serum was used for the analyses of total triglyceride (TG), total cholesterol (C), blood glucose (S), alanine transferase (ALT), creatinine, urea, high density (HDL) and low density lipoproteins (LDL) level.

Histopathological examination of liver

At the end of the experiment, rabbits were slaughtered according to the method described by Hussain and others (Hussain et al., 2022) and their liver was isolated and preserved in formalin buffer (10%). Tissues sectioning were made, stained and histopathologically examined as described by Khan and others (Khan et al., 2022). Prepared slides were observed under the light microscope, model no. M 7000 D (SWIFT, Japan) and images were taken by a digital camera coupled with a microscope with a resolution of 2.4 Mpixel.

Statistical analysis

Data were analyzed by one way analysis of variance (ANOVA) and Tukey test using online statistical software, prism demo version 05 (www.graphpad.com). Data were presented as mean from triplicate results (n=3) with standard deviation. The mean and standard deviation were sorted out for each parameter.

Results

Effects on hematological parameters

Results showed no significant effect (p<0.05) on the Hb level; however, the platelet count remarkably decreased in all groups after the 21st day. The platelet count was the lowest in the CO3 group. Similarly, no significant change was observed for neutrophils and lymphocyte count. The monocyte count doubled in both CO2 and CO3 groups. The eosinophile number increased in all groups, but such increase was not remarkably high. The results from all measurements (day 0, 11th and 21st day) were combined in Table 1. Different biochemical parameters of the rabbits were shown in Fig. 1.

Effects on biochemical parameters

The glucose level was significantly (p<0.05) high in the oxidized oil (OO) group; however, groups C and CO1 also reflected substantial elevated sugar level. The urea level remained the same in all groups except for CO3 that showed a significant decrease at 21st day. The SGPT/ALT level were significantly (p<0.05) increased in all groups, especially among OO, C, CO1, CO2, and CO3 variants. After the 21st day, the cholesterol levels in NC, NO and CO2 groups remained almost like day 0 values. However, in the OO group, the cholesterol level was high, and the result was the opposite in the C group, wherein cholesterol levels decreased. In CO1 variant, the total cholesterol level significantly (p<0.05) increased, as compared to CO3, where the cholesterol level decreased on the 21st day. The triglyceride values were significantly (p<0.05) elevated in all groups, but they were remarkably high in OO, CO1, and CO2 variants. The HDL levels in all groups showed no significant change. The LDL levels increased in all groups except CO3 wherein, a significant decrease was noted; pertinent to mention, the OO group showed the most negatively correlated LDL values. The results of biochemical parameters on days 0, 11 and 21 were combined in Table 2.

Histopathological pattern

At the end of the experiment, the rabbits were slaughtered, and liver samples were collected for the histopathological analysis. Liver slides were studied under 10X and 40X magnitude for detailed histopathological changes. Results have been presented in Fig 2. Liver histopathology of control variant showed that endothelial linings of central veins had normal morphology, and no evidence of pericentral fibrosis was noted. Kupffer cells were non-reactive. The orientation of the hepatic cord was very good. Hepatic portal veins and arteries showed a normal structure (Fig 2A). On the contrary, in the oxidized oil fed group, cholestasis was present on both sides (bile pigment secretion). Zone 3 necrosis with mild inflammatory cells was noted. Mild hepatocytic vacuolization was also observed (Fig 2B). However, in the normal oil fed group, there was mild zone 2 necrosis especially central vein inflammation and fatty changes (Fig 2C). In Chia seed fed group, there was hepatocyte vacuolization and visible signs of mild necrosis (Fig 2D).

Discussion

It is common practice to repeatedly heat vegetable oils at high temperatures during cooking. Oils are heated during food preparation or deep frying. The present study aimed to sort out the effect of Chia seed against oxidized sunflower oil in rabbits. In our findings, lymphocyte count was significantly increased, while there was a low number of platelets, neutrophils, and monocytes after feeding with oxidized oil as compared to control. The results for these parameters were similar to control ones when fed with Chia seeds. Similar to our results, fresh palm oil was fed to rats, and it was observed that heated oil decreased PCV, Hb level, RBCs and increased WBCs (Mesembe et al., 2004). Similarly, oxidized olive oil significantly altered hematological parameters in rats (Khan et al., 2017). No effects of repeatedly heating cooking oil were observed on hematological parameters after its administration to Wistar rats (Shue et al., 1968; Perumalla et al., 2016), which is not in accordance with our findings. In our study, we heated the sunflower oil for 5 hours at 100oC which may have led to the accumulation of free radicals and altered the hematological parameters. The consumption of Chia has shown good digestibility, hypoglycemic effects, improved lipid and glycemic profiles, and reduced fat deposition in the animal liver.

There was a statistically significant increase in creatinine and urea level in rabbits that were fed with oxidized sunflower oil, and the Chia seed reduced their levels to normal. The co-administration of oxidized oil and Chia seed decreased the creatinine and urea level in a dose-dependent manner. Much of the toxicity of severely heated food oils has been associated with a non-urea-adducting fatty acid (urea filtrate) fraction (Shue et al., 1968; Billek et al., 2000). Certain fractions of the heated oils, the total polar materials cause growth retardation, increased liver and kidney weights and disorders of the enzyme system, but only if fed in high doses (Billek et al., 2000; Ani et al., 2015).

The significant increase in serum creatinine concentration in the group fed with thermo-oxidized palm oil may be suggestive of possible renal system damage. Creatinine levels in plasma are usually measured to determine acute or chronic renal insufficiency. They are usually raised in renal disease (Ani et al., 2015; Toscano et al., 2014). Chia seeds contain high fiber and linolenic fatty acid which may reduce the creatinine level of the rabbits. Salvia hispanica controls blood pressure (BP) and its associated cardiometabolic factors. Also, Chia flour could reduce ambulatory and clinical BP in both treated and untreated hypertensive individuals (Toscano et al., 2014; Al-Othman et al., 2006).

For rabbits fed with un-oxidized sunflower oil, the ALT level was normal. Oxidized sunflower oil increased ALT level in rabbits. In the group which was fed with Chia seeds, ALT level was in normal range. Oxidized oil promotes serum ALT level significantly (Khan et al., 2017; Al-Othman et al., 2006; Zeb et al., 2019), leading to hepatotoxicity. Unoxidized oil is beneficial for liver as it has oleuropein that protects hepatocytes from damage. It had been observed that oxidative stress induced by rancid oils leads to liver injury, which caused an increase in the ALT level (Zeb et al., 2019; Aguilera et al., 2002). In our study, unoxidized oil had no effect on cholesterol level, while oxidized sunflower oil elevated the cholesterol level in rabbits. Chia seeds had a positive effect on lowering blood cholesterol level. Similar to our results, blood cholesterol levels were increased by oxidized olive oil in rats (Khan et al., 2017; Kritchevsky et al., 2000) and by oxidized sunflower in rabbits (Khan et al., 2022). The Chia seed significantly decreased serum cholesterol level when compared to oxidized and un-oxidized sunflower oil treatments. Oxidized sunflower oil elevates the blood cholesterol level, while chia seed decreases its level. After absorption, it increases serum cholesterol level and may lead to atherosclerosis (Lou et al., 2012).

The oxidized sunflower oil fed group significantly increased the triglyceride level of rabbits, while Chia seed decreased the triglyceride level in rabbits. Thermally oxidized oils keep users at risk to arteriosclerosis and cardiovascular diseases due to the depletion of phenolic as well as antioxidants in its constituents. A significant (p < 0.05) increase in the triglyceride level was observed when rabbits were fed with the oxidized olive oil (Khan et al., 2017), oxidized sunflower oil (Zeb et al., 2017) and mustard oil (Carmena et al., 1996). The HDL concentration after feeding with oxidized sunflower oil was significantly decreased, whereas LDL values were increased as compared to normal group. Chia seed significantly increased HDL and decreased LDL level in rabbits. It has been suggested that HDL cholesterol and its constituents is increased after feeding sunflower oil, which helps in prevention of heart diseases (Lou et al., 2012; Carmena et al., 1996; Quiles et al., 1998). A significantly higher LDL susceptibility to oxidation was observed after sunflower oil intake in comparison with virgin olive oil, despite an increase in LDL α-tocopherol concentration in sunflower oil group (Aguilera et al., 2004). Histological studies revealed that un-oxidized sunflower oil caused no significant effect on the liver morphology and functions. Oxidized sunflower oil caused necrosis in centrilobular regions. It has also been reported that thermally oxidized ground nut oil leads to some liver diseases (Aguilera et al., 2004; Jimoh et al., 2004; Abdel Raouf et al., 2012). On the other hand, Chia seed caused no significant changes, as hepatocyte vacuolization and mild necrosis were present in the liver.

Conclusions

In comparison to unoxidized oil, it has been determined that oxidized sunflower oil significantly affects hematological and biochemical parameters of serum and alters liver histological pattern in rabbits. Chia seed on the other hand, minimizes harmful effects of oxidized oil and display antioxidant potential.

Declarations

Author contribution. Conceptualization, T.A and A.A.K.; Original draft, G.Y.Z, S.R and F.I ; Methodology, M.A and F.I.; Data curation: M.A and A.A.S.; Writing- review & editing, A.A.K, and T.A.; Visualization, T.A; Resources, M.A.; Project administration, T.A and A.A.K ; Funding acquisition, T.A.; Validation, F.A .; Investigation, S.R and A.A.K ; Formal analysis, T.A and S.R.; Supervision, T.A and A.A.K

Funding: This research work received no external funding.

Conflicts of Interest: The authors declare no conflict of interest.

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