Palm olein, a derivative of palm oil, is widely used in the deep-fat frying process. It contains oleic acid, palmitic acid, and linoleic acid at 42.1%, 38.3%, and 10.6%, respectively.[1,2] Palm olein undergoes oxidative and thermal degradation during repeated heating to high temperatures (158–185 °C) in the presence of air and moisture, thus producing oxidation changes and polymerized compounds. The oxidation of lipids in food takes place through a free radical mechanism called auto-oxidation. This process has three stages including initiation, propagation and termination. Lipid oxidation is a series of reactions which produced primary and secondary products during oxidation including, peroxides, free fatty acids, hydroperoxides, aldehyde, alcohol and ketones. The stages of lipid oxidation are occurred at different rates depending on the light, oxygen, temperature, moisture, type of foods and oils. Deep frying foods absorbed substantial amount of oxidised oil in which the safety and nutrition value were affected.[3,4,11] These compounds are absorbed from the small intestine and distributed in the body. Consumption of diets rich in saturated fatty acids and cholesterol generates free radicals which disrupt homeostasis and lead to oxidative stress in hepatocytes.[5,6] Under physiological conditions, oxidative stress leads to the production of active intermediate molecules, namely reactive oxygen and nitrogen species (ROS and NRS).[7,8] Active intermediates react with body proteins, carbohydrates, and lipids affecting cellular homeostasis and leading to damage to DNA, lipoproteins, membrane lipids, and eventual cell death.[9,10] Poly-unsaturated fatty acids in cellular membrane are highly targeted to reactive oxygen species which caused damaging in cellular membrane. Several researches have reported the relationship between oxidative stress and various diseases, including cancer, heart failure, diabetes, hemochromatosis, and neurodegenerative diseases.[12‐14] An antioxidant defence system protects the body against free radicals (ROS and RNS) by inhibiting oxidation, preventing the formation of active molecules, and decreasing the concentration of oxidants.
Pteropyrumscoparium is a medicinal plant and rich in phytochemical compounds, including saponins, glycosides, tannins, terpenoids, flavonoids, omega-3 fatty acids, and alkaloids.P. scoparium is a medicinal plant belongs to the Polyonaceae family. It is available in tropical and sub-tropical countries in wadis and mountains including, Oman and United Arab Emirates. It is growing after the rain season in February and locally known as sidaf. Omanis people used fresh leaves to make a traditional cuisine by mixing them with bread, lemon, chili, onion, ghee, and dried fish. P. scoparium leaves used for dyspepsia and blood purification treatment. Crushed stalks are used to prepare a tea which is used to treat liver diseases.P. scoparium extract is reported to exhibit antibacterial activity against chloroform and butanol strains and antioxidant activity. This extract could be used as food additives, dietary supplements, and nutraceuticals.P. scoparium is highly branched plant with a grey-white stems up to 1.5 m length. The leaves are placed in bundles of three to six leaves, fleshy in consistency, rectangular to flat, and up to 2 cm long. The flowers are hermaphroditic, small, and pink or brown. Leaves of P. scoparium have various phytochemicals such as, saponins, glycosides, coumarins, tannins, phenols, resins, flavonoids, steroids (omega-3), and alkaloids. Thus far, few studies have investigated the protective effect of PSE on oxidative stress. Therefore, this study aimed to assess the potential protective effect of PSE against heated palm olein (HPO)-induced oxidative stress in rat liver.[20,21]
Materials and Methods
P. scoparium plant collection and leaves extract preparation P. scoparium plant was collected from its natural habitat (Wilayat Al-Hamra, Oman) and then cleaned from impurities and dust using distilled water. The identification of the plant material was evaluated by an expert botanist at the Crop Sciences Department at Sultan Qaboos University. The collected plant was left to dry at room temperature, and the leaves were freeze-dried for 5 days at −40 °C using a free zone 6-L benchtop freeze dry system (LABCONCO, USA). Then, the freeze-dried leaves were ground into fine powder by an electrical grinder (Moulinex, AR1043-UK0, France). The extracted powder was macerated in aqueous methanol and shaken at 200 rpm at room temperature for 6 hours. The supernatant was filtered by Whatman No. 1 filter paper and the methanol was evaporated from the filtrates by a rotary evaporator (G3 Heidolph, Germany) at 40 °C, yielding 100 g dry solids powder per 1000 g of fresh P. scoparium leaves and kept at −40 °C till use for subsequent animal experiments.
Palm olein sampling
Crude palm olein (PO) was purchased from a local company (Jabal Al-Akhdar, Oil Company, Muscat, Oman). The frying process was done in a deep-fat fryer (Prestige, China) with a 12 L capacity. French fries were prepared by frying potato strips (50 g) in palm olein (2 L) in a cycle of 15 minutes of heating and resting for 5 hours daily at 180 ±1 °C with a total frying time of 25 hours over 5 continuous days. At the end of the fifth day of frying, the oil was collected and kept at −18 °C for further use and for animal studies.[19,20]
Determination of acid value
The acid value of the palm olein oil was detected based on the American Oil Chemists’ Society Method Cd 3d-63 (AOCS 5a-40, 1999). A mixture of absolute alcohol (25 mL) and diethyl ether (25 mL) was titrated with sodium hydroxide (0.1 M). About 1% of phenolphthalein solution was used to neutralize the mixture and was used as an indicator. Oil (1.0 g) was mixed with the neutral solvent and titrated with sodium hydroxide (0.1 M) until the pink color appeared. The following equation was used to calculate the acid value (mg KOH/g).
Determination of peroxide value
The peroxide value of the palm olein was evaluated referring to the method done by the American Oil Chemists’ Society Cd 8-53. An equal amount (1.0 g) of palm olein and potassium iodide powder was mixed in a test tube. The solvent mixture was prepared by mixing 200 mL of glacial acetic acid and 100 mL of chloroform (2:1 v/v). Then, 20 mL of the prepared mixture was added to the test tube. After that, the test tube was kept for 30 seconds in boiling water. The solution was transferred from the test tube in a conical flask, and the test tube was rinsed with 50 mL of distilled water and 20 mL of 5% potassium iodide. Then, the solution was titrated with sodium thiosulfate (0.02 M) and starch was used as an indicator. The peroxide value was expressed as milliequivalents of active oxygen per kilogram of oil (mEq/kg oil). The following equation was used to calculate the peroxide value:
Peroxide value = titration (mL) × 2
Determination of total polar compounds
A spectrophotometric method was used to determine the total polar compounds in palm olein samples. First, non-heated and heated olein samples were placed in a standard disposable cuvette. Then, oil samples were warmed in the oven for 15 minutes at 60 °C. After that, the spectrophotometric absorbance of oil was measured by (UV/vis Spectrometer, USA) at 490 nm. The following equation was used to convert absorbance to total polar compound contents:
y = −2.7865x2 + 23.782x + 1.0309
where, y is the total polar compounds and x is oil absorbance.
Fatty acid analysis
The fatty acid composition of palm olein was determined according to the Association of Official Analytical Chemists official method (969.33, 1996). First, 0.3 to 0.5 g of palm olein was added to the boiling flask with a boiling chip. After that, 6.0 mL of methanolic NaOH solution was added to the flask with an attached condenser. The oil was boiled until fat globules disappeared, usually for 5 to 10 minutes, then 7 mL of boron trifluoride methanol solution (BF3) was added from the automatic pipet through the condenser and continued boiling for 2 minutes. After 2 minutes, 5 mL of heptane was added through the condenser and boiled for 1 minute longer. Then, the heat and condenser were removed, and 30 mL of saturated NaCl solution was added and shaken for 15 seconds. The upper layer of oil was taken by dropper and transferred into a glass-stoppered test tube. A small amount of anhydrous Na2SO4 was added to filter the water. About 1 mL of the extracted fatty acid was injected into a gas chromatograph-mass spectrometer (GCMS-QP2010) for analysis. GCMS real-time analysis software was used to analyze the result.
GC-MS analysis was achieved on a Shimadzu GC-2010 Plus, fitted with an SP-2560 Supelco capillary column which was 100 m long, 0.25 mm internal diameter with a thin film (0.25 μm thickness) coupled to GCMS-QP2010 ULTRA MS. As carrier gas, helium was used as it has an ultra-high purity (99.9999%) with a constant flow rate of 1.0 mL/min. The injection line had a temperature of 250 °C, while the transfer line and ion source had a temperature of 240 °C. The ionizing energy was 70 eV. Electron multiplier (EM) voltage was obtained from autotune. The full-scan mass spectra were collected within the scan range of 35 to 500 amu to obtain all the data. The volume of the injected sample into GC-MS was 1 µL with a split ratio of 10:1. The temperature program for the oven was 50 °C which was held for 5 minutes. Then, the temperature was accelerated at a rate of 4 °C/min and held at 250 °C for 10 minutes. There was a comparison between mass spectrum libraries (NIST 2011 v.2.3 and Wiley, 9th edition) and spectra to identify unknown compounds and confirm them using Supelco 37 component FAME mixture (cat. # 47885-U).
Experimental animal design
Forty-eight male adult Fischer 344 rats with a weight range of 325 to 400 g were used in this study. The animals were divided randomly into four equal groups (12 rats/group) and housed in individual polypropylene cages in a well-ventilated room. All cages were kept in standard conditions of temperature (22 ± 2 °C), humidity (60%), and 12 hour light/dark cycle. The rats were given water and an experimental diet ad-libitum. The experiments were conducted in accordance with guidelines and protocols approved by the Institutional Animal Ethics Committee of Sultan Qaboos University (SQU/AEC/2018-19/1).
As presented in Table 1, the experimental diet (g/kg diet) contained: 140 g protein, 40 g dietary fat (non-heated or heated palm olein), 100 g sucrose, 620.692 g corn starch, 50 g fiber (α-cellulose), 35 g mineral mixture (AIN-93M-MX) and 10 g vitamin mixture (AIN-93-VX), 2.50 g choline bitartrate, 0.008 g tert-Butylhydroquinone, and 1.80 g DL-methionine. Casein, dietary fat (palm olein), sucrose, and corn starch were obtained from the national companies in Muscat, Oman. Meanwhile, mineral mixture (AIN-93M-MX) and vitamin mixture (AIN-93-VX) were purchased from Dytes Inc., Bethelhem, PA, USA. Other dietary components (α-cellulose, choline bitartrate, tert-Butylhydroquinone, and L-Cysteine) were purchased from Sigma Chemical Co., St. Louis, MI, USA. All the dietary ingredients were mixed together, homogenized and sieved by the normal house hold sieve (25 mesh/cm2). The processed diets were packed in opaque plastic containers and stored at 4 °C. The exposure to the light was minimized throughout the experiment.
Table 1 -
Composition of the experimental diet fed to rats
||Amount (g/kg diet)
|Dietary fat (non-heated or heated palm olein)
|Mineral mixture (AIN-93M-MX)
|Vitamin mixture (AIN-93-VX)
The Pteropyrum scoparium leaves extract (PSE) was used based on the study theme of using herbal plants as a medicinal intervention not as a pharmaceutical agent, was administrated through oral gavages with an oral dose of (0.1 mg dry solid extract/1 mL water/day) for the treated group(s); the oral dose was selected similar to earlier works by our research group.[15,22] While non-treated groups were given distilled water orally to maintain the similarity among all groups. All animals were continuously fed ad-libitum. Body weight and food intake (each rat consumed 10–12 g diet/day) were recorded every week throughout the experiment. The experiment was conducted during 8 weeks because it replicates a chronic effect based on previous experimental animal trials by our research group. After 8 weeks, rats were anesthetized by mixing 75 mg of Ketamil/kg of rat weight with 5 mg of Xylazil/kg of rat weight. Then, the liver of each rat was promptly removed, of which a portion was placed in 10% formalin for histopathological examination. Another portion of liver tissue was homogenized by mixing 2 g of the liver with 10 mL of phosphate-buffered saline buffer (pH 7.4) using a homogenizer (IKA ULTRA TURRAX, Germany) then centrifuged (4000 × g/4 °C/20 min), CL30R Centrifuge, Germany. The supernatant was used to determine protein content and to evaluate glutathione and total antioxidant measurements.
Glutathione (GSH) measurement
Aliquots of the liver tissue homogenate supernatant (20 µL) were mixed with 10 µL of monochlorobimane (25 mmol/L) and 10 µL of Glutathione-S-transferase reagent as provided by a commercial kit (Biovision, Catalogue # K251-100). The volume was made up to 100 µL using a cell lysis buffer. After 20 minutes of incubation in a 96-well plate at 37 °C, the samples were read in an ELISA reader (Multiskan Go Microplate Spectrophotometer, Finland) at 620 nm. The GSH content was detected by comparison with values from a standard curve using freshly prepared GSH and normalized to the protein content of the assayed liver tissue homogenates.
Total antioxidant capacity (TAC) measurement
A colorimetric Assay Kit (Biovision, Catalogue # K274-100) was used to detect the TAC; 20 µL of the supernatant was mixed with 10 µL of Cu2+ reagent and 10 µL of protein, then assay diluent was added to make the volume reach 100 µL, after that, the samples kept in the incubator for 30 minutes at 37 °C. The absorbance was detected at 620 nm. Trolox was used as a standard to compare the TAC of the samples. The assay results were normalized to the protein content of the assayed liver tissue homogenates.
Protein content analysis
Protein content of liver tissue homogenates was performed using a method described by Lowry et al., the protein content was expressed as mg/mL of sample.
Liver histopathological examination
The liver tissues were fixed in 10% formalin for 24 hours, dehydrated in graded ethanol and cleared in xylene, embedded in paraffin wax, and sectioned at 5 μm then stained with Hematoxylin and Eosin. Liver sections were examined by an Olympus BX51 microscope and an Olympus camera DP70 (Olympus, USA).
Determination of liver enzymes
Blood samples were taken by cardiac puncture, collected into non-heparinized tubes, and then centrifuged (4000 × g/4 °C/10 min) and the serum enzyme activities of aspartate aminotransferase (AST; EC 18.104.22.168) and alanine aminotransferase (ALT; E.C 22.214.171.124) were measured to assess hepatic function. ALT and AST activities were measured spectrophotometrically (cobas® 6000, Roche Clinical Chemistry analyzer), using Biosystems kits, according to the manufacturer’s instructions. ALT and AST activities were expressed as U/L.
The data were expressed as mean ± standard deviation (SD). The results were analyzed by one-way ANOVA followed by Tukey test, Student unpaired t-test, the differences were considered statistically significant when P value <0.05. GraphPad software (version 8) was employed for the statistical analysis.
Results and Discussion
Table 2 illustrates that the frying process altered the palm olein properties (peroxide value, acid value, and total polar compounds). The peroxide value is used as an indicator of oil degradation and provides an indication of the formation of hydroperoxides during oxidation. The peroxide values were significantly increased for HPO (11.45 ± 0.10) compared with the value of NHPO (0.54 ± 0.03), this is expected because of fatty acid peroxidation during heating. Leong et al., reported that the peroxide value of the palm olein was increased significantly with the frying period and it reached 11.76 mEqO2/kg oil after heating 10 times and similar result was found in a different study. Fan et al. observed that the peroxide value for palm olein was increased from 6.80 to 34.55 mEqO2/kh on the fifth day of repeated frying. There was a significant difference with P < 0.05 in the acid value and total polar compounds after repeated frying. Higher content of total polar compounds and acid value in palm olein are due to the content of diglycerides and the presence of lipase, which decompose oil to free fatty acids.
Table 2 -
Chemical properties of non-heated and heated palm oil
||1.37 ± 0.06a
||2.63 ± 0.10b*
||0.54 ± 0.03a
||11.45 ± 0.10b*
|Total polar compounds
||2.90 ± 0.08a
||8.12 ± 0.08b*
Values (means ± SD, n = 3) within a row with different superscript letters are significantly different. HPO, heated palm olein; NHPO, non-heated palm olein; acid value: mg KOH/g; peroxide value: mEqO2/kg oil. *P < 0.05, Student unpaired comparison t-test.
Table 3 shows the fatty acid composition of non-heated and heated palm olein. Oleic acid (18:1) had the highest proportion in both samples (NHPO 49.61 ± 0.62% and HPO 50.34 ± 0.01% of total fatty acids) followed by palmitic acid (16:0) and linoleic (18:2); NHPO 30.68 ± 0.57, HPO 27.86 ± 0.11, NHPO 17.37 ± 0.52, HPO 18.16 ± 0.61, respectively. Similar fatty acid composition of heated and non-heated palm olein was found in several studies regardless of frequency of frying.[25,26] There was no significant difference in fatty acid composition before and after frying for the same sample (P > 0.05). The results seem to show that heating did not cause an increase in the amount of saturated fatty acids present in the palm olein.
Table 3 -
Effect of heating on fatty acid content of palm
|Fatty acid (mg/g)
|Caprylic acid (C8:0)
||0.01 ± 0.01a
||0.01 ± 0.01a
|Capric acid (C10:0)
||0.03 ± 0.00a
||0.03 ± 0.00a
|Lauric acid (C12:0)
||0.33 ± 0.01a
||0.34 ± 0.01a
|Myristic acid (C14:0)
||2.02 ± 0.07a
||1.65 ± 0.03b*
|Palmitic acid (C16:0)
||30.68 ± 0.57a
||27.86 ± 0.11b*
|Stearic acid (C18:0)
|Oleic acid (C18:1)
||49.61 ± 0.62a
||50.34 ± 0.60a
|Linoleic acid (C18:2)
||17.37 ± 0.52a
||18.16 ± 0.61a
|Linolenic acid (C18:3)
|Arachidonic acid (C20:0)
||1.21 ± 0.08a
||0.89 ± 0.03b*
Values (means ± SD, n = 3) within a row with different superscript letters are significantly different. n.d., not detectable. *P < 0.05, Student unpaired comparison t-test.
Figure 1 depicts the changes in body weight in the experimental groups. The initial body weight increased gradually throughout the experimental period for all the groups. The findings revealed that the initial body weight of all rats was 325 ± 10 g, and there was a trend of increment in body weight in all groups. However, there was a significant reduction in body weight for rats that received heated palm olein fed with PSE extraction (P < 0.05). A remarkable observation was that the PSE supplementation in HPO-fed rats showed a recovery in the weekly body weight, reaching 369 g at the end of the experiment. There was a reduction in the weight of the PSE treated groups due to the effect of oral gavage on the esophagus, which decreased the appetite. Also, prolonged feeding with a non-heated or heated palm olein diet decreased food consumption as a result of the taste changes. Previous studies have shown an association with consumption of high-fat diet-induced and low body weight in rats.[27,28]
Figures 2 and 3 outline the effects of PSE on GSH and TAC in liver tissues. The results exhibited that concomitant treatment of HPO and PSE significantly restored GSH and TAC to their basal levels (P < 0.01). On the other hand, in the absence of PSE, GSH depletion and TAC impairment were significantly greater in the HPO-fed groups than in the NHPO-fed groups (P < 0.05). Previous study conducted by Al-Attabi et al. reported that P. scoparium extract provided free radical scavenging activity against H2O2 by improving GSH level.
AST or ALT levels are enzymes found mainly in the liver and considered as a valuable aid primarily in the diagnosis of liver disease. However, when liver tissue is diseased or damaged, additional AST and ALT are released into the bloodstream, causing levels of the two enzymes (AST and ALT) to rise. Therefore, the amount of AST and ALT in the blood is directly related to the extent of the liver tissue damage. Heating of palm olien produces lipid peroxidation which induces hepatic injuries. The HPO-treated rats developed extensive hepatic damage evidenced by a significant rise in AST and ALT activities in comparison with the normal control group (NHPO), Table 4. On the other hand, a lower level AST and ALT were observed after oral PSE administration in HPO group [Table 4]. PSE administered orally significantly reduced serum AST and ALT activities, restoring altered biochemical parameters to normal levels in comparison to the HPO-treated group indicating its potential antioxidant potential properties and this hepatic protective effect observed suggests that the active compounds (polyphenolic and flavonoids) compounds present in the PSE extract play an important role in plasma membrane stabilization, as well as repairing liver damage caused by HPO to prevent leakage of intracellular enzymes. PSE with its antioxidant capacity was able to quenches free radicals and lipids peroxides, in this regard, previous studies have reported that antioxidant activities in plant extracts such as those identified in the PSE exhibit a hepatocellular improvement function due to the presence of antioxidant compounds.[29‐33]
Table 4 -
Liver enzyme levels
||126.25 ± 6.9
||63.65 ± 4.9
|NHPO + PSE
||126.11 ± 7.4
||62.84 ± 3.8
||562.81 ± 23.4a*
||141.23 ± 7.2a*
|HPO + PSE
||255.49 ± 16.8b*
||80.74 ± 6.9b*
HPO, heated palm olein group; NHPO, non-heated palm olein group. n = 6, values expressed as mean ± standard deviation. a Significantly higher as compared to NHPO group. b Significantly lower as compared to HPO group. *P < 0.05, ANOVA-Tukey.
The histopathological examinations of liver tissues for experimental groups are shown in Figure 4. The groups that were fed with NHPO and those fed with NHPO with PSE showed normal histological structures and architecture of the hepatic parenchyma. A study was done by Jaarin et al. counters this finding. They found that feeding fresh palm oil, induced microvesicular steatosis in rate hepatocytes after 16 weeks; that is twice the duration of our study. In contrast, the group fed with HPO showed few hepatic cells with fatty infiltration. This result is due to the saturation of palm olein oil. A study showed that rats fed with fresh palm oil (4.5 g/kg/day) had normal liver histology at 24 weeks, while rats that fed with reused palm oil induced hepatic fat accumulation at the same duration. In this study, the amount of oil (15%), administration method, and feeding duration (8 weeks) were different. The group fed with HPO with PSE showed normal structures of the hepatic parenchyma. That indicates the positive effect of PSE against oxidizing oil in preventing any harm to hepatocytes due to the presence of bioactive compounds.
Experimental findings confirmed the hepatoprotective effect of PSE, demonstrating its antioxidant action against HPO-induced hepatotoxicity. The PSE significantly restored AST and ALT levels, as confirmed by histopathological observations. In addition, the activities of TAC and GSH were significantly enhanced in HPO-treated rats after oral PSE-treatment, in comparison to NHPO treated group. The resulting antioxidant and hepatoprotective activities of PSE extract might be attributed to phytochemicals content which inhibited the HPO-mediated oxidative stress in liver tissue. The antioxidant efficiency observed in the PSE extract is likely due to the active constituents (phenolic compounds and flavonoids). Further studies are needed to identify the active constituents at PSE, and to validate the PSE usage as a medicinal plant in primary health care.
Financial support and sponsorship
The authors merit Sultan Qaboos University Internal Grant Fund (IG/AGR/FOOD/20/01) awarded to the authors for providing financial support to carry out this research project.
Conflicts of interest
There are no conflicts of interest.
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