Food Chemistry xxx (2015) xxx–xxx
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Sea buckthorn seed oil protects against the oxidative stress produced by thermally oxidized lipids Alam Zeb ⇑, Sana Ullah Department of Biotechnology, University of Malakand, Chakdara, Pakistan
a r t i c l e
i n f o
Article history: Received 22 December 2014 Received in revised form 12 March 2015 Accepted 15 March 2015 Available online xxxx Keywords: Sea buckthorn oil Oxidized vegetable ghee Oxidative stress Serum lipids Rabbits Liver
a b s t r a c t Thermally oxidized vegetable ghee was fed to the rabbits for 14 days with specific doses of sea buckthorn seed oil (SO). The ghee and SO were characterized for quality parameters and fatty acid composition using GC–MS. Rabbits serum lipid profile, hematology and histology were investigated. Major fatty acids were palmitic acid (44%) and oleic acid (46%) in ghee, while SO contains oleic acid (56.4%) and linoleic acid (18.7%). Results showed that oxidized vegetable ghee increases the serum total cholesterol, LDLcholesterols, triglycerides and decrease the serum glucose. Oxidized ghee produced toxic effects in the liver and hematological parameters. Sea buckthorn oil supplementation significantly lowered the serum LDL-cholesterols, triglycerides and increased serum glucose and body weight of the animals. Sea buckthorn oil was found to reduce the toxic effects and degenerative changes in the liver and thus provides protection against the thermally oxidized lipids induced oxidative stress. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Sea buckthorn (SBT) is a famous medicinal and aromatic plant, widely admired for its role to possess multiple health promoting properties. Sea buckthorn has been found to be a rich source of carotenoids, vitamin E, C, and different phenolic compounds. The seed oil had been found to help the recovery of skin injuries and skin diseases (eczema, burns, and wounds). It also helps from the damaging effects of sun on the skin, and during the radiation therapy and cosmetic laser surgery (Zeb & Khan, 2008, 2009). It had also been used in digestive ulcers and cardiovascular disease (Yang & Kallio, 2002; Zeb, 2004). The scientific studies during the recent decade confirmed the medicinal and nutritional values of sea buckthorn. The most important of these studies are the antioxidant and anti-carcinogenic properties. In animal studies, sea buckthorn products showed significant antioxidant potential. For example, Geetha et al. (2008) showed that the sea buckthorn leaf extract has a significant hepato-protective effect which was attributed to the antioxidant compounds present in the extract. Similarly,
Abbreviations: SO, sea buckthorn oil; OG, oxidized ghee; GC–MS, gas chromatography–mass spectrometry; LDL, low density lipoproteins; HDL, high density lipoproteins; PV, peroxide value; AV, anisidine value; FFA, free fatty acids; Hb, hemoglobin; RBC, red blood cells; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin. ⇑ Corresponding author. Tel.: +92 945 761622. E-mail address: [email protected] (A. Zeb).
Gumustekin et al. (2010) studied the effects of nicotine-induced oxidative stress in rat heart with vitamin E and sea buckthorn extract. The authors showed that supplementation of extract increased superoxide dismutase and glutathione S-transferase activities, which may be contributing to the prevention of nicotine-induced oxidative stress in rat heart. They also suggested that the antioxidant activity of the sea buckthorn extract was due to the presence of quercetin and isorahmnetin, tocopherols, and carotenoids. Arimboor and Arumughan (2012) observed that SBT has a considerably high antioxidant and xanthine oxidase inhibitory potential shown by proanthocyanidins of the seed. Similarly, the Kim et al. (2012) findings suggested that the sea buckthorn seed extract may be a potential therapeutic agent for preventing and treating skin photo-aging. The results of Hsu, Tsai, Chen, and Lu (2009) showed that oral administration of SBT seed oil for eight weeks significantly lowered serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), triglyceride (TG), and cholesterol, which were elevated by CCl4 (1 mL/kg) in mice. Thus, different products of sea buckthorn have significant ameliorating properties. Vanaspati ghee is one of the important components for the preparation of various kinds of foods. During frying or cooking the thermal stress oxidizes vanaspati ghee and produce toxic substances. These toxic substances subsequently enter the food matrix and ingested by the human (Zeb & Rahman, 2012). Experiments had shown that thermally oxidized lipids are potentially carcinogenic (Yang et al., 1998) and enhance the growth of
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Please cite this article in press as: Zeb, A., & Ullah, S. Sea buckthorn seed oil protects against the oxidative stress produced by thermally oxidized lipids. Food Chemistry (2015), http://dx.doi.org/10.1016/j.foodchem.2015.03.053
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hepato-carcinoma in experimental animals (Jurek et al., 2005). Recently we have shown that thermally oxidized vanaspati ghee had produced significant effects in the serum lipid profile as well as peroxidation in different tissue (Zeb & Mehmood, 2012). It is therefore necessary to find out the possible remedies to counterbalance the oxidative stress produced by oxidized lipids in our daily foods. Various plant extracts have been used to possess the antioxidant potential. The studies of Yeh, Hsieh, Lee, and Shen (2012) showed that oxidative stress induced by oxidized cholesterol can be minimized using a supplement of 0.1% of dietary Sea buckthorn. Similarly, Zeb and Hussain (2014) showed that the sea buckthorn seed powder provides protection against the toxic properties of thermally oxidized sunflower oil. The study, however does not provide information about the similar potential of the most widely used products of sea buckthorn such as seed oil. This study was therefore aimed to find out further correlation of the oxidative stress induced by thermally oxidized lipids with higher saturation and sea buckthorn oil in animal models. 2. Materials and methods 2.1. Materials Sea buckthorn seed oil and vanaspati ghee was obtained from the commercial market. All other chemicals and reagents were of ACS grade from Sigma Aldrich (USA) or otherwise mentioned. 2.2. Thermal oxidation Vanaspati ghee was thermally oxidized on the hot plate at 160 °C for successive 10 h in the open air to mimic the real frying conditions. After thermal oxidation the samples were stored in the refrigerator at 20 °C. 2.3. Characterization of sea buckthorn oil The peroxide value (PV), p-anisidine value (AV) and free fatty acids (FFA) of ghee and SBT oil samples were determined using the AOCS official methods (AOCS., 1998). All samples were measured in triplicate or otherwise mentioned. Total phenolic contents (TPC) were measured using Folin–Ciocalteu reagent and quantification was carried out with the help of Gallic acid calibration curve. Fatty acids were converted to their respective fatty acid methylesters (FAMEs). Briefly, a sample of 20 mg (±0.5 mg) was weighted in 20 mL vial. Then 6 mL of 0.5 M methanolic NaOH was added and stirred at 80 °C for 30 min. After the samples were cooled, BF3/ methanol was added and stirred further at 80 °C for 15 min. Water and n-heptane phases were separated. The heptane phase was injected into gas chromatography coupled with mass spectrometry (Agilent 5975, Agilent Germany). Fatty acids and sterol were identified from their relative and absolute retention times and also by the MS library database. The values were expressed as g/100 g determined from the peak area.
animal house facility. Feed and water were provided ad libitum. The rabbits were fed with pellets from the commercial market and labeled chemical composition of the pellets showed that they contained 17.5% protein, 14.0% fiber, 2.7% fat. All animals were placed one week ahead to get familiarized with the environment. Rabbits were classified into six groups, consisting of three animals per group. Each group is designated as the individual treatment. The rabbits were fed with oxidized ghee and sea buckthorn seed oil for 14 days, according to the feeding scheme given below: Group C: control fed on normal food; Group SO: 2 g/kg bwt SO; Group OG: 1 g/kg bwt OG; Group OGSO1: 1 g/kg bwt OG and 1 g/kg bwt SO; Group OGSO2: 1 g/kg bwt OG and 2 g/kg bwt SO; and Group OGSO3: 1 g/kg bwt OG and 3 g/kg bwt SO. The blood samples were collected in falcon tubes and centrifuge for 10 min to obtain serum for further analysis.
2.5. RSA of liver lipids The liver tissues of each individual rabbit were taken and stored in formalin. Lipids were extracted using the modified procedure of Folch, Lees, and Sloane-Stanley (1957). The radical scavenging assays (RSA) of the lipids extracted from the tissues were measured using DPPH free radicals (Zeb & Mehmood, 2012).
2.6. Serum biochemical analysis Serum biochemical parameters like cholesterol, HDL-cholesterol, LDL-cholesterol, ALT, and glucose were measured using HUMAN (Human Diagnostics, Germany) kits, while total triglycerides were measured using DiaSys (DiaSys Diagnostic Systems GmbH, Germany) kit.
2.7. Histopathological studies of liver A section of the median lobe of the liver was dissected and fixed in 10% formalin buffered for at least 14 h. The section was then dehydrated with ethanol solutions and processed for embedding in paraffin. Sections of 8–10 mm in thickness were cut, deparaffinized, rehydrated, and stained. The slides were studied by binocular microscope (model M 7000 D series, Swift Instruments, Inc., Japan) and the pictures were documented using the digital camera (DCM 130) of the microscope with the resolution of 1.3 MP.
2.8. Hematological studies For hematological examination 2 mL blood was collected from the jugular vein of rabbits in EDTA tubes. Hematology or blood profile was carried out by automatic digital machine (CELL-DYN 3200 Abbott Diagnostic Division, Canada) for the following parameters; Blood Hemoglobin Level (Hb), Blood Total RBC Level (RBC), Blood HCT Level (HCT %), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Blood Platelets Concentration, and Blood White blood cell Concentration.
2.4. Experimental animals All experiments were carried out according to the approved guidelines for the care and proper use of the animals of the Department of Biotechnology, University of Malakand. Rabbits selected for the study because of the highly selective and developed organ system and were easily available animals for these experiments. Rabbits were grouped by random distribution into six groups, irrespective of the gender and placed in the same
2.9. Statistical analysis All analyses were carried out in triplicate or otherwise mentioned. Data were analyzed by one-way analysis of variance (ANOVA) and Holm–Sidak method of multiple comparison method at a = 0.05 using SigmaPlot for windows version 12.0 (Systat Software, Inc., 2011).
Please cite this article in press as: Zeb, A., & Ullah, S. Sea buckthorn seed oil protects against the oxidative stress produced by thermally oxidized lipids. Food Chemistry (2015), http://dx.doi.org/10.1016/j.foodchem.2015.03.053
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3. Results 3.1. Characterization of lipids The total phenolic contents (TPC) of sea buckthorn seed oil were 3.38 ± 0.28 mg of GAE/g, PV was 0.9 meq/kg, free fatty acid contents were 0.97%, and para-anisidine value was 1.0. The PV of the oxidized ghee was 155.0 meq/kg, AV of 35.0 and FFA contents of the oxidized ghee was 6.3%. The PV of the control ghee was 2.6 meq/kg, AV was 11.0 and FFA was 0.87%. The RSA values of the control ghee and sea buckthorn seed oil were 58%. The RSA value of the oxidized ghee was 29.0% as shown in Table 1. The fatty acid composition of the un-oxidized ghee showed palmitic acid 35.9 g/100 g, linoleic acid 7.02 g/100 g, oleic acid 37.2 g/100 g and stearic acid 3.3 g/100 g. It also contains b-sistosterol of 8.8 g/100 g. In oxidized ghee, palmitic acid was 44.3 g/100 g, linoleic acid 3.6 g/100 g, oleic acid 46.3 g/100 g and stearic acid 5.7 g/100 g. Similarly, in sea buckthorn seed oil, palmitoleic acid 6.7 g/100 g, palmitic acid 11.8 g/100 g, linoleic acid 18.7 g/100 g, oleic acid 56.4 g/100 g and stearic acid 2.0 g/100 g (Table 1). The amount of squalane was 1.7 g/100 g. 3.2. Effects on the serum biochemistry The serum cholesterol levels of the control group were 69.6 and 66.0 mg/dL corresponds to the 7 and 14 days studies respectively. In SO group the serum total cholesterol were 76.4 and 68.0 mg/dL corresponds to the 7 and 14 days doses respectively. The values were close to the serum cholesterol level of the control group. In the OG group the serum cholesterol level increased significantly (p < 0.05) by comparing the 7 and 14 days levels and also as compared to control group. The OGSO1 group showed increased levels of the total cholesterol as compared with a control group. The serum cholesterol of OGSO3 group were 91.0 and 79.5 mg/dL corresponds to the 7 and 14 days doses, respectively, which shows a significant (p < 0.05) decrease as compared with OG group (Fig. 1A). The serum triacylglycerols (TGs) of the control group were 65.6 and 65.0 mg/dL corresponds to 7 and 14 days studies, respectively. In SO group TGs were 63.7 and 61.3 mg/dL corresponds to 7 and 14 days doses respectively, with no significant (p < 0.05) difference as compared to the control group. In OG group, TGs increased significantly (p < 0.05) as compared to the control group. The OGSO1 group showed an increase as compared to the control, while a decrease in the TGs was highly significant (p < 0.05) in OGSO3 group with respect to the OG group (Fig. 1B). The serum HDL-cholesterol levels of the control group were 38.0 and 37.0 mg/dL corresponds to 7 and 14 days studies respectively. The SO group shows a little increase as compared to the control group. The other groups showed no significant increase or decrease in serum HDL-cholesterol (Fig. 1C). The serum LDLcholesterol levels of the control group were 14.0 and 15.0 mg/dL corresponds to 7 and 14 days studies respectively. In the SO group, the serum LDL-cholesterol showed no significant (p < 0.05)
difference as compared to the control group. The OG group showed a significant (p < 0.05) increase as compared to the control group. In OGSO1 group, the serum LDL-cholesterol levels of 40.0 and 28.5 mg/dL corresponds to 7 and 14 days doses, respectively, showed a significant (p < 0.05) increase as compared to the control group. Similarly the OGSO3 group also showed a significant (p < 0.05) decrease as compared to OG group. (Fig. 1D). The serum glucose of the control group was 85.0 and 90.0 mg/ dL corresponds to 7 and 14 days studies respectively were close to the SO group. The OG and OGSO1 groups showed a significant (p < 0.05) decrease as compared to the control group. Further supplementation of SO showed significant (p < 0.05) increase as compared to OG group (Fig. 1E). The serum ALT in the SO group showed no significant (p < 0.05) difference as compared to the control group, while there was a significant (p < 0.05) increase of ALT in OG group as compared to the control group. In the OGSO1, ALT increased significantly (p < 0.05) as compared to the control group. Similarly the serum ALT of the OGSO3 group showed significant (p < 0.05) decrease as compared to the OG group (Fig. 1F). These results show that when SO supplementation for 14 days produces significant effects toward normalization of these parameters. A significant correlation of the serum ALT with serum LDL-cholesterol was also observed (supplementary materials). 3.3. Radical scavenging activity of liver lipids Fig. 2 shows a higher RSA value of the control liver lipids, while the values was significantly decreased with supplementation of SO. The supplementation of OG significantly lowered the RSA value of liver lipids as compared to the control group. The administration of SO along with OG, increases the RSA values with an increase of doses exceeding the value of the control RSA level of liver lipids. 3.4. Effects on hematological parameters The hemoglobin concentrations of the control group were 12.0 and 12.0 g/dL corresponds to 7 and 14 days studies respectively. The SO group showed no significant (p < 0.05) difference as compared to the control group, while the OG group shows a significant (p < 0.05) decrease. The OGSO1 group, showed a non-significant decrease as compared to the control group, in OGSO2–3 groups show an increase as compared to OG group (Table 2). The RBC of the control group was 5.94 and 5.8 106 cells per lL corresponds to 7 and 14 days studies respectively. In the SO group, the red blood cell count showed no significant (p < 0.05) increase as compared to control group. The OGSO1, OGSO2 and OGSO3 groups showed a significant (p < 0.05) increase in the total red blood cell count corresponds to 7 and 14 days doses as compared to the OG group (Table 2). The WBC of the control group was 7.1 and 6.8 103 cells per lL corresponds to 7 and 14 days studies respectively. The SO group shows no significant difference with respect to the control group. In the OG group levels of the WBC showed significant (p < 0.05)
Table 1 Characteristics and fatty acid composition of vegetable ghee and sea buckthorn oil. Sample
PV (meq/kg)
AV
FFA (%)
TPC (mg/g)
RSA (%)
Sea buckthorn oil Control ghee Oxidized ghee
0.9 ± 0.05 2.6a ± 0.47 155.0b ± 7.5
1.0 ± 0.1 11.0a ± 0.25 35.0b ± 0.36
0.97 ± 0.15 0.87a ± 0.15 6.3b ± 0.46
3.38 ± 0.28 – –
58.0 ± 0.21 58.0a ± 0.41 29.0b ± 1.0
Fatty acids (g/100 g)* C16:0
C16:1
C18:0
C18:1
C18:2
11.8 35.9 44.3
6.7 0.3 0.1
2.0 3.3 5.7
56.4 37.2 46.3
18.7 7.0 3.6
Values are expressed as mean ± SD of n = 3. Mean with different subscript letter differ significantly (p < 0.05). PV, peroxide value; AV, anisidine value; FFA, free fatty acids; TPC, total phenolic contents; RSA, radical scavenging activity. * Values expressed are the composition (g/100 g) obtained from the percent peak area of GC–MS chromatogram of the respective sample.
Please cite this article in press as: Zeb, A., & Ullah, S. Sea buckthorn seed oil protects against the oxidative stress produced by thermally oxidized lipids. Food Chemistry (2015), http://dx.doi.org/10.1016/j.foodchem.2015.03.053
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Fig. 1. Effects of supplementation of SBT oil on the serum biochemical parameters of rabbits. Different letters (a–e) represent significance at (p < 0.05) (Holm–Sidak test).
Fig. 2. Effects of the supplementation of sea buckthorn seed oil on the radical scavenging activity (RSA) of the liver lipids. Different letters (a–f) represent significance at (p < 0.05) (Holm–Sidak test).
increase as compared to the control group, while the OGSO1 group showed non-significant increase compared with control and SO groups. In the OGSO2 and OGSO3 groups no significant difference was observed with respect to control as shown in (Table 2).
The blood platelet level of the control group was 141 and 163.6 103 cell per lL corresponds to 7 and 14 days studies respectively. The increase in blood platelets of the SO and OG in the 7 and 14 days treatment was highly significant (p < 0.05) as compared to the control. Similarly, a significant (p < 0.05) increase in the blood platelets of OGSO1 and OGSO2 groups was observed as compared to control group, while the OGSO3 group showed significant (p < 0.05) decrease as compared to the OG group (Table 2). The mean corpuscular hemoglobin (MCH) of the control group were 20.0 and 20.0 pg/cell corresponds to 7 and 14 days studies, respectively were close to the SO group (Table 2). The OG, OGSO1, OGSO2 and OGSO3 groups showed a small non-significant increased as compared to the control group (Table 2). Similar observations were recorded for mean corpuscular volume (MCV) and hematocrit values. 3.5. Effects on the liver pathology The changes in biochemical and hematological parameters were further confirmed by a comparative histo-architectural
Please cite this article in press as: Zeb, A., & Ullah, S. Sea buckthorn seed oil protects against the oxidative stress produced by thermally oxidized lipids. Food Chemistry (2015), http://dx.doi.org/10.1016/j.foodchem.2015.03.053
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A. Zeb, S. Ullah / Food Chemistry xxx (2015) xxx–xxx Table 2 Effects of supplementation of sea buckthorn oil on the hematological parameters of rabbits. Group
Hb (g/dL)
lL) 106
RBC (cells/
WBC (cells/ lL) 103
Platelets (cells /lL) 103
MCH (pg/cell)
MCV (fL/cell)
HCT (%)
Treatment days
Control SO OG OGSO1 OGSO2 OGSO3
7
14
7
14
7
14
7
14
7
14
7
14
7
14
a
a
5.94 5.8 4.9 4.9 5.0 5.4
5.8 5.9 3.9 5.6 5.3 5.8
7.1 7.7 9.4 6.3 10.1 10.3
6.8 6.4 12.0 7.5 7.0 5.03
141.0 170.3 212.0 235.6 155.0 179.0
163.6 210.6 513 251.3 321.7 246.7
a
a
a
a
a
a
12.0 ± 0.2 a 12.0 ± 2.0 a 11.0 ± 0.8 a 9.9 ± 0.6 a 11.0 ± 0.7 a 11.0 ± 0.8
12.0 ± 0.5 a 13.0 ± 2.0 b 8.4 ± 0.6 a 12.0 ± 1.1 a 12.0 ± 0.4 a 13.0 ± 1.5
20.0 ± 1.7 a 20.0 ± 1.4 a 22.0 ± 2.2 a 20.0 ± 1.2 a 22.0 ± 0.6 a 21.0 ± 1.7
20.0 ± 2.0 a 21.0 ± 2.1 a 22.0 ± 1.4 a 21.0 ± 2.4 a 23.0 ± 1.5 a 23.0 ± 2.4
78.0 ± 7.0 a 79.0 ± 4.0 b 87.0 ± 8.3 a 81.0 ± 3.2 b 87.0 ± 2.7 a 82.0 ± 5.3
79.0 ± 7.3 a 81.0 ± 5.1 b 91.0 ± 5.7 a 81.0 ± 7.6 a 87.0 ± 5.3 a 86.0 ± 7.4
46.0 ± 0.75 a 46.0 ± 6.1 b 42.0 ± 2.6 b 40.0 ± 2.0 a 43.0 ± 2.3 a 44.0 ± 2.5
46.0 ± 1.7 48.0 ± 6.5 b 35.0 ± 2.0 a 45.0 ± 3.4 a 46.0 ± 1.2 a 50.0 ± 4.5 a
Values are expressed as mean ± SD of n = 3 or otherwise mentioned. Hb, hemoglobin; RBC, red blood cells; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; HCT, hematocrit. SO, sea buckthorn oil; OG, oxidized ghee; OGSO1, oxidized ghee (1 g/kg bwt) with sea buckthorn oil (1 g/kg bwt); OGSO2, oxidized ghee (1 g/ kg bwt) with sea buckthorn oil (2 g/kg bwt) and OGSO3, oxidized ghee (1 g/kg bwt) with sea buckthorn oil (3 g/kg bwt). Different letters in each column represent significant at (p < 0.05, Holm–Sidak test).
examination of the liver sections from rabbits fed on oxidized ghee and sea buckthorn seed oil. The gross morphology of liver in control (Fig. 3A) and SO (Fig. 3B) fed rabbits showed normal structure. In case of OG treated group, the section shows a disarray of hepatic cord and lobular architecture. The central veins were disoriented (shown by arrow) and the portal tract showed increased infiltrate of lymphocytes; the later have crossed the limiting plates and piece meal necrosis was seen in periportal areas. At focal areas porto-portal fibrosis with associated lymphocytic infiltrate was also seen. Kupffer cells were reactive and plump. Fatty changes near the portal tracts as shown in the Fig. 3C. In case of OGSO1 group, the central vein showed abnormal endothelial lining with some evidence of pericentral fibrosis. The hepatic cords are regularly oriented with intervening reactive plump. Portal area shows increased lymphocytes. The lymphocytes have crossed over the limiting flats of the lobule and have caused piece meal necrosis of the adjacent hepatocytes. The supporting tissue of the portal tracts shows mild infiltrate of lymphocytes only. The cholangiolar epithelia show proliferative activity (Fig. 3D). In OGSO2 treated group, the gross morphology of the liver shows well oriented hepatic cords and reactive kupffer cells. The portal tracts show increase infiltration of lymphocytes; the later have crossed the limiting plates and piece meal necrosis is seen in periportal areas. At focal areas bridging necrosis is seen in the porto-portal
pattern. The cholangioles were increased and show proliferative activity. No evidence of malignancy was seen as shown in Fig. 3E. The gross morphology of the liver of the OGSO3 group shows central veins showed normal endothelial linings with no evidence of pericentral fibrosis. Hepatic cords were well oriented and kupffer calls were non-reactive. The portal tracts showed branches of the hepatic artery and portal vein with normal morphology. No evidence of malignancy was seen as shown in Fig. 3F.
4. Discussion Fat frying is a traditional method for the preparation of food. In restaurants, fast food chains as well as in industrial frying operations, fat frying is the main procedure. Due to the high price of vegetable oils, deep ghee processed food products are used by people in Pakistan and across several countries in Asia. During frying of food, the ghee is exposed to high temperature in the presence of air and moisture. The oxidized ghee thus mixes with the food and cause toxic effects (Kamal-Eldin, Makinen, & Lampi, 2003; Ramadan, Amer, & Sulieman, 2006). Humans and animals are susceptible to oxidative damage, which depends on the balance between oxidative stress and antioxidant defense capacity. Many different kinds of antioxidants are found in various plants.
Fig. 3. Effects of the supplementation of sea buckthorn seed oil on the liver histology of rabbits. Pictures of the liver specimens were taken with light microscope, (A) control group, (B) SO group, (C) OG group, (D) OGSO1 group, (E) OGSO2 group, and (F) OGSO3 group. Arrow indicates the disoriented central vein.
Please cite this article in press as: Zeb, A., & Ullah, S. Sea buckthorn seed oil protects against the oxidative stress produced by thermally oxidized lipids. Food Chemistry (2015), http://dx.doi.org/10.1016/j.foodchem.2015.03.053
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Antioxidants have the potential to inhibit the oxidative breakdown of the lipid component of food and are also important for the defense of living cells against oxidative damage. Sea buckthorn (Hippophae rhamnoides L.) is a rich source of antioxidants was therefore used in this study. Sea buckthorn seed oil contains 338 mg of GAE/100 g of TPC. Arora, Sunil, Ashish, Ravi, and Tsering (2012) reported 162.56 mg GAE/g total phenolic contents in the methanolic seed extract of sea buckthorn, which is much higher than our reported value. Similarly Gao, Ohlander, Jeppsson, Bjork, and Trajkovski (2000) reported that the total phenolic content of sea buckthorn fruit range from 114 to 244 mg GAE/100 g. The variation in TPC may be due to types of extraction, methodology and variety. The peroxide values of the vanaspati ghee increase significantly after oxidation, and subsequently reduce the RSA values. The present result is in agreement with Zeb and Mehmood (2012) who reported an increase in the peroxide value of vanaspati ghee with increasing time of oxidation. The secondary oxidation products were measured in terms of anisidine value, which was increased significantly with oxidation compared to the control in vanaspati ghee. The increase in p-anisidine value and FFA is in agreement with Ahmed, Atta, Sohail, Khan, and Akhtar (2011) who reported that p-anisidine value and FFA values of vanaspati ghee increase with oxidation. Sea buckthorn seed oil showed lower p-anisidine value compared to oxidized ghee and thus may be helpful in oxidation and more beneficial to health. The present results for fatty acid composition are in agreement with Ahmed et al. (2011) who showed that oxidation of vanaspati ghee decrease the linoleic acid and increase palmitic acid, oleic acid and stearic acid contents. Fatty acid profile of sea buckthorn seed oil showed higher concentrations of oleic acid and linoleic acid, while palmitoleic acid and stearic acid in low quantity were in accordance with previous study (Abid, Hussain, & Ali, 2007). Recent studies showed that oleic acid is the main fatty acid responsible for stability against the thermal stress (Zeb, 2012). The serum biochemical parameters like TG, total cholesterol, LDL-c showed an increase while a decrease in HDL-c was observed with feeding oxidized ghee, while the supplementation of SO at different doses reduced the toxic level by positively affecting the above parameters. The results are in agreement with the finding of SBT oil against CCl4 induced hepatotoxicity in rats (Hsu et al., 2009). Recent results by Zeb and Mehmood (2012) showed a dose dependent increase in triglycerides, total cholesterol and LDLcholesterol, while a statistically non-significant increase in the HDL-cholesterol of the rabbits. Similarly Shila et al. (2011) observed that the serum total cholesterol, HDL-cholesterol and malondialdehyde increased significantly (p < 0.05) by feeding the oxidized oil as compared to fresh oil fed group. Basu et al. (2007) reported that sea buckthorn oil fed with high cholesterol diet has shown to restrict the further rise of total cholesterol and thus caused a significant decline of triglycerides and LDL-cholesterol. The HDL-cholesterol levels of the sea buckthorn seed oil treated animals were significantly higher than the non-treated animals showing more protection. The increase in the Hb and RBC contents was due to the feeding of oxidized ghee, while the supplementation of SO is helpful in recovery. A significant increase was observed in the WBC and platelet count in all treated groups with the exception of control and SO groups. This shows that supplementation of oxidized ghee produces significant stress on these blood markers. The present results are in agreement with the studies of Mesembe, Ibanga, and Osim (2004) which shows that oxidation of lipids cause decrease in the hemoglobin concentration. Igiri, Ibegbu, and Osim (1994) stated that intestinal mucosa is severely damaged by thermally oxidized palm oil in rats. This damage may lead to a lower absorption of iron by the intestinal mucosa which results into a decrease
bioavailability of iron in the system. Similarly Chen, Zhong, Liu, and Ge (2003) reported an increase in red blood cell count of the mice fed on sea buckthorn seed oil. These results suggest that SO has the ability to maintain normal hematological parameters under oxidative stress induced by oxidized lipids. Sea buckthorn seed oil protects the liver and kidney against the toxicity of oxidized ghee and increased the level of serum glucose to normal. Similarly the serum ALT was elevated by OG and maintained by supplementation of SO. It suggested that sea buckthorn seed oil is able to improve the condition of hepatocytes, and thus the liver function. This was observed in RSA value of the lipids, which was higher in the sea buckthorn seed oil fed group. The hepto-protective potential of sea buckthorn oil against the oxidized lipids might be due to anti-lipid peroxidation and antioxidants (tocopherols, vitamin C, and phenolic compounds) present in the oil. The mechanism may be the regeneration of parenchymal cells, thus protecting against membrane fragility and reduce the levels of marker enzymes into the blood by the sea buckthorn oil. 5. Conclusions Sea buckthorn seed oil has strong antioxidative activity. The present study shows the protective effects of sea buckthorn seed oil against the side effects of thermally oxidized ghee. Oxidation of ghee decreases the level of radical scavenging activity and increases the peroxide, p-anisidine and free fatty acid values of the ghee. Oxidation of ghee also decreases the level of unsaturated fatty acids and increases the level of saturated fatty acids. Oxidized consumption increases the serum total cholesterol, LDL-cholesterol, TGs, ALT and decreases the serum glucose. Oxidized ghee also causes reduction in RBC, Hb, hematocrit and increase in the WBC and platelets counts as well as degenerative changes in liver and decreases the radical scavenging activity level of the lipids in the liver. Sea buckthorn seed oil supplementation with thermally oxidized ghee significantly lower the serum LDL-cholesterol, ALT, triglycerides and increase the RSA and serum glucose. Hematological parameters were optimized by sea buckthorn seed oil supplementation. The results of the present study showed that sea buckthorn seed oil supplemented with thermoxidized vanaspati ghee reduce the side effects of vanaspati ghee. Sea buckthorn seed oil maintains the stability of the immune system and provides protection to the liver against oxidative stress. Conflict of interest The authors have declared no conflict of interest to anybody or institution. Acknowledgment We are grateful to the University of Malakand for financial support to conduct this study. The financial support of Higher Education Commission (HEC) Pakistan for presenting this work at 11th Euro Fed Lipid Congress, Antalya, Turkey is gratefully acknowledged. We are also grateful of Prof. Dr. Tufail Ahmad Shirazi of National Centre of Excellence in Analytical Chemistry, University of Sindh Jamshoro, Pakistan for his help in fatty acid analysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2015. 03.053.
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Kamal-Eldin, A., Makinen, M., & Lampi, A. (2003). The challenging contribution of hydroperoxides to the lipid oxidation mechanism. In A. Kamal-Eldin (Ed.), Lipids oxidation (pp. 1–35). Illinois: AOCS Press. Kim, H., Cho, H., Seo, Y., Kim, S., Yoon, M. Y., Kang, H., et al. (2012). Inhibitory effects of sea buckthorn (Hippophae rhamnoides L.) seed on UVB-induced photo-aging in human dermal fibroblasts. Biotechnology & Bioprocess Engineering, 17, 465–474. Mesembe, O. E., Ibanga, I., & Osim, E. (2004). The effects of fresh and thermoxidized palm oil diets on some haematological indices in the rat. Nigerian Journal of Physiological Science, 19, 86–91. Ramadan, M. F., Amer, M. M. A., & Sulieman, A. R. M. (2006). Correlation between physicochemical analysis and radical-scavenging activity of vegetable oil blends as affected by frying of French fries. European Journal of Lipid Science & Technology, 108, 670–678. Shila, S., Jalal, J., Ali, A., Leila, A., Roghayeh, R., Narges, K., et al. (2011). The effect of consuming oxidized oil supplemented with fiber on lipid profiles in rat model. Journal of Research in Medical Science, 16, 1541–1549. Yang, B., & Kallio, H. (2002). Composition and physiological effects of sea buckthorn (Hippophae) lipids. Trends in Food Science and Technology, 13, 160–167. Yang, C. M., Kendall, C. W., Stamp, D., Medline, A., Archer, M. C., & Bruce, W. R. (1998). Thermally oxidized dietary fat and colon carcinogenesis in rodents. Nutrition and Cancer, 30, 69–73. Yeh, Y., Hsieh, Y., Lee, Y., & Shen, Y. (2012). Dietary sea buckthorn (Hippophae rhamnoides L.) reduces toxicity of oxidized cholesterol in rats. e-SPEN Journal, 7, e69–e77. Zeb, A. (2004). Important therapeutic uses of sea buckthorn (Hippophae); a review. Journal of Biological Science, 4, 687–693. Zeb, A. (2012). Triacylglycerols composition, oxidation and oxidation compounds in camellia oil using liquid chromatography-mass spectrometry. Chemistry and Physics of Lipids, 165, 608–614. Zeb, A., & Hussain, S. (2014). Sea buckthorn seed oil provides protection in the oxidative stress produced by thermally oxidized sunflower oil in rabbits. Journal of Food Biochemistry, 38, 498–508. Zeb, A., & Khan, I. (2008). Composition and medicinal properties of sea buckthorn juice. In H. Kallio & B. Yang (Eds.), Sea Buckthorn – A multipurpose wonder plant. Vol. III – Advances in research & development (pp. 205–214). N. Delhi: Daya Publishing House. Zeb, A., & Khan, I. (2009). Pharmacological applications of sea buckthorn (Hippophae). In M. Eddouks (Ed.), Advances in phytotherapy research (pp. 1–16). N. Delhi: Research Signpost. Zeb, A., & Mehmood, A. (2012). Effects of oxidized vanaspati ghee on the serum lipids profile and radical scavenging activity of the in vitro lipids of liver, brain and muscles. Turkish Journal of Biochemistry, 37, 417–423. Zeb, A., & Rahman, W. (2012). Effect of oxidized tallow on rabbit serum lipids and antioxidant activity of the in-vitro lipids. Toxicological Research, 28, 151–157.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Sea buckthorn seed oil protects against the oxidative stress produced by thermally oxidized lipids Alam Zeb ⇑, Sana Ullah Department of Biotechnology, University of Malakand, Chakdara, Pakistan
a r t i c l e
i n f o
Article history: Received 22 December 2014 Received in revised form 12 March 2015 Accepted 15 March 2015 Available online xxxx Keywords: Sea buckthorn oil Oxidized vegetable ghee Oxidative stress Serum lipids Rabbits Liver
a b s t r a c t Thermally oxidized vegetable ghee was fed to the rabbits for 14 days with specific doses of sea buckthorn seed oil (SO). The ghee and SO were characterized for quality parameters and fatty acid composition using GC–MS. Rabbits serum lipid profile, hematology and histology were investigated. Major fatty acids were palmitic acid (44%) and oleic acid (46%) in ghee, while SO contains oleic acid (56.4%) and linoleic acid (18.7%). Results showed that oxidized vegetable ghee increases the serum total cholesterol, LDLcholesterols, triglycerides and decrease the serum glucose. Oxidized ghee produced toxic effects in the liver and hematological parameters. Sea buckthorn oil supplementation significantly lowered the serum LDL-cholesterols, triglycerides and increased serum glucose and body weight of the animals. Sea buckthorn oil was found to reduce the toxic effects and degenerative changes in the liver and thus provides protection against the thermally oxidized lipids induced oxidative stress. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Sea buckthorn (SBT) is a famous medicinal and aromatic plant, widely admired for its role to possess multiple health promoting properties. Sea buckthorn has been found to be a rich source of carotenoids, vitamin E, C, and different phenolic compounds. The seed oil had been found to help the recovery of skin injuries and skin diseases (eczema, burns, and wounds). It also helps from the damaging effects of sun on the skin, and during the radiation therapy and cosmetic laser surgery (Zeb & Khan, 2008, 2009). It had also been used in digestive ulcers and cardiovascular disease (Yang & Kallio, 2002; Zeb, 2004). The scientific studies during the recent decade confirmed the medicinal and nutritional values of sea buckthorn. The most important of these studies are the antioxidant and anti-carcinogenic properties. In animal studies, sea buckthorn products showed significant antioxidant potential. For example, Geetha et al. (2008) showed that the sea buckthorn leaf extract has a significant hepato-protective effect which was attributed to the antioxidant compounds present in the extract. Similarly,
Abbreviations: SO, sea buckthorn oil; OG, oxidized ghee; GC–MS, gas chromatography–mass spectrometry; LDL, low density lipoproteins; HDL, high density lipoproteins; PV, peroxide value; AV, anisidine value; FFA, free fatty acids; Hb, hemoglobin; RBC, red blood cells; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin. ⇑ Corresponding author. Tel.: +92 945 761622. E-mail address: [email protected] (A. Zeb).
Gumustekin et al. (2010) studied the effects of nicotine-induced oxidative stress in rat heart with vitamin E and sea buckthorn extract. The authors showed that supplementation of extract increased superoxide dismutase and glutathione S-transferase activities, which may be contributing to the prevention of nicotine-induced oxidative stress in rat heart. They also suggested that the antioxidant activity of the sea buckthorn extract was due to the presence of quercetin and isorahmnetin, tocopherols, and carotenoids. Arimboor and Arumughan (2012) observed that SBT has a considerably high antioxidant and xanthine oxidase inhibitory potential shown by proanthocyanidins of the seed. Similarly, the Kim et al. (2012) findings suggested that the sea buckthorn seed extract may be a potential therapeutic agent for preventing and treating skin photo-aging. The results of Hsu, Tsai, Chen, and Lu (2009) showed that oral administration of SBT seed oil for eight weeks significantly lowered serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), triglyceride (TG), and cholesterol, which were elevated by CCl4 (1 mL/kg) in mice. Thus, different products of sea buckthorn have significant ameliorating properties. Vanaspati ghee is one of the important components for the preparation of various kinds of foods. During frying or cooking the thermal stress oxidizes vanaspati ghee and produce toxic substances. These toxic substances subsequently enter the food matrix and ingested by the human (Zeb & Rahman, 2012). Experiments had shown that thermally oxidized lipids are potentially carcinogenic (Yang et al., 1998) and enhance the growth of
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Please cite this article in press as: Zeb, A., & Ullah, S. Sea buckthorn seed oil protects against the oxidative stress produced by thermally oxidized lipids. Food Chemistry (2015), http://dx.doi.org/10.1016/j.foodchem.2015.03.053
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hepato-carcinoma in experimental animals (Jurek et al., 2005). Recently we have shown that thermally oxidized vanaspati ghee had produced significant effects in the serum lipid profile as well as peroxidation in different tissue (Zeb & Mehmood, 2012). It is therefore necessary to find out the possible remedies to counterbalance the oxidative stress produced by oxidized lipids in our daily foods. Various plant extracts have been used to possess the antioxidant potential. The studies of Yeh, Hsieh, Lee, and Shen (2012) showed that oxidative stress induced by oxidized cholesterol can be minimized using a supplement of 0.1% of dietary Sea buckthorn. Similarly, Zeb and Hussain (2014) showed that the sea buckthorn seed powder provides protection against the toxic properties of thermally oxidized sunflower oil. The study, however does not provide information about the similar potential of the most widely used products of sea buckthorn such as seed oil. This study was therefore aimed to find out further correlation of the oxidative stress induced by thermally oxidized lipids with higher saturation and sea buckthorn oil in animal models. 2. Materials and methods 2.1. Materials Sea buckthorn seed oil and vanaspati ghee was obtained from the commercial market. All other chemicals and reagents were of ACS grade from Sigma Aldrich (USA) or otherwise mentioned. 2.2. Thermal oxidation Vanaspati ghee was thermally oxidized on the hot plate at 160 °C for successive 10 h in the open air to mimic the real frying conditions. After thermal oxidation the samples were stored in the refrigerator at 20 °C. 2.3. Characterization of sea buckthorn oil The peroxide value (PV), p-anisidine value (AV) and free fatty acids (FFA) of ghee and SBT oil samples were determined using the AOCS official methods (AOCS., 1998). All samples were measured in triplicate or otherwise mentioned. Total phenolic contents (TPC) were measured using Folin–Ciocalteu reagent and quantification was carried out with the help of Gallic acid calibration curve. Fatty acids were converted to their respective fatty acid methylesters (FAMEs). Briefly, a sample of 20 mg (±0.5 mg) was weighted in 20 mL vial. Then 6 mL of 0.5 M methanolic NaOH was added and stirred at 80 °C for 30 min. After the samples were cooled, BF3/ methanol was added and stirred further at 80 °C for 15 min. Water and n-heptane phases were separated. The heptane phase was injected into gas chromatography coupled with mass spectrometry (Agilent 5975, Agilent Germany). Fatty acids and sterol were identified from their relative and absolute retention times and also by the MS library database. The values were expressed as g/100 g determined from the peak area.
animal house facility. Feed and water were provided ad libitum. The rabbits were fed with pellets from the commercial market and labeled chemical composition of the pellets showed that they contained 17.5% protein, 14.0% fiber, 2.7% fat. All animals were placed one week ahead to get familiarized with the environment. Rabbits were classified into six groups, consisting of three animals per group. Each group is designated as the individual treatment. The rabbits were fed with oxidized ghee and sea buckthorn seed oil for 14 days, according to the feeding scheme given below: Group C: control fed on normal food; Group SO: 2 g/kg bwt SO; Group OG: 1 g/kg bwt OG; Group OGSO1: 1 g/kg bwt OG and 1 g/kg bwt SO; Group OGSO2: 1 g/kg bwt OG and 2 g/kg bwt SO; and Group OGSO3: 1 g/kg bwt OG and 3 g/kg bwt SO. The blood samples were collected in falcon tubes and centrifuge for 10 min to obtain serum for further analysis.
2.5. RSA of liver lipids The liver tissues of each individual rabbit were taken and stored in formalin. Lipids were extracted using the modified procedure of Folch, Lees, and Sloane-Stanley (1957). The radical scavenging assays (RSA) of the lipids extracted from the tissues were measured using DPPH free radicals (Zeb & Mehmood, 2012).
2.6. Serum biochemical analysis Serum biochemical parameters like cholesterol, HDL-cholesterol, LDL-cholesterol, ALT, and glucose were measured using HUMAN (Human Diagnostics, Germany) kits, while total triglycerides were measured using DiaSys (DiaSys Diagnostic Systems GmbH, Germany) kit.
2.7. Histopathological studies of liver A section of the median lobe of the liver was dissected and fixed in 10% formalin buffered for at least 14 h. The section was then dehydrated with ethanol solutions and processed for embedding in paraffin. Sections of 8–10 mm in thickness were cut, deparaffinized, rehydrated, and stained. The slides were studied by binocular microscope (model M 7000 D series, Swift Instruments, Inc., Japan) and the pictures were documented using the digital camera (DCM 130) of the microscope with the resolution of 1.3 MP.
2.8. Hematological studies For hematological examination 2 mL blood was collected from the jugular vein of rabbits in EDTA tubes. Hematology or blood profile was carried out by automatic digital machine (CELL-DYN 3200 Abbott Diagnostic Division, Canada) for the following parameters; Blood Hemoglobin Level (Hb), Blood Total RBC Level (RBC), Blood HCT Level (HCT %), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Blood Platelets Concentration, and Blood White blood cell Concentration.
2.4. Experimental animals All experiments were carried out according to the approved guidelines for the care and proper use of the animals of the Department of Biotechnology, University of Malakand. Rabbits selected for the study because of the highly selective and developed organ system and were easily available animals for these experiments. Rabbits were grouped by random distribution into six groups, irrespective of the gender and placed in the same
2.9. Statistical analysis All analyses were carried out in triplicate or otherwise mentioned. Data were analyzed by one-way analysis of variance (ANOVA) and Holm–Sidak method of multiple comparison method at a = 0.05 using SigmaPlot for windows version 12.0 (Systat Software, Inc., 2011).
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3. Results 3.1. Characterization of lipids The total phenolic contents (TPC) of sea buckthorn seed oil were 3.38 ± 0.28 mg of GAE/g, PV was 0.9 meq/kg, free fatty acid contents were 0.97%, and para-anisidine value was 1.0. The PV of the oxidized ghee was 155.0 meq/kg, AV of 35.0 and FFA contents of the oxidized ghee was 6.3%. The PV of the control ghee was 2.6 meq/kg, AV was 11.0 and FFA was 0.87%. The RSA values of the control ghee and sea buckthorn seed oil were 58%. The RSA value of the oxidized ghee was 29.0% as shown in Table 1. The fatty acid composition of the un-oxidized ghee showed palmitic acid 35.9 g/100 g, linoleic acid 7.02 g/100 g, oleic acid 37.2 g/100 g and stearic acid 3.3 g/100 g. It also contains b-sistosterol of 8.8 g/100 g. In oxidized ghee, palmitic acid was 44.3 g/100 g, linoleic acid 3.6 g/100 g, oleic acid 46.3 g/100 g and stearic acid 5.7 g/100 g. Similarly, in sea buckthorn seed oil, palmitoleic acid 6.7 g/100 g, palmitic acid 11.8 g/100 g, linoleic acid 18.7 g/100 g, oleic acid 56.4 g/100 g and stearic acid 2.0 g/100 g (Table 1). The amount of squalane was 1.7 g/100 g. 3.2. Effects on the serum biochemistry The serum cholesterol levels of the control group were 69.6 and 66.0 mg/dL corresponds to the 7 and 14 days studies respectively. In SO group the serum total cholesterol were 76.4 and 68.0 mg/dL corresponds to the 7 and 14 days doses respectively. The values were close to the serum cholesterol level of the control group. In the OG group the serum cholesterol level increased significantly (p < 0.05) by comparing the 7 and 14 days levels and also as compared to control group. The OGSO1 group showed increased levels of the total cholesterol as compared with a control group. The serum cholesterol of OGSO3 group were 91.0 and 79.5 mg/dL corresponds to the 7 and 14 days doses, respectively, which shows a significant (p < 0.05) decrease as compared with OG group (Fig. 1A). The serum triacylglycerols (TGs) of the control group were 65.6 and 65.0 mg/dL corresponds to 7 and 14 days studies, respectively. In SO group TGs were 63.7 and 61.3 mg/dL corresponds to 7 and 14 days doses respectively, with no significant (p < 0.05) difference as compared to the control group. In OG group, TGs increased significantly (p < 0.05) as compared to the control group. The OGSO1 group showed an increase as compared to the control, while a decrease in the TGs was highly significant (p < 0.05) in OGSO3 group with respect to the OG group (Fig. 1B). The serum HDL-cholesterol levels of the control group were 38.0 and 37.0 mg/dL corresponds to 7 and 14 days studies respectively. The SO group shows a little increase as compared to the control group. The other groups showed no significant increase or decrease in serum HDL-cholesterol (Fig. 1C). The serum LDLcholesterol levels of the control group were 14.0 and 15.0 mg/dL corresponds to 7 and 14 days studies respectively. In the SO group, the serum LDL-cholesterol showed no significant (p < 0.05)
difference as compared to the control group. The OG group showed a significant (p < 0.05) increase as compared to the control group. In OGSO1 group, the serum LDL-cholesterol levels of 40.0 and 28.5 mg/dL corresponds to 7 and 14 days doses, respectively, showed a significant (p < 0.05) increase as compared to the control group. Similarly the OGSO3 group also showed a significant (p < 0.05) decrease as compared to OG group. (Fig. 1D). The serum glucose of the control group was 85.0 and 90.0 mg/ dL corresponds to 7 and 14 days studies respectively were close to the SO group. The OG and OGSO1 groups showed a significant (p < 0.05) decrease as compared to the control group. Further supplementation of SO showed significant (p < 0.05) increase as compared to OG group (Fig. 1E). The serum ALT in the SO group showed no significant (p < 0.05) difference as compared to the control group, while there was a significant (p < 0.05) increase of ALT in OG group as compared to the control group. In the OGSO1, ALT increased significantly (p < 0.05) as compared to the control group. Similarly the serum ALT of the OGSO3 group showed significant (p < 0.05) decrease as compared to the OG group (Fig. 1F). These results show that when SO supplementation for 14 days produces significant effects toward normalization of these parameters. A significant correlation of the serum ALT with serum LDL-cholesterol was also observed (supplementary materials). 3.3. Radical scavenging activity of liver lipids Fig. 2 shows a higher RSA value of the control liver lipids, while the values was significantly decreased with supplementation of SO. The supplementation of OG significantly lowered the RSA value of liver lipids as compared to the control group. The administration of SO along with OG, increases the RSA values with an increase of doses exceeding the value of the control RSA level of liver lipids. 3.4. Effects on hematological parameters The hemoglobin concentrations of the control group were 12.0 and 12.0 g/dL corresponds to 7 and 14 days studies respectively. The SO group showed no significant (p < 0.05) difference as compared to the control group, while the OG group shows a significant (p < 0.05) decrease. The OGSO1 group, showed a non-significant decrease as compared to the control group, in OGSO2–3 groups show an increase as compared to OG group (Table 2). The RBC of the control group was 5.94 and 5.8 106 cells per lL corresponds to 7 and 14 days studies respectively. In the SO group, the red blood cell count showed no significant (p < 0.05) increase as compared to control group. The OGSO1, OGSO2 and OGSO3 groups showed a significant (p < 0.05) increase in the total red blood cell count corresponds to 7 and 14 days doses as compared to the OG group (Table 2). The WBC of the control group was 7.1 and 6.8 103 cells per lL corresponds to 7 and 14 days studies respectively. The SO group shows no significant difference with respect to the control group. In the OG group levels of the WBC showed significant (p < 0.05)
Table 1 Characteristics and fatty acid composition of vegetable ghee and sea buckthorn oil. Sample
PV (meq/kg)
AV
FFA (%)
TPC (mg/g)
RSA (%)
Sea buckthorn oil Control ghee Oxidized ghee
0.9 ± 0.05 2.6a ± 0.47 155.0b ± 7.5
1.0 ± 0.1 11.0a ± 0.25 35.0b ± 0.36
0.97 ± 0.15 0.87a ± 0.15 6.3b ± 0.46
3.38 ± 0.28 – –
58.0 ± 0.21 58.0a ± 0.41 29.0b ± 1.0
Fatty acids (g/100 g)* C16:0
C16:1
C18:0
C18:1
C18:2
11.8 35.9 44.3
6.7 0.3 0.1
2.0 3.3 5.7
56.4 37.2 46.3
18.7 7.0 3.6
Values are expressed as mean ± SD of n = 3. Mean with different subscript letter differ significantly (p < 0.05). PV, peroxide value; AV, anisidine value; FFA, free fatty acids; TPC, total phenolic contents; RSA, radical scavenging activity. * Values expressed are the composition (g/100 g) obtained from the percent peak area of GC–MS chromatogram of the respective sample.
Please cite this article in press as: Zeb, A., & Ullah, S. Sea buckthorn seed oil protects against the oxidative stress produced by thermally oxidized lipids. Food Chemistry (2015), http://dx.doi.org/10.1016/j.foodchem.2015.03.053
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Fig. 1. Effects of supplementation of SBT oil on the serum biochemical parameters of rabbits. Different letters (a–e) represent significance at (p < 0.05) (Holm–Sidak test).
Fig. 2. Effects of the supplementation of sea buckthorn seed oil on the radical scavenging activity (RSA) of the liver lipids. Different letters (a–f) represent significance at (p < 0.05) (Holm–Sidak test).
increase as compared to the control group, while the OGSO1 group showed non-significant increase compared with control and SO groups. In the OGSO2 and OGSO3 groups no significant difference was observed with respect to control as shown in (Table 2).
The blood platelet level of the control group was 141 and 163.6 103 cell per lL corresponds to 7 and 14 days studies respectively. The increase in blood platelets of the SO and OG in the 7 and 14 days treatment was highly significant (p < 0.05) as compared to the control. Similarly, a significant (p < 0.05) increase in the blood platelets of OGSO1 and OGSO2 groups was observed as compared to control group, while the OGSO3 group showed significant (p < 0.05) decrease as compared to the OG group (Table 2). The mean corpuscular hemoglobin (MCH) of the control group were 20.0 and 20.0 pg/cell corresponds to 7 and 14 days studies, respectively were close to the SO group (Table 2). The OG, OGSO1, OGSO2 and OGSO3 groups showed a small non-significant increased as compared to the control group (Table 2). Similar observations were recorded for mean corpuscular volume (MCV) and hematocrit values. 3.5. Effects on the liver pathology The changes in biochemical and hematological parameters were further confirmed by a comparative histo-architectural
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A. Zeb, S. Ullah / Food Chemistry xxx (2015) xxx–xxx Table 2 Effects of supplementation of sea buckthorn oil on the hematological parameters of rabbits. Group
Hb (g/dL)
lL) 106
RBC (cells/
WBC (cells/ lL) 103
Platelets (cells /lL) 103
MCH (pg/cell)
MCV (fL/cell)
HCT (%)
Treatment days
Control SO OG OGSO1 OGSO2 OGSO3
7
14
7
14
7
14
7
14
7
14
7
14
7
14
a
a
5.94 5.8 4.9 4.9 5.0 5.4
5.8 5.9 3.9 5.6 5.3 5.8
7.1 7.7 9.4 6.3 10.1 10.3
6.8 6.4 12.0 7.5 7.0 5.03
141.0 170.3 212.0 235.6 155.0 179.0
163.6 210.6 513 251.3 321.7 246.7
a
a
a
a
a
a
12.0 ± 0.2 a 12.0 ± 2.0 a 11.0 ± 0.8 a 9.9 ± 0.6 a 11.0 ± 0.7 a 11.0 ± 0.8
12.0 ± 0.5 a 13.0 ± 2.0 b 8.4 ± 0.6 a 12.0 ± 1.1 a 12.0 ± 0.4 a 13.0 ± 1.5
20.0 ± 1.7 a 20.0 ± 1.4 a 22.0 ± 2.2 a 20.0 ± 1.2 a 22.0 ± 0.6 a 21.0 ± 1.7
20.0 ± 2.0 a 21.0 ± 2.1 a 22.0 ± 1.4 a 21.0 ± 2.4 a 23.0 ± 1.5 a 23.0 ± 2.4
78.0 ± 7.0 a 79.0 ± 4.0 b 87.0 ± 8.3 a 81.0 ± 3.2 b 87.0 ± 2.7 a 82.0 ± 5.3
79.0 ± 7.3 a 81.0 ± 5.1 b 91.0 ± 5.7 a 81.0 ± 7.6 a 87.0 ± 5.3 a 86.0 ± 7.4
46.0 ± 0.75 a 46.0 ± 6.1 b 42.0 ± 2.6 b 40.0 ± 2.0 a 43.0 ± 2.3 a 44.0 ± 2.5
46.0 ± 1.7 48.0 ± 6.5 b 35.0 ± 2.0 a 45.0 ± 3.4 a 46.0 ± 1.2 a 50.0 ± 4.5 a
Values are expressed as mean ± SD of n = 3 or otherwise mentioned. Hb, hemoglobin; RBC, red blood cells; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; HCT, hematocrit. SO, sea buckthorn oil; OG, oxidized ghee; OGSO1, oxidized ghee (1 g/kg bwt) with sea buckthorn oil (1 g/kg bwt); OGSO2, oxidized ghee (1 g/ kg bwt) with sea buckthorn oil (2 g/kg bwt) and OGSO3, oxidized ghee (1 g/kg bwt) with sea buckthorn oil (3 g/kg bwt). Different letters in each column represent significant at (p < 0.05, Holm–Sidak test).
examination of the liver sections from rabbits fed on oxidized ghee and sea buckthorn seed oil. The gross morphology of liver in control (Fig. 3A) and SO (Fig. 3B) fed rabbits showed normal structure. In case of OG treated group, the section shows a disarray of hepatic cord and lobular architecture. The central veins were disoriented (shown by arrow) and the portal tract showed increased infiltrate of lymphocytes; the later have crossed the limiting plates and piece meal necrosis was seen in periportal areas. At focal areas porto-portal fibrosis with associated lymphocytic infiltrate was also seen. Kupffer cells were reactive and plump. Fatty changes near the portal tracts as shown in the Fig. 3C. In case of OGSO1 group, the central vein showed abnormal endothelial lining with some evidence of pericentral fibrosis. The hepatic cords are regularly oriented with intervening reactive plump. Portal area shows increased lymphocytes. The lymphocytes have crossed over the limiting flats of the lobule and have caused piece meal necrosis of the adjacent hepatocytes. The supporting tissue of the portal tracts shows mild infiltrate of lymphocytes only. The cholangiolar epithelia show proliferative activity (Fig. 3D). In OGSO2 treated group, the gross morphology of the liver shows well oriented hepatic cords and reactive kupffer cells. The portal tracts show increase infiltration of lymphocytes; the later have crossed the limiting plates and piece meal necrosis is seen in periportal areas. At focal areas bridging necrosis is seen in the porto-portal
pattern. The cholangioles were increased and show proliferative activity. No evidence of malignancy was seen as shown in Fig. 3E. The gross morphology of the liver of the OGSO3 group shows central veins showed normal endothelial linings with no evidence of pericentral fibrosis. Hepatic cords were well oriented and kupffer calls were non-reactive. The portal tracts showed branches of the hepatic artery and portal vein with normal morphology. No evidence of malignancy was seen as shown in Fig. 3F.
4. Discussion Fat frying is a traditional method for the preparation of food. In restaurants, fast food chains as well as in industrial frying operations, fat frying is the main procedure. Due to the high price of vegetable oils, deep ghee processed food products are used by people in Pakistan and across several countries in Asia. During frying of food, the ghee is exposed to high temperature in the presence of air and moisture. The oxidized ghee thus mixes with the food and cause toxic effects (Kamal-Eldin, Makinen, & Lampi, 2003; Ramadan, Amer, & Sulieman, 2006). Humans and animals are susceptible to oxidative damage, which depends on the balance between oxidative stress and antioxidant defense capacity. Many different kinds of antioxidants are found in various plants.
Fig. 3. Effects of the supplementation of sea buckthorn seed oil on the liver histology of rabbits. Pictures of the liver specimens were taken with light microscope, (A) control group, (B) SO group, (C) OG group, (D) OGSO1 group, (E) OGSO2 group, and (F) OGSO3 group. Arrow indicates the disoriented central vein.
Please cite this article in press as: Zeb, A., & Ullah, S. Sea buckthorn seed oil protects against the oxidative stress produced by thermally oxidized lipids. Food Chemistry (2015), http://dx.doi.org/10.1016/j.foodchem.2015.03.053
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Antioxidants have the potential to inhibit the oxidative breakdown of the lipid component of food and are also important for the defense of living cells against oxidative damage. Sea buckthorn (Hippophae rhamnoides L.) is a rich source of antioxidants was therefore used in this study. Sea buckthorn seed oil contains 338 mg of GAE/100 g of TPC. Arora, Sunil, Ashish, Ravi, and Tsering (2012) reported 162.56 mg GAE/g total phenolic contents in the methanolic seed extract of sea buckthorn, which is much higher than our reported value. Similarly Gao, Ohlander, Jeppsson, Bjork, and Trajkovski (2000) reported that the total phenolic content of sea buckthorn fruit range from 114 to 244 mg GAE/100 g. The variation in TPC may be due to types of extraction, methodology and variety. The peroxide values of the vanaspati ghee increase significantly after oxidation, and subsequently reduce the RSA values. The present result is in agreement with Zeb and Mehmood (2012) who reported an increase in the peroxide value of vanaspati ghee with increasing time of oxidation. The secondary oxidation products were measured in terms of anisidine value, which was increased significantly with oxidation compared to the control in vanaspati ghee. The increase in p-anisidine value and FFA is in agreement with Ahmed, Atta, Sohail, Khan, and Akhtar (2011) who reported that p-anisidine value and FFA values of vanaspati ghee increase with oxidation. Sea buckthorn seed oil showed lower p-anisidine value compared to oxidized ghee and thus may be helpful in oxidation and more beneficial to health. The present results for fatty acid composition are in agreement with Ahmed et al. (2011) who showed that oxidation of vanaspati ghee decrease the linoleic acid and increase palmitic acid, oleic acid and stearic acid contents. Fatty acid profile of sea buckthorn seed oil showed higher concentrations of oleic acid and linoleic acid, while palmitoleic acid and stearic acid in low quantity were in accordance with previous study (Abid, Hussain, & Ali, 2007). Recent studies showed that oleic acid is the main fatty acid responsible for stability against the thermal stress (Zeb, 2012). The serum biochemical parameters like TG, total cholesterol, LDL-c showed an increase while a decrease in HDL-c was observed with feeding oxidized ghee, while the supplementation of SO at different doses reduced the toxic level by positively affecting the above parameters. The results are in agreement with the finding of SBT oil against CCl4 induced hepatotoxicity in rats (Hsu et al., 2009). Recent results by Zeb and Mehmood (2012) showed a dose dependent increase in triglycerides, total cholesterol and LDLcholesterol, while a statistically non-significant increase in the HDL-cholesterol of the rabbits. Similarly Shila et al. (2011) observed that the serum total cholesterol, HDL-cholesterol and malondialdehyde increased significantly (p < 0.05) by feeding the oxidized oil as compared to fresh oil fed group. Basu et al. (2007) reported that sea buckthorn oil fed with high cholesterol diet has shown to restrict the further rise of total cholesterol and thus caused a significant decline of triglycerides and LDL-cholesterol. The HDL-cholesterol levels of the sea buckthorn seed oil treated animals were significantly higher than the non-treated animals showing more protection. The increase in the Hb and RBC contents was due to the feeding of oxidized ghee, while the supplementation of SO is helpful in recovery. A significant increase was observed in the WBC and platelet count in all treated groups with the exception of control and SO groups. This shows that supplementation of oxidized ghee produces significant stress on these blood markers. The present results are in agreement with the studies of Mesembe, Ibanga, and Osim (2004) which shows that oxidation of lipids cause decrease in the hemoglobin concentration. Igiri, Ibegbu, and Osim (1994) stated that intestinal mucosa is severely damaged by thermally oxidized palm oil in rats. This damage may lead to a lower absorption of iron by the intestinal mucosa which results into a decrease
bioavailability of iron in the system. Similarly Chen, Zhong, Liu, and Ge (2003) reported an increase in red blood cell count of the mice fed on sea buckthorn seed oil. These results suggest that SO has the ability to maintain normal hematological parameters under oxidative stress induced by oxidized lipids. Sea buckthorn seed oil protects the liver and kidney against the toxicity of oxidized ghee and increased the level of serum glucose to normal. Similarly the serum ALT was elevated by OG and maintained by supplementation of SO. It suggested that sea buckthorn seed oil is able to improve the condition of hepatocytes, and thus the liver function. This was observed in RSA value of the lipids, which was higher in the sea buckthorn seed oil fed group. The hepto-protective potential of sea buckthorn oil against the oxidized lipids might be due to anti-lipid peroxidation and antioxidants (tocopherols, vitamin C, and phenolic compounds) present in the oil. The mechanism may be the regeneration of parenchymal cells, thus protecting against membrane fragility and reduce the levels of marker enzymes into the blood by the sea buckthorn oil. 5. Conclusions Sea buckthorn seed oil has strong antioxidative activity. The present study shows the protective effects of sea buckthorn seed oil against the side effects of thermally oxidized ghee. Oxidation of ghee decreases the level of radical scavenging activity and increases the peroxide, p-anisidine and free fatty acid values of the ghee. Oxidation of ghee also decreases the level of unsaturated fatty acids and increases the level of saturated fatty acids. Oxidized consumption increases the serum total cholesterol, LDL-cholesterol, TGs, ALT and decreases the serum glucose. Oxidized ghee also causes reduction in RBC, Hb, hematocrit and increase in the WBC and platelets counts as well as degenerative changes in liver and decreases the radical scavenging activity level of the lipids in the liver. Sea buckthorn seed oil supplementation with thermally oxidized ghee significantly lower the serum LDL-cholesterol, ALT, triglycerides and increase the RSA and serum glucose. Hematological parameters were optimized by sea buckthorn seed oil supplementation. The results of the present study showed that sea buckthorn seed oil supplemented with thermoxidized vanaspati ghee reduce the side effects of vanaspati ghee. Sea buckthorn seed oil maintains the stability of the immune system and provides protection to the liver against oxidative stress. Conflict of interest The authors have declared no conflict of interest to anybody or institution. Acknowledgment We are grateful to the University of Malakand for financial support to conduct this study. The financial support of Higher Education Commission (HEC) Pakistan for presenting this work at 11th Euro Fed Lipid Congress, Antalya, Turkey is gratefully acknowledged. We are also grateful of Prof. Dr. Tufail Ahmad Shirazi of National Centre of Excellence in Analytical Chemistry, University of Sindh Jamshoro, Pakistan for his help in fatty acid analysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2015. 03.053.
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