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In vitro Anticancer Activity of Quinoa and Safflower Seeds and Their Preventive Effects on Non-alcoholic Fatty Liver



Doha A. Mohamed, Karem Aly Fouda and Rasha S. Mohamed
 
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ABSTRACT

Background and Objective: Non-alcoholic fatty liver disease (NAFLD) is not only the most common cause of liver diseases in humans but also it may complicate and become a risk factor for liver cancer. The present work aimed to evaluate the anticancer activity (in vitro) of quinoa and safflower seeds powder and their beneficial effects against NAFLD (in vivo). Materials and Methods: Proximate analysis, fatty acids profile, total phenolic and phytic acid of quinoa and safflower seeds were assessed. Also their anticancer activities (in vitro) against liver cancer were evaluated. The preventive effect of both seeds on NAFLD was evaluated using twenty four male rats. NAFLD was induced in rats by high fructose diet (HFD) for 4 weeks. The effects of HFD and HFD supplemented with 20% quinoa or safflower powder on plasma and liver lipids, lipid peroxidation, total protein, albumin as well as liver and kidney functions were determined. Results: Quinoa seeds powder was promising in cytotoxicity against hepatocarcinoma cell line HEPG2 (IC50 was 14.6 μg). Feeding rats on HFD produced dyslipidemia and significant increase in liver functions and lipid peroxidation with significant elevation in liver triglycerides and total cholesterol. Quinoa and safflower seeds powder produced improvement in the biochemical parameters with different degrees. Conclusion: Quinoa and safflower seeds powder possessed cytotoxicity against hepatocarcinoma cell line HEPG2 and afford hepato-protection against NAFLD.

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Doha A. Mohamed, Karem Aly Fouda and Rasha S. Mohamed, 2019. In vitro Anticancer Activity of Quinoa and Safflower Seeds and Their Preventive Effects on Non-alcoholic Fatty Liver. Pakistan Journal of Biological Sciences, 22: 383-392.

DOI: 10.3923/pjbs.2019.383.392

URL: https://scialert.net/abstract/?doi=pjbs.2019.383.392
 
Copyright: © 2019. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver diseases in humans. NAFLD is characterized by extravagant aggregation of triglycerides in the hepatocyte not due to the alcohol consumption1. A wide variety of complications may be accompanied to NAFLD, from simple steatosis to non-alcoholic steatohepatitis (NASH) and can eventually lead to cirrhosis, liver failure and/or liver cancer2,3. It was reported that NASH might develop to liver cirrhosis in about 10-29% of patients and liver cirrhosis might progress to hepatocellular carcinoma (HCC) in about 4-27% of patients4. NAFLD is associated with hyperlipidemia, overweight/or obesity and type 2 diabetes, so it is considered the hepatic component of the metabolic syndrome (MetS)5. The exact mechanism of starting and advancement of NAFLD till now unclear; inflammation and elevation of oxidative stress play an important role in the progression of NAFLD6. Fructose is a monosaccharide sugar found in fruits, honey, sucrose and high fructose corn syrup (HFCS). Fructose is able to elevate either plasma triglycerides or liver fat, which differentiate it from glucose. Fat accumulation in the liver due to fructose consumption (via the lipogenesis activation and the blocking in the fatty acid oxidation) is the key in elevation of triglycerides in liver7. Food can play an important role in treatment and prevention of different diseases. One of the newest concept in the prevention and treatment of NAFLD is the intake of dietary supplements that possess various health benefits besides to their nutritional value. Fruits, vegetables, spices, herbs are among these dietary supplements that contain high levels of biologically active compounds/or nutrients such as omega-3 fatty acids, phenolic compounds, antioxidant vitamins, fibers and minerals which play an important role in diseases prevention and treatment8,9. Biologically active compounds present in dietary supplements possess different biological activities such as anti-inflammatory, hypoglycemic, hypolipidemic, hypotensive, anti-atherosclerotic, anti-thrombotic, hepatoprotective and hypocholesterolemic effects10,11. Liver lipid-lowering activity of biologically active compounds may be attributed to their ability to regulate the expression of genes that implicated in de novo lipogenesis and fatty acid oxidation12. Quinoa (Chenopodium quinoa Willd.) is a pseudo-cereal, which used like rice and wheat13. Quinoa seeds are rich source of carbohydrates, high-quality protein and dietary fiber14,15. Recently, the biological activities of quinoa seeds have been studied through their effective role as antimicrobial16 and anti-inflammatory17. Phenolic compounds and saponins were the most studied phytochemicals in quinoa seeds18. Safflower (Carthamus tinctorius L.) belongs to Asteraceae family. The Carthamus species have been cultivated in China, India, Iran and Egypt almost from prehistoric times. The seeds of safflower contain oil, protein and crude fiber by 30, 20 and 35%, respectively. Safflower seeds are rich source of minerals, tocopherols (α, β and δ), carotenoids and thiamine19. Safflower seeds have been used in the folk medicine for prevention and treatment of different diseases such as diabetes, cardiovascular diseases and bone diseases20,21. Safflower seeds powder possessed anti-androgenic effect in rats22 and ameliorated benign prostate hyperplasia in rats23. Due to the plenty of biologically active compounds in quinoa and safflower seeds, it was hypothesized that these plants may protect liver from NAFLD and cancer. So, the present research aimed to evaluate the anticancer activity of these plants and their preventive effect on NAFLD in rats.

MATERIALS AND METHODS

The present study was carried out in the National Research Centre, Egypt. It was started in March, 2018 and end in August, 2018.

Quinoa seeds were purchased from local markets, while safflower seeds (Giza 1) were purchased from Agriculture Research Centre, Cairo, Egypt.

Cancer cells: Liver human tumor derived cell lines (HEPG2) were supplied from National Cancer Institute, Cairo University, Egypt and used for cytotoxicity study.

Animals: Male Sprague Dawley rats of 129.8±12.17 g as Mean±SD were used in the present study. Animals were obtained from Animal house of National Research Centre, Cairo, Egypt. Animals were kept individually in stainless steel cages; water and food were given ad libtium.

Preparation of plant materials: Quinoa and safflower seeds were dried separately in an air-circulated oven at 40°C till complete dryness and then they were reduced into powder form and stored in airtight containers and kept at 5-7°C until used.

Chemical analysis of seeds powder: Quinoa and safflower seeds powder samples were sieved through 100-mesh sieve. The samples were analyzed for moisture, protein, fat, crude fiber and ash contents using standard AOAC24 procedures. Different chemical analysis were carried out in triplicate and averaged.

Assessment of fatty acids of quinoa and safflower seed oils: Oil of both seeds was extracted using Soxhlet apparatus; petroleum ether 40-60°C was used for extraction of oil. The solvent was evaporated under vacuum using rotary evaporator. Fatty acid methyl esters of the both oils were prepared according to AOAC24 to be subjected to GLC analysis of fatty acids. Assessment of the methyl ester was carried out by injecting 2 μL into a Hewlett Packard HP-system 6890 gas chromatograph equipped with FID. HP-5 capillary column (30 m×0.32 mm i.d.; 0.25 μm film thickness) was used to separate the different methyl esters. The chromatographic analysis conditions were: initial temperature 70°C with a hold for 1 min, then rose to 120°C at a rate of 40°C min1 with 2 min hold then the temperature was finally raised to 220°C at a rate of 4°C min1 with another 20 min hold. The injector and detector temperatures were 250 and 280°C, respectively. Identification of the fatty acid methyl esters was carried out by direct comparison of retention times of each of the separated compounds with standards of the fatty acid methyl esters analyzed under the same conditions. Quantization was based on peak area integration.

Determination of total phenolics: Quinoa and safflower seeds powder were extracted twice with 80% ethanol according to the optimized extraction conditions described in a previous work25. Total phenolics were determined using Folin-Ciocalteu reagent26. Absorbance was measured at 765 nm using UVPC spectrophotometer. The results were expressed as milligrams of gallic acid equivalents (GAE) per100 g on dry weight basis. Three replicates were measured and the results were expressed as Mean±SE.

Determination of phytic acid (PA): Phytic acid content was determined in the quinoa and safflower seeds powder according to the method of Gao et al.27. A series of calibration standards containing 0, 1.12, 2.24, 3.36, 5.6, 7.84, or 11.2 mg L1 PA were prepared from sodium phytate (Sigma, St. Louis, MO). The PA content of the sample was calculated from the standard curve and was represented as PA content/100 g sample. Three replicates were measured and the results were expressed as Mean±SE.

Anticancer activity: Anticancer activity of quinoa and safflower seeds was tested using the cell line technique according to Cordero et al.28. Cells of HEPG2 (liver carcinoma cell) were plated in 96-multiwell plate (104 cells/well) for 24 h before treatment to be attached to the wall of plate. All plants powder was dissolved in dimethyl sulfoxide (DMSO) at 10 mM as a stock solution. Different concentrations of the plants powder (0, 5, 12.5, 25 and 50 μg mL1) were added to the cell monolayer, triplicate wells were prepared for each individual dose of each plant powder. Plates were incubated for 48 h at 37°C and in atmosphere of 5% CO2. After 48 h, cells were fixed, washed and stained with Sulfo-Rhodamine-B stain, then excess stain was washed with acetic acid and attached stain was recovered with Tris EDTA buffer. Color intensity was measured in an ELISA reader. The relation between surviving fraction of cancer cell and plants powder dose was plotted. IC50 was obtained from the curves.

Preventive effect of quinoa and safflower seeds on NAFLD in rats
Diets:
Diets were formulated according to the following scheme given in Table 1. High fructose diet was prepared similar to Chang et al.29 for inducing of fatty liver. Twenty grams from quinoa and safflower seeds powder were mixed with the high fructose diet to give quinoa diet and safflower diet, respectively. The contents of protein, fat and carbohydrate of the 20 g powder were reduced from casein protein, corn oil and starch, respectively without affecting fructose levels.

Table 1:
Composition of different experimental diets (g/100 g)
*12 casein has been estimated to contain 10 g protein using AOAC24

Animals' treatments: Twenty-four rats were divided into four groups, each of six rats. The first group was considered as the normal healthy group where rats received a balanced diet. The second group was named control NAFLD where rats were fed on high fructose diet. Rats of group three and four were fed on high fructose diet supplemented with 20% quinoa or safflower powder. During the experiment, body weight and food intake were recorded weekly. After four weeks (end of the study) total food intake, body weight gain and feed efficiency ratio (Body weight gain/total food intake) were calculated.

Analysis of blood and tissues: Blood samples were collected from all rats after an overnight fast for the determination of plasma total cholesterol (T-Ch)30, high density lipoprotein cholesterol (HDL-Ch)31, low density lipoprotein cholesterol (LDL-Ch)32 and triglycerides (TG)33. The T-Ch/HDL-Ch ratio was calculated as indicator of cardiovascular disease risk. Plasma total protein34 and albumin35 were determined. Plasma malondialdehyde (MDA)36 was estimated as an indicator of lipid peroxidation. Plasma uric acid37 was determined. The activity of plasma transaminases38 aspartate transaminase (AST) and alanine transaminase (ALT) were estimated as an indicator of liver functions. Plasma levels of creatinine39 and urea40 were determined to study any possible changes in kidney functions. Liver was immediately removed, weighed and stored at -20°C till analyzed. Total hepatic lipids were extracted and weighed according to the procedure of Folch et al.41. In brief, liver tissues were homogenized with chloroform:methanol (2:1) for lipids extraction. After the extraction and evaporation, tissue lipids were re-dissolved in isopropanol and liver cholesterol30 and triglyceride33 levels were estimated enzymatically.

This study has been carried out according to the Medical Research Ethics Committee, National Research Centre, Cairo, Egypt and followed the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).

Statistical analysis: The results of animal experiments were expressed as the Mean±SE and they were analyzed statistically using the one-way analysis of variance ANOVA followed by Duncan’s test. In all cases p<0.05 was used as the criterion of statistical significance.

RESULTS

Phytochemical analysis of quinoa and safflower seeds: Table 2 summarized the chemical composition of quinoa and safflower seeds powder respectively. Safflower seeds contained high amount of protein (17.3%) and fat (30.1%) than quinoa seeds.

Table 2:
Chemical composition of quinoa and safflower seeds powder
*Calculated by differences

Table 3:
Fatty acids contents of quinoa and safflower seeds oils (as percentage of total fatty acids)

Table 4:
IC50 dose of the different plants powders under study

Quinoa seeds were found to contain the highest amount of carbohydrate (72.3%) than safflower seeds. Total phenolic compounds (Table 2) were present in safflower and quinoa seeds by 176 and 36.4 as mg GAE/100 g, respectively. Phytic acid was present in safflower seeds by 2.2% and in quinoa seeds by 1.03%.

Fatty acid profile of quinoa and safflower seeds oil: The identified FAMEs percentages of quinoa and safflower seeds oil were presented in Table 3. Quinoa and safflower contained four major fatty acids, including palmitic, oleic, linoleic and α-linolenic acids. These results showed that approximately 82.33 and 77% of total fatty acids are represented by unsaturated fatty acids. Linoleic acid was the highest fatty acid in both seeds it was present by 46.7% and 66.8% in quinoa and safflower seeds oil, respectively. Palmitic acid was the highest saturated fatty acid present in both seeds oil. α-Linolenic acid was present in quinoa and safflower seeds oil by 8.83 and 5.1%, respectively which may provide favorable nutritional implications and beneficial physiological effects.

Anticancer activity of quinoa and safflower seeds: The cytotoxicity effect of quinoa and safflower seeds powder was shown in Table 4 and Fig. 1. Data in Table 4 showed the IC50 dose (concentration which reduces survival of the exposed cancer cells to 50%) of plants powder and Doxorubicin, drug used for treatment of cancer.

Table 5:
Plasma lipid profile and liver fat, total cholesterol and triglycerides of normal and different fatty liver groups
In each row same letter means non-significant difference while different letter means significant difference at 0.05 probability. The data are expressed as mean values±standard error

Table 6:
Plasma malondialdehyde, total protein and albumin of the different experimental groups
In each row same letter means non-significant difference while different letter means significant difference at 0.05 probability. The data are expressed as mean values±standard error

Fig. 1:
Survival curve of liver carcinoma cell line (HEPG2)

Figure 1 illustrated the survival curve of HEPG2 cells which were exposed to various concentrations (5-50 μg mL1) of quinoa and safflower seeds powder for 48 h. Quinoa seeds powder was more efficient than safflower seeds powder where IC50 was 14.6 and 19.4 μg, respectively.

Protective effect of quinoa and safflower seeds against NAFLD
Effect of quinoa and safflower seeds on plasma and liver lipids: Plasma lipid concentrations in the different groups were shown in Table 5. NAFLD control rats exhibited significant increase in T-Ch, TG and LDL-Ch compared with normal control rats. In addition, significant elevations in total fat, T-Ch and TG in the liver tissues recorded by NAFLD control rats (Table 5) in comparison to normal control rats. The ratio of T-Ch/HDL-Ch elevated significantly in rats NAFLD control compared with normal rats, which means elevation of the risk of cardiovascular disease (CVD) that is associated with reduction of HDL-Ch plasma levels. Supplementation of high fructose diet (HFD) with 20% quinoa or safflower seeds powder improved plasma lipid profile and liver total fat, T-Ch and TG contents with variable degrees (Table 5). Feeding rats on HFD supplemented with quinoa or safflower seeds powder showed significant reduction of CVD risk as observed by reduction of the T-Ch/HDL-Ch ratio.

Effect of quinoa and safflower seeds on MDA, total protein and albumin: Plasma MDA levels as an indicator of lipid peroxidation elevated significantly in NAFLD control rats in comparison with normal control (Table 6). Plasma levels of MDA reduced significantly in rats fed on HFD supplemented with 20% quinoa or safflower seeds powder when compared with NAFLD control. Plasma total protein and albumin reduced significantly in NAFLD control rats. On the other hand, plasma total protein elevated in rats fed on HFD supplemented with 20% quinoa or safflower seeds.

Effect of quinoa and safflower seeds on liver and kidney functions: Increased plasma activities of AST and ALT were noticed in NAFLD control (Table 7). Treatment with quinoa and safflower seeds powder produced significant reduction in AST and ALT activities compared to NAFLD control rats. Significant elevations in kidney functions, plasma creatinine and urea, were recorded by NAFLD control (Table 7). Plasma uric acid elevated significantly in rats fed on HFD compared with normal rats.

Table 7:
Liver and kidney functions of the different experimental groups
In each row same letter means non-significant difference while different letter means significant difference at 0.05 probability. The data are expressed as mean values±standard error

Table 8:
Nutritional parameters of the different experimental groups
In each row same letter means non-significant difference while different letter means significant difference at 0.05 probability. The data are expressed as mean values±standard error

Feeding rats on high fructose diet supplemented with quinoa and safflower seeds powder attenuated the deterioration in liver and kidney functions with different degrees and reduced the elevation in plasma uric acid significantly compared with NAFLD control group.

Effect of quinoa and safflower seeds on the nutritional parameters: Nutritional parameters of all the experimental groups were presented in Table 8. Non-significant changes were observed in final body weight, body weight gain and total food intake between NAFLD control and normal control rats. Final body weight and body weight gain of rats fed on HFD supplemented with 20% of quinoa seeds powder reduced significantly when compared with NAFLD group. Rats feeding on diet supplemented with safflower seeds powder showed significant reduction in feed efficiency ratio compared with other rats groups. Liver relative weight of NAFLD control increased significantly when compared with normal rats groups. Liver relative weight of rats fed on HFD supplemented with 20% of quinoa seeds powder reduced significantly when compared with NAFLD control rats or rats of HFD containing safflower seeds powder.

DISCUSSION

In the present research quinoa and safflower seeds powder showed anticancer activity against HEPG2 cancer cell line that may be imputed to the presence of phytic acid, phenolic compounds and polyunsaturated fatty acids (PUFAs) in these seeds. It was reported previously that phytic acid prevented and inhibited tumor development42. The anti-carcinogenic activity of phytic acid may be due to its suppression of forming of oxygen free radicals through prevention of the Fenton reaction in iron chelation43. Feruloylserotonin is bioactive component present in safflower seeds, which possess antibacterial, anti-inflammatory and free radical scavenging activities20. Choi et al.44 clarified the cytoprotective activity of feruloylserotonin based on its ability to prevent oxidative stress-induced cell death, lipid peroxidation and phosphorylation of histone H2AX in H2O2 treated HEPG2 and HACAT cells.

Accumulation of fat is the implicit cause of NAFLD which induce disturbance in lipid metabolism in the liver45. NAFLD in many patients ultimately progresses to liver cirrhosis and hepatocellular carcinoma46. NAFLD is developed after hepatic steatosis and is associated with cellular stresses, such as oxidative stress, gut-derived lipopolysaccharide stress, etc.,3,47. High intake of fructose is one of the most important causes of NAFLD48 since fructose increases fat synthesis and suppresses fat oxidation which stimulates fat accumulation in liver49. Fructose also induced elevation of oxidative stress and inflammation50. In the present research feeding rats on high fructose diet for four weeks proved induction of NAFLD, which characterized by accumulation of fat in liver tissue, dyslipidemia, elevation of liver function (AST and ALT) and elevated MDA in addition to reduction in plasma total protein and albumin. High fructose diet also elevated kidney dysfunction which may be used as an indicator of hepatorenal syndrome. Fan et al.51 and Al-Okbi et al.52 also found that rats feeding on diet rich in fructose showed renal damage which associated with inflammation and lipid accumulation. Glomerular hypertrophy has occurred after fructose intake for short (four weeks) or long-term53,54. Fructose intake induced glomerular hypertension and reduced renal blood flow which associated with preglomerular vascular disease55. In the present study HFD elevated uric acid significantly in rats. Higher levels of uric acid due to fructose consumption were observed also in liver tissues56. Synthesis of uric acid from amino acid precursors is stimulated by high fructose diets, which elevates fasting serum uric acid levels7.

Rats fed on HFD supplemented with quinoa and safflower powder as dietary supplements exhibited significant improvement in plasma lipid profile and liver fat in addition to the reduction in plasma MDA and uric acid. This improvement may be due to the affluent of quinoa and safflower seeds powder in polyunsaturated fatty acids, phenolic compounds and phytic acids. Intake of PUFAs is inversely associated with the incidence of heart disease via the reduction of plasma cholesterol and triacylglycerol57. In the present study quinoa seeds contained 7.1% oil; this result is coinciding with those recorded by Vega Galvez et al.58 who found that oil content in quinoa ranged from 2-10%. Vitamin E presented in quinoa oil protects PUFAs from oxidation. Omega-3 and omega-6 play important roles in human health such as membrane function, immunity and brain development59. Safflower seeds contain phenolic compounds such as lignans, glucosides, flavonoids and serotonins60. Safflower seeds contain arctigenin, which is considered plant lignan and acts as precursor for the mammalian lignans61. Arctigenin displayed inhibition activity of TNF-α (inflammatory marker) production62,63 and hepatoprotective effect in primary cultures of rat cells which may be due to its antioxidant activity64,65. It was reported that the main phenolic in quinoa seeds are flavonol glycosides66. Quercetin and kaempferol are the main flavonol glycosides in quinoa seeds which present at the level of approximately 839 μg g1 dry weight67. Lutz et al.68 identified isoflavones in quinoa seeds; genistein and daidzein presented by 0.05-0.41 and 0.7-2.05 mg/100 g, respectively. Quinoa and safflower seeds contain fibers as shown in the proximate analysis of both seeds. Satiety feeling, reduction of cholesterol and lipid absorption, management of the postprandial insulin level, catalyzing the turning endogenous cholesterol to bile acids as well as enhancement of the gut microbiota can be achieved via the consumption of dietary fiber69. Diet supplemented with quinoa seeds powder elevated plasma total protein and albumin similar to normal control rats. Quinoa is rich in high quality protein and amino acids especially lysine and methionine14 to which it can be attributed the increments in both parameters.

The non-significant change in body weight gain between NAFLD control rats and control normal rats is coincide with the work of Chang et al.29 that demonstrated that the induction of fatty liver didn’t change the body weight. Reduction of total food intake in the present study in rats feeding on diet supplemented with 20% of quinoa seeds powder indicated that these pseudo-cereals have awesome ability to decrease food intake and are able to keep on standard body weight upon regular consumption. The present results are coinciding with those obtained previously by Mithila and Khanum70.

CONCLUSION

It was concluded that the dietary supplements, a hopeful area of research for discovery alternative medicine for treatment and demonstrated to protect from diseases such as NAFLD. Quinoa and safflower seeds as rich sources of phenolic compounds, polyunsaturated fatty acids and phytic acid possessed anti-cancer activity against HEPG2 cancer cell line and exhibited protective impact against NAFLD. So, these seeds especially quinoa seeds can be used as dietary supplements for weight loss and prevention from NAFLD and liver cancer.

SIGNIFICANCE STATEMENT

This study confirmed that quinoa and safflower powder as dietary supplement are good strategy for prevention of NAFLD in rats fed on high fructose diet. Both plants powder reduced weight gain, improve dyslipidemia, reduced lipid peroxidation and prevent hepatic lipid accumulation effectively. Quinoa and safflower powder were effective as anti-cancer against hepatic cancer in cell line technique using HEPG2 cell.

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