J Med Allied Sci 2017; 7(1):20-25 DOI: https://doi.org/10.5455/jmas.248536

Original article

Antihyperlipidemic effect of crude extract of saffron (Crocus sativus) stigma in healthy male rats

Iliass Lahmass1, Sabir Ouahhoud1, Assia Sabouni1, Mohammed Elyoubi1, Redouane Benabbas1, Rachid Elmoussaoui2, Mohammed Choukri2, Ennouamane Saalaoui1

Affiliation(s):

1Laboratory of Biochemistry and Biotechnology, Department of Biology, Faculty of Sciences, University Mohamed Ist, Oujda-60000, Morocco.

2Unité de Biochimie, Centre Hospitalier Universitaire Mohammed VI, Oujda, Morocco.

Corresponding author: Dr. Iliass Lahmass, Laboratory of Biochemistry and Biotechnology, Department of Biology, Faculty of Sciences, University Mohamed Ist, Oujda-60000, Morocco.

Phone: +212-629530036 Email: iliass.lahmass@gmail.com

Abstract

In this study, we investigated for the first time the antihyperlipidemic effects of crude extract of stigmas from Crocus sativus (saffron) against hyperlipidemia induced by tartrazine (synthetic dye) in normal male rats. Thirty adult male albino rats weighing about 150 - 200 g, were divided into 5 groups (n = 6) and daily treatment was given orally. Clinical biochemistry and metabolic parameters were evaluated at the end of the experiment and after 105 days. (n=6, for all groups). Our data revealed that the metabolic parameters like consumption of food and water, pH and urine volume have not been affected; also the difference between liver, right kidney and heart weight was not significant. The levels of cholesterol and triglyceride were significantly increased in group 2 and group 3 compared to control group. There was no significant difference in the level of cholesterol and triglyceride in group 4. Treatment with saffron alone did not have any significant effects on the level of fat compared to control group. The oral administration of the crude extract of saffron revealed good hypolipidemic effects in adult male albino rats. These results suggest that aqueous saffron extract reduced plasma cholesterol and decreased triglyceride. Therefore, it could conceivably lead to suitable changes in blood lipid profiles.

Keywords: Antihyperlipidemic effect, Crocus sativus, Saffron, Tartrazine

Running title: Antihyperlipidemic effect of extract of saffron

Introduction

Hyperlipidemia, raised cholesterol and triglycerides, contribute significantly in the causation of atherosclerosis and coronary artery diseases (CAD). Several factors contribute to the emergence of cardiovascular disease such as age, hereditary, diet, life style and hypertension. Likewise high level of cholesterol in blood particularly low density lipoprotein (LDL) cholesterol is mostly responsible for the commencement of CAD1. To reduce the level of cholesterol in blood, numerous medicinal plants were reported to have a potential to decrease fat in the body.

Saffron (Crocus sativus) is one of the highest priced and the most used spice around the world as both flavouring and colouring agent2. Numerous studies have demonstrated that saffron have antispasmodic, gingival sedative, nerve sedative, carminative, diaphoretic and expectorant3, anti-oxidant4,5 and have beneficial effects in the treatment of neurodegenerative disorders such as Alzheimer’s disease6, also saffron or its active constituents has demonstrated an antinociceptive effect, as well as acute and/or chronic anti-inflammatory activity7. Saffron and its constituent, crocin, have an overall protective effect against hyperlipidemic manifestation in rat8 likewise aqueous extract of saffron and its constituent showed an aphrodisiac activity in normal male rats9. Recently, it was found that saffron extract, exhibited significant decrease of blood cholesterol10 and glucose level11,12.

Tartrazine is an orangecoloured, water soluble powder used worldwide as food colouring agent. This food additive is most often responsible for allergic reactions in specific human populations13,14. The results of some studies showed that Tartrazine has the carcinogenic and mutagenic effects15-19. Tartrazine also increase blood glucose level and plasma creatinine, protein, cholesterol and triglyceride20.

The aim of this study was to evaluate the anti-hyperlipidemic effect of crude extract of stigmas from Crocus sativus linnaeus.

Materials and methods

Plant materials

Crocus sativus (Saffron) was obtained from Taliouine (Taroudant Province, Souss-Massa-Drâa, Morocco). Three specimens of the plant have been deposited at the plant section of Herbarium University Mohammed Premier, Oujda, Morocco (HUMPOM), under the voucher number (HUMPOM210). The identification of the plant has been done and confirmed by a professional botanist, Professor Fennane Mohammed from Scientific Institute in Rabat, Morocco. Dried milled powder of stigmas of Crocus sativus was macerated for 12 hours in distilled water before usage and crude extract was used to treat male rats.

Chemicals

Tartrazine (CAS 1934210, Purity 86.7%), was purchased from Alfa Aesar (Germany), SigmaAldrich (Japan) and was dissolved in distilled water 12 hours before use.

Qualitative determination

One hundred µL of extracts samples were injected into a liquid chromatography (HPLC) to determine the chemical compounds of the saffron extract. A Waters Symetry® C18 (4,6µm x 250mm) column. A linear gradient of methanol (10–100%) in water (15% of acetonitrile) was used as a mobile phase with a flow-rate of 1 ml/min for a maximum elution time of 60 min at room temperature. The sample size was 20 µl of the test solution21. The analyses were triplicated for each sample.

Animals

Maintenance and handling of rats were in accordance to the international conventional standard guidelines and with the Helsinki declaration for use of laboratory animals. 30 male Wistar rats weighing 150 – 200 g were housed in individual cage under standard laboratory conditions in a 12 h/12 h light/dark cycle and at a temperature of 21 - 25°C (animal house of the Department of Biology, Faculty of Sciences, Oujda, Morocco) and were given free access to water and dry rat pellets feeds (SONABETAIL Society, Oujda, Morocco). 

Experimental design

Animals were arbitrarily separated to five groups of equal number and weight (six animals each). All animals were treated by daily oral gavage for 105 days with a volume of 10 ml/kg body weight (b. w.).

Group 1 (Control group): Rats were given distilled water.

Group 2 (Tartrazine-Saffron group): Animals were treated with Tartrazine (10 mg/kg) for 60 days and then administered with saffron (120 mg/kg) until the last day of treatment.

Group 3 (Tartrazine group): Rats were administered only with Tartrazine (10 mg/kg) for all period of treatment.

Group 4 (Saffron-Tartrazine group): Animals were treated with saffron (120 mg/kg) for 60 days and then administered with Tartrazine (10 mg/kg) until the last day of treatment.

Group 5 (Saffron group): Rats were administered only with saffron (120 mg/kg) for all period of treatment.

On the day of necropsy, blood samples were collected via the abdominal aorta for measurements of biochemical parameters. Cholesterol levels in plasma wawes estimated by the method used by Allain et al22. Level of triglyceride in plasma was determined by the Trinder method23 and biochemistry determinations were performed by using ILab 300 (Instrumentation Laboratory Corporate Headquarters, Barcelona, Spain).

Statistical analysis

All data are expressed as means ± SEM. Significant differences among control and experimental groups was determined by one-way analysis of variance (ANOVA) followed by Tukey post-test using Graph Pad Prism 5.

Results

As shown in the table 1, treatment with Tartrazine and Saffron did not affect metabolic parameters like pH and urine volume and the difference was significant on consumption of food and water; also, the difference between liver, right kidney and heart weight is not significant (Table 2).

Table 1: Metabolic parameters of Wistar rats feeding with Tartrazine and saffron and sacrificed after 105 days of treatment

Metabolic parameters

Control group

Tartrazine (10 mg/kg)

Tartrazine (10mg)+ Saffron (120mg)

Saffron (120mg)+ Tartrazine (10 mg)

Saffron (120mg/kg)

Water consumption

37.5±1.71

46.00±3.65*

37.5±4.79

36.67±6.15

30.00±5.63

Food consumption

32.21±1.56

20.34±2.04*

25.72±4.91

16.51±2.68*

35.28±3.39

pH

8.71±0.07

8.69±0.09

8.64±0.1

8.44±0.13

8.38±0.27

Urine volume

13.0±1.63

15.16±1.46

12.17±0.87

15.17±3.91

11.33±1.2

Note: values represent the mean ± SEM of six rats; *p<0.05. Significantly different from controls.

Table 2: Organ weight of Wistar rats sacrificed on day 105 of subchronic treatment and feeding with Tartrazine and Saffron

Organ weight

Control group

Tartrazine (10 mg/kg)

Tartrazine (10mg)+ Saffron (120mg)

Saffron (120mg)+ Tartrazine (10 mg)

Saffron (120mg/kg)

Liver

6.63±0.15

6.68±0.21

6.9±0.18

6.25±0.58

6.51±0.29

Heart

1.00±0.03

0.96±0.04

0.98±0.04

0.85±0.05

0.79±0.03

Right kidney

0.92±0.03

0.92±0.05

0.92±0.04

0.91±0.04

0.91±0.05

Note: values represent the mean ± SEM of six rats

The levels of cholesterol and triglycerides were significantly increased in all groups treated with 10 mg/kg b. w. of Tartrazine compared to control group. The levels of triglycerides were significantly increased in group treated with 10 mg/kg b. w. of Tartrazine + 120 mg/ kg b. w. of Saffron. There was no significant difference in the level of cholesterol and triglyceride among all groups treated with 120 mg/kg b. w. of saffron + 10 mg/kg b. w. of Tartrazine. Treatment with 120 mg/kg b. w. of Saffron did not have any significant effects on the level of cholesterol and triglycerides (Fig 1 & 2).

Fig 1. Effects of Tartrazine and Saffron on plasma triglyceride level. ED: treated with distilled water; TART: treated with 10 mg/kg b. w. of Tartrazine; TART+SAFR: treated with 10 mg/kg b. w. of Tartrazine + 120 mg/kg b. w. of Saffron; SAFR+TART: treated with 120 mg/kg b. w. of Saffron + 10 mg/kg b. w. of Tartrazine; and SAFR: treated with 120 mg/kg b.w of Saffron.

Note: values represent the mean ± SEM of six rats; +++ p<0.001 highly significantly different from group 2. + p<0.05 significantly different from group 4. *** p<0.001 highly significantly different from controls. (+ symbol of comparison with other groups; * symbol of comparison with control group).

Fig 2. Effects of Tartrazine and Saffron on plasma cholesterol level. ED: treated with distilled water; TART: treated with 10 mg/kg b. w. of Tartrazine; TART+SAFR: treated with 10 mg/kg b. w. of Tartrazine + 120 mg/kg b. w. of Saffron; SAFR+TART: treated with 120 mg/kg b. w. of Saffron + 10 mg/kg b. w. of Tartrazine; and SAFR: treated with 120 mg/kg b.w of Saffron.

Note: values represent the mean ± SEM of six rats; + p<0.05 significantly different to group 4.

Discussion

One hundred µL of extract samples were injected into a liquid chromatography (HPLC) to determine the chemical compounds of the saffron extract. The carotenoid compounds were identified based on their retention times and quantified according to the respective standard calibration curves (Fig 3). The HPLC chromatogram of the saffron extract indicated crocin as the major compound present in the extract.

Fig 3. HPLC chromatograms of extract of saffron with different peaks of various components of the stigma. A Waters Symetry® C18 column, a linear gradient of methanol (10–100%) in water (15% of acetonitrile), and a flow rate of 1 ml/min were used for qualitative determinations.

The peak identification is as follows: number 1 was crocin 1 trans, peak 2 was crocin 3 trans, peak 3 was standard crocin pure, peak 4 was crocin 3 cis, peak 5 was crocin 1 cis and peak 6 was safranal. According to our analysis, different forms of crocins were detected in our saffron samples.

As shown in figure 1, the day before experiment and until the end of treatment with Tartrazine and saffron extract in the control group, no significant difference in triglyceride plasma was found compared to the group 2, group 3 and group 4 (p > 0.05). There was significant difference on plasma triglyceride between control groups compared to group 1 (p < 0.05). The level of plasma triglyceride of the group 2 significantly increase compared to group 4 and significantly decrease compared to group 1. No significant difference of the group 3 compared to group 4.

For the plasma cholesterol (Fig 2), there was no significant difference between the control group compared to the group 1, group 2, group 3 and group 4 (p > 0.05). There was no statistically significant difference between the group 1, group 2 and group 3 (p > 0.05). Significant difference was observed in plasma cholesterol between the group 1 compared to the group 4 (p < 0.05).

The differences in mean body weight, organ weights and metabolic parameters between control and groups treated with Tartrazine and saffron were not significant.

For groups treated with Tartrazine, these results are in accordance with the data from Himri and Borzelleca15,20 who suggested that the decrease of body weight is not toxicological but it was due to decreased caloric intake because of the Tartrazine component of the diet. For groups treated with saffron, our results are in agreement with Elgazer12 who observed that oral administration of saffron extract caused significant increase in body weight.

Our work showed that the treatment with Tartrazine for 105 days exhibited a significant increase in plasma triglyceride and cholesterol concentration when compared with control rats. This result was in agreement with Himri20 who observed a significant elevation in serum triglyceride in rats which consumed Tartrazine in different doses. While our results are in a contrast with study of Amin et al which demonstrated that a high dose of Tartrazine decreases serum triglyceride concentration when rats consumed (500 mg/kg b. w.) or low dose of Tartrazine (15 mg/kg b. w.) and with Ashour and Abdelaziz24 who obtained a significant reduction in serum total cholesterol and triglycerides level when food colour azo dye (fast green) was consumed orally by male albino rats for 35 days.

For group treated with saffron, our study showed that the level of fat (triglyceride, cholesterol) is low compared to control group, these results are in accordance with Elgazer who demonstrated that oral administration of saffron extract caused important differences in body weight, serum levels of blood glucose and insulin and lipid profile as well as the improvement in liver and kidney functions12. Mohajeri demonstrated that saffron extract is effective in the reduction of blood sugar and fat amount25, also Zheng reported that in hyperlipidemic rabbits, crocetin prevented atherosclerosis disease26. Sheng suggested that crocin had hypolipidemic properties27. Furthermore, He et al28 indicated that saffron especially crocin inhibit the formation of atherosclerosis in quails, whereas all of his results are in agreement with our study.

The level of plasma triglyceride of the group treated with 10 mg of Tartrazine followed by 120 mg of saffron significantly decreased compared to the group treated with Tartrazine only, this result showed the hypolipidemic effect of consumption of crude extract of saffron for 105 days. This effect could be attributed firstly to the scavenging activity of crocin and safranal and to regenerative properties of the extract.

Conclusion

We can determine from this data that oral administration of crude extract of stigmas from Crocus sativus linnaeus has significant beneficial effects. In fact, saffron extract contains crocin and safranal and has shown good antihyperlipidemic effect and could be effective in reducing plasma cholesterol and triglyceride against hyperlipidemia induced by Tartrazine. Further studies are necessary to elucidate in detail the mechanism of action of this medicinal plant. Therefore, saffron may be regarded as a useful therapy for hyperlipidemia.

Acknowledgements: This research is financially sponsored by the ARES “Coopération au développement”. We are very thankful to unit of biochemistry in the hospital of Al Farabi, Oujda, Morocco. We thank also El Mostapha Bedraoui for helping in animal care.

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

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