Mortality from cardiovascular disease is the second leading cause of death
worldwide (Malach and Imperato, 2006). Hypercholesterolemia
is generally accepted as an independent risk factor contributing to the development
of coronary heart disease (Altman, 2003; Jalali-khanabadi
et al., 2006). While controlled diet is the primary treatment of
hyperlipidemia (Henley et al., 2002). It is more
practicable to decrease the elevated lipid levels by treatment with anti-hyperlipidemic
drugs. However, treatment by drugs is not always satisfactory due to side effects
of chemically synthesized drugs (Yanovski and Yanovski,
2002; Garg and Simha, 2007). Phytopharmaceuticals
are gaining importance in allopathic as well as traditional medicine owing to
their non-addictive and non-toxic nature (Jenkins et
al., 1983; Raskin et al., 2002).
Fenugreek (Trigonella foenum-graecum L.) is an herbaceous annual plant
from the family of Leguminosae, cultivated in Mediterranean countries, North
Africa and India. It seeds have long been known as a herbal remedy for various
pathological conditions (Sharma, 1986; Subhashini
et al., 2011). Fenugreek is commonly used against diabetes in Pakistan
(Karim et at., 2011).
Steroidal Saponin obtained from fenugreek seeds are composed mainly of diosgenin.
A number of pharmacological and clinical studies have shown glucose- and lipid-lowering
properties of the seed itself, seed extracts or purified components (Sauvaire
et al., 2000; Yadav et al., 2008).
Diosgenin has been shown to have favorable effects on glucose lowering (McAnuff
et al., 2005), antioxidant activity (Son et
al., 2007), lipid metabolism and myocardial infarction (Chiang
et al., 2007; Jayachandran et al., 2009).
Chromium (Cr+3) is an essential element for carbohydrate and lipid
metabolism in animals and humans (El-Hommosany, 2008).
Chromium has been shown to potentiate the action of insulin and accordingly
to modulate carbohydrate, protein, and lipid metabolism (Vincent,
Some researchers highlighted its role with regard to the metabolism of lipids
and the correlations between chromium status and cardiovascular disease (Rajpathak
et al., 2004) in which low chromium level leads to high cholesterol
and brings the risk of developing cardiovascular disorders. Furthermore, chromium
chloride administration causes a substantial reduction of coronary lipid deposits,
aortic lipid deposits and serum cholesterol concentration in rabbits (Bakhiet
and Elbadwi, 2007; Price-Evans et al., 2009).
This study was carried out to investigate the combined effect of natural saponin (diosgenin) extracted from fenugreek seeds, in comparison with the pure standard diosgenin and chromium chloride (CrCl3) supplementation on high-cholesterol fed Japanese quails.
MATERIALS AND METHODS
Extraction of diosgenin from fenugreek seeds: Fenugreek seeds were purchased
from the local market and their botanical identity was confirmed by a botanist
from our University. Diosgenin was alcohol-extracted from fenugreek seeds according
to the method of Prasanna (2000).
Chromatographic investigation: The bioactive extract of fenugreek seeds,
diosgenin, and standard diosganin (≥99.0% purity, purchased from Fluka, BioChemika,
West Germany) were subjected to chromatographic identification using thin layer
chromatography (TLC) (National Research Council, 1994).
Animals and diets: Male Japanese quail (Coturnix japonica) 45 days old
were purchased. They were housed in wire cages (5 birds/pen) at 22±2°C
with a controlled 12-hr daylight cycle. The quails had ad libitum access
to basal diet (Table 1) and water and kept under observation
for 7 days before treatment was initiated. The basal diet was formulated using
the National Research Council, (Yuan et al. 1998)
guidelines and contained 20% crude protein and 12.9% MJ kg-1 Metabolizable
Energy (ME). One hundred and five (105) of these quails were then fed an experimental
diet (diet contained basal diet supplemented with 1% cholesterol) and continued
throughout the experiment.
|| Ingredients of basal diet
|Vitamin premix provides the following per kilogram (kg) of
diet: all-trans retinyl acetate: 1.8 mg; cholecalciferol: 0.025 mg; all-rac-α-tocopheryl
acetate: 1.25 mg; menadione sodium bisulfite: 1.1 mg; thiamine-hydrochloride:
1.1 mg; riboflavin: 4.4 mg; Ca-pantotheenate: 10 mg; pyridoxine hydrochloride:
2.5 mg; vitamin B-6: 2.2 mg; vitamin B-12: 0.02 mg; folic acid: 0.55 mg;
niacin: 35 mg; d-biotin: 0.1 mg; Mineral premix provides the following per
kg:manganese: 40 mg; iron: 12.5 mg; zinc:25 mg; copper: 3.5 mg; iodine:
0.3 mg; selenium: 0.15 mg; choline chloride: 175 mg
||Effects of dietary diosgenin (either extracted or pure standard)
and chromium chloride supplementation on performance of Japanese quails
fed high-cholesterol for 12 days.
|Values are expressed as Mean±SEM
Quails were randomly divided into four groups. Group one (n=25) fed experimental
diet only (control), group 2 (n = 20) fed experimental diet supplemented with
chromium chloride only (CrCl3, 400 μg kg-1 of body
weight), groups 3 and 4 (n = 30 per group) were fed experimental diet supplemented
with either 0.5% (w/w) of extracted diosgenin (Son et
al., 2007) plus CrCl3, (400 μg kg-1 of body
weight) or pure standard diosgenin (0.5%) plus CrCl3 (400 μg
kg-1 of body weight) respectively for 12 days.
Diosgenin, either extracted or pure standard that fed to groups 3 and 4 was dissolved in chloroform, added to the basal diet on metal tray, air dried and mixed mechanically. The 1% cholesterol was added to the mixed diet. The feed intake was measured daily throughout the experiment (Table 2). Daily replacement of diets minimized exposure of birds to oxidized dietary lipids. Chromium chloride was dissolved in 1 ml distilled water and administered by oral gavage. The control group received water as placebo. The study was approved by the university committee for research.
Blood sampling: Fasting blood samples were collected, at day 0 and day
12 of experimental dietary treatments, from jugular vein of Japanese quail and
placed in tubes containing either no anticoaggulant for measuring serum levels
(mg dL-1) of Total Cholesterol (TC), Triglyceride (TG), high density
lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C)
and very LDL-C (VLDL-C), or EDTA for superoxide dismutase (SOD) enzyme activity
in erythrocytes (measured at day 12 only).
Determination of serum lipid profile: The serum TC, TG and HDL-C, levels
were measured using assay kits (Linear chemicals, Spain) based on enzymatic
calorimetric methods. All other chemicals were of reagent grade. Serum LDL-C
and VLDL-C levels were calculated according to Friedewald's formula (Guemouri
et al., 1991) in which LDL-C = TC-HDL-C-TG/5 and VLDL-C = TG/5.
Erythrocytes superoxide dismutase extraction:: Blood samples (5 mL)
were collected in EDTA tubes. The plasma and the majority of white blood cells
were removed by centrifugation at 1000 rpm for 10 min. The red blood cells were
washed twice with saline and hemolyzed by adding approximately 1.5 volume of
distilled water. Five hundred micro-litters (500 μL) of hemolysate were
then added to 4 mL of ice cold distilled water followed by 1 mL of ethanol and
0.6 mL of chloroform. The mixture was then shacked, centrifuged for 10 min at
3000 rpm and the clear top layer which contains the enzyme SOD was separated
and stored at -20°C until assayed. The SOD activity in erythrocytes was
assayed by the method described by Guemouri et al. (1991)
using commercially available kits (Randox Lab. Ltd., Ireland) based on the photochemical
Nitrobluetetrazolium (NBT) method.
Statistical analysis: Data are expressed as the Mean±SEM. The significance of differences between the groups was assessed by ANOVA using SPSS 11.5 statistical software. Value of (p<0.05) was considered significant.
Extraction of diosgenin from fenugreek seeds and TLC analysis: Thin layer chromatography (TLC) revealed that extracted diosgenin which appears as a dark pink spots after development with 50% H2SO4 has rate of flow (Rf) value equal 0.66 which is identical to the Rf value of the standard diosgenin as illustrated in the chromatogram (Fig. 1).
Body weights and feed intake: The effects of dietary diosgenin (either extracted or pure standard) and the chromium chloride supplementation on performance of Japanese quails fed high-cholesterol for 12 days are shown in Table 2. Final body weights and feed intake of birds were not significantly affected by dietary supplementation of either chromium chloride alone or with diosgenin among all the groups.
||Chromatogram from thin layer chromatography (TLC) analysis
using (Chloroform: Methanol) as mobile phase and developed by 50% H2SO4
S: Standard, E: Extract
||Serum total cholesterol (TC) and triglyceride (TG) levels
in Japanese quails fed different experimental diets for 12 days
|Values are expressed as Mean±SEM. *Significant difference
(p<0.01) compared with control d0. **Significant difference (p<0.01)
compared with control d12
Serum total cholesterol (TC) and triglyceride (TG): The effect of supplemented CrCl3 alone or CrCl3 with diosgenin either extracted or pure standard in Japanese quails on serum TC and TG I is summarized in Table 3.
The mean levels of TC in quails fed with experimental diet only (control) for twelve days (induced hypercholesterolemia) was significantly (p<0.01) increased compare to those at pretreated (d0). The mean levels of TC in control quails at d12 (432.0±62.3 mg dL-1) was 82.9% more than that of d0 (236.0±14.3 mg dL-1).
At d12, the mean levels of TC in quails supplemented with CrCl3 alone or CrCl3 with diosgenin either extracted or pure standard was also increased by 17.1% (259.0± 30.6 mg dL-1), 22.3% (282.0±23.2 mg dL-1) and 13.3% (276.0±19.3 mg dL-1) above that determined at d0 (221.0±10.8 mg dL-1) (231.0±28.2 mg dL-1) and (244.0±27.1 mg dL-1) respectively, such increase did not carry statistical significance.
Supplementation of CrCl3 alone or CrCl3 with diosgenin either extracted or pure standard to the hypercholesterolemic quails produced a significant (p<0.01) drop in serum TC compared with the control group. Serum TC levels were significantly (p<0.01) decreased by 39.9% (259.0±30.6 mg dL-1) in quails supplemented with CrCl3 alone, 34.6% (282.0±23.2 mg dL-1) in quails supplemented with extracted diosgenin plus CrCl3 and by 36% (276.0±19.3 mg dL-1) in those supplemented with pure standard diosgenin plus CrCl3 as compared to control group (432.0±62.3 mg dL-1).
Although there is no significant difference in levels of TC between the three supplemented groups at d12, quails supplemented with CrCl3 alone shows the higher reduction in mean values of TC when compared with other groups supplemented with diosgenin (either pure standard or extracted) plus CrCl3. It should be mentioned that the supplementation of hypercholesterolemic quails with CrCl3 alone or CrCl3 with diosgenin either extracted or standard is effective in reducing TC values and succeeded to normalize them nearly to the base line.
As shown in Table 3, the mean serum levels of TG for quails supplemented with CrCl3 alone or CrCl3 with diosgenin either extracted or pure standard for 12 days (132.0±5.26, 151.0±26.6 and 148.0±21.3 mg dL-1; respectively) were not significantly (p>0.05) different from those in control quails (124.0±8.85 mg dL-1). Furthermore, there is no significant difference in the mean levels of TG at d12 when compared to those at d0 in all groups.
Serum lipid profile: The changes of lipid profile in the serum quails are presented in Table 4. At d0, the mean levels of HDL-C were not significantly different between control and supplemented groups.
The levels of HDL-C are significantly (p<0.01) increased at d12 in all groups when compared with those determined at d0. The level of HDL-C of the control quails fed only experimental diet for 12 days (90.9± 14.2 mg dL-1) was 86.6% higher than those in d0 (48.7±1.93 mg dL-1).
However, the serum levels of HDL-C of the quails supplemented with CrCl3 alone or CrCl3 with diosgenin either extracted or standard for 12 days (102.0±15.3, 135.0±20.8 and 126.0±15.7 mg dL¯1; respectively) were 129.0%, 199.0% and 156.0% higher than those determined at d0 (44.6±3.34, 45.2±3.86 and 49.1±6.18 mg dL-1; respectively).
Supplementation of the quails with diosgenin either extracted or standard plus CrCl3 for 12 days had a significant (p<0.01) effect on increasing HDL-C levels by 48.6 and 38.3%; respectively as compared to those in the control quails. However, supplementation of the quails with CrCl3 alone increase HDL-C levels by 12.4%, yet such increase did not carry statistical significance.
At d0, in a pattern similar to that of HDL-C, the serum levels of LDL-C were not significantly changed in all supplemented groups compared with those of controls.
In control quails, the mean levels of LDL-C at d12 was significantly (p<0.01) above (92.2%) the mean levels at d0 (316.0±42.8 vs 164.0±13.3 mg dL-1).
The serum levels of LDL-C in quails supplemented with CrCl3 alone or CrCl3 with diosgenin either extracted or pure standard for 12 days (131.0±18.4, 117.0±20.8 and 120.8±22.7 mg dL-1; respectively) were decreased by 13.4%, 24.8% and 29.2% below than those at d0 (151.0±18.1, 156.0±24.4 and 171.0±21.1 mg dL-1; respectively). Yet these differences were not statistically significant.
Supplementation of diet with CrCl3 alone or CrCl3 with
diosgenin either extracted or pure standard to quails fed high cholesterol for
12 days showed a significant (p<0.01) decrease in LDL-C by 58.6, 62.9 and
61.7%, respectively, below the mean values for control quails.
|| Serum HDL-C, LDL-C, VLDL-C levels in Japanese quails fed
different experimental diets for 12 days
|Values are expressed as Mean±SEM. *Significant difference
(p<0.01) compared with d0 of the same group. **Significant difference
(p<0.01) compared with control d12
||Erythrocytes superoxide dismutase (SOD) activities in Japanese
quails fed different experimental diets for 12 days
|Values are expressed as Mean±SEM. *Significant difference
(p<0.01) compared with control d12
It should be note that there is no significant difference in serum LDL-C levels
between all supplemented groups.
On day 12, the ratio of HDL:LDL was higher in the quails fed diet supplemented with CrCl3 alone (78.2 vs 29.5%) or CrCl3 with diosgenin either extracted (116.0 vs 29.1%) or pure standard (104.0 vs. 28.8%), when compared to day 0.
As shown in Table 4, serum levels of VLDL-C on d12 not differ from those in d0 in all groups. Furthermore, VLDL-C levels following 12 days supplementation with CrCl3 alone or CrCl3 with diosgenin either extracted or standard were not significantly differ compared to control quails (26.3±2.03, 30.1±7.08 and 29.6±4.48 vs 24.8±6.20 mg dL-1; respectively).
Erythrocytes SOD enzyme activity: The activities of SOD enzyme erythrocytes of the control and all supplemented quails are represented in Table 5. The antioxidative enzyme activity (SOD) was not only affected by CrCl3 supplementation but also by diosgenin plus CrCl3 supplementation. There was a significant (p<0.01) increase in the activities of SOD in all supplemented groups as compared to those in control group. The SOD activities in erythrocytes of quails supplemented with CrCl3 alone for 12 days was 4.88±0.76 U mg-1 Hb and showed a 52.5% above the mean values for control quails (3.20±0.71 U mg-1 Hb). However, this activity in erythrocytes of quails supplemented with extracted diosgenin plus CrCl3 was 5.34±0.73 U mg-1 Hb and showed a 66.9% above the mean values for control quails. The SOD activity in erythrocytes of quails supplemented with pure standard diosgenin plus CrCl3 was more pronounced and reach 72.8% (5.53±0.82 U mg-1 Hb) as compared to the mean value of controls. The mean SOD activity in erythrocytes of quails supplemented with CrCl3 alone did not show significant variation when compared p to those supplemented with diosgenin either extracted or pure standard lus CrCl3.
Using of quail model in this study has numerous advantages over others, including
their small size, ease of management, their short life span, the rabidity of
atherosclerosis plaque development. In addition, quails are naturally deficient
in apolipoprotein E and exhibiting structural features (e.g., focal hemorrhage,
calcification and fibrosis) that closely resemble those in the human disorders
(Ojerio et al., 1972).
A recent study reports that Vytorin and Zetia, two major drugs in the treatment
of high cholesterol, failed to decrease the incidence of heart disease (Mitka,
2008). Also, uses of anti-obesity drugs are severely restricted due to accompanying
side effects (Wasan and Looije, 2005). Thus, there is
still a need for efficient, safe and economic means to combat dyslipidemia and
associated metabolic disorders.
One of naturally cholesterol absorption inhibitors is the saponins, the utility
of the naturally occurring plant saponins as hypolipidemic agents is limited
by difficulties in obtaining large quantities from natural sources. This is
due in part to the presence of multiple saponins of differing structure and
biological activity within a given species (Price et
al., 1987). Thus, in the present study we used one fraction of saponin
which is diosgenin in order to clear its individual effect and not to interfere
with effects of other sapogenins. The present study demonstrated for the first
time the hypocholesterolemic and antioxidative effect of natural saponin (diosgenin)
extracted from fenugreek seeds, in comparison with the pure standard, plus CrCl3
supplementation on high-cholesterol fed Japanese quails.
The dose used in our pilot study (0.5%) was within (Rajpathak
et al., 2004) or below than that used in previous studies. Previous
investigators have been used diosgenin at dose of 80 mg kg-1 in rats
(Jayachandran et al., 2009).
The Rf value for diosgenin recorded in this study was approximately similar
to that reported by Li et al. (2002) which was
0.65. It is important to mention that the normal range for Rf value of diosgenin
is between 0.20-0.70 (National Research Council, 1994).
Although using of TLC, rather than high performance liquid chromatography, for
the chromatographic determination of diosgenin in the extract is not the most
adequate technique but it can be considered as an auxiliary technique in our
Trivalent chromium (Cr3+) is an essential trace element for animals
and humans. It was suggested that deficiency of chromium could cause hypoglycemia
and high level of cholesterol which are critical cardiovascular risk factors
(Rajpathak et al., 2004). Chromium supplementation
lowers blood levels of proinflammatory cytokines, oxidative stress, and lipids
levels in diabetic rats (Jain et al., 2007).
The changes of total cholesterol and triglycerides levels during the experimental
period are presented in Table 3. Feeding quails with the high-cholesterol
diet for 12 days resulted in hypercholesterolemia, as was evident from the significant
increase in serum total cholesterol level of control group at d12 compared to
d0 and does not induce hypertriglyceridemia. Supplementation of the high-cholesterol
diet with diosgenin either extracted or pure standard plus CrCl3 reduced
serum TC by about 34.6% and 36.0% respectively compared to the quails fed on
the high-cholesterol diet alone. These results are consistent with recent studies
revealed that dietary diosgenin (Temel et al., 2009)
has a marked hypocholesterolemic effect.
It is noteworthy to mention that the potent effect of reducing elevated TC and LDL-C levels were similar in both diosgenin supplemented groups (extracted and pure standard) which conforms diosgenin purity and successful extraction steps.
In the present study, reduction of TC levels was not statistically different between extracted and standard diosgenin (plus CrCl3), though as can be seen in Fig. 1 (TLC chromatogram), the extract has a much lower concentration of diosgenin than the standard. The possible explanation is that the level of supplementation of both extract and standard were well above the minimum, thus both supplements were capable of returning TC values nearly to base line. Moreover, it is possible that the lack of difference is in part due to the high dosage of CrCl3.
The reduction in serum TC reported in the present study was reflected by significant
reduction in the serum levels of LDL-C. These results are in agreement with
other studies observed that the reduction in serum cholesterol was in LDL (Sowmya
and Rajyalakshmi, 1999). Furthermore, the significant increase in the serum
level of HDL-C associated with the reduction in serum TC of quails fed diet
supplemented with CrCl3 alone or CrCl3 with diosgenin
either extracted or standard for 12 days contradict the recent study carried
by Niu et al. (2009) finding that the serum level
of HDL was decreased in hyperlipidemic rats after administration niacin and
chromium for 12 weeks comparing with high-fat group.
Yet in the present study, the results indicated that the supplemented diets, CrCl3 alone or CrCl3 with diosgenin either extracted or pure standard, offered to quails fed high cholesterol for 12 days, provided a better serum lipid profiles when compared to the control diet, as was evident from HDL:LDL ratio.
It is well known that diosgenin lowers plasma cholesterol by increasing faecal
cholesterol excretion. Until recently it was believed that this effect was due
both to an inhibition of intestinal absorption of cholesterol as a stimulation
of its biliary secretion (by increasing LDL and HDL hepatic clearance). However,
Temel et al. (2009) demonstrated that stimulation
of faecal cholesterol excretion by diosgenin is independent of Niemann-Pick
C1-Like 1 (NPC1L1)-mediated cholesterol absorption (NPC1L1 is the protein with
major role in intestinal cholesterol absorption). Therefore, the hypocholesterolemic
effect of dietary diosgenin by increasing of faecal cholesterol excretion is
primarily attributable to its impact on hepatic cholesterol metabolism rather
than intestinal cholesterol absorption.
Furthermore, we found that the diosgenin (0.5%) and CrCl3 (400 μg kg-1 of body weight) supplementation had no effect on the serum triglyceride level. This result may be explained by the short duration of the supplementation.
In this study, supplementation of the high-cholesterol diet with CrCl3
alone reduced serum TC by about 39.9% which is in agreement with previous
reports showed that dietary Cr3+ lowers circulating cholesterol concentrations
in rats (Vincent, 2000; Jain et
al., 2007) and birds (Kim et al., 1996).
However, others failed to find the effects of Cr3+ on blood cholesterol
concentrations (Kim et al., 2010).
The dose of CrCl3 used in this study (400 μg CrCl3 kg-1
of body weight) is similar to the previous studies using quail (Sahin
et al., 2005). Thus, CrCl3 supplementation dose used per
body weight in the present study is much higher than that used in human clinical
trials. Whether or not there are any differences in the absorption of Cr3+
between humans and quails is not known.
The biological effects of free radicals are controlled in vivo by a wide range
of antioxidants such as vitamins E and C, carotenoids, glutathione and anti-oxidative
enzymes. Among these enzymes, SOD catalyzes dismutation of the superoxide anion
into hydrogen peroxide (Ojerio et al., 1972).
A reduced antioxidative defense status in the plasma and erythrocytes might
result in increased peroxidation of cell membrane lipids and hence an increased
plasma concentration of lipid peroxides (Buczynski et
al., 1993), thus, it might play an important role in atherogenesis.
Under hypercholesterolemic conditions, the red blood cells are known to accumulate
cholesterol from LDL by a nonreceptor-mediated process which is enhanced by
oxidation of LDL (Panasenko et al., 1991). Red
blood cells may therefore provide a useful experimental model system to investigate
influences of hyperlipidemia on tissue antioxidant status.
In this study, we examined the possible preventive role of combined effect
of two hypocholesterolemic and antioxidant agents, diosgenin (either extracted
or pure standard) plus CrCl3 or CrCl3 alone on high-cholesterol
fed Japanese quails. Present results revealed higher erythrocyte SOD activities
in all supplemented groups compared to those in the control group and was more
pronounced in erythrocytes of quails supplemented with pure standard diosgenin
plus CrCl3. Therefore, diosgenin may play a role like that of a lipid
peroxidation chain-breaking antioxidant in cell membrane and might provide protection
against the oxidative damaging effects of polyunsaturated fatty acid.
On the other hand, it might be assumed that the increased activity of antioxidative
enzymes in all supplemented groups played an important role in restricting the
production of the active oxygen species induced by hypercholesterolemia. This
dogma further supported by previous study showed that an excess of trivalent
chromium can act as a prooxidant (Terpilowska and Zaporowska,
In conclusion, the present study revealed that the combined diosgenin and chromium chloride supplementation to high-cholesterol fed quails produced greater decrease in cholesterol and LDL-C and greater increase in HDL-C than did chromium chloride alone by both improving the lipid profile and modulating oxidative stress. Thus, suggests its potential usefulness as an agent for treating human hypercholesterolemia.