INTRODUCTION
Hyperlipidemia is characterized by elevated serum total cholesterol, low
density and decreased high-density lipoprotein levels. It is a metabolic disorder
and is closely related to coronary heart disease and diabetes1.
According to global survey on non-communicable diseases in 2010, approximately
2.8 million people die per annum of being overweight or obese which is also
one of the risk factor for developing cardiovascular diseases and around 2.6
million people die globally every year due to raised cholesterol which develops
heart diseases and stroke2.
Hyperlipidemia is a condition, characterized by abnormal elevation of lipid
such as (triglyceride and cholesterol) and lipoproteins such as (LDL, VLDL)
levels in the blood. Elevated plasma triglyceride concentrations contribute
to increased risk of metabolic cardiovascular diseases, both directly and due
to these elevations. This is associated with risk factors like obesity, proinflammatory
markers, metabolic syndrome, biomarkers and type II diabetes mellitus.
Cholesteryl esters and triglycerides, water insoluble lipids, are the core
components and apoproteins, phospholipids and unesterified cholesterol, water
soluble components, are located on the surface and perform regular functions3.
But due to defect in the lipid metabolism whether genetically or due to sedentary
lifestyle, there is a disturbance in the lipoprotein lipase activity or due
to absence of the surface Apoprotein C-II, the end result is hyperlipidemia4.
Statins and fibrates are the well-established treatments for hyperlipidemias
and the prevention of vascular events. However, if both i.e., statins and fibrates
are used together as previous studies have made known, it results in development
of rhabdomyolysis if gemfibrozil is taken originally with lovastatin and recently,
even with cerivastatin. Again, clofibrate causes cholesterol gallstones and
inhibition of glucuronidation of statins. Other alternative of niacin also causes
problems with patient compliance.
Despite of several available interventions to counteract the disease, epidemiological
data are witnessing the growing trend of the problem, reflecting both the multiple
etiologies as well as scarce compliance of patients to established therapies5.
Lagenaria siceraria fruit (LSF); Molina commonly known as bottlegourd,
has composition of all the essential constituents like choline, vitamin B complex,
fibers and proteins like lagenin-A novel ribosome-inactivating protein and also
as a fair source of vitamin C, β-carotene, cucurbitacins and saponins.
Since the saponins can be very helpful in faster removal of free fatty acids
from the circulation that causes in turn a decrease in total cholesterol by
enhancing the lipoprotein lipase activity (LPL) as reported in the previous
studies. In addition to this LSF also contains the soluble dietary fibers such
as pectin which helps in promotion of the bile acid formation and in the inhibition
of cholesterol absorption or their excretion in stool6-11.
Another such anti-hyperlipidemic plant Trigonella foenum graecum (TFG)
usually called as fenugreek or Methi belongs to Leguminosae family has been
used in foods or as a medicine since ages. Fenugreek seeds contain proteins
rich in lysine and L-tryptophan, infrequent chemical constituents such as saponins,
fenugreekine, coumarin, nicotinic acid, sapogenins, phytic acid, scopoletin,
trigonelline and mucilaginous fiber which are thought to account for many of
its presumed therapeutic effects12-16.
The present study was done with an objective to study the effect of the combinatorial
extract for the enhanced anti-hyperlipidemic activity in triton induced model
of hyperlipidemia. The activity was evaluated by estimation of biochemical parameters:
Cholesterol, triglycerides, LDL and HDL plasma levels.
MATERIALS AND METHODS
Standardization of plant material: From the local market of Mumbai, India,
in the months of February-March, the fruits of Lagenaria siceraria and
the seeds of Trigonella foenum-graecum were procured. For authentication,
the specimen samples were sent to Ramnarain Ruia College, Matunga (E), Mumbai.
Extraction procedures: The Lagenaria siceraria extract was prepared
by initially defatting the fruits using petroleum ether (60%) and subsequently
extracted with ethanol (95%) using soxhlet apparatus and finally concentrated
using rotary evaporator. The dried seeds of Trigonella foenum-graecum
were used to prepare the extract. This was done by heating at reflux with 95%
ethanol for 6-12 h. The mixture was filtered by suction filtration and then
the filtrate was concentrated by rotary evaporator.
Characterization of Lagenaria siceraria and Trigonella foenum-graecum
extract: Phytochemical tests were performed on the extracts of Lagenaria
siceraria and Trigonella foenum-graecum, to check the presence of
different valuable phytoconstituents like carbohydrates, alkaloids, glycosides,
steroids, phenolics, flavonoids and proteins. This was done with the aid of
standard phytochemical models17-20.
The identification of compounds was carried on and confirmed using TLC. A mobile
phase consisting of ethyl acetate: Chloroform: Formic Acid (70:29.5:0.5) was
developed for Lagenaria siceraria extract and the Rf (Retention factor)
values obtained were compared to the reported Rf values in Ayurvedic Pharmacopoeia
of India. Similarly for Trigonella foenum-graecum extract the composition
of mobile phase for the TLC optimized based on higher resolution was N-butanol:
acetic acid: water (3:1:0.5).
In vitro model for evaluation of antioxidant activity using DPPH
(1, 1–dipheny-2-picryl hydrazyl) method: The evaluation of free radical
scavenging activities of the two extracts along with the combinatorial extract
was carried out using DPPH method. Ascorbic acid served as standard for comparison.
Different concentrations of standard and extract were tested for their antioxidant
activity. Absorbance was measured at 517 nm21.
Animal studies
Toxicity studies: Acute oral toxicity studies were carried out on female
albino mice in accordance with OECD guidelines 423. The mice were divided into
three groups consisting of three mice each. Lagenaria siceraria fruit
extract at dose 2 g kg-1 was tested for toxicity and similarly, Trigonella
foenum-graecum extract at dose 2 g kg-1 and combinatorial extract
(1 g and 1 g kg-1) were tested. The treatments were given once and
the animals were observed over a period of 14 days.
Induction of hyperlipidemia: The Wistar albino rats were obtained from
Haffkine Institute, Parel, Mumbai. All the animals were acclimatized for a week
prior to commencement of the study while maintaining the required humidity and
temperature conditions as stated by the guidelines (temperature 25±2°C and
humidity 75±5%) (CPCSEA approval number: CPCSEA/IAEC/SPTM/P-56/2011). Hyperlipidemia
was induced in Wistar albino rats by single intraperitoneally injection of freshly
prepared solution of triton WR 1339 (300 mg kg-1) in 0.5% CMC (Carboxy
Methyl Cellulose); after overnight fasting for 18 h22.
Experimental design: The rats were divided into six groups, each group
consisting of six animals. The experimental design and schedule of treatment
was followed as:
Group I: |
Normal control |
Group II: |
Positive control (hyperlipidemic rats) |
Group III: |
Atorvastatin tablets (10 mg kg-1); p.o. (standard drug) |
Group IV: |
Lagenaria siceraria fruit extract (200 mg kg-1); p.o. |
Group V: |
Trigonella foenum-graecum extract (200 mg kg-1); p.o. |
Group VI: |
Combinatorial extract (100 and 100 mg kg-1); p.o. |
The standard and experimental treatment was given 24 and 48 h post-hyperlipidemia
induction.
Biochemical analysis of plasma: At the end as well as at the initiation
of the experiment, rats were kept fasting overnight, they were anaesthetized
under light ether and blood samples were collected from retro orbital plexus
using glass capillary in heparin coated tubes at 0, 24 and 48 h time interval.
Plasma was separated by centrifugation at 4000 rpm for 5 min at 4°C and
was then analyzed for total cholesterol, triglycerides, LDL and HDL using ERBA
semi-auto analyser21.
Statistical analysis: One way analysis of variance (ANOVA) followed
by Bonferroni’s test were applied for comparison of values for control and treated
groups. All the values were expressed in terms of Mean±SEM (n = 6). Significant
difference between control and experimental groups were assessed by student’s
t-test. ***p<0.001 were considered as statistically significant. The statistical
analysis was carried out by the Graph Pad 3.0 software.
RESULTS AND DISCUSSION
Characterization of Lagenaria siceraria fruit (LSF) and Trigonella
foenum graecum (TFG)
Physiochemical parameters: Foreign organic matter, ash values and
extractive values were found to be within Pharmacopoeial limits (Ayurvedic Pharmacopoeia
of India).
In Lagenaria siceraria, the total ash was 5.92% w/w and acid insoluble
ash 0.19% w/w. The alcohol soluble extract was 12.7% w/w while water soluble
extract was found to be 28.9% w/w. The moisture content was found to be 4.1%
w/w. In Trigonella foenum graecum, foreign matter was found to be 1.18%
w/w. The total ash was 3.95% w/w and acid insoluble ash 0.25% w/w. The alcohol
soluble extract was 13.6% w/w while water soluble extract was found to be 36.2%
w/w. The moisture content was found to be 3.9% w/w. The physicochemical standards
of both the plant materials were found to be within the Pharmacopoeial limits.
Characterization of extracts by phytochemical tests: Tests for LSF extract
confirmed the presence of carbohydrates, steroids, flavonoids, glycosides and
phenolic compounds. Carbohydrates, flavonoids, glycosides and alkaloids were
found to be present in the TFG extract.
Preliminary phytochemical screening was performed for all the herbal extracts
in order to determine the organic as well as inorganic chemical constituents
present in the plant extract. The tests indicated the presence of carbohydrates,
steroids, flavonoids, phenolic compounds, glycosides in the LSF extract and
the TFG extract in addition to these constituents was found to have alkaloids.
In vitro evaluation of antioxidant activity using DPPH (1, 1-dipheny-2-picryl
hydrazyl) method: The radical scavenging action of combinatorial extract
was observed to have a significant increase than the herbal extracts used alone
(Table 1).
Acute oral toxicity studies: The two extracts LSF and TFG alone as well
as the combinatorial extract did not cause any mortality or symptoms of toxicity
through the 14-day period.
|
Table 1: DPPH free radical scavenging
activity of ascorbic acid standard solution |
|
Table 2: DPPH free radical scavenging
activity of combinatorial extract of LSF and TFG |
In accordance to OECD 423 guidelines, the dose of 2 g kg-1
was selected. No mortality or any sign of toxicity were observed for the extracts
given alone or in the combinatorial extract. Thus the extracts and their combinatorial
extract were found to be safe as there was no mortality and no significant change
in weight or feed-water intake was observed.
Antihyperlipidemic activity of combinatorial extract of LSF extract and
TFG extract in triton induced hyperlipidemia: The acute administration of
triton WR-1339 (300 mg kg-1) caused marked increase (p<0.001)
in the plasma levels of cholesterol, triglycerides (TG), Low Density Lipoprotein
(LDL) 356.52±11.03, 1254.5±11.08 and 299.48±8.74 mg dL-1 at
24 h. In addition to this, there occurred significant reduction in High Density
Lipoprotein (HDL) levels in hyperlipidemic animals as compared with normal control
group. The HDL levels after 24 and 48 h time points were elevated in the animals
treated with combinatorial extract as compared to positive control animals at
the same time points (Table 2).
Effect of combinatorial extract of Lagenaria siceraria fruit extract
and Trigonella foenum graecum extract on plasma lipid profile in rats:
On treating the triton WR-1339 induced hyperlipidemic rats with LSF extract
(200 mg kg-1), TFG extract (200 mg kg-1) and the combinatorial
extract (200 mg kg-1), caused decrease (p<0.001) in the levels
of triglyceride (TG), total cholesterol, Low Density Lipoproteins (LDL) and
increase in High Density Lipoproteins (HDL) as compared to the positive control
(triton induced) animals (Fig. 1a-d).
Triton WR-1339, a nonionic surfactant is used to block the clearance of triglyceride
rich lipoproteins and hence aid in development of hyperlipidemia in several
animals. These acute models are highly used in evaluation of the newer herbal
or synthetic anti-hyperlipidemic drugs. In this study, triton-1339 was administered
i.p., in rats to facilitate elevation of plasma cholesterol, triglyceride, LDL
and reduction of plasma HDL levels. This action is generally due to association
of surfactants with triglycerides in the plasma which reduces their rate of
hydrolysis by the enzyme, lipoprotein lipase or clearing factor lipase, thus
interfering with the uptake of triglycerides from the circulation by the extra-hepatic
tissues. It has been studied that the triton induced significant increase in
the plasma cholesterol and triglyceride levels is due to increase in VLDL secretions
by liver along with a significant reduction of LDL and VLDL catabolism23.
Lagenaria siceraria is accounted for its cardio protective, antihyperlipidemic,
antioxidant and antihyperglycemic, analgesic, anti-inflammatory, immunomodulatory
and hepatoprotective functions in humans24.
Saponins are among the major constituents in LSF extract, it can be very helpful
in faster removal of free fatty acid from circulation that causes in turn a
decrease in total cholesterol by enhancing the lipoprotein lipase activity (LPL)
as reported in the previous studies. Saponins are known to increase the permeability
of intestinal cells in vitro and also to inhibit active mucosal transport
and increase absorption of substances. Across the small intestines of rats they
are known to lower the transmural potential difference. Saponins have hemolytic
activity which can be attributed to their affinity towards aglycone moiety for
membrane sterols especially cholesterol, thus forming a insoluble complex25.
LSF contains the parts of soluble dietary fibers such as pectin which helps
in promotion of the bile acid formation and their excretion in the stool or
in the blockage of cholesterol absorption.
TFG seeds are also responsible for the anti-hyperlipidemic activity due to
the presence of steroidal saponins (diosgenin, yamogenin, tigogenin and neotigogenin),
alkaloids (mainly trigonelline) and free amino acids. TGF seeds are an important
source of diosgenin, a precursor for the production of steroidal drugs and hormones
such as testosterone, glucocorticoids and progesterone. These steroidal sapogenins
are effective mediators for the management of hypocholesterolemia. Isoleucine,
an amino acid is known to modulate the secretion of insulin26.
The protective efficacy of the TGF seeds extract against artherogenesis was
demonstrated by a significant reduction in the triglyceride and total cholesterol
level after 24 and 48 h of triton injection in TGF extract treated group.
|
Figure 1 (a-d): Effect of combinatorial
extract of LSF extract, TFG extract on plasma (a) Triglyceride, (b) Cholesterol,
(c) LDL and (d) HDL in triton induced hyperlipidemia. Values are Mean±S.E.M.
of six rats ***p<0.001, **p<0.01 and *p<0.05 versus control hyperlipidemic+Lagenaria
siceraria extract, Trigonella foenum graecum extract, their
combinatorial extract and standard drug (10 mg kg-1) were compared
with the hyperlipidemic (positive) control |
As a result the efficiency of TGF extract in lipid regulating can be taken
advantage in prevention of plaque formation leading to artherogenesis and congestive
heart failure. High content of soluble fiber in fenugreek can be attributed
to its anti-hyperlipidemic activity. The soluble fiber acts by decreasing the
gastric emptying rate and thus delays the absorption of lipids from small intestine.
The result of the study reveal that the seed extract of fenugreek can effectively
control the blood levels in dyslipidemic conditions by interfering with biosynthesis
of cholesterol and utilization of lipids27.
In this study, it was observed that the combinatorial extract of LSF extract
and TFG extract at the dose of 200 mg kg-1 significantly decreased
(p<0.001) the levels of triglyceride (TG), Low Density Lipoproteins (LDL),
cholesterol and showed an increase in High Density Lipoproteins (HDL) levels.
The observations suggested that the cholesterol lowering activity of the combinatorial
extract can be a result of the enhanced catabolism of LDL cholesterol through
its hepatic receptors for the final elimination in the form of bile acids. Even
Lecithin-cholesterol acyltransferase (LCAT) and tissue lipases are activated
in the biological system. From the results of this study it can be concluded
that the elevated levels of triglycerides, LDL and total cholesterol were significantly
decreased by using the combinatorial extract of LSF extract and TFG extract
than using the two extracts alone. Thus the antihyperlipidemic activity as well
as anti-hyperlipidemic activity of this combinatorial extract can be attributed
to the presence of polyphenols and flavanoids for their diverse pharmacological
actions which also includes anti-artherogenic activity.
CONCLUSION
The present data revealed the medicinal use of the combinatorial extract
of Lagenaria siceraria fruit and Trigonella foenum graecum can
effectively modulate triton induced hyperlipidemia. Further studies to explore
the exact mechanism attributing to the effect are needed to ascertain the potential
of the combinatorial extract as an antihyperlipidemic agent.
ACKNOWLEDGMENT
Sincere thanks to Shobhaben Pratapbhai Patel School of Pharmacy and Technology
Management, SVKM’S NMIMS for providing the opportunity and facilities to conduct
the study.
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