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www.wjpr.net Vol 8, Issue 9, 2019. 1
Umaru et al. World Journal of Pharmaceutical Research
ANTIOXIDANT AND ANTIHYPERLIPIDAEMIC ACTIVITIES OF
METHANOLIC EXTRACT OF CASSIPOUREA CONGOENSIS FRUIT
IN TRITON X-100 INDUCED HYPERLIPIDAEMIC RATS
Umaru Hauwa Aduwamai*, Samson Ezekiel and Dahiru Daniel
Department of Biochemistry, School of Life Sciences Modibbo Adama University of
Technology, Yola, P.M.B. 2076 Adamawa State, Nigeria.
ABSTRACT
Cardiovascular diseases have become the leading and major clinical
and public health problem. It is one of the major killer diseases in the
world. The anti-hyperlipidaemic effect of methanol extract of
Cassipourea congoensis fruit was tested in triton X-100 induced
hyperlipidaemic rats. The hyperlipidemia was induced by a single
intraperitoneal injection of Triton X-100 (100mg/kg body weight). The
phytochemical investigation indicated the presence of flavonoids,
Phenols, glycosides, alkaloids, terpenoids and tannins. The GC-MS
analysis of the fruit extract revealed the presence of squalene, 4-
mercaptophenol, ascorbyl-palmitate, farnesol, octadecadienoic acid,
oleic acids, Gallein, trans-Farnesol and 9,12-Octadecadienoic acid. The
antioxidant activities of methanol extract were evaluated by various
methods. Results obtained revealed that the methanol extract of
Cassipourea congoensis fruit exhibited strong antioxidant activity measured using Hydrogen
Peroxide (H2O2), 2, 2-Diphenyl-l-Picryl Hydrazyl (DPPH) and Ferric Reducing Antioxidant
Power (FRAP) assay at different concentrations of the methanol extract (20, 40, 60, 80 and
100 mg/mL). Treatment of hyperlipidaemia with methanol extract of Cassipourea congoensis
fruit at different concentrations (100, 200, 300 and 400 mg/kg body weight) significantly (p ≤
0.05) decreased the levels of serum total cholesterol, triglycerides, low density lipoprotein
cholesterol and very low density lipoprotein cholesterol in a dose dependent manner
compared to experimental control. However a significant (p≤0.05) increase in serum high
density lipoprotein cholesterol was observed with extract administration. The
antihyperlipedaemic activity of the extract at 400mg/kg was found to be comparable to the
World Journal of Pharmaceutical Research SJIF Impact Factor 8.074
Volume 8, Issue 9, 1-19. Research Article ISSN 2277– 7105
Article Received on
25 May 2019,
Revised on 14 June 2019,
Accepted on 05 July 2019
DOI: 10.20959/wjpr20199-15396
*Corresponding Author
Umaru Hauwa Aduwamai
Department of
Biochemistry, School of
Life Sciences Modibbo
Adama University of
Technology, Yola, P.M.B.
2076 Adamawa State,
Nigeria.
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Umaru et al. World Journal of Pharmaceutical Research
standard drug Atorvastatin (10 mg/kg). The results demonstrated that methanol extract of
Cassipourea congoensis fruit possessed significant antihyperlipidaemic activity.
KEYWORDS: Hyperlipidaemia, Cassipourea congoensis, Triton X-100, Antioxidant
activity, Lipid profile.
1.0: INTRODUCTION
Hyperlipidaemia is a predisposing factor to the development of atherosclerosis, coronary
artery disease and several cardiac manifestations such as myocardial infarction, ischemia and
angina. It is specifically characterized by alterations in serum lipid and lipoprotein profile. It
has been reported that abdominal obesity, impaired postprandial lipid metabolism and insulin
resistance are all interrelated risk factors for coronary heart diseases.[1]
Hyperlipidaemia is
caused by either genetic (primary hyperlipidemia) or from a poor diet and other specific
factors (secondary hyperlipidaemia).[2]
Cardiovascular disease (CVD) is a worldwide, health problem currently, growing in
developing countries as significant parameter of non-communicable disease burden. CVD
causes more than 4 million deaths each year across Europe, accounting for 45% of all deaths.
CVDs account for one third of total deaths around the world, it is believed that CVDs will
turn out to be the main cause of death and disability worldwide by the year 2020.[3,4]
In the
last ten years in Nigeria according to Gabriel et al.,[5]
cardiovascular diseases have become
the leading and major clinical and public health problem. It is associated with high rates of
disability, case fatality, unnecessary and sudden death particularly in Nigeria. The national
survey in Nigeria and previous studies reports revealed that the prevalence of cardiovascular
diseases is escalating in all parts of Nigeria. Hyperlipidaemia has been one of the major killer
diseases in the world, accounting to 80% worldwide of most cases of cardiovascular
diseases.[6]
In spite of many advancements in the pharmaceutical preparation, studies
available support the uses of herbal and homeopathic methods for management of diseases
such as hyperlipidaemia, dyslipidaemia associated cardiovascular disorders and others life
threaten ailments due to its affordable properties with significant effectiveness.[7]
The high cost of drugs and the inability of many people in developing countries particularly
Africa to purchase synthetic drugs have forced many communities to look for products in the
form of medicinal plants that have proven to be effective, inexpensive and culturally
acceptable to treat certain diseases.[8]
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Cassipourea congoensis fruits constitute an important part of a balanced diet as they are
naturally source of food nutrient needed by man and animals. Cassipourea congoensis plant
has many important uses which range from nutritional usage to medicinal purposes. Such
food nutrient includes protein, carbohydrate, minerals and dietary fibre. Cassipourea
congoensis is a wild fruit which is frequently found in Northern Nigeria especially in rural
areas where a variety of edible fruits bearing trees are available.[9]
It is called tsamiya dutse in
hausa language of northern Nigeria. Several of these species bear fruits during the dry season
when cultivated fruits are scarce.[10]
Cassipourea congoensis is particularly used as a
substitute for tamarind (Tamarindus indica) in preparing local pap in many areas.
Cassipourea congoensis fruit is used for seasoning as a food component, to flavour
confections, curries and sauces. It is a main component in juice and certain beverages.
Cassipourea congoensis is eaten fresh and often made into a juice, infusion or brine and can
also be processed into jam and sweets. The refreshing drinks are popular in many countries
around West Africa and sometimes it is fermented into an alcoholic beverage.[11]
An infusion
of its roots is used to treat miscellaneous diseases such as stomach ache, throat infections,
bronchitis and syphilis.[12]
The seeds are known to contain oil. In addition to the traditional uses, the plant is reported for
anti-inflammatory, anti-diabetic, anti-microbial, anti-parasitic and anti-helmentic activities.[13]
It is a multipurpose plant, almost every part has at least some uses either nutritional or
medicinal.[14]
Herbal medicine represents one of the most important fields of traditional
medicine. WHO recognized that medicinal plants played an important role in health care.
About 80% of the world population in developing countries depend largely on traditional
medicine.[15]
This project is aimed at evaluating the antioxidant and invivo
antihyperlipidaemic activities of methanol extract of Cassipourea congoensis in triton x-100
induced hyperlipidaemia.
2.0 MATERIALS AND METHODS
2.1 Plant Material
Plant sample was collected from Tiri village, Michika Local government area of Adamawa
State, Nigeria. The sample was identified and authenticated at the Department of Plant
Sciences, Modibbo Adama University of Technology Yola, Nigeria.
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2.2 Experimental Animals
Adults male wistar rats (42) weighing (120±10g) were obtained from National Veterinary
Research Institute (NVRI) Vom Jos, Plateau state. The animals were grouped and housed in a
plastic cage and maintained under standard laboratory condition with dark and light cycle.
The animals were acclimatized for seven (7) days before the commencement of the
experiment. They were allowed free access to standard dry pellet diet (finished vital feeds
Jos) and water ad libitum. The animals were handled in accordance with the National
Institute of Health guide for the care and use of laboratory animals.
2.3 Preparation of Extract
The sample was shade dried for three (3) weeks and was coarsely made to powder with a
mechanical grinder. The powdered sample was soaked with methanol for about 72hrs at room
temperature in a conical flask container, extraction was carried out using soxhlet method.
After completion of the extraction, the solvent was removed by rotary evaporator where a
methanol extract of the plant was obtained. The residue was concentrated and stored in an
airtight container pending use.
2.4 Pytochemical Test
2.4.1 Qualitatative Phytocemical Test
The extract was subjected to phytochemical investigation. Tannins, alkaloids, flavonoids,
glycosides, saponins, terpenoids and phenols were determined using the standard methods
described by Trease and Evans.[16]
2.4.2 Quantitative Pytochemical Test
Alkaloid, phenols and terpenoids were determined using the method of Harborne.[17]
Flavonoid was determined using the method of Bohm and Kocipai- Abyazan.[18]
Determination of tannins was carried out using the method of Anchana et al.,[19]
2.4.3 Quantification of Phytochemicals
Method: The GC-MS analysis was carried out according to the method of Vengaiah et al.,[20]
Principle: A combination of two different analytical techniques, Gas Chromatography (GC)
and Mass Spectrometry (MS), is used to analyze complex organic and biochemical mixtures.
The GC-MS instrument consists of two main components. The gas chromatography portion
separates different compounds in the sample into pulses of pure chemicals based on their
volatility by 23 flowing through an inert gas (mobile phase), which carries the sample,
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through a stationary phase fixed in the column. Spectra of compounds are collected as they
exit a chromatographic column by the mass spectrometer, which identifies and quantifies the
chemicals according to their mass-to-charge ratio (m/z).[21]
Procedure: The analysis was performed using agilent Gas Chromatography mass
spectrometer (GC-MS): GC Model 7890A, MS 5975C (Inert MSD) equipped with 7638B
Auto sampler. Helium was used as the carrier gas at a constant flow rate of 1 ml/min and an
injection volume of 2 μl was employed (split ratio of 10:1). Injector temperature was 250°C;
Ion- source temperature 280°C. The oven temperature was programmed from 110°C
(isothermal for 2 min.), with an increase of 10°C/min, to 200°C, then 5°C/min to 280°C,
ending with a 9 min. isothermal at 280°C. Mass spectra of compounds in sample obtained by
electron ionization (EI) at 70 eV; a scan interval of 0.5 seconds and fragments from 45 to 450
Da. Total GC running time was 36 min. The relative % amount of each component was
calculated by comparing its average peak area to the total areas.[21]
2.5 Antioxidant Activities
2.5.1 Hydrogen Peroxide Scavenging Assay
Method: Hydrogen peroxide scavenging activity of the fruit extract was determined using the
method of Jayaprakasha.[22]
Principle: Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes
directly, usually by oxidation of essential thiol (-SH) groups. It can cross cell membranes
rapidly and enter the cell. H2O2 probably reacts with Fe2+
and possibly Cu2+
ions to form
hydroxyl radical which is the origin of many of its toxic effects.
Procedure: A solution of hydrogen peroxide (20 mM) was prepared in phosphate buffer
saline (PBS at pH 7.4). Various concentrations of the extract and standards in methanol (1
ml) were added to 2 ml of hydrogen peroxide solutions in PBS. After 10 min, the absorbance
was measured at 230nm against a blank solution that contained extracts in PBS without
hydrogen peroxide. The percentage scavenging of hydrogen peroxide and standard
compounds was calculated using the following equation:
% Scavenged H2O2 = × 100
2.5.2 DPPH Free Radical Scavenging Assay
Method: The scavenging activity was carried out according to the method of Patil et al.,[23]
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Principle: The stable 1, 1-diphenyl-2-picryl hydrazyl (DPPH) is a free radical and accepts an
electron or hydrogen radical to become a stable diamagnetic molecule which is widely used
to investigate radical scavenging activity. In DPPH radical scavenging assay, antioxidants
react with DPPH (deep violet color) and convert it to yellow coloured α,α-diphenyl-β-
picrylhydrazine. The degree of discoloration indicates the radical-scavenging potential of the
antioxidant.
Procedure: About 1ml of various concentrations of the extracts in methanol was added to 4
ml (0.004% w/v) methanol solution of DPPH. After 30 minutes the absorbance of the
preparations was taken at 517nm by a UV spectrophotometer which was compared with the
corresponding percentage inhibition of standard ascorbic acid. The free radical scavenging
activity (FRSA) was calculated using;
DPPH radical scavenging activity (%) =
2.5.3 Ferric Reducing Antioxidant Power (FRAPAssay)
Method: The Ferric reducing antioxidant power (FRAP Assay) was carried according to the
method described by Banerjee et al.,[24]
Principle: Ferric reducing antioxidant power (FRAP Assay) assay is a widely used method
that uses antioxidants as reductants in a redox-linked colorimetric reaction, where Fe3+ is
reduced to Fe2+. Ferric (Fe3+) to ferrous (Fe2+) ion reduction at low pH causes formation of
coloured ferrous-probe complex from a colourless ferric-probe complex.
Procedure: In ferric reducing antioxidant power assay, 1 ml of test sample extract in
different concentration were mixed with 1 ml of 0.2M sodium phosphate buffer (pH 6.6) and
1 ml of 1% potassium ferricyanide in separate test tubes. The reaction mixtures were
incubated in a temperature-controlled water bath at 500C for 20min, followed by addition of
1 ml of 10% trichloroacetic acid. The mixtures were then centrifuged for 10 min at room
temperature. The supernatant obtained (1 ml) was added with 1 ml of deionised water and
200 μl of 0.1% FeCl3. The blank was prepared in the same manner as the samples except that
1% potassium ferricyanide was replaced by distilled water. The absorbance of the reaction
mixture was measured at 700 nm. The reducing power was expressed as an increase in A700 nm
blank subtraction.
FRAP radical scavenging activity (%) =
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2.6 Experimental Design
2.6.1 Induction of Hyperlipidaemia
Triton X-100(100mg/kg body weight) single dose was administered interperitoneally to rats
to induce hyperlipidaemia.
2.6.2 Grouping of Experimental Animal
Forty two (42) rats were grouped into seven (7) groups of 6 rats per each group.
I. Group (1) received normal feed + water (normal control)
II. Group (2) received Triton X-100(100mg/kg body weight), experimental control-no
treatment
III. Group (3) received Triton X-100(100mg/kg body weight)+Atovarstatin 10mg (standard
control)
IV. Group 4, 5 6, and 7 received Triton X-100 (100mg/kg body weight) and were treated for
seven (7) days with 100, 200, 300 and 400 mg/kg body weight of the Cassipourea congoensis
fruit extract respectively.
Experimental Design
Group Treatment
1 Normal
2 Experimental control
3 Standard control (drug)
4 Treatment I
5 Treatment II
6 Treatment III
7 Treatment IV
Normal control
Experimental control -Triton X-100(100mg/kg bwt)
Triton X-100(100mg/kg bwt) + Atorvastatin 10mg
Triton X-100(100mg/kg bwt) + 100 mg C. congoensis
Triton X-100(100mg/kg bwt) + 200 mg C. congoensis
Triton X-100(100mg/kg bwt) + 300 mg C. congoensis
Triton X-100(100mg/kg bwt) + 400 mg C. congoensis
2.6.3 Biochemical Analysis
After seven days of treatment, rats were fastened for 18 hours. The rats were then
anesthetized using chloroform, the blood samples were collected by cardiac puncture. The
serum was separated by centrifugation of blood at 3000 rpm/10mins for estimation of
biochemical parameters (lipid profile) which includes TC, TG, HDL-C, LDL-C and VLDL-
C.[25]
2.6.3.1 Estimation of Serum total cholesterol (TC)
Method: The Method of CHOD-PAP for estmation of serum cholesterol was used as
described by Safiullah et al.,[26]
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Principle: The total cholesterol is measured enzymatically in serum or plasma in a series of
coupled reactions that hydrolyze cholesteryl esters and oxidize the 3-OH group of
cholesterol. One of the reaction products is measured quantitatively in a peroxidase catalyzed
reaction that produces a colour. Absorbance is measured at 500nm. The colour intensity is
proportional to cholesterol concentration.
Procedure: The samples were pipetted into the reaction vessel using a micro pipette. Test
samples (T): 0.02 ml serum, 2.00 ml reaction solution; the standard sample (S): 0.02ml
standard and 2.00 ml reaction solution, while for the blank sample (B): 0.02 ml DW and
2.00ml reaction solution. The mixture were mixed well and incubated for 10 minutes at +20
to 25 0C or 5 minutes at 370C. The absorbance was read at 670 nm against the reagent blank.
2.6.3.2. Estimation of serum triglycerides (TG)
Method: GPO-PAP method as described by Safiullah et al.,[26]
was used to estimate the
serum triglycerides.
Principle: Sample triglycerides incubated with lipoprotein lipase (LPL) liberate glycerol and
free fatty acids. Glycerol is converted to glycerol-3-phosphate (G3P) and adenosine -5-
diphosphate (ADP) by glycerol kinase. Glycerol-3-phosphate (G3P) is then converted by
Glycerol phosphate dehydrogenase (GPO) to dihydroxyacetone phosphate (DAP) and
hydrogen peroxide (H2O2). In the last reaction, hydrogen peroxide (H2O2) reacts with 4-
aminophenazone (4-AP) and p-chlorophenol in the presence of peroxidase (POD) to give a
red coloured dye. The intensity of the colour formed is proportional to the triglycerides
concentration in the sample.
Procedure: For this analysis 0.01 ml of serum was taken in a test tube (T) in which 1ml
reaction solution was added. In another test tube (S) 0.01ml standard and 1ml reaction
solution was added. The solution was mixed well and incubated at 25oC for 10 min. The
absorbance of standard and test against reagent blank was read at 540 nm.
2.6.3.3. Estimation of HDL-cholesterol
Method: CHOD-PAP method was used to estimate the serum HDL cholesterol level as
described by Safiullah et al.,[26]
Principle: During the first phase, LDL, VLDL particles and Chylomicrons generate free
cholesterol, which through an enzymatic reaction, produce hydrogen peroxide. The generated
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peroxide is consumed by a peroxidase reaction with DSBmT yielding a colorless product.
During the second phase, specific detergent solubilizes HDL-Cholesterol. In conjunction with
CO and CE action, POD + 4-AAP develop a colored reaction which is proportional to HDL-
cholesterol concentration.
Procedure: For this analysis 2ml of serum was taken in a test tube and 0.5 ml of precipitation
reagent was added. The mixture was shaken thoroughly and left to stand for 10min at 25oC
and then centrifuged for 15min at 4000rpm. Within 2hr after centrifugation, the clear
supernatant was used for the determination of HDL-C. One ml of the supernatant was taken
in a test tube (T) and 1 ml of reaction solution was added to it. In another test tube 0.1 ml DW
was taken and 1ml reaction solution (B) was added. The mixtures were mixed thoroughly,
incubated for 5min at 37OC and measured the absorbance of the sample against reagent blank
at 546 nm.
2.6.3.4. Estimation of LDL cholesterol
Method: The Estimation method described by Friedwald et al.,[27]
was used in determining LDL-C
LDL cholesterol was estimated using Friedwald’s formula as follows:
LDL in mg % Total cholesterol–HDL cholesterol – (triglycerides/5).
2.6.3.5 Estimation of VLDL cholesterol:
Method: The Estimation method of Friedwald et al.,[27]
was used in determining VLDL-C
VLDL cholesterol was estimated by using the following formula:
VLDL in mg %=
2.6.3.6 Atherogenic Index (AI): was calculated using the formula:
AI=TC/HDL-C
2.7 Statistical Analysis
Experimental data were analyzed using one-way analysis of variance (ANOVA) and LSD
multiple range test to determine significant differences between means. The difference
between the means was regarded as significant at p<0.05 using SPSS software version 23.
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3.0: RESULTS
3.1: Table 1 Shows the results of qualitative phytochemicals screening of methanolic extract
of Cassipourea congoensis fruit. Alkaloids, flavonoids, tannins, terpenoids, phenols, steroids
and glycosides were all found to be present.
Table 1: Qualitative Phytochemical Content of Methanolic Extract of Cassipourea
congoensis Fruit.
S/N Phytochemicals Result
1. Flavonoid +
2. Saponins -
3. Phenols +
4. Terpenoid +
5. Tannins +
6. Steroid +
7. Alkaloid +
8. Glycoside +
Key:
(+)= Positive
(-) = Negative
3.2: Table 2 Shows some quantitative phytochemical content of methanolic extract of
Cassipourea congoensis fruit. Phenols had the highest value (17%) closely followed by
flavonoids (16%). Terpernoids had the least value (1%).
Table 2: Quantitative Phytochemical Constituents of Methanolic Extract of Cassipourea
congoensis Fruit.
S/N Phytochemical constituents Percentage % (w/w)
1. Flavonoid 16
2. Phenols 17
3. Terpenoid 1
4. Alkaloid 6
5. Tannins 3
3.3: Table 3 shows GC-MS Analysis of Cassipourea congoensis fruit. Results obtained
revealed the presence of squalene, 4-mercaptophenol, ascorbyl-palmitate, farnesol,
octadecadienoic acid, oleic acids gallein, trans-farnesol and 9,12-octadecadienoic acid.
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8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0
5 0 0 0 0 0 0
1 e + 0 7
1 . 5 e + 0 7
2 e + 0 7
2 . 5 e + 0 7
3 e + 0 7
3 . 5 e + 0 7
4 e + 0 7
4 . 5 e + 0 7
5 e + 0 7
5 . 5 e + 0 7
T im e - - >
A b u n d a n c e
T I C : 0 1 0 1 0 0 3 . D \ d a t a . m s
7 . 2 8 0 7 . 3 5 3 7 . 5 6 4 7 . 6 6 0 7 . 7 4 0 7 . 8 8 3 7 . 9 9 6 8 . 1 3 0 8 . 3 2 5 8 . 4 6 6 8 . 5 3 7 8 . 5 7 6 8 . 6 2 9 8 . 7 2 9 8 . 8 3 3 8 . 9 4 6 9 . 1 8 5 9 . 3 6 9
9 . 7 9 9 9 . 9 4 71 0 . 0 8 91 0 . 4 4 41 0 . 4 8 11 0 . 6 7 71 0 . 9 0 31 1 . 0 0 21 1 . 1 1 5
1 1 . 9 0 41 2 . 0 8 91 2 . 1 3 81 2 . 2 6 21 2 . 2 8 91 2 . 3 9 11 2 . 5 2 81 2 . 6 0 61 2 . 6 5 11 2 . 6 9 81 2 . 8 0 81 2 . 9 7 81 3 . 1 5 21 3 . 2 5 01 3 . 3 7 31 3 . 5 8 51 3 . 7 2 81 3 . 9 3 11 4 . 0 4 71 4 . 4 5 51 4 . 5 3 11 4 . 5 8 91 4 . 9 0 41 5 . 2 6 11 5 . 4 0 41 5 . 8 7 51 5 . 9 8 01 6 . 1 3 6
1 6 . 2 6 01 6 . 4 3 21 6 . 5 1 01 6 . 5 6 41 6 . 7 2 7
1 7 . 0 8 3
1 7 . 8 0 81 8 . 0 5 51 8 . 1 9 81 8 . 3 1 31 8 . 4 8 31 8 . 6 4 11 8 . 8 6 41 9 . 0 4 91 9 . 3 1 6
1 9 . 6 7 01 9 . 8 2 91 9 . 8 8 42 0 . 0 0 62 0 . 0 7 22 0 . 3 0 32 0 . 4 1 62 0 . 4 9 92 0 . 7 1 92 0 . 9 4 9
2 1 . 5 3 0
2 2 . 3 9 6
2 2 . 9 4 6
2 3 . 7 0 7
2 4 . 5 6 72 4 . 7 5 52 4 . 8 4 62 5 . 0 3 92 5 . 1 1 82 5 . 3 8 42 5 . 8 2 3
2 6 . 1 1 22 6 . 4 1 12 6 . 5 3 02 6 . 5 6 72 6 . 6 7 22 6 . 7 6 42 6 . 8 2 12 6 . 9 6 12 7 . 0 1 62 7 . 1 3 22 7 . 1 7 42 7 . 2 7 62 7 . 4 2 02 7 . 5 4 92 7 . 6 8 82 7 . 7 8 52 7 . 8 3 32 7 . 9 3 22 8 . 0 4 32 8 . 1 6 32 8 . 2 3 22 8 . 4 6 52 8 . 5 6 52 8 . 6 8 02 8 . 7 2 82 8 . 7 8 42 9 . 0 2 92 9 . 5 2 62 9 . 5 8 12 9 . 8 9 42 9 . 9 7 23 0 . 0 0 23 0 . 0 4 1
Figure 1: GC-MS Spectral Chromatogram of Methanolic Extract of Cassipourea
congoensis Fruit.
Table 3: GC-MS Analysis of Cassipourea congoensis fruit.
Name of compound Retention
time (min)
Peak
area
Percentage
peak area
(%)
Molecular
weight
(g/mol)
Molecular
formula
AscorbylPalmitate 20.072 244721 0.24 414.533 C22H38O7
Phloroglucinol, tris
trifluoroacetate 26.570 244484 0.28 126.11 C6H6O3
Squalene 20.947 243219 1.14 410.73 C30H50
l-(+)-Ascorbic acid 2,6-
dihexadecanoate 15.980 274387 0.29 414.583 C23H42O6
4-Mercaptophenol 9.948 11357 0.46 126.173 C6H6OS
Oleic Acid 17.082 142069 4.11 282.468 C18H34O2
Ergost-25-ene-3,5,6,12-
tetrol, (Gallein) 25.821 256291 2.17 364.309 C20H12O7
trans-Farnesol 19.671 85703 0.29 222.372 C15H26O
9,12-Octadecadienoic acid 17.082 140138 4.11 294.435 C18H30O3
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3.4: Table 4 Shows the hydrogen peroxide activity of methanol extract of Cassipourea
congoensis fruit. The antioxidant activity of the fruit extract was significantly higher (p≤0.05)
compared to ascorbic acid at 80 and100mg/ml. Extract activity was found to be dose
dependent.
Table 4: Hydrogen Peroxide Antioxidant Activity of Methanolic Extract of Cassipourea
congoensis Fruit.
Concentration (µg/ml) Methanolic extract (%) Ascorbic acid (%)
20 38.38±0.20 38.62±0.23
40 39.50±0.38 41.25±0.12
60 39.74±0.15 41.50±0.32
80 45.63±0.90 a 42.38±0.30
100 49.50±0.15a 45.34±0.35
All values are mean ± SEM for 3 determinations.
a=significantly (P<0.05) higher compared to ascorbic acid.
3.5: Table 5 shows the DPPH radical scavenging activities of methanol extract of
Cassipourea congoensis fruit. The highest percentage inhibition of the fruit was 55.45±0.65
at concentration of 100mg/ml. Percentage inhibition of the fruit extract was significantly
higher at 80 and 100mg/ml compared to ascorbic acid at the same dose. Increase in
antioxidant activity was found to be dose dependent.
Table 5: DPPH Radical Scavenging Activities of Methanol Extract of Cassipourea
congoensis Fruit.
Concentration (µg/ml) Metahnolic extract (%) Ascorbic acid (%)
20 27.25±0.12 29.95±0.60
40 32.10±0.30 32.24±0.17
60 41.50±0.29 40.88±0.35
80 51.90±0.30 a 47.80±0.62
100 55.45±0.65 a 51.35±0.38
All values are mean ± SEM for 3 determinations.
a=significantly (P<0.05) higher compared to ascorbic acid.
3.6: Table 6 shows the ferric reducing antioxidant power (FRAP) of methanolic extract of
Cassipourea congoensis fruit. The antioxidant activity was found to be dose dependent with
the highest activity observed at the concentration of 100mg/ml.
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Table 6: Ferric Reducing Antioxidant Power Activity of Metabolic Extract of
Cassipourea congoensis Fruit.
Concentration (µg/ml) Methanolic extract (%) Ascorbic acid (%)
20 28.73±0.17 31.82±0.19
40 32.65±0.15 34.80±0.17
60 37.62±0.21 a 35.58±0.20
80 42.50±0.41a 39.15±0.12
100 52.90±0.21a 49.54±0.34
All values are mean ± SEM for 3 determinations.
a=significantly (P<0.05) higher compared to ascorbic acid.
3.7: Table 7 shows the effect of Cassipourea congoensis fruit extract on serum lipid
parameters in triton X-100 induced hyperlipidaemic rats. Induction with triton X-100 causes
significant increase (P<0.05) in the levels of LDL, TG, TC and VLDL with decrease in the
level of HDL. Administration of methanol extract of Cassipourea congoensis at various
concentrations (100, 200,300 and 400mg/kg/bw) significantly decreased the levels of TC,
TG, LDL and VLDL while HDL level increased significantly at (P<0.05) with administration
of the extract in a dose dependent manner.
Table 7: Effect of Cassipourea congoensis Fruit Extract on Serum Lipid Parameters in
Triton X-100 Induced Hyperlipidaemic Rats.
GROUPS TC(mg/dl) TG(mg/dl) HDL(mg/dl) LDL(mg/dl) VLDL(mg/dl) AI
Normal control 88.47±7.32 b
76.70±3.90 b
51.43±0.84d
21.70±1.80 b
15.34±1.42 b
1.72±0.04 b
Hyperlipidaemic
control 194.50±2.90
a 105.68±4.44
a 30.24±2.82
c 143.12±1.16
a 21.14±0.32
a 6.43±0.28
a
Atorvastatin
(10mg/kg/b.w) 96.94±0.78
b 78.56±1.72
b 47.62±1.64
d 34.01±1.67
ab 15.31±0.35
b 2.04±0.12
b
CC(100mg/kg/b.w) 157.35±1.60ab
100.44±1.20a 34.48±1.80
c 101.89±0.58
ab 20.89±0.80
a 4.56±0.34
a b
CC(200mg/kg/b.w) 134.28±1.70ab
94.23±1.15a b
40.10±0.70d
75.33±1.85ab
18.85±0.26ab
3.35±0.05ab
CC(300mg/kg/b.w) 102.67±2.10ab
82.10±1.62 b
46.80±1.05d
39.45±3.16ab
16.42±0.34b
2.19±0.16a b
CC(400mg/kg/b.w) 76.84±1.00 bce
74.35±1.34 bc
54.60±0.92ad
7.37±0.06 bce
14.41±0.14bc
1.41±0.08bce
Values are means± SEM of 6 replicates
a= Significantly higher (p≤0.05) compared to normal
b= Significantly lower (p≤0.05) compared to experimental control
c= Significantly lower (p≤0.05) compared to different extracts
d= Significantly higher (p≤0.05) compared to experimental control
e= Significantly lower (p≤0.05) compared to standard drug
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4.0 DISCUSSION
The results of phytochemical analysis of Cassipourea congoensis fruit extract showed the
presence of different phytochemical constituents which includes alkaloids, flavonoids,
glycosides, steroids, terpenoids, phenols and tannins. The quantification analysis in terms of
percentage revealed that the fruit extract possesses phenols and flavonoids in high amounts
(17% and 16% respectively).
The presence of high amount of flavonoids, phenols and alkaloids in the methanol extracts of
Cassipourea congoensis fruit extract may be responsible for the antihylipidaemic activity of
this fruit. The extract might have mobilized cholesterol from the extra hepatic tissues to the
liver for bile acid synthesis. This is often indicated by an increase in HDL–cholesterol.
Dietary management with fruits has been recommended as part of the scrupulous controls
necessary to prevent and/or manage dyslipidaemia.[28]
Flavonoids have been identified as a potent hypolipidaemic agent in experimental studies.
Flavonoids promote an increase in faecal sterol which in turn leads to a decreased absorption
of dietary cholesterol. Flavonoids are known to improve cardiac function, decrease anginas
and lowers cholesterol levels. These compounds act by regulation of inflammation
mediators.[29]
Flavonoids and plyphenols contribute to hypolipidaemic activity by increasing
cholesterol metabolism and by modulating the enzymes involve in cholesterol metabolism
such as HMG- coA; lecithin cholesterolacyl transference, cholesterol 7α-hydroxylase and
acyl coA: cholesterol acyl transferase. It has been reported that flavonoids intake can
decrease LDL-C and increase HDL-C which may hasten the removal of cholesterol from
peripheral tissue to the liver for catabolism and excretion.[30]
Flavonoids are also responsible
for the antioxidant activities of plants through their scavenging or chelating activity.[31]
Phenolic compounds are found in both edible and non edible plants with several biological
effects. Tannins are stronger in their antioxidant and antiradical activities because tannins
possess more numbers of hydroxyl groups than flavonoids.[30)]
Tannins like others phenolic
compounds also possess a variety of other biological activities such as reduction of plasma
lipids resulting from the up regulation of LDL, inhibition of hepatic lipid synthesis and
increase in cholesterol elimination via bile acids.[32]
The effects of tannins on human health
have been attributed mainly to their strong free radical-scavenging and antioxidant
activities.[33]
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Umaru et al. World Journal of Pharmaceutical Research
Ascorbyl palmitate acted as an antioxidant and as an anti-inflammatory agent.[34]
Therefore, it
plays a vital role in hyperlipidaemia management since it confers antioxidant activity. The
consumption of saturated fatty acids has been shown to increase plasma LDL-cholesterol in
man and the increase of LDL-cholesterol has been correlated with coronary heart disease.[35]
The reduction of the low-density lipoprotein could be due to protection on cellular oxidative
stress, thrombogenicity, and atheroma plaque formation.[36]
Other compounds of great
interest are citronellol which lowers the blood pressure in the treatment of cardiovascular
diseases. Farnesol are precursors of steroids in plants and it is a starting compound of natural
organic synthesis and intermediate metabolite in the synthesis of antioxidants.[37]
The methanol extract of Cassipourea congoensis fruit showed that the fruit has high
antioxidant activity. Its potential antioxidant activity was tested using H2O2, DPPH and
FRAP antioxidant assay. Results obtained in this study revealed that Cassipourea congoensis
fruit has significantly higher (p≤0.05) antioxidant activity compared to ascorbic acid.
Oxidants and free radicals are harmful to the body health when their overload cannot steadily
be destroyed and consequently generate an occurrence called oxidative stress.[38]
This
disproportionate production of free radicals plays a key role in the formation and
development of chronic diseases such as cancer, rheumatoid arthritis, cardiovascular and
autoimmune disorders or even aging.[38]
In this study, we conclude that the methanolic extract
of Cassipourea congoensis has good antioxidant property and its constituent can be of use as
an easily accessible source of natural antioxidant and as a food supplement or in
pharmaceutical industry.
Treatment of hyperlipidaemia with Cassipourea congoensis fruit extract (100, 200, 300 and
400mg/kg/b.w) for 7 days successfully decreased the elevated levels of serum total
cholesterol, triglycerides, low density lipoproteins and very low density lipoproteins
cholesterol in triton x-100 induced hyperlipidaemic rats. Triton X-100 is a non-ionic
surfactant that accelerates hepatic cholesterol synthesis and increases intestinal lipid
absorption by the emulsification process. It suppresses the action of lipoprotein lipase and
blocks the uptake of lipoproteins from circulation by the extrahepatic tissues resulting in
increased blood lipid concentrations.[39]
The experimental control group had the highest value of atherogenic index. Elevated levels of
total cholesterol are associated with increased risk of atherosclerosis. High level of
triglycerides and LDL are associated with coronary artery disease. Administration of the
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Umaru et al. World Journal of Pharmaceutical Research
extract at 400mg significantly decreased the lipid profile parameters very closely to the
standard drug atorvastatin. A considerable increment in the level of HDL-cholesterol was
also observed in a dose depedent manner. The combined effect of the phytochemicals in the
extract might have synergistically accounted for the observed decrease in total cholesterol,
triglycerides, LDL-cholesterol and very low density lipoprotein.
4.2 CONCLUSION
The study revealed that Cassipourea congoensis fruit possesses high antioxidant activities
comparable to vitamin C. The high antioxidant activity may be attributed to the presence of
alpha tocopherol and some of the phytochemicals present in the fruit exact. Administration of
Cassipourea congoensis extract produced significant improvement in lipid profile by
lowering TC, TG, and LDL and increasing HDL level. The atherogenic index was
significantly lower (p≤0.05) in group administered 400mg of the extract compared to the
standard drug atorvastatin. Result obtained from this study indicates that the fruit
Cassipourea congoensis can be used in the management and treatment of hyperlipidemia and
its complications.
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