antioxidant and antihyperlipidaemic activities of

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.



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]



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.



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,



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]



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 (%) =



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




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]



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



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.



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.



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



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



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.



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



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



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]



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



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.

REFERENCES

1. Setty, S.K., Patel J.S., Chakraborty, M. and Kamath J.V. Antihyperlipidemic Activities of

Divya methipachak Against Triton x-100 iInduced hHyperlipidemia in Rats. International

Research Journal of Pharmacy, 2012; 3(8): 2230-8407.

2. Harikumar, K., Niveditha, B. and Reddy, P. K. Anti-Hyperlipidemic activity of Alcoholic

And Methonolic extracts of Crotolaria juncea in Triton-Wr 1339 Induced

Hyperlipidemia. International Journal of Phytopharmacology, 2012; 3(3): 256-262.

3. Ginghina, C., Bejan, I. and Ceck, C. D. Modern risk stratification in coronary heart

Diseas Journal of Medical Life., 2011; 4(4): 377-86.

4. Jorgensen, T., Capewell, S., Prescott, E., Allender, S., Sans, S. and Zdrojewski I.

Population-Level Changes to Promote cardiovascular Health. Europe Journal of

Prevalence Cardiology, 2013; 20(3): 409-21.

5. Gabriel, U. P., Chuku A., Obiegbu N. P., Ofoedu J. N., Ikwudinma A. O. Frequency of

Cardiovascular Risk Factors in Adult Nigerians with Family History of Non-

Communicable Cardiovascular Disease in a Primary CareClinic of a Tertiary Hospital in

a Resource-Constrained Environment of Eastern Nigeria. American Journal of Health

Research, 2013; 1(1): 17-25.



6. WHO, (2010): Guidelines for the Treatment of hyperlipidemia (Report) 2nd

ed. World

Health Organization. ISBN 978-9-2415-4792-5.

7. Lateef, T., Riaz, A., Zehra, A. and Qureshi, S.A. Antihyperlipidemic effect of Adonis

vernalis. Journal of the Dow University of Health Sciences Karachi., 2012; 8.6(2): 48-52.

8. Berhanu, A., and Adefa M. Ethnobotanical Survey of Traditional Medicinal Plants in

Tehuledere District, South Wollo, Ethopia. Journal of Medicinal Plants Research, 2011;

5(26): 6233-6242.

9. Nkafamiya, I. I., Manju A. J., Modibbo U.U., and Umaru, H. A. Biochemical Evaluation

of Cassipourea congoensis. African Journal of Biotechnology, 2006; 6(19): 2461-2463.

10. Nadro, M.S., and Umaru, H.A. Comparative chemical evaluation of locust bean (Parkia

biglobosa) Fruits Pulp Harvested During the Dry and Wet Seasons. Nigerian Journal of

Biotechnology, 2004; 15(4): 42-47.

11. El-Siddig, K., Gunasena, H.P.M., Prasa, B.A., Pushpakumara, D.K.N.G., Ramana,

K.V.R., Vijayanand. P. and Williams, J.T. Tamarind – Tamarindus indica L. Fruits for

the future 1. Southampton Centre for Underutilized Crops, Southampton, UK, 2006; 188.

12. Christine, O. and Hannington O. Analysis of the Fresh Pulps of Borassus aethiopum

Fruits of Gulu District, Uganda. American Journal of Food and Nutrition, 2016; 4(6):

177-181.

13. Sadiq, I.S., Duruminiya, N. I., Kwada, D., and Izuagie, T. Nutritional and Anti-

Nutritional Value of Some Wild Fruits. International Journal of Applied Research and

Technology, 2016; 5(3): 50-56.

14. Kumar, C.S. and Bhattacharya, S. Tamarind Seed: Properties, Processing and Utilization.

Critical Reviews in Food Science and Nutrition, 2008; 48: 1-20.

15. Reddy, S.N., Swetha, A., Arijum, M. And Ganga, R.m. Antihyperlipidemic Activities of

Methanolic Extract of Syzgium alternifolium Bark against High Fat Diet Dexamethasone

Induced Hyperlipidemia in rats. Asian Journal of Pharmaceutical Clinical Research,

2015; 8: 165-168.

16. Trease G.E., and Evans W.C. Textbook of Pharmacognosy 14th Edition W. B. Saunders

Company Limited, New York, 1999; 1-340.

17. Harborne, J. B. Textbook of phytochemical methods, 1st Edition. Champraan and Hall

Ltd. London, 1973; 110-113.

18. Boham, B.A. and Kocipai-Abyazan R. Flavonoids and condensed tannins from leaves of

Hawaiian Vaccinium vaticulatum and V. calycinium”. Pacific Science, 1974; 48:

458- 463.



19. Anchana, C., Aphiwat T. and Nuansri R. Screening of antioxidant activity and antioxidant

compounds of some edible plants of Thailand” Food Chemistry, 2005; 92: 491– 497.

20. Vengaiah, P.C., Vijayakumari B. and Kiranmayi P. GC-MS Analysis of Bioactive

Compounds in Ethanolic Root Extract of Palmyra Palm (Borassus Flabellifer L.).

America journal of pharmaceutical research, 2015; 5(3): 2231-6876.

21. Syed, Z. H. and Khushnuma M. GC-MS: Principle, Technique and its Application in

Food Science. International Journal of Current Science, 2014; (13): 116-126.

22. Jayaprakasha, G. K., Jaganmohan, R. L. and Sakariah, K. K. "Antioxidant activities of

flavidin in different invitro model systems." Bioorganic Medical Chemistry, 2004;

12(19): 5141-5146.

23. Patil, S.M., KadamV.J. and Ghosh R. In- vitro Antioxidant Activity of Methanolic

Extract of Stem Bark of Gmelina arborea roxb. International Journal of Pharmaceutical

Resource, 2009; 1(4): 1480-1484.

24. Banerjee, D., Chakrabarti S., Hazra A. K., Banerjee S., Ray J. and Mukherjee B.

Antioxidant Activity and Total Phenolics of Some Mangroves in Undarbans” African

Journal of Biotechnology, 2008; 7: 805–810.

25. VijayKvijay, G. and Devanna N. Antihyperlipidemic Activity of Leaf Extracts of Leucas

aspera Linn. Against Dexamethasone-induced Hyperlipidemia in Rats. Asian Journal of

Pharmaceutics, 2016; 10(3): 413.

26. Safiullah, G. S., Rizwan K., Kaab E. and AbidHussain A. Evaluation of

antihyperlipidemic activity of ethanolicextact of Glycosmis pentaphylla in

hyperlipidemicwistarrats. International Journal of Pharma Sciences and Research, 2015;

6(2): 282-292.

27. Friewald, W.T., Levy R.I. and Fredrickson D.S. Estimation of the Concentration of Low

Density Lipoprotein Cholesterol in Plasma without the use of Preparative Ultracentrifuge.

Clinical Chemistry, 1972; 18: 499-502.

28. Mirmiran, P., Noori N., Zavarch, M. B. and Azizi, F. Fruits and Vegetables Consumption

and Risk Factors for Cardiovascular Disease. Journal of Metabolism, 2009; 58: 460-468.

29. Jiang, H., Ma Y., Chen X., Pan S. And Sun B. Genistein synergizes with arsenic trioxide

to suppress human hepatocellular carcinoma. Cancer Science, 2010; 101: 975-983.

30. Walid, H. E. Biochemical Effects, Hypolipidemic and Anti-inflammatory Activities of

Artemisia vulgaris Extract in Hypercholesterolemic Rats. Journal of Clinical

Biochemistry and Nutrition, 2005; 57(1): 33-38.



31. Kessler, M., Ubeaud G. and Jung L. Anti-and Prooxidant Activity of Rutin and Quercetin

derivatives. Journal of Pharmacy and Pharmacology, 2003; 55: 131-142.

32. Del-Bas, J. M., Fernandez-Larrea, J., Blay, M., Aedvol A., Salvado M. J., Arola I. and

Blade C. Grape Seed Procyanidins Improve Atherosclerotic Risk Index and Induce Liver

CYP7AI and SHP expression in healthy Rats. Journal of the Federation of American

Societies of Experimental Biology, 2005; 19: 479-481.

33. Mona, S. M., Wadah, J.A., Elrashied A.E., Garelnabi, Z. O., Bashier O., Hassan S.,

Khalid, M. A. and Mohamed O. Secondary Metabolites as Anti-inflammatory Agents.

The Journal of Phytopharmacology, 2014; 3(4): 275-285.

34. Hira, K., Akhtar N., Khan H.M.S., Arshad A.I., Naeem M., Ali A., Rasool F. and Nawaz

Z. Synergistic Effects of Ascorbyl Palmitate and Sodium Ascorbyl Phosphate Loaded in

Multiple Emulsion on Skin Melanin and Erythema Content. Biomedical Research, 2016;

27(2): 30-139.

35. Lee, H., Le, S.C., OH,, Y. G., Kim K.H., Kim, B. H., Park, Y.H., Chaem S. H. and Chung

I. B. Effects of Rumen Protected Oleic Acid in the Diet on Animal Performances, Carcass

Quality and Fatty Acid Composition of Hanwoo Steers. Asian-Aust. Journal of Animal

Science, 2003; 16(7): 1003-1010.

36. Perona, J.S., Cabello-Moruno R. and Ruiz-Gutierrez V. The Role of Virgin Olive Oil

Components in the Modulation of Endothelial Function. Journal of Nutrition

Biochemistry, 2006; 17: 429–445.

37. Visioli, F., Bogani P., Grande S., Detopoulou V., Manios Y. and Galli C. Local food and

cardioprotection: the role of phytochemicals. Forum Nutrition, 2006; 59: 116–129.

38. Sima, G., Sima R., Maryam J., Hossein K., H. and Soheila N. A. Review on antioxidants

and their Health eEffects. Journal of Nutrition and Food Security, 2018; 3(2): 106-112.

39. Parwin, A., Najmi A.K., Ismail, M. V., Kaundal M. and Akhtar M. Protective Effects of

Alendronate in Triton X-100-Induced Hyperlipidemia in Rats. Turkish Journal

Gastroenterology, 2019. DOI:10.5152/18076

antioxidant and antihyperlipidaemic activities of

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