FOOD AND NUTRITION OPEN ACCESS (ISSN:2517-5726)

Female albino wistar rats (n=54) weighing 80-90 g at 6 weeks (42 days) of age were used for this study. Nine experimental treatments involving the random distribution of six (6) rats in each group were carried out. The serum lipid profile of the rats revealed that group 2 (negative control) had significantly high serum TCH, TG, LDL-C and VLDL-C and very low serum HDL-C (p ≤ 0.05) when compared with group 1 (control) while groups 3, 4, 5, 7, 8 and 9 had reduced serum TCH, TG, LDL-C and VLDL-C when compared with groups 1 and 2. The latter groups (i.e., groups 3, 4, 5, 7, 8, and 9) maintained elevated serum HDL-C concentrations when compared with groups 1 and 2. Group 6 had same TCH, TG, and VLDL-C concentrations with group 1 (p>0.05). 

Aim: To investigation the effect of Cola lepidota seeds on the serum lipid profile of high fat fed albino Wistar rats.

Conclusion: Diets high in fat tend to promote overweight, obesity and other fat induced conditions, therefore, inhibiting digestion and absorption of dietary fats becomes a logical remedy to either prevent or ameliorate such conditions. Results have shown that Cola lepidota seeds possess hypolipidaemic and anti-atherosclerotic activities, hence, become good repository for new drug discovery.

Lipid profile; High fat diet; Cholesterol; Lipoprotein; Cola lepidota

Perturbed intravascular lipid and lipoprotein metabolism is a common feature of obesity leading to a dyslipidemia involving elevated plasma concentrations of triacylglycerols and apoliproptein B-containing lipoproteins (very low density lipoprotein: VLDL and low density lipoprotein: LDL) and subnormal levels of high density lipoprotein: HDL), a lipid phenotype associated with cardiovascular risk [1].

Obesity is the most prevalent nutritional disease and a growing public health problem worldwide [2]. This disease according to Gamboa-Gomez et al. [2], is a causal component of the metabolic syndrome related with abnormalities, including hyperglycemia, dyslipidemia, hypertension, inflammation, among others. Malik et al. [3] reported that in the year 2015, obesity had acquired epidemic proportions projected to reach 2.3 billion of overweight adults and 700 million obese adults. WHO (2015) [4] also reported that about 1.9 billion adults worldwide and 42 million children below 5 years are obese while morbidity and mortality associated with obesity-related diseases, such as type 2 diabetes mellitus (T2DM), cardiovascular disease (CVD), hyperlipidemia and hypertension, have increased exponentially [5,6].

Some research findings have revealed that the use of ethnopharmacological knowledge is one attractive way to reduce empiricism and enhance the probability of success in new drug finding efforts [7,8].

According to World Health Organization (WHO), more than 80% of the world’s population depends on traditional medicine for their primary healthcare needs [9].

Most plant materials contain fiber and the fiber content of fruits have been reported to possess certain blood cholesterol lowering action and aid in the prevention of large bowel disease [10].

Ene-Obong et al. (2016) [11] reported that fruits are low in calorie and can as well help reduce one’s calories intake as part of a weight loss diet and enlisted Cola lepidota as an under-utilized fruit. All these research findings point to the fact that plant constituents have therapeutic potentials.

Cola lepidota  is a member of the family of Sterculiaceae and belongs to a group called drupes [12]. The pod of Cola lepidota  is yellowish and roundish and is also called Yellow Monkey Kola, while the white variety which is Cola parchycarpa has more cylindrical shape and is also called White Monkey Kola. Cola lepidota  is cultivated throughout the  regions of the world. It is commonly found in Southern Nigeria between the months of June to November [13]. Cola lepidota  fruits are highly nutritious and medicinal [12] and Cola lepidota  (having yellow pod), Cola parchycarpa (having white pod) and Cola lateritia (having red pod) all belong to the family of monkey kola [14].

Cola lepidota is mostly consumed fresh and according to Okudu et al. (2015) [14], it has a very short life span probably due to its high moisture content and its hard texture which limits its consumption particularly among the vulnerable group (i.e., children and aged) due to poor dentition.

Cola lepidota seed is selected specie for this study because of its traditional use in Isiala Ngwa North and South L. G. A of Abia State as a weight reducer and research findings have shown that it contains significantly higher phytochemical constituents than other species and it is more widely distributed [14-16]. Okudu et al. (2015) [14] reported that Cola lepidota juice contains significantly higher phytochemical constituents than Cola parchycarpa. Also, Okudu et al. (2015) [14] were able to investigate the phytochemical constituents of the membranes and seeds of Cola lepidota and revealed that B-vitamins, particularly riboflavin and niacin were found in significant amount in Cola lepidota membrane and both C. lepidota and C. parchycarpa had substantial amounts of phytochemicals (particularly alkaloids, phenols, flavonoids and saponins. Essien et al. (2015) [16] detected from their phytochemical screening, alkaloids, saponins, terpenoids, carbohydrates, and flavonoids in the seeds and fruit pulp extracts of C. lepidota, K. Schum and C. rostrata.

Not much has been done on Cola lepidota seed with respect to its effects on the lipid profile of high fat fed albino rats. Okudu et al. (2015) [14] have reported that the high phytochemical values obtained in Cola lepidota seed makes it an important seed that needs to be fully exploited. Some research works on the other hand have reported that C. lepidota is employed in Nigerian folk medicine as febrifuges, for pulmonary problems and cancer related ailments [15,17].

The leaf and stem bark extracts of C. lepidota have been studied [16] with respect to its antioxidant activity [15], anticancer [17], and acute toxicity [18,19].

There is paucity of information about the effects of C. lepidota seeds on serum lipid profile of high fat fed albino wistar rats irrespective of its use by local herbal medicine practitioners in Isiala Ngwa North and South L. G. As of Abia State as weight reducing agents and this forms the basis for this investigation. The pictures of Cola lepidota fruits and seeds (Figures 1-3).

The Figure 1 shows the mature, intact Cola lepidota fruit. It shows the scaly exocarp that is usually hard but can be easily cut open with a knife. The fruit does not have a definite shape. Its shape comes from the shape and size of the seed inside it. The exocarp is usually brownish in colour and covered with tiny hairs. This portion of the exocarp must be removed to get to the edible yellowish mesocarp.

The Figure 2 shows two slightly torn scaly exocarps, revealing the edible yellow pulps as well as two yellow pulps completely devoid of the scaly exocarp. It is these yellow pulps that are often relished.

The Figure 3 shows three Cola lepidota seeds which are obliquely ovoid with two flattered surfaces and are usually rough with either reddish-brown or greenish colour. The seeds contain hairy spines within the interior of the opposing faces. These hairy spines could be the major reason why earlier people preferred consuming its closer specie, Cola nitida.

With urbanization, changing lifestyles, diminished assess/ availability of fresh vegetables as well as increased consumption of processed foods, the number of people with obesity and/or dyslipidaemia tends to increase. Therefore, a critical management of traditional medicinal plant resources has become a matter of urgency [20]. Cola lepidota seed has been primitively used as a local weight reducing remedy in Isiala Ngwa North and South Local Government Areas of Abia State by some herbal medicine practitioners. The present study remains the first to investigate the effects of Cola lepidota seeds on serum lipid profile of high fat fed Wistar albino rats.

The aim of this study is to investigate the effect of Cola lepidota seeds on the serum lipid profile of high fat fed albino Wistar rats.

Collection of plant materials

Cola lepidota K. Schum fruits were purchased from ‘New Market’, Aba, Abia State, Nigeria and were identified in the Forestry Department of Michael Okpara University of Agriculture, Umudike by Mr. Ibe Ndukwe and the seed specimen stored in the Department’s herbarium.

Preparation of plant seed extract for oral administration in rats 

The seeds were removed from their pods and sun-dried and ground to fine powder and stored in an air-tight container till when needed for the experiment.

Hot continuous extraction with soxhlet extractor (Fisher Scientific Laboratory, Ireland) was used to obtain the organic compounds from the dry ground seed powder and the solvent used was pure ethanol (99%) (Sigma Aldrich, Germany) in order to obtain polar lipids. The temperature was maintained at 40°C (so as not to degrade certain compounds in the seed) for 8 hours in order to obtain the complete extraction of the sample.

The procedure involved weighing 200 g of the powdered sample into a cellulose thimble in the soxhlet extractor containing 600 ml of the pure ethanol. The sample was refluxed for 8 hours at 40°C using a condenser (with running cold water) attached to the top of the soxhlet extractor. This condenser dropped the temperature quickly, enabling the condensation of the solvent on the sides of the glass to drop back into the cellulose thimble. The solvent was allowed to cool to room temperature and filtered with Whatman No. 1 filter paper (Whatman International Ltd, England) to remove any particulate matter. The filtrate was concentrated using a rotary evaporator (RE-52A, Union Laboratories, England) and kept in a refrigerator (Thermocool, England) at about 4°C prior to oral administration (Table 1).

Formula for determining the appropriate extract or drug concentrations required per body weight of rats is as follows: 

Body weight of rat (g) ÷ Average body weight of man (70 kg) × Conc. of extract or drugs

Fifty four (54) female albino wistar rats (Rattus norvergicus) weighing 80-90 g at 6 weeks (42 days) of age were used for this study and were purchased from Animal Laboratory Unit of the Department of Biochemistry, University of Nigeria Nsukka. The animals were acclimatized for 7 days in a 12 h light and 12 h dark cycle and animals were housed in wire mesh cages in an environmentally friendly animal house in the Department of Veterinary Medicine, Michael Okpara University of Agriculture Umudike (MOUAU) and animals were handled according to MOUAU ethics on animal handling. Water and food given ad libitum.

Nine (9) experimental groups involving the distribution of six (6) female rats in each group were also carried out

High Fat Diet (HFD) feed formulation

HFD feed formulation was done according to the method of Nna et al., 2014 [21].

According to Nna et al. [21], 20 g of unrefined palm karnel oil (PKO) were mixed with 180 g of normal rat feed. In this study, 20 g of saturated oil (in this case, PKO) which was manufactured locally by grinding palm kernels and extracting the oil was added to 180 g of normal poultry feed manufactured by Livestock Feeds PLC, No. 1 Henry Carr Street, Ikeja Lagos State, Nigeria and properly mixed together with the aid of a blender (Eurosonic, China) and was pelleted before being oven-dried (Vickas Ltd. England) at 28°C. The pellets were served fresh daily throughout the experiment.

Animal grouping and treatment

Group 1 (control): Rats in this group received exclusively, fresh 200 g of normal rat chow (Livestock Feeds Plc., Ikeja, Lagos State, Nigeria) daily, throughout the experimental period of 80 days (approximately, 3 months).

Group 2 (exclusive High Fat Diet (HFD) group) (negative control): Rats in this group received exclusively, fresh 200 g of HFD daily, throughout the experimental period of 80 days (approximately, 3 months). 

Group 3 (HFD+extract): Rats in this group received exclusively, fresh 200 g of HFD daily without oral seed extract administration for only a period of 30 days (approximately, 1 month) in order to increase the rats’ overall adiposity and establish an increased serum lipid in the rats. After this period, the rats continued on 200 g fresh daily HFD with a concomitant daily oral dose of 300 mg/kg seed extract throughout the remaining 50 days (approximately, 2 months) of the experimental period. 

Group 4 (HFD+ORLISTAT): Rats in this group received exclusively, fresh 200 g of HFD daily without oral orlistat (manufactured by Micro labs limited, India) administration for only a period of 30 days (approximately, 1 month) in order to increase the rats’ overall adiposity and establish an increased serum lipid in the rats. After this period, the rats continued on 200 g fresh daily HFD with a concomitant daily oral dose of 120 mg/kg orlistat throughout the remaining 50 days (approximately, 2 months) of the experimental period. 

Group 5 (HFD+SIMVASTATIN): Rats in this group received exclusively, fresh 200 g of HFD daily without oral simvastatin (manufactured by Bristol Laboratories Ltd, Unit 3, Canalside, Northbridge Road, Berkhamsted, Hertfordshire, HP41EG, United Kingdom) administration for only a period of 30 days (approximately, 1 month) in order to increase the rats’ overall adiposity and establish an increased serum lipid in the rats. After this period, the rats continued on 200 g fresh daily HFD with a concomitant daily oral dose of 20 mg/kg simvastatin throughout the remaining 50 days (approximately, 2 months) of the experimental period.  

Group 6 (HFD+Ascorbic acid (Vitamin C)): Rats in this group received exclusively, fresh 200 g of HFD daily without oral ascorbic acid (manufactured by Orange Drugs Limited, 66/68 Town Planning Way Ilupeju, Lagos) administration for only a period of 30 days (approximately, 1 month) in order to increase the rats’ overall adiposity and establish an increased serum lipid in the rats. After this period, the rats continued on 200 g fresh daily HFD with a concomitant daily oral dose of 300 mg/kg ascorbic acid throughout the remaining 50 days (approximately, 2 months) of the experimental period. 

Group 7 (HFD+Extract+ORLISTAT): Rats in this group received exclusively, fresh 200 g of HFD daily without oral seed extract and orlistat combination dose administration for only a period of 30 days (approximately, 1 month) in order to increase the rats’ overall adiposity and establish an increased serum lipid in the rats. After this period, the rats continued on a daily 200 g fresh HFD with a daily oral combination therapy of 300 mg/kg of seed extract and 120 mg/kg of orlistat throughout the remaining 50 days (approximately, 2 months) of the experimental period.

Group 8 (HFD+Extract+SIMVASTATIN): Rats in this group received exclusively, fresh 200 g of HFD daily without oral seed extract and simvastatin combination dose administration for only a period of 30 days (approximately, 1 month) in order to increase the rats’ overall adiposity and establish an increased serum lipid in the rats. After this period, the rats continued on a daily 200 g fresh HFD with a daily oral combination therapy of 300 mg/kg of seed extract and 20 mg/ kg of simvastatin throughout the remaining 50 days (approximately, 2 months) of the experimental period.  

Group 9 (HFD+Extract+ASCORBIC ACID): Rats in this group received exclusively, fresh 200 g of HFD daily without oral seed extract and ascorbic acid combination dose administration for only a period of 30 days (approximately, 1 month) in order to increase the rats’ overall adiposity and establish an increased serum lipid in the rats. After this period, the rats continued on a daily 200 g fresh HFD with a daily oral combination therapy of 300 mg/kg of seed extract and 300 mg/kg of ascorbic acid throughout the remaining 50 days (approximately, 2 months) of the experimental period.

After 80 days of experiment, rats were fasted overnight (12-14 hours) before sacrificed by first anaesthetizing them with ethyl ether (Merck Chemicals, UK) inhalation. The rats remained in terminal anaesthetics and thoracotomy was conducted with a surgical blade and blood was collected in 5 ml sterile syringes (Dana Jet, Nigeria) via heart puncture and blood was drawn directly from the heart ventricle to avoid heart collapse and also to obtain good quality blood devoid of oxidation especially by atmospheric oxygen. Blood was allowed to clot for 1 hour to obtain the serum. Serum was separated by centrifugation (Vickas Ltd., England) at 1400 rev/min for 10 minutes according to Novelli et al., 2007 [22].

Serum lipid profile assay

Serum lipid profile which includes total cholesterol (TCH) (mg/ dL), triacylglycerol (TG) (mg/dL), low-density lipoprotein cholesterol (LDL-C) (mg/dL), very low density lipoprotein cholesterol (VLDL-C) (mg/dl), and high-density lipoprotein cholesterol (HDL-C) (mg/dL) were assayed with commercial kits as follows:

Serum total cholesterol (TCH) concentration 

Determination of serum TCH was done using enzymatic end-point method, a method employed by Trinder (1969). 

Principle: In this coupled enzymatic system, cholesterol esters are enzymatically hydrolyzed to cholesterol and fatty acids. The cholesterol produced and the free cholesterol in the sample are enzymatically oxidized to form cholest-4-en-3-one and hydrogen peroxide. A quinoneimine chromogen with an absorption maximum at 500 nm is produced when phenol is oxidatively coupled with 4-aminoantipyrine in the presence of hydrogen peroxide and the enzyme peroxidase. The absorbance is proportional to the amount of total cholesterol in the test sample.

Procedure: To the tubes labeled test (T), standard (S) and blank (B), 0.01 mL of serum, standard and distilled water were added as test, standard and blank respectively. A known volume (1 mL) of the reagent was dispensed into each of the test tubes. The content of each test tube was mixed and incubated at 38°C for 5 min. Absorbance of the sample and standard were measured against the reagent blank at 546 nm within 60 min.

Where:

Sample abs = Absorbance of sample or test

Standard abs = Absorbance of standard

Conc. of standard = 2.29 mmol/L or 2.29 mM

Determination of High-Density Lipoprotein (HDL)  

cholesterol concentration: The method of Assmann (1979) was used in the determination of high-density lipoprotein (HDL) cholesterol concentration. 

Principle: HDL fraction was precipitated in the presence of phosphotungstic acid and magnesium ions. After centrifugation, the cholesterol concentration in the HDL fraction, which remains in the supernatant, was determined by the below cholesterol assay.

Procedure: Serum (200 µL) was pipetted into a centrifuge tube, 500 µL of phosphotungstic acid (precipitant) was also added and mixed thoroughly, and allowed to stand for l0 minutes at room temperature. It was then centrifuged for 15 min at 4000 rpm. 100 µL of the separated supernatant (sample) was pipetted into a test tube labeled ‘T’ and 100 µL of distilled water pipetted into the test tube blank labeled B. l00 µL of HDL-cholesterol reagent was pipetted into all the test tubes, mixed and incubated for 5 minutes at 38°C. The absorbances of the sample and standard were measured spectrophometrically at 500 nm against the blank.

Where:

Sample abs = Absorbance of sample or test

Standard abs = Absorbance of standard.

Determination of Triacylglycerol (TG) concentration: The method of Assmann (1979) was used in the determination of triacylglycerol concentration.

Principle: The triacylglycerols are determined after enzymatic hydrolysis of the ester with lipases. The indicator is a quinoneimine formed from hydrogen peroxide, 4-aminophenazone and 4-chlorophenol, a reaction catalyzed by peroxidase.

Where:

GPO = glycerol-3-phosphate oxidase

POD = peroxidase

Procedure: A quantity of the serum (0.5 mL) was pipetted into a clean test tube labeled sample and 0.5 ml of trichloroacetic acid (TCA) was added to it, mixed and then centrifuged at 250 rpm for 10 mins. The supernatant was decanted and reserved for use.

The assay procedure was carried out as shown Table 2. The mixtures were allowed to stand for 20 mins at 25°C and the absorbances of the sample and standards read against the blank at 540 nm.

Calculation: The concentration of triacylglycerol in serum was calculated as follows: 

Determination of Low-Density Lipoprotein (LDL) cholesterol concentration: The Low density lipoprotein (LDL) Cholesterol was determined using the formula below according to Harvard Medical School, 2018 [23].

LDL-cholesterol = total cholesterol (TCH) – (HDL-cholesterol + triacylglycerol (TG)/5)

Where, 5 represent the value of very low density lipoprotein (VLDL) cholesterol

Statistical analysis

Statistical analyses were processed using Statistical Program of Social Sciences (SPSS) for windows version 23.0. Values of the measured parameters were expressed as mean value ± standard error of mean (SEM) and the differences between groups were determined using one-way analysis of variance (ANOVA) and the variant mean was separated by least significant difference (LSD) of the different groups and significance was considered at p value ≤ 0.05.   

Serum lipid profile of rats

The Figure 4 shows that group 2 displayed significantly (p ≤ 0.05) highest serum TCH when compared with other groups while groups 3, 4, 5, 7, 8 and 9 had reduced TCH concentration when compared with groups 1 and 2. Group 6 had same TCH concentration with group 1 (p>0.05).

The Figure 5 shows that group 2 had significantly (p ≤ 0.05) highest serum TG concentration while groups 3, 4, 5, 7, 8 and 9 had reduced TG concentration whereas group 6 had same TG concentration as group 1 (p>0.05).

The Figure 6 shows that group 2 had significantly (p ≤ 0.05) least serum HDL-C concentration when compared with the rest of the groups. Groups 3, 4, 5, 7, 8 and 9 maintained elevated HDL-C concentration while group 6 was higher than groups 1 and 2.

Groups 2 and 6 in Figure 7 had significantly high serum LDL-C concentration while groups 3, 4, 5, 7, 8, 9 maintained reduced serum LDL-C concentration when compared with groups 1 and 2.

The Figure 8 shows that group 2 displayed significantly (p ≤ 0.05) highest serum VLDL-C when compared with other groups while groups 3, 4, 5, 7, 8 and 9 had reduced VLDL-C concentration. Group 6 and 1 were the same (p>0.05).

The Figure 9 represents the atherogenic index (TCH/HDL-C ratio) of the rats. Group 2 had highest TCH/HDL-C ratio concentration whereas group 9 had the highest HDL-C/TCH ratio than the rest of the groups.

The Figure 10 represents the atherogenic index (TG/HDL-C ratio) of the rats. Group 2 had highest TG/HDL-C ratio concentration when compared with the rest of the groups while groups 3, 4, 5, 6, 7, 8 and 9 had favourable atherogenic index (HDL-C/TG ratio). 

Observations suggest that the ethanolic seed extract of Cola lepidota has hypolipidemic activities on the rats as indicated in the serum lipid profile and this is supported by the study of Ekweogu et al. [24], which reveals that the administration of extract of Cola lepidota seeds produced hypolipidemic effects in experimental animals. Also, it is observed in the present study that a daily combination therapy of the extract and the standard drugs (i.e., 300 mg/kg extract+120 mg/ kg orlistat, 300 mg/kg extract+20 mg/kg simvastatin, and 300 mg/kg extract+300 mg/kg ascorbic acid) improved the HDL-C concentration more than daily single doses of extract, orlistat, simvastatin or ascorbic acid as seen in Figure 6 thus, suggesting that a synergy between the extract and the drugs could have resulted in the elevated HDL-C concentration. The significant reductions (p ≤ 0.05) of LDL-C for groups 3, 4, 5, 7, 8, and 9 when compared to groups 1 and 2 in Figure 7 suggest that a daily dose of 300 mg/kg C. lepidota seed has an antiatherosclerotic effect on the experimental animals while combination therapy of extract and test drugs also showed synergistic effects in LDL-C reduction especially with that of extract and ascorbic acid combination. This is clearly displayed in appendix. Figure 7 reveals that ascorbic acid was the only test drug that did not show favourable effect on serum LDL-C concentration of the rats. Therefore, ascorbic acid is not a good anti-atherosclerotic agent. The atherogenic indices obtained and presented in Figures 9 and 10 showed that group 2 had the highest TCH/HDL-C ratio and TG/HDL-C ratio respectively when compared to the other groups, suggesting that this group of rats were more prone to developing cardiovascular disease (CVD) than the rest of the rats. This is largely due to the unrefined PKO that was used in the HFD formulation. PKO according to Rakel (2012) [25] is high in lauric acid which has been shown to raise blood cholesterol levels. Current evidence has shown that different types of dietary fatty acids have divergent effects on CVD risk [26]. The scientific report of the 2015 dietary guideline advisory committee, US, place greater emphasis on types of dietary fat than total amount of dietary fat and recommend replacing saturated fats (SFAs) with unsaturated fats, especially polyunsaturated fatty acids (PUFAs) for CVD prevention (Scientific Report of the 2015 Dietary Guideline Advisory Committee Rep, USDA US Dep. Health Hum. Serv., Washington, DC [27]).

Diets high in fat tend to promote overweight, obesity and other fat induced conditions, therefore, inhibiting digestion and absorption of dietary fats becomes a logical remedy to either prevent or ameliorate such conditions. Results have shown that Cola lepidota seeds possess hypolipidaemic and anti-atherosclerotic activities, hence, become good repository for new drug discovery. 

1. Ståhlman M, Fagerberg B, Adiels M, Ekroos K, Chapman JM, et al.Dyslipidemia, but not hyperglycemia and insulin resistance, isassociated with marked alterations in the HDL lipidome in type2 diabetic subjects in the DIWA cohort: impact on small HDLparticles. Biochim Biophys Acta. 2013 Nov;1831(11):1609-1617.
2. Gamboa-Gómez CI, Rocha-Guzmán NE, Gallegos-Infante JA,Moreno-Jiménez MR, Vázquez-Cabral BD, et al. Plants withpotential use on obesity and its complications. EXCLI J. 2015Jul;14:809-831.
3. Malik VS, Willett WC, Hu FB. Global obesity: trends, risk factorsand policy implications. Nat Rev Endocrinol. 2013 Jan;9(1):13-27.
4. WHO. Obesity and Overweight. World Health Organization.2015.
5. Kopelman PG. Obesity as a medical problem. Nature. 2000Apr;404(6778):635-643.
6. Boden G. Obesity, insulin resistance and free fatty acids. CurrOpin Endocrinol Diabetes Obes. 2011 Apr;18(2):139-143.
7. Patwardhan B. Ethnopharmacology and drug discovery. JEthnopharmacol. 2005 Aug;100(1-2):50-52.
8. Cordell GA, Colvard MD. Some thoughts on the future ofethnopharmacology. J Ethnopharmacol. 2005 Aug;100(1-2):5-14.
9. WHO. Quality Control Methods for medicinal plant materials.World Health Organization. 1992.
10. Williams YJ, Popovski S, Rea SM, Skillman LC, Toovey AF, et al. Avaccine against rumen methanogens can alter the compositionof archaeal populations. Send to Appl Environ Microbiol. 2009Apr;75(7):1860-1866.
11. Ene-Obong HN, Okudu HO, Asumugha UV. Nutrient andphytochemical composition of two varieties of Monkey kola(Cola parchycarpa and Cola lepidota): An underutilised fruit.Food Chem. 2016 Feb;193:154-159.
12. Pamplona-Roger GD. Encyclopedia of foods and their healing power. In: Umeh AS, Nwadialu MA (eds). Production and proximate analysis of jam (food spread) prepared from Cola parchycarpa. J HER. 2008;13:152-158.
13. Ogbu JU, Essien BA, Kadurumba CH. Nutritional value of wild Cola spp. (monkey kola) fruits of southern Nigeria. Nig J Hort Sci. 2007;12:113-117.
14. Okudu HO, Ene-Obong HN, Asumugha VU. The chemical andsensory properties of juice developed from two varieties ofmonkey kola (Cola parchycarpa, Cola lepidota). Afri J Food SciTechnol. 2015;6(5):149-155.
15. Oghenerobo VI, Falodun A. Antioxidant activities of the leafextract and fractions of Cola lepidota K. Schum (Sterculiaceae).Nig J Biotech. 2013;25(2013):31-36.
16. Essien EE, Peter NS, Akpan SM. Chemical Composition andAntioxidant Property of Two Species of Monkey Kola (Colarostrata and Cola lepidota K. Schum) Extracts. Euro J Med Plants.2015;7(1):31-37.
17. Engel N, Oppermann C, Falodun A, Kragl U. Proliferative effectsof five traditional Nigerian medicinal plant extracts on humanbreast and bone cancer cell lines. J Ethnopharmacol. 2011Sep;137(2):1003-1010.
18. Imieje V, Igbe I, Falodun A. Phytochemical screening, proximateanalysis and acute toxicity studies of leaves of Cola lepidota K.Schum (Sterculiaceae). J Pharma Allied Sci. 2013;10(1):1684-1689.
19. Imieje V, Erharuyi O, Engel N, Falodun A. Antiprotective andapoptotic activities of Cola lepidota against estrogen receptorpositive breast cancer cells. Scientia Africana. 2014;13(1):1-8.
20. Zschocke S, Rabe T, Taylor JL, Jäger AK, van Staden J. Plant partsubstitution--a way to conserve endangered medicinal plants? JEthnopharmacol. 2000 Jul;71(1-2):281-292.
21. Nna UV, Essien NM, Bassey SC, Ofem OE. Comparative effect ofchronic consumption of some edible vegetable oils on lipid profileand some haematological parameters in rats. Annals of BiologicalResearch. 2014;5(7):16-21.
22. Novelli EL, Diniz YS, Galhardi CM, Ebaid GM, Rodrigues HG, et al.Anthropometrical parameters and markers of obesity in rats. LabAnim. 2007 Jan;41(1):111-119.
23. Harvard Medical School. The pros and cons of total cholesterol,HDL, LDL, and triglyceride testing. Harvard Health Publishing.2018.
24. Ekweogu CN, Nwankpa P, Egwurugwu JN, Etteh CC, UgwuezumbaPC, et al. Effects of ethanol extract of Cola lepidota seed on lipidprofile and haematological parameters of Albino wistar rats. Int JCurr Microb Appl Sci. 2018;7(3):3178-3186.
25. Rakel D. Integrative Medicine. Elsevier Health Sciences. 2012;381.
26. Wang DD, Hu FB. Dietary Fat and Risk of Cardiovascular Disease:Recent Controversies and Advances. Send to Annu Rev Nutr. 2017Aug;37:423-446.
27. USDA. Scientific report of the 2015 dietary guideline advisorycommittee rep. USDA US Dep. Health Hum. Serv., Washington, DC.