Study Area
The research was carried out at the Department of Laboratory Animals, Enugu State University of Science and Technology, Enugu (ESUT). The draft proposal was reviewed and approved by the Department of Pharmacology and Therapeutics, College of Medicine University of Nigeria Enugu Campus. The study protocol was approved by the college of medicine research ethics committee.
Study Animals
60 male Wistar rats approximately of same aged (12 -16week old) and weight (160 – 273g) (Supplementary Table 1 and Supplementary Table 2) were sourced from ESUT animal house. Only male rats were used for the study due to considerable gender differences in glucose regulation and insulin sensitivity (42) (43).
They were kept in 12 cages (5 rats per cage), weighed and fed with standard laboratory diet, and had free access to water for 1 month. The rodent cages were temperature, humidity and light-controlled housing.
The study was carried out in line with conventional laboratory principles on the use of animals based on the European Community Guidelines 1986.
Study Plant
Fresh grapefruit peels were obtained from one of the grapefruit trees in the garden of Ofodire Emeka (the principal investigator) for the study.
The LD50 of grapefruit peel and seed extract is 5,000mg/kg (44). The No-observed-adverse-effect-level (NOAEL) for male rats is 600mg/kg/day (45) .
Sample Preparation: Samples of grapefruit peels were first dried in air. The dried samples were coarse ground to particles less than 0.5mm to improve the extraction efficiency and weighed 1.187kg.
Extraction of Coumarins from Grapefruit Peel was carried out in Department of Chemistry Laboratory, University of Nigeria, Nsukka.
Cold maceration method of extraction was used as studies have shown it to be suitable and convenient method for coarse ground plant materials (46) and phenolic compounds (46) (47). Methanol was chosen as the solvent of extraction as studies reported it to have the highest extractability among other solvents for phenolic compounds (47) and coumarins (48).
90% methanol was added to the sample at menstruum: sample ratio 4:1. The mixture was constantly agitated and left for 72 hours. Filtration was carried out using number 1 Whatman filter paper.
The micelle was concentrated using Rotary evaporator under vacuum to remove the menstruum from the extract, and water bath used for cooling. The crude extract was weighed and measured 212g, and the marc discarded.
Separation of the Extract into Factions: was carried out using Vacuum Liquid Chromatography (VLC) in Department of Chemistry Laboratory, University of Nigeria, Nsukka.
The crude extract was mixed with silica gel adsorbent (solid phase) of size 100 – 200 mesh to form homogeneous mixture. The mixture was allowed to dry and was further ground into fine powder using porcelain (ceramic) mortar. Then, it was gently and neatly packed into glass column of length 1m and diameter 4cm.The column was connected to a vacuum pump and the pump switched on.
The fractionation took place based on choice solvent (mobile phase), N-Hexane, Ethyl Acetate and methanol in increasing order of polarity. The fractions were concentrated by exposing them to dry air. The weights of the concentrated fractions were as follows: n-Hexane (dry fraction) = 0.02g, Ethyl Acetate (dry fraction) = 1.01g, Methanol (oily, waxy, viscous fraction) = 78.20g.
Gas Chromatography – Mass Spectrometry (GCMS): was carried out at Ebic Integrated Services Limited Laboratory Port Harcourt Nigeria (Supplementary figure 1).
The analyzed results for the 3 fractions reported the most abundant constituents as follows:
a) Ethyl Acetate Fraction:2H-1-Benzopyran-2-one, 5,7-dimethoxy- (Citropten Coumarin C11 H10 O4) 85.66%, Phytol (Diterpenes) 1.96%, 7H – Furo(3,2-g)(1) benzopyran – 7- one, 4-methoxy- (bergapten Furanocoumarin C12 H8 O4) 1.05%, Carveol (Tarpenoid alcohols) 0.63%.
b) Methanol Fraction:2H-1-Benzopyran-2-one, 5,7-dimethoxy- (Citropten Coumarin) 70.60%, 7H – Furo(3,2-g)(1) benzopyran – 7- one, 4-methoxy- (bergapten Furanocoumarin) 4.70%, Diisooctyl phthalate (Phthalate esters) 1.16%, Phytol (Diterpenes) 0.64%.
c) n-Hexane Fraction: Bis(2-ethylhexyl) phthalate (Phythalate esters) 51.03%, beta-Bisabolene (sesquiterpenes) 8.70%.
Further purification of fractions was not possible due to unavailability of specified High Performance Liquid Chromatography and Nuclear Magnetic Resonance equipment in Nigeria at time of study.
Study Design
The study was Randomized Controlled Blinded and divided into experiments. Because we were dealing with homogenous rat population that were in cages (clusters), multistage cluster sampling method was used for selection. The mean values of obtained results sets were used. Heparinized sample bottles were used for blood samples collection. Plasma was used for assessing hormones due to their short lifespan in serum (49).
0.5% polysorbate 20 excipient (Tween 20) served as diluents and vehicle for the methanol and ethyl acetate fractions of 5,7 – dimethoxycoumarin and the controls (50).
Intraperitoneal Glucose Tolerance Tests (IPGTT) was used in the study 1) to produce hyperglycemia, 2) for on-the-spot determination of glucose levels with Glucometer, 3) for simultaneous injection of glucose (50% dextrose 2g/kg), grapefruit peel fractions and drugs, 4) for determination of plasma parameters at 0min, 30min, 60min and 120min intervals of time, after overnight fast (12 hours), 5) as an alternative route of administering coumarins. About 3mls of blood was collected from retro-orbital plexus puncturing of each animal for the entire study. The samples were immediately centrifuged after collection and plasma separated from blood cells. The plasma was stored at – 20degrees Celsius and analysis started immediately.
Primary Experiments on All Parameters:
1) 5 rats (Rats 1 – 5) were used as negative control on IPGTT. 1ml of sterile water was given simultaneously (in quick succession) with 2g/kg of glucose intraperitoneally over 30 seconds.
2) 5 rats (Rats 6 -10) as positive control on IPGTT. 0.2mg/kg of glimepiride (equivalent of 2mg daily dosing in humans) (51) given simultaneously with 2g/kg of glucose. Glimepiride was chosen as positive control drug because has better safety profile, does not interfere with insulin and glucagon homeostasis and is less prone to profound hypoglycemia compared to other sulfonylureas (52). Oral LD50 of glimepiride in rats is >10000mg/kg, intraperitoneal LD50 in rats is 3950mg/kg (53).
3) 5 rats (Rats 11 -15) test for direct insulin releasing effects of ethyl acetate fractions containing 5,7-dimethoxycoumarin on IPGTT. 2g/kg of glucose was given simultaneously with ethyl acetate fraction containing 20mg/kg of 5,7-dimethoxycoumarin. (1g of ethyl acetate fraction contains 856.6mg of 5,7-dimethoxycoumarin).
4) 5 rats (Rats 16 – 20) test for direct insulin releasing effects of methanol fractions containing 5,7-dimethoxycoumarin on IPGTT. 2g/kg of glucose was given simultaneously with methanol fraction containing 20mg/kg of 5,7-dimethoxycoumarin.
5) 5 rats (Rats 21 – 25) test for pro-oxidant actions of H2O2 and high dose vitamin C on effects of ethyl acetate fractions containing 5,7-dimethoxycoumarin on IPGTT.
H2O2 is an oxidizing agent implicated in the pathogenesis of oxidative stress. The LD50 is 600 – 1617mg/kg (Y.Li, unpublished; Ito et al., 1976). It is generally safe at 3%.
Household concentration varies from 3 – 9% (Nelson AL, et al., 2023). 2g/kg of glucose was given simultaneously with 1ml of 6% H2O2 and 20mg/kg of 5,7-dimethoxycoumarin, intraperitoneally in Rat 23. Repeat with 0.6% H2O2 for Rats 21 – 22.
Vitamin C acts as antioxidant at low doses (30 – 100 mg/kg) and pro-oxidant at high doses (1000mg/kg) (54). The LD50 of vitamin C is 11,900mg/kg in rats (55). 2g/kg of glucose was given simultaneously with 1000mg/kg of vitamin C and 20mg/kg of 5,7-dimethoxycoumarin, in Rats 24 - 25.
Secondary (Supplemental) Experiments on Plasma Glucose Effect Only:
6) 5 Rats (Rats 26 – 30) test for ethyl acetate fraction containing 10mg/kg 5,7-dimethoxycoumarin on IPGTT.
7) 5 Rats (Rats 31 – 35) test for methanol fraction containing 10mg/kg 5,7-dimethoxycoumarin on IPGTT.
8) 5 Rats (Rats 36 – 40) negative control on Oral Glucose Tolerance Test (OGTT).
9) 5 Rats (Rats 41 – 45) positive (glimepiride) control on OGTT.
10) 5 Rats (Rats 46 – 50) test for ethyl acetate fraction containing 20mg/kg 5,7-dimethoxycoumarin on OGTT.