Chemicals and equipment
Gallic acid, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-Diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich Co., St Louis, USA. Sodium acetate anhydrous and Alloxan Monohydrate were obtained from BDH (BDH Laboratory Supplies, England), Formaldehyde Solution from (Laboratory Rasayan, Boisar); Folin–Ciocalteu phenol reagent (FC) from Merck (KGaA, Germany), Glibenclamide (Daonil; Aventis Pharma. Ltd., India), Cholesterol reagent (Teco Diagnostics., USA), Triglyceride GPO reagent (Teco Diagnostics., USA) and HDL Cholesterol reagent (Agape Diagnostics., Switzerland). Other chemicals were of analytical grade. All spectrophotometric measurements were taken using the double beam Shimadzu UV spectrophotometer, UV-1800 (Shimadzu Scientific Instruments Inc, Nakagyo-ku, Kyoto, Japan) while centrifugation was performed using High Speed Refrigerated Centrifuge, LR10-2.4A (Hunan Kecheng Instrument and Equipment Co., Ltd, Changsha city, China).
Processing and extraction of plant materials
The leaves of Nelsonia canescens (Acanthaceae) were harvested from the vicinity of Bosso campus of Federal University of Technology Minna, Niger State, Nigeria. The plant was identified and authenticated at the Plant Biology Department of Federal University of Technology Minna and a specimen of the plant sample was deposited at the herbarium with a voucher number: FUT/PLB/ACA/001. Plant sample was cleaned and washed with distilled water before air drying at room temperature for 2 weeks before crushing the leaves into smaller particles and blending into fine powder using a clean blender. A portion of the powdered sample (500 g) was refluxed at 40 °C with distilled water for 2 h to obtain the aqueous extract (AQ) which was filtered using a muslin cloth and Whatmann No 1 filter paper. The extract was further concentrated in a water bath at 30 °C and a semi-solid brownish like paste was finally obtained as the extract. The dried extract was stored in the refrigerator at 4 °C prior to usage [23, 25].
Partitioning of the crude extract
Fifty grams (50 g) of the aqueous extract was reconstituted in distilled water and solvent partitioned using ethylacetate (EA) and methanol (ME) in a separating funnel at 25 °C. The fractions were concentrated in a water bath at 30 °C to obtain a solid paste.
Quantitative phytochemical analysis of the extract
Quantitative phytochemical analysis of the crude aqueous extract (AQ), EA and ME fractions were performed using the method described by Chang et al. [26] and Singleton et al. [27].
Total flavonoid content
A colorimetric method using aluminum chloride (AlCl3) was used to quantify the flavonoid content in the crude extract, EA and ME fractions of Nelsonia canescens plants. A 1 mL aliquot of crude extract (1 mg/mL), fractions (EA and ME) or quercetin (12.5–100 mg/mL; standard) were mixed with 0.1 mL of 10% (w/v) AlCl3 in methanol, 0.1 mL of 1 M sodium acetate and 2.8 mL of distilled water. The reaction mixture was incubated at 25 °C for 30 min and absorbance measured at 415 nm against a blank. The results were expressed as mg/g quercetin equivalents.
Total phenolic content
Total phenolic content of the crude extract and fractions (EA and ME) was determined using the Folin–Ciocalteu method. For the crude extract (1 mg/mL) and each fraction, a 0.5 mL aliquot was mixed with 2.5 mL of 10% Folin–Ciocalteu reagent (v/v). This mixture was oxidized with 2 mL of 7.5% Na2CO3 (w/v) and incubated at 45 °C for 40 min. The absorbance was read at 765 nm and the amount of phenols in the crude extract and fractions was calculated using the Gallic acid standard curve expressed as mg/g of gallic acid equivalents.
Alkaloids content
For the measurement of alkaloid content in the crude extract and separate fractions (EA and ME), 0.5 g of each sample was dissolved (separately) in 5 mL of 50% ethanol in sulfuric acid and filtered using Whatmann No 1 filter paper. An aliquot (1 mL) of the filtrate was mixed with 5 mL of 60% H2SO4 (v/v) and incubated for 5 min. After incubation 5 mL of 0.5% of formaldehyde solution (v/v) was added to the sample mixture and incubated for another 3 h and the absorbance measure at 565 nm. The molar extinction coefficient of Vincristine (ɛ = 15,136 mol/cm) was used to estimate the alkaloids content of the crude extract and fractions (EA and ME).
Saponin content
The total saponin content in crude extract and fraction of Nelsonia canescens leaves was determined using a colorimetric assay. 0.5 g of the crude extract and fractions was reconstituted separately in 20 mL of 1 M HCl, at 80 °C for 4 h and then filtered using Whatmann No 1 filter paper. Fifty milliliters of petroleum ether was added to the filtrate in a separating funnel to obtain the ether layer which was evaporated in a water bath. The sample was dissolved in 5 mL of 50% acetone in ethanol, 6 mL ferrous sulfate (FeSO4) reagent and 2 mL of concentrated. H2SO4 were added to it and incubated for 10 min. The absorbance of each sample was measured at 490 nm and the amount of saponins extrapolated from a saponin standard curve.
Tannin acid content
The amount of tannins present in the extract and fraction was calculated using the tannic acid standard curve. 0.2 g each of the crude extract and fractions were weighed separately into a beaker and containing 20 mL of 50% methanol (v/v), covered with a foil paper and placed in a water bath at 80 °C for 1 h. 2.5 and 20 mL Folin–Ciocalteu reagent and distilled water, respectively, were added to the mixture and oxidized with 10 mL of 17% Na2CO3 (w/v). The mixture was incubated at room temperature for 20 min for color development and the absorbance was read at760 nm.
In vitro antioxidant activity of the crude extract and fractions
ABTS radical scavenging assay
This test was based on the samples’ capacity to scavenge the radical cation produced when ABTS solution was reacted with a potent oxidizing agent like potassium permanganate (KMnO4) or potassium persulfate (K2S2O8) [28]. Equal volume of 2.45 mM K2S2O8 and 7.4 mM ABTS stock solution were mixed together to generate the free radicals' persulfate in the dark for 12 to 16 h at room temperature. To get an absorbance of 0.700 ± 0.02 at 734 nm, the ABTS+ solution was diluted with ethanol (0.90 mL ABTS and 25 mL of ethanol). Aliquot of different concentration of the samples (0.1 mL) and 0.9 mL of the ABTS•+ were mixed and allowed to react for 30 min in the dark before reading the absorbance at 734 nm. Percentage inhibition of the radicals was calculated using the formula below:
$$\% {\text{ inhibition }} = \frac{A0 - A1}{{A0}} \times 100$$
(1)
where A0 and A1 are the absorbance of control (without sample) and test samples, respectively.
DPPH radical scavenging assay
The DPPH radical scavenging potentials of the extract and fractions were evaluated as described by Gyamfi et al. [29]. A total of 1 mL of the crude extract and fractions in a concentration dependent dose (12.5–100 µg/mL) and 1 mL of 0.4 mM methanolic DPPH were mixed and incubated for 30 min in the dark. The absorbance was measured at 516 nm and the percentage inhibition of DPPH radical was calculated relative to the blank (0.4 mM methanolic DPPH) using the equation as follows:
$${\text{Inhibition }}\left( \% \right) \, = \frac{A0 - A1}{{A0}} \times 100$$
(2)
where A0 is the absorbance of control (without sample) and A1 is the absorbance of the extract and DPPH solution.
Experimental animals and ethics
Forty-five (45) male wistar rats (100–150 g) were obtained from the University of Jos animal farm. The rats were acclimatized for 2 weeks at 28 ± 2 °C, 45–55% of relative humidity on a 12 h light/12 h dark cycle, with access to water and pelletized standard guinea feed ad libitum. The rats were kept for 2 weeks to acclimatize to the environmental conditions. The present study was approved by the Ethical Committee on the use of animals for research at the Federal University Technology, Minna, Nigeria with NO: 000018EAU. Handling of the rats was in accordance with the standard principles of laboratory animal care of the United States National Institutes of Health (NIH, 1978) as reported by Belayneh et al. [10].
Induction of diabetes mellitus
A single intraperitoneal (i.p.) injection of 90 mg/kgbwt of alloxan monohydrate (Sigma, St. Louis, USA) in phosphate buffer saline (PBS) with a pH of 7.4 was used to induce diabetes mellitus in the rats. Animals were selected for the study if their fasting plasma glucose concentration (FPGC) was greater than 111 mg/dL for five consecutive days as determined by the Fine test Auto-coding Premium Blood Glucose Monitoring System for self-testing. Nine groups of five male wistar rats each were created from a total of 45 male wistar rats. Before the start of the treatment, the animals were deprived of food and water for an extra 16 h [30].
Experimental design
The experimental design consists of 9 different groups which was based on the treatment received for 21 days as shown as follows:
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Group I: normal control (non-diabetic) received1 mL PBS
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Group II: positive control and treated with 5 mg/kgbwt of Glibenclamide (reference drug)
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Group III: negative control (Diabetic control)
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Group IV: treated with 50 mg/kgbwt of AQ
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Group V: treated with 50 mg/kgbwt of EA
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Group VI: treated with 50 mg/kgbwt of ME
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Group VII: treated with 300 mg/kgbwt of AQ
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Group VIII: treated with 300 mg/kgbwt of EA
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Group IX: treated with 300 mg/kgbwt of ME
The rats were weighed every 7 days to monitor their body weight.
Collection and preparation of blood and tissues
After 21 days, rats who had been fasting overnight were sacrificed by heart puncture anesthetized with 150 mg/kgbwt of sodium pentobarbitone. Blood samples were then taken from each rat and put into a plain sample bottle. The blood samples were centrifuged at 2000 rpm for 10 min after being allowed to clot at room temperature for 2 h. With a fresh Pasteur pipette, the serum was separated. Prior to further biochemical analysis, serum samples were kept at − 80 °C. For liver enzyme analyses, the livers of the rats in each experimental group were removed, washed in normal saline, and stored in 10% formalin (v/v) [10].
Biochemical assays
Using different assay kits, the biochemical indices of the rats after 21-day of treatment with the extract and fractions to evaluate the level of high-density lipoprotein (HDL), cholesterol concentration Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total proteins, albumins, creatinine total cholesterol, and triglyceride. Additionally, the serum LDL-C concentration was determined according to the formula described by Kumari et al. [17] and previously reported by Oluba et al. [31].
$$[\text{LDL-C}]=\left[\mathrm{TC}\right]-\left[\text{HDL-C}\right]-\frac{\mathrm{TAG}}{5}$$
(3)
Serum VLDL-C concentration was estimated using the methods of Burnstein and Sammaille [32] where the ratio of serum VLDL-C to triglyceride concentrations was fixed at 1:5 in fasting animals.
$$[\text{VLDL-C}]=\frac{\mathrm{TAG}}{5}$$
(4)
In vivo antioxidant enzyme activity
After sacrifice, the livers of the rats in each treatment group were immediately removed, washed with chilled saline solution, homogenized in ice-cold sucrose (10% w/v) and centrifuged at 10,000 g for 20 min. at 4 °C [33]. The activity of catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH) were assessed in the resulting supernatant.
Catalase activity
According to a method described by Luck (1974) and reported by Ahmad et al. [34], the activity of catalase (CAT) was assessed. 50 µL of the liver's supernatant was mixed with 2.5 mL of a buffer containing 30% H2O2 and vortexed for two minutes. After 30 s and subsequently 90 s, the reaction mixture's absorbance at 240 nm was measured. The blank contained only 2.5 mL of 30% H2O2 buffer. The CAT activity was measured using the formula as follows:
$${\text{CAT activity }}\left( {{\text{U}}/{\text{mins per mL of serum}}} \right) \, = \frac{{\Delta {\text{Abs}}}}{0.0008} \times 1 \;\min$$
(5)
where ΔAbs is the difference in sample absorbance after 30 and 90 secs.
Superoxide dismutase activity
The activity of the superoxide dismutase (SOD) enzyme was measured using the method described by Misra and Fridovich [35]. The reaction mixture consists of 20 µL of liver homogenate, 960 mL of sodium carbonate buffer (50 mM, pH 10.2), and 0.1 mM EDTA. Furthermore, 20 µL of 30 mM epinephrine, dissolved in 0.05% v/v acetic acid, were added to the mixture to initiate the reaction. The blank and control contained sodium carbonate buffer and distilled water, respectively. The increase in absorbance was measured at 480 nm for 4 min and activity calculated as follows:
$$\% {\text{ inhibition}} = 100 - \left( {\frac{{\Delta {\text{Abs control}} - \Delta {\text{Abs sample}}}}{{\Delta {\text{Abs control}}}} \times 100} \right)$$
(6)
$${\text{SOD Activity }}\left( {{\text{U}}/{\text{mL}}} \right) \, = \, \% {\text{ inhibition }} \times \, 3.75$$
(7)
where ΔAbs control is the difference in control absorbance at different times and ΔAbs sample is the difference in control absorbance at different times.
Reduced glutathione concentration
Reduced GSH content was assayed following the method described by Jollow et al. [36]. The liver homogenate (50 μL) was mixed with 150 μL of sulfosalicylic acid (SSA) and centrifuged at 5000 × g for 10 min at 4 °C. The amount of GSH was determined by mixing 66 μL of supernatant with 66 μL of 0.01 M 5,5-dithiobis-2-nitrobenzoic acid (DTNB) and 865 μL of potassium phosphate buffer (0.1 M, pH 7.4). After 5 min, the absorbance was measured against SSA as blank at 412 nm and the concentration calculated as follows:
$${\text{GSH concentration }}\left( {{\text{U}}/{\text{mL}}} \right) \, = \frac{{{\text{Abs}}}}{0.416} \times 2$$
(8)
where Abs is the sample absorbance.
Statistical and data analyses
The data collected were analyzed using one-way analysis of variance (ANOVA) while treatment means was separated by the least significance difference (LSD) incorporated in the statistical package for social sciences (SPSS) version 20.
