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Abstract

Health forecasters predict that cases of diabetes will double in 2030; hence proactive action is required to salvage this problem. Thus, this study was undertaken to evaluate the toxicological and anti-diabetic potential of n-hexane extract of T. bangwensis leaves on α-amylase and α-glucosidase activity. The phytochemical screening, antioxidant activity as well as the inhibitory effect of the plant extract was determined by UV-spectrophotometry method while brine shrimp and Allium cepa methods were used for the toxicity study. Preliminary phytochemical screening detected the presence of flavonoid, phenol, tannin, alkaloid and cardiac glycoside whereas phlobatanin, steroid, terpenoid and saponin were absent. The result also showed that flavonoid concentration was the highest compared to others. The 2,2-diphenyl-1-picrylhydrazine (DPPH) and nitric oxide (NO) results showed that the plant extract exhibited significant antioxidant activity particularly at the highest concentration (100 µg/ml). Brine shrimp lethality result showed that the highest mortality rate of nauplii and median inhibition concentration (IC₅₀) are 97% and 7.46 ± 0.33 µg/ml respectively. Furthermore, the results also revealed that mitotic index, root growth length and mitotic division (cytotoxicity indicators) decreased as concentration increases. Finally, the results showed that the plant extract exhibited significant inhibitory effect on α-amylase and α-glucosidase activities at 100 µg/ml; nevertheless, the effect was higher on α-amylase than α-glucosidase activity. In summary, the significant antioxidant and inhibitory effects may be attributed to the presence of the phytochemicals mentioned above. It can therefore be concluded that T. bangwensis leaves may demonstrate potent anti-diabetic effect.

Keywords

toxicity inhibitory effect α-amylase α-glucosidase

Introduction

The international diabetes federation (IDF) predicts that cases of diabetes mellitus which is the third killer disease threatening global population beside cancer and cardiovascular disease will double in 2030.^1,2 Diabetes mellitus is a chronic metabolic disorder or an endocrine systemic problem caused by either low insulin secretion and release, insulin resistance/insensitivity of pancreatic β-cells or insulin action to down-regulate blood glucose level resulting in hyperglycaemic condition thereby triggering several diabetic complications/abnormalities such as hyperglycaemia, hyperlipidaemia, ophthalmopathy, neuropathy, atherosclerosis, renal failure, ulceration, reproductive disorder to mention but a few.^3
–5 Furthermore, diabetes mellitus also occur due to imbalance between glycaemic index and glucose intolerance which increases the risk of individual’s progression to Type 2 diabetes (T2D) (also called non-insulin dependent diabetes mellitus (NIDDM) or maturity onset diabetes mellitus) and contributes approximately 90–95% of all cases of diabetes mellitus.^2,6

Since elevated blood glucose level is the major implicated factor of diabetes mellitus therefore inhibition of carbohydrate metabolizing enzymes such as alpha amylase and alpha glucosidase reduces the rate of glucose influx into the circulatory bloodstream thereby preventing postprandial rise/increase in blood glucose and invariably reducing drastically the effects associated with diabetes mellitus complications.^7,8 Several reports have established that oral hypoglycaemic drugs are effective in the treatment and management of cases of diabetes mellitus, however, they show undesirable adverse side effects such as gastrointestinal discomfort, oedema, renal failure, overweight and so forth.^9
–11 It is based on these toxicity outcomes that encouraged researchers to inquire about natural products with comparative advantage and little or no adverse side effect.

Some natural products have been isolated and screened which afterwards were found to be effective in the treatment and management of diabetes mellitus.^12
–14 Nevertheless, there is a need for further discoveries. Interestingly, studies showed that T. bangwensis leaf is ethno-medicinally beneficial as anti-inflammatory, hypolipidemic, antimicrobial, anticancer, hypotensive agent and so on. Mistletoe is a woody shoot parasite that belongs to the Loranthaceae family and consists of the genera namely: Phragmanthera, Oncocalyx, Tapinanthus, Plicosepathus, Globumetula, Agelanthus, Helixanthera, Berhauria, and Engderina.^15,16 Previous studies documented that Tapinanthus species such as aqueous-ethanol extract of Tapinanthus globiferus leaves and aqueous leaf extract of Tapinanthus butungii possess hypoglycaemic property.^17,18 However, information on the anti-diabetic property of T. bangwensis is scanty, therefore, the present study was undertaken to screen for the toxicological and anti-diabetic potential of n-hexane extract of T. bangwensis leaves so as to establish the safety status and therapeutic effect of the plant in the treatment of diabetes mellitus. African mistletoe (also known as T. bangwensis or Loranthus bangwensis) (Engl & K. Krause) Danser which is a citrus-dependent plant of African origin is called by the Hausas, Igbos and Yorubas as Kauci (Kanchi), Awurusie (or Apari) and Afomo onisana while the English acronyms are ‘all heal tree’ ‘bird lime’ or ‘tree of life’. ¹⁹

Materials and methods

Chemical reagents

Methanol, ethylacetate, n-hexane, sodium carbonate, (Na₂CO₃), Iron chloride (FeCl₃), ammonium hydroxide, sodium hydroxide (NaOH) and hydrochloric acid (HCl) were bought from Chimex chemical Limited, Lagos, Nigeria. Ascorbic acid, Folin-ciocalteu reagent, gallic acid, rutin, α-amylase, α-glucosidase, 2,2-diphenyl-1-picrylhydrazine (DPPH), Dinitrosalicylic acid (DNSA), 4-nitrophenyl-d-glucopyroinoside (pNPG) acetic acid, orcein stain, picric acid, glacial acetic acid and polyvinyl polypyrrolidone (PVPP) were purchased from Sigma-Aldrich company, St Louis, USA while starch soluble was obtained from J. T. Baker Inc., Phillipsburg, USA

Authentication of plant material

Fresh leaves of T. bangwensis bought from Mushin market in Lagos were authenticated by Mr Adeleke of the Department of Pharmacognosy, University of Lagos. The unique characteristic that distinguished it from other Tapinanthus species is the presence of match-like flower with red/purple corolla tip. The plant leaves were then deposited in the University’s herbarium for documentation and a voucher number (LUH 3856) was assigned.

Extraction of plant material

The leaves of the plant were washed with distilled water and air-dried for 5days at 25–28°C. They were blended using electric blender and 1.5 kg powdered mass of T. bangwensis leaves was obtained. Approximately, 500 g of the powdered leaves of T. bangwensis was then extracted with 1.5 L of 100% methanol solvent. The content was left to stand and after 2days, it was filtered using Whatman filter paper and the filtrate concentrated by evaporation at room temperature. The final weight obtained was 40.85 g. Furthermore, approximately 25.0 g of methanol extract of T. bangwensis was extracted by solvent partitioned method using separating funnel in a resulting mixture of ethylacetate-water. The upper portion which was ethylacetate extract was collected, air-dried by evaporation and the mass yield was 10.2 g. Subsequently, 7.0 g of ethylacetate extract of T. bangwensis was further subjected to column chromatography extraction using 100% n-hexane as the eluting solvent. A total of five fractions in 100 ml bottle were collected and concentrated by evaporation at room temperature. The mass yield of solid n-hexane extract obtained was 1.58 g which was then used for this study (Figure 1).

Figure 1.

Schematic diagram of extraction of T. bangwensis.

Phytochemical screening of plant extract

Qualitative phytochemical screening

Preliminary phytochemical screening of alkaloid, terpenoid, saponin, tannin, flavonoid, steroid, phlobatanin phenol and cardiac glycoside were determined systematically using the method described by Geetha and Geetha.²⁰

Quantitative phytochemical screening of plant extract

Quantification of phenolic content

Total phenolic content was quantified using Folin-Ciocalteu reagent (FCR) described by Siddhuraju and Becker.²¹ 100 µl of 1 mg/ml of the extract dissolved in methanol was mixed with 750 µl of FCR (diluted in 10-fold) and allowed to stand for 5 mins at 25°C. 750 µl of 0.57 M Na₂CO₃ solution was then added to the mixture and after 60 mins, the absorbance was measured at 725 nm. The result obtained was expressed as gallic acid equivalent (mgGAE/100 g).

Quantification of tannin content

The tannin content was estimated by the method described by Siddhuraju and Becker.²¹ Briefly, 1.0 ml of distilled water was added to 100 mg of polyvinyl polypyrrolidone (PVPP) followed by addition of 100 µl of 1 mg/ml of extract dissolved in methanol. The reaction mixture was vortexed for 15 mins at 4°C. After 10 mins, it was then centrifuged at 3000 rpm at 25°C. The absorbance of the supernatant which contains the simple phenolic was then measured at 725 nm. The result was expressed as non-tannin phenolic and the tannin content was calculated as follows:

T a n n i n = T o t a l p h e n o l i c s - (N o n - t a n n i n p h e n o l i c s)

Quantification of flavonoid content

Flavonoid content was determined according to the method described by Kumaran and Karunakaran.²² 100 µl of the extract in methanol (1 mg/ml) was mixed with 100 µl of 20% AlCl₃ in methanol, followed by addition of a drop of acetic acid and then made up to 5 ml with methanol. After 40 mins, the absorbance was then taken at 415 nm. Blank sample contains 100 µl of extract, a drop of acetic acid and then made up to 5 ml with methanol. The absorption of standard rutin solution (0.5 mg/ml) in methanol was measured under the same condition. The amount of flavonoid in the extract in rutin equivalent was calculated as:

F l a v o n o i d c o n t e n t = \frac{Absorbance of extract \times weight of rutin in solution (mg)}{Absorbance of standard \times weight of extract (mg)}

Quantification of alkaloid content

The alkaloid content in the plant extract was estimated by the method described by Herin et al.²³ Approximately, 0.2 g of the extract was weighed into a conical flask and 100 ml of 10% acetic acid in ethanol was added and allowed to stand for 4hours. The solution was then filtered and the filtrate obtained was concentrated on a water bath at 40°C. 1.0 ml 0.5 M NH₄OH was added dropwise to the solution until a precipitate was formed. The precipitate was then collected, washed with dilute ammonium hydroxide and the content filtered. The residual sample which contains the alkaloid was dried and weighed.

Quantification of cardiac glycoside

The concentration of cardiac glycoside was quantified by the method of Yassa et al.²⁴ 10% of extract was mixed with 10 ml freshly prepared Baljet’s reagent (95 ml of 1% picric acid and 5 ml of 10% NaOH solution). After 1hour, the mixture was diluted with 20 ml of distilled water and the absorbance was read at 495 nm. The standard curve was plotted by preparing 10 ml of different concentrations (12.5–100 mg/ml) of securidaside. Total cardiac glycoside was expressed as mg of securidaside per g of extract.

Antioxidant activity of the plant extract

2,2-Diphenyl-1-picrylhydrazine (DPPH) assay

The antioxidant property of n-hexane extract of T. bangwensis was evaluated using the DPPH free radical scavenging assay described by Nithiananthan et al.²⁵ and Zuraini et al.²⁶ Different concentrations of the extract were prepared (25, 50, 75 and 100 µg/ml) and 5 ml of 0.004% (w/v) solution of DPPH (or 4 mg of DPPH in 100 ml of methanol) was added. The solution obtained was vortexed and incubated at 25°C for 30 mins in a relatively dark place and afterwards the absorbance was measured at 517 nm. The blank contains 80% (v/v) methanol only. The absorbance was measured in triplicates for each concentration. Ascorbic acid was used for comparison as positive standard. DPPH scavenging free radical activity was calculated as:

% s c a v e n g i n g f r e e r a d i c a l a c t i v i t y = \frac{Absorbance of Blank - Absorbance of extract}{Absorbance of Blank} \times 100

Nitric oxide antioxidant assay

The antioxidant activity of the plant extract using nitric oxide assay was determined by the method described by Alisi and Onyeze.²⁷ Briefly, 5.0 ml of the reaction mixture which contains 5 mM sodium nitroprusside (SNP) in phosphate buffered saline (pH = 7.3) with or without the extract at different concentrations (25, 50, 75 and 100 µg/ml) was incubated at 25°C for 180 mins under a polychromatic light source. The nitric oxide free radicals generated interact with oxygen to produce the nitrite ion which was assayed at 30 min intervals by mixing 1.0 ml of incubation mixture with an equal amount of Griess reagent (1% sulphanilamide in 5% phosphoric acid and 0.1% naphthylethylene diaminedihydrochloride). The absorbance of the chromophore formed (purple azo dye) was measured at 546 nm in triplicates for each concentration and the percentage inhibition calculated as:

% i n h i b i t o r y a c t i v i t y = \frac{Absorbance of Blank - Absorbance of extract}{Absorbance of Blank} \times 100

Toxicological study of the plant extract

Brine shrimp Lethality assay

The brine shrimp lethality assay used in this study was that described by Geethaa et al.²⁸ Artemia salina eggs were obtained from the Aquatic animal research centre, University of Lagos while salt water was collected from the Bar beach in Victoria Island, Lagos. 300 eggs were placed into 500 ml of salt water in a 1000 ml volumetric flask. After 36 hrs, the larvae (nauplii) hatched at 25°C in an aerated condition and continuous illumination. The different concentrations of the plant extract (10, 100 and 1000 µg/ml), and control (salt water) were prepared in test tubes and 10 nauplii were added into each of the test tube. After 24 hrs, the number of dead nauplii was counted under an intensive illumination. The percentage death was calculated as below while inhibition concentration (IC₅₀) was determined.

% d e a t h o f n a u p l i i = \frac{Number of dead nauplii}{Total number of nauplii} \times 100

Cytotoxicity study using Allium cepa assay

The cytotoxic effect of n-hexane extract of T. bangwensis leaves using Allium cepa was determined by the method of Fiskesjo.²⁹ Thirty pieces of onion bulbs (Allium cepa Linn) were used. The outer scales were removed carefully before initiating the test to avoid destroying the root primordia. The onion bulbs were then placed in tap water and after 24 hours the five best growing bulbs were exposed to the test solutions (0.01, 0.03, 0.06 and 0.1 mg/ml) for 4days in the dark at 25°C. The root growth length were then measured 24hourly using a ruler. For chromosomal aberration evaluation 5–7 mm of the onion root tips were excised and fixed in ethanol-glacial acetic acid solution (3:1 v/v). After 60 mins, the root tips were then hydrolysed in 1 N hydrochloric acid at 60°C and subsequently rinsed with distilled water. The root tip was then squashed in a glass slide and stained with 2% aceto-orcein for 10 mins. It was covered carefully with a slide cover to avoid air bubbles and sealed with fingernail polish. Ten slides were prepared for each concentration and the control and were then viewed under a microscope mounted on a camera. The mitotic index was then calculated as:

% m i t o t i c i n d e x = \frac{P + M + A + T}{Total number of cells} \times 100

where P, M, A and T represents the number of dividing cells in prophase, metaphase, anaphase and telophase respectively.

Inhibitory effect of the plant extract

Inhibitory effect on α-amylase activity

The inhibitory effect of n-hexane extract of T. bangwensis on α-amylase was determined by the method of Kazeem et al.³⁰ A 250 µl of the extract/acarbose at different concentrations (25, 50, 75 and 100 µg/ml) were incubated with 500 µl of 2U/ml α-amylase in 100 mM phosphate buffer (pH = 6.8) for 20 mins at 37°C. 250 µl of 1% starch dissolved in 100 mM phosphate buffer was then added to the reaction mixture and incubated for 1hour at 37°C. 500 µl of dinitrosalicylic acid (DNSA) (colour forming agent) was then added and boiled for 10 mins. The mixture was cooled and diluted with 5 ml of distilled water. The control was prepared in the same manner as the test sample with distilled water replacing the extract. The absorbance was measured at 540 nm and the inhibitory activity calculated as:

% i n h i b i t o r y a c t i v i t y = \frac{Absorbance of control - Absorbance of extract}{Absorbance of control} \times 100

Inhibitory effect on α-glucosidase activity

The inhibitory effect of n-hexane extract of T. bangwensis was determined by the method of Kazeem et al.³⁰ A 250 µl solution of the extract/acarbose at different concentrations (25, 50, 75 and 100 µg/ml) were incubated with 500 µl of 1.0U/ml α-glucosidase solution in 100 mM phosphate buffer (pH = 6.8) at 37°C for 15 mins. 5000 µl of 3 mM of 4-nitrophenyl-d-glucopyronoside (pNPG) solution in 100 mM phosphate buffer was added and the mixture was further incubated at 37°C for 25 mins and 5% w/v Na₂CO₃ was added. The mixture was cooled at 25°C and 5 ml of distilled water added and vortexed for 10 mins. The control contained all the component of the test sample except the extract. The absorbance of the released p-nitrophenol was measured at 405 nm and the percentage inhibitory activity calculated as:

% i n h i b i t o r y a c t i v i t y = \frac{Absorbance of control - Absorbance of test}{Absorbance of control} \times 100

Statistical analysis

All data obtained from this study were statistically expressed as Mean ± SD. The significant difference was compared with ANOVA and Tukey’s post hoc test while the confidence limit was considered at p < 0.05.

Results

Preliminary phytochemical screening of n-hexane extract of T. bangwensis revealed the presence of flavonoid, phenol, tannin, alkaloid and cardiac glycoside while phlobatanin, steroid, terpenoid and saponin were not detected (Table 1). Furthermore, the result also showed that the concentration of these phytochemical compounds were remarkable, nonetheless, flavonoid concentration was the highest compared to other phytochemicals that were quantified. (Figure 2)

Table 1.

Qualitative Phytochemical Screening of n-hexane extract of T. bangwensis.

Phlobatanin	Steroid	Flavonoid	Phenol	Tannin	Alkaloid	Terpenoid	Saponin	Cardiac glycoside
–	–	+	+	+	+	–	–	+

Abbreviation: (+) indicates detected while (–) indicates not detected.

Figure 2.

Concentration of phytochemical compounds in n-hexane extract of T. bangwensis.

The results of the antioxidant activity of the plant extract using 2,2-diphenyl-1-picrylhydrazine (DPPH) and nitric oxide assays showed that the plant extract exhibits significant antioxidant activity with the highest inhibition of DPPH free radicals observed at the highest concentration (100 µg/ml) (Figure 3 and 4). However, the antioxidant activity of ascorbic acid was higher compared to that of the plant extract in both assays.

Figure 3.

Antioxidant activity of n-hexane extract of T. bangwensis using 2,2-diphenyl-1-picrylhydrazine antioxidant model (DPPH).

Figure 4.

Antioxidant activity of n-hexane extract of T. bangwensis using nitric oxide antioxidant model.

Table 2 and 3 shows the results of the toxicological effect of the plant extract of T. bangwensis using Brine shrimp lethality assay and Allium cepa test. The brine shrimp lethality result showed toxic effect characterized by increase in nauplii mortality rate as concentration increases with the highest mortality rate (97%) observed at 1000 µg/ml. The median inhibition concentration (IC₅₀) was found to be 7.46 ± 0.33 µg/ml (Table 2). There was significant difference between the normal control (saltwater) and the plant extract at different concentrations at p < 0.05. Also, the result of the Allium cepa test showed evidence of cytotoxicity such as decrease in root growth length (Figure 5) as well as decrease in mitotic index and mitotic division (Table 3) as concentration increases compared to the control (distilled water). From Table 3, it was observed that there was significant difference between the control (distilled water) and the plant extract at different concentrations at p < 0.05. The predominant chromosomal aberrations observed from Table 3 were bridged and sticky chromosomes compared to other chromosomal aberrations while laggard and multipolar chromosomes were absent. The various chromosomal aberrations are shown in Figure 6.

Table 2.

Toxicological effect of n-hexane extract of T. bangwensis using Brine shrimp model.

concentrations	Log Concentration	No of nauplii	No of dead nauplii	% death of nauplii	Probit value	IC₅₀ (µg/ml)
Salt water (Normal control)	0	10	0.67 ± 0.58^*	6.7	3.72	7.46 ± 0.33
10 µg/ml	1	10	7.6 ± 0.58^**	76	5.71
100 µg/ml	2	10	8.7 ± 0.58^**	87	6.13
1000 µg/ml	3	10	9.7 ± 0.58^**	97	7.05

Values were in triplicates and were statistically expressed as Mean ± SD. Different superscript shows significant difference between the normal control and n-hexane extract of T. bangwensis at different concentrations at p < 0.05. Abbreviation: IC50 = Median inhibition concentration.

Table 3.

Cytotoxic effect of n-hexane extract of T. bangwensis using Allium cepa test.

Different concentrations of n-hexane extract of T. bangwensis
Parameters	Distilled water (control)	0.01 mg/ml	0.03 mg/ml	0.06 mg/ml	0.1 mg/ml
Total No of cells	500	479	473	467	461
Prophase	7	14	1	0	0
Metaphase	17	15	13	13	11
Anaphase	12	11	10	10	09
Telophase	15	12	14	11	10
Dividing cells	51	42	38	34	30
% Mitotic index	10.2 ± 3.14^*	8.77 ± 2.83^**	8.04 ± 2.24^**	7.32 ± 2.19^**	6.53 ± 1.83^**
Attached chrom	0	4	2	2	3
Bridged chrom	0	5	7	5	4
C-Mitosis	0	2	0	1	0
Sticky chrom	0	6	6	5	4
Vagrant chrom	0	5	6	4	3
Laggard chrom	0	0	0	0	0
Binuclei chrom	0	1	1	1	1
Multipolar	0	0	0	0	0
Total aberrations	0	23	22	18	15
% Aberrations	0	4.80	4.65	2.83	3.25

Values were in Pentaplates (n = 5) and were statistically expressed as Mean ± SD. Different superscript shows significant difference between control (distilled water) and n-hexane extract of T. bangwensis at different concentrations at p < 0.05.

Figure 5.

Effect of n-hexane extract of T. bangwensis on root growth length on Allium cepa.

Figure 6.

Photomicrograph of different chromosomal aberrations showing the effect of n-hexane extract of T. bangwensis leaves. Abbreviations: a1 = Prophase a2 Anaphase, a3 Metaphase, a4 Telophase, b = Bridged Anaphase, c = Sticky Telophase, d = C-Mitosis, e = Attached chromosome, f = Vagrant chromosome, g = Bridged Telophase and h = Binuclear chromosome.

The results of the inhibitory effect on α-amylase and α-glucosidase activities demonstrate that the plant exhibited significant inhibitory effects on both α-amylase and α-glucosidase activities particularly at the highest concentration (100 µg/ml). On the other hand, the inhibitory effect was higher on α-amylase activity compared to α-glucosidase activity. The median inhibition concentration (IC₅₀) determined shows that the inhibitory effect of the plant extract on α-amylase activity was 59.15 ± 0.21 µg/ml while that of α-glucosidase was 73.29 ± 0.52 µg/ml. Meanwhile, the inhibition concentration at 90% (IC₉₀) on α-amylase activity was 122.30 ± 0.45 µg/ml whereas that of α-glucosidase activity was found to be 139.52 ± 1.45 µg/ml. Nonetheless, the inhibitory activity of acarbose (standard drug) was higher compared to the plant extract. The IC₅₀ of acarbose on α-amylase and α-glucosidase activities are 35.42 ± 0.22 µg/ml and 40.51 ± 0.11 µg/ml while the IC₉₀ for α-amylase and α-glucosidase activities are 87.88 ± 0.20 µg/ml and 89.90 ± 0.34 µg/ml respectively. The results also showed significant difference between the control (acarbose) and n-hexane extract of T. bangwensis at p < 0.05 (Tables 4 and 5).

Table 4.

Inhibitory effect of n-hexane extract of T. bangwensis on α-amylase activity.

Percentage inhibition at different concentrations
	25 µg/ml	50 µg/ml	75 µg/ml	100 µg/ml	IC₅₀(µg/ml)	IC₉₀ (µg/ml)
Control (Acarbose)	64.45 ± 0.41^*	74.15 ± 0.35^*	78.91 ± 0.41^*	88.08 ± 0.18^*	35.42 ± 0.22^*	87.88 ± 0.21^*
n-hexane extract	42.44 ± 0.25**	48.91 ± 0.31**	58.69 ± 0.25**	71.02 ± 0.30**	59.15 ± 0.21**	122.30 ± 0.45**

Values were in triplicates and were statistically expressed as Mean ± SD. Different superscript shows significant difference between control and n-hexane extract of T. bangwensis at each concentration at p < 0.05. Abbreviations: IC50 = Median inhibition concentration, IC90 = Inhibition concentration at 90%.

Table 5.

Inhibitory effect of n-hexane extract of T. bangwensis on α-glucosidase activity.

Percentage inhibition at different concentrations
	25 µg/ml	50 µg/ml	75 µg/ml	100 µg/ml	IC₅₀(µg/ml)	IC₉₀ (µg/ml)
Control (Acarbose)	55.59 ± 0.38^*	64.89 ± 0.70^*	77.79 ± 1.15^*	90.15 ± 0.40^*	40.51 ± 0.11^*	89.90 ± 0.34^*
n-hexane extract	28.73 ± 0.58**	35.43 ± 0.38**	51.24 ± 1.44**	64.24 ± 0.17**	73.29 ± 0.52**	139.52 ± 1.45**

Discussion

The present study was undertaken to evaluate the toxicological and anti-diabetic potential of n-hexane extract of T. bangwensis leaves as a possible alternative remedy for the treatment and management of hyperglycaemic conditions. Phytochemical screening revealed the presence of flavonoid, phenol, tannin, alkaloid and cardiac glycoside. Studies have shown that the mechanistic action of phytochemical compounds as antioxidant agent is basically either by inhibiting/reducing oxidant species (free radicals) generation or stabilizing their effect by donating electron for them to attain a stable configuration.^31,32 For instance, flavonoid and phenolic compounds scavenge free radicals by transferring hydrogen to the electron deficient molecule (free radical) thereby transforming it into a stable molecule.³³ The remarkable antioxidant activity exhibited by the plant extract may be attributed to the high amount of flavonoid and phenol content. Therefore, the positive correlation obtained between antioxidant activity and high flavonoid and phenol content in this study is in agreement with previous research finding.³⁴

An investigation into toxicity study using Ames/salmonella test, cellular response test, micronucleus test, chromosomal aberration assay, cytotoxicity/genotoxicity assay, chronic and sub-chronic assay as well as brine shrimp lethality and Allium cepa test reveals that many plants used as food or in traditional medicine contain mutagenic, cytogenic, genotoxic and carcinogenic agents. ^35,36

–39 The brine shrimp lethality assay which is a useful preliminary bioassay (or first-line toxicity tool) used for evaluating the toxicological effect of fungal toxins, plant extracts, heavy metals, pesticides, effluents was developed by Michael et al and one advantage of this assay is its rapidity, inexpensiveness and simplicity.^40
–42 It is noteworthy that the results obtained from this assay is generally acceptable and correlates with cytotoxic and anti-tumour properties.⁴³ Furthermore, the Allium cepa model studies the cytotoxic, inhibitory/mitodepressive and chromosomal aberration (abnormalities/changes in the DNA structure) effects which probably could be due to either chromatin dysfunction, disturbance in the cell cycle induced by the interaction of alkaloid with the DNA the inhibition of DNA synthesis or blocking of the G2 phase of the cell cycle from entering mitosis or chromatin dysfunction (DNA interaction).^44
–46 For instance, chromosome fragments indicate chromosome breaks and could be a consequence of anaphase/telophase bridges.⁴⁷ Subsequently, sticky anaphase/telophase chromosome observed in this study may be attributed to the influence of mutagens on the physical chemical properties of DNA, protein or formation of complexes with phosphate groups in DNA which leads to loss in the normal chromosomal appearance thereby resulting in ‘stick-surface’ causing chromosome agglomeration.^48

–52 This suggest that the plant extract may possess mutagenic potential which may possibly outweigh its therapeutic potential. The results of the brine shrimp lethality and Allium cepa test demonstrate that n-hexane extract of T. bangwensis leaves shows cytotoxic effect characterized by high mortality rate and decrease in root growth length, mitotic index (%MI) and mitotic division respectively.

Several strategic approaches have been explored in the treatment and management of diabetes mellitus such as stimulation of adenosine monophosphate dependent-protein kinase (AMPK), blockage of ATP gated potassium ion channels in β cells, stimulation of peroxisome proliferator-activated receptors activities (PPAR γ), glucagon-like peptide-1 (GLP-1) modulation among others.^11,53,54 These mechanisms of actions are directed at either enhancing insulin secretion/release, insulin sensitivity or reducing glycolysis by the liver. Currently, a new approach has been developed in the management of postprandial hyperglycaemia (PPH) by inhibiting α-amylase and α-glucosidase activity. The reduction in postprandial hyperglycaemia is important because it reduces advanced glycation and products formation which has been identified as a major risk factor for cardiovascular complications of diabetes mellitus.⁵⁵ Previous studies reported that the inhibition of these carbohydrate digesting enzymes may be beneficial to diabetic patients with impaired insulin tropic response particularly when used in combination with other hypoglycaemic agents.⁵⁶ Interestingly, the present study produced a significant inhibitory effect against the activities of α-amylase and α-glucosidase particularly at the highest concentration (100 µg/ml) and the results obtained is in agreement with other findings.^57,58 The inhibitory effect observed may be due to high flavonoid, phenol and alkaloid content which agrees with previous findings. Furthermore, the inhibitory action may probably be either due to the protective role of antioxidant phytochemicals against attack by free radicals on pancreatic/intestinal membrane thereby preventing elevated enzyme activity or increase in the concentration of the plant extract resulting in increase in the inhibition of the enzymes activities.^59,60,56

Conclusion

The present research findings showed that n-hexane extract of T. bangwensis leaves exhibited significant antioxidant activity and inhibitory effect on α-amylase and α-glucosidase activity particularly at 100 µg/ml. Furthermore, the antioxidant and inhibitory effects observed in this study may be attributed to high concentration of the phytochemical compounds mentioned above. It can, therefore, be concluded that T. bangwensis leaves may demonstrate potent anti-diabetic property. Within the scope of the toxicological models used, the results suggest that the plant extract may be cytotoxic.

Footnotes

Acknowledgements

We acknowledge the technical expertise of Mr Obu Francis and Mr Sunday Adenekan, Department of cell Biology and genetics and Department of Biochemistry respectively. All of the University of Lagos. Appreciation also goes to Ndam Shaifa, Department of Biochemistry and Forensic Science, Nigeria Police Academy, Wudil., Kano.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

The ethical approval for the use of Artemia salina eggs in this research work was obtained from the ethics committee for the care and use of laboratory animals of Nigeria Police Academy, Wudil, Kano, Nigeria.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Godwin O Ihegboro

References

International Diabetes Federation (IDF) Diabetes Atlas. 5th edn, Brussels: International Diabetes Federation, 2011.

Ononamadu

Alhassan

Imam

, et al. In vitro and in vivo anti-diabetic and anti-oxidant activities of methanolic leaf extracts of Ocimum Canum. Caspian J Intern Med 2019; 10(2): 162–175.

Ardisson-Karat

Willnet

. Diet lifestyle and genetic risk factors for Type 2 diabetes: a review from the Nurses’ health study. Nurses’ health study 2 and health professionals follow-up study. Curr Nutr Rep 2014; 3: 345–354.

Panti

Convera

. The role of mitochondria in the pathogenesis of Type 2 diabetes. Endocr Rev 2010; 31: 364–395.

Koneri

Samaddar

Ramaiah

. Antidiabetic activity of a triterpenoid Saponin isolated from Momordica cymbalaria Fenzl. Indian J Exp Biol 2014; 52: 46–52.

Wild

Roglic

Green

, et al. Global prevalence of diabetes: estimates for year 2000 and projections for 2030. Diabet Care 2004; 27: 1047–1053.

Kumar

Narwal

Kumar

, et al. α-Glucosidase inhibitors from plants: a manual approach to treat diabetes. Pharmacogen Rev 2011; 5: 19–29.

Telagari

Hullarti

. In vitro α-amylase and α-glucosidase inhibitory activity of A. caudonum Linn and C. argentia Linn extracts and fractions. Indian J Pharmacol 2015; 47: 425–429.

Ali

Jehanger

Hussein

, et al. Inhibition of α-glucosidase by oleantotolic acid and its synthetic derivatives. Phytochemistry 2002: 60: 295–299.

10.

Gayathri

Konnabiran

. Antidiabetic and ameliorative potentials of T. bangwensis bark extract in STZ induced diabetic rats. Indian J Clinical Biochem 2008; 23: 394–400.

11.

Ganesan

Rama

MBM

Sultan

. Oral hypoglycemic medications. Treasure Island: Statpearls Publishing Company. 2020. Updated May 28, 2020.

12.

Nishiokat

Kawahata

Aoyama

. Baicalenin, an α-glucosidase inhibitor from S. baicalensis. J Nat Prod 1998; 61: 1413–1415.

13.

Ortiz-Anadrade

Garcia-Jimenez

Castillo-Espana

, et al. α-glucosidase inhibitory activity of the methanolic extract of T. harwegiana: an anti-hyperglycemic agent. J Ethnopharmacol 2007; 109: 48–53.

14.

Fenell

Lindsay

K.L

McGraw

, et al. Assaying African medicinal plants for efficacy and safety: pharmacological screening and toxicology. J Ethnopharmacol 2004; 94: 205–217.

15.

Omar

Ramzi

Adnan

, et al. Phytochemical analysis and anti-diabetic, anti-inflammatory and antioxidant activities of Loranthus acacia Zucc Grown in Saudi Arabia. Saudi Pharm J 2019; 27: 724–730.

16.

Simeon

Lloh

Imoh

, et al. African mistletoe (Loranthaceae): ethnopharmacology, chemistry and medicinal values: an update. Afr J Tradit Complement Altern Med 2013; 10(4): 161–170.

17.

Ogbonnia

Anyika

Mbaka

, et al. Anti-hyperglycemic and hyperlipidemic effects of aqueous-ethanol extract of Tapinanthus globiferus leaves and Treculia Africana root bark and their mixture in alloxan diabetic rats. Agric Biol J North Am 2012; 3(6): 237–246.

18.

Osinubi

Ajayi

Adesiyun

. Evaluation of the anti-diabetic effect of aqueous leaf extract of Tapinanthus butungii in male Sprague Dawley rats. Med J Islamic World Acad Sci 2016; 16(1): 41–47.

19.

Ihegboro

Alhassan

Ononamadu

, et al. Identification of bioactive compounds in ethylacetate fraction of Tapinanthus bangwensis that ameliorate CCl₄-induced hepatotoxicity in wistar rat. Toxicol Res Appl 2020; 4: 1–14.

20.

Geetha

Phytochemical screening, quantitative analysis of primary and secondary metabolites of Cymbopogan citratus (DC) staft. Leaves from Kodaikanal hills, Tamilnadu. Int J PharmeTech Res 2014; 6(2): 521–529.

21.

Siddhuraju

Becker

. Antioxidant properties of various solvent extracts of total phenolics constituents from the different origins of Drumstick tree (Moringa oleifera Lam) leaves. J Agric Food Chem 2003; 51: 2144–2155.

22.

Kumaran

Karunakaran

. In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India. LWT-Food Sci Technol 2006; 40: 344–352.

23.

Herin

SGD

John De Britto

Benjamin Jeya

RKP

. Quantitative and qualitative analysis of phytochemicals in five Pteris species. Int J Pharm Pharm 2013; 5(1): 105–107.

24.

Yassa

Tofighi

Ghazi saeidi

, et al. Determination of cardiac glycosides and total phenols in different generations of Securigera securidaca suspension culture. Res J Pharmacogn 2016; 3(2): 25–31.

25.

Nithiananthan

Shyamala

Chen

, et al. Hepatoprotective potential of Clitoria termatea leaf extract against paracetamol-induced damage in mice. Molecules 2011; 16: 10134–10145.

26.

Zuraini

Rais

Yoga-Latha

, et al. Antioxidant activity of Coleus blumel, Orthosiphon stanineus, Ocimium basilicum and Mentha arvensis from Lanthaceae family. Int J Nat Eng Sci 2008; 2: 93–95.

27.

Alisi

Onyeze

GOC

. Nitric oxide scavenging ability of ethylacetate fraction of methanolic leaf extracts of Chromolaena odorata (Linn.). Afr J Biochem Res 2008; 2: 145–150.

28.

Geethaa

Surash

Sreenivasan

, et al. Brine shrimp lethality and acute toxicity studies on Swietenia mathagoni (Linn) Jacq. Seed methanolic extract. Pharmacognosy Res 2010; 2(4): 215–220.

29.

Fiskesjo

. The Allium test as a standard in environmental monitoring. Herediteas 1985; 102(1985): 92–112.

30.

Kazeem

Dansu

Adeola

. Inhibitory effects of Azadirachta indica A juss leaf extract on the activities of α-amylase and α-glucosidase. Pakistan J Biol Sci 2013; 16: 1358–1362.

31.

Abbas

Saggu

Sakaran

, et al. Phytochemical, antioxidant and mineral composition of hydroalcoholic extract of dicoyl (Cichorium intybus L) leaves. Saudi J Biol Sci 2014; 22(3): 322–326.

32.

Hudda-Faujan

Noriham

Norrakiah

, et al. Antioxidant activity of plants methanolic extracts containing phenolic compounds. Afr J Biotechnol 2004; 8(3): 484–489.

33.

Huang

Cai

Zhang

Natural phenolic compounds from medicinal herbs and dietary plants: potential used for cancer prevention. Nutr cancer 2010; 62(1): 1–20.

34.

Dzoyem

Eloff

. Anti0inflammatory, anticholinesterase and antioxidant activity of leaf extracts of twelve plants used in traditionally to alleviate pain and inflammation in South Africa. J Ethnopharmacol 2015; 160(3): 194–201.

35.

Eisenbrand

Pool-Zobel

Baker

, et al. Methods of in vitro toxicology. Food Chem Toxicol 2002; 40: 193–236.

36.

Higashimoto

Purintrapiban

Karaoka

. Mutagenicity and anti-mutagenicity of extracts of three species and a medicinal plant in Thailand. Mutat Res 1993; 303(3): 135–142.

37.

Schimmer

Kruger

Paulini

, et al. An evaluation of 55 commercial plant extracts in the Ames mutagenicity test. Pharmazle 1994; 49(6): 448–451.

38.

Kassie

Purzefall

Musk

. Genotoxic effects of crude juices from Brassica vegetables and juices and extracts from phytopharmaceutical preparations and species of Cruciferous plant origin in bacterial and mammalian cells. Chemico-Biological Infections 1996; 102(1): 1–6.

39.

Askin-Celik

Aslanturk

. Cytotoxic and genotoxic effects of Lovandulo stoechas aqueous extracts. Biologia 2007; 62(3): 292–296.

40.

Vein

Pushpananthan

. Comparison of the Artemia salina and Artemia fransiscana bioassays for toxicity of Indian medicinal plants. J Coastal Life Med 2004; 2: 453–457.

41.

Mayorga

Perez

Crutz

, et al. Comparison of bioassays using the anostracan crustaceans, Artemia salina and Thamnocephathus platyurus for plant extract toxicity screening. Bras J Pharm 2010; 20: 897–903.

42.

Michael

Thompson

Abramovitz

. Artemia salina as a test organism for bioassay. Science 1956; 123: 464.

43.

McLaughlin

Chang

Smith

. Simple bench-top bioassays (Brine shrimp and Potato discs) for the discovery of plant antitumor compounds. Am Chem Soc Symp Ser 1993; 534: 112–134.

44.

Sudhakar

Ninge-Gowda

Venu

. Mitotic abnormalities induced by silk dyeing industry effluents in the cells of Allium cepa. Cytologia 2001; 66(3): 235–239.

45.

Tulay

Ozlem

. Evaluation of cytotoxicity and genotoxicity of Inula viscosa leaf extracts with Allium test. J Biomed Biotechnol 2010; 2010: 189252.

46.

Ihegboro

Alhassan

Owolarafe

, et al. Evaluation of the Biosafety potentials of Tapinanthus bangwensis and Moringa oleifera Leaves using Allium cepa Model. Toxicol Rep 2020; 7: 671–679.

47.

Singh

. Plant cytogenetics. Boca. Raton, FL: CRC Press, 2003.

48.

Rancuzogullan

Ila

Kayraldiz

, et al. Chromosome observations and sister chromatid exchanges in cultured human lymphocytes treated with sodium metabisulfite, a food preservative. Mutat Res 2001; 490(2): 109–112.

49.

EL-Ghanmery

EL-Kholy

EL-Youssar

MAA

. Evaluation of biological effects of Zn²⁺ in relation to germination and root growth of Nigello sativa L and Triticum aestivum. Mutat Res 2003; 537: 29–41.

50.

Gomurgen

. Cytological effect of the potassium metabisulphite and potassium nitrate food preservatives on root tips of Allium cepa L. Cytologia 2005; 70(2): 119–128.

51.

Turkoglu

. Genotoxicity of five food preservatives treated on root tips of Allium cepa L. Mutat Res 2007; 626(1-2): 4–14.

52.

Babich

Segall

Fox

. The Allium test—a simple, eukaryote genotoxicity assay. Am Biol Teach 1997; 59(9): 580–583.

53.

Chaudhury

Duvood

Reddy-Dendi

. Clinical review of anti-diabetic drugs. Implications for type 2 diabetes mellitus management. Front Endocrinol 2017; 8(6): 1–12.

54.

Alhandramy

. A review of oral therapies for diabetes mellitus. J Tadbah Univ Med Sci 2016; 11(4): 317–329.

55.

Geriello

Davidson

Hansefeld

. Postprandial hyperglycemia and cardiovascular complications of diabetes in update. Nutr Metabol Dis 2006; 18(7): 453–456.

56.

Mahomoodall

Subratty

Gurb-Fakim

, et al. Traditional medicinal herbs and food plants have the potential to inhibit key carbohydrate hydrolysing enzymes in vitro and release postprandial blood glucose peaks in vivo. Sci World J 2012; 2012: 285284.

57.

Olaokan

McGraw

Eloff

, et al. Evaluation of the inhibition of carbohydrate hydrolysing enzymes, antioxidant activities and polyphenolic content of extracts of ten African Ficus species (Moraceae) used traditionally to treat diabetes. Evid Based Complement Altern Med 2013; 13(1): 2–12.

58.

Picot

Subrartty

Mahomoodally

. Inhibitory potential of traditionally used native anti-diabetic medicinal plants on α-amylase and α-glucosidase, glucose entrapment and amylolysis kinetics in vitro. Adv Pharmacol Sci 2014; 2014: 1–7.

59.

Spimola

Castiho

PC.

Evaluation of Asteraceae herbal extracts in the management of diabetes and obesity. Contribution of caffeoylquinic acids on the inhibition of digestive enzymes activity and formation of advanced glycation end production in vitro. Phytotherepy 2017; 143: 29–35.

60.

Kupeti

Arhan

Gurbuz

, et al.

Evaluation of in vivo biological activity profile of isoorientin.

Z Naturforsch C J Biosci 2004; 59: 787–790.

Screening for toxicological and anti-diabetic potential of n-hexane extract of Tapinanthus bangwensis leaves

Abstract

Keywords

Introduction

Materials and methods

Chemical reagents

Authentication of plant material

Extraction of plant material

Phytochemical screening of plant extract

Qualitative phytochemical screening

Quantitative phytochemical screening of plant extract

Quantification of phenolic content

Quantification of tannin content

Quantification of flavonoid content

Quantification of alkaloid content

Quantification of cardiac glycoside

Antioxidant activity of the plant extract

2,2-Diphenyl-1-picrylhydrazine (DPPH) assay

Nitric oxide antioxidant assay

Toxicological study of the plant extract

Brine shrimp Lethality assay

Cytotoxicity study using Allium cepa assay

Inhibitory effect of the plant extract

Inhibitory effect on α-amylase activity

Inhibitory effect on α-glucosidase activity

Statistical analysis

Results

Discussion

Conclusion

Footnotes

Acknowledgements

Declaration of conflicting interests

Ethical approval

Funding

ORCID iD

References