Polyphenolic-rich extracts of Andrographis paniculata mitigate hyperglycemia via attenuating β-cell dysfunction, pro-inflammatory cytokines and oxidative stress in alloxan-induced diabetic Wistar albino rat

Abstract

Background

This study sought to investigate anti-hyperglycemic potentials of free and bound phenolic-rich extracts of Andrographis paniculata (A. paniculata) leaves, commonly called “king of the bitter”, a plant locally employed in folkloric alternative medicine.

Method

In vitro antioxidant potentials such as total phenolic and flavonoid contents were evaluated in addition to phosphomolybdenum reducing total antioxidant activity in bound and free polyphenol-rich extracts of A. paniculata. Also, following induction of diabetes through a single intraperitoneal injection of freshly prepared alloxan monohydrate (150 mg/kg body weight, b.w), diabetic rats were divided into seven (7) treatment groups with six rats each (n = 6) i.e. group 1 (normal control), 2 (diabetic untreated), 3 (5 mg/kg glibenclamide -treated control), while 4–7 were administered 50 and 100 mg/kg b.w of free and bound phenolic extracts of A. paniculata, respectively for twenty-one (21) days.

Results

There was a significant (p < 0.05) difference in hematological indices, hepatic biomarkers, total protein, antioxidant enzymes activities, total thiol and fasting blood glucose levels of diabetic groups administered polyphenolic-rich extracts of A. paniculata compared to diabetic untreated control. Similarly, serum insulin levels, hexokinase and glucose-6-phoshatase activities were significantly (p < 0.05) improved in phenolic-rich extracts of A. paniculata-treated diabetic groups compared to diabetic untreated control. A significant (p < 0.05) reduction was as well observed in the levels of inflammatory biomarkers such as interleukin-6 (IL-6) and tumor necrosis factor (TNFα) among extract of A. paniculata administered diabetic groups compared diabetic untreated group.

Conclusions

Anti-hyperglycemic activities demonstrated by polyphenolic-rich extracts of A. paniculata when compared to glibenclamide and normal control, could possibly have been occasioned by β-cell protection, restoration of glycolytic enzymes as well as mitigation of inflammatory markers via antioxidant defensive/protective properties of the extracts.

Introduction

Diabetes mellitus (DM) is a metabolic disorder caused by impairment in carbohydrate and lipid metabolism [1, 2]. It’s usually characterized by; impaired β-cell function, subsequently with a relative insulin deficiency, followed by insulin resistance with a decrease in glucose uptake in the muscle and fat cells, as well as an unrestrained hepatic glucose production [3, 4]. Recent statistics indicate that about 451 million people have diabetes globally in 2017 and the figure is estimated to increase by 242 million by 2045 [2, 5]. The various complications attributed to DM have continued to pose major medical problems despite the introduction of synthetic oral hypoglycemic drugs [6], hence, the need for alternatives becomes imperative in the management of the menace.

Oxidative stress is believed to play an important role in the pathogenesis of DM and related complications, particularly type 2 diabetes [7]. The formation of reactive oxygen specie (ROS) in DM via processes that involve non-enzymatic glycation of proteins, glucose oxidation and increased lipid peroxidation, has been reported [8, 9]. Study has also implicated excessive generation of free radicals in severe depletion of cellular antioxidant enzymes as well as exacerbating insulin resistance in experimental diabetic subjects [10].

However, the use of bioactive natural plants has recently gained relevance attention in the quests for an alternative therapy in the management of DM [11, 12]. These medicinal plants have been reported to demonstrate substantial ability to inhibit the production/generation of free radicals [13], whose activities underlie the onset of major human diseases with much credits being attributed primarily to their naturally endowed bioactive polyphenolic constituents [14]. Plant polyphenols (majorly phenolic acids and flavonoids) are secondary metabolites in plant-based foods, such as fruits, vegetables, legumes etc. [15]. Several biological activities and beneficial properties of dietary polyphenols have been documented [16], some of these include antioxidant, anti-inflammatory, anti-allergic, among others [16]. Similarly, some of these polyphenolic-endowed natural plants have revealed antihyperglycemic properties and have locally been engaged among folks, for the purpose of managing DM and other diseases with little or no side effects [17].

Andrographis paniculata (A. paniculata), commonly called the king of the bitter, is one of the locally used medicinal plants for several therapeutic purposes, especially in the folkloric management therapy of DM [18]. Also, the plant has been literatured to be effective in the treatment of health-threatening medical conditions such as; myocardial infarction, hypertension and malaria [19, 20]. Similarly, Chao and Lin [21] reported various polyphenolic bioactive components in the plants [22]. Therefore, this study sought to investigate anti-hyperglycemic potentials of free and bound phenolic-rich extracts of A. paniculata leaf via in vitro antioxidant capacity assessment and evaluation of their administration on hematological indices, enzymatic and non-enzymatic antioxidants, insulin levels, glucose metabolizing enzymes activities and inflammatory markers in alloxan-induced diabetic rat.

Materials and methods

Chemicals and reagents used

Alloxan monohydrate, sodium hydroxide, hydrochloric acid, gallic acid, sodium carbonate and other reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA), ELISA kits (Crystal Chem Inc., IL, US). All other chemicals used were of analytical grades and prepared in all glass apparatus using distilled water.

Collection of plant sample

Fresh aerial leaves of A. paniculata were purchased from a local market in Ibadan, Oyo State and was identified with a voucher specimen number: No. SP- 98, by a taxonomist in the Department of Plant Science, Ekiti State University, Ado-Ekiti.

Sample preparation

Fresh aerial leaves of A. paniculata were air-dried at room temperature (37 °C) to a constant weight and then ground to powdery form using an automated laboratory blender (Jenway product).

Extraction of free phenolic extract of A. paniculata

Obtention of the soluble free phenols was carried out according to the method of Chu et al. [22]. The powdery plant sample (250 g) was extracted with 80% acetone (1:5 w/v) for 72 h at room temperature. The mixture was thereafter filtered with Whatman no 2 filter paper on a Buchner funnel under vacuum. The residue obtained was kept for the extraction of bound phenolic extract (Section 1). The filtrate was then evaporated to dryness at 45 °C.

Extraction of bound phenolic extract from A. paniculata

The bound phenolic extract from A. paniculata leaf was extracted from residue obtained from section 1 (above) according to the method described by Afolabi et al. [14]. The residue was drained off and hydrolyzed directly with 20 mL 4 M NaOH at room temperature for 1 h with continuous auto-agitation. The mixture was acidified to pH 2.0 with concentrated hydrochloric acid (HCl) and extracted six times (×6) with 200 mL ethylacetate. The ethylacetate fractions were pooled together and evaporated at 45 °C under vacuum to dryness.

In vitro quantification of phytochemical potentials of free and bound phenolic-rich extracts of A. paniculata

Determination of total phenolic content

The total phenolic content of free or bound phenolic-rich extract of A. paniculata was determined by the method of Singleton et al. [23]. 0.2 mL of the extract was mix with 2.5 mL 10% folin-ciocalteau’s reagent and 2 mL 7.5% sodium carbonate. The reaction mixture was subsequently incubated at 45 °C for 40 min, and the absorbance was read at 700 nm against the reagent blank. Phenolic content extrapolated and expressed as mg gallic acid equivalent/g of dried sample (mg GAE/g dried sample).

Determination of total flavonoid content

The total flavonoid content of free or bound phenolic-rich extract of A. paniculata was determined using a method described by Bao [24]. 0.2 mL of extract was added to 0.3 mL 5% NaNO₃ at zero time. After 5 min, 0.6 mL 10% AlCl₃ was added and after 6 min, 2 mL NaOH was added to the mixture followed by the addition of 2.1 mL of distilled water. Absorbance was read at 510 nm against the reagent blank and the flavonoid content was extrapolated expressed as mg quercetin equivalent/ g of dried sample (mg QE/g dried sample).

In vitro antioxidant activity assays

Phosphomolybdenum reduction assay

Determination of the total antioxidant activity of free or bound phenolic-rich extract of A. paniculata via phosphomolybdenum reduction assay, was carried out according to the method described by Prieto et al. [25]. Briefly, 0.3 mL of sample was mixed with 3 mL of reagent solution (600 mmol/L H₂SO₄, 28.0 mmol/L sodium phosphate and 4.0 mmol/L ammonium molybdate). The mixture was incubated at 95 °C for 90 min and the absorbance of the green phosphomolybdenum complex was measured at 695 nm against the reagent blank. The result was determined from the mean ± standard deviation of triplicate values (n = 3) and the total antioxidant activity expressed as mg ascorbic acid equivalent per gram of the dried sample (mg AAE/g dried sample).

Study protocol

Eight (8) weeks old Albino rats (Male) of weight between 150 and 180 g were used in this experimental study. Animals were procured from the animal house of Afe Babalola University, Ado–Ekiti, Ekiti State, Nigeria. They were acclimatized for a period of 2 weeks before the commencement of the experiment at room temperature of 37 °C in a plastic cage and had free access to pelletized animal feed and water ad libitum. The animal handling protocol engaged in the study was in accordance with the guidelines of the animal care of Afe Babalola University (ABUAD).

Induction of diabetes

On the first day (Day one), rats were administered alloxan monohydrate (150 mg/ kg b.w) dissolved in normal saline intraperitoneally (IP), according to the method described by Nagappa et al. [26]. Diabetes was thereafter confirmed after 72 h (Fourth day) of injecting alloxan monohydrate by checking the fasting blood glucose levels of the rats. The blood samples used were collected from the tail vein and read using a portable glucometer (ACCU-CHECK Active-Roche Diabetes Care, Germany). Animals with a FBG level ≥ 250 mg/dl were considered diabetic. Thereafter, treatment started exactly at the fourth day (4th day) and ended at twenty-fifth day (25th day) (3 weeks).

Animal grouping

The experimental rats were randomly divided into seven (7) treatment groups of six (6) rats each as follow;

• Group 1 – Normal control, fed with pelletized animal feed and distilled water ad libitum for 21 days;

• Group 2 – Untreated diabetic control, fed with pelletized animal feed and distilled water ad libitum for 21 days;

• Group 3 – Diabetic rats administered with oral gavage of 5 mg/kg b.w glibenclamide, received pelletized animal feed and distilled water ad libitum for 21 days;

• Group 4 – Diabetic rats administered with oral gavage of 50 mg/kg free phenolic extract of A. paniculata, received animal pelletized feed and distilled water ad libitum for 21 days;

• Group 5– Diabetic rats administered with oral gavage of 100 mg/kg free phenolic extract of A. paniculata, received animal pelletized feed and distilled water ad libitum for 21 days;

• Group 6– Diabetic rats administered with oral gavage of 50 mg/kg bound phenolic extract of A. paniculata, received pelletized animal feed and distilled water ad libitum for 21 days;

• Group 7– Diabetic rats administered with oral gavage of 100 mg/kg bound phenolic extract of A. paniculata, received pelletized animal feed and distilled water ad libitum for 21 days.

Determination of relative body weight

The weight of rats in each experimental groups were taken on the first day of the experiment before the start of various treatments and also, following overnight fast on the last day of the treatment.

Isolation and preparation of tissues

Preparation of the blood sample

Animals were euthanized by a mild exposure to di-ethyl ether briefly following an overnight fasting. Blood samples were rapidly collected via cardiac puncture (with the aid of sterilized syringe) into both the plain and EDTA-containing sample bottles (Section A). The EDTA-containing blood samples were for hematological analyses while blood samples in the plain bottles were subsequently centrifuged at 3000 rpm for 10 min using a table top centrifuge to obtain sera used for various biochemical analyses.

Preparation of tissue homogenate

Hepatic tissues were extracted and rapidly placed on ice and weighed. Tissue homogenates were prepared according to the method of Bamikole et al. [27]. The tissues were homogenized in cold Tris-HCl buffer (1/10 w/v, pH 7.4) and the homogenate centrifuged at 3000 rpm for 10 min, thereafter, supernatant was separated and kept for further bioassays.

Hematological analyses

EDTA-containing blood samples from section A (above) were used for the hematological studies performed to determine the following parameters: packed cell volume (PCV), hemoglobin (Hb), white blood count (WBC), red blood cell (RBC), neutrophils (N), lymphocytes (L), monocytes (M), and eosinophils (E) [28].

Biochemical assays

Biochemical analyses were carried out on the activities of liver function markers such as alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP), as well as serum total protein to assess level oxidative damage on the liver according to the method described by Reitman and Frankel [29]. Also, serum lipid profile was carried out by analyzing the levels of serum HDL, triglyceride (TG) and total cholesterol (TC) by colorimetric method described by Reitman and Frankel [29] using commercially available test kits, products of Randox Laboratories (Crumlin, United Kingdom); while antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and GPx were determined using colorimetric method described by Alía et al. [30], Sinha [31] and Ellman [32], respectively. Total thiol (TSH) was determined using the method of Ellman [32], oxidative stress markers, malondialdehyde (MDA) by the method of Ohkawa et al. [33]; fasting blood glucose was determined using the Accu-chek Advantage II Clinical Glucose meter [34]; serum insulin was assayed using an enzyme linked immunosorbent assay (ELISA) kit (Crystal Chem Inc., IL, US); glucose-6-phosphatase was determined by the method of Koide and Oda [35]; hepatic activity of hexokinase was measured using the modified method of Branstrup et al. [36]; serum cytokines (IL-6 and TNFα) analyses were performed using enzyme-linked immunosorbent assay (ELISA) kit (Elabscience Rat Insulin ELISA from Mercodia) as described by Voller [37].

Statistical analyses

Data were analyzed using one-way ANOVA, followed by Tukey’s test for post-hoc analysis and graphical representation of results by GraphPad Prism 5 Program (GraphPad Software, San Diego, CA, USA). All values were expressed as mean ± SEM, except where otherwise stated. Statistical differences were considered at p < 0.05.

Results

In vitro antioxidant capacity of free and bound phenolic-rich extracts of A. paniculata

Figure 1 represents quantified antioxidant parameters such as total phenolic content (TPC), total flavonoid content (TFC) and phosphomolybdenum reducing total antioxidant capacity of free and bound phenolic extracts of A. paniculata. The results revealed that free phenolic extract demonstrated a higher phenolic content (74.56 ± 0.82 mg GAE/g dried sample) than the bound phenolic extract (64.13 ± 0.64 mg GAE/g dried) of A. paniculata. In the same manner, quantified total flavonoid content was higher in free phenolic extract (67.20 ± 0.87 mg QE/g dried sample) compared to bound phenolic extract (46.71 ± 0.19 mg QE/g dried sample) of A. paniculata. Similarly, total antioxidant capacity through phosphomolybdenum reduction was higher in free phenolic extracts (92.29 ± 0.59 mg AAE/g dried sample) than in bound phenolic extracts (85.01 ± 0.54 mg AAE/g dried sample) of A. paniculata.

Fig. 1.

Antioxidant capacity of free and bound phenolic-rich extracts of A. paniculata. Values were expressed as mean ± standard deviation (SD) of triplicate trials (n = 3).

Effects of phenolic-rich extracts of A. paniculata on relative body weight in alloxan-induced diabetic rats

Figure 2 represents the effect of free and bound phenolic leave extracts of A. paniculata on the body weight (g) of Alloxan-induced diabetic rats after 21 days treatment. In this study, it was evident that diabetic control (group 2) rats showed a significant (p < 0.05) reduction in body weight (139.4 ± 0.50) compared to normal control (group 1) (195.1 ± 0.03). Similarly, a 21-day administration of 50 and 100 mg/kg b.w of free and bound phenolic extracts of A. paniculata caused a significant (p < 0.05) increase in final body weight among the treated diabetic groups compared to diabetic control. However, this observation was compared favorably to normal control and 5 mg/kg b.w glibenclamide-treated control.

Fig. 2.

Effect of phenolic-rich extracts of A. paniculata on body weight in alloxan-induced diabetic rats. Results were expressed as mean ± SEM of six trials (n = 6). Different symbols on bars indicate statistical difference at p < 0.05. *p < 0.05 vs normal control, ^#p > 0.05 vs normal control and p < 0.05 vs diabetic control

Effects of phenolic-rich extracts of A. paniculata on hematological indices in alloxan-induced diabetic rats

Table 1 represents the effect of free and bound phenolic extracts of A. paniculata on hematological indices of alloxan-induced diabetic rats after 21 days administration. In this study, diabetic control rats showed a significant (p < 0.05) reduction in parameters such as PCV (40.03 ± 0.10), Hb (13.25 ± 2.75) and RBC (3.65 ± 0.92) compared to normal control (PVC, 46.00 ± 0.66; Hb, 16.8 ± 0.71 and RBC, 4.85 ± 0.02). In a similar manner, all diabetic groups administered 50 and 100 mg/kg b.w free and bound phenolic extracts of A. paniculata revealed a significant (p < 0.05) improvement in their RBC, Hb and PCV levels compared to untreated diabetic control after 21 days. Similarly, administration of 50 and 100 mg/kg b.w free phenolic extracts of A. paniculata among the treated groups revealed a significant (p < 0.05) difference when compared to normal control and 5 mg/kg b.w glibenclamide- treated control.

Table 1.

Effect of phenolic-rich extracts of A. paniculata on hematological parameters in alloxan-induced diabetic rats

Groups	PCV (%)	Hb (g/d)	WBC (mm3)	RBC (×1012/l)	N (%)	L (%)	M (%)	E (%)
Group 1	46.00 ± 0.66	16.8 ± 0.71	29.15 ± 2.01	4.85 ± 0.02 a	48.50 ± 0.01 a	41.5 ± 1.20a	7.54 ± 0.04a	2.50 ± 0.10a
Group 2	40.03 ± 0.10c	13.25 ± 2.75d	22.05 ± 0.82d	3.65 ± 0.92 c	45.01 ± 0.24d	40.04 ± 0.03d	7.01 ± 1.41d	1.71 ± 0.03d
Group 3	54.5 ± 0.13b	18.01 ± 0.21c	23.03 ± 0.01b	4.95 ± 1.10 a	45.50 ± 0.03c	44.00 ± 1.01c	7.45 ± 0.71b	2.00 ± 0.01b
Group 4	53.12 ± 0.61b	17.80 ± 0.63c	23.20 ± 0.42b	4.95 ± 0.35 a	47.02 ± 1.41a	43.01 ± 1.14b	7.52 ± 3.01a	3.08 ± 2.20c
Group 5	45.50 ± 0.03a	15.20 ± 0.42b	34.00 ± 0.23c	4.60 ± 0.14 a	50.12 ± 0.42b	42.82 ± 0.04b	7.51 ± 2.12a	3.00 ± 0.19c
Group 6	46.01 ± 0.08a	15.25 ± 2.07b	29.02 ± 0.02 c	4.02 ± 0.08b	49.00 ± 0.06 a	41.02 ± 0.37a	8.03 ± 0.01c	2.13 ± 0.04b
Group 7	49.50 ± 0.03a	16.45 ± 2.01a	28.00 ± 0.22 c	4.34 ± 1.13b	49.50 ± 0.95 a	43.00 ± 0.39b	7.44 ± 0.11a	2.50 ± 0.07b

Values were compared down the column. Statistical differences at p < 0.05 are represented with different superscripts. Each value indicates mean of six (n = 6) determinations ± SEM. Key: Group 1, normal control; Group 2, diabetic control; Group 3, diabetic +5 mg/kg glibenclamide; Group 4, diabetic +50 mg/kg free phenolic extract; Group 5, diabetic +100 mg/kg free phenolic extract; Group 6, diabetic +50 mg/kg bound phenolic extract; Group 7, diabetic +100 mg/kg bound phenolic extract

Effects of phenolic-rich extracts of A. paniculata on biochemical parameters in alloxan-induced diabetic rats

Liver function biomakers and total protein

Table 2 represents the effect of 21 days administration of free and bound phenolic extracts of A. paniculata (at doses of 50 and 100 mg/kg b.w) on hepatic tissue-bound biomarkers and total protein in alloxan-induced diabetic rats. As revealed from the Table 2, a significant (p < 0.05) decrease was indicated in hepatic tissue ALT, AST, ALP activities as well as TP level of the untreated diabetic control compared to normal control only. Whereas, a significant (p < 0.05) increase was markedly noted in ALT, AST, ALP activities and TP level of diabetic groups administered free and bound phenolic extracts of A. paniculata for 21 days compared to untreated diabetic control. However, the effect observed in the extract-treated diabetic groups revealed no significant (p < 0.05) difference compared to normal control as well as a favorable comparison with 5 mg/kg b.w glibenclamide-treated control.

Table 2.

Effect of phenolic-rich extracts of A. paniculata on liver function markers in alloxan-induced diabetic rats

Groups	ALT (U/l)	AST (U/l)	ALP (U/l)	T. Protein (g/dl)
Normal Control	241.10 ± 0.53	92.90 ± 0.67	51.05 ± 0.53	7.51 ± 0.56
Diabetic Control	20.21 ± 0.15c	34.80 ± 0.11c	21.48 ± 1.34b	5.85 ± 2.56d
Diabetic+5 mg/kg Gliben.	220.71 ± 0.03b	80.65 ± 0.84b	52.69 ± 2.51a	7.89 ± 2.32a
Diabetic+50 mg/kg FPEAP	251.04 ± 0.07a	87.40 ± 2.57b	53.41 ± 0.87a	8.59 ± 1.08b
Diabetic+100 mg/kg FPEAP	238.85 ± 0.95a	89.50 ± 0.05a	53.93 ± 0.02a	7.61 ± 0.01a
Diabetic+50 mg/kg BPEAP	236.43 ± 1.55a	81.10 ± 0.59b	51.91 ± 0.56a	8.18 ± 0.57b
Diabetic+100 mg/kg BPEAP	227.00 ± 0.14b	81.40 ± 0.60b	53.00 ± 0.41a	7.13 ± .007c

Values represent mean ± SEM of six determinations (n = 6). Different superscripts indicate levels of significant difference down the column at p < 0.05. Note: T. Protein, total protein; Gliben., glibenclamide. Key: FPEAP, free phenolic extract of A. paniculata; BPEAP, bound phenolic extract of A. paniculata

Serum lipid profile

Figure 3 represents the effect of free and bound phenolic extracts of A. paniculata administration on lipid profile in alloxan–induced diabetic rats. In the results, there was a significant (p < 0.05) decrease in HDL and increase significantly (p < 0.05) in TC and TG in the untreated diabetic control compared to normal control only. However, diabetic groups administered free and bound phenolic extracts of A. paniculata (at doses of 50 and 100 mg/kg b.w), revealed a significant (p < 0.05) increase in HDL with a decrease significantly (p < 0.05) in TC and TG compared to untreated diabetic control. However, the effect observed in the extract-treated diabetic groups revealed no significant (p < 0.05) difference compared to normal control as well as a favorable comparison with 5 mg/kg b.w glibenclamide-treated control.

Fig. 3.

Effect of phenolic-rich extracts of A. paniculata on lipid profile in alloxan-induced diabetic rats. Results were expressed as mean ± SEM of six trials (n = 6). Different alphabets on bars indicate levels of significant difference at p < 0.05

Antioxidants enzymes activities

Figure 4i-iii represent the effects of phenolic–rich extracts of A. paniculata on activities of antioxidant enzyme such as SOD CAT and GPx in alloxan-induced diabetic rats. As shown in Fig. 4i-iii, there was a significant (p < 0.05) decrease in the activities of SOD, CAT and GPx of untreated diabetic control compared to normal control only. Whereas, a significant (p < 0.05) increase was occasioned in SOD, CAT and GPx activities among diabetic groups administered 50 and 100 mg/kg b.w free and bound phenolic extracts of A. paniculata for 21 days compared to untreated diabetic control. Also, in Fig. 4i-ii, no significant (p > 0.05) difference was observed in SOD and CAT activities of extract-treated groups compared to normal control. However, there was a significant (p < 0.05) difference in GPx activities of extract-treated diabetic groups compared to normal control, as well as 5 mg/kg b.w glibenclamide-treated control.

Fig. 4.

i, ii, iii Effect of phenolic-rich extracts of A. paniculata on antioxidant enzyme activities in alloxan-induced diabetic rats. Results were expressed as mean ± SEM of six trials (n = 6). *^{, #} indicate significant difference at p < 0.05. *p < 0.05 vs normal control, ^#p > 0.05 vs normal control and p < 0.05 vs diabetic control

Total thiols and MDA levels

Figure 5i-ii show the effects of phenolic–rich extracts of A. paniculata on total thiols and MDA levels in alloxan-induced diabetic rats. In Fig. 5i, a significant (p < 0.05) depletion was markedly observed in the level of total thiol of the untreated diabetic control compared to normal control only. However, 21 days administration of 50 and 100 mg/kg b.w free phenolic extracts of A. paniculata significantly (p < 0.05) increased total thiols among extract- treated diabetic groups compared to normal control. More so, from Fig. 5ii, a significant (p < 0.05) increase was evident in lipid peroxidation marker (MDA) level of untreated diabetic control compared to normal control only. Conversely however, following 21 days administration of 50 and 100 mg/kg b.w free phenolic and bound extracts of A. paniculata, a significant (p < 0.05) decrease was noted among the treated diabetic groups compared to untreated diabetic control. However, this observation was not significantly (p < 0.05) different compared to normal control and favorably in comparison to 5 mg/kg b.w glibenclamide-treated control.

Fig. 5.

i, ii Effect of the phenolic-rich extracts of A. paniculata on total thiol and MDA levels in alloxan-induced diabetic rats. Results were expressed as mean ± SEM of six trials (n = 6). *indicate significant difference at p < 0.05 vs normal control and ^# p < 0.05 vs diabetic control

Serum glucose levels

Figure 6 represents the effect of 21 days administration of free and bound phenolic extracts of A. paniculata on fasting blood glucose (mg/dL) in alloxan-induced diabetic rats. In the results, a significant (p < 0.05) increase was noticeable in the fasting blood glucose among the experimental groups after 72 h of exposure to 150 mg/kg b.w alloxan monohydrate. However, after 21 days treament, a significant (p < 0.05) increase was observed in the fasting blood glucose level of the untreated diabetic control (255.08 ± 0.24) compared to normal control (79.68 ± 0.38) only. Contrarily, a significant (p < 0.05) reduction was markedly noted in the diabetic groups administered 50 and 100 mg/kg b.w free and bound phenolic extracts of A. paniculata compared to untreated diabetic control, however, there was no significant (p > 0.05) difference in this observation compared to normal control and 5 mg/kg b.w glibenclamide-treated control.

Fig. 6.

Effect of phenolic-rich extracts of A. paniculata on the fasting blood glucose (FBG) levels of alloxan-induced diabetic rats. Results were expressed as mean ± SEM of six trials (n = 6). Different alphabets on bars indicate levels of significant difference at p < 0.05

Serum insulin levels

Figure 7 represents the effect of 21 days administration of free and bound phenolic extracts of A. paniculata on serum insulin levels (ƿmol/L) in alloxan-induced diabetic rats. As shown in the results, there was a significant (p < 0.05) reduction in the insulin level of diabetic untreated control compared to normal control only. However, administration of 50 and 100 mg/kg b.w free and bound phenolic extracts of A. paniculata triggered a significant (p < 0.05) rise in insulin level of the extract-treated groups compared to untreated diabetic control. Whereas, observation in the extract-treated groups was not significantly (p > 0.05) different compared to normal control and 5 mg/kg b.w glibenclamide-treated control.

Fig. 7.

Effect of phenolic-rich extracts of A. paniculata on serum insulin levels in alloxan-induced diabetic rats. Results were expressed as mean ± SEM of six trials (n = 6). *indicate significant difference at p < 0.05 vs normal control and ^# p < 0.05 vs diabetic control

Carbohydrate-metabolizing enzymes

Figure 8 represents- the effect of 21 days administration of free and bound phenolic extracts of A. paniculata on hepatic hexokinase (mmol/min/mg protein) and glucose-6-phosphatase (G6P) (mU/gHb) activities in alloxan-induced diabetic rats. As presented in the result (Fig. 8), hexokinase activity was significantly (p < 0.05) lower with a significant (p < 0.05) increase in G6P in the hepatic tissue of untreated diabetic control when compared to normal control only. However, treatment with 50 and 100 mg/kg b.w free and bound phenolic-rich extracts of A. paniculata caused a significant (p < 0.05) increase in hexokinase activity as well as a decrease significantly (p < 0.05) in G6P activity when compared to diabetic untreated control. In the same manner, there was a significant (p < 0.05) different in the activities of these carbohydrate-metabolizing enzyme among extract-treated groups compared when compared to the observation in normal control and 5 mg/kg b.w glibenclamide treated control.

Fig. 8.

Effect of phenolic-rich extracts of A. paniculata on the carbohydrate-metabolizing enzyme levels in alloxan-induced diabetic rats. Results were expressed as mean ± SEM of six trials (n = 6). Different alphabets on bars indicate significant difference at p < 0.05

Serum inflammatory markers

Figure 9 represents the effect of 21 days administration of free and bound phenolic extracts of A. paniculata on the levels of serum inflammatory markers such as IL-6 and TNFα in alloxan-induced diabetic rats. As shown in the Fig. 9, a significant (p < 0.05) increase was observed in IL-6 and TNFα levels in the untreated diabetic control compared to normal control only. Whereas, administration of 50 and 100 mg/kg free and bound phenolic-rich extracts of A. paniculata significantly (p < 0.05) attenuated IL-6 and TNFα levels in the treated diabetic groups compared to untreated diabetic control. More so, no significant (p > 0.05) difference was established in IL-6 and TNFα levels among extract-treated group compared to normal control but a significant (p < 0.05) difference was noticeable compared to 5 mg/kg glibenclamide-treated control.

Fig. 9.

Effect of phenolic-rich extracts of A. paniculata on inflammatory markers in alloxan-induced diabetic rats. Results were expressed as mean ± SEM of six trials (n = 6). *indicate significant difference at p < 0.05 vs normal control and ^# p < 0.05 vs diabetic control

Discussion

DM has recently been described to pose a threat on global health [38] with a report on an increasing number of people suffering from the disorder according to the statistical evaluation of World health organization (WHO) [38]. However, natural plants that are rich in polyphenols have been considered to exhibit strong antihyperglycemic activities [39], by demonstrating wholesome ability to remove free radicals, chelate metal catalysts and reduce alpha-tocopherol radicals etc. [40]. In our study (Fig. 1), free and bound phenolic-rich extracts of A. paniculata revealed considerable amounts of flavonoid and phenolic components as well as phosphomolybdenum reduction total antioxidant activities that have been reported crucial and helpful in attenuating hyperglycemia, most especially in type II DM [41, 42].

More so, according to literatured evidence [43], a significant reduction in body weight has strongly been associated with DM, due to depletion/degradation of structural proteins in order to make available amino acids for gluconeogenetic pathway during insulin deficiency [44, 45]. Sufficiently enough, in the case of this study (Fig. 2), a significant loss in body weight was seen in alloxan-induced diabetic control rats. However, administration of free and bound phenolic extracts of A. paniculata meaningfully demonstrated a reversal in weight loss. This observation suggests the enriching effect of the polyphenolic-rich extracts of A. paniculata to possibly have enhanced proper utilization of glucose and thereby causing reduction in protein breakdown [45].

Similarly, blood and various components play a crucial role in homeostasis [46]. However, a substantial decrease was noted in the blood parameters of alloxan-induced diabetic control (Table 1). Though, possible mechanism by which hyperglycemia could result in altered RBC is yet unknown. However, documented literatures have implicated ROS occasioned by glucose enolisation as one of the possible factors underlie diabetic anaemia, a secondary disorder in DM [47]. Also, a reduction in RBC and PCV could be attributed to an increase in non-enzymatic glycosylation of RBC membrane proteins due to the damaging effect of ROS which may also be accompanied by a fall in Hb [47, 48]. Alteration in hematologic profile has been marked as indication of severe diseases and disorders [46, 49]. It is evident in this study that, administration of phenolic-rich extracts of A. paniculata triggered improved hematological parameters such RBC, PCV and Hb, probably due to the ability of the extracts to inhibit proliferation of ROS that has been implicated in diabetic anaemia [50]. Also, it suggests that the extracts could possibly stimulate the secretion of erythropoietin in the bone marrow (stem cells) to form red blood cells in the treated diabetic rats [51] and as well aids restoration of oxygen carrying capacity of the blood [52].

Hepatocellular injury commonly measured by increased activities of serum liver-bound biomarkers (proteins) following insulin resistance is an important consideration in DM [53]. Aminotransferases (AST and ALT) involves in the catalysis of amino-transfer reactions [54], while ALP is responsible for phosphate group transfer from nucleotides and proteins [54]. Leakage of these enzymes from hepatic cytosol into the blood stream, has been recognized as an indication of liver damage [55]. In this study, increase in serum activities of these biomarker proteins which probably signals liver necrotic was seen in the untreated diabetic control rats (Table 2). Previous studies have reported similar observation [56, 57]. However, a reversal to these proteins efflux was noted among groups administered phenolic-rich extracts of A. paniculata (Table 2). This suggests that, phenolic-rich extracts of A. paniculata could be hepatoprotective via ameliorating hepatic injury and/or restore cellular permeability and thereby reducing lethal effect of liver toxicity and as well preventing leakage of liver-localized enzymes [53, 58].

Increase in plasma cholesterol, triglycerides (TG) and decrease in HDL has frequently been reported to accompany hyperglycemia [59]. According to the findings of this study, TC and TG levels were observed increased in diabetic untreated control with a decrease in HDL level (Fig. 3). A recent report has shown that, excessive production of free fatty acids from adipose tissue during hyperglycemia is associated with the activation of hormone-sensitive lipase (HSL) [60], which promotes generation of phospholipids and cholesterol in the liver and subsequently their release in form lipoproteins in the serum [61]. A double- fold increase in de novo synthesis of cholesterol has also been implicated in diabetic state [62]. However, a decrease in TC and TG levels observed following treatment with phenolic-rich extracts of A. paniculata, might be due to the ability of the extracts to cause inhibition of the hormone–sensitive lipase (EC 3.1.1.79) in the adipose tissue [63], thereby hindering the mobilization of fatty acid from the adipose tissues in to the serum [64].

Hyperglycemia-induced ROS generation has been implicated in the cellular antioxidant defense depletion in DM [14, 65]. In this study (Fig. 4 (i-iii)), diabetic control rats revealed a decrease in the activities of different enzymatic antioxidant profile such as SOD, CAT, GPx and total thiol with an increase in the levels of generated MDA (Fig. 5 ii). The observed reduction in antioxidant defense mechanism may be due to direct inactivation of these enzymes by hyperglycemia triggered oxidative stress [66]. More so, a direct glycation of enzymes such as SOD anion which causes inactivation of CAT, that is involved in the detoxification of hydrogen peroxide has been reported in DM [67, 68]. Thiols constitute a major portion of the total cellular antioxidant system and play a significant role in defense against ROS [69]. Glutathione (GSH) and free thiol (SH) groups present on albumin form a major part of total thiol pool [70]. GSH participates in the cellular defense against oxidative stress by scavenging free radicals and ROS intermediates [69]. However, a decrease in serum thiols have been established in both forms of DM, due to several factors that include; metabolic-, inflammatory alterations, among others [71]. Whereas, in this study Figs. 4 (i-iii) and 5 (i), administration of phenolic-rich extracts of A. paniculata demonstrated anti-oxidative effect as it’s evident by the improved enzymatic antioxidant, total thiols status as well decreased level of generated MDA (Lipid peroxidation). This observation suggests that phenolic-rich extracts of A. paniculata could probably have exhibited protection on hepatic tissue in alloxan-induced hyperglycemia by scavenging the generated free radicals [72, 73], thereby averting deleterious effect of ROS, giving credence to the presence of phytochemical constituents revealed in the Fig. 1. This finding corroborates the earlier report of Sivakumar and Rajeshkumar [65].

In type II diabetes, skeletal muscle uptake of glucose is greatly impaired thus, resulting in hyperglycemia [74]. In this study (Fig. 6), the progressive reduction in serum glucose levels observed after treatment with phenolic-rich extracts of A. paniculata suggests glucose homeostasis enhancing ability of the extracts. This finding correlates with the earlier report of Ajiboye et al. [75]. Insulin aids proper glucose homeostasis by stimulating glucose transport into muscle and adipose cells, thereby reducing hepatic production of glucose and maintaining normoglycemia [76]. The chronic exposure of β-cells to hyperglycemia has been highlighted in the progressive loss of β-cells, deterioration of function and possibly β-cell failure leading to insulin deficiency [77]. Deficiency in serum insulin has been implicated in the activation of hepatic gluconeogenic enzymes during DM [76]. The administration of phenolic-rich extracts of A. paniculata prompted an increase in serum insulin level (Fig. 7). This suggests that phenolic extracts of A. paniculata could possibly possess ability to initiate regeneration of β-cells in Langerhans islet and thereby causing an increase in pancreatic insulin secretion and action [78, 79].

Hexokinase is an important enzyme in the oxidative phosphorylation of glucose to glucose-6-phosphate during the catabolism of glucose [80]. However, studies have reported a decrease in hexokinase activity during DM [81]. In our study (Fig. 8), administration of phenolic-rich extracts of A. paniculata improved the activity of this rate limiting and ATP-requiring enzyme possibly by stimulating proper glucose uptake by peripheral tissues, thus enhancing glycolytic processes [81–83]. Similarly, latest report has shown an increase in the activity of glucose-6-phosphatase (G6P) enzyme which catalyzes the terminal step in both gluconeogenesis and glycogenolysis in hyperglycemic state [84]. However, diabetic groups administered free and bound phenolic extracts of A. paniculata revealed a reduction in the activity of G6P (Fig. 8). This observation suggests that the extracts could possibly have inhibited gluconeogenesis and glycogenolysis, thereby promoting glycolysis or by modulating the activities of these enzymes through the regulation of cyclic adenosine monophosphate (cAMP) [85].

Direct link with different roles of inflammation in metabolic disorder has recently been elucidated with an elevated levels of pro-inflammatory markers such as TNFα and IL-6 [86]. Elevated serum levels of these markers were observed in the alloxan-induced diabetic rats (Fig. 9), which is in agreement with a report by AlAmri et al. [87]. This observation perhaps could be the result of chronic hyperglycemia-induced oxidative stress or macrophage stimulation by high glucose, resulting in insulin resistance and ultimate progression of hyperglycemia [88]. However, administration of phenolic-rich extracts of A. paniculata mitigated progressive inflammatory roles of these markers which could be a direct biological response to the attenuated free radicals generated in alloxan-induced hyperglycemia (Fig. 5 ii) [89], this finding however, corroborates the earlier report of Low et al. [90].

Conclusion

The findings of this study showed that administration of phenolic-rich leaf extracts of A. paniculata markedly improved parameters assayed for in alloxan-induced diabetic rat and this observation was found comparable with glibenclamide. It therefore suggests that, phenolic-rich leaf extracts of A. paniculata might have demonstrated anti-hyperglycemic effect via enhancing glycolytic enzymes activities, insulin levels and mitigating oxidative stress as well as causing reduction in the levels of pro-inflammatory biomarkers such TNFα and IL-6. However, free phenolic extract of A. paniculata revealed a promising antidiabetic effects than bound phenolic extract in most parameters evaluated probably due to its high antioxidant contents.

Funding

The authors hereby declared no funding/grant was received for this study either from governmental/NGO or any foreign bodies.

Data availability

They will be provided on request.

Compliance with ethical standards

Conflict of interest

The authors of the manuscript declare no conflict of interest concerning the work.

Ethical approval

This study was approved with ethical code: 16/SCI03/1003.

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Code availability

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