Accepted by the Acta Poloniae Pharmaceutica Drug Research
3.2 Phytochemistry, anti-oxidative and anti-diabetic effects of various solvent fractions from fruit ethanolic extract of
Aframomum melegueta in vitro
Aminu Mohammed,
1,3Neil Anthony Koorbanally
2and Md. Shahidul Islam
1*1Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, (Westville Campus), Durban, 4000, South Africa
2Department of Chemistry, School of Chemistry and Physics, University of KwaZulu-Natal, (Westville Campus), Durban, 4000, South Africa
3Department of Biochemistry, Faculty of Science, Ahmadu Bello University, Zaria-Nigeria
3.2.1 Abstract
Objectives: This study was aimed to investigate the anti-oxidative potentials and α-glucosidase and α- amylase inhibitory effects of various fractions derived from crude ethanolic (EtOH) extract of A.
melegueta fruit. Additionally, possible bioactive compounds from the fraction with higher activity were analyzed using GC-MS.
Methods: Fruit EtOH extract was fractionated using solvents of increasing polarity. They were subjected and investigated for 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity, ferric reducing power, inhibition of hemoglobin glycosylation, α-amylase and α-glucosidase activities as markers of in vitro anti-diabetic effects at various doses (30-240 µg/mL). Possible compounds were analyzed using Gas Chromatography-Mass Spectroscopic (GC-MS) analysis.
Results: Our findings revealed that the ethyl acetate (EtOAc) fraction had the highest anti-oxidative activities (IC50 values, DPPH: 15.49 ± 4.38 µg/mL; Hemoglobin glycosylation: 111.33 ± 6.46 µg/mL) and inhibition of α-amylase (IC50 values: 68.69 ± 6.05µg/mL) and α-glucosidase (IC50 values: 40.44 ± 5.77 µg/mL) actions compared to other fractions and the respective standards used in this study. GC- MS analysis revealed that the EtOAc fraction contained some compounds aromatic in nature.
Conclusion: Data obtained from the study suggest that the EtOAc fraction derived from the EtOH extract of A. melegueta fruit possessed potent anti-oxidative as well as α-glucosidase and α-amylase inhibitory activities than other solvent fractions.
Keywords: Aframomum melegueta, Anti-oxidative, Ethyl acetate fraction, Type 2 diabetes
3.2.2 Introduction
Type 2 diabetes (T2D) is a heterogeneous disorder characterized by insulin resistance and partially dysfunctional pancreatic β-cell to properly secrete insulin in response to elevation of blood glucose levels (Hui et al. 2007). The inability of pancreatic β-cell to secret insulin disrupts the regulation of hepatic gluconeogesis, muscles glucose uptake and lipolysis in adipose tissues. The consequence is postprandial hyperglycemia which results into T2D (Gastaldelli, 2011). Moreover, in chronic uncontrolled hyperglycemia, there is an increased production of reactive oxygen species (ROS) and a declined of in vivo anti-oxidant defense system, a term referred as oxidative stress (Maritim et al. 2003).
The ROS are derived from the normal physiological processes and become highly deleterious if the levels increases and not arrested by complex anti-oxidant systems in the body (Chang and Chuang, 2010). The most common ROS include superoxide (O2·-), hydrogen peroxide (H2O2), peroxyl (ROO·) and reactive hydroxyl (OH·) radicals (Niki, 2010). Furthermore, increase intake of anti-oxidants has good correlation in amelioration of complication caused by ROS. However, people are now aware of the high risk caused by the synthetic anti-oxidants due to their chemical instability and hence depend on plant-derived anti-oxidants for food preservations and disease controls such as T2D.
On the other hand, inhibition of the activities of carbohydrate hydrolysing enzymes such as α- glucosidase has been used as one of the recent alternatives for the treatment and control of T2D (Adefegha and Oboh, 2012). This reduces the rate of intestinal glucose absorption which apparently decreases the postprandial hyperglycemia (Bhadari et al. 2008). Currently, some synthetic α- glucosidase inhibitors such as acarbose and miglitol are available for the control of T2D. But the undesirable adverse effects with the use of these drugs cause a shift to look for options from natural sources such as plants that are to cause fewer or no adverse side effects.
Interestingly, in most of the recent studies, plant-derived extracts and/or fractions have shown potent anti-oxidant as well as inhibition of α-glucosidase activities and therefore could possibly be a potential candidates for the treatment and control of T2D (Ibrahim et al. 2014; Kong et al. 2014;
Adamson and Oboh, 2012; Adefegha and Oboh, 2012a).
Aframomum melegueta K. Schum. (Zingiberaceae) commonly known as guinea or alligator pepper is a native to west and central parts of Africa (Iwu, 2014). The seeds, leaf or fruit are consumed as flavoring agent in various traditional foods and are used locally to treat diseases such as diabetes, microbial infections, diarrhea, abdominal pain, snakebite and wounds healing in Nigeria, Kenya and Gabon (Iwu, 2014; Soladoye et al. 2012; Gbolade, 2009; Akendengue and Louis, 1994; Kokwaro, 1993). Previously, Sugita et al. (2013) have reported that A. melegueta seed alcoholic extract stimulates brown adipose tissue and increases whole-body energy expenditure in human subjects which is directly linked with the pathogenesis of T2D. Additionally, in a number of recent studies reported that various solvent extracts from A. melegueta seed have demonstrated potent anti-oxidant as well as anti-diabetic actions in vitro (Adefegha and Oboh 2012; Kazeem et al. 2012; Adefegha and Oboh 2011; Etoundi et
al. 2010) and in vivo (Onoja et al. 2014). Furthermore, in our recent study, we have reported that fruit ethanolic (EtOH) extract demonstrated higher anti-oxidant and anti-diabetic actions in vitro compared to other solvent extracts from the various parts of A. melegueta (Mohammed et al. 2015a). Therefore, in our effort to find potent anti-diabetic plant-derived formulations, we further partitioned and fractionated the crude EtOH extract from A. melegueta fruit with the hope to obtain the best fraction with highest anti-diabetic activities.
Therefore, our present study was aimed to investigate the anti-oxidative potentials and inhibitory effects of various fractions derived from crude EtOH extract of A. melegueta fruit on the activities of α-glucosidase and α-amylase enzymes. Additionally, possible bioactive compounds from the fraction with highest activities was also analysed using GC-MS.
3.2.3 Materials and methods
For sample collection, identification and preparation of the fractions, including the detail experimental protocols, please refer to chapter 2, sub-sections 2.3 and 2.4.
3.2.4 Results
The results of the percentage yield recovered, total polyphenols and flavonoids contents of various fractions from the fruit EtOH extract are presented in Table 3.4. It was observed from the data that the least polar solvent (hexane) recovered higher yield whereas the acetone, which is the most polar recovered the least amount. Similarly, the most polar fractions (ethyl acetate (EtOAc) and acetone) have demonstrated higher polyphenols and flavonoids contents compared to the less polar solvent fractions (hexane and dichloromethane). Furthermore, within the most polar, the polyphenols and flavonoids contents of the EtOAc fraction were relatively higher compared to that of acetone fraction (Table 3.4).
The total polyphenols content was in the order; EtOAc > acetone > dichloromethane > hexane.
Table 3.4. Percentage yield, total polyphenol and flavonoid contents of various fractions from ethanolic fruit extract of A. melegueta
Solvent Yield (%) Total polyphenols content (mg/gGAE)
Total flavonoids content (mg/gQE)
Hexane 17.80 1.46 ± 0.16a 0.53 ± 0.41a
Dichloromethane 10.54 3.64 ± 0.41b 0.52 ± 0.14a
Ethyl acetate 5.60 18.98 ± 0.06c 6.06 ± 0.06b
Acetone 1.70 16.21 ± 0.70c 5.02 ± 0.34b
Data are presented as mean±SD values of triplicate determinations. a-cDifferent superscripted letters for a given value within a column are significantly different from each other (Tukey’s-HSD multiple range post hoc test, p < 0.05).
The results of ferric (Fe3+) reducing total anti-oxidant power (expressed as gallic acid equivalents) of the various fractions are presented in Figure 3.8. It was observed that EtOAc fraction showed a significantly (p < 0.05) higher and concentration dependent reducing power compared to other solvent fractions as well as the standard ascorbic acid (Figure 3.8). Hexane and acetone fractions demonstrated similar Fe3+ to Fe2+ reducing abilities while dichloromethane fraction was the least active among the fractions tested (Figure 3.8).
Figure 3.8. Total reducing power (relative to gallic acid) of various fractions from fruit ethanolic extract of A. melegueta. Data are presented as mean±SD of triplicate determinations. a-d Different letters presented over the bars for a given concentration of each extract are significantly different from each other (Tukey’s-HSD multiple range post hoc test, p < 0.05).
The data of DPPH radical scavenging activities and the calculated IC50 values of various fractions from the fruit EtOH extract are presented in Figure 3.9 and Table 3.5, respectively. All the solvent fractions have shown variable scavenging abilities. The EtOAc fraction exhibited significantly (p < 0.05) lower IC50 value (15.49 ± 4.38 µg/mL) for DPPH scavenging activities compared to other solvent fractions and comparable to standards used in this study (Ascorbic acid: 25.34 ± 6.19 µg/mL;
Gallic acid: 20.01 ± 4.42 µg/mL) (Table 3.5).
Figure 3.9: DPPH Radical scavenging activity (%) of various fractions from fruit ethanolic extract of A. melegueta. Data are presented as mean±SD of triplicate determinations. a-e Values with different letters presented for a given concentration for each extract are significantly different from each other (Tukey’s-HSD multiple range post hoc test, p < 0.05).
Table 3.5: IC50 values of various solvent fractions from ethanolic fruit extract of A. melegueta in different anti-oxidative and anti-diabetic models.
Solvent
IC50 (µg/mL)