Inhibition of key enzymes linked to type 2 diabetes and sodium nitroprusside-induced lipid peroxidation in rat pancreas by water extractable phytochemicals from some tropical spices

Abstract

Context: Spices have been used as food adjuncts and in folklore for ages. Inhibition of key enzymes (α-amylase and α-glucosidase) involved in the digestion of starch and protection against free radicals and lipid peroxidation in pancreas could be part of the therapeutic approach towards the management of hyperglycemia and dietary phenolics have shown promising potentials.

Objective: This study investigated and compared the inhibitory properties of aqueous extracts of some tropical spices: Xylopia aethiopica [Dun.] A. Rich (Annonaceae), Monodora myristica (Gaertn.) Dunal (Annonaceae), Syzygium aromaticum [L.] Merr. et Perry (Myrtaceae), Piper guineense Schumach. et Thonn (Piperaceae), Aframomum danielli K. Schum (Zingiberaceae) and Aframomum melegueta (Rosc.) K. Schum (Zingiberaceae) against α-amylase, α-glucosidase, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and sodium nitroprusside (SNP)-induced lipid peroxidation in rat pancreas - in vitro using different spectrophotometric method.

Materials and methods: Aqueous extract of the spices was prepared and the ability of the spice extracts to inhibit α-amylase, α-glucosidase, DPPH radicals and SNP-induced lipid peroxidation in rat pancreas - in vitro was investigated using various spectrophotometric methods.

Result: All the spice extracts inhibited α-amylase (IC₅₀ = 2.81–4.83 mg/mL), α-glucosidase (IC₅₀ = 2.02–3.52 mg/mL), DPPH radicals (EC₅₀ = 15.47–17.38 mg/mL) and SNP-induced lipid peroxidation (14.17–94.38%), with the highest α-amylase & α-glucosidase inhibitory actions and DPPH radical scavenging ability exhibited by X. aethiopica, A. danielli and S. aromaticum, respectively. Also, the spices possess high total phenol (0.88–1.3 mg/mL) and flavonoid (0.24–0.52 mg/mL) contents with A. melegueta having the highest total phenolic and flavonoid contents.

Discussion and conclusion: The inhibitory effects of the spice extracts on α-amylase, α-glucosidase, DPPH radicals and SNP-induced lipid peroxidation in pancreas (in vitro) could be attributed to the presence of biologically active phytochemicals such as phenolics and some non-phenolic constituents of the spices. Furthermore, these spices may exert their anti-diabetic properties through the mechanism of enzyme inhibition, free radicals scavenging ability and prevention of lipid peroxidation.

Keywords::

• α-Amylase
• α-glucosidase
• lipid peroxidation
• pancreas
• tropical spices
• inhibition

Introduction

Type 2 diabetes is regarded as the second most common noncommunicable disorder, after hypertension, in terms of public health significance (WHO, 1999). The high burden of diabetes mellitus (DM) in Nigeria is largely attributable to cardiovascular diseases which account for 15% of all DM deaths (Ogbera, 2007; Ogbera et al., 2007). Type 2 diabetes is a metabolic disorder resulting from the body’s inability to produce enough or properly utilize insulin. Type 2 diabetes patients have hyperglycemia but are ketosis resistant (WHO, 1999). Previous studies indicate that hyperglycemia-induced vascular dysfunction may be triggered by reactive oxygen species (ROS) produced in the mitochondrial electron transport chain (Kawamura et al., 1994; Brownlee, 2005). ROS such as the superoxide anion radical (O₂) hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH) are physiological metabolites formed as a result of respiration in aerobic organisms. Uncontrolled generated ROS are very unstable, and react rapidly with other substances including DNA, membrane lipids and proteins. In humans, this is believed to be involved with DM (Niedowicz & Daleke, 2005).

Starch is the major source of carbohydrates in most diets and plays a crucial role in energy supply. The dietary carbohydrates are first broken down to monosaccharides by some gastrointestinal enzymes, since only monosaccharides can be absorbed from intestinal lumen (Abesundara et al., 2004; Kwon et al., 2006). α-Glucosidase and α-amylase are the key enzymes involved in the digestion of carbohydrates (Ali et al., 2006). α-Amylase degrades complex dietary carbohydrates to oligosaccharides and disaccharides that are ultimately converted into monosaccharides by α-glucosidase. Liberated glucose is then absorbed by the gut and results in postprandial hyperglycemia (Kim et al., 2000; Shim et al., 2003). The inhibition of enzymes involved in the digestion of carbohydrates can significantly decrease the postprandial increase of blood glucose after a mixed carbohydrate diet by delaying the process of carbohydrate hydrolysis and absorption (Kwon et al., 2006; Oboh et al., 2010a). The control of postprandial hyperglycemia is an important strategy in the management of DM, especially type II diabetes, and reducing chronic complications associated with the disease (Kim et al., 2000; Ali et al., 2006). This is done by retarding the absorption of glucose through the inhibition of the carbohydrate-hydrolysing enzymes α-glucosidase and α-amylase in the digestive tract (Kim et al., 2000; Shim et al., 2003; Oboh et al., 2010a). Inhibitors of these enzymes could cause delay in carbohydrate digestion, prolong overall carbohydrate digestion time, reduce the rate of glucose absorption and consequently blunting the postprandial plasma glucose rise (Bhadari et al., 2008). Several drugs such as acarbose, miglitol, voglibose, nojirimycin and 1-deoxynojirimycin had been developed and are currently in use. These drugs could act by either blocking or inhibiting these enzymes, however, they come with financial constraints and their attendant side effects, hence, alternative treatments need to be evaluated. Therefore, effective, nontoxic and cheap natural dietary inhibitors of α-amylase and α-glucosidase with little or no side effects could be potentially promising and highly desirable. Moreover, there is strong evidence that dietary factors could be involved in the regulation and prevention of type 2 diabetes (Kwon et al., 2007).

In recent times, research on medicinal plant and plant foods for the management of diabetes has attracted the interest of scientists (McCue et al., 2005; Ali et al., 2006) and quite a number of plants are known to exert their antihyperglycemic activity via inhibition of carbohydrate hydrolyzing enzymes (Matsumoto et al., 1993; Matsui et al., 2001; Johnston et al., 2003; McDougall et al., 2005; Cheplick et al., 2007; Oboh et al., 2010a; Pinto et al., 2010; Ranilla et al., 2010). Many of the health-promoting characteristics of plant food are partly linked to the presence of an array of important phenolic phytochemicals including phenolic acids and flavonoids (Chu et al., 2002; Sun et al., 2002; Cheplick et al., 2007; Oboh & Rocha, 2007; Oboh et al., 2008, 2010a,b; Wang et al., 2010). Phenolic compounds or phenolic phytochemicals are secondary metabolites of plants and are present in diets (Bravo, 1998). They exhibit strong antioxidant, free radical scavenging activity, and play crucial role in health promotion and disease prevention (Scalbert et al., 2005; Oboh & Rocha, 2007). Spices are esoteric food adjuncts that have been used as flavoring agents and as preservatives for thousands of years. Spices have also been recognized to possess medicinal properties and their use in traditional systems of medicine has been on record for a long time. This study seeks to evaluate the inhibitory effects of the water extractable phytochemicals from some tropical spices [Monodora myristica (Africa nutmeg), Xylopia aethiopica (Ethiopian pepper), Syzygium aromaticum (Cloves), Piper guineense (Black pepper), Aframomum danielli (Bastered melegueta) and Afromomum melegueta (Alligator pepper or grains of paradise)] on some key enzymes (α-amylase and α-glucosidase) linked with type 2 diabetes as well as their inhibitory effect on sodium nitroprusside (SNP)-induced lipid peroxidation in rat pancreas with the aim of investigating their potential as dietary intervention in the management or control of postprandial hyperglycemia associated with type 2 diabetes.

Materials and methods

Materials

Fresh samples of some tropical spices (M. myristica, X. aethiopica, S. aromaticum, P. guineense, A. danielli and A. melegueta) were purchased from Akure main market in Ondo State, Nigeria. Authentication of the samples was carried out by Dr. A. E Ayodele, a plant taxonomist in the Department of Botany and Microbiology, University of Ibadan, Oyo State, Nigeria. The different parts of the spices used for the analyses are described in Table 1. All the chemicals used were of analytical grade, while the water was glass distilled.

Table 1.  Description of some commonly consumed Nigerian species.

Sample & code	Common name	Part used
Xylopia aethiopica (XA)	African/ethiopia pepper	Fruit
Piper guineense (PG)	Ashanti/black pepper	Seed
Aframomum melegueta (AM)	Alligator pepper or grains of paradise	Seed
Monodora myristica (MM)	African nutmeg	Seed
Aframomum danielli (AD)	Bastered melegueta	Seed
Syzygium aromaticum (SA)	Clove	Bud

Handling and use of animal

Approval was obtained from the relevant University ethics committee responsible for the use of laboratory animals. The handling and the use of the animals were in accordance with NIH Guide for the care and use of laboratory animals. In the experiment, Wistar strain albino rats weighing 200–230 g were purchased from the breeding colony of Department of Veterinary Medicine, University of Ibadan, Nigeria. Rats were maintained at 25°C, on a 12 h light/12 h dark cycle, with free access to food and water. They were acclimatized under these conditions for two weeks before the commencement of the experiment.

Aqueous extract preparation

The inedible portions of the seeds, fruits, flower and leaves were removed from the edible portions; the edible portions were subsequently washed in distilled water. The fruits, flower and leaves were chopped into small pieces by table knife, air dried and milled. Each milled sample was soaked in 100 mL distilled water for about 24 h. The mixture was filtered and later centrifuged at 400g for 10 min to obtain a clear supernatant which was then used for subsequent analysis (Oboh et al., 2007).

α-Amylase inhibition assay

The aqueous extracts dilution (500 µL) of spices and 500 µL of 0.02 mol/L sodium phosphate buffer (pH 6.9 with 0.006 mol·L⁻¹ NaCl) containing hog pancreatic α-amylase (EC 3.2.1.1) (0.5 mg/mL) were incubated at 25°C for 10 min. Then, 500 µL of 1% starch solution in 0.02 mol·L⁻¹ sodium phosphate buffer (pH 6.9 with 0.006 mol/L NaCl) was added to the reaction mixture. Thereafter, the reaction mixture was incubated at 25°C for 10 min and stopped with 1.0 mL of dinitrosalicylic acid color reagent. The mixture was then incubated in a boiling water bath for 5 min, and cooled to room temperature. The reaction mixture was then diluted by adding 10 mL of distilled water, and absorbance measured at 540 nm in the UV–Visible spectrophotometer (Model 6305; Jenway, Barloworld Scientific, Dunmow, United Kingdom). The α-amylase inhibitory activity was expressed as percentage inhibition (Bernfield, 1951).

Where,

Abs_ref = Absorbance of reference

Abs_sam = Absorbance of sample

α-Glucosidase inhibition assay

Appropriate dilution of the aqueous extracts (50 µL) of spices and 100 µL of α-glucosidase solution (1.0 U/mL) in 0.1 mol/L phosphate buffer (pH 6.9) were incubated at 25°C for 10 min. Thereafter, 50 µL of 5 mmol/L p-nitrophenyl-α-d-glucopyranoside solutions in 0.1 mol/L phosphate buffer (pH 6.9) was added. The reaction mixture was then incubated at 25°C for 5 min, and then absorbance was measured at 405 nm in the UV–Visible spectrophotometer (Model 6305; Jenway, Barloworld Scientific, Dunmow, United Kingdom). The α-glucosidase inhibitory activity was expressed as percentage inhibition (Apostolidis et al., 2007).

Where,

Abs_ref = Absorbance of reference

Abs_sam = Absorbance of sample

Lipid peroxidation assay

Preparation of tissue homogenates

The rats were decapitated under mild diethyl ether anaesthesia and the pancreas was rapidly excised, placed on ice and weighed. This tissue was subsequently homogenized in cold saline (1/10, w/v) with about 10 up and down strokes at approximately 1200 rpm in a Teflon glass homogenizer. The homogenate was centrifuged for 10 min at 3000× g to yield a pellet that was discarded, and a low-speed supernatant (S1) containing mainly water, proteins and lipids (cholesterol, galactolipid, individual phospholipids, gangliosides) was kept for lipid peroxidation assay (Belle et al., 2004).

Lipid peroxidation and thiobarbituric acid reactions

The lipid peroxidation assay was carried out by the modified method of Ohkawa et al. (1979). Briefly, 100 µL of S1 fraction was mixed with a reaction mixture containing 30 µL of 0.1 M Tris-HCl buffer (pH 7.4), extract (0–100 µL) and 30 µL of the pro-oxidant (7 µM sodium nitroprusside). The volume was made up to 300 µL by water before incubation at 37°C for 2 h. The color reaction was developed by adding 300 µL of 8.1% sodium duodecyl sulphate to the reaction mixture containing S1, followed by the addition of 600 µL of acetic acid/HCl (pH 3.4) and 600 µL of 0.8% thiobarbituric acid. This mixture was incubated at 100°C for 1 h. The absorbance of thiobarbituric acid reactive species produced were measured at 532 nm in UV–Visible spectrophotometer (Model 6305; Jenway, Barloworld Scientific, Dunmow, United Kingdom). Malondialdehyde (MDA) produced was expressed as % Control.

Phytochemical screening

The methods described by Trease and Evans (1983) were used for phytochemical screening of spice extracts for the presence of bioactive compound.

Determination of total phenol content

The total phenol content was determined on the extracts using the method reported by Singleton et al. (1999). Appropriate dilutions of the extracts were oxidized with 2.5 mL of 10% Folin–Ciocalteau’s reagent (v/v) and neutralized by 2.0 mL of 7.5% sodium carbonate. The reaction mixture was incubated for 40 min at 45°C and the absorbance was measured at 765 nm in the UV–Visible spectrophotometer (Model 6305, Barloworld Scientific, Dunmow, Essex, United Kingdom). The total phenol content was subsequently calculated using gallic acid as standard.

Determination of total flavonoid content

The total flavonoid content of the spice extracts was determined using a slightly modified method reported by Meda et al. (2005). Briefly, 0.5 mL of appropriately diluted sample was mixed with 0.5 mL methanol, 50 µL of 10% AlCl₃, 50 µL of 1 mol/L potassium acetate and 1.4 mL water, and allowed to incubate at room temperature for 30 min. Thereafter, the absorbance of the reaction mixture was subsequently measured at 415 nm in the Jenway UV–Visible spectrophotometer. The total flavonoid was calculated using quercetin as standard.

1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging ability

The free radical scavenging ability of the spice extracts against DPPH free radical was evaluated as described by Gyamfi et al. (1999). Briefly, an appropriate dilution of the spice extracts (1 mL) was mixed with 1 mL of 0.4 mmol/L methanol solution containing DPPH radicals. The mixture was left in the dark for 30 min and the absorbance was measured at 516 nm in the UV–Visible spectrophotometer (Model 6305; Jenway, Barloworld Scientific, Dunmow, United Kingdom). The DPPH free radical scavenging ability was subsequently calculated with respect to the reference, which contained all the reagents without the test sample.

Where,

Abs_ref = Absorbance of reference

Abs_sam = Absorbance of sample

Data analysis

The result of the replicates were expressed as mean ± standard deviation (Zar, 1984). A one-way analysis of variance (ANOVA) and the least significance difference were carried out. Significance was accepted at p ≤ 0.05.

Results and discussion

The control of postprandial hyperglycemia is recognized to be an effective therapeutic approach in the management of type 2 diabetes and in reducing chronic vascular complications (Ortiz-Andrade et al., 2007). Hence, inhibition of enzymes involved in the digestion of polysaccharides by dietary phytochemicals has shown promising potentials (Shim et al., 2003; Pinto et al., 2009; Oboh et al., 2010a).

α-Amylase and α-glucosidase are key enzymes involved in starch breakdown and intestinal absorption, respectively. In humans, the diabetogenic process may be caused by immune destruction of the β cells in the Islets of Langerhans in the pancreas and this is apparently mediated by white blood cell production of ROS (Oberley, 1988). Diabetes can be induced in animals by the drugs alloxan and streptozotocin; the mechanism of action of these two drugs is different, but both result in the production of ROS. Scavengers of oxygen radicals are effective in preventing diabetes in these animal models. Not only are oxygen radicals involved in the cause of diabetes, they also appear to play a role in some of the complications seen in long-term treatment of diabetes (Oberley, 1988). It is believed that inhibition of the enzymes involved in the digestion and uptake of carbohydrates can significantly decrease the postprandial increase of blood glucose level after a mixed carbohydrate diet and therefore can be an important strategy in the management of hyperglycemia linked to type 2 diabetes (Shim et al., 2003; Ortiz-Andrade et al., 2007; Pinto et al., 2009). The ability of aqueous extract of some commonly consumed spices in Nigeria to inhibit α-amylase activity was investigated (Figure 1) and the result revealed that all the extracts inhibited α-amylase in a concentration-dependent manner (0–3 mg/mL); with the 50% inhibition concentration by (IC₅₀) values shown in Table 2. However, X. aethiopica extract had the highest inhibitory activity on α-amylase (IC₅₀ = 2.81 mg/mL), while A. melegueta extract showed the least inhibition against α-amylase (IC₅₀ = 4.83 mg/mL). The trend in the determined α-amylase inhibitory activity followed similar trend with some earlier reports where plant phytochemicals in six Allium species inhibited α-amylase activity (Nickavar & Yousefian, 2009). The result agrees with our recent work, where red and white ginger inhibited α-amylase activity in vitro (Oboh et al., 2010a).

Figure 1.  α-Amylase inhibitory activities of aqueous extracts of some tropical spices. Values represent mean ± standard deviation, n = 3. AD, A. danielli; AM, A. melegueta; MM, M. myristica; PG, P. guineense; SA, S. aromaticum; XA, X. aethiopica.

Subsequently, α-glucosidase inhibitory activities of the spice extracts was assessed and shown in Figure 2. All the spice extracts inhibited α-glucosidase activities in a concentration-dependent manner (0–3 mg/mL). However, the highest inhibitory activity on α-glucosidase was shown by A. danielli (IC₅₀ = 2.02 mg/mL), while M. myristica had the least α-glucosidase inhibitory activity (IC₅₀ = 3.52 mg/mL). Also, the result of α-glucosidase inhibitory activities of spice extracts follow similar trend with recent reports on the inhibitory potentials of commonly used medicinal plants, herbs and spices in Latin America against key enzymes relevant to hyperglycemia (Ranilla et al., 2010) and that of common constituents from some traditional Chinese medicine used for DM (Ye et al., 2010).

Table 2.  EC₅₀ /IC₅₀ values of DPPH radical scavenging ability, α-amylase and α-glucosidase inhibitory activity of aqueous extract of some tropical spices

Samples	EC50 of DPPH radical scavenging ability (mg/mL)	IC50 of α-amylase inhibitory activity (mg/mL)	IC50 of α-glucosidase inhibitory activity (mg/mL)
XA	16.77 ± 2.10	2.81 ± 0.79	2.57 ± 1.15
PG	17.05 ± 3.70	3.23 ± 1.05	2.49 ± 0.96
MM	16.88 ± 1.70	3.80 ± 1.00	3.52 ± 1.22
AM	17.38 ± 2.00	4.83 ± 0.56	2.14 ± 1.08
AD	16.39 ± 2.50	4.30 ± 1.04	2.02 ± 1.13
SA	15.47 ± 2.25	3.34 ± 0.95	2.69 ± 0.75

Values represent mean ± standard deviation, number of samples n = 3.

AD, A. danielli; AM, A. melegueta; MM, M. myristica; PG, P. guineense; SA, S. aromaticum; XA, X. aethiopica.

Figure 2.  α-Glucosidase inhibitory activities of aqueous extracts of some tropical spices. Values represent mean ± standard deviation, n = 3. AD, A. danielli; AM, A. melegueta; MM, M. myristica; PG, P. guineense; SA, S. aromaticum; XA, X. aethiopica.

Furthermore, it is worth noting that all the spice extracts showed higher α-glucosidase inhibitory activities (IC₅₀ = 2.02–3.52 mg/mL) than their respective α-amylase inhibitory activities (IC₅₀ = 2.81-4.83 mg/mL). This agrees with previous reports that plant phytochemicals are mild inhibitors of α-amylase and strong inhibitors of α-glucosidase activities (McDougall et al., 2005; Kwon et al., 2007; Pinto et al., 2009; Oboh et al., 2010a). This action may pose an advantage over synthetic drugs presently in use for the management of postprandial blood glucose, such as acarbose, which strongly inhibit α-amylase. There are indications that excessive pancreatic amylase inhibition could result into abnormal bacterial fermentation of undigested saccharides in the colon (Horii et al., 1986; Bischoff, 1994). Hence, stronger inhibition of α-glucosidase activity and mild inhibition of α-amylase activity of the spice extracts could minimize the major setback of currently used α-glucosidase and α-amylase inhibitory drugs with side effects such as abdominal distention, flatulence, meteorism and possibly diarrhea (Bischoff, 1994). Therefore, this study supports the assertion that natural inhibitors from some plant foods have lower inhibitory effect against α-amylase activity and stronger α-glucosidase inhibitory activity, and could be effectively used in the management of postprandial hyperglycemia with minimal side effects (Kwon et al., 2007; Oboh et al., 2010a; Pinto et al., 2010).

Increased oxidative stress is widely accepted participant in the development and progression of diabetes (Maritim et al., 2003). Although, the mechanisms by which increased oxidative stress is involved in the diabetic complications are partly known, including glucose oxidation, non-enzymatic glycation of proteins, and the subsequent oxidative degradation of glycated proteins (Maritim et al., 2003). Abnormally high levels of free radicals and the simultaneous decline of antioxidant defense mechanisms could lead to damage of cellular organelles and enzymes, increased lipid peroxidation and development of insulin resistance. Insulin resistance is a major contributor to progression of type 2 DM and its complications (Weyer et al., 2001). This could have originated from certain oxidative stress related defect(s) in oxidative phosphorylation machinery and mitochondrial β-oxidation leading to excess accumulation of intracellular triglyceride in muscle and liver and subsequently insulin resistance (Rosca et al., 2005). The pancreatic β cells are no longer able to compensate for insulin resistance because of increased insulin production and impaired glucose tolerance, characterized by excessive postprandial hyperglycemia (Gerich, 2003). This assertion is in line with the work carried out by Evans et al. (2003) that pancreatic β-cell dysfunction could result from prolonged exposure to high glucose, elevated free fatty acid levels, or a combination of both. The pancreatic β cells could be susceptible to ROS because they are low in antioxidant enzymes such as catalase, glutathione peroxidase, and superoxide dismutase. Therefore, the ability of the spice extracts to inhibit SNP-induced lipid peroxidation in isolated rat pancreas homogenates was carried out and presented in Figure 3. SNP is a potent and rapidly acting intravenous hypotensive agent. It is a peripheral vasodilator that acts directly on the smooth muscle fibers of blood vessels (Kaisserlian et al., 2005). This direct action is thought to be due to the release of the nitroso group in form of nitric oxide (NO). NO has also been implicated as an important signaling molecule in the contraction-mediated glucose uptake pathway. Nevertheless, SNP may also cause cytotoxicity due to the release of cyanide, which produces a real complication (Varon & Marik, 2000). Incubation of the rat pancreas in the presence of 7 µM SNP (induced) caused a significant increase (p < 0.05) in the MDA content of the pancreas (122.50%) as presented in Figure 3. However, the introduction of the water extractable phytochemicals from the spices caused a significant decrease (p < 0.05) in the MDA content of sodium nitroprusside-stressed pancreas homogenates (14.17–94.38%) in a concentration-dependent manner (0.16–0.96 mg/mL) with Aframomum melegueta showing the highest inhibition of MDA production, while X. aethiopica had the least inhibition of MDA production in a concentration-dependent manner. The result of the inhibition of MDA production agrees with Oboh et al. (2010a), where red and white ginger extract inhibited some prooxidants induced lipid peroxidation in rat brain. The inhibition of SNP-induced lipid peroxidation in pancreas tissues by the spice extracts (Figure 3) could be attributed to the ability of the extractable phytochemicals to scavenge NO˙ radical produced by the SNP, thus protecting the pancreas against oxidative insults and possibly upregulating the antioxidant enzymes.

Figure 3.  Inhibition of sodium nitroprusside (SNP) induced lipid peroxidation in rat pancreas by aqueous extracts of some commonly consumed spices. Values represent mean ± standard deviation, n = 3; Basal = lipid peroxidation without SNP as prooxidants and no spice extracts; Induced = Lipid peroxidation with SNP as prooxidants and no spice extracts. AD, A. danielli; AM, A. melegueta; MM, M. myristica; PG, P. guineense; SA, S. aromaticum; XA, X. aethiopica.

Free radical scavenging is one of the known mechanisms by which antioxidants inhibit lipid oxidation and DPPH is frequently used in its determination. The free radical DPPH possesses a characteristic absorption at 517 nm (purple in color), which decreases significantly on exposure to radical-scavengers (by providing hydrogen atoms or by electron donation). Therefore, the capacity of the spice extracts to scavenge the ‘‘stable’’ free radical DPPH radical was monitored and shown in Figure 4. The extract concentration causing 50% inhibition (EC₅₀) values obtained from the dose-response curve (Table 2) revealed that S. aromaticum had the highest DPPH scavenging effects (15.47 mg/mL), while the least effect was exhibited by A. melegueta (17.38 mg/mL). The trend in the results of the ability of the individual spice extracts to inhibit the key enzymes linked to type 2 diabetes did not follow a similar trend with that of the spices in scavenging the DPPH radicals.

Figure 4.  DPPH free radical scavenging ability of aqueous extract of some tropical spices. Values represent mean ± standard deviation, n = 3. AD, A. danielli; AM, A. melegueta; MM, M. myristica; PG, P. guineense; SA, S. aromaticum; XA, X. aethiopica.

The presence of phytochemicals and phenolic compounds has been shown to contribute immensely to the protective potential against degenerative diseases, therapeutic effects essential to preventing diseases and nutritional quality of food and food products (Chu et al., 2002; Oboh et al., 2010b). Thus, the preliminary phytochemical screening tests may be useful in the detection of the bioactive principles and could subsequently lead to the development of functional foods and nutraceuticals. In order to ascertain the phytochemicals responsible for the biological activities of these spices in vitro, the spices were screened for the presence of various phytochemicals. The result of the phytochemical screening as shown in Table 3 revealed the presence of flavonoid and cardiac glycoside in all the spices tested, tannin in X. aethiopica and S. aromaticum, phlobatanin, and anthraquinone in S. aromaticum and saponin in all spices under investigation (except in M. myristica). Previous studies have shown that these spices are rich in phenols and flavonoids, hence their disease preventing and health promoting abilities have been attributed to the presence of these phytochemicals (Fasoyiro et al., 2006; Olonisakin et al., 2006; Wojdyło et al., 2007; Uwakwe & Nwaoguikpe, 2008; Ezekwesili et al., 2010; Doherty et al., 2010). Dietary polyphenols are the most abundant antioxidants in human diets and represent a group of secondary metabolites which widely occur in fruits, vegetables, spices, wine, tea, extra virgin olive oil, chocolate and other cocoa products. They exhibit many biologically significant functions, such as protection against oxidative stress, and degenerative diseases (Bravo, 1998; Scalbert et al., 2005). Phenolic acids account for about one third of the total intake and flavonoids account for the remaining two thirds. Flavonoids are the most abundant polyphenols in human diets and flavanols (catechins plus proanthocyanidins), anthocyanins and their oxidation products have been shown to be the most abundant flavonoids in the diet (Scalbert et al., 2005). Phenolic compounds can remove free radicals, chelate metal catalysts, activate antioxidant enzymes, and reduce ions and radicals (Amic et al., 2003). The result of the total phenol content of spices is presented in Table 4. A. melegueta had the highest total phenol content (2.28 mg/g), followed by A. danielli (1.65 mg/g) and M. myristica (0.60 mg/g) having the least total phenol content. These values were generally higher than the phenolic content reported for red and white ginger (Oboh et al., 2010a) except M. myristica that was lower. Also, the values were generally higher than that reported by Oboh et al. 2007 for hot pepper. In the same vein, the total flavonoid contents of the spices are also shown in Table 4 with A. melegueta (0.55 mg/g) having the highest flavonoid content, followed P. guineense (0.41 mg/g), A. danielli (0.29 mg/g), S. aromaticum (0.26 mg/g), X. aethiopica (0.24 mg/g) and M. myristica (0.21 mg/g). The result of the phytochemical screening alongside the total phenol and total flavonoids contents showed clearly that all the spices tested are rich sources of polyphenols and flavonoids; which of late have aroused great interest owing to their antioxidant activities and enzyme inhibitory properties.

Table 3.  Phytochemical constituents of some tropical spices.

Spices	Alkaloids	Saponins	Tannins	Phlobatanins	Anthraquinones	Flavonoids	Cardiac glycosides
							Legal	Liebamans	Salkowki	Keller killani
XA	−	+	+	−	−	+	+	+	+	+
PG	−	+	−	−	−	+	+	−	+	+
MM	−	−	−	−	−	+	+	−	+	−
AM	−	+	−	−	−	+	+	−	+	+
AD	−	+	−	−	−	+	+	−	+	−
SA	−	+	+	+	+	+	+	−	+	+

+ signifies present; - signifies absent.

AD, A. danielli; AM, A. melegueta; MM, M. myristica; PG, P. guineense; SA, S. aromaticum; XA, X. aethiopica.

Table 4.  Phenolic distribution of some tropical spices (mg/g).

Sample	Total phenol	Total flavonoid
Xylopia aethiopica (XA)	1.38 ± 0.20	0.24 ± 0.03
Piper guineense (PG)	1.25 ± 0.12	0.41 ± 0.05
Monodora myristica (MM)	0.60 ± 0.09	0.21 ± 0.05
Aframomum melegueta (AM)	2.28 ± 0.02	0.55 ± 0.00
Aframomum danielli (AD)	1.65 ± 0.05	0.29 ± 0.07
Syzygium aromaticum (SA)	0.88 ± 0.30	0.26 ± 0.07

Note: + signifies present; − signifies absent.

Conclusions

The results of the enzyme (α-amylase and α-glucosidase) inhibitory assays and that of the antioxidant assays did not follow a similar trend with the phenolic contents of the spices. The high inhibitory effect of aqueous extracts of the spices on α-amylase and α-glucosidase activities could be attributed to the presence of some non-phenolic phytochemicals that are potent enzyme inhibitors, exhibiting an additive or synergistic effect with the phenolics present in the spices. Furthermore, these spices may exert their anti-diabetic properties through the mechanism of enzyme inhibition, free radicals scavenging ability and prevention of lipid peroxidation.

Declaration of interest

The authors report no conflicts of interest.