Aqueous Extracts of Roselle ( Hibiscus sabdariffa Linn.) Varieties Inhibit α -Amylase and α -Glucosidase Activities In Vitro

Abstract

This study sought to investigate the inhibitory effect of aqueous extracts of two varieties (red and white) of Hibiscus sabdariffa (Roselle) calyces on carbohydrate hydrolyzing enzymes (α-amylase and α-glucosidase), with the aim of providing the possible mechanism for their antidiabetes properties. Aqueous extracts were prepared (1:100 w/v) and the supernatant used for the analysis. The extracts caused inhibition of α-amylase and α-glucosidase activities in vitro.The IC₅₀ revealed that the red variety (25.2 μg/mL) exhibited higher α-glucosidase inhibitory activity than the white variety (47.4 μg/mL), while the white variety (90.5 μg/mL) exhibited higher α-amylase inhibitory activity than the red variety (187.9 μg/mL). However, the α-glucosidase inhibitory activities of both calyces were higher than that of their α-amylase. In addition, the red variety possessed higher antioxidant capacity as exemplified by the ^•OH scavenging abilities, Fe²⁺ chelating ability, and inhibition of Fe²⁺-induced pancreatic lipid peroxidation in vitro. The enzyme inhibitory activities and antioxidant properties of the roselle extracts agreed with their phenolic content. Hence, inhibition of α-amylase and α-glucosidase, coupled with strong antioxidant properties could be the possible underlying mechanism for the antidiabetes properties of H. sabdariffa calyces; however, the red variety appeared to be more potent.

Introduction

D iabetes mellitus is a chronic metabolic disease constituting major health problems worldwide. It has been reported that about 171 million people worldwide have diabetes and the numbers could increase to 366 million by 2030.¹ Type 2 diabetes is the most common form of diabetes; it is characterized primarily by insulin resistance in target tissues leading to hyperglycemia and ultimately malfunctions of the β-cells. Prolonged hyperglycemia could lead to increased generation of reactive oxygen species (ROS) and alteration of endogenous antioxidants.² Oxidative stress resulting from the hyperglycemic condition in type 2 diabetes has been implicated in the destruction of the β-cells and diabetes complications such as neuropathy and retinopathy. Hence, augmenting the body antioxidant status through dietary means may help reduce oxidative stress. Postprandial hyperglycemia could also induce nonenzymatic glycosylation of various proteins and biomolecules, resulting in the development of chronic complications.³ Therefore, control of postprandial hyperglycemia is critical for early treatment, management of diabetes mellitus, and reduction of chronic complications. This could be achieved through the inhibition of α-amylase and α-glucosidase, which would help prevent hyperglycemia-induced increase in free radical generation and oxidative stress associated with diabetes mellitus. However, one practical approach for the control of postprandial hyperglycemia is to slow down glucose absorption through the inhibition of carbohydrate-hydrolyzing enzymes (α-amylase and α-glucosidase) along the digestive tract.⁴ Clinically available inhibitors of these enzymes delay carbohydrate digestion and prolong its overall digestion time, causing reduction in the rate of glucose absorption and consequently reducing postprandial plasma glucose rise.

Vegetables and herbs have been used by humans as food and to treat ailments for generations. Scientific evidence is accumulating, proving that many of these plants and plant materials have medicinal properties that alleviate symptoms or prevent diseases.⁵ Studies have reported inverse correlation between consumption of plant foods and incidences of some degenerative diseases.^6,7 Plants and plant materials have been employed in folklore and ethnomedicinally for the treatment of diabetes in the form of concoctions, decoctions, and infusions. Research on medicinal plants for the management of diabetes in recent years has attracted the interest of scientists⁸ and a number of them are known to exert antihyperglycemic activity via inhibition of carbohydrate hydrolyzing enzymes. This property has been attributed to the presence of physioactive phytochemicals such as phenolics and flavonoids.⁹ Phenolic compounds are also strong antioxidants due to the redox properties of their hydroxyl groups.

Hibiscus sabdariffa (Roselle) is a plant species of the Malvaceae family, which probably originated in West Africa and has a wide distribution throughout the tropics.¹⁰ This vegetable is widely cultivated for its calyx (flowers) in the northeastern and middle-belt regions of Nigeria.¹¹ The calyx of the white variety, popularly called “Isakpa” by the Yorubas of southwestern Nigeria, is the major component of a local soup preparation. However, the calyx of the red variety finds various uses in traditional medicine. It is reported to be an antiseptic, digestive, diuretic, emollient, and purgative agent. Roselle is a folk remedy for abscesses, bilious conditions, cancer, and hypertension.¹² Recent reports have also established hypolipidemic properties of H. sabdariffa ^13,14 and isolation of α-amylase inhibitors from H. sabdariffa tea.¹⁵

Nevertheless, despite the known antidiabetic and hypoglycemic properties of the roselle plant in folklore and experimental animals in vivo,¹⁶ there is a dearth of information on its possible mechanism of antidiabetic action. Hence, this present work sought to investigate the inhibitory effect of aqueous extracts of two varieties of H. sabdariffa (red and white) on carbohydrate-hydrolyzing enzymes (α-amylase and α-glucosidase) coupled with their antioxidant properties with the aim of revealing the possible underlying mechanism of the antidiabetic properties of the plant.

Materials and Methods

Materials

H. sabdariffa calyces (red and white varieties) were purchased from Oja–Oba market, Akure, Nigeria. The authentication of the plants was done by Mr. K. Oladunjoye at the Herbarium of the Department of Biology, Federal University of Technology (Akure), where voucher specimens (no. 32186a and 32186b) were deposited. The red and white roselle varieties are genetically distinct and are from clonally propagated lines. The rat intestinal α-glucosidase and porcine pancreatic α-amylase were purchased from Sigma Aldrich Chemical Co. Unless stated otherwise, all chemicals used were of analytical grade and were purchased from Sigma Chemical Co.

Sample preparation

The samples (calyces) were washed under running water, sun dried after which the dried samples were ground to powder and kept dry in an air-tight container prior to the extraction. 1 g each of the dried samples was weighed into 100 mL of distilled water and was left for 24 h. The mixture after 24 h was filtered and the filtrate lyophilized (LGJ-12MC, Ningbo Scientz Bio-tech Co., Ltd.) to yield a powdery solid that was reconstituted in distilled water and then used for the assay.

Phytochemical screening

The methods described by Trease and Evans¹⁷ were used for phytochemical screening of H. sabdariffa calyces extracts for the presence of bioactive compound. The test for tannins was carried out by subjecting 3 g of each plant extract in 6 mL of distilled water, filtered and ferric chloride reagents added to the filtrate. The test for alkaloids was carried out by subjecting 0.5 g of aqueous extract in 5 mL of 1% (v/v) HCl, boiled, filtered, and Mayer's reagent added. The extract was subjected to frothing test for the identification of saponin. The presence of flavonoids was determined using 1% (v/v) aluminum chloride solution in methanol-concentrated HCl, magnesium turnings, and potassium hydroxide solution. These qualitative tests were based on the color change as indication of a positive test. Deposition of a red precipitate when the aqueous extracts were boiled in 1% (v/v) aqueous hydrochloric acid was taken as evidence for the presence of phlobatannins.

Determination of extractable phenol content

The phenol content of the extracts was determined according to Singleton et al.¹⁸ Appropriate dilutions of the extracts were oxidized with 2.5 mL 10% (v/v) Folin-Ciocalteau's reagent and neutralized by 2.0 mL of 7.5% (v/v) sodium carbonate. The reaction mixture was incubated for 40 min at 45°C and the absorbance was measured at 765 nm in the spectrophotometer. The total phenol content was subsequently calculated as gallic acid equivalent using gallic acid standard curve.

Determination of extractable flavonoid content

The flavonoid content of the extracts was determined using a slightly modified method of Meda et al. ¹⁹ Briefly 0.5 mL of appropriately diluted sample was mixed with 0.5 mL methanol, 50 μL 10% (v/v) AlCl₃, 50 μL 1 M potassium acetate and 1.4 mL water, and allowed to incubate at room temperature for 30 min. The absorbance of the reaction mixture was subsequently measured at 415 nm and the total flavonoid content was calculated using quercetin standard curve.

Determination of vitamin C

Vitamin C content of the aqueous extract was determined using the method of Benderitter et al.²⁰ Briefly, 75 μL DNPH (2 g dinitrophenyl hydrazine, 230 mg thiourea, and 270 mg CuSO₄.5H₂O in 100 mL of 5 M H₂SO₄) were added to 500 μL reaction mixture containing 300 μL of appropriate dilution of the extracts with 100 μL 13.3% (v/v) trichloroacetic acid (TCA) and water. The reaction mixture was subsequently incubated for 3 h at 37°C, thereafter 0.5 mL of 65% (v/v) H₂SO₄ was added to the mixture and the absorbance was measured at 520 nm. The vitamin C content of the extracts was calculated using ascorbic acid as standard.

Fe²⁺ chelation assay

The Fe²⁺ chelating ability of the extracts were determined using a modified method of Minotti and Aust²¹ with a slight modification by Puntel et al.²² Freshly prepared 500 μM FeSO₄ (150 μL) was added to a reaction mixture containing 168 μL 0.1 M Tris-HCl (pH 7.4), 218 μL saline, and the extracts (0–120 μL). The reaction mixture was incubated for 5 min, before the addition of 13 μL 0.25% (w/v) 1,10-phenanthroline. The absorbance was subsequently taken at 510 nm; the Fe²⁺-chelating ability was then calculated and expressed as a percentage: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} \begin{split} \% \ &\hbox{Antioxidant or Inhibition} = \\& [({\rm Abs}_{\rm Control} - {\rm Abs}_{\rm Samples})/{\rm Abs}_{\rm Control}] \times 100 \%\end{split} \tag{1}\end{align*} \end{document}

where Abs_Control is absorbance of the reference and Abs_Samples is absorbance of the test samples.

Fenton reaction (degradation of deoxyribose)

The extract (0–200 μL) was added to a reaction mixture containing 120 μL of 20 mM deoxyribose, 400 μL of 0.1 M phosphate buffer, and 40 μL of 500 μM of FeSO₄, and the volume were made up to 800 μL with distilled water. The reaction mixture was incubated at 37°C for 30 min and the reaction was then stopped by the addition of 0.5 mL of 2.8% (v/v) TCA. This was followed by further addition of 0.4 mL of 0.6% (v/v) thiobarbituric acid (TBA) solution. The tubes were then incubated in boiling water for 20 min.²³ The absorbance was taken at 532 nm and percentage ^•OH scavenging ability was subsequently calculated [Eq. (1)].

Lipid peroxidation assay

Experimental animals

Male Wistar albino rats weighing between 190 and 250 g were purchased from the Central Animal House, Department of Biochemistry, University of Ilorin, Ilorin, Nigeria. They were housed in stainless steel cages under controlled conditions of a 12 h light/dark cycle, 50% humidity, and 28°C temperature. The rats were allowed access to food and water ad libitum. The animals were used in accordance with the procedure approved by the Animal Ethics Committee of the Federal University of Technology.

Preparation of tissue homogenates

The rats were decapitated by cervical dislocation and the pancreas (tissue) was rapidly dissected and 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 ∼35.4 g in a Teflon glass homogenizer (Mexxcare, mc14 362; Aayushi Design Pvt. Ltd. India). The homogenate was centrifuged (KX3400C Kenxin Intl. Co.) for 10 min at 3000 g to yield a pellet that was discarded and a low-speed supernatant (SI), which was kept for lipid peroxidation assay.

Lipid peroxidation and thiobarbibutric acid reactions

The lipid peroxidation assay was carried out using the modified method of Ohkawa et al.²⁴ Briefly, 100 μL of the SI fraction was mixed with a reaction mixture containing 30 μL of 0.1 M (pH 7.4) Tris-HCl buffer, extract (0–100 μL), and 30 μL of freshly prepared pro-oxidant (25 μM FeSO₄). The volume was made up to 300 μL with water before incubation at 37°C for 2 h. The color reaction was developed by adding 300 μL 8.1% (v/v) sodium dodecyl sulfate (SDS) to the reaction mixture containing SI and subsequently followed by the addition of 600 μL of acetic acid/HCl (pH 3.4) mixture and 600 μL of 0.8% (v/v) TBA. This mixture was incubated at 100°C for 1 h. Thiobarbituric acid reactive species produced was measured at 532 nm and expressed as malondialdehyde (MDA) produced (% control) using MDA standard curve (0–0.035 mM).

α-Amylase inhibition assay

Briefly, appropriate dilutions (0–200 μL) of the extracts and 500 μL of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) containing porcine pancreatic α-amylase (EC 3.2.1.1; 0.5 mg/mL) was incubated at 25°C for 10 min. Then, 500 μL of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) was added to the reacting mixture. Thereafter, the reaction mixture was incubated at 25°C for 10 min and 1.0 mL of dinitrosalicylic acid was added. Then, the reaction was stopped by incubating in boiling water bath for 5 min and later cooled to room temperature. The reaction mixture was then diluted by adding 10 mL of distilled water, and absorbance was measured at 540 nm by a spectrophotometer (JENWAY 6305; Barloworld Scientific).²⁵ The reference sample included all other reagents and the enzyme with the exception of the test sample. The α-amylase inhibitory activity was expressed as percentage inhibition [Eq. (1)].

α-Glucosidase inhibition assay

Appropriate dilutions of the extracts (0–200 μL) and 100 μL of α-glucosidase (EC 3.2.1.20) solution (1.0 U/mL) in 0.1 M phosphate buffer (pH 6.9) were incubated at 25°C for 10 min. Then, 50 μL of 5 mM p-nitrophenyl-α-D-glucopyranoside solution in 0.1 M phosphate buffer (pH 6.9) was added. The mixtures were incubated at 25°C for 5 min, before reading the absorbance at 405 nm in the spectrophotometer.²⁶ The reference sample included all other reagents and the enzyme with the exception of the test sample. The α-glucosidase inhibitory activity was expressed as percentage inhibition [Eq. (1)].

Data analysis

All experiments were carried out in triplicate. Data were expressed as mean±standard deviation (SD). Differences were evaluated by one-way analysis of variance followed by Duncan multiple test. Significance was accepted at P<.05. The IC₅₀ was calculated using nonlinear regression analysis.

Results and Discussion

Type 2 diabetes is a metabolic disorder characterized by hyperglycemia and insulin insensitivity or secretion. However, hyperglycemia has been implicated in increased free-radical generation leading to oxidative stress in major organs and tissues, the underlying principle behind diabetes and diabetic complications. Hence, augmenting the body antioxidant status through dietary means may help reduce oxidative stress. Further, control of hyperglycemia through the inhibition of α-amylase and α-glucosidase would help prevent hyperglycemia-induced increase in free radical generation and oxidative stress associated with diabetes mellitus.

The result of the phytochemical screening revealed the presence of saponin and flavonoids (except tannin present only in red calyx) in both calyces of red and white roselle varieties. However, as shown in Table 1, the total phenol and vitamin C content of the red roselle are significantly (P<.05) higher than that of the white variety, while there is no significant (P<.05) difference in their flavonoid content. Phenolic compounds and vitamin C are plant phytochemicals with known antioxidant properties and their high content in red roselle calyx compared with white roselle may be responsible for the higher antioxidant properties exhibited by the red roselle and its protective effect against pancreas lipid peroxidation. Polyphenols participate in the prevention of several major chronic diseases such as cardiovascular diseases, diabetes, cancers, or neurodegenerative diseases whose pathogenesis and progression have been linked to the action of free radicals. Previous study has shown rapid absorption and bioavailability of anthocyanindin-3-glycosides of H. sabdariffa extract with peak plasma concentration at 1.5 h after intake,²⁷ suggesting the bioavailability of roselle polyphenol constituents in organs and tissues where they could exert their health promoting effects. Report also suggested that flavonoids may prevent the progressive impairment of pancreatic β-cells function due to oxidative stress thus, reducing the occurrence of type 2 diabetes.²⁸

Table 1.

Total Phenol, Total Flavonoids, and Vitamin C Content (mg/100 g) of the Aqueous Extracts of Red and White H. sabdariffa (Roselle) Calyces

	Total phenol (mg/100 g)	Total flavonoid (mg/100 g)	Vitamin C (mg/100 g)
Red roselle	2.3^a±0.8	0.1^c±0.0	12.4^b±1.5
White roselle	1.7^b±0.3	0.2^c±0.1	1.5^a±0.1

Values represent mean±standard deviation of replicate readings (n=3).

abc

Values with the same superscript letter within the same column are not significantly different (P<.05)

The roselle extracts (red and white) chelate Fe²⁺ in vitro in a concentration dependent (20–100 μg/mL) pattern (figure not provided). However, as revealed by the IC₅₀ (Table 2) the red roselle exhibited significantly (P<.05) higher Fe²⁺ chelating ability than the white roselle. Further, the trend in the Fe²⁺ chelating ability of the roselle extracts is in agreement with their ^•OH scavenging ability (Table 2) and their phenolic content (Table 1). Nevertheless, the IC₅₀ values are significantly (P<.05) higher than that of Quercetin (a flavonoid) This finding agreed with earlier studies that showed that the antioxidant property of plant foods is a function of their total phenol content.²⁹ Thus, highlighting the possible role played by the roselle antioxidant phenolic constituents in the prevention of Fe²⁺-induced ^•OH production and its contribution to lipid peroxidation through Fenton reaction. Hence, ^•OH scavenging and Fe²⁺ chelating abilities are possible mechanisms through which roselle antioxidant phytochemicals prevent oxidative damage to pancreas.

Table 2.

IC₅₀ Values for the α-Amylase Inhibition, α-Glucosidase Inhibition, Inhibition of Lipid Peroxidation, Fe²⁺-Chelating Ability, and OH-Scavenging Ability of Aqueous Extracts of Red and White H. sabdariffa (Roselle) Calyces

	IC₅₀ values (μg/mL)
	Red roselle	White roselle	Acarbose	Quercetin
α-Amylase inhibition	187.9^a±10.2	90.5^b±5.2	59.8^c±4.1
α-Glucosidase inhibition	25.2^b±2.4	47.4^a±2.0	3.5^c±0.6
Inhibition of Fe²⁺-induced pancreatic lipid peroxidation	21.4^d±1.5	22.1^d±3.1		14.8^a±1.0
Fe²⁺-chelating ability	30.7^d±4.3	47.5^c±2.3		12.5^b±1.8
^•OH-radical scavenging ability	80.1^a±2.2	135.9^b±8.0		7.3^c±0.7

Values represent mean±standard deviation of replicate readings (n=3).

abcd

Values with the same superscript letter along the same row are not significantly different (P<.05)

Incubation of the isolated rat pancreatic tissue in the presence of 25 μM freshly prepared FeSO₄ solution caused a significant (P<.05) increase in its MDA content (185.4%) as against the basal (in the absence of FeSO₄), which is 100% (figure not provided). Previous study has shown which incubation of rat tissues (liver and brain) in the presence of 25 μM FeSO₄ solution caused significant increase in their MDA content.³⁰ However, the introduction of roselle calyx extracts (red and white) caused a significant (P<.05) decrease in the MDA content of the incubated pancreatic tissue homogenate concentration-dependently (figure not provided). Nevertheless, as revealed by the IC₅₀, no significant (P<.05) difference in the decrease in MDA content was observed between roselle varieties. Fe²⁺ catalyzes one-electron transfer reactions that generate ROS, such as the reactive ^•OH, which is formed from H₂O₂ through the Fenton reaction. Iron also decomposes lipid peroxides, generating peroxyl and alkoxyl radicals, which favor the propagation of lipid peroxidation. Therefore, the decrease in the pancreatic MDA content in the presence of the roselle extracts could be attributed to their Fe²⁺ chelating properties and their ability to scavenge the ^•OH produced (Table 2). Nevertheless, their inhibition of Fe²⁺-induced lipid peroxidation compare favorably with that of quercetin (Table 2). Pancreatic β-cells are extremely vulnerable to damage caused by ROS due to their limited antioxidant defence system.³¹ Thus, antioxidants could help protect against oxidative damage to the pancreas and delay diabetic complications. Similarly, earlier research demonstrated long-term preservation of rats' pancreatic islets under physiological conditions by polyphenols in a dose-dependent manner.³²

Oxidative stress is important in diabetes, not only for its role in the development of diabetic complications; rather persistent hyperglycemia may induce free radical production and contribute to β-cell destruction in type 2 diabetes.³³ Halliwell and Gutteridge³⁴ reported that glucose in the presence of transition metals (Fe) can be oxidized to produce ROS. It has also been demonstrated that reducing sugars trigger oxidative modification and apoptosis in pancreatic β-cells by provoking oxidative stress through glycation reaction.³⁵ Hence, control of hyperglycemia remains the most efficient approach to manage and/or slowdown the disease progression and the development of its complications arising from oxidative stress. Inhibition of enzymes involved in carbohydrate hydrolysis (α-amylase and α-glucosidase) has been exploited as therapeutic approaches for lowering hyperglycemia.⁴ Pancreatic α-amylase breaks down starch into disaccharides and oligosaccharides before intestinal α-glucosidase catalyzes the breakdown of disaccharides to liberate glucose, which is later absorbed into blood. Inhibition of these enzymes would slow down the overall breakdown of starch in the gastrointestinal tract, thus reducing postprandial hyperglycemia.³⁶ The α-amylase inhibitory properties of the roselle varieties (red and white) was in a concentration-dependent (10–80 μg/mL) pattern (figure not provided); however, as revealed by the IC₅₀ (Table 2), the white variety (90.5 μg/mL) caused a significantly (P<.05) higher inhibition of α-amylase activity than the red (187.9 μg/mL). The reason for this cannot be categorically stated, however, it may be due to additive/synergistic effects of its constituent phytochemicals. However, these values were lower than that of acarbose (59.8 μg/mL). Nevertheless, the α-amylase inhibitory ability of these roselle extracts agreed with earlier studies on some plant extracts.^2,37

Further, the α-glucosidase inhibitory activity of the roselle varieties also followed a concentration-dependent (10–80 μg/mL) pattern (figure not provided). However, as revealed by the IC₅₀ (Table 2), the red roselle (25.2 μg/mL) caused a significantly (P<.05) higher inhibition than the white (47.4 μg/mL), which is in agreement with their phenolic content (Table 1). Nevertheless, these values were significantly (P<.05) lower than that of acarbose (3.5 μg/mL). A previous report has attributed the α-glucosidase inhibitory activity of medicinal plants or food to their phenolic content.⁹ Nevertheless, the α-glucosidase inhibitory activity of both roselle extracts are significantly (P<.05) higher than their corresponding α-amylase inhibitory activity. This finding supports earlier reports that plant phytochemicals are mild inhibitors of α-amylase and strong inhibitors of α-glucosidase. Strong α-glucosidase and mild α-amylase inhibitory properties of the roselle extracts could help address major drawbacks (flatulence, abdominal distention, meteorism, and diarrhea) associated with synthetic inhibitors. Excessive inhibition of pancreatic α-amylase causes abnormal bacterial fermentation of undigested saccharides in the colon.³⁸

Aqueous extracts of roselle protected the pancreas from Fe²⁺-induced lipid peroxidation in vitro and demonstrated potent free radical scavenging ability. Inhibition of α-amylase and α-glucosidase activities is suggested as a possible mechanism through which roselle spp. exhibit their hypoglycemic properties. Protection of the pancreas from oxidative damage may be due to their Fe²⁺-chelating properties and scavenging of ^•OH produced. Therefore, roselle spp. may be good dietary sources of extractable phytochemicals for the prevention/management of type 2 diabetes and postprandial hyperglycemia–induced complications arising from oxidative stress. Nevertheless, this is an in vitro study with possible physiological relevance.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

References

Wild

, Roglic

, Green

, Sicree

, King

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

Ohkuwa

, Sato

, Naoi

. Hydroxyl radical formation in diabetic rats induced by streptozotocin. Life Sci, 1995; 56:1789–1798.

Negre-Salvayre

, Salvayre

, Auge

, Pamplona

, Portero-Otin

. Hyperglycemia and glycation in diabetic complications. Antioxid Redox Signal, 2009; 11:3071–3109.

Shim

, Doo

, Ahn

, Kim

, Seong

, Park

, Min

. Inhibitory effect of aqueous extract from the gall of Rhuz chinensis on alpha-glucosidase activity and postprandial blood glucose. J Ethnopharmacol, 2003; 85:283–287.

Loizzo

, Tundis

, Menichini

, Statti

, Menichini

. Influence of ripening stage on health benefits properties of capsicum annuum var. acuminatum l.: in vitro studies. J Med Food, 2008; 11:184–189.

La Vecchia

, Altieri

, Tavani

. Vegetables, fruit, antioxidants and cancer: a review of Italian studies. Eur J Nutr, 2001; 40:261–267.

Hung

, Joshipura

, Jiang

, Hu

, Hunter

, Smith-Warner

, Colditz

, Rosner

, Spiegelman

, Willett

. Fruit and vegetable intake and risk of major chronic disease. J Natl Cancer Inst, 2004; 96:1577–1584.

Ali

, Houghton

, Soumyanath

. Alpha amylase inhibitory activity of some Malaysian plants used to treat diabetes; with particular reference to Phyllanthus amarus. J Ethnopharmacol, 2006; 107:449–455.

Randhir

, Kwon

, Shetty

. Effect of thermal processing on phenolics, antioxidant activity and health-relevant functionality of select grain sprouts and seedlings. Innov Food Scid Emerg Technol, 2008; 9:355–364.

10.

Purseglove

. Tropical Crops Dicotyledons 1. Longman Green and Co.: London, 1968; 171–180.

11.

Akanya

, Oyeleke

, Jigam

, Lawal

. Analysis of sorrel drink (Zoborodo) Nigerian J Biochem Mol Biol, 1997; 12:77–81

12.

Morton

. Roselle. Fruits of Warm Climate. Dowlins

. Media, Inc.: Greensboro, NC, and Miami, FL, 1987; 281–286.

13.

Carvajal-Zarrabal

, Waliszewski

, Barradas-Dermitz

, Orta-Flores

, Hayward-Jones

, Nolasco-Hipolito

, Angulo-Guerrero

, Sanchez-Ricano

, Infanzon

, Trujillo

. The consumption of Hibiscus sabdariffa dried calyx ethanolic extract reduced lipid profile in rats. Plant Foods Hum Nutr, 2005; 60:153–159.

14.

Agoreyo

, Agoreyo

, Onuorah

. Effect of aqueous extracts of Hibiscus sabdariffa and Zingiber officinale on blood cholesterol and glucose levels of rats. Afr J Biotechnol, 2008; 7:3949–3951.

15.

Hansawasdi

, Kawabata

, Kasai

. α-Amylase inhibitors from roselle (Hibiscus sabdariffa Linn.) tea. Biosci Biotechnol Biochem, 2000; 64:1041–1043.

16.

Ogundipe

, Moody

, Akinyemi

, Raman . Hypoglycemic potentials of methanolic extracts of selected plant foods in alloxanized mice. Plant Foods Hum Nutr, 2010; 58:1–7

17.

Trease

, Evans

. Pharmacognosy, 12th. Bailliere Tindal: London, 1983; 469–470.

18.

Singleton

, Orthofer

, Lamuela–Raventos

. Analysis of total phenols, other oxidation substrates, antioxidants by means of Folin–Ciocalteu reagent. Packer

. Methods in Enzymology, 299. Oxidants and Antioxidants, Part A. Academic Press: San Diego, CA, 1999; 152–178

19.

Meda

, Lamien

, Romito

, Millogo

, Nacoulma

. Determination of the total phenolic, flavonoid and proline contents in Burkina Faso honey, as well as their radical scavenging activity. Food Chem, 2005; 91:571–577.

20.

Benderitter

, Maupoil

, Vergely

, Dalloz

, Briot

, Rochette

. Studies by electron paramagnetic resonance of the importance of iron in the hydroxyl scavenging properties of ascorbic acid in plasma: effects of iron chelators. Fundam Clin Pharmacol, 1998; 12:510–516.

21.

Minotti

, Aust

. An investigation into the mechanism of citrate-Fe²⁺-dependent lipid peroxidation. Free Radic Biol Med, 1987; 3:379–387.

22.

Puntel

, Nogueira

, Rocha

JBT

. Krebs cycle intermediates modulate thiobarbituric acid reactive species (TBARS) production in Rat Brain in vitro. Neurochem Res, 2005; 30:225–235.

23.

Halliwell

, Gutteridge

JMC

. Formation of a thiobarbituric-acid-reactive substance from deoxyribose in the presence of iron salts: the role of superoxide and hydroxyl radicals. FEBS Lett, 1981; 28:347–352.

24.

Ohkawa

, Ohishi

, Yagi

. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Ann Biochem, 1979; 95:351–358.

25.

Worthington

. Alpha amylase. Worthington Enzyme Manual. Worthington

. Worthington Biochemical Corp: Freehold, NJ, 1993; 36–41.

26.

Apostolidis

, Kwon

, Shetty

. Inhibitory potential of herb, fruit, and fungal enriched cheese against key enzymes linked to type 2 diabetes and hypertension. Innov Food Sci Emerg Technol, 2007; 8:46–54.

27.

Frank

, Jansen

, Netzel

, Stras

, Kler

, Kriesl

, Bitsch

. Pharmacokinetics of anthocyanidin-3-glycosides following consumption of Hibiscus sabdariffa L. extract. J Clin Pharmacol, 2005; 45:203–210.

28.

Song

, Manson

, Buring

, Sesso

, Liu

. Association of dietary flavonoids with risk of type 2 diabetes, and markers of insulin resistance and systemic inflammation in women: a prospective study and cross sectional analysis. J Am Coll Nutr, 2005; 24:376–384.

29.

Chu

, Sun

, Wu

, Liu

. Antioxidant and antiproliferative activity of common vegetables. J Agric Food Chem, 2002; 50:6910–6916.

30.

Oboh

, Puntel

, Rocha

JBT

. Hot pepper (Capsicum annuum, Tepin and Capsicum chinese, Habanero) prevents Fe²⁺-induced lipid peroxidation in brain–in vitro. Food Chem, 2007; 102:178–185.

31.

, Frigerio

, Maechler

. The sensitivity of pancreatic β-cells to mitochondrial injuries triggered by lipotoxicity and oxidative stress. Biochem Soc Trans, 2008; 36:930–934.

32.

Hyon

, Kim

. Long-term preservation of rat pancreatic islets under physiological conditions. J Biotechnol, 2001; 85:241–246.

33.

Maria

, Maria

, Corina

, Carmen

, Alexandra

. The sources and the targets of oxidative stress in the etiology of diabetic complications. Rom J Biophy, 2007; 17:63–84.

34.

Halliwell

, Gutteridge

JMC

. Non-enzymatic glycation, glycoxidation. Free Radicals in Biology and Medicine. Oxford University Press: Oxford, United Kingdom, 2007; 510–514.

35.

Kaneto

, Fujii

, Myint

et al. Reducing sugars trigger oxidative modification and apoptosis in pancreatic β-cells by provoking oxidative stress through the glycation reaction. Biochem J, 1996; 320:855–863.

36.

Kwon

, Apostolidis

, Kim

, Shetty

. Health benefits of traditional corn, beans and pumpkin: in vitro studies for hyperglycemia and hypertension management. J Med Food, 2007; 10:266–275.

37.

Nickavar

, Yousefian

. Inhibitory effects of six Allium species on α-amylase enzyme activity. Iran J Pharm Res, 2009; 8:53–57.

38.

Horii

, Fukasse

, Matsua

, Kame da

, Asano

, Masui

. Synthesis and α-D-glucosidase inhibitory activity of N-substituted valiolamine derivatives as potent oral antidiabetic agents. J Med Chem, 1987; 29:1038–1046.

Aqueous Extracts of Roselle ( Hibiscus sabdariffa Linn.) Varieties Inhibit α -Amylase and α -Glucosidase Activities In Vitro

Abstract

Introduction

Materials and Methods

Materials

Sample preparation

Phytochemical screening

Determination of extractable phenol content

Determination of extractable flavonoid content

Determination of vitamin C

Fe2+ chelation assay

Fenton reaction (degradation of deoxyribose)

Lipid peroxidation assay

Experimental animals

Preparation of tissue homogenates

Lipid peroxidation and thiobarbibutric acid reactions

α-Amylase inhibition assay

α-Glucosidase inhibition assay

Data analysis

Results and Discussion

Footnotes

Author Disclosure Statement

References

Fe²⁺ chelation assay