Aqueous Extracts of Two Varieties of Ginger ( Zingiber officinale ) Inhibit Angiotensin I–Converting Enzyme,Iron(II),and Sodium Nitroprusside-Induced Lipid Peroxidation in the Rat Heart In Vitro

Abstract

Ginger has reportedly been used in folk medicine for the management and prevention of hypertension and other cardiovascular diseases. Therefore, this study sought to investigate the inhibitory effect of aqueous extracts of two varieties of ginger on a key enzyme linked to hypertension (angiotensin I–converting enzyme [ACE]), and on pro-oxidants [Fe²⁺ and sodium nitroprusside (SNP)] which have been shown to induce lipid peroxidation in the rat's isolated heart in vitro. Aqueous extracts (0.05 mg/mL) of red ginger (Zingiber officinale var. Rubra) and white ginger (Zingiber officinale Roscoe) were prepared and the ability of the extracts to inhibit ACE along with Fe²⁺- and SNP-induced lipid peroxidation was determined in rat's heart in vitro. Results revealed that both extracts inhibited ACE in a dose-dependent manner (25–125 μg/mL). However, red ginger extract (EC₅₀=27.5 μg/mL) had a significantly (P<.05) higher inhibitory effect on ACE than white ginger extract (EC₅₀=87.0 μg/mL). Furthermore, incubation of the rat's heart in the presence of Fe²⁺ and SNP caused a significant increase (P<.05) in the malondialdehyde (MDA) content of the heart homogenates, while the introduction of the ginger extracts (78–313 μg/mL) caused a dose-dependent decrease in the MDA content of the stressed heart homogenates. This suggests that the possible mechanism through which ginger exerts its antihypertensive properties may be through inhibition of ACE activity and prevention of lipid peroxidation in the heart. Furthermore, red ginger showed stronger inhibition of ACE than white ginger. Additionally, it should be noted that these protective properties of the ginger varieties could be attributed to their polyphenol contents.

Introduction

H ypertension is a metabolic disease in which the blood pressure is chronically elevated¹ and is one of the long-term complications of type 2 diabetes. Persistent hypertension has been linked to several cardiovascular diseases (CVDs), such as stroke, heart attack, and heart failure.² The oxidative stress associated with these diseases can result in inflammation of the heart.^3,4 Evidence has shown that these diseases are caused by free radicals.⁵ Oxidative stress results from either a decrease in natural cell antioxidant capacity or an increase in the amount of reactive oxygen species (ROS) in organisms. However, consumption of foods rich in antioxidant phytochemicals may help fight degenerative diseases caused by oxidative stress of the heart by improving the body's antioxidant status.

High levels of both Cu and Fe have been implicated in several degenerative diseases.⁶ Although Fe is necessary physiologically as a component of many enzymes and proteins, free Fe in the cytosol and mitochondria can cause considerable oxidative damage by acting catalytically in the production of ROS, which have the potential to damage cellular lipids, nucleic acids, proteins, and carbohydrates, resulting in wide-ranging impairment in cellular function and integrity.⁷ ROS can directly attack the polyunsaturated fatty acids of the cell membranes and induce lipid peroxidation, the process in which ROS degrade polyunsaturated fatty acids. Malondialdehyde (MDA) is the end product of lipid peroxidation. This compound is a reactive aldehyde and is one of the many reactive electrophile species that cause toxic stress in cells and form advanced glycation end products. The production of this aldehyde is used as a biomarker to measure the level of oxidative stress in an organism.⁸

The renin–angiotensin system plays a pivotal role in blood pressure regulation, salt and water balance, and in the pathophysiology of CVDs such as congestive heart failure and hypertension.⁹ Renin produces angiotensin I from angiotensinogen, after which it is converted to a potent vasoconstrictor, angiotensin II, by the angiotensin I–converting enzyme (ACE). Inhibition of ACE is considered a useful therapeutic approach in the management and treatment of high blood pressure in both diabetic and nondiabetic patients,¹⁰ and several drugs have been designed for this purpose. However, recent findings have shown phenolic phytochemicals to have promising potential as well in mitigating this process.^11,12

Polyphenols are common constituents of the human diet, present in most foods and beverages of plant origin. They are considered to contribute to the prevention of various degenerative diseases such as diabetes and hypertension. This assumption originally came from in vitro studies, showing the antioxidant properties of several polyphenols and their ability to modulate the activity of various enzymes. Research suggests that many flavonoids are more potent antioxidants than vitamins C and E.^13,14

Ginger (Zingiber officinale Roscoe, Zingiberacae family) is widely used around the world in foods as a spice. For centuries, it has been an important ingredient in Chinese, Ayurvedic, and Tibb-Unani herbal medicines for the treatment of catarrh, rheumatism, nervous diseases, gingivitis, toothache, asthma, stroke, constipation, hypertension, and diabetes.^15
–17 Several reviews have appeared in literature about this plant, and this may reflect its popularity and common use as a spice and medicinal plant.^18,19 Further, many reviews have documented specific aspects of ginger as an anti-inflammatory agent²⁰ and its cancer prevention properties.²¹ Recently, Oboh et al. ^22,23 provided some possible mechanism for the use of ginger in the management of type 2 diabetes and Alzheimer's disease. Studies have shown that the active ingredients in ginger root include volatile oils, pungent phenol compounds known as gingerols, sesquiterpenoids, and shogaols, with high levels of anthocyanin and tannin in its root bark.²⁴ Ghayur and Gilani²⁵ reported that crude extract of ginger induced a dose-dependent (0.3–3 mg/kg) fall in the arterial blood pressure of anesthetized rats. Although ginger extracts have reportedly been used in traditional medicine for the management and prevention of hypertension, there is a dearth of information on the possible mechanism of action by which they exert this antihypertensive property. Hence, the objective of this study is to investigate the inhibitory effect of two varieties of ginger (Zingiber officinale) on ACE (a key enzyme linked to hypertension) activity and on Fe²⁺ and sodium nitroprusside (SNP), which are pro-oxidants involved in inducing oxidative stress of heart tissue. We hope to provide information on the possible mechanism by which ginger extracts exert their antihypertensive property.

Materials and Methods

Sample collection

Fresh samples of white ginger (Z. officinale Roscoe) and red ginger (Z. officinale var. Rubra) were purchased from the local market, in Akure metropolis, Nigeria. Authentication of the plants was carried out by Mr. K. Adejobi at the Department of Biology, Federal University of Technology, Akure, Nigeria, where voucher specimens (nos. 2360a and 2360b) was deposited at the herbarium.

Chemicals and reagents

Chemicals and reagents used, such as Hippuryl-histidyl-leucine substrate, thiobarbituric acid (TBA), gallic acid, and Folin–Ciocalteau's reagent were procured from Sigma-Aldrich, Inc. (St. Louis, MO, USA); acetic acid and quercetin were sourced from BDH Chemicals Ltd., (Poole, United Kingdom). Tris-HCl buffer, sodium dodecyl sulfate, and FeSO₄ were of analytical grade, while the water was glass-distilled.

Aqueous extract preparation

The inedible parts of the rhizomes were removed from the edible parts. The edible portions were thoroughly washed in distilled water to remove any contaminants, chopped into small pieces, sun-dried, and then milled. The aqueous extract of the rhizomes were subsequently prepared by soaking 5 g of the milled samples in 100 mL distilled water for∼24 h. The mixtures were filtered and later centrifuged at 357.8 g for 10 min to obtain a clear supernatant, and the filtrates were lyophilized by freeze-drying. The yields were 5.8% and 7.1% for red and white ginger varieties, respectively. The ginger extracts were then reconstituted in distilled water and used for subsequent analysis.

ACE inhibition assay

Appropriate dilutions of the extracts (0–200 μL) and 50 μL ACE (EC 3.4.15.1) solution (4 mU/mL) were incubated at 37°C for 15 min. After pre-incubation, the enzymatic reaction was initiated by adding 150 μL of 8.33 mM Hippuryl-histidyl-leucine (Bz-Gly-His-Leu) in 125 mM Tris-HCl buffer (pH 8.3) to the mixture and incubating at 37°C for 30 min. After incubation, the reaction was arrested by adding 250 μL of 1 M HCl. The Gly–His bond was then cleaved and the hippuric acid produced by the reaction was extracted with 1.5 mL ethyl acetate. Thereafter, the mixture was centrifuged to separate the ethyl acetate layer; then 1 mL of the ethyl acetate layer was transferred to a clean test tube and evaporated. The residue was redissolved in distilled water and its absorbance was measured at 228 nm. The control experiment was performed without the test sample and the ACE inhibitory activity was expressed as percentage inhibition:²² % Inhibition=[(Abs_Control−Abs_Samples)/Abs_Control]×100. ACE removes C-terminal dipeptides from susceptible substrates such as N-Hippuryl-His-Leu (1 unit will produce 1.0 μmole/min of hippuric acid from Hippuryl-His-Leu in 125 mM Tris-HCl at pH 8.3 at 37°C).

Determination of extractable phenol content

The phenol content in the extracts was determined using the method reported by Singleton et al. ²⁷ Briefly, 1 mL of ginger extract was transferred into a test tube and mixed with 5 mL of distilled water. To each sample, 2.5 mL 10% (v/v) Folin–Ciocalteau's reagent was added and mixed. After 5 min, 2 mL of 7.5% sodium carbonate was added to the reaction mixture and incubated for 40 min at 45°C, and the absorbance was measured at 765 nm in the spectrophotometer (JENWAY 6305, Barloworld Scientific, Dunmow, United Kingdom). The absorbance was extrapolated on a gallic acid standard curve and the values for total phenolics expressed as milligrams of gallic acid equivalents (GAE) per gram of the sample extracts.

Determination of extractable flavonoid content

The flavonoid content of the extracts was determined using a slight modification of the method reported by Meda et al. ²⁸ Briefly, 0.5 mL of appropriately diluted sample was mixed with 0.5 mL methanol, 50 μL 10% AlCl₃, 50 μL 1 M potassium acetate, and 1.4 mL water, and it was 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.

Lipid peroxidation assay

Experimental animals

Five male wistar albino rats weighing between 190 and 250 g were purchased from the Central Animal House, Department of Biochemistry, University of 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, Akure, Nigeria.

Preparation of heart homogenates

The rats were decapitated under mild diethyl ether anesthesia and the heart (tissue) was rapidly dissected and placed on ice and weighed. This tissue was subsequently homogenized in cold saline (0.1 g/mL) with ∼10 up and down strokes at ∼1200 rpm in a Teflon glass homogenizer (Mexxcare mc14 362; Aayushi Design Pvt. Ltd., New Delhi, India). The homogenate was centrifuged (KX3400C, Kenxin Intl. Co., Hong Kong, China) for 10 min at 3000 g to yield a pellet, which was discarded, and a low-speed supernatant (SI), which was kept for lipid peroxidation assay.²⁹

Lipid peroxidation and thiobarbibutric acid reactions

The lipid peroxidation assay was carried out using the modified method of Ohkawa et al. ³⁰ Briefly, 100 μL of the SI fraction was mixed with a reaction mixture containing 30 μL of 0.1 M Tris-HCl buffer (pH 7.4), extract (100 μL), and 30 μL of the pro-oxidant solution (250 μM freshly prepared FeSO₄ and 5 mM Sodium nitroprusside [SNP]). 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 8.1% Sodium dodecyl sulfate (SDS) to the reaction mixture containing SI, and this was followed by the addition of 600 μL of acetic acid/HCl (pH 3.4) mixture and 600 μL of 0.8% TBA. This mixture was incubated at 100°C for 1 h. TBA reactive species (TBARS) produced were measured at 532 nm and expressed as MDA produced (% control) using MDA standard curve (0–0.035 mM).

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 (ANOVA) followed by Duncan's multiple test.³¹ Significance was accepted at P<0.05.

Results and Discussion

The renin-angiotensin system plays a pivotal role in blood pressure regulation, salt and water balance, and in the pathophysiology of CVDs, such as congestive heart failure and hypertension.⁹ Renin produces angiotensin I from angiotensinogen, after which it is converted to a potent vasoconstrictor, angiotensin II, by ACE. ACE cleaves angiotensin I to produce angiotensin II, a powerful vasoconstrictor that has been identified as a major factor in hypertension.³² As such, inhibition of ACE is considered a useful therapeutic approach in the management/treatment of high blood pressure. As shown in Figure 1, our results revealed that aqueous extracts of the ginger varieties inhibited ACE activity in a dose-dependent manner (25–125 μg/mL). Red ginger extract (EC₅₀=27.5 μg/mL) showed stronger inhibition of ACE activity than white ginger extract (EC₅₀=87.0 μg/mL), as shown in Table 1. This ACE-inhibitory property of both gingers clearly showed that extracts of ginger could inhibit ACE in vitro, and this could explain the possible mechanism for its use in the management and treatment of hypertension in folklore. The inhibition of ACE by the ginger extracts agreed with earlier reports on phenolic extracts of bitter leaf ³³ and soybeans.³⁴ ACE inhibitors have been widely developed to prevent angiotensin II production in CVD patients, and have been utilized in clinical applications since the discovery of ACE inhibitors in snake venom.³⁵

FIG. 1.

Angiotensin 1 converting enzyme (ACE) inhibitory activity of aqueous extract of two varieties of ginger (Zingiber officinale). n=3.

Table 1.

The Total Phenol, Flavonoid Content, and EC₅₀ of Angiotensin 1–Converting Enzyme Inhibitory Activity of Two Varieties of Ginger (Zingiber officinale)

	Phenol content (mg/100 g)	Flavonoid content	EC₅₀ of ACE (μg/mL)
Red ginger	95.3b±8.4	53.7a±0.3	27.5c±0.0
White ginger	61.9c±9.1	34.6b±0.6	87.0d±0.0

Values represent mean±standard deviation of triplicate experiments. n=3

abcd

Values with the same superscript letter along the same column are not significantly different (P<0.05).

EC₅₀, extract concentration causing 50% enzyme inhibition.

Enzyme-inhibitory activities of plant foods has been attributed to their phenol content.^36,37 Hence, the phenolic content of the rhizome extracts was determined. Phenolic compounds can protect the human body from free radicals, whose formation is associated with the normal natural metabolism of aerobic cells. The phenol and flavonoid content of both red ginger (Z. officinale var. Rubra) and white ginger (Z. officinale Roscoe) are shown in Table 1. These results reveal that the phenol content of red ginger (95.3 mg/100 g) was significantly (P<.05) higher than that of white ginger (61.9 mg/100 g). Also, the flavonoid content of the gingers, as revealed in Table 1, indicate that red ginger (53.7 mg/100 g) had significantly (P<.05) higher flavonoid content than white ginger (34.6 mg/100 g). The antiradical activity of flavonoids and phenols is principally based on the structural relationship between different parts of their chemical structure.³⁸ Natural polyphenols are capable of removing free radicals, chelating metal catalysts, activating antioxidant enzymes, reducing α-tocopherol radicals, and inhibiting oxidases.^39,40

Lipid peroxidation in biological membranes is considered as one of the major mechanisms of cell injury in aerobic organisms subjected to oxidative stress.⁴¹ The inhibition of Fe²⁺-induced lipid peroxidation in isolated rat's heart homogenates by the extracts is presented in Figure 2. Incubation of the rat's heart in the presence of Fe²⁺ caused a significant increase (P<.05) in the MDA content of the heart (111.5%). These findings agree with our earlier reports on the interaction of Fe²⁺ with the brain,⁴² in which Fe²⁺ was shown to be a very potent initiator of lipid peroxidation (a pro-oxidant) in the brain. The increased lipid peroxidation in the presence of Fe²⁺ could be attributed to the fact that Fe²⁺ can catalyze 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, thus generating peroxyl and alkoxyl radicals, which favors the propagation of lipid oxidation.⁴³ However, the introduction of water-extractable phytochemicals (78–313 μg/mL) from the gingers caused a significant dose-dependent decrease (P<.05) in the MDA content of the Fe²⁺-stressed heart homogenates (white ginger, from 59.4% down to 24.0%; red ginger, from 88.0% down to 21.0%) with the least MDA production occurring at the introduction of the highest concentration of the ginger extracts (313 μg/mL). The mode of inhibition of Fe²⁺-induced lipid peroxidation cannot be categorically stated; however, there is the possibility that the water-extractable phytochemicals could have formed complexes with the Fe²⁺, thereby preventing them from catalyzing the initiation of lipid peroxidation, or perhaps the phytochemicals could have scavenged the free radicals produced by the Fe²⁺-catalyzed reaction.⁴⁴

FIG. 2.

Inhibition of Fe²⁺-induced lipid peroxidation in isolated rat's heart homogenates by aqueous extract of two varieties of ginger (Zingiber officinale). n=3.

Likewise, incubation of rat's heart tissue homogenates in the presence of SNP also caused a significant increase (P<.05) in the rat heart MDA content, as shown in Figure 3; however, both extracts inhibited MDA content in both tissues in a dose-dependent manner (78–313 μg/mL).We found that aqueous extract of red ginger had a higher inhibitory effect on MDA production in the heart in vitro (decreasing from 64.7% to 42.9%) than did white ginger (a decrease from 69.2% to 51.1%). SNP, a component of antihypertensive drugs, causes cytotoxicity through the release of cyanide and nitric oxide (NO).⁴⁵ The protective properties of the gingers against SNP-induced lipid peroxidation in the heart could be because of the ability of the antioxidant phytochemicals present in the aqueous extract to scavenge the nitrous and Fe radicals produced from the decomposition of SNP.

FIG. 3.

Inhibition of Sodium nitroprusside (SNP)-induced lipid peroxidation in isolated rat's heart homogenates by aqueous extract of two varieties of ginger (Zingiber officinale). n=5.

Summary

The two ginger varieties inhibit ACE and also protect the heart from Fe²⁺- and SNP-induced lipid peroxidation in vitro. However, these properties could be attributed to the polyphenol content of the spices. Therefore, a possible mechanism by which ginger extracts exert antihypertensive properties could be through inhibition of ACE activity and prevention of lipid peroxidation in the heart. In addition, red ginger extract showed stronger inhibition of ACE than white ginger extract.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

References

WHO. Diabetes Mellitus: World Health Organization Study Group Technical ReportSeries 727Geneva: World Health Organization, 1985.

Sowers

, Epstein

. Diabetes mellitus and associated hypertension, vascular disease, and nephropathy: an update. Hypertension, 1995; 26:869–879.

Martinon

. Signaling by ROS drives inflammasome activation. Eur J Immunol, 2010; 40:616–619.

Huang

, Glass

. Nuclear receptors and inflammation control molecular mechanisms and pathophysiological relevance. Arterioscler Thromb Vasc Biol, 2010; 30:1542–1549.

Halliwell

, Gutterdge

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; 128:347–352.

Johnson

. The possible crucial role of iron accumulation combined with low tryptophan zinc and manganese in carcinogenesis. Med Hypothesis, 2001; 57:539–543.

Britton

, Leicester

, Bacon

. Iron toxicity and chelation therapy. Int J Hematol, 2002; 76:219–228.

Murray

, Granner

, Mayes

, Rodwell

. Harper's Biochemistry 25th Edition. Lange Medical Books/McGraw-Hill Medical Publishing Division: New York, 2000.

Brunner

, Laragh

, Baer

, Newton

, Goodwin

, Krakoff

, Bard

, Buhler

. Essential hypertension: renin and aldosterone, heart attack and stroke. New Eng J Med, 1972; 286:441–449.

10.

Erdos

, Skidgel

. The angiotensin I-converting enzyme. Lab Investig, 1987; 56:345–348.

11.

Kwon

, Jang

, Shetty

. Evaluation of Rhodiola crenulata and Rhodiola rosea for management of type II diabetes and hypertension. Asian Pacific J Clin Nutri, 2006; 15:425–432.

12.

Kang

, Lee

, Kim

, Lee

. Angiotensin converting enzyme inhibitory phenylpropanoid glycosides from Clerodendron trichotomum. J Ethnopharmacol, 2003; 89:151–154.

13.

Oboh

. Effect of blanching on the antioxidant property of some tropical green leafy vegetables. Lebens Wissens und Technol, 2005; 38:513–517.

14.

Oboh

, Akindahunsi

. Change in the ascorbic acid, total phenol and antioxidant activity of some sun-dried green leafy vegetables in Nigeria. Nutri Health, 2004; 18:29–36.

15.

Awang

DVC

. Ginger. Can Pharm J, 1992; 125:309–311.

16.

Wang

, Wang

. Studies of commonly used traditional medicine-ginger. Zhongguo Zhong Yao Za Zhi, 2005; 30:1569–1573.

17.

Tapsell

, Hemphill

, Cobiac

, Patch

, Sullivan

, Fenech

, Roodenrys

, Keogh

, Clifton

, Williams

, Fazio

, Inge

. Health benefits of herbs and spices: the past, the present, the future. Med J Aust, 2006; 185:4–24.

18.

Afzal

, Al-Hadidi

, Menon

, Pesek

, Dhami

. Ginger: an ethnomedical, chemical and pharmacological review. Drug Metab Drug Interact, 2001; 18:159–190.

19.

Chrubasik

, Pittler

, Roufogalis

. Zingiberis rhizoma: a comprehensive review on the ginger effect and efficacy profiles. Phytomed, 2005; 12:684–701.

20.

Grzanna

, Lindmark

, Frondoza

. Ginger—an herbal medicinal product with broad anti-inflammatory actions. J Med Food, 2005; 8:125–132.

21.

Shukla

, Singh

. Cancer preventive properties of ginger: a brief review. Food Chem Toxicol, 2007; 45:683–690.

22.

Oboh

, Akinyemi

, Ademiluyi

, Adefegha

. Inhibitory effects of aqueous extract of two varieties of ginger on some key enzymes linked to type-2 diabetes in vitro. J Food Nutr Res, 2010; 49:14–20.

23.

Oboh

, Ademiluyi

, Akinyemi

. Inhibition of acetylcholinesterase inhibition activities and some prooxidant induced lipid peroxidation in rat brain by two varieties of ginger (Zingiber officinale) Exp Toxicol Pathol, 2012; 64:315–319.

24.

, Zhang

, Wu

, Tian

. Anti-inflammatory effect and mechanism of proanthocyanidins from grape seeds. Acta Pharmacol Sin, 2001; 22:1117–1120.

25.

Ghayur

, Gilani

. Ginger lowers blood pressure through blockade of voltage-dependent calcium channels. J Cardiovasc Pharmacol, 2005; 45:74–80.

26.

Cushman

, Cheung

. Spectrophotometric assay and properties of the angiotensin I converting enzyme of rabbit lung. Biochem Pharmacol, 1971; 20:1637–1648.

27.

Singleton

, Orthofer

, Lamuela-Raventos

. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteu reagent. Methods Enzymol, 1999; 299:152–178.

28.

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.

29.

Belle

NAV

, Dalmolin

, Fonini

, Rubim

, Rocha

JBT

. Polyamines reduces lipid peroxidation induced by different pro-oxidant agents. Brain Res, 2004; 1008:245–251.

30.

Ohkawa

, Ohishi

, Yagi

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

31.

Zar

. Biostatistical Analysis. Prentice-Hall, Inc., Upper Saddle River: NJ, USA, 1984.

32.

Ahnfelt-Ronne

. Enzyme inhibitors as drugs. A Textbook of Drug Design and Development. Krogsgaard-Larsen

, Bundgaard

. Harwood Academic Publishers: Chur, Switzerland, 1991; 302–307.

33.

Saliu

, Ademiluyi

, Akinyemi

, Oboh

. In vitro antidiabetes and antihypertension properties of phenolic extracts from bitter leaf (Vernonia amygdalina Del.) J Food Biochem, 2012; 36:569–576.

34.

Ademiluyi

, Oboh

. Soybean phenolic-rich extracts inhibit key-enzymes linked to type 2 diabetes (α-amylase and α-glucosidase) and hypertension (angiotensin I converting enzyme) in vitro. Exp Toxicol Pathol, 2013; 65:305–309.

35.

Villar

, Paya

, Terencio

. Plants with antihypertensive action. Fitoterapia, 1986; 57:131–145.

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.

Kwon

, Apostolidis

, Shetty

. In vitro studies of eggplant (Solanum melongena) phenolics as inhibitors of key enzymes relevant for type 2 diabetes and hypertension. Bioresource Technol, 2008; 99:2981–2988.

38.

Rice-Evans

, Miller

, Paganga

. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Rad Biol Med, 1996; 20:933–956.

39.

Amic

, Davidovic-Amic

, Beslo

, Trinajstic

. Structure-radical scavenging activity relationship of flavonoids. Croatia Chem Acta, 2003; 76:55–61.

40.

Alia

, Horcajo

, Bravo

, Goya

. Effect of grape antioxidant dietary fibre on the total antioxidant capacity and the activity of liver antioxidant enzymes in rats. Nutri Res, 2003; 23:1251–1267.

41.

Oboh

, Rocha

JBT

. Antioxidant in Foods: A New Challenge for Food Processors: Leading Edge Antioxidants Research. Nova Science Publishers Inc.: New York, 2007; 35–64.

42.

Oboh

, Akinyemi

, Ademiluyi

. Antioxidant and inhibitory effect of red ginger (Zingiber officinale var. Rubra) and white ginger (Zingiber officinale var Roscoe) on Fe²⁺ induced lipid peroxidation in rat brain in vitro. Exp Toxicol Pathol, 2012; 64:31–36.

43.

Zago

, Verstraeten

, Oteiza

. Zinc in the prevention of Fe²⁺ initiated lipid and protein oxidation. Biological Res, 2000; 33:143–150.

44.

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.

45.

Oboh

, Rocha

JBT

. Antioxidant and neuroprotective properties of sour tea (Hibiscus sabdariffa, calyx) and green tea (Camellia sinensis) on some pro-oxidants induced lipid peroxidation in brain—in vitro. Food Biophys, 2008; 3:382–389.