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Journal of Oleo ScienceCopyright ©2017 by Japan Oil Chemists’ Societydoi : 10.5650/jos.ess16029J. Oleo Sci. 66, (1) 51-63 (2017)

Essential Oil Composition, Antioxidant, Antidiabetic and Antihypertensive Properties of Two Afromomum SpeciesStephen Adeniyi Adefegha1,＊ , Tosin Abiola Olasehinde2 and Ganiyu Oboh1

1 Functional foods and Nutraceutical unit, Department of Biochemistry, Federal University of Technology, Akure, P.M.B. 704, Akure 340001, NIGERIA

2 Nutrition and Toxicology Division Food Technology Department, Federal Institute of Industrial Research, Oshodi, P.M.B. 21023, NIGERIA

1 IntroductionOxidative stress plays a crucial role in the development

and progression of type-2 diabetes and its complications due to increased radical generation and reduced antioxi-dant defense system1, 2）. Previous investigations have re-vealed the presence of pro-oxidants and oxidative stress biomarkers at elevated levels in serum, plasma and pancre-atic tissues of type-2 diabetic patients3）. Type-2 diabetes is characterized by elevated blood glucose level which can induce the generation of reactive oxygen species（ROS）. ROS are capable of attacking pancreatic β-cells due to low antioxidant capacity thereby causing β-cells insufficiency and insulin resistance3, 4）. As hyperglycemia aggravates, the β-cell function becomes impaired and secretion of insulin declines. This process eventually leads to the development of hypertension, diabetic nephropathy, neuropathy and cardiovascular diseases5）.

＊Correspondence to: Stephen Adeniyi Adefegha, Functional foods and Nutraceutical unit, Department of Biochemistry, Federal University of Technology, Akure, P.M.B. 704, Akure 340001, NIGERIAE-mail: saadefegha@futa.edu.ngAccepted August 12, 2016 (received for review February 3, 2016)Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 onlinehttp://www.jstage.jst.go.jp/browse/jos/　　http://mc.manusriptcentral.com/jjocs

ROS have also been linked to the development of high blood pressure which is a risk factor of cardiovascular dis-eases6）. High levels of ROS such as superoxides and hydro-gen peroxides have been observed in hypertensive pa-tients7）. Angiotensin II is a major bioactive product of the renin angiotensin system and has been implicated in oxida-tive stress8）. Increased levels of Angiotensin II stimulates the production of superoxides which combines with nitric oxide to form peroxynitrite- a highly reactive radical capable of initiating oxidation of cellular proteins and lipids9－11）. This process leads to loss of NO required for blood pressure regulation thereby causing hypertension12）.

Modern therapeutic approach for the treatment and management of diabetes and hypertension using natural sources involves scavenging of free radicals, inhibition of lipid peroxidation and enzymes linked to diabetes（α-amylase and α-glucosidase）and hypertension（angioten-

Abstract: This study was designed to assess the antioxidant, antidiabetic and antihypertensive effects of essential oils from A. melegueta and A. danielli seeds. The essential oils were extracted via hydrodistillation, dried with anhydrous Na2SO4 and characterized using gas chromatography-mass spectrometry (GC-MS). Antioxidant properties and inhibition of some pro-oxidant induced lipid peroxidation in rats’ pancreas and heart homogenates were also determined. The results revealed that eugenol, eucalyptol, α-terpineol, α-caryophyllene and β-caryophyllene were the most abundant components in A. melegueta and A. danielli seeds. The essential oils inhibited α-amylase, α- glucosidase and angiotensin-I-converting enzyme in vitro. A. melegueta oil showed a higher α-amylase and α- glucosidase inhibitory activities with EC50 values of 139.00 µL/mL and 91.83 µL/mL respectively than A. danielli. However, A. danielli oil (EC50 = 48.73 µL/mL) showed the highest ACE inhibitory acivity. The highest NO radical scavenging ability was observed in A. melegueta oil while A. danielli had the highest OH radical scavenging and Fe2+- chelating ability. Furthermore, both essential oils inhibited SNP and Fe2+- induced lipid peroxidation in rats’ pancreas and heart respectively in a dose dependent manner. This study reveals the biochemical principle by which essential oils from A. danielli and A.melegueta seed elicits their therapeutic effects on type-2 diabetes and hypertension.

Key words: Afromomum melegueta, Afromomum danielli, essential oil, type-2 diabetes, hypertension, antioxidants

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sin I converting enzyme［ACE］）. Dietary approach to the treatment and management of diabetes and hypertension using plant phytochemicals has gained interest recently as it prevents cellular oxidative damage, cardiovascular and renal diseases13）.

Afromomum species belongs to the Zingiberaceae family and are very common in tropical and subtropical regions14）. Afromomum danielli is a large, robust perenni-al plant 3-4 m tall which grows in central and west African countries15）. The seeds of this plant are used for flavouring traditional dishes and the essential oil is used in perfumery, flavouring and dye preparations. Afromomum melegueta is commonly referred to as “Alligator pepper”16）. It is a common spice which has been used for alleviating stomach ache and diarrhea as well as hypertension. Although there are limited reports on its use for the treatment of tubercu-losis, snakebites and scorpion stings17－19）. Recently there has been some research works on the chemical composi-tion, antioxidant and antimicrobial activities of essential oils from A. danielli and A. melegueta. However, to the best of our knowledge there is little or no information on the antidiabetic and antihypertensive properties of the es-sential oils from the seeds of these plants. This study in-vestigated the chemical composition, antioxidant, antidia-betic and antihypertensive properties of essential oils from A. melegueta and A. danielli seeds.

2 Materials and methods2.1 Sample collection

Afromomum melegueta and Afromomum danielli seeds were collected from Akure main market, southwest Nigeria. The seeds were ground to fine powder using Warring Commercial heavy Duty Blender（Model 37BL18; 24ØCB6）. Authentication of the samples was carried out at the Department of Crop, Soil and Pest management（CSP）, Federal University of Technology, Akure, Nigeria.

2.2 Essential oil isolationOne hundere grams（100 g）of each seed powder was

subjected to hydrodistillation for 3 h in an all glass Clev-enger – type apparatus according to the method recom-mended by European Pharmacopoeia20）. The oil samples obtained were passed over anhydrous sodium sulfate and stored in sealed vials at 4℃ for further analysis.

2.3 Chemicals and reagentsChemicals and reagents used in this study include thio-

barbituric acid TBAR, 1,10-phenanthroline, deoxyribose, gallic acid, Folin–Ciocalteaus reagent were procured from Sigma-Aldrich, Inc., St Louis, MO, trichloroacetic acid TCA. These chemicals were sourced from Sigma-Aldrich, Chemie GmbH Steinheim, Germany. Hydrogen peroxide, methanol,

acetic acid, thiourea, Copper sulfate, sulfuric acid, sodium carbonate, aluminum chloride, potassium acetate, sodium dodecyl sulfate, Iron（II）sulfate, potassium ferricyanide and ferric chloride used in this study were sourced from BDH Chemicals Ltd., Poole, England, Porcine pancreatic α-amylase and rat intestinal α-glucosidase were purchased from Sigma Chemical Co.（St. Louis, MO）. Except stated otherwise, all other chemicals and reagents were of analyti-cal grades and the water was glass distilled.

2.4 Determination of total phenol contentThe total phenol content was determined according to

the method of Singleton et al.21）. Briefly, appropriate dilu-tions of the essential oils were oxidized with 2.5 mL 10％ Folin-Ciocalteu reagent and neutralized by 2.0 mL of 7.5％ sodium carbonate. The reaction mixture was incubated for 40 min at 45℃ and the absorbance was measured at 765 nm in the spectrophotometer. The total phenol content was subsequently calculated as gallic acid equivalent.

2.5 Determination of total �avonoid contentThe total flavonoid content of the oils was determined

using a slightly modified method reported by Meda et al.22）. Briefly, 0.5 mL of appropriately diluted sample was mixed with 0.5 mL methanol, 50 μL 10％ AlCl3, 50 μL 1 M potassi-um acetate and 1.4 mL water and allowed to incubate at room temperature for 30 min. The absorbance of the reac-tion mixture was subsequently measured at 115 nm and the total flavonoid content calculated as quercetin equiva-lent.

2.6 α-Amylase inhibition assayThe essential oil（500 μL）and 500 μL of 0.02 M sodium

phosphate buffer（pH 6.9 with 0.006 M NaCl）containing Hog pancreatic α-amylase（EC 3.2.1.1）（0.5 mg/mL）were incubated at 25℃ 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 each tube. The reaction mix-tures was incubated at 25℃ for 10 min and stopped with 1.0 mL of dinitrosalicylic acid colour reagent. Thereafter, the mixture was 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 nm23）.

2.7 α-Glucosidase inhibition assayThe essential oil（50 μL）and 100 μL of α-glucosidase

solution（1.0 U/mL）in 0.1 M phosphate buffer（pH 6.9）was incubated at 25 ℃ for 10 min. Then, 50 μL of 5 mM p-ni-trophenyl-α-D-glucopyranoside solution in 0.1 M phos-phate buffer（pH 6.9）was added. The mixtures were incu-bated at 25℃ for 5 min, before reading the absorbance at 405 nm in the spectrophotometer. The α-glucosidase in-hibitory activity was expressed as percentage inhibition24）.

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2.8 Angiotensin I converting enzyme（ACE）inhibition as-say

The oil extracts（50 μL）and ACE solution（50 μL, 4 mU）was incubated at 37℃ for 15 min. The enzymatic reaction was initiated by adding 150 μL of 8.33 mM of the substrate Bz–Gly–His–Leu in 125 mM Tris– HCl buffer（pH 8.3）to the mixture. After incubation for 30 min at 37℃, the reaction was arrested by adding 250 μL of 1 M HCl. The Gly–His bond was then cleaved and the Bz–Gly produced by the re-action 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 re-dissolved in distilled water and its absorbance was mea-sured at 228 nm. The ACE inhibitory activity was ex-pressed as percentage inhibition25）.

2.9 Gas chromatography analysisThe analytical GC was carried out by Hewlett-Packard

5890 gas chromatograph Hewlett-Packard Corp., Palo Alto, CA）equipped with Flame Ionization Detectors（FID）with DB-5 column（30 m length, 0.25 mm column id., 0.25 μm film thickness）. The following conditions were applied: In-jection temperature: 290℃. Injection volume: 1.0 μL. Injec-tion mode: Split（1:50）. Temperature program: 50℃ for 4 min, rising at 3℃ /min to 240℃, then rising at 15℃/min to 300℃, held at 300℃ for 3 min. FID（290℃）: H2 flow: 50 mL/min; air flow: 400 mL/min.

2.10 Nitric oxide radical scavenging assayThe scavenging effect of the extract on nitric oxide

（NO・）radical was measured according to the method of Mercocci et al.26）. The essential oil（100–400 μL）was added in the test tubes to 1mL of sodium nitroprusside solution（25 mM）and incubated at 37℃ for 2 h. An aliquot（0.5mL）of the incubation was removed and diluted with 0.3 mL Griess reagent（1％ sulfanilamide in 5％ H3PO4 and 0.1％ naphthlethylene diaminedihydrochloride）. The absorbance of the chromophore formed was immediately read at 570 nm against distilled water as blank.

2.11 Fenton reaction（Degradation of deoxyribose）The method of Halliwell and Gutteridge27） was used to

determine the ability of the extract to prevent Fe2＋/H2O2 induced decomposition of deoxyribose. The essential oil（0-100 μL）was added to a reaction mixture containing 120 μL of 20 mM deoxyribose, 400 μL of 0.1 M phosphate buffer, 40 μL of 500 mm of FeSO4, and the volume were made up to 800 μL with distilled water. The reaction mixture was incubated at 37℃ for 30 min and the reaction was then stopped by the addition of 0.5 mL of 28％ trichlo-roacetic acid. This was followed by addition of 0.4 mL of 0.6％ thiobarbituric acid solution. The tubes were subse-quently incubated in boiling water for 20 min. The absor-

bance was measured at 532 nm in a spectrophotometer.

2.12 Fe2＋-chelation assayThe Fe2＋- chelating ability of the volatile oil was deter-

mined using a modified method of Minotti and Aust28） with a slight modification by Puntel et al.29）. 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 – 25 μL）. The reaction mixture was in-cubated for 5 min, before the addition of 13 μL 0.25％ 1,10-phenanthroline（w/v）. The absorbance was subse-quently measured at 510 nm in a spectrophotometer. The Fe2＋-chelating ability was subsequently calculated.

2.13 Lipid peroxidation assay2.13.1 Preparation of tissue homogenates

The rats were decapitated under mild diethyl ether an-aesthesia and the pancreas and heart were rapidly isolated and placed on ice and weighed. Each tissue was subse-quently homogenized in cold saline（1/10 w/v）with about 10 up and down strokes at approximately 1200 rev/min in a Teflon glass homogenizer. The homogenate was centrifuged for 10 min at 3000xg to yield a pellet that was discarded, and a low-speed supernatant（S1）was kept for lipid peroxi-dation assay30）.2.13.2 Lipid peroxidation and thiobarbibutric acid reac-

tionsThe lipid peroxidation assay was carried out using the

modified method of Ohkawa et al.31）. Briefly, 100 μL S1 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 250 μM freshly prepared FeSO4（the procedure was also carried out using 7 mM sodium nitroprusside）. The volume was made up to 300 μL by water before incu-bation at 37℃ for 2 h. The colour reaction was developed by adding 300 μL 8.1％ SDS（Sodium dodecyl sulfate）to the reaction mixture containing S1, which was subsequent-ly followed by the addition of 600 μL of acetic acid/HCl（pH 3.4）mixture and 600 μL 0.8％ TBA（Thiobarbituric acid）. This mixture was incubated at 100℃ for 1 h. TBARS（Thio-barbituric acid reactive species）produced were measured at 532 nm and the absorbance was compared with that of standard curve using MDA（Malondialdehyde）.

2.14 Data analysisThe result of three replicate experiments were pooled

and expressed as mean±standard deviation（SD）. Student t- test was carried out to analyze the result. Significance was accepted at p ≤ 0.05.

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3 Results and Discussion3.1 Chemical constituents of essential oils

The results of the total phenolic and flavonoid contents as depicted in Table 1 revealed significantly（p＜0.05）higher total phenolic content in essential oil from A. me-legueta than the oil obtained from A. danielli. However, there was no significant（p＞0.05）difference in the flavo-noid content of the oils. The quantitative and qualitative analyses of the essential oils in the order of elution are shown in Table 2. The results revealed the presence of vol-atile compounds such as limonene, eugenol, eucalyptol, α-pinene, β-pinene, β-cadinene, β-caryophyllene, α-terpineol, germacrene D etc. A. melegueta oil had higher concentration of eugenol（82.2％）and α-caryophyllene（3.35％）when compared to A. danielli as presented in Table 2. However, A. danielli oil had higher concentration of β-caryophyllene（5.11％）, eucalyptol（10.93％）, α-terpineol（5.56）, Caryophyllene oxide（4.94％）, limonene（4.50％）than A. melegueta etc. Gamma elemene, Tau cadinol, and 3-Allyl-6- methoxyphenol were present in A. melegueta but were not detected in A. danielli as shown in Figs. 9 and 10 respectively. However, α-pinene, β-pinene, myrtenol, thymol, P-menthene, peruviol, Gamma gurjene, α-Bisabolene epoxide and camphene were present in A. danielli and were not detected in A. melegueta.

3.2 Antidiabetic properties of essential oils from A. me-legueta and A. danielli seeds

The effect of the essential oils on α-amylase and α-glucosidase activities was investigated in vitro. These enzymes are carbohydrate-hydrolyzing enzymes that are commonly used as therapeutic targets to modulate post-prandial hyperglycemia. Synthetic inhibitors of these enzymes such as acarbose, voligbose and miglitol reduce postprandial hyperglycemia via inhibition of enzymatic breakdown of complex carbohydrates which can delay ab-sorption of glucose into the bloodstream32, 33）. However these inhibitors are limited in use due to their side effects and failure to alter the course of diabetic complications. Previous scientific investigations have suggested plant phytochemicals as an alternative strategy for the treatment and management of type-2-diabetes and its complica-

tions34）. The inhibition of α-amylase and α-glucosidase by the essential oils are shown in Figs. 1 and 2 respectively. Acarbose and the essential oils inhibited the enzymes in vitro. Although acarbose（18.63 μg/mL）had the highest enzyme inhibitory activities, A. melegueta oil（EC50＝ 139.04 μL/mL and 91.83 μL/mL）had significantly（p＜0.05）higher α-amylase and α-glucosidase inhibitory activities than the essential oil obtained from A. danielli（EC50＝ 156.43 μL/mL and 114.65 μL/mL）respectively as shown in Table 3 . Moreover both samples have s t ronger α-glucosidase inhibition than α -amylase. These results correlates with the work of Reshma et al.32） who reported a stronger α-glucosidase inhibition than α -amylase Previous findings have also revealed a stronger inhibition of α-glucosidase and mild inhibition of α-amylase as a good therapeutic strategy for the management of type-2 diabe-tes35）. The inhibition of these enzymes can be attributed to the presence of phenolic monotepernes and sesquiterpenes in the essential oils. Moreover, eugenol, limonene, α-pinene and β-pinene have been reported to have high inhibitory effects against α-amylase and α- glucosidase activities36）. The higher inhibitory effects observed in A. melegueta could be attributed to the synergistic or additive effects of the different components and varying biological activities of the chemical constituents present in the oil.

3.3 In vitro Anti-hypertensive activity of essential oilsType-2 diabetes mellitus has been linked to hypertension

which is a risk factor of cardiovascular diseases. Inhibition of ACE activity by ACE-inhibitors has been considered as a good therapeutic approach for the treatment and manage-ment of hypertension37）. Consequently, the inhibition of ACE activity by the essential oils used in this study sug-gests that these oils could possess the ability to control blood pressure by reducing the production of angiotensin II- a vasoconstrictor. In this study, captoril and the essen-tial oils inhibited angiotensin-I-converting enzyme activity as shown in Fig. 3. The result in Table 3 shows that captoril（2.81 μg/mL）had the highest ACE inhibitory activity while the oil extracted from A. danielli had significantly（p＜0.05）higher ACE inhibitory activity than that of A. melegu-eta with EC50＝48.73 μL/mL and 65.53 μL/mL respectively. To the best of our knowledge, there are few reports on ACE inhibitory activity of plant essential oils however; the inhibitory activity of A. melegueta and A. danielli can be attributed to the chemical constituents of the oil38）. Chaud-hary et al.13） established that eugenol isolated from the oil obtained from Ocimum sanctum L. inhibited ACE activity; however, the percentage inhibition was lower than that of the essential oil. This finding further confirms the claim that the observed inhibitory effect could be attributed to the synergistic or additive effects of the bioactive compo-nents present in the oils.

Table 1　 The total phenolic content reported as gallic acid equivalent and total flavonoid content reported as quercetin equivalent.

Parameter (unit) A. danielli A. melegueta

Total phenol (mg/GAE/L) 4.23±0.33a 5.54±0.50b

Total flavonoid (mg/QE/L) 2.35±0.67a 3.40±0.88a

Values represent means of triplicate readings. Values with different superscripts letter along the same row are significantly different (p < 0.05).

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Table 2　Chemical composition of essential oil from A. danielli and A. melegueta.

Sample A. melegueta A. danielli

Composant RT % RT %β-Thujene 5.49 0.11 5.49 0.067α-Pinene ND ND 6.23 0.32Camphene ND ND 6.54 0.027β-pinene ND ND 7.11 1.14-2-Carene ND ND 7.92 0.064Eucalyptol 8.23 0.099 8.26 10.933- Carene 8.55 0.07 8.81 0.0964-carene 9.44 0.15 9.45 0.121,6 Octadiene- 3- ol 9.65 1.05 9.64 0.85Cyclopentosiloxane 10.79 0.08 10.78 0.15Terpinen-4-ol 11.36 0.27 11.36 0.48α-Terpineol 11.63 0.67 11.66 5.562-Myrtenol ND ND 11.77 0.37Thymol ND ND 13.82 0.19P-Menthene ND ND 14.20 0.21Eugenol 15.04 82.2 15.11 51.143 Allyl 6 methoxyphenol 15.38 0.30 ND NDHexamethylene 15.65 0.30 15.66 0.41B-Caryophyllene 16.23 3.27 16.25 5.112-Norphinene 16.38 0.097 16.42 0.251,6,10- dodecatriene 16.71 0.14 16.72 0.03A- Caryophyllene 16.84 3.35 16.85 2.21Naphthalene, 1,2 17.20 0.37 17.20 0.65Germacrene D 17.33 1.26 17.33 1.38β-Eudesma-4 (14), 11-diene 17.44 0.47 17.44 1.05β-Cadinene 17.58 0.27 17.58 0.60Limonene 17.72 2.19 17.72 4.50Naphthalene 17.88 0.10 17.88 0.18epi-bicyclosesquiphellandrene 18.00 0.82 18.01 1.29Peruviol ND ND 18.61 1.32gamma elemene 18.67 0.54 ND NDSpathulenol ND ND 19.03 0.46Caryophyllene oxide 19.13 1.62 19.15 4.94Tau cadinol 20.05 0.21 ND NDgamma Gurjunene ND ND 20.39 0.62α-Bisabolene epoxide ND ND 20.53 1.06Thujopsene ND ND 20.79 0.11(-) Isokaurene ND ND 26.23 0.16

RT: retention time. Compounds are identified on the basis of comparison with MS database spectra and listed in order of elution.

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3.4 Radical scavenging and chelating abilities of essen-tial oils from Afromomum sp.

Several clinical and experimental investigations have re-vealed that oxidative stress can contribute to the develop-ment of diabetes and hypertension via generation of excess free radicals which are capable of mediating oxidative damage to cells39, 40）. Free radical scavengers commonly known as antioxidants prevent radical induced-oxidative damage. Although Nitric oxide（NO）act as an oxidative sig-naling molecule in physiological processes. Overproduction of NO induces nitrosative stress which can lead to nitrosyl-ation reactions that can alter protein structure11）. However, the essential oils used for this study scavenged NO radicals in a dose dependent manner as shown in Fig. 4. Essential oil from A. melegueta（EC50＝128.46 μL/mL）had signifi-

cantly higher NO radical scavenging ability than A danielli（EC50＝145.94 μL/mL）（Table 3）. Furthermore, OH radical is a product of the breakdown of hydrogen peroxide in the presence of metals such as Fe2＋. OH radical can abstract electron from polyunsaturated fatty acids thereby generat-ing lipid radical which can induce lipid peroxidation11）. Moreover, accumulation of Fe2＋ in the cells can induce the generation of free radicals and the production of malondi-aldehyde-a deleterious product of lipid peroxidation. The depletion of Fe2＋ can prevent radical induced-oxidative damage to cells. The results obtained in this study shows that the essential oils inhibited OH radicals and were able to chelate Fe2＋ as revealed in Figs. 5 and 6 respectively. A. danielli oil（EC50＝343.87 and 263.85 μL/mL）had signifi-cantly（p＜0.05）higher OH radical scavenging and Fe2＋-

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Acarbose A. Danielli A. melegueta

Fig. 1　 α-amylase inhibition by acarbose, essential oil from A. melegueta and A. danielli seeds. The essential oil extract concentrations for the plot are 0, 20 μL/mL, 40 μL/mL, 80 μL/mL and 180 μL/mL. The concentrations of acarbose used in the plot are 0, 10 μg/mL, 20 μg/mL, 30 μg/mL and 40 μg/mL. Values represent means±of standard deviation of triplicate readings.

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Fig. 2　 α-glucosidase inhibition by Acarbose, essential oil from A. melegueta and A. danielli seeds. The essential oil extract concentrations for the plot are 0, 20 μL/mL, 40 μL/mL, 80 μL/mL and 180 μL/mL. The concentrations of acarbose used in the plot are 0, 10 μg/mL, 20 μg/mL, 30 μg/mL and 40 μg/mL. Values represent means±of standard deviation of triplicate readings.

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chelating abilities than A. melegueta（EC50＝370.64 μL/mL and 297.62 μL/mL）（Table 3）. The chelating ability of the oils could prevent the formation of OH radicals and initia-tion of lipid peroxidation. The radical scavenging and che-lating ability of the essential oils could be attributed to phenolic monoterpenes and oxygenated monoterpernes present in the oil. Moreover antioxidant properties of es-sential oils have been attributed to the presence of Pheno-lic compounds attached to the terpene moiety of the con-stituents41, 42）.

3.5 Inhibition of Pro-oxidant induced lipid peroxidationLipid peroxidation in biological membranes induces oxi-

dative damage and cell death. This process involves con-stant generation of free radicals which initiate further per-oxidation43）. The ability of the essential to inhibit sodium nitroprusside（SNP）- induced lipid peroxidation in rats’ pancreas was investigated. SNP releases NO which reacts with super oxides to produce peroxynitrites, a highly reac-tive oxidative molecule capable of initiating lipid peroxida-tion and causing injury to pancreatic β-cells44）. It was ob-served that A. melegueta oil effectively inhibited SNP-

Table 3　 EC50 of inhibition of α-amylase, α-glucosidase and ACE activities, inhibition of Fe2＋ and SNP - induced lipid peroxidation in rat’s pancreas homogenates and radicals（NO, OH）scavenging and Fe2＋ chelating abilities of clove bud essential oils（μL/L）.

Parameter A. danielli A. meleguetaα-Amylase 156.43± 5.01a 139.00± 8.24b

α-glucosidase 114.65± 8.26a 91.83± 6.3 b

ACE 48.73± 3.45a 65.53± 6.21b

Lipid PeroxidationFe2＋ - induced 111.23± 7.14a 126.98± 5.62b

SNP - induced 162.83±12.11a 131.76±10.51b

Radicals scavenging abilityOH・ 343.87±10.52a 370.64±12.32b

NO・ 145.94± 8.68a 128.46± 6.32b

Fe2＋- chelating ability 263.85±14.50a 297.62±13.42b

Values represent means of triplicate readings. Values with different superscripts letter along the same row are significantly different (p < 0.05).

Fig. 3　 Angiotensin-I-converting enzyme inhibition by Acarbose, essential oil from A. melegueta and A. danielli seeds. The essential oil extract concentrations for the plot are 0, 20 μL/mL, 40 μL/mL, 80 μL/mL and 180 μL/mL. The concentrations of captopril used in the plot are 0, 1.5 μg/mL, 2.5 μg/mL, 5.0 μg/mL and 6.5 μg/mL. Values represent means±of standard deviation of triplicate readings.

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Fig. 4　 NO radical scavenging ability of essential oil from A. danielli and A. melegueta seeds. Values represent means±of standard deviation of triplicate readings.

Fig. 5　 OH radical scavenging ability of essential oil from A. danielli and A. melegueta seeds. Values represent means±of standard deviation of triplicate readings.

Fig. 6　 Fe2＋- chelating ability of essential oil from A. danielli and A. melegueta seeds. Values represent means±of standard deviation of triplicate readings.

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induced lipid peroxidation than A. danielli. This correlates with the NO radical scavenging ability of the oils. The result in Fig. 7 shows that the incubation of rat pancreas in the presence of SNP caused a significant increase（p＜0.05）in malondiadehyde（MDA）content. However, the es-sential oils caused a significant（p＜0.05）decrease in MDA levels in the pancreas in a dose dependent manner. Never-theless, essential oil from A. melegueta（EC50＝131.76 μL/mL）had significantly higher inhibition of SNP-induced lipid peroxidation in rats’ pancreas than the oil from A. danielli（EC50＝162.83 μL/mL）.

Similarly, the incubation of rats’ heart homogenates in presence of Fe2＋ caused a significant increase（p＜0.05）in the MDA content as shown in Fig. 8. However, the essential oils caused a significant（p＜0.05）decrease in the MDA content of the heart in a dose dependent manner. Table 3 shows that A. danielli had significantly higher inhibition of Fe2＋- induced lipid peroxidation in rats heart homogenates

than A. melegueta with EC50 values of 111.23 μL/mL and 126.98 μL/mL respectively. Moreover, it was observed that A. danielli oil was more effective in inhibiting Fe2＋

-induced lipid peroxidation in rats’ heart than A. melegue-ta. This result is similar to what was obtained for the OH radical scavenging and Fe2＋- chelating ability of the oils. Therefore the inhibition of pro-oxidant induced lipid per-oxidation in rats’ pancreas and heart by A. melegueta and A. danielli essential oils could prevent radical induced-ox-idative damage to cells and reduce the risk for the develop-ment of diabetes and hypertension.

4 ConclusionThis study revealed the chemical composition, antioxi-

dant, antidiabetic and antihypertensive activities of essen-tial oil from A. melegueta and A. danielli seeds. The es-

Fig. 7　 Inhibition of SNP-induced lipid peroxidation in rat’s pancreas homogenates by essential oils from A. danielli and A. melegueta seeds. Values represent means±of standard deviation of triplicate readings.

Fig. 8　 Inhibition of Fe2＋-induced lipid peroxidation in rat’s heart homogenates by essential oils from A. danielli and A. melegueta seeds. Values represent means±of standard deviation of triplicate readings.

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sential oils inhibited α-amylase, α-glucosidase and ACE activities and some pro-oxidant induced lipid peroxidation in rats’ pancreas and heart in vitro. While A. melegueta oil had higher antidiabetic activity than A. danielli, the latter had higher antihypertensive activity than the former. The observed biological activities of these oils were attrib-uted to the synergistic and/or additive effects of the chemi-cal constituents. However, further test could be carried out

to characterize the active principles responsible for these activities.

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