This study was designed to evaluate antioxidant and antihemolytic activities of Hyssopus angustifolius flower, stem and leaf methanol extracts by employing various in vitro assays. The leaf extract showed the best activity in DPPH (63.2 ± 2.3 μg mL-1) and H2O2  (55.6 ± 2.6 μg mL-1) models compared to the other extracts. However, flower extract exhibited the highest Fe2+ chelating activity (131.4 ± 4.4 μg mL-1). The extracts exhibited good antioxidant activity in linoleic acid peroxidation and reducing power assays, but were not comparable to vitamin C. The stem (23.58 ± 0.7 μg mL-1) and leaf (26.21 ± 1 μg mL-1) extracts showed highest level of antihemolytic activity than the flower extract

Alinezhad, H.

Azimi, R.

Baharfar, R.

Hellio, Claire

Nabavi, S.

Zare, M.

English

Portsmouth University Research Portal (Pure)

Antioxidant activities of fi ve different mulberry cultivars in Korea LWT - Food Sci.

Antioxidant activity of selected phenols and herbs used in diets for medical conditions.

Changes in total phenols, total fl avonoids, and antioxidant activities of common beans and pinto beans after soaking, cooking, and in vitro digestion process.

Interaction of fl avonoids with red blood cell membrane lipids and proteins: Antioxidant and antihemolytic effects.

Protection against hydrogen peroxide induced oxidative damage in rat erythrocytes by Mangifera indica L. peel extract. Food Chem.

Radical scavenging and iron-chelating activities of some greens used as traditional dishes in Mediterranean diet.

Toxicity and carcinogenicity studies of quercetin, a natural component of foods.

Antioxidant and antihemolytic activities of methanol extract of Hyssopus angustifolius

Fazel Nabavi, Seyed

Nabavi, Seyed Mohammad

Alinezhad, Heshmatollah

Zare, Mahboobeh

Azimi, Razieh

Bahafar, Robabeh

JKI Open Journal Systems (Julius Kühn-Institut)

Antioxidant activity of Hyssopus angustifoliusJournal of Applied Botany and Food Quality 85, 198 - 201 (2012)1Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran 2National Elites Foundation of Iran, Tehran, Iran3School of Biological Sciences, University of Portsmouth, King Henry Building, Portsmouth, UK 4Museum National d`Histoire Naturelle, Department Milieux et Peuplement Aquatiques, Paris, France5Department of Organic Chemistry, Faculty of Chemistry, University of Mazandaran, Babolsar, IranAntioxidant and antihemolytic activities of methanol extract of Hyssopus angustifolius Seyed Fazel Nabavi 1, 2, Seyed Mohammad Nabavi 1, 2*, Claire Hellio 3, 4, Heshmatollah Alinezhad 5, Mahboobeh Zare 5, Razieh Azimi 5, Robabeh Baharfar 5(Received August 9, 2012)* Corresponding authorSummaryThis study was designed to evaluate antioxidant and antihemolytic activities of Hyssopus angustifolius fl ower, stem and leaf methanol extracts by employing various in vitro assays. The leaf extract showed the best activity in DPPH (63.2 ± 2.3 µg mL-1) and H2O2 (55.6 ± 2.6 µg mL-1) models compared to the other extracts. However, fl ower extract exhibited the highest Fe2+ chelating activity (131.4 ± 4.4 µg mL-1). The extracts exhibited good antioxidant activity in linoleic acid peroxidation and reducing power assays, but were not comparable to vitamin C. The stem (23.58 ± 0.7 µg mL-1) and leaf (26.21 ± 1 µg mL-1) extracts showed highest level of antihemolytic activity than the fl ower extract. Introduction  There are increasing evidences that the consumption of polyphenolic compounds from natural sources may lower the risk of serious oxidative injuries such as atherosclerosis, infl ammatory processes, cancer and cardiovascular diseases as a result of their antioxidant activity (SURH, 2002).  Chelation therapy is one of the most known and reported therapeutic usages of antioxidants (GRAZUL and BUDZISZ, 2009). Chelation therapy reduces iron-related complications and improves survival time in patients with various diseases such as thalassemia major, cancer, HIV and Wilson’s disease (GRAZUL and BUDZISZ, 2009; HEBBEL et al., 1990). Numerous studies have demonstrated that chelators, such as desferrioxamine, have anti-proliferative effects against different type of cancers (RICHARDSON, 2002). Moreover, different synthetic chelators usage may have different side effects and therefore it remains an urgent need to identify new natural chelators with reduced side effects (PORTER, 1997).Previous studies demonstrate the direct role of nitric oxide radical in pathogenesis of different diseases, such as cancer, infl ammation, burn, and etc. (SREEJAYAN and RAO, 1997). The antioxidant may have the property to quench NO formation and prevent illnesses induced by excessive NO.Due to the safety and limitations of synthetic antioxidants, naturally originated antioxidants provide an interesting alternative to minimize the oxidative damage caused by NO and other oxidant agents (GHASEMZADEH et al., 2010). Hyssopus angustifolius is one of the most famous medicinal plants from the Lamiaceae family and is widely cultivated in several European countries such as Russia, Spain, France and Italy. It is used in tea blends for its antitussive and antispasmodic properties, and, to relieve catarrh (OMIDBAIGI, 2000). It appears that there is yet no scientifi c report about antioxidant and antihemolytic activities of methanol extracts of different parts of H. angustifolius. Thus, this study was carried out to determine antioxidant and antihemolytic activities of methanol extracts of H. angustifolius leaf, stem and fl ower, in order to better understand the medicinal uses of this plant.Materials and methods ChemicalsTrichloroacetic acid (TCA), Folin Ciocalteau, 1, 1-diphenyl-2-picryl hydrazyl (DPPH), hydrochloric acid, linoleic acid, ferrozine, butylated hydroxyanisole (BHA), quercetin, ascorbic acid, hydrogen peroxide, gallic acid, sodium nitroprusside and methanol were purchased from Sigma-Aldrich Chemical Company, USA. Sodium carbonate, potassium acetate, aluminium chloride (AlCl3), potassium ferricyanide, sulfanilamide, N-(1-naphthyl) ethylenediamine di-hydrochloride, EDTA and ferric chloride (FeCl3) were purchased from Merck Compagny, Germany. Other chemicals were purchased at analytical grade or purer.Plant materialAerial parts of Hyssopus angustifolius M. Bieb. has been collected from Veresk area (central Elburz Mountains Latitude: 35° 54’ N, Longitude: 52° 59’ E, altitude: 1900 above sea level,), Iran through spring 2010. The plant was authenticated at the Herbarium of Department of Biology in the University of Mazandaran (voucher specimen No 975).Extraction procedures Plant powder (100 g) was placed in a soxhlet extractor and extracted with methanol (3 liter) for 8 h. The solvent was recovered by distillation in vacuo, and the residue, stored in the desiccator, was used for subsequent experiments.Determination of total phenolic content Briefl y, 0.5 mL of sample (1.6 mg mL-1) was incubated with 2.5 mL of Folin-Ciocalteau reagent (0.2 N) for 5 min and then 2.0 mL aqueous solution of sodium carbonate (75 mg mL-1) was added. The absorbance of reactions mixture was measured at 760 nm after 2 h of incubation at room temperature. Total phenol content was calculated using gallic acid standard curve (AKILLIOGLU and KARAKAYA, 2010). Determination of fl avonoid contentBriefl y, extract (0.5 mL of a solution at 1.6 mg mL-1) was incubated with methanol (1.5 mL), aluminum chloride (0.1 mL, 10%), potassium acetate (0.1 mL, 1 M) and distilled water (2.8 ml) at room temperature for 30 min. Then, absorbance of sample was recorded at 415 nm by spectrophotometer (AKILLIOGLU and KARAKAYA, 2010). Quercetin was used for calibration curve preparation.Antioxidant activity of Hyssopus angustifolius 1991, 1-diphenyl-2-picryl hydrazyl (DPPH) radical scavengingEqual volumes of sample (25-400 µg mL-1) were added to methanol solution of DPPH (100 µM). Absorbance of reaction mixture was recorded at 517 nm after 15 min incubating at room temperature. The experiment was performed triplicate. Vitamin C, BHA and quercetin were used as standard antioxidants (VILLAÑO et al., 2007).Reducing powerFor determination of the reducing power ability of extracts, 2.5 mL of sample (25-400 µg mL-1) was mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1%), and, were incubated for 20 min (50°C). Then, 2.5 mL of trichloroacetic acid (10%) was added to the sample in order to  stop the reaction. Reaction mixture was the centrifuged (10 min, 1000 g). The upper layer of reaction mixture (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of FeCl3 (0.1%). Finally, absorbance of the sample was recorded at 700 nm (FERREIRA et al., 2007). Metal chelatingIron ions chelating by the extracts was examined according to the method of EL and KARAKAYA (2004). Sample (25-400 µg mL-1, 1 mL) was mixed with 0.05 mL of FeCl2 (2 mM) and 0.2 mL of ferrozine (5 mM). After 10 min incubation at room temperature, absorbance of the sample was recorded at 562 nm. Nitric oxide scavengingExtract (25-400 µg mL-1) was mixed with sodium nitroprusside (10 mM) and incubated for 150 min at 25°C. Then, 0.5 mL of Griess reagent (1% sulfanilamide, 2% phosphoric acid and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride) was added to the reaction mixture. Finally, absorbance of the sample was recorded at 546 nm (KUMARAN and KARUNAKARAN, 2006). Hydrogen peroxide scavengingExtract (2 mL, 0.1-1 mg mL-1) was mixed with 0.6 mL of 40 mM of hydrogen peroxide solution in phosphate buffer (pH 7.4). The absorbance of reaction mixture was recorded at 230 nm against a blank (GÜLÇIN et al., 2010). Hemoglobin-induced linoleic acid modelReaction mixture (2 mL) containing extract (25-400 µg mL-1), phosphate buffer (40 mmol L-1, pH 6.5), hemoglobin suspension (0.0016%) and linoleic acid emulsion (1 mmol L-1). Reaction mixture was incubated for 45 min (37°C). Then, 2.5 mL of ethanolic solution of hydrochloric acid (0.6%) was added to the sample for stopping lipid peroxidation process. The level of peroxidation was evaluated via thiocyanate method by recording the absorbance at 480 nm after adding 100 µl of FeCl2 (0.02 mol/L) and 50 µl of ammonium thiocyanate (0.3 g mL-1) (BAE and SUH, 2007). Preparation of rat erythrocytesMale Wistar rats (180-220 g) were purchased from Pasteur Institute of Iran, Amol research center. The animals were anesthetized via intraperitoneally administration of ketamine (60 mg/kg) and xylazine (5 mg/kg). Blood samples were collected through cardiac puncture. Erythrocytes of the blood samples were isolated and further stored at -60°C.Protective role of extracts against H2O2 induced hemolysisProtective role of the extracts against H2O2 induced hemolysis was evaluated according to the method of AJILA and PRASADA RAO (2008). Different concentrations of extracts (0.5 mL, 10-100 µg·mL-1) were mixed with 2mL of erythrocyte suspension (4%) and the volume of reaction mixture was made up to 5 mL with phosphate buffered saline. After 5 min incubation at 25°C, 0.5 mL of H2O2 solution was added to the reaction mixtures. After 240 min incubation at 25°C, reaction mixtures were centrifuged (2500 g, 10 min). Absorbance of the reaction mixture was recorded at 540 nm.Statistical analysis of the dataExperimental results are expressed as means ± SD. All measurements were replicated three times. The data were analyzed by an analysis of variance (p < 0.05) and the means separated by Duncan’s multiple range tests. The IC50 values were calculated from linear regression analysis.Results and discussion The total phenolic content of the methanol extract of fl owers, leaves and stems were respectively of 445 ± 22.1, 663.9 ± 33.1 and 349.6 ± 18.7 mg gallic acid equivalent g-1 of extract. Also, total fl avonoid contents of methanol extract of fl owers, leaves and stems were in the order of 122 ± 6.1, 131.7 ± 6.5 and 88.4 ± 4.3 mg quercetin equivalent g-1 of extract powder, respectively. Leaf extract revealed better DPPH radical scavenging activity (IC50 = 63.20 ± 2.32 µg mL-1) than others extracts tested, while in Fe2+ chelating activity, fl ower extract displayed the highest activity (IC50 = 131.40 ± 4.43 µg mL-1). The inhibition percentage of nitric oxide radical increased concomitantly with the concentration. In this assay, the leaf extract showed the highest nitric oxide-scavenging activity Tab. 1:  Antioxidant activities of fl owers, stems and leaves extracts of Hyssopus angustifoliusSamples DPPH free radical Nitric oxide H2O2 scavenging  Fe2+ chelating Antihemolytic   scavenging scavenging activity ability activityfl ower 149.6 ± 5.5 196 ± 6.4 177.9 ± 7.8 131.4 ± 4.4 65.3 ± 2.5leaf 63.2 ± 2.3 194.1 ± 4.1 55.6 ± 2.6 154.6 ± 7.1 26.2 ± 1stem 197.3 ± 6 259.7 ± 7.9 123.3 ± 3.9 211 ± 9.7 23.6 ± 0.7Vitamin C 5.05 ± 0.1 - 21.4 ± 1.1 - 235 ± 9EDTA - - - 18 ± 1.5 -Quercetin 5.3 ± 0.2 20 ± 0.1 52 ± 2.6 - -There is no signifi cant difference between quercetin and leaf extract in H2O2 scavenging models (p > 0.05) and between leaf and stem extract in antihemolytic activity (p > 0.05). IC50 (µg/mL)200 S.F. Nabavi, S.M. Nabavi, C. Hellio, H. Alinezhad, M. Zare, R. Azimi, R. Baharfar Antioxidant activity of Hyssopus angustifolius(IC50 = 194.13 ± 4.12 µg mL-1 vs. quercetin 20 ± 0.01 µg mL-1). Quercetin showed good NO radical scavenging activity but it has carcinogenic activity (DUNNICK and HAILEY 1992), therefore search for new natural NO quenching substances has been increased recently. The extracts were capable of scavenging hydrogen peroxide in a concentration dependent manner (Tab. 1). Vitamin C shows better activity than others. In reducing power assay, increasing absorbance of reaction mixture at 700 nm indicates an increase in reductive ability. Fig. 1 shows the dose dependent reducing power of the extracts, however no signifi cant differences (p> 0.05) were observed. Tested extracts showed good activity in hemoglobin-induced linoleic acid system (Fig. 2) with no signifi cant differences among them (p > 0.05). In addition, extracts did not show any side effects on erythrocytes. The best antihemolytic activity was observed while assessing the leaf and stem extracts (Tab. 1). Any substances which can donates hydrogen or electron, can change DPPH color from violet to yellow can be considered as a DPPH radical scavengers and, therefore as antioxidant (NABAVIet al., 2012b). Previously, CHAPOVA et al. (2010) reported potent DPPH radical scavenging activity for another species of Hyssopusgenus (Hyssopus offi cinalis L.) that was higher than the results in the present study, where H. angustifolius showed a moderate DPPH scavenging ability. This difference of activity may be associated with phenolic and fl avonoid contents (NABAVI et al., 2012b). Another assay that is related to the electron donating ability of this plant is the reducing power assay. In this assay, phenols or other electron donor compounds in the sample can reduce Fe3+ to Fe2+. The amount of Fe2+ complex monitored through reading absorbance of sample at 700 nm (FERREIRA et al., 2007). Fig. 1 showed the dose-response curves for the reducing power of extracts. They demonstrated very good activity, which was comparable to the vitamin C (p < 0.01). In iron ion chelating model, extracts and EDTA compete with ferrozine in capturing iron ion, and thus inhibited ferrozine-iron complex formation. Abnormally excessive NO radical production has a crucial role in the pathogenesis of numerous diseases such as sepsis and renal failure and therefore elimination of nitric oxide have therapeutic effects (SHAH et al., 2004). Also, NO scavengers inhibited chain reactions initiated by NO radicals that are harmful for human health. Hydrogen peroxide is not very reactive but it causes cytotoxicity through increasing of hydroxyl radicals in the cell. Therefore, hydrogen peroxide removal can help to protect human health (CHAUDHURI et al., 2007). Membrane lipids are rich in unsaturated fatty acids that are most susceptible to oxidative processes, mainly the linolenic and arachidonic acids, which are targets for lipid peroxidation (NABAVI et al., 2012C). Erythrocytes are known as main targets oxidative stress due to the presenting of membrane polyunsaturated fatty acids and also existence of the oxygen transport joined to hemoglobin molecules. Antihemolytic activity of fl avonoids has been previously reported and the good activity of extracts may be the result of high fl avonoid content(CHAUDHURI et al., 2007). Conclusion  In conclusion, Hyssopus angustifolius extracts can be used as a powerful herbal antioxidant. This activity may be associated with the presence of polyphenolic compounds. These results may explain some of the medicinal uses of Hyssopus angustifolius since the excessive production of reactive oxygen species have important role in the pathogenesis of several diseases.Author Disclosure StatementNo competing fi nancial interests exist.References AJILA, C.M., PRASADA RAO, U.J.S., 2008: Protection against hydrogen peroxide induced oxidative damage in rat erythrocytes by Mangifera indica L. peel extract. Food Chem. Toxicol. 46, 303-309.AKILLIOGLU, H.G., KARAKAYA, S., 2010: Changes in total phenols, total fl avonoids, and antioxidant activities of common beans and pinto beans after soaking, cooking, and in vitro digestion process. Food Sci. Biotechnol. 19, 633-639. BAE, S.H., SUH, H.J., 2007: Antioxidant activities of fi ve different mulberry cultivars in Korea LWT - Food Sci. Technol. 40, 955-962.CHAUDHURI, S., BANERJEE, A., BASU, K., SENGUPTA, B., SENGUPTA, P.K., 2007: Interaction of fl avonoids with red blood cell membrane lipids and proteins: Antioxidant and antihemolytic effects. Int. J. Biol. Macromol. 41, 42-48.CHAPOVA, D., KOURIMSKA, L., GORDON, M.H., HERMANOVA, V., ROUBICKOVA, I., PANEK, J., 2010: Antioxidant activity of selected phenols and herbs used in diets for medical conditions. Czech J. Food Sci. 28, 317-325.Fig. 1: Reducing power activity of methanolic extracts of leaf, stem and fl owers of Hyssopus angustifolius. Vitamin C is used as standard. Fig. 2: Antioxidant activity of leaf, stem and fl owers of Hyssopus angus-tifolius against linoleic acid peroxidation induced by hemoglobin. Vitamin C is used as standard.  Antioxidant activity of Hyssopus angustifolius 201DUNNICK, J.K., HAILEY, J.R., 1992: Toxicity and carcinogenicity studies of quercetin, a natural component of foods. Fund. Appl. Toxicol. 19, 423-431. EL, S.N., KARAKAYA, S., 2004: Radical scavenging and iron-chelating activities of some greens used as traditional dishes in Mediterranean diet. Int. J. Food Sci. Nutr. 55, 67-74.FERREIRA, I.C.F.R., BAPTISTA, P., VILAS-BOAS, M., BARROS, L., 2007: Free-radical scavenging capacity and reducing power of wild edible mushrooms from northeast Portugal: Individual cap and stipe activity. Food Chem. 100, 1511-1516.GHASEMZADEH, A., JAAFAR, H.Z.E., RAHMAT, A., 2010: Antioxidant activities, total phenolics and fl avonoids content in two varieties of Malaysia Young Ginger (Zingiber offi cinale Roscoe). 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Toxicol. 88, 486-490 NABAVI, S.M., NABAVI, S.F., ALINEZHAD, H., ZARE, M., Azimi, R., 2012b: Biological activities of fl avonoid-rich fractions of Eryngium Caucasicum Trautv. Eur. Rev. Med. Pharmacol. Sci. 16 Suppl 3, 81-87.NABAVI, S.M., NABAVI, S.F., ESLAMI, S., MOGHADDAM, A.H., 2012c: In vivo protective effects of quercetin against sodium fl uoride-induced oxidative stress in the hepatic tissue. Food Chem. 132, 931-935 OMIDBAIGI, R., 2000: Production and processing of medicinal plants, Vol. 3, Astan Quds Razavi Publications, Behnashr Co. Mashad, Iran.PORTER, J.B., 1997: A risk-benefi t assessment of iron-chelation therapy. Drug Safety 17, 407-421.RICHARDSON, D.R., 2002: Iron chelators as therapeutic agents for the treatment of cancer. Crit. Rev. Oncol. Hematol. 42, 267-281.SHAH, V., LYFORD, G., GORES, G., FARRUGIA, G., 2004: Nitric oxide in gastrointestinal health and disease. Gastroenterology 126, 903-913.SREEJAYAN, N., RAO, M.N.A., 1997:  Nitric oxide scavenging by curcumi-noids. J. Pharm. Pharmacol. 49, 105-107.SURH, Y.J., 2002: Anti-tumor promoting potential of selected spice ingredients with antioxidative and anti-infl ammatory activities: a short review. Food Chem. Toxicol. 40, 1091-1097.VILLANO, D., FERNANDEZ-PACHON, M.S., MOYA, M.L., TRONCOSO, A.M., GARCIA-PARRILLA, M.C., 2007: Radical scavenging ability of polyphenolic compounds towards DPPH free radical Talanta 71, 230-235.Address of the corresponding author:Seyed Mohammad Nabavi, Applied Biotechnology Research Center, Baqi-yatallah University of Medical Sciences, Tehran, Iran, P.O. Box 19395-5487; E-mail: Nabavi208@gmail.com

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Antioxidant and antihemolytic activities of methanol extract of Hyssopus angustifolius

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