Scholarly article on topic 'Polyphenols content, antioxidant and antiviral activities of leaf extracts of Marrubium deserti growing in Tunisia'

Polyphenols content, antioxidant and antiviral activities of leaf extracts of Marrubium deserti growing in Tunisia Academic research paper on "Biological sciences"

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{"Antioxidant activity" / "Antiviral activity" / " Marrubium deserti de Noé" / "Total phenolic content"}

Abstract of research paper on Biological sciences, author of scientific article — H. Edziri, M. Mastouri, M. Aouni, L. Verschaeve

Abstract Marrubium deserti de Noé is used in traditional medicine, for example as a remedy for asthma and diabetes, and was also found to have antibacterial properties. It is of particular interest in Tunisia and this is why we continued research on this plant. We here report on an investigation of its antioxidant and antiviral activities of petroleum ether, ethyl acetate, chloroform, butanol and methanol extracts. Total phenolic content of the extracts was determined by the Folin–Ciocalteu colorimetric method. The antioxidant activity was evaluated with the DPPH assay, the ABTS test and ammonium thiocyanate method. The antiviral activity was evaluated against human cytomegalovirus (HCMV) strain AD-169 (ATCC Ref. VR 538) and Coxsackie B virus type 3 (CoxB-3) using a cytopathic effect (CPE) reduction assay. With regards to IC50 values (50% inhibitory concentration) of scavenging abilities of the DPPH radical, methanol extract exhibited important antioxidant activities with IC50 of 0.150mg/ml. The ethyl acetate extract showed the highest antioxidant activity using the linoleic acid emulsion system (79.23%). The butanol, methanol and ethyl acetate extracts showed significant antiviral activity against Coxsackie B3 virus. In conclusion, these results can be considered very promising and justify further research, amongst others on the identification of antioxidant and antiviral components in the active extracts.

Academic research paper on topic "Polyphenols content, antioxidant and antiviral activities of leaf extracts of Marrubium deserti growing in Tunisia"

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South African Journal of Botany 80 (2012) 104 - 109

www.elsevier.com/locate/sajb

Polyphenols content, antioxidant and antiviral activities of leaf extracts of

Marrubium deserti growing in Tunisia

H. Edziri a *, M. Mastouri b, M. Aouni a, L. Verschaeve c,d

a Laboratoire des Maladies Transmissibles et des Substances Biologiquement Actives, Faculté de Pharmacie, 5000 Monastir, Tunisia b Laboratoire de Microbiologie, CHU Fattouma Bouguiba, 5000 Monastir, Tunisia c Scientific Institute of Public Health, O.D. Public Health and Surveillance, Laboratory of Toxicology, Brussels, Belgium d University of Antwerp, Department of Biomedical Sciences, Antwerp, Belgium

Received 5 December 2011; received in revised form 17 February 2012; accepted 7 March 2012

Abstract

Marrubium deserti de Noe is used in traditional medicine, for example as a remedy for asthma and diabetes, and was also found to have antibacterial properties. It is of particular interest in Tunisia and this is why we continued research on this plant. We here report on an investigation of its antioxidant and antiviral activities of petroleum ether, ethyl acetate, chloroform, butanol and methanol extracts. Total phenolic content of the extracts was determined by the Folin-Ciocalteu colorimetric method. The antioxidant activity was evaluated with the DPPH assay, the ABTS test and ammonium thiocyanate method. The antiviral activity was evaluated against human cytomegalovirus (HCMV) strain AD-169 (ATCC Ref. VR 538) and Coxsackie B virus type 3 (CoxB-3) using a cytopathic effect (CPE) reduction assay. With regards to IC5o values (50% inhibitory concentration) of scavenging abilities of the DPPH radical, methanol extract exhibited important antioxidant activities with IC50 of 0.150 mg/ml. The ethyl acetate extract showed the highest antioxidant activity using the linoleic acid emulsion system (79.23%). The butanol, methanol and ethyl acetate extracts showed significant antiviral activity against Coxsackie B3 virus. In conclusion, these results can be considered very promising and justify further research, amongst others on the identification of antioxidant and antiviral components in the active extracts. © 2012 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Antioxidant activity; Antiviral activity; Marrubium deserti de Noe; Total phenolic content

1. Introduction

Lipid peroxidation is a complex process occurring in aerobic cells which reflects the interaction between molecular oxygen and polyunsaturated fatty acids. Formation of free radicals plays an important role in ageing and cancer promotion (Ashok and Ali, 1999) which means that antioxidants are also important in health protection. Several synthetic antioxidants such as buty-rated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) may be inappropriate for chronic human consumption as recent publications have mentioned their possible toxic properties for human health and environment (Ito et al., 1986; Stich,

* Corresponding author. Tel.: +216 73629254; fax: +216 73460830. E-mail address: jaziri_hayet@yahoo.fr (H. Edziri).

1991). Therefore, the interest in natural (non toxic) antioxi-dants, especially of plant origin, has greatly increased in recent years (Jayaprakasha and Jaganmohan, 2000). Natural antioxi-dants can protect the human body free radicals that may cause some chronic diseases (Kinsella et al., 1993; Lai et al., 2001). Research conducted on the antioxidant activities of some plants generally focused on the herbs and aromatic plants (Miliauskas et al., 2004; Pizzale et al., 2002). The antioxidant properties of plant extracts have been attributed to their polyphenol contents (Lu and Foo, 2001; Murthy et al., 2002; Revilla and Ryan, 2000).

Moreover, the presence and growth of pathogenic microorganisms (bacteria, mould, viruses, fungi) in food may cause its spoilage and result in a reduction in its quality and quantity (Soliman and Badeaa, 2002). These microbial contaminations

0254-6299/$ -see front matter © 2012 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2012.03.001

still cause important public health and economic concerns for human society. Many medicinal and aromatic plants have good antimicrobial activity (Candan et al., 2003; Nychas, 1995).

The genus Marrubium is represented by 97 species which are widely spread over the temperate and warm regions. Marrubium species are indigenous in Europe, the Mediterranean area and in Asia (Wattand and Breyer-Brandwizk, 1962). Many Marrubium species are reported in the literature to be used in folk medicine (Lewis, 1977; Mabberly, 1997). Marrubium deserti is a common plant in Tunisia and is used in traditional medicine in the form of a decoction as a remedy for asthma, diabetes and as a diuretic. Recent research demonstrated the antibacterial activity of M. deserti extracts (Edziri et al., 2007). Antimicrobial and antioxidant activities were also described in a few other studies (e.g., Laouer et al., 2008; Zaabat et al., 2011). The aim ofthis study was to further investigate the antioxidant and antiviral properties of leaf extracts of M. deserti de Noe growing in Tunisia.

2. Material and methods

2.1. Plant materials

Fresh leaves from M. deserti de Noe (Lamiaceae) were collected in June 2009 in Hamma (South Tunisian). The plant was identified by Pr. Mohamed Chaieb, botanist at the University of Science (Sfax, Tunisia). A voucher specimen (M.D.01) was deposited in the biological laboratory of the faculty of pharmacy of Monastir.

2.2. Extraction

250 g of dried M. deserti de Noe was extracted at room temperature with methanol (MeOH) through maceration for five days. The unfiltered resulting extract was evaporated at reduced pressure to obtain a green residue (119 g) which was successively further extracted with petroleum ether, chloroform and ethyl acetate. All extracts were concentrated to dryness and kept at - 20 °C until use.

2.3. Determination of total phenolic content

Total phenolic content in each extract was determined using Folin-Ciocalteus reagent according to the method of Singleton and Rosi (1965). Forty microliters of extract (1 mg/ml) was mixed with 200 pl (1N) Folin-Ciocalteus reagent (Sigma-Aldrich, Germany) and 1160 pl of distilled water, followed by 600 pl 20% sodium carbonate (Na2CO3) 3 min later. The mixture was shaken for 2 h at room temperature and absorbance was measured at 765 nm. All tests were performed in triplicate. Gallic acid (Sigma-Aldrich, Germany) was used as a standard. The concentration of total phenolic compounds (TPC) was determined as pg gallic acid equivalents (GAE)/mg extract using the following equation obtained from a standard gallic acid graph (R2 =0.9877; Singleton and Rossi, 1965):

absorbance = 0.0012 x gallic acid(pg)-0.0034

2.4. Antioxidant activity

2.4.1. Scavenging ability on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical

Antioxidant activity was determined spectrophotometrically according to the slightly modified method of Burits and Bucar (2000) using stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical as a reagent. A 0.5 ml aliquot of the test sample in a specified concentration of ethanol (EtOH) was added to a 0.003% (w/v) EtOH solution of DPPH (0.5 ml). The mixture was shaken vigorously and left to stand for 30 min in the dark, and the absorbance was then measured at 517 nm against a blank. The inhibitory concentration (mg extract/ml) at which 50% DPPH radicals were scavenged (IC50) was obtained by interpolation from linear regression analysis. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid ) was used as a positive control.

2.4.2. Scavenging ability on ABTS radical cation

The Trolox Equivalent Antioxidant Capacity (TEAC) assay is based on the scavenging of the radical ABTS+ [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)], which is converted into a colorless product. The standard TEAC assay described by Re et al. (1999) was used with minor modification. This assay assesses the ability with phosphate-buffer saline (pH 7.4) to give a final absorbance of 0.7+0.1 at 734 nm at 37 °C. Stock Trolox and stock extract solutions were prepared in EtOH. An aliquot (10 pl) of the test solution (or pure solvent, for the blank) was added to 7.4 mM ABTS+ Solution (990 pl) and the absorbance at 734 nm was measured. Antioxidant capacity of test samples was expressed as IC50 (defined as the concentration necessary for 50% reduction in ABTS + concentration) and as TEAC. In this assay Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-car-boxylic acid) was used as a positive control.

2.4.3. Antioxidant activity by ammonium thiocyanate method

The lipid peroxidation inhibition activity was measured by

ammonium thiocyanate method (Mitsuda et al., 1966). Each sample (1.3 mg/ml) in 0.5 ml of absolute ethanol was mixed with a linoleic acid emulsion (2.5 ml, 0.02 mol, pH 7.0) in phosphate buffer (2 ml, 0.2 mol, pH 7.0). The linoleic acid emulsion was prepared by mixing and homogenizing 0.2804 g of linoleic acid, 0.2804 g of Tween 20 as emulsifier, and 50 ml phosphate buffer. The reaction mixture was incubated at 37 °C. Aliquots of 0.1 ml were taken at several intervals during incubation. The degree of oxidation was measured by sequentially addition of ethanol (4.7 ml, 75%), ammonium thiocyanate (0.1 ml, 30%), sample solution (0.1 ml), and ferrous chloride (0.1 ml, 0.02 mol in 3.5% HCl). After the mixture was incubated for 3 min, the peroxide value was determined by monitoring absorbance at 500 nm with a spectrophotometer. This was done every 24 h until a day after that the absorbance of the control reached its maximum. The control consisted of linoleic acid without sample. The % lipid peroxidation inhibition (LPI) was calculated as:

LPI(%) = [1-(Absorbance of sample/Absorbance of control)] x 100

2.5. Antiviral activity

2.5.1. Cell line and growth condition

Human diploid embryonic lung fibroblasts (MRC-5) (Bio-merieux-France) were grown in MEM-D (Seromed, Germany, Ref. T041-05) supplemented with 10% fetal calf serum (FCS Ref. S0115, Seromed, Germany), 100 units/ml penicillin, 100 |g/ml streptomycin. FCS was reduced to 2% for the experiments involving viral infection. The cells were incubated for 3 days at 37 °C in a humidified atmosphere of 5% CO2 until formation of a confluent monolayer of cells.

2.5.2. Cell toxicity assay

The evaluation is based on the reduction of MTT, (3-(4.5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) by mi-tochondrial dehydrogenase of viable cells, to give a blue forma-zan product which can be measured spectrophotometrically. The MTT colorometric assay was performed in 96-well plates (Polydoro et al., 2004). Cells were seeded in a 96-wells plate at a concentration of 5 x 104 cells/well and incubated for 24 h at 37 °C in a 5% CO2 enriched atmosphere. After treatment with various concentration of each extract (100, 200, 400 and 800 |g/ml), the cells were incubated for an additional 48 h at 37 °C. After that, the medium was removed and cells in each well were incubated with 50 |l of MTT solution (5 mg/ml) for 4 h at 37 °C. MTT solution was then discarded and 50 |l dimethyl sulfoxide (DMSO) were added for 20 min to dissolve insoluble formazan crystal. Optical density was measured at 540 nm after removal of the DMSO. Data were obtained from triplicate wells. The concentration (|ig/ml) of substrate that causes 50% death of cells (CC50) was calculated and analyzed according to the Litchfield and Wilcoxon test (1949).

2.5.3. Titration of viruses

Human cytomegalovirus (HCMV) strain AD-169 (ATCC Ref. VR 538) and coxsakie virus type B3 were used to investigate the antiviral activity of the extracts. Serial 10-fold dilution (10-1-10-5) were prepared in MEM containing 2% FCS. Virus dilutions were inoculated into confluent cells in quadruplicate wells of 96-well plates and incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 3-5 days. When a cytopathic effect CPE in the virus-infected cells was observed microscopically, TCID50 (the 50% tissue culture infective dose) was determined by the method of Reed and Muench (1938).

2.5.4. Antiviral activity assay

We performed a cytopathic effect (CPE) reduction assay for screening the antiviral activities of the plant extracts. In brief to confluent cell monolayers in a 96-well plate, 100 TCID50 (50% tissue culture-infective dose) virus suspension and serial twofold dilutions of crude extracts were added simultaneously. As a positive control, cells were infected with the same concentration of virus but without the addition of extract, and as a negative control, only MEM-D was added to the cells. The plates were incubated at 37 °C in a humidified CO2 atmosphere for 3-5 days. The concentration that reduced 50% of CPE in respect to the virus control was estimated from the plots of the

data and was defined as the 50% inhibitory concentration (IC50). The selective index (SI) was calculated from the ratio CC50/IC50 (Kujumgiev etal., 1999).

3. Results and discussion

3.1. Yield and antioxidant activity

The antioxidant activity of plant extracts has been correlated to their total phenolic content (TPC) due to their ability to scavenge free radicals (Revilla and Ryan, 2000). The total phenolic content of the different extracts of M. deserti is given in Fig. 1. The total phenolic content varied from 118 to 26.6 p,gGAE/ mg. We can conclude that methanol and butanol extracts had the highest total phenolic contents.

The inhibitory concentrations of each fraction and reference compound (Trolox) required to scavenge 50% of the DPPH radical, the IC50 values in antioxidant activity and the yield of each plant extract are presented in Table 1. It shows that methanol, butanol and ethyl acetate extracts have important antioxi-dant potencies with IC50 values of 0.150, 0.850 and 0.950 mg/ ml, respectively. Furthermore, methanol extracts showed to be the most potent free radical scavenger (IC50 = 0.35 p,g/ml).

The TEAC of each fraction at 20 min was also determined. Since TEAC measures the effective antioxidant activity of the extract, a higher TEAC would imply greater antioxidant activity of the sample. The methanol fraction showed the highest TEAC value (0.834 mM; Table 1).

The antioxidant effects of the extracts from M. deserti and a-tocopherol on the peroxidation of linoleic acid are presented in Fig. 2. Oxidation of linoleic acid was effectively inhibited by the ethyl acetate fraction (79.23 ±0.12%) and butanol and petroleum ether fractions (77.13±0.12, 77.00±0.02) respectively. These values were comparable to those obtained with a-tocopherol (82.51 ±0.14).

Antioxidants are believed to play a significant role in the body's defense system against free radicals. Numerous reports have described antioxidants and other compounds with radical-scavenging activity present in fruits, vegetables, herbs and cereals

UJ 120 <

2> 100-

01 ■C

Fig. 1. Total phenolic content of M. deserti extracts (|ig gallic acid equivalents (GAE)/ml extract. M: methanol extract; But: butanol extract; Ch: chloroform extract; AE: ethyl acetate extract; EP: petroleum ether extract.

Table 1

Effects of extracts of Marrubium deserti on the in vitro free radical (DPPH, ABTS).

Fractions Yield (%) IC50 (mg/ml)

DPPH ABTS (20 min) TEAC at

20 min

Petroleum ether 2.71 2.50±0.5 > 20 0.248

Chloroform 10.71 1.25±0.12 13±1.13 0.367

Ethyl acetate 0.42 0.95±0.01 14± 1.30 0.380

Butanol 0.57 0.85±0.02 17±0.50 0.231

Methanol 2.57 0.15±0.05 0.35±1.2 0.834

Trolox - 23.1±0.01 0.12±0,02 -

TEAC: The Trolox Equivalent Antioxidant Capacity. ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). nd: not determined. a IC50: concentration of sample required to scavenge 50% of free radicals

extracts (Gray et al., 2002; Nuutila et al., 2003). The DPPH radical was widely used to evaluate the free-radical scavenging capacity of antioxidants (Cotelle et al., 1996). We can conclude that the methanol extract was the most effective in this respect.

Antioxidant activity of natural antioxidants has been shown to be involved in termination of free radical reaction (Tanaka et al., 1988). Phenols were known to be effective antioxidants (Madhavi et al., 1996) due to their scavenging abilities on free radicals and chelating abilities on ferrous ions (Lotito and Fraga, 1988). As a matter of fact, antioxidant activities of plant extracts are generally ascribed to their phenolic content (Lu and Foo, 2001; Miliauskas et al., 2004; Murthy et al., 2002). As methanol, chloroform and ethyl acetate extracts showed high-antioxidant activity also, this activity may be directly correlated to their high-phenolic contents. The key role of phenolic compounds as scavengers of free radicals is emphasized in several reports (Katalinic et al., 2006; Theriault et al., 2006).

Oxidation of linoleic acid was effectively inhibited by the ethyl acetate-, butanol- and petroleum ether fractions. It seems that this activity is probably also related to the presence of phenolic compounds in M. deserti extracts such as phenolic acids.

Fig. 2. Antioxidant activity of M. deserti extracts by ammonium thiocyanate method. aEach value is the mean±SD of triplicate measurements; TCP: a-tocopherol; M: methanol extract; But: butanol extract; CH: chloroform extract; AE: ethyl acetate extract; EP: petroleum ether extract.

3.2. Antiviral activity

Viral infections are accompanied with profound changes in cell/tissue metabolism, which lead to generation of reactive oxygen species. The latter may enhance the pathogenesis of the infection. It was recently found that the main cause of mortality from influenza virus-induced pneumonia is cytotoxicity, which is determined by a considerably increased level of O2 before viral replication in the bronchial epithelial cells occurs (Akaike et al., 1996). Therefore the use of antioxidants can be of great value in preventing the inception or the progression of the disease.

Several studies made a comparative evaluation of the antioxi-dant and the antiviral activities of plant extracts and naturals substances. Aruoma et al. (1996) proved that extracts from rosemary and Provencal herbs showed potential antioxidant and anti-HIV activities. Euphorbia thymifolia L. was shown to possess antioxidant and anti-HSV-2 activity (Lin et al., 2002a, 2002b). Some fractions and flavonoids and proanthocyanidins, obtained from Crataegus sinaica were also found to possess anti HSV-1 and antioxidant activities (Shahat et al., 2002).

Sokmen et al. (2004) found that various extracts from Origanum acutidens had good antioxidant and anti-influenza virus activities. The observed anti-Coxsackie B3 activity may be due to the higher amount of phenolic compounds particularly flavonoids and tannins known to possess good antiviral activities (Fukuchi et al., 1989; Namba et al., 1998).

Estimation of the antiviral activity was in this study based on the cytopathic effect (CPE) of the virus-infected confluent monolayer of MRC 5 cells. The mean IC50, CC50 and SI (CC50/IC50) values are shown in Table 2. We can conclude that all extracts were not toxic against MRC5 cells (IC50 > 500 pg/ml). Methanol, butanol and ethyl acetate extracts showed significant anti-coxsakie B3 activity with IC50 values ranging from 100 to 135 pg/ml and with a selective index (SI) above 3. All extracts showed weak anti HCMV activity with IC50 values from 200 up to 450 pg/ml.

Table 2

Antiviral activity of M. deserti extracts.

Anti-CMV Anti-CoxB3

Extracts aIC50 bCC50 SIc IC50 SIc

(pg/ml)a (pg/ml)b (pg/ml)

Petroleum ether 450 >500 >1.1 200 >2.5

Ethyl acetate 250 >500 >2 135 >3.7

Chloroform 250 >500 >2 150 >3.3

Butanole 200 >500 >2.5 100 >5

Methanol 200 >500 >2.5 100 >5

Ribavirine d Nt >250 nt 130 > 1.9

Ganciclovire d 0.8 >200 >250 Nd nd

a IC50 is the concentration of the sample required to inhibit 50% virus-induced CPE. The blank parts in the table denote no inhibitory effect on virus-induced CPE in MNCC.

b CC50 is the concentration of the 50% cytotoxic effect. c SI (selective index) is the ratio CC50/IC50. d Not determined.

4. Conclusion

In conclusion, our study on the in vitro antioxidant, and antiviral properties of extracts prepared from M. deserti de Noe leaves shows that butanol and ethyl acetate extracts showed effective antioxidant properties by the DPPH assay. Methanol extract had the lowest IC50 according to the ABTS method and ethyl acetate extract showed the highest antioxidant activity with the linoleic acid emulsion system. We also found that bu-tanol, methanol and ethyl acetate extracts exhibit significant antiviral activity against Coxsackie B3 virus. All these activities were correlated with the higher amount of TPC in these active extracts. These promising results justify further research. We are presently trying to isolate and identify the antioxidant and antiviral components in these active extracts.

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Edited by J Van Staden