Antimicrobial and Antioxidant Activities of Various Extractsof Verbascum antiochium Boiss. (Scrophulariaceae)

Birgul Ozcan, Miray Yilmaz, and Mahmut Caliskan

Biology Department, Sciences and Letters Faculty, Mustafa Kemal University, Hatay, Turkey

ABSTRACT Verbascum antiochium Boiss., a member of the Scrophulariaceae family, is endemic to Turkey. The extracts

obtained from V. antiochium by increased polarity and direct methanol extraction were tested by the agar well diffusion

method against various Gram-positive and Gram-negative bacteria and one fungus. The methanol/water extract exhibited a

larger inhibition zone against both the Gram-negative and Gram-positive bacteria than the other extracts. Haemophilus

influenzae was found to be the most sensitive bacterium among the bacteria tested. The antioxidant activities of the methanolic

extract of V. antiochium were examined by two complementary test systems. The 50% inhibition activity of the methanolic

extract of V. antiochium against the free radical 2,2-diphenyl-1-picrylhydrazyl was determined as 4.80 mg/mL. In the case of

the linoleic acid system, oxidation of linoleic acid was inhibited by the methanolic extract of V. antiochium with 79.92%

inhibition, which is close to the value of the synthetic antioxidant reagent, tert-butylated hydroxytoluene. The total phenolic

components of V. antiochium were determined to be 92.71 mg of gallic acid equivalents/g. Iridoid glycosides, flavonoids, and

saponins were detected as the major chemical constituents in the extract.

KEY WORDS: � antimicrobial � antioxidant � total phenols � Verbascum antiochium

INTRODUCTION

Agreat number of plants contain chemical com-pounds that exhibit antioxidant and antimicrobial

properties.1–3 Various preparations of these plants have beenused as expectorants, mucolytics, sudorifics, sedatives, di-uretics, and constipates in traditional Turkish medicine.4

Many plants are used in rural areas of Turkey for the treat-ment of several diseases, including microbial infections, foremetic and strengthening effects, and for increasing theexcretion of urine and decreasing blood pressure.5

Based on the literature, there are approximately 9,000plant species in Turkey’s flora, of which more than 500 arewidely used in folkloric medicine.6 The genus Verbascumbelonging to the Scrophulariaceae family is the richestgenus, represented in Turkish flora by 232 species, of which185 are endemic.6 Some species of the Verbascum genushave been used to treat internal and external infections bymany societies in all around world.7 Verbascum species arewidespread in Turkey and have been used for their ethno-pharmacological effects for centuries.8 Seeds of Verbascumspecies are poisonous because of the saponins they contain.People in Turkey use these poisonous seeds for huntingfish.5 Verbascum thapsus (mullein), one of the best-known

Verbascum species, has been used as a remedy for irritatingand dry coughs, respiratory tract, tuberculosis, asthma, andhoarseness.8 Verbascum phlomoides and Verbascum densi-florum have been used for their ethnopharmacological ef-fects among the people in Turkey. In particular, their flowershave been used for diuretic and expectorant effects, and theirleaves have also been used for their diuretic, expectorant,and sedative effects.9

Although several studies have been conducted to displaythe antimicrobial and antioxidant activity of several Ver-bascum species,10–13 Verbascum antiochium has not beenstudied yet in terms of its antimicrobial and antioxidantactivities. In this study, the extracts obtained from V. anti-ochium by increased polarity and direct methanol extractionwere tested by the agar well diffusion method against var-ious Gram-positive and Gram-negative bacteria and onefungus. The possible antioxidant activity of the methanolicextract of V. antiochium was assessed with two comple-mentary test systems, the 2,2-diphenyl-1-picrylhydrazyl(DPPH) and b-carotene-linoleic acid assays. Furthermore,total amounts of phenolics and chemical constituents of theextracts were determined.

MATERIALS AND METHODS

Plant material

V. antiochium Boiss. plants were collected between Juneand July 2007 from Cevlik-Batiayaz, Samandag, Hatay,

Manuscript received 15 September 2009. Revision accepted 22 November 2009.

Address correspondence to: Asst. Prof. Dr. Birgul Ozcan, Biology Department, Sciencesand Letters Faculty, Mustafa Kemal University, Hatay, Turkey, E-mail: birgulozcan@gmail.com

JOURNAL OF MEDICINAL FOODJ Med Food 13 (5) 2010, 1147–1152# Mary Ann Liebert, Inc. and Korean Society of Food Science and NutritionDOI: 10.1089/jmf.2009.0213

1147

Turkey. The voucher specimen was identified by Dr. HikmetYolcu at the Department of Biology, Mustafa Kemal Uni-versity, Hatay.

Preparation of extracts

Air-dried herbal parts of V. antiochium were subjected tothe different extraction procedures given below.

Preparation of increased polarity extracts (hexane,dichloromethane, and methanol extracts). The plant partswere air-dried, and then dry powdered plant material (20 g)was extracted with 150 mL of hexane, followed by di-chloromethane (150 mL) and methanol (150 mL), in aSoxhlet apparatus for 6 hours for each solvent. The metha-nolic extract was suspended in water and partitioned withchloroform to obtain polar and nonpolar subfractions.14

Preparation of direct methanol extracts. The plant partswere air-dried, and then dry powdered plant material (20 g)was extracted with 150 mL of methanol in a Soxhlet appa-ratus for 8 hours. All extracts were filtered, and the filtrateswere then evaporated under reduced pressure and driedusing a rotary evaporator at 508C.

Antioxidant activity

DPPH assay. The DPPH test was carried out as ex-plained before.15,16 Fifty microliters of various dilutions ofthe extract was mixed with 5 mL of 0.004% methanol so-lution of DPPH. After an incubation period of 30 minutes,the absorbance of the samples was read at 517 nm in aBiospec-mini Shimadzu spectrophotometer (ShimadzuBiotech, Kyoto, Japan). tert-Butylated hydroxytoluene(BHT) and ascorbic acid were used as positive controls.Inhibition of free radical DPPH as a percentage (I%) wascalculated as follows:

I%¼ ([Ablank�Asample]=Ablank) · 100

where Ablank is the absorbance of the control reaction(without test compound) and Asample is the absorbance of thetest compound. The concentration of extract that provide the50% inhibition was calculated from the graph plotting in-hibition percentage against extract concentration.

b-Carotene/linoleic acid assay. This assay was used tovisualize instrumentally bleaching of the carotene/linoleicacid solution.17 The model test mixture was prepared bydissolving 0.5 mg of b-carotene in 1 mL of chloroform; 25mLof linoleic acid and 200 mg of Tween-40 were added to the b-carotene solution. Chloroform was removed using a rotaryevaporator at 508C. Distilled water (100 mL) saturated withoxygen for 30 minutes (flow rate, 100 mL/minute) was added,and the mixture was vigorously shaken. Two hundred fiftymicroliters of this model test mixture was distributed into eachof the test tubes. Thirty-five microliters of an ethanolic solu-tion of the extract was added to each test tube. The test tubeswere then placed in an incubator at 558C for 48 hours. After

the incubation period the absorbance was measured at490 nm. An equal amount of ethanol was used for the blanksamples, and BHT was used as the synthetic antioxidant.

Phytochemical screening of the methanolic extracts

The screening of chemical constituents was carried outwith the methanol extracts using chemical methods and thin-layer chromatography. Precoated thin-layer chromatogra-phy silica gel 60F254 (10 cm�10 cm, Merck, Darmstadt,Germany) and different mixtures of organic solvents asmobile phases, e.g., toluene:ethyl acetate:formic acid,chloroform:methanol:H2O, were used in the analyticalprotocol. Several chemical reagents, e.g., DPPH-, vanillin-H2SO4, and Naturstoff’s, Dragendorf’s, and Borntraeger’sreagents, among others, were used in the detection accord-ing to the methodology given by Wagner and Bladt.18

Moreover, the high-performance liquid chromatographyfingerprint of the extracts was established using a Restek(Bellefonte PA, USA) Pinnacle II C18 (particle size, 5 mm)column (250 mm�4.6 mm�5 mm) with the isocratic elutionsolvent system composed of an acetonitrile-water solution(20%). The detection wavelength was 210 nm, and the col-umn temperature was 308C.

Determination of total phenolics. Total phenolic com-pounds in the V. antiochium extract were determined by theFolin-Ciocalteu colorimetric method.19 The amount of totalphenolics in the methanolic extract of V. antiochium wasexpressed as gallic acid equivalents (GAE) (in mg of GAE/gof extract). Measurements were taken in triplicate.

Antimicrobial activity

Microorganisms. The various extracts of V. antiochiumwere individually tested on different microorganisms, in-cluding Staphylococcus aureus ATCC 29213, S. aureusATCC 25923, S. aureus ATCC 43300 (methicillin-resistant), Bacillus subtilis ATCC 6633, Enterococcusgallinarium CDC-NJ-4, Enterococcus faecium NJ-1, En-terococcus faecalis ATCC 29212, Streptococcus pyogenesATCC 19615, Listeria monocytogenes ATCC 7644, Es-cherichia coli O157:H7, E. coli ATCC 25922, E. coliATCC35218, Salmonella typhi NCTC 8394, Haemophilusinfluenzae ATCC 49247, Pseudomonas aeruginosa ATCC27853, and Candida albicans ATCC 10231.

Agar well diffusion method. The dried plant extractswere dissolved in dimethyl sulfoxide to a final concentrationof 200 mg/mL. Each well inoculated with 50mL of extract(10 mg per well) at a concentration of 200 mg/mL.20 Theinocula were suspended in sterile saline and diluted ac-cording to 0.5 McFarland standards. They were ‘‘flood-inoculated’’ onto the surface of Mueller-Hinton agar. Thewells (6 mm) were cut from the agar surface, and 50mL ofvarious extracts was inoculated into them. After the incu-bation period (24 hours at 378C), diameters of the zoneswere measured (in mm). All tests in the present study wereperformed in triplicate.

1148 OZCAN ET AL.

RESULTS AND DISCUSSION

Antimicrobial activity

There are plenty of publications revealing the antimi-crobial properties of various Verbascum species. Some ofthese species include Verbascum olympicum, Verbascumprusianum, Verbascum bombyciferum, Verbascum bellum,Verbascum cilicium, Verbascum chianophyllum, Verbascumtrapifolium, Verbascum meinckeanum, Verbascum lyr-atifolium, Verbascum undulatum, and Verbascum leptos-tychum.7,21–24 According to our literature survey, V.antiochium, the material of the current study, has not beenexamined previously for its antimicrobial capacities.

The antimicrobial activities of various extracts of V. an-tiochium and the inhibition zones formed by standard anti-biotic disks are indicated in Tables 1 and 2, respectively.From the results of this screening it has been concluded thatthe extracts, except for the one obtained by direct methanolextraction, generally have strong antibacterial activityagainst Gram-positive bacteria compared to Gram-negativebacteria (Table 1). As can be seen from Table 1, the variousextracts of V. antiochium exhibited antimicrobial activity atdifferent levels against the microorganisms tested. Only thedirect methanol extract displayed antibacterial activityagainst E. coli strains. The fungus, C. albicans, was sensi-tive only to the hexane extract of V. antiochium. Althoughprevious studies have revealed higher antibacterial activityof Verbascum species against Gram-positive bacteria,22,25,26

in this study we found that only the direct methanol extractcontained higher antibacterial activity against Gram-positive bacteria (Table 1). Methanol/water and directmethanol extracts of V. antiochium have been found to bemore effective against the bacteria tested. Among the bac-teria tested, H. influenzae was determined to be the mostsensitive one with all extracts; in particular, the methanol/

water extract displayed a larger inhibition zone than theantibiotics tested. It is also noteworthy that methanol/waterand direct methanol extracts of V. antiochium have re-markable antibacterial activity against methicillin-resistantS. aureus (Table 1).

Verbascum species have a broad variation in terms oftheir antimicrobial activity. For instance, Dulger25 reportedthat V. cilicium exhibited higher antibacterial activity than

Table 1. Antimicrobial Activity of Various Extracts of V. antiochium

Inhibition zone diameter (mm) with extract

Microorganism HE DCM M/C M/W D/M

E. gallinarium CDC-NJ-4 NA NA 9 9.7 10.7E. faecium NJ-1 7 7 7 18.3 10.7E. faecalis ATCC 29212 NA 7 8 8 10E. coli O157:H7 NA NA NA NA 10.7E. coli ATCC 25922 NA NA NA NA 10E. coli ATCC 35218 NA NA NA NA 10.3S. pyogenes ATCC 19615 NA NA 8 8.3 9S. aureus ATCC 29213 NA 7 11 16.7 14.7S. aureus ATCC 25923 8.3 NA 12 17.7 17.3S. aureus ATCC 43300 (MRSA) NA NA 13 20 17.3P. aeruginosa ATCC 27853 NA 7.3 7 7.3 10.7B. subtilis ATCC 6633 7.7 NA 10 NA 8S. typhi NCTC 8394 NA 7 7.3 11.7 14H. influenzae ATCC 49247 7 7 11.3 27 24.7L. monocytogenes ATCC 7644 NA NA NA 15 10.7C. albicans ATCC 10231 12 NA NA NA NA

The diameter of the inhibition zone includes the well diameter of 6 mm.

HE, hexane; DCM, dichloromethane; M/C, methanol/chloroform; M/W, methanol/water; D/M, direct methanol; NA, not active; MRSA, methicillin-resistant S. aureus.

Table 2. Antimicrobial Susceptibility Testing

of Microorganisms

Inhibition zone diameter(mm) with

MicroorganismCiprofloxacin

(10 mg)Streptomycin

(25 mg)Penicillin

(10 U)

E. gallinarium CDC-NJ-4 24 NA 18E. faecium NJ-1 20 NA NAE. faecalis ATCC 29212 24.8 20 21E. coli O157:H7 25 19 NAE. coli ATCC 25922 20.8 17.3 NAE. coli ATCC 35218 21.8 NA NAS. pyogenes ATCC 19615 25.8 21 23.8S. aureus ATCC 29213 24 17 33.5S.aureus ATCC 25923 25 16.3 30S.aureus ATCC 43300

(MRSA)18 15.3 9.8

B. subtilis ATCC 6633 30 20.66 21P. aeruginosa

ATCC 2785327 20.7 NA

S. typhi NCTC 8394 30 20.7 22H. influenzae

ATCC 4924725 20.7 20

L. monocytogenesATCC 7644

26 21.3 30

The diameter of the inhibition zone includes the well diameter of 6 mm.

V. ANTIOCHIUM: ANTIMICROBIAL AND ANTIOXIDANT 1149

other Verbascum species and that among the bacteria testedS. aureus was more susceptible to the extracts of the Ver-bascum species studied. In the current study, however, H.influenzae was found to be more sensitive to all extracts ofV. antiochium and displayed the largest inhibition zones(Table 1). Verbascum L. species owe their antimicrobialactivities to a wide range of compounds, such as glyco-sides,27–29 alkoloids,30 and saponins.31 Magiatis et al.21

identified a novel macrocyclic dimmer lactone, namedverbalactone, isolated from the roots of V. undulatum; thiscompound exhibited strong antibacterial activity. Similarly,verbascoside isolated from Verbascum L. species27 has beenshown to have antimicrobial activity against Gram-positivebacteria.32 It is a matter of further research to determinewhether the antibacterial activity of V. antiochium extractsobtained in this study is due to the above compounds.

Antioxidant activity

Two types of antioxidants, synthetic and natural, arewidely used by humans to protect themselves against ac-tivity of reactive oxygen species.33 Synthetic antioxidantssuch as butylated hydroxyanisole and BHT have been usedas antioxidants since the beginning of the 20th century.Because of the carcinogenic risk of the synthetic antioxi-dants, interest in natural antioxidants has increased consid-erably.34 In the current study the potential antioxidantactivity of the methanolic extract of V. antiochium wasdetermined by using two complementary tests, namely, theDPPH free radical scavenging and the b-carotene/linoleicacid test systems. Assessment of total antioxidant activity ofthe plant extracts is recommended to be carried out by usingat least two or more methods.35 Therefore, in the currentstudy two complementary test systems were used to evaluatethe antioxidant properties of the methanolic extract of V.antiochium. In both of the test systems the methanolic ex-tract exhibited some antioxidant properties (Table 3). Thefree radical scavenging capacity of the methanolic extract ofV. antiochium was measured by the DPPH assay to be4.80 mg/mL (Table 3). Free radical scavenging activity ofthe extracts is concentration dependent, and a lower 50%inhibition concentration value means better protective ac-

tivity. On the other hand, in the b-carotene/linoleic acidassay, the inhibition value of linoleic acid oxidation by theV. antiochium methanolic extract was detected as 79.92%,which is close to the value of the synthetic antioxidant re-agent BHT of 92.46% (Table 3).

The iridoid and phenylethanoid glycosides are widelydistributed in the genus Verbascum, and in particular phe-nylethanoid glycosides were reported to possess antioxidantactivity.36 Verbascum lesianthum was shown to containsome chemical groups (verbascoside, phenylethanoid gly-cosides, etc.) that display antioxidant properties.37 Theneolignan glucosides and the phenylethanoid glycosidesisolated from Verbascum salviifolium have been shown tobe potential natural free radical scavengers based on theexperiment of DPPH.38 Although there are plenty of pub-lications about the antimicrobial activity of various Ver-bascum species, in our literature survey we found only onestudy assessing the antioxidant properties of Verbascumextracts. In that study, Mothana et al.39 revealed high freeradical scavenging activity in the DPPH assay for the me-thanolic extract of Verbascum bottae grown in Yemen. Asfar as our literature survey could ascertain, the current studyis the first report about the antioxidant activity of V. anti-ochium species.

Phytochemical screening of the methanolic extractand the amount of total phenolics

In this study, together with the detection of antimicrobialand antioxidant properties, the total phenolic content of theextract was determined as well, because phenolic com-pounds are reported to be major contributors to the overallantioxidant activities of various plant extracts.40,41 The totalphenolic component of V. antiochium was determined byusing the literature methods involving the Folin-Ciocalteureagent and gallic acid as a standard.19 According to thismethod, the phenolic component of V. antiochium was de-termined to be 92.71 mg of GAE/g (the data are means ofthree different experiments).

Verbascum L. species were reported to contain eight mainchemical groups: saponins, iridoid and phenylethanoidglycosides, monoterpene glucoside, neolignan glucosides,flavonoids, steroids, and spermine alkaloids.27,42 Ourphytochemical screening revealed the presence of threetypes of natural compounds, namely, iridoid glycosides,flavonoids, and saponins, in V. antiochium extracts. Thepresence of saponins and iridoids as major componentscould be the reason for the antimicrobial activity as reportedbefore.29,31 The iridoids and flavonoids found through thephytochemical investigation might be responsible for thenoteworthy antimicrobial and antioxidant activities detectedin this study.

ACKNOWLEDGMENTS

This research was supported partially by the ResearchCouncil of Mustafa Kemal University. The authors wish tothank Dr. Hikmet Yolcu for supplying plant materials.

Table 3. Antioxidative Capacity of the Methanolic Extract

of V. antiochium Measured in 2,2-Diphenyl-1-Picrylhydrazyl

and b-Carotene/Linoleic Acid Assays

Extract and controlsDPPH

(mg/mL)ab-Carotene/

linoleic acidb

V. antiochium extract 4.80 79.92BHT 1.53 92.46Ascorbic acid 0.46 NT

Data are means of three different experiments.a50% inhibition concentration values in the 2,2-diphenyl-1-picrylhydrazyl

(DPPH) assay.bPercentage inhibition rate of linoleic acid oxidation.

BHT, tert-butylated hydroxytoluene; NT, not tested.

1150 OZCAN ET AL.

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

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