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Journal of Ethnopharmacology 115 (2008) 327–329

Ethnopharmacological communication

Antimicrobial activity of some important Adiantum speciesused traditionally in indigenous systems of medicine

Meenakshi Singh a, Neha Singh a, P.B. Khare b, A.K.S. Rawat a,∗a Pharmacognosy & Ethnopharmacology Division, National Botanical Research Institute, Lucknow 226001, India

b Pteridology Division, National Botanical Research Institute, Lucknow 226001, India

Received 26 September 2006; received in revised form 3 July 2007; accepted 24 September 2007Available online 29 September 2007

bstract

Adiantum Linn. of Adiantaceae family is one of the most common and widely distributed species. Ethnomedicinally, the genus is important andopularly known as “Hansraj” in Ayurvedic System of Medicine. It has been used in cold, tumors of spleen, liver and other viscera, skin diseases,ronchitis and inflammatory diseases. It is also considered as tonic and diuretic. In the present study its four important species, i.e. Adiantumapillus–veneris, Adiantum peruvianum, Adiantum venustum and Adiantum caudatum were collected and extracted with methanol. These extractsere tested for their antimicrobial agents against five gram positive, six gram negative (including multiresistant bacteria Staphylococcus aureus)

nd eight fungal strains using standard microdilution assay. The maximum activity was exhibited by the methanolic extract of Adiantum venustumollowed by Adiantum capillus–veneris, Adiantum peruvianum and Adiantum caudatum. The methanolic extract of Adiantum capillus–venerisad very low MIC value (0.48 �g/ml) against Escherichia coli whereas, Adiantum venustum extract against Aspergillus terreus with MIC of

.97 �g/ml. Total phenolic constituents of Adiantum species viz. Adiantum venustum, Adiantum capillus–veneris, Adiantum peruvianum anddiantum caudatum were 0.81% (w/w), 0.83% (w/w), 0.71% (w/w) and 0.52% (w/w), respectively (as gallic acid equivalent); implying that thebserved activity could be related to the amount of phenolics.

2007 Elsevier Ireland Ltd. All rights reserved.

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eywords: Pteridophytes; Antimicrobial; Phenols; MIC; Phytochemical

. Introduction

As folk medicine, the pteridophytes which constitute fernsnd ferns allies, have been known to man for more than 2000ears, and also been mentioned in ancient literature (Kirtikarnd Basu, 1935; Nayar, 1957; Chopra et al., 1958; Kumar andoy, 1972; Watt, 1972; Dixit and Bhatt, 1975; Sharma and Vyas,985).

It has been observed that pteridophytes are not infected byicrobial pathogens, which may be one of the important fac-

ors for the evolutionary success of pteridophytes and the facthat they survived for more than 350 million years. A systematicurvey of the antimicrobial activity of pteridophytes has been

carcely undertaken. Khare (1996) and Vasudeva (1999) had dis-ussed the traditional uses of some potential Adiantum speciesor the treatment of various infectious diseases. Therefore, the

∗ Corresponding author. Tel.: +91 522 2207219; fax: +91 522 2207219.E-mail address: pharmacognosy1@rediffmail.com (A.K.S. Rawat).

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378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2007.09.018

onfirmation of traditional uses of Adiantum species (Adiantumapillus–veneris, Adiantum peruvianum, Adiantum venustumnd Adiantum caudatum), methanolic extracts were screeneds microbial agents against eleven bacterial and eight fungaltrains by calculating the minimum inhibitory concentrationMIC), minimum bacterial concentration (MBC) and minimumycotic concentration (MMC). Besides to get an insight on to

he components of extracts responsible, a preliminary phyto-hemical screening along with the total phenolic content werelso evaluated and calculated.

. Materials and methods

.1. Extraction of plant material

Plant samples of each species were individually extractedy cold percolation in methanol (consecutively three times) atoom temperature for 24 h the resultant extracts were filtered andoncentrated to dryness under reduced pressure below 40 ◦C in

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otary evaporator. The concentrated extracts thus obtained wereyophilized for further investigation. The yields of methanolicxtracts of Adiantum species viz. Adiantum venustum, Adiantumapillus–veneris, Adiantum peruvianum and Adiantum cauda-um were 9.32%, 8.50%, 10.2% and 9.34%, respectively.

.2. Assay for total phenolics

The total phenolic content of the methanolic extract ofelected species was determined employing the methods oflinkard and Singleton (1977) and Chandler and Dodds (1983)

nvolving the Folin-Ciocalteu reagent and gallic acid as standard.otal phenolic constituents of Adiantum species viz. Adiantumenustum, Adiantum capillus–veneris, Adiantum peruvianumnd Adiantum caudatum were 0.81% (w/w), 0.83% (w/w),.71% (w/w) and 0.52% (w/w), respectively.

.3. Antimicrobial assay by microdilution method

The test microorganisms used for the antimicrobial screen-

ng were 11 bacteria—Micrococcus luteus (MTCC-106),acillus subtilis (MTCC-121), Bacillus cereus (MTCC-30), Enterobacter aerogenes (MTCC-111), Escherichiaoli (MTCC-443), Klebsiella pneumoniae (MTCC-109),

GMUs

able 1IC and MBC values of methanolic extracts of some Adiantum species against some

acterial strains Acv Ap Av

MIC MBC MIC MBC M

ram positiveBc 15.62 7 × 10−7 62.50 7 × 10−5 31Ml 15.62 3 × 10−7 3.90 5 × 10−9 62Sa 31.25 2 × 10−6 15.62 3 × 10−7 15Bs 62.50 3 × 10−5 7.81 2 × 10−8 31Sp 7.81 2 × 10−8 3.90 2 × 10−9 15

ram negativeKp – – – – 7Ec 0.48 4 × 10−12 – – 15Pa – – – – 15Pm – – – – 31St – – 31.25 2 × 10−6 62Ea – – 15.62 4 × 10−7 62

ungal strains Acv Ap Av

MIC MMC MIC MMC MIC

a 3.90 5 × 10−9 7.81 3 × 10−8 3.9r 7.81 2 × 10−8 15.62 2 × 10−7 1.9ry A 15.62 7 × 10−7 15.62 1 × 10−7 15.6n 7.81 5 × 10−8 7.81 4 × 10−8 31.2f 15.62 6 × 10−7 15.62 3 × 10−7 15.6s 7.81 4 × 10−9 – – 15.6t 7.81 2 × 10−8 7.81 3 × 10−8 0.9ni 7.81 2 × 10−8 15.62 2 × 10−7 1.9

C: Bacillus cereus; Ml: Micrococcus luteus; Sa: Staphylococcus aureus; Bs; Bacscherichia coli; Pa: Pseudomonas aeruginosa; Pm: Proteus mirabilis; St: Salmonespergillus flavus; An: Aspergillus niger; Cry A: Cryptococcus albidus; As: Aspergilluapillus–veneris; Ap: Adiantum peruvianum; Av: Adiantum venustum; Ac: Adiantumoncentration; MMC: minimum mycotic concentration.

macology 115 (2008) 327–329

roteus mirabilis (MTCC-1429), Pseudomonas aeruginosaMTCC-424), Staphylococcus aureus (MTCC-96), Salmonellayphimurium (MTCC-98) and Streptococcus pneumoniaeMTCC-2672), and 8 fungi viz. Candida albicans (MTCC-183)nd Cryptococcus albidus (MTCC-2661), Trichophyton rubrumMTCC-296), Aspergillus niger (MTCC-16404), Aspergillusavus (MTCC-1973), Aspergillus spinulosus (MTCC-16919),spergillus terreus (MTCC-1782) and Aspergillus nidulans

MTCC-11267).These organisms were procured from Institutef Microbial Technology (IMTECH) Chandigarh, India.

The MIC values of plant extracts against pathogenic strainsere determined based on a micro dilution method (Zgoda

nd Porter, 2001) with some modifications. The inoculums oficroorganisms were prepared from 6 h broth (MHB/PDB) cul-

ure and suspensions were adjusted to 0.5 McFarland standardurbidity (NCCLS, 1999). The plant extracts dissolved in 2.5%imethyl sulfoxide (DMSO) were first dilution to the highestoncentration (1000 mcg/ml) to be tested, and then serial twoold dilution was made in concentration range 0.48–1000 �g/mln 10ml sterile test tubes containing 2.5% DMSO. Antibiotics

entamicin and Ketonzole (Sigma) used as positive control.icrobial growth was determined by using Ultraspec 200V/visible spectrophotometer and confirmed by plating 1ml

ample from clear plate on MHA/PDA. The minimum inhibitory

bacterial and fungal pathogenic strains (�g/ml)

Ac Gantamicin

IC MBC MIC MBC MIC MBC

.25 2 × 10−6 31.25 3 × 10−6 3.90 2 × 10−9

.50 4 × 10−5 15.62 3 × 10−7 1.95 4 × 10−10

.62 3 × 10−7 – – 0.97 3 × 10−11

.25 4 × 10−6 31.25 7 × 10−6 0.48 2 × 10−12

.62 2 × 10−7 – – 3.90 2 × 10−9

.81 2 × 10−8 – – 0.48 3 × 10−12

.62 4 × 10−7 – – 7.81 4 × 10−8

.62 2 × 10−7 – – 0.97 3 × 10−11

.25 3 × 10−6 – – 0.24 4 × 10−13

.50 4 × 10−5 – – 0.97 2 × 10−11

.50 7 × 10−5 – – 3.90 4 × 10−9

Ac Ketaconzole

MMC MIC MMC MIC MBC

0 4 × 10−9 3.90 2 × 10−9 0.48 1 × 10−12

5 4 × 10−10 15.62 4 × 10−7 7.81 3 × 10−8

2 2 × 10−7 7.89 5 × 10−8 15.62 2 × 10−7

5 2 × 10−6 – – 3.90 3 × 10−9

2 4 × 10−7 15.62 7 × 10−7 1.45 2 × 10−10

2 3 × 10−7 15.62 4 × 10−7 3.90 3 × 10−9

7 3 × 10−11 31.25 3 × 10−6 15.62 2 × 10−7

5 3 × 10−10 31.25 2 × 10−6 1.95 2 × 10−10

illus subtilis; Sp: Streptococcus pneumoniae; Kp: Klebsiella pneumonia; Ec:lla typhimurium; Ea: Enterobacter aerogenes; Tr; Trichophyton rubrum; Af;s spinulosus; Ani: Aspergillus nidulans; Ca: Candida albicans; Acv: Adiantum

caudatum; MIC: minimum inhibitory concentration; MBC: minimum bacterial

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oncentration (MIC) values of extract/compound of antimicro-ial agent was determined by lack visual turbidity (matching theegative growth control). The minimum bacterial concentrationMBC) and minimum mycotic concentration (MMC) of extractnd standard drugs was determined by streaking a loopful/5 �lample of each wells (well showing minimum turbidity) onto anver-dried agar (MHA/PDA) plates and then incubating 37 ◦Cor 18–24 h (bacteria) and 48–96 h (28 ◦C) for fungi. The MBCnd MMC were then recorded, as the concentration at whichhere was minimum bacterial/fungal growth or colony.

. Results and discussion

The minimum inhibitory concentration (MIC) and min-mum bacterial concentration (MBC) values of methanolicxtracts of four Adiantum species were evaluated and sum-arized in Table 1. The methanolic extracts of Adiantum

apillus–veneris gave low MIC values against Streptococ-us pneumoniae, Escherichia coli and Candida albicans withoncentration of 7.81 �g/ml, 0.48 �g/ml and 3.90 �g/ml. How-ver, the Adiantum peruvianum, Adiantum venustum anddiantum caudatum against Micrococcus luteus (3.90 �g/ml),treptococcus pneumoniae (7.81 �g/ml), Aspergillus terreus0.97 �g/ml) and Micrococcus luteus (15.62 �g/ml), Candidalbicans (7.81 �g/ml). The data showed that the activity of theethanolic extracts of Adiantum species was better to standard

ntibiotics Gentamicin and Ketoconzole (Table 1).Preliminary phytochemical screening showed the presence of

avonoid and tannin along with reducing sugar in all the speciesnvestigated, while triterpenoids, steroids and glycosides werebsent in all the species. Saponin was present only in Adiantumaudatum and Adiantum venesutum. The total phenolic contentnd other phytochemical constituents present in these speciesay responsible for potent antimicrobial activity. Some of the

igher phenols like ellagic acid and gallic acid have also beeneported for potent antimicrobial activity (Gyamfi and Aniya,002).

This is the first comparative assessment report of four mostommon species of Adiantum found in India. All selected Adi-

ntum species especially Adiantum venustum and Adiantumapillus–veneris showed potent antimicrobial activity againstscherichia coli, Trichophyton rubrum and Aspergillus terreusnd proved to be a good and new source as antimicrobial agents.

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macology 115 (2008) 327–329 329

he observation confirmed the evidence of traditional and eth-obotanical uses of these species. The preliminary results ofresent investigation appear to indicate that a number of Indianedicinal plants especially lower groups (pteridophytes) have

igher potential antimicrobial properties.

cknowledgements

The authors are thankful to the Director, National Botanicalesearch Institute, for his kind support and encouragement. Onef the authors, Meenakshi Singh is thankful to Department ofcience Technology (DST), New Delhi for providing Womencientist Fellowship.

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