Antimicrobial activity of Saussurea lappa Clarke Roots

 

 

Patil SM1, Patil MB2 Sapkale GN1 and Umbare RP1

1Department of Pharmacognosy and Phytochemistry, A.S.P.M’s K. T. Patil College of  Pharmacy, Osmanabad - 413501. Maharashtra, India.

2Department of Pharmacognosy and Phytochemistry, K.L.E.S’s College of Pharmacy, Belgaum - 590010. Karnataka, India.

 

ABSTRACT

The Antimicrobial potential of Saussurea lappa Clarke. against certain microorganisms was studied using agar diffusion method. The aqueous and ethanolic extract of S. lappa roots significantly inhibited the growth of microorganisms as compared to standard bactericide and fungicide drugs. Diethyl ether fraction exhibited prominent fungicidal activity against Candida albicans. Gentamycin, Streptomycin and Fluconazole were used as positive controls.

 

Keywords: Microorganisms, Candida albicans, Bactericide, Gentamycin, Saussurea lapp.a.

 

INTRODUCTION

Saussurea lappa Clarke (Family -Composite) commonly known as liquorice, perennial herbs or sub-shrubs growing to a height of 2 m with horizontal underground stems. It is native to the Mediterranean and near East; distributed in the sub-tropical and warm temperature regions of the World (Anonymous, 1998).

 

Mitra S. K. et al., (1996) investigated UL-409, herbal formulation (containing S. lappa as one of the constituents) for its possible ulcero-protective activity in Wistar rats of either sex and male guinea pigs. UL-409 was found to possess antiulcer activity which may be due to the modulation of defensive factors by improvement in gastric cytoprotectionetc. Have been reported for roots of S. lappa .

 

These activities are reported due to two kinds of main constituents, the Volatile oils and Tannins present in S. lappa. Topical applications of such compounds, with free-radical-scavenging properties, have shown to improve significantly wound healing and protect tissues and antimicrobial activity from oxidative damage.

 

In this context, antimicrobical potential of S. lappa against various microorganisms (including two gram positive, two gram negative and one fungus spp.) was studied in present research work.

 
EXPERIMENTAL METHODS

Collection of plant material

The roots of S. lappa was procured from M/s. Pragati Pharma, Belgaum, Karnataka, India and were authenticated by Dr. A. V. Kulkarni, Professor, Department of Botany, G. S. Institute, Belgaum, Karnataka, India. The voucher specimen of the plant has been kept in Dept. of Pharmacognosy and Phytochemistry, K. L. E. S’s College of Pharmacy, Belgaum, Karnataka, India for further reference.

 

Extraction and phytochemical screening

The coarse powder of shade dried roots of S. lappa (1.5 kg) was extracted with ethanol (95%) in a soxhlet extractor. The ethanolic extract was then concentrated on rotary flash evaporator to 1/10th volume.

 

Table 1: Preliminary phytochemical evaluation of Saussurea lappa Clarke. Roots

Phytoconstituents

Ethanolic extract

Pet. Ether fraction

Diethyl ether Fraction

Butanol Fraction

Chloroform fraction

Alkaloids

+

-

+

-

-

Amino acids

-

-

-

-

-

Carbohydrates

+

-

-

+

+

Flavonoids

+

+

-

-

-

Glycosides

+

-

-

+

+

Proteins

-

-

-

-

-

Saponins

-

-

-

-

-

Tannins

+

+

+

+

-

Triterpenoids

-

-

-

-

-

 

 

 

 

 

 

 

 

 

 

‘+’: Present; ‘-’: Absent

 

Table 2: Antimicrobial activity of different extracts and fractions of Saussurea lappa Clarke Roots

Sample

Concentration

Mean ± SEM of diameter of inhibition zone (in mm)

Gram positive bacteria

Gram negative bacteria

Fungi

B. subtilis

S. aureus

P. aeruginosa

E. coli

C. albicans

Ethanolic extract

1 mg/disc

17.00±

9.82a†b‡

21.00±

12.13 a# b#

20.33±

11.75 a*b*

16.67±

9.63 a†b†

17.33±

10.02 c‡

Pet.Ether fraction

1 mg/disc

18.33±

10.59 a†b‡

18.67±

10.78 a*b#

18.33±

10.59 a‡b‡

17.00±

9.82 a†b†

15.67±

9.05 c‡

Diethyl ether fraction

1 mg/disc

12.67±

7.32 a†b†

10.67±

6.17 a†b‡

13.67±

7.9 a†b†

14.00±

8.09 a†b†

10.67±

6.17 c†

Butanol fraction

1 mg/disc

17.33±

10.02 a†b‡

15.67±

9.05 a‡ b*

16.00±

9.24 a‡b‡

18.67±

10.78 a†b†

12.00±

6.93 c†

Chloroform fraction

1 mg/disc

15.33±

8.86 a†b†

15.33±

8.86 a‡ b*

18.67±

10.78 a‡b‡

16.00±

9.24 a†b†

14.33±

8.28 c‡

DMF

-

-

-

-

-

-

Gentamycin

10 mcg/disc

26.67±

15.4

28.33±

16.37

26.33±

15.21

27.67±

15.98

-

Streptomycin

10 mcg/disc

25.33±

14.64

25.67±

14.83

27.00±

15.59

27.67±

15.98

-

Fluconazole

10 mcg/disc

-

-

-

-

23.34±13.48

n=3; Symbols represent statistical significance: † p<0.001,  ‡   p<0.01, * p<0.05, #  p>0.05; Comparisons are made with: a Gentamycin, b Streptomycin and c Fluconazole, ‘-’: Not Applicable. DMF: Dimethyl Formamide

 

Table 3: MIC values (μg/disc) of different extract of S. lappa roots and its various fractions against certain microorganisms

Test Sample

MIC value (in μg/disc) against

Gram positive bacteria

Gram negative bacteria

Fungi

B. subtilis

S. aureus

P. aeruginosa

E. coli

C. albicans

Ethanolic extract

100

25

50

100

50

Pet. Ether fraction

100

50

100

100

100

Diethyl ether fraction

>200

>200

200

200

>200

Butanol fraction

50

200

100

50

>200

Chloroform fraction

100

100

50

100

100

 

 

 

The concentrated ethanolic extract was then fractionated with n-butanol, ethyl acetate, solvent ether and petroleum ether (40-600C) in succession. An aqueous extract was also prepared by maceration method using chloroform water I.P. as vehicle. The aqueous extract was concentrated to dryness under vacuum using rotary flash evaporator. An attempt was also made to observe the presence and absence of different phytochemical constituents, viz. alkaloids (Dragendorff’s test), anthraquinones, saponins (foam formation), flavonoids (using magnesium and dil. HCl), sesquiterpenes and terpenes (Liebermann–Burchard’s test) according to standard methods (Trease and Evans, 1987).

 

Microorganisms used

The test microorganisms used for the antimicrobial activity screening were four bacterial spp. (two Gram positive and two Gram negative) - Bacillus subtilis MTCC (121), Staphylococcus aureus MTCC (96), Pseudomonas aeruginosa MTCC (429), Escherichia coli MTCC (443) and one fungal spp. - Candida albicans MTCC (183). These organisms were identified and procured from

 

Institute of Microbial Technology (IMTECH-CSIR), Chandigarh, India.

 

Antimicrobial activity

The agar diffusion method (Murray et al., 1995) was used to evaluate the antimicrobial activity. Bacteria were cultured overnight at 370C in Mueller Hinton Broth (MHB) and fungus at 280C for 72 h in Potato Dextrose Broth (PDB) and used as inoculum. A final inoculum, using 100 μl of suspension containing 108 CFU/ml of bacteria and 104 spore/ml of fungus spread on Mueller Hinton Agar (MHA) and Potato Dextrose Agar (PDA) medium, respectively.

 

The disc (6 mm in diameter) was impregnated with 10 μl of 100 mg/ml (1 mg/disc) extracts placed on seeded agar. Gentamycin (10 μg/disc) and Streptomycin (10 μg/disc) were used as positive controls for bacteria and Fluconazole (10 μg/disc) for fungus. The test plates were incubated at 370C for 24 h for bacteria and at 280C for 72 h for fungi depending on the incubation time required for a visible growth.

 

MIC values were also studied for microorganisms, which were determined as sensitive to the extract in disc diffusion assay. Sterile filter paper discs (6 mm in diameter) containing 2.5 – 1000 μg/disc of plant extracts were placed on the surface of a medium. MIC was defined as the lowest concentration of extract that inhibited visible growth on agar.

 

Statistical analysis

The values are represented as mean ± standard error of mean (SEM) for triplicate set of experiments.

 

 
RESULTS AND DISCUSSION

Preliminary phytochemical screening of S. lappa roots showed the presence of saponins, tannins, flavonoids and triterpenoids mainly in aqueous and ethanolic extracts and partially in its various fractions; while, alkaloids, proteins and amino acids were absent.  The results of preliminary phytochemical screening tests are tabulated in  Table 1.

 

The disc diffusion method was used to determine the inhibition zones of S. lappa extracts and its various fractions. The plant showed significant antibacterial and antifungal activity against almost all the organisms (Table 2).

 

A stronger and broader spectrum of antimicrobial activity was observed in aqueous an ethanolic extracts of S. lappa, as compared to vehicle, dimethyl formamide (DMF). Also, the diethyl ether fraction of ethanolic extract of S. lappa showed significant antifungal and antibacterial activity.

 

Increased inhibition was found at higher levels of extract concentration. MIC of these extracts is summarized in Table 3. The ethanolic extract of S. lappa showed very low MIC values of 25 μg/disc; while, its diethyl ether fraction and aqueous extract showed MIC values of 50 μg/disc.

 

It may be concluded that the antimicrobial activity of S. lappa may be due to the presence of, Carbohydrates, Tannins and Volatile oils. Further, study is needed to characterize the active principles in accordance

 

ACKNOWLEDGEMENTS

We thank Dr. A. V. Kulkarni, Professor, Department of Botany, G. S. Institute, Belgaum, Karnataka, India for authentication of the plant material. Supportive help from Institute of Microbial Technology (IMTECH-CSIR), Chandigarh, India, is gratefully acknowledged.

 

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Received on 10.05.2009

Accepted on 26.05.2009     

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Research Journal of Pharmacognosy  and Phytochemistry. 1(1): July.-Aug. 2009, 51-53