Antimicrobial assessment of marine brown algae Sargassum whitti against UTI pathogens and its phytochemical analysis

 

J. Sangeetha1, S.Gayathri1, S. Rajeshkumar2

1 Department of Microbiology, Sri Akilandeswari Women’s college, Wandiwash – 604 408, TN, India

2 School of Bio-Sciences and Technology (SBST), Vellore Institute of Technology University, Vellore – 632014, TN, India

*Corresponding Author E-mail: ssrajeshkumar@hotmail.com

 

ABSTRACT:

The present work aimed screen the bioactive potential of Sargassum wightii. The acetone, methanol and aqueous extract prepared from S. wightii screened for antimicrobial activity against UTIs causing bacteria. Qualitative phytochemical analysis was performed to find the presence of tannins, saponins, flavanoids, terpenoids, alkaloids and glycosides. In phytochemical studies, aqueous extract has glycosides, saponins, tannins and carbohydrates. Glycosides, saponins, teroenoids, steroids, and tannins are present in acetone extract. Methanol extract has flavonoids, saponins, terpenoids, steroids, tannins and carbohydrates.  Acetone extract shows maximum inhibition zone diameter was obtained in P. aeuroginosa and in Enterococcus sp with diameter 24.3±0.42 mm 21.5±0.52 mm, respectively. Similarly, Methanol extract showed maximum inhibition zone with diameter of 26.5±0.86 mm in E. coli and 25.1±0.33 mm P.mirbilis. The aqueous extract (8-13 mm) showed restrained and minimum activity, respectively. The present studies concluded the bioactive potential of methanolic extract of Sargassum wightii effective against various urinary tract infecting pathogens.

 

KEYWORDS: Sargassum wightii, Brown algae, Antimicrobial activity, FT-IR.

 

 

 


INTRODUCTION:

Urinary tract infection is a common disease that occurs when bacteria enter into urinary bladder [1, 2] and invade into urinary tract.  The bacterium was grow and multiplied in urine within the renal tract. This common disease infected in persons aged 20 – 50 years and mostly pregnant ladies. The common uropathogens are mostly belongs to Enterobacteriaceae family which are gram negative bacteria with E.coli being the most common followed by the Proteus mirabilis, Klebsiella sps and Enterococcus.  

 

The UTI infections are two types based on attacking site of the urinary system are lower UTI and upper UTI. The lower UTI is cystitis and urethritis and the upper UTI is Pyelonephritis cause flank pain, fever, blood in the urine[3].

 

Marine atmosphere is natural resources that have considered as reservoirs of biologically active natural products with medicinal properties. However, secondary metabolites produced by marine living organisms, have been exploited by people for a variety of activities including their uses as medicines [4, 5]. In marine environment seaweed was the important biomass which is classified under macro algae. The crude extract from seaweed thus obtained is subjected to biological screening for antifungal, antiviral, antibacterial, antimalarial and antifertility activity [6]. Most of the seaweeds possess bio-active components as secondary metabolites which inhibit the growth of some of the Gram positive and Gram negative bacterial pathogens.

The seaweed Sargassum wightti belongs to the class Phaeophyceae and the Sargassaceae. Sargassum species are found throughout tropical areas of the world [7]. This species may grow to a length of several meters and they are commonly brown or dark green in color. The structure of marine algae consists of a holdfast, a stipe, and a frond. It has special branches like oogonia and antheridia occur in conceptacles embedded in receptacles [8]. The aim of this study is to collect and extract the crude compounds from Seaweeds by using different solvents like aqueous, acetone and methanol. Urine samples were collected and to identify their antimicrobial activity by disc diffusion method and to determine the minimum inhibitory concentration (MIC).

 

MATERIALS AND METHODS:

Sample collection:

Fresh marine seaweeds Sargassum whitti were collected from Mandapam (Latitude 8°35’- 9°25’ N; Longitude 78°08’-79°30’ E), Rameshwaram and South East coast of Tamil Nadu. Collected samples were washed with tap water in order to remove epiphytes and other marine organisms and then washed with distilled water and dried at 45 °C and ground.

 

Preparation of extract using different solvents:

About 10 g of powdered leaves were mixed with 100 ml of sterile double distilled water and incubated on a water bath shaker for 12 h at 40°C. The organic solvents of S. whitti were prepared by using acetone and methanol. To this extraction process, 25 g of powdered leaves were mixed with 80% acetone and 70% methanol at 60°C for 48 h in soxhlet apparatus. Then the mixture was filtered through Whatman No 1 filter paper, and then the filtrate was collected. The collected filtrate used for preliminary phyto-chemical analysis and antimicrobial activity against UTI causing microorganism.

 

Phytochemical screening:

The three extracts thus obtained were analyzed to preliminary phytochemical screening such as alkaloids, tannins (ferric chloride test), saponons, glycosides, flavonoids, terpenoids, triterpenoids, phenol, steroids, protein and sugars etc following the standard protocols [9-13].

 

Isolation of UTI causing strains:

Urine sample was collected from UTI infected person. The isolated strains from the sample were identified based on staining and biochemical test. Identification of isolates were done based on morphological and biochemical test like colony morphology (Grams’ staining), motility, catalase test, oxidase test, coagulase test and biochemical tests like Tripal sugar iron agar, Hydrogen sulfide test, Carbohydrate fermentation test, Phenylalanine deaminase test, Methyl red test, Nitrate reduction test, Urease test, Voges proskauer, Citrate utilization test, Indole test From this, multidrug resistant strains were identified by conducting antimicrobial susceptibility test of bacteria against standard antibiotics and three different solvent extracts [14].

 

Antibacterial activity by disc diffusion method:

Antibacterial activity of the seaweed crude extract of different solvents was tested by disc diffusion method. In this method, sterile filter paper discs were prepared with 5 mm diameter and the sterile discs were impregnated with three different solvent extracts of seaweed at different concentrations (25 – 100 mg/mL) and allowed to dry. The fresh cultures of isolated and identified UTI causing microorganisms were spread on sterilized Muller Hinton Agar plates. Then, extracts impregnated discs were placed on the culture swabbed MH agar plates. The commercial Amphicilin antibiotic disc (10 mg) was used as a positive control and solvent alone impregnated discs were considered as negative control. All the plates were incubated at 35 °C for 24 h and then the bactericidal activity was assessed by measuring zone of inhibition around the discs and expressed in mm in diameter [15].

 

Minimum Inhibitory Concentration:

The minimum inhibitory concentration (MIC) of aqueous, acetone and methanol extracts of seaweed was analysed by growth inhibition method. MIC is considered as the lowest amount of bactericidal agents is required to inhibit the growth of pathogenic microorganisms. To this assay fresh sterile 100 ml of Lueria Bertani broth was supplemented with different concentrations of three different solvent extracts in separate flasks (25 – 100 mg/ml). Then the fresh bacterial cultures of UTI pathogens E.coli, Klebsiells sp, P. aeruginosa,, Enterococcus sp and P. mirabilis were inoculated into the broth and incubated in shaker incubator for 24 hours at 37°C and 120 rpm. Extract free broth was used as a control. After 24 hours of incubation the microbial growth was measured by turbidity of the each culture and recorded by recording the absorbance at 600 nm. Sterilized LB broth was used as a blank.

 

RESULTS AND DISCUSSION:

Phytochemical characterization:

In the phytochemical analysis, the chemical compounds such as alkaloids, steroids, tannins, flavanoids, glycosides, phenols, saponins, phlorotannins, terpenoids were tested in aqueous extract and different solvent extracts of S. wightii. Among the three different solvent extracts, methanol extract showed the presence of maximum phytochemical compounds such as steriods, tannin, flavanoids, phenols, glycosides, saponin and terpenoids and the results were tabulated in Table 1. The solvent methanol and acetone extracts of S. wightii indicated the presence of a number of metabolites. Seaweed extracts are considered to be a rich source of phenolic compounds [16, 17]. Steroids are widely distributed in marine algal weeds [18]. S. wightii showed the maximum presence of steroids in two different extracts except aqueous, followed by flavonoids in methanol extract saponins compounds in all the three extracts. Glycosides are present only in acetone extract of S. wightii. Among the three different extracts tested, methanol extracts showed the presence of maximum number (6/14) of compounds. Aqueous and acetone extracts showed the presence of 4 and 5 compounds each, respectively.

 

Table 1: Preliminary phytochemical evaluation of different solvent extract of S. Wighti

Phytoconstituents

Aqueous

Acetone

Methanol

Alkaloids

_

_

_

Phenols

_

_

_

Flavonoids

_

_

+

Glycosides

+

+

_

Saponins

+

+

+

Proteins

_

_

_

Quinones

_

_

_

Terpenoids

_

+

+

Steroids

_

+

+

Tannins

+

+

+

Phlorotannins

_

_

_

Carboxylic acids

_

_

_

Coumarins

_

_

_

Carbohydrates

+

_

+


Table 2: Biochemical characters of the isolates

S.No

Tests

Klebsiella sp

Proteus mirbilis

Pseudomonas aeuroginosa

Enterococci sp

E.coli

1

Gram staining

(-) ve rod

(-) ve rod

(+) ve cocci chain forms

(+) ve cocci

(-) ve rod

2

Motility

Non motile

Non- Motile

Motile 

Non motile

Motile

3

Spore

-

_

_

-

-

4

Catalase

+

_

 +

_

+

5

Oxidase

_

_

+

_

_

6

Indole

_

+

_

_

+

7

Methyl red

_

+

_

_

+

8

Voges proskauer

+

_

_

+

_

9

Citrate utilization

+

+

+

_

_

10

Gelatin hydrolysis

_

_

+

+/-

_

11

Urease

+

+

_

_

_

12

H2S production

_

+

_

_

_

13

Glucose

+

+

_

+

+

14

Sucrose

+

-

_

+/-

+/-

15

Lactose

+

-

_

+

+

16

Maltose

+

+

_

_

A+

17

Coagulase test

 Positive

_

_

_

_

18

TSI

A/A

 

_

+

_

Cultural character on media

 

 

 

19

EMB agar

_

_

_

_

Green metallic sheen

20

Macconkey agar

Positive 

_

_

_

_

21

Pseudomonas agar

_

 

Bluish green colonies

_

_

 


Isolation and identification of UTI causing organisms:

The isolated microorganisms were tested by various cultural and biochemical test for identification (Table. 2). Based on the morphological and cultural and biochemical characters the isolated organisms were identified as Klebsiellsa sp, Proteus mirabilis, Pseudomonas aeuroginosa, Enterococci sp, and E. coli

 

Antimicrobial activity by disc diffusion method:

The antimicrobial potential of the experimental seaweed was evaluated according to their zone of inhibition against urinary tract infecting pathogens and the results (zone of inhibition) were compared with the activity of the standard Ampicillin (10 mg/disc). The results revealed that all the extracts are potent antimicrobials against all the UTI microorganisms studied. For all the tested microorganisms ethanol and Acetone showed maximum antibacterial activity in S. wightii. In Acetone extract maximum inhibition zone diameter was obtained in P. aeuroginosa and in Enterococcus sp with diameter 24.3±0.42 mm 21.5±0.52 mm, respectively. Similarly, Methanol extract showed maximum inhibition zone with diameter of 26.5±0.86 mm in E. coli and 25.1±0.33 mm P.mirbilis. The aqueous extract (8-13 mm) showed restrained and minimum activity, respectively. More specifically, aqueous extract represented higher susceptibility to all bacterial strains (Table 3; Fig.1 -3).


 

Figure 1: Antibacterial activity of aqueous S. wightii extracts against UTI causing bacteria.

 

Figure 2: Antibacterial activity of acetone S. wightii extracts against UTI causing bacteria.

 

Figure 3: Antibacterial activity of methanol S. wightii extracts against UTI causing bacteria


Minimum inhibitory concentration (MIC) growth after incubation was taken as MIC of particular extract against the respective pathogen. Aqueous extract showed highest MIC value of 32.0±0.23 mg/mL against Proteus mirabilis. Acetone extract showed the low MIC value against Enterococcus sp (28.5±0.42 mg/ mL). Methanol extract showed the lowest MIC value against E.coli and Proteus mirabilis (3.12 mg/ mL) (Table 6).


 

Table 3: Antibacterial activity of aqueous S. wightii extracts against UTI causing bacteria.

 

Zone of inhibition  (mm in diameter)

Concentration (mg/ml)

E.coli

Pseudomonas aeruginosa

Klebsiella pneumonia

Enterococcus sp

Proteus mirabilis

25mg

11.9±0.41

10.6±0.25

8.5±0.56

10.3±0.62

09.3±0.40

50 mg

15.2±0.51

12.2±0.91

9.2±0.41

13.8±0.11

10.4±0.43

75 mg

19.3±0.81

17.8±0.86

9.8±0.41

12.2±0.32

11.2±0.57

100 mg

21.5±0.11

22.3±0.13

10.5±0.51

18±0.65

12.1±0.15

Antibiotic

12.3±0.54

21.52±0.31

20.5±0.35

17.4±0.25

20.8±0.52

 

Table 4: Antibacterial activity of acetone S. wightii extracts against UTI causing bacteria.

 

Zone of inhibition  (mm in diameter)

Concentration (mg/ml)

E.coli

Pseudomonas aeruginosa

Klebsiella pneumonia

Enterococcus sp

Proteus mirabilis

25mg

10.2±0.51

12.6±0.64

11.3±0.51

11.3±0.12

10.3±0.24

50 mg

11.5±0.81

15.8±0.25

13.5±0.82

14.8±0.33

13.4±0.33

75 mg

13.4±0.64

20.5±0.81

15.6±0.52

18.2±0.35

16.2±0.39

100 mg

18.6±0.11

24.3±0.42

19.5±0.31

22.5±0.52

21.1±0.25

Antibiotic

12.3±0.32

21.52±0.91

18.5±0.55

21.4±0.38

20.8±0.75

 

Table 5: Antibacterial activity of methanol S. wightii extracts against UTI causing bacteria.

 

Zone of inhibition  (mm in diameter)

Concentration (mg/ml)

E.coli

Pseudomonas aeruginosa

Klebsiella pneumonia

Enterococcus sp

Proteus mirabilis

25mg

13.8±0.23

13.0±0.52

12.1±0.33

12.3±0.42

13.5±0.42

50 mg

18.2±0.33

14.5±0.54

14.0±0.63

15.8±0.62

16.5±0.33

75 mg

22.5±0.61

19.7±0.76

18.5±0.15

17.9±0.50

20.5±0.39

100 mg

25.5±0.86

24.5±0.62

22.5±0.33

22.5±0.52

25.1±0.33

Antibiotic

12.3±0.52

21.52±0.91

20.5±0.55

21.4±0.38

20.8±0.75

 

Table 6: MIC of Aqueous, acetone and methanol extract of S. wightii

Solvent

E.coli

Pseudomonas aeruginosa

Klebsiella pneumonia

Enterococcus sp

Proteus mirabilis

Aqueous  (mg/ml)

24.5±0.50

21.50±0.57

24.5±0.32

22.0±0.33

32.0±0.23

Acetone (mg/ml)

19.12±0.47

13.16±0.45

23.3±0.54

28.5±0.42

24.5±0.12

Methanol (mg/ml)

03.12±0.63

4.50±0.33

4.20±0.50

4.80±0.54

3.12±0.42

Data are expressed as mean±SD. Data are mean of triplicate determinations.

 

Figure 4: FT-IR spectrum of S. Wightii


Though, the mechanism of the action of these plant constituents is not yet fully known it is clear that the effectiveness of the extracts largely depends on the type of solvent used. Many studies revealed that the organic extracts provided more antimicrobial activity potential while compared to aqueous extracts. This study also revealed that acetone extract shows moderated and aqueous extract shows minimum activity as compared with methanol solvent extract. This observation clearly indicates that the existence of non-polar residues in the extracts which have higher bactericidal activities against UTI pathogens. Preethi et al [19] and Seyydnejad et al [20] reported that similar results showing that the alcoholic extract having the best antimicrobial activity. In the present study the MIC value of the active seaweed extracts obtained in this study was lower suggesting that the seaweed extracts were bacteriostatic at lower concentration but bactericidal activity at higher concentration.

 

FT-IR analysis of S. wightii

The figure 4 and table 7 shows FT-IR spectrum and functional groups present in the marine brown seaweed Sargassum wightii. There is so many phytochemicals present in the marine brown seaweeds is responsible for the zone of inhibition in antimicrobial activity.

 

Table 7: FT-IR analysis of S. wighti

S. No

Peak

Chemical group

1

3406.6

Amines—Secondary

N-H Stretch

2

2529.6

Nitriles

3

1647.3

Alkenes

C=C-H Asymmetric Stretch

4

1417.0

Alkanes  H-C-H Bend

5

1324.8

Nitro Groups

N=O Bend

6

1030.4

Ethers

C-O Stretch)

7

823.2

Ethers

 

CONCLUSION:

Antimicrobial drugs from marine sources have received much consideration and efforts have been put in to identify compounds that can act as alternative antimicrobials agent which replace synthetic drugs. In the present study, different solvents derived extracts of seaweed S. wightii was evaluated for investigation of their antimicrobial activity against urinary tract infection causing microorganisms. Susceptibility of each plant extract was tested by disc well diffusion method and MIC was determined. It revealed that all acetone, Methanol, and aqueous extracts were active against the urinary tract infection causing pathogens like Escherichia coli, Pseudomonas aeruginosa, Enterococcus sp, K. pneumonia, and Proteus mirabilis. The methanol extracts showed significant antimicrobial activity against multi-drug resistant clinically isolated microorganisms.

 

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Received on 13.04.2017          Modified on 17.05.2017

Accepted on 31.05.2017        © RJPT All right reserved

Research J. Pharm. and Tech. 2017; 10(6): 1905-1910.

DOI: 10.5958/0974-360X.2017.00334.1