Antimicrobial activity of Averrhoa carambola L. leaf extract and its Phytochemical Analysis

 

Basanti Majhi1, Kunja Bihari Satapathy2*, Sagar Kumar Mishra3

1,2Post Graduate Department of Botany, Utkal University, Vani Vihar, Bhubaneswar

3University Department of Pharmaceutical Science, Utkal University, Vani Vihar, Bhubaneswar

*Corresponding Author E-mail: basantimicro@rediffmail.com

 

ABSTRACT:

Growing drug resistance of microorganisms and adverse result of antibiotic usage have led to the search for alternative medicines from nature. Many plants have been exploited to cure infectious diseases from time immemorial. The current investigation deals with the evaluation of the antimicrobial activity, preliminary phytochemical screening and Thin Layer Chromatography (TLC) analysis of Averrhoa carambola L. leaf extracts. The antimicrobial activity was done by agar well diffusion method. Preliminary phytochemical screening was done by standard method. Plant extracts were found active against all the bacteria. Aqueous extract of A. carambola leaves showed higher antibacterial activity as compared with methanol and petroleum ether extract at the concentration of 25 mg/ml. Highest antibacterial activity was exhibited by the aqueous extract against Salmonella typhi (29.23±0.02 mm). The highest antifungal potential was exhibited by the methanol extract against Candida krusei (21.09±0.08 mm). Plant extracts were observed to be active against all the fungal pathogens except Aspergillus niger. Minimum inhibitory concentration (MIC) was determined using serial dilution method. The preliminary phytochemical analysis revealed the presence of alkaloids, tannins and polyphenolic compounds, flavonoids, phytosterol, proteins and amino acids, carbohydrates, glycosides etc. while saponins was absent in both the extract. The petroleum ether and methanolic extracts were subjected to TLC study and the Rf value of different spots was observed in various solvent systems. The outcomes of the present study indicate that A. carambola leaf is endowed with potential antimicrobial activity and hold promise for development of pharmaceuticals and antimicrobial agents in future.

 

KEYWORDS: Averrhoa carambola L., Phytochemicals, MIC, Antibacterial, Antifungal, TLC.

 

 


INTRODUCTION:

Medicinal plants have been used for a wide variety of purposes for many years in India and all over the World 1. In India herbal medicines have been the basis of treatment and cure for various diseases or physiological conditions in traditional methods practised such as Ayurveda, Unani and Siddha. The history of plants being used for medicinal purpose is probably as old as the history of mankind. Extraction and characterization of several active phytocompounds from these green factories have given birth to some high activity profile drugs2.

 

Simultaneous with population explosion virulent strains of microorganisms become more common and their increased attack accounts for increased mortality3. Ethno pharmacologists, botanists, microbiologists and natural product chemists have been exploring the earth for phytochemicals and “leads” which could be developed for the treatment of infectious diseases4. Clinical microbiologists have two reasons to be interested in the topic of antimicrobial plant extracts; (i), it is very likely that these phytochemicals will find their way into the arsenal of antimicrobial drugs prescribed by physicians; several are already being tested in humans. (ii) plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids and flavonoids which have been found to have in vitro antimicrobial properties.

 

Averrhoa carambola L. (Oxalidaceae), locally called ‘Karamanga’ and commonly known as Star fruit because of its peculiar shape. It is a small, multistemmed, slow growing evergreen tree, 5-7 m high or, rarely, 10 m high. At the base, the trunk reaches a diameter of 15 cm, the bark is light brown, smooth or finely fissured. Leaves are 15-25 cm long, alternate, spirally arranged, pinnate, shortly petiolate with 7-9 pendant leaflets5,6. It is cultivated extensively in India for its edible fruits5,6. In India the ripe fruit or the juice may be taken to counteract fever. It has widely been used in Ayurveda; A. carambola   leaves and fruit are used to pacify impaired kapha, pitta, skin diseases, pruritis, vomiting, diarrhoea, worm infestations, general debility and intermittent fever7,8. The leaves of Averrhoa carambola L. are anti-puritic, antipyretic, anti-helmintic and are also usefull in scabies, various types of poisoning, intermittent fever and intestinal worm. It has also been reported to be useful for treating factured bones9.  Averrhoa carambola L. is also widely used in traditional medicines in countries like India, China, Phillipines, Brazil for various ailments7,8.

 

In the present study, an attempt was made to investigate the antimicrobial activity and preliminary phytochemicals from the leaves of Averrhoa carambola L.

 

 

1(a)

 

1(b)

Fig: 1 (a, b) representing the leaf, flower, fruit and bark of Averrhoa carambola L.

 

MATERIALS AND METHODS:

Plant materials:

Fresh and matured leaves of Averrhoa carambola L. [Fig. 1(a, b)] were collected from Utkal University campus, Bhubaneswar, Odisha, during the month of March 2015. Plant materials was identified and authenticated by following the ‘Flora of Orissa’10 and matching the voucher specimen with the herbarium present in the Post Graduate Department of Botany, Utkal University, Bhubaneswar, Odisha. Freshly collected leaves were washed under running tap water, and then cut into small pieces and shade dried for 15-20 days at room temperature until it is completely gets dried.  The dried plant material was ground into coarse powder with the help of mechanical grinder.

 

Preparation of extracts:

50 grams of dried leaf powder was extracted by using soxhlet extraction (60 °C to 80 °C) process with 500 ml of petroleum ether, methanol and aqueous (Dimethyl Sulfoxide) as solvent separately for 72 hours. The solvent of the dilute extracts were evaporated under reduced pressure by using rotary evaporator (IKA RV IOV Digital) and kept inside the desiccator for further studies. Extracts were subsequently concentrated under reduced pressure to obtain solid extracts. For aqueous extract, 10 g of powdered leaf sample, one hundred millilitres (100 ml) of Dimethyl Sulfoxide were added in sterile conical flask and allowed to soak at room temperature for 24 h. The soaked sample were periodically shaken to ensure complete extraction. The extracts were then filtered using filter paper No.1 and the residue obtained were dried and stored at 4 °C in a freezer for further use. Four different concentrations i.e., 6.25 (mg/ml), 12.50 (mg/ml), 25 (mg/ml) and 50 (mg/ml) of petroleum ether, methanol and aqueous extracts were prepared by dissolving solid extracts in dimethyl sulfoxide (DMSO). Extracts so prepared were stored in a refrigerator at 4 °C for further use. Percentage of yield for each extract was calculated as:

 

                              Final weight of extract     

Yield % = --------------------------------------------- X 100 

                         Total weight of ground plant

 

Test micro-organisms:

Ten bacterial strains including four Gram- positive bacteria namely Bacillus subtilis (MTCC-1305), Bacillus circulans (MTCC-490), Staphylococcus aureus (MTCC-1430), Streptococcus mitis (MTCC- 2897) and six Gram negative bacteria namely Escherichia coli (MTCC- 614), Pseudomonas aeruginosa (MTCC-1035), Klebsiella pneumoniae (MTCC-109), Salmonella typhimurium (MTCC-733), Salmonella paratyphi (MTCC-3220) and Proteus vulgaris (MTCC- 9543) were employed for antibacterial assay. Six fungal strains, namely Aspergillus niger (MTCC-478), Candida albicans (MTCC-227), Candida tropicalis (MTCC-230), Candida krusei (MTCC-9215), Fusarium oxysporum (MTCC-2087) and Trichophyton mentagrophytes (MTCC-8476) were employed for antifungal assay. The Bacterial and fungal strains were obtained from Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India. Bacterial and fungal strains were maintained by sub-culturing them on Nutrient Agar (Himedia) and Potato Dextrose Agar (Himedia) respectively after every fifteen days and then stored at 4°C. Ampicillin, Ciprofloxacin, Streptomycin, Tetracycline and Fluconazole, Terbinafine, Itraconazole, Clotrimazole powder (0.5 mg/ml) was obtained from Himedia Laboratories, India and served as positive controls for antibacterial and antifungal assays respectively. Dimethyl sulfoxide (Himedia) was used as negative control.

 

Antimicrobial assay:

Antimicrobial activity of the leaf extracts was tested by agar-well diffusion method using Nutrient Agar and Potato Dextrose Agar (PDA) medium 11. Each experiment was performed triplicate and the average value of inhibition and standard deviation were calculated. Growth medium (30 ml) was poured into Petri dishes and it was left to solidify under ultraviolet (UV) light (265 nm wavelength) for 15 min. Subsequently, a sterile cotton swab was dipped into overnight bacterial or fungal suspensions of indicator strains (adjusted to turbidity of 0.5 McFarland Standard). An agar plate was inoculated by evenly streaking cotton swab over the agar medium. Then wells with a diameter of 6 mm were cut in the medium with a sterile cork borer. Stock solutions of the samples were diluted in DMSO to get 6.25, 12.5,25 and 50 mg/ml concentrations. The tested samples and controls (50 μl) were dispensed into the wells by using micro pipettes and allowed to diffuse for 15 minutes. The plates were incubated at 37 °C for 24 h for bacteria and 28 °C for 48-72 h for fungi. Then the diameters of growth inhibition zones around the wells were measured

 

Minimum Inhibitory Concentration (MIC):

The minimum inhibitory concentration (MIC) of the plant extract was determined by broth dilution technique 12. The different plant extracts such as petroleum ether, methanol and aqueous were taken (50, 25, 12.5 and 6.25 mg/ml,) and serial dilution of the extract with nutrient broth for bacterial culture and for fungal species, potato dextrose broth medium with respective inoculums was used. The tubes were incubated for bacteria at 37 °C for 24 h and 72 h at 28 oC for fungi, respectively. After incubation, each test tube was observed for turbidity. The lowest concentration of the extract for which no turbidity was recorded was the minimum inhibition concentration13.

 

Phytochemical analysis:

The leaf extracts were undergone for the preliminary phytochemical screening for the secondary metabolites using standard methods14,15.

 

Thin layer chromatography analysis (TLC):

The TLC plates were trimmed to strips and the position of the origin marked by a straight line. The petroleum ether, methanol extract was spotted on the origin and put in a lidded tank containing a solvent system. The procedure was repeated with various solvent systems viz., chloroform: methanol, n-hexane: ethyl acetate, n-hexane: acetone. The TLC plate was sprayed with sulfuric acid: methanol (5:95) reagent dried then heating on hot plate and spots were observed and Rf values was calculated16.

 

RESULTS AND DISCUSSION:

Antimicrobial activity:

The results of antibacterial activity studies was performed by agar well diffusion assay by using crude petroleum ether, methanol and aqueous extracts of leaf parts of Averrhoa carambola analysed against 10 human pathogenic bacteria compared with standard antibiotics viz., Ampicillin, Ciprofloxacin, Streptomycin and Tetracycline (0.5 mg/ml). It was found that the plant extracts were active against all the bacteria. Aqueous extract of A. carambola leaves showed higher antibacterial activity as compared with methanol and petroleum ether extract at the concentration of 25 mg/ml. Highest antibacterial activity was exhibited by the aqueous extract against Salmonella typhi (29.23±0.02 mm) followed by Proteus vulgaris (21.18±0.03 mm), Streptococcus mitis (17.22±0.01 mm), Klebsiella pneumonia (17.2±0.02 mm), Escherichia coli (17.2±0.01 mm), Staphylococcus aureus (17.03±0.06 mm), Pseudomonas aeruginosa (16.26±0.02 mm), Salmonella paratyphi (16.19±0.02 mm), Bacillus circulans (15.28±0.01 mm). Petroleum ether extract showed highest antibacterial activity against Escherichia coli and Salmonella paratyphi with a zone of inhibition of each 15.20 mm at the concentration of 50 mg/ml and methanol extract showed highest antibacterial activity against Klebsiella pneumoniae (14.19 ± 0.02 mm) at the same concentration. The result of antifungal activity studies against 6 human pathogenic fungi were compared with standard antibiotics viz., Fluconazole, Terbinafine, Itraconazole, chloramphenicol (0.5 mg/ml). It was found that the highest antifungal potential was exhibited by the methanol extract against Candida krusei (21.09±0.08 mm) followed by Fusarium oxysporum (18.12±0.02 mm) and Trichophyton mentagrophytes (14.12±0.02 mm), while the petroleum ether extract showed highest antifungal activity against Fusarium oxysporum (16.13±0.02 mm) followed by Candida krusei (14.07±0.06 mm), Candida tropicali (13.14±0.01 mm), Candida albicans (12.13±0.02 mm). Aqueous extract showed highest antifungal activity against Candida krusei (13.14±0.02 mm) followed by Candida tropicalis (13.12±0.02 mm). Plant extracts were active against all the fungal pathogens except Aspergillus niger. Table 1 and Table 2 show the results of antibacterial and antifungal activity of Averrhoa carambola leaf crude extract.


Table 1: Antibacterial activity (zone of inhibition - ZOI) of different extracts of Averrhoa carambola leaf

 

Concentration of plant extract

 

Bacterial strain

Solvent

6.25 mg/ml

12.5 mg/ml

25 mg/ml

50 mg/ml

Negative control

Antibiotics

A

C

S

T

Escherichia coli

PE

10.03±0.05

10.09±0.01

10.14±0.03

15.20±0.01

00±0.00

15

 

29

 

25

 

27

 

METH

9.07±0.07

14.26±0.02

9.60±0.53

13.30±0.30

AQE

10.03±0.05

10.1±0.01

17.2±0.01

10.15±0.04

Bacillus subtilis

PE

6.06±0.05

6.03±0.05

6.03±0.06

6.07±0.06

00±0.00

15

26

27

29

METH

4.15±0.17

4.96±0.19

4.96±0.06

5.81±0.31

AQE

11.03±0.06

11.13±0.04

13.23±0.02

10.19±0.01

Bacillus circulans

PE

10.02±0.04

10.03±0.05

10.06±0.05

10.07±0.06

00±0.00

13

28

24

27

METH

9.08±0.08

10.11±0.10

11.28±0.15

10.38±0.17

AQE

11.11±0.01

11.16±0.03

15.28±0.01

12.24±0.03

Pseudomonas aeruginosa

PE

6.04±0.04

6.05±0.04

6.03±0.05

6.07±0.06

00±0.00

40

33

16

33

METH

6.05±0.05

11.04±0.08

11.04±0.05

11.15±0.13

AQE

10.06±0.05

10.15±0.02

16.26±0.02

12.24±0.03

Klebsiella pneumoniae

PE

6.00±0.00

10.02±0.04

8.03±0.05

7.09±0.01

00±0.00

15

28

25

26

METH

7.05±0.08

13.10±0.09

11.04±0.06

14.19±0.02

AQE

13.11±0.01

13.16±0.02

17.2±0.02

10.07±0.06

Staphylococcus aureus

PE

6.00±0.00

6.00±0.00

6.00±0.00

6.00±0.00

00±0.00

35

35

23

35

METH

6.00±0.00

5.00±0.00

5.04±0.08

6.03±0.05

AQE

10.07±0.06

10.03±0.05

17.03±0.06

10.03±0.05

Salmonella typhi

PE

10.04±0.06

10.06±0.05

11.12±0.02

11.18±0.01

00±0.00

19

30

25

27

METH

10.00±0.00

10.03±0.05

10.06±0.06

10.15±0.02

AQE

15.20±0.02

10.08±0.07

29.23±0.02

12.21±0.01

Salmonella paratyphi

PE

15.11±0.01

14.19±0.00

15.18±0.02

15.20±0.02

00±0.00

13

28

28

27

METH

14.03±0.06

10.00±0.00

10.03±0.06

11.10±0.09

AQE

11.07±0.06

13.15±0.01

16.19±0.02

10.13±0.03

Streptococcus mitis

PE

6.00±0.00

9.03±0.05

12.07±0.06

10.07±0.06

00±0.00

16

 

29

 

29

30

METH

5.99±0.01

5.99±0.01

6.00±0.00

6.03±0.06

AQE

11.07±0.06

11.05±0.09

17.22±0.01

11.06±0.05

Proteus vulgaris

PE

10.00±0.00

10.03±0.03

10.03±0.05

10.09±0.07

00±0.00

18

22

25

29

METH

11.04±0.06

11.08±0.07

11.19±0.01

11.19±0.03

AQE

11.03±0.06

14.14±0.02

21.18±0.03

12.09±0.08

Note: Minus sign (-) indicates no activity

Each value represents the mean ± Standard deviation of three independent experiments. PE: petroleum ether extracts, METH: Methanol extracts, AQE: Aqueous extracts, Negative control: DMSO - dimethyl sulfoxide and Standard: A=Ampicillin, C=Ciprofloxacin, S=Streptomycin, T=Tetracycline (for comparative study, the maximum activity given extracts standard values are mentioned in the table).

 

Table 2: Antifungal activity (zone of inhibition - ZOI) of different extracts of Averrhoa carambola leaf

 

Concentration of plant extract

 

Fungaal strain

Solvent

6.25 mg/ml

12.5 mg/ml

25 mg/ml

50 mg/ml

Negative control

Antibiotics

F

T

I

C

Aspergillus niger

PE

00±0.00

00±0.00

00±0.00

00±0.00

00±0.00

24

40

25

 

25

 

METH

00±0.00

00±0.00

00±0.00

00±0.00

AQE

00±0.00

00±0.00

00±0.00

00±0.00

Candida albicans

PE

12.13±0.02

11.08±0.07

8.07±0.06

7.96±0.06

00±0.00

29

30

28

 

27

 

METH

10.03±0.06

10.11±0.01

12.17±0.02

11.07±0.06

AQE

10.06±0.05

10.07±0.06

10.03±0.06

10.07±0.06

Candida tropicalis

PE

13.14±0.01

12.07±0.06

11.07±0.06

00±0.00

00±0.00

16

25

 

24

 

26

 

METH

00±0.00

00±0.00

11.07±0.06

00±0.00

AQE

10.07±0.06

11.11±0.01

13.12±0.02

12.07±0.06

Candida krusei

PE

13.03±0.05

13.07±0.06

14.07±0.06

15.13±0.03

00±0.00

13

27

23

 

17

 

METH

16.07±0.06

14.08±0.07

19.03±0.06

21.09±0.08

AQE

11.07±0.06

13.11±0.01

13.14±0.02

10.12±0.02

Fusarium oxysporum

PE

6.00±0.00

16.08±0.07

13.07±0.06

16.13±0.02

00±0.00

14

 

15

 

14

 

13

 

METH

15.11±0.01

17.11±0.09

18.12±0.02

15.09±0.08

AQE

11.06±0.08

11.07±0.06

11.12±0.02

10.13±0.02

Trichophyton mentagrophytes

PE

11.11±0.01

0.00±0.00

0.00±0.00

0.00±0.00

00±0.00

12

 

13

 

10

 

17

 

METH

14..12±0.02

12.07±0.06

11.11±0.01

13.07±0.06

AQE

00±0.00

11.07±0.06

00±0.00

00±0.00

Note: Minus sign (-) indicates no activity

Each value represents the mean ± Standard deviation of three independent experiments. PE: petroleum ether extracts, METH: Methanol extracts, AQE: Aqueous extracts, Negative control: DMSO - dimethyl sulfoxide and Standard: F-Fluconazole, T-Terbinafine, I-Itraconazole, C-Clotrimazole (for comparative study, the maximum activity given extracts standard values are mentioned in the table).

 


 

 

Table 3: Minimum Inhibitory Concentration (MIC) of different extracts of Averrhoa carambola leaf against pathogenic organisms

MIC in mg/ml

Microorganisms

Petroleum ether

Methanol

Aqueous

Escherichia coli

50 mg/ml

50 mg/ml

25 mg/ml

Bacillus subtilis

50 mg/ml

50 mg/ml

25 mg/ml

Bacillus circulans

50 mg/ml

50 mg/ml

25 mg/ml

Pseudomonas aeruginosa

50 mg/ml

50 mg/ml

25 mg/ml

Klebsiella pneumoniae

12.5 mg/ml

50 mg/ml

25 mg/ml

Staphylococcus aureus

50 mg/ml

50 mg/ml

25 mg/ml

Salmonella typhi

12.5 mg/ml

50 mg/ml

25 mg/ml

Salmonella paratyphi

50 mg/ml

6.25 mg/ml

25 mg/ml

Streptococcus mitis

25 mg/ml

50mg/ml

25 mg/ml

proteus vulgari

50 mg/ml

50 mg/ml

25 mg/ml

Aspergillus niger

-

-

-

Candida albicans

6.25 mg/ml

25 mg/ml

50 mg/ml

Candida tropicalis

6.25 mg/ml

25 mg/ml

25 mg/ml

Candida krusei

25 mg/ml

25 mg/ml

25 mg/ml

Fusarium oxysporum

50 mg/ml

25 mg/ml

25 mg/ml

Trychophyton mentagrophytes

6.25 mg/ml

6.25 mg/ml

12.5 mg/ml

MIC Minimum Inhibitory Concentration (mg/ml), -: Not detected

 

Preliminary phytochemical screening

The preliminary phytochemical analysis revealed the presence of alkaloids, tannins and polyphenolic compounds, flavonoids, phytosterol, proteins and amino acids, carbohydrates, glycosides etc. Glycosides, Proteins and amino acids are present in petroleum ether leaf extracts only (Table 4) while saponins are absent in both the extracts.

 

Table 4: It shows Phytochemical screening of leaf extracts of Averrhoa carambola

Phytoconstituents

Fruit Extracts in different solvent media

Methanol

Petroleum ether

Alkaloids

+

+

Poly phenolic compounds

+

-

Flavonoids

+

+

Saponins

-

-

Tannins

+

-

Phytosterol

+

+

Proteins and amino acids

-

+

Carbohydrates

+

+

Glycosides

-

+

+ = Present and - =Absent

 

TLC profile of Averroha carambola leaves:

Thin layer chromatography of the petroleum ether and methanol extracts was carried out separately using solvent systems viz., Chloroform: Methanol, N-hexane: Ethyl acetate and N-hexane: Acetone and the Rf values were recorded in Table 5. TLC finger printing studies on petroleum ether and methanol extract showed presence of various phytoconstituents with their respective Rf values.

 

Table 5: Thin layer chromatography of Averrhoa carambola leaf against different extracts

Sl. No.

Leaves extracts

Solvent system

Number of spots

Colour

Rf value

1

Petroleum ether

Chloroform: Methanol (90:10)

Spot-1

pink

0.47

Spot-2

pink

0.75

Spot-3

violet

0.85

Spot-4

pink

0.92

N-hexane: Ethyl acetate (90:10)

Spot-1

pink

0.21

Spot-2

violet

0.38

Spot-3

pink

0.52

Spot-4

yellow

0.83

Spot-5

pink

0.95

N-hexane: Acetone (95:5)

Spot-1

pink

0.79

2

Methanol

Chloroform: Methanol (90:10)

Spot-1

pink

0.45

Spot-2

Pink

0.68

Spot-3

yellow

0.75

Spot-4

pink

0.77

Spot-5

violet

0.79

Spot-6

pink

0.88

N-hexane: Ethyl acetate (90:10)

Spot-1

pink

0.31

Spot-2

violet

0.36

Spot-3

violet

0.56

Spot-4

pink

0.93

N-hexane: Acetone (95:5)

Spot-1

violet

0.66

 

CONCLUSION:

Present study has justified the claimed use of this plant in the traditional medicinal systems for the treatment of various types of diseases. From the outcome of the study, it is concluded that the crude extracts of the Averrhoa carambola L. leaf possess considerable antimicrobial activity, against all the pathogenic strains except Aspergillus niger used in the study. The plant may contain potent antimicrobial compounds, effective in the treatment of various fungal and bacterial infections. TLC finger printing studies on petroleum ether and methanol extract showed the presence of various phytoconstituents with their respective Rf values. However, further investigation is needed in the direction of pure compound isolation, toxicological studies and clinical trials so as to use the promising compounds as effective antimicrobial agents.

 

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Received on 31.10.2018         Modified on 19.11.2018

Accepted on 21.12.2018      © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(3): 1219-1224.

DOI: 10.5958/0974-360X.2019.00202.6