Antimicrobial Activity of Euryale ferox.

Salisb:- A Threatened Aquatic Plant of Kashmir Himalaya

By
Javid Ahmad Parray M.Phil Research scholar

Supervisor: Prof.(Dr.) Azra N. Kamili

Co-Supervisor: Dr. Raies A. Qadri
P.G Department of Environmental Science University of Kashmir

INTRODUCTION

Medicinal Plants

Medicinal plants contain chemical substances which produce a definite physiological action on human body and animal system.

Chemical substances

Essential oils

Tannins

Glycosides

Phenols and Mucilages

Steroids

Flavnoids

Secondary metabolites

pharmacological activities

Spasmolytic

Antiinflammatory

Antimicrobial Stimulatory Sedative

Anti cancerous

The main objective of the work was to determine the Antibacterial and Antifungal activity of Euryale ferox.

Study Area

Study Area- IRSP6-LISS III October Image of Mansabal lake

Source: Department of Geology and Remote Sensing, University of Kashmir.

PLANT PROFILE

Euryale ferox

Parts Studied
Seeds, Rhizome, Petiole and Leaves.

Euryale ferox

Scientific Classification
Domain Kingdom Eukaryota Plantae

Subkingdom
Phylum Sub-phylum Infra-phylum Class Sub-class Super- order Order Family Sub-family Tribe Genus

Viridaeplantae
Tracheophyta Euphyllophytina Radiatopses Magnoliopsida Nymphaeidae Nymphaeanae Nymphaeales Nymphaeaceae Euphorbioideae Euphorbie Euryale

Species E. Ferox Source: http://Plants.gov/java/Euryale f erox/Classification Servelet

Different names of Euryale ferox

Bihar English Madhubani Fox nut

Kashmir
Punjabi Hindi Korean China Japan Sanskrit

Jewer
Juwar Makhana Ga-si-yeon-kot Qianshi Onibasu Mukhauna, Padma

Vietnamese

Khiem Thuc

Source: http: //www. herbalextractplus.com/Euryale.cfm

Botanical Description

An annual aquatic herb with rhizomatous stems, rhizome erect or repent and unbranched. Leaves directly arise from rhizomes and are of 410 cm in length with long petiolete. Floating leaves are prickly on petioles and along veins. Leaf blade is abaxially dark purple and adaxially green and is 1.3-2.7 mm in diameter and primary veins are prickly on both surfaces . Seeds are very hard, remains dormant for year.

Distribution
China, Korea, Japan, Russia, Bangladesh, India and grows wild in J&K and is an non endemic threatened plant. (Khan et al., 2000; Dar et al., 2002).

Medicinal Uses
The plant has been traditionally used though out the world to cure many diseases including chronicdiarrhea, kidney problem, leucorrhea and hypofunction of spleen. (Brown, 1995). used as an analgesic, aphrodisiac, astringent, oxytonic and tonic in China (Duke et al., 1985). Leaves are used in the case of difficult parturition (Duke and Ayensu, 2003). The plant is internally taken to treat vaginaldischarge, impotence etc.(Brown, 1995).

 In Indian traditional system of medicines the dry seeds of the plant are being used as immuno-stimulant for mothers after child birth with relatively poor immune status (Puri et al., 2000). E. ferox also has been shown to enhance the activities of superoxide dismutase, catalase and glutathione peroxidase in V79-4 cells (Lee et al., 2002) Seed of this plant have cardio protective properties which may link with ability of makhana to induce TRP32 and TrX-1 protein and to scavenge ROS (Samarajit et al., 2006).

Euryale ferox

Seeds of Euryale ferox

METHODOLOGY

METHODOLOGY

Methodology involved: Selection and collection of the medicinal plant. Solvent extraction. Selection of specific bacterial and fungal strains. Antimicrobial assay Qualitative phyto-chemical screening

Identification and Collection

Identification: Plant was identified at KASH, Department of Botany, University of Kashmir under Acc. No.1015.
Collection: August -September 2008

Crude extraction of the sample

The powder form of different plant parts (leaf, rhizome, petiole and seeds) collected were subjected to hot extraction (Soxhlet extraction). Solvents: Petroleum ether, Chloroform, and Methanol.

Soxhlet extractor

Whole plant extraction

Fifty grams of dried powder of each plant part (leaf, rhizome, petiole and seed) were used.

Extracts collected were later dried and weighed and kept for further process.

Preparation of Discs Dissolve 200 mg of extract in 25 ml of solvent. (1l=8g)

Preparation of media
Nutrient Agar Medium

: Nutrient Broth
Muller Hinton Agar

Dissolve 37.0 gm in 1000 ml of distilled water Suspend 8.0 gm in 1000 ml of distilled water. Dissolve 38.0 gm in 1000 ml of distilled water

Muller Hinton Broth

Blood Agar

Sabourand Dextrose agar

Suspend 21g of the powder in 1000ml of distilled water. Suspend 40 g of the blood agar base in 1000 ml of distilled water. Dissolve 64 g in 1000ml of distlled water

Test Microorganisms
Gram Positive Bacterial Strains
Staphylococcus aureus I Staphylococcus aureus II Staphylococcus aureus III Pnemococcus spp.

Gram Negative Bacterial Strains

E. Coli I E. Coli II Pseudomonas aeruoginosa I Pseudomonas aeruoginosa II

Proteus vulgaris
Salmonella typhii Salmonella typhimurium Klebsiella pneumonia Acintobacter sp Citrobacter fruendi Shigella flexneri

Strains were provided by Bacteriological and Mycological section ( Department of Microbiology, SKIMS Soura. Srinagar.

SENSITIVITY TEST FOR ANTIMICROBIAL ASSAY

Antimicrobial susceptibility Tests: The primary purpose of antimicrobial susceptibility testing is to determine the potency of crude drug.
Disc diffusion method: (Nutrient Agar , Muller Hinton Agar and Saboarnd Dextroser agar, Hi Media)

Kirby-Bauer (Bauer et al , 2002) method was followed.

Sterile discs with antimicrobial agents are poured on microbial cultures and inhibition zone diameters (mm) around the discs are measured as the potency of crude plant extracts. Mixed culture of bacteria obtained from the polluted drain (that was spread on the nutrient agar plate) and twelve extracts ( 3 of each plant parts) were tested for antibacterial activity.
Extracts which shows maximum activity ( methanol seed and methanol leaf extract ) were than tested against specific bacterial and fungal strains.

Dilution Susceptibility Tests

Minimum inhibitory concentration (MIC)

Minimum bactericidal concentration (MBC).

Total activity (ml)

Standard ATCC Bacterial Strains

S. aureus ATCC.25923 E. coli ATCC.25922

Pseudomonas aeroginosa ATCC.27853 Shigella flxmeri ATCC.12022

Proteus vulgaris*

Salmonella typhi*

Strains were provided by Bacteriological and Mycological section, Department of Microbiology, SKIMS Soura. Srinagar.

Medium

Muller Hinton Broth (Hi Media ) (NCCLS, 1990)

Preparation of dilutions of extracts for use in MIC determination

Antimicrobial Conc. mg/l 10000 10000 100 100 10000 10000 Volume of solution (ml) 1 0.1 1 0.1 1 0.512 Distilled water (ml) 9 9.9 9 9.9 0 0.488 Antimicrobial Conc. mg/l 1000 100 10 1 10000 5120 Final Conc. at 1:20 dilution(g/ml) _ _ _ _ 500 256

10000 10000
1000 1000 1000 100 100 100 10 10 10 10 1 1 1

0.256 0.128
0.64 0.32 0.16 0.8 0.4 0.2 1 0.5 0.25 0.125 0.625 0.313 0.156

0.744 0.872
0.36 0.68 0.84 0.2 0.6 0.8 0 0.5 0.75 0.875 0.375 0.687 0.844

2560 1280
640 320 160 80 40 20 10 5 2.5 1.25 0.625 0.313 0.156

128 64
32 16 8 4 2 1 0.5 0.25 0.125 0.06 0.03 0.015 0.008

(As per Philips et al ., 1991)

Phyto-chemical Screening Qualitative Tests

Physiochemical standards of Unani formulations- 1987

Alkaloi ds Dragen dorffs test

Flavonoids NaOH and conc. HCl test

Phenols Ferric chloride test

Glycosides Fehlings test

Steroids LiebermanBurchards test

Tannins Ferric chlorid e test

Saponins Frothing test.

Isolation of Phyto-constituents

By

Thin Layer chromatography

RESULTS

Antimicrobial activity of Euryale ferox extracts against mixed culture of GPC & GNB bacteria.

Strains

Inhibition Zone Diameter (mm) Rhizome Petiole Leaf Seed

Pt
Mixed culture of GPC & GNB bacteria. _

C
_

M
_

Pt
_

C
_

M
_

Pt
_

C
_

M Pt C
+ _ _

M
+

GPC= Gram Positive Cocii GNB= Gram Negative Bacillus + = Inhibitory activity - = No activity Pt = Petroleum ether extract C = chloroform extract M = methanol extract

Solvent Control
Strains Inhibition Zone Diameter (mm) Petroleum ether
Mixed culture of GPC & GNB bacteria. _

Chloroform
_

Methanol
_

GPC= Gram Positive Cocii GNB= Gram Negative Bacillus; + = Inhibitory activity - = No activity.

FIG. 1a: Solvent extracts of E. ferox

GPC=Gram Positive Cocci GNB= Gram Negative Bacilli M=methanol Pt=Petroleum ether C=chloroform SM=Seed methanol SC=Seed chloroform SP= Seed pet ether LM=Leaf methanol LC=Leaf Chloroform LP=Leaf pet ether PM=Petiole methanol PC=Seed chloroform PP= Seed pet ether RM=Leaf methanol RC=Leaf Chloroform RP=Leaf pet ether

FIG. 1b: Solvent Control

Plate 1: Mixed culture of GPC & GNB

Fig 2a : E. coli

Fig 2b: Staphylococcus aureus

Plate 2: Effect of different concentrations of methanol seed extracts of E.ferox on E. coli and Staphylococcus aureus SI = 5 l and S11 =55 l (seed methanol extract)

Antibacterial activity of methanolic seed extracts of Euryale ferox against Gram Positive Bacterial strains. S. No.
01 02

Strains

Inhibition Zone Diameter (mm)*

160 g 320 g
S. aureus I S. aureus II 120.64 120.81 23 0.41 18 0.70

Antibiotics
Erythromycin(25) Vancomycin (20)

03
04

S. aureus III
Pnemococcus spp

101.63
151.29

18 0.81
20 0.81

Cotrimaxozole (24)
Lenzolid (22)

* Results are mean diameter of zone of inhibition of three replicates followed by standard deviation; Methanol was taken as negative control and was found resistant in all strains.

Antibacterial activity of methanolic leaf extracts of Euryale ferox against Gram Positive Bacterial strains.

S. No

Strains

Inhibition Zone Diameter (mm) *

160 g 320 g
NA 151.28 201.73 160.40

Antibiotic
Erythromycin (25) Vancomycin (20) Cotrimaxozole (24) Lenzolid (22)

01 02 03 04

S. aureus I S. aureus II S. aureus III Pnemococcus spp

NA 90.00 171.28 121.07

*Results are mean diameter of zone of inhibition of three replicates followed by standard deviation; Methanol was taken as negative control and was found resistant in all strains

Fig. 1: Comparative assessment of antibacterial activity of methanolic seed and leaf Extract of E. ferox against Gram Positive Bacterial Strains (320g)

Inhibition zones Diameter (mm) Gram Positive Bacterial Strains

Antibacterial activity of methanolic seed extracts of Euryale ferox against Gram Negative Bacterial strains.
S. No.
01 02 03 04 05 06 07 08 09 10 11

Strains
E .coli I E. coli II Pseudomonas aeroginosa I Pseudomonas aeroginosa II Acintobacter spp Citrobacter freundii Proteus vulgaris Shigella flexmeri Klebsiela pneumonia Salmonella typhi Salmonella typhimurum

Inhibition Zone Diameter (mm) * 160 g

12 1.15 16 2.16 18 2.82 16 0.00 10 2.16 NA NA 141.41 NA 18 0.81 NA

320 g
15 0.00 22 1.63 28 0.81 27 1.29 14 1.15 NA 19 1.41 231.61 NA 25 0.57 15 1.28

Antibiotics
Ceftazidime (30) Gentamycin (20) Amikacin (15) Gatifloxcin (20) Piperacilin (0) Vancomycin (22) Cefixime Ciprofloaxcin (29) Gentamycin (25) Imipenin (28) Gatifloxcin (0)

*Results are mean diameter of zone of inhibition of three replicates followed by standard deviation; Methanol was taken as negative control and was found resistant in all strains.

Antimicrobial activity of methanolic leaf extracts of Euryale ferox against Gram Negative Bacterial strains.
S. No.
01 02 03 04 05 06 07 08 09 10 11

Strains
E. coli I E. coli II Pseudomonas aeroginosa I Pseudomonas aeroginosa II Acintobacter spp Citrobacter freundii Proteus vulgaris Shigella flxmeri Klebsella pneumoniae Salmonella typhi Salmonella typhimurum

Inhibition Zone Diameter (mm) * 160 g 320 g Antibiotics

8 1.22 13 0.42 16 1.10 17 1.24 10 0.16 NA NA 12 0.74 NA 11 0.08 NA 100.16 200.41 22 0.24 21 0.81 16 0.40 NA 10 0.00 18 0.21 NA 21 0.14 10 0.16 Ceftazidime (30) Gentamycin (20) Amikacin (15) Gatifloxcin (20) Piperacilin (0) Vancomycin (22) Cefixime Ciprofloaxcin (29) Gentamycin (25) Imipenin (28) Gatifloxcin (0)

*Results are mean diameter of zone of inhibition of three replicates followed by Standard deviation; Methanol was taken as negative control and was found resistant in all strains.

Inhibition Zones Diameter (mm) Gram Negative Bacterial Strains Fig. 2: Comparative assessment of antibacterial activity of methanolic seed and leaf extract of E. ferox against Gram Negative Bacterial Strains (320 g)

Fig. 3a: S. aureus I

Fig. 3b: Pneumococcus Spp.

Plate 3: Antibacterial activity of methanolic seed and leaf extract of E. ferox against S.aureus I and Pneumococcus spp SI=160 g, S2=320 g= Seed methanol extract. L1=160 g, L2=320 g = Leaf methanol extract, Ab= Antibiotic; M= methanol

Fig. 4a: S. aureus II

Fig. 4b: S. aureus III

Plate: 4 Antibacterial activity of methanolic seed and leaf extract of E. ferox against S.aureus II and S.aureus III SI=160 g, S2=320 g= Seed methanol extract. L1=160 g, L2=320 g = Leaf methanol extract, Ab= Antibiotic; M= methanol

Fig. 5a: P. aureoginosa I

Fig. 5b: P. aureoginosa II

Plate 5: Antibacterial activity of methanolic seed and leaf extract of E. ferox against P. aureoginosa I and P. aureoginosa II SI=160 g, S2=320 g = Seed methanol extract. L1=160 g, L2=320 g = Leaf methanol extract, Ab= Antibiotic; M= methanol

Fig. 6a: S. typhi

Fig. 6b: E. coli I

Plate 6: Antibacterial activity of methanolic seed and leaf extract of E. ferox against S. typhi, E. coli I and E. coli II. SI=160 g, S2=320 g = Seed methanol extract. L1=160 g, L2=320 g = Leaf methanol extract, Ab=Antibiotic; M=methanol

Fig. 6c: E. coli II

Fig. 7a: Shigella flexnerii

Fig. 7b: Acintobacter spp.

Plate 7: Antibacterial activity of methanolic seed and leaf extract of E. ferox against Shigella flexnerii and Acintobacter spp. SI=160 g, S2=320 g = Seed methanol extract. L1=160 g, L2=320 g = Leaf methanol extract, Ab= Antibiotic; M=methanol

Fig. 8a: Citrobacter freundii

Fig. 8b: Klebsiella pneumonia

Plate 8: Antibacterial activity of methanolic seed and leaf extract of E. ferox against Citrobacter freundii and Klebsiella pneumonia

SI=160 g, S2=320 g= Seed methanol extract. L1=160 g, L2=320 g = Leaf methanol extract, Ab= Antibiotic; M= methanol

Fig. 9a: Salmonella typhimurium

Fig. 9b: Proteus vulgaris

Plate 9: Antibacterial activity of methanolic seed and leaf extract of E. ferox against Salmonella typhimurium and Proteus vulgaris

SI=160 g, S2=320 g= Seed methanol extract. L1=160 g, L2=320 g = Leaf methanol extract, Ab= Antibiotic; M= methanol

Minimum Inhibitory Concentration (MIC) of methanolic seed extract of Euryale ferox against standard bacterial strains (g/ml)
S. No. Strains MIC MBC MIC Index Total Activity (ml) 593.75 296.87 593.75 _ 76 1.95 148.43 Gt. 10 g/ml MIC MBC

01 02 03 04 05 06

S. aureus ATCC.25923 E. coli ATCC.25922 Pseudomonas aeroginosa ATCC. 27853 Shigella flxmeri ATCC.12022 Proteus vulgaris* Salmonella typhi*

64 128 64 ND 500 256

128 256 256 ND ND 500

2.0 2.0 4.0 _

32 128 16 ND 256 ND

128 500 64 ND 500 ND

Methanol was used as negative control and exhibited no activity against tested organisms; ND = Non detectable within the range tested; * = isolated Strains; Gt. = Gentamicin MIC index = 4 (bactericidal) and = 4 (bacteriostatic) Total activity =highest value= highest inhibitory activity

Minimum Inhibitory Concentration (MIC) of methanolic leaf extract of Euryale ferox against standard bacterial strains (g/ml).
S. No. Strains MIC MBC MIC Total Index Activity (ml) 3.90 1.95 1.95 ND ND 737.5 368.75 368.75 188.8 _ 188.8 Gt 10 g/ml MIC 32 128 16 ND 256 MBC 128 500 64 ND 500 ND

01 02 03 04 05 06

S. aureus ATCC.25923 E. coli ATCC.25922 Pseudomonas aeroginosa ATCC.27853 Shigella flxmeri ATCC.12022 Proteus vulgaris* Salmonella typhi*

128 256 256 500 ND 500

500 500 500 ND ND ND

Methanol was used as negative control and exhibited no activity against tested organisms; ND = non detectable within the range tested; * = isolated Strains; Gt. = Gentamicin. MIC index = 4 (bactericidal) and = 4 (bacteriostatic) Total activity =highest value= highest inhibitory activity

MIC (g/ml)

Standard Bacterial Strains Fig. 3: Comparative assessment of MIC of methanolic seed and leaf extract of E. ferox against standard bacterial strains (g/ml)

g/ml) MBC (g/ml)

Standard Bacterial Strains

Fig. 4: Comparative assessment of MBC of methanolic seed and leaf extract of E. ferox against standard bacterial Strains (g/ml)

Total activity (ml)

Standard Bacterial Strains

Fig. 5: Comparative assessment of Total activity of methanolic seed and leaf Extract of E. ferox against standard bacterial Strains (ml)

Antifungal activity of methanolic leaf and seed extract of Euryale ferox.

S. No. Fungal Strains Leaf extract Seed extract

200 g
01 02 03 04 05 Crytococcus neoformans Aspergilus fumigates Candida albicans C. kruesie C.Stealoidea NA NA 9 0.81 8 0.48 10 0.62

400 g
NA NA 100.28 150.34

200 g
NA NA 70.3 80.2

400 g
NA NA 12 0.81 9 0.00 11 0.5

16 0.70 70.3

06
07 08 09

C. tropicals
C. parapsilosis Rhizopus spp Pencillum notatum

NA
NA NA 10 0.81

150.34
NA NA 150.60

NA
NA NA 80.2

18 0.14
NA NA 19 0.84

*Results are mean diameter of zone of inhibition of three replicates followed by standard deviation; Nystatin was used as positive control and Methanol as negative control in all strains.

Inhibition Zone Diameter (mm) Fungal Strains Fig. 6: Comparative assessment of antifungal activity of methanolic Seed and Leaf extract of E. ferox against fungal Strains (400 g).

Fig. 10a: Aspergillus fumigates

Fig. 10b: Cryptoccocus neoformans

Plate 10: Antifungal activity of methanolic seed and leaf extract of E. ferox against Aspergillus fumigates and Cryptoccocus neoformans SI=200 g, S2=400 g = Seed methanol extract. L1=200 g, L2=400 g = Leaf methanol extract, Ab= Antibiotic; M=methanol

Fig. 11b: Rhizopus spp Plate. 11: Antifungal activity of methanolic seed and leaf extract of E. ferox against C. parapsilosis and Rhizopus spp. SI=200 g, S2=400 g = Seed methanol extract. L1=200 g, L2=400 g = Leaf methanol extract, Ab= Antibiotic; M=methanol

Fig. 11a: C. parapsilosis

Fig. 12a: C. albicans

Fig. 12b: C. Kruesie

Plate 12: Antifungal activity of methanolic seed and leaf extract of E. ferox against C. albicans and C. kruesie SI=200 g, S2=400 g = Seed methanol extract. L1=200 g, L2=400 g = Leaf methanol extract, Ab= Antibiotic; M=methanol

Fig. 13a: P. notatum

Fig. 13b: C. stealoidea

Plate 13: Antifungal activity of methanolic seed and leaf extract of E. ferox against P. notatum C. stealoidea and C. tropicals . SI=200g, S2=400 g = Seed methanol extract.L1=200g,L2=400 g = Leaf methanol extract, Ab= Antibiotic; M=methanol
Fig. 13c: C. tropicals

Phyto-chemical Constituents of Methanolic Extracts of Euryale ferox

Constituents tested for Leaf extract Seed extract

Alkaloids
Steroids Glycosides Phenols Tannins Saponins Flavonoids
- = Not present , + = Present.

+
_ + + + _ +

+
+ + + + + +

C D

A= Petiole methanol extract B= Seed methanol extract C= Rhizome methanol extract D= Leaf methanol extract Solvent front =16.5cm

Rf values of Methanolic extracts of Euryale ferox

Methanolic Extracts
Seed

Rf. value
0.176, 0.41, 0.58, 0.63 and 0.84

Leaf
Petiole Rhizome

0.176, 0.35, 0.45 and 0.59

0.26 0.11

Solvent front = 17 cm solvents used = Chloroform: Methanol (7:3)

Extraction yield and macroscopic characteristics of the crude extracts of Euryale ferox
Name of the Common plant Name Solvent Methanol Chloroform Petroleum ether Methanol Chloroform Petroleum ether Methanol Chloroform Petroleum ether Methanol Chloroform Petroleum ether Part used Macroscopic characteristics Seed Seed Seed Black crystalline powder Brown crystalline powder Dark green shiny surface Extract/ yield (%) 3.84 3.07 0.3 9.44 1.74 0.6 7.88 0.38 1.76 10.9 0.98 1.66

Leaves Leaves Leaves

Euryale ferox

Jewer

Dark green shiny surface Dark green powder Yellowish green powder Dark black powder Dark green shiny surface A dark brown shiny surface Dark green powder Brown crystalline powder Yellowish blue powder

Rhizome Rhizome Rhizome Petiole Petiole Petiole

CONCLUSION

 Only methanolic extracts of seed and leaf exhibited highest antimicrobial activity. Seed extracts exhibited higher inhibitory activity than leaf extracts. Gram Positive strains were more susceptible than gram negative strains. Among the gram positive strains, S.aureus shows maximum zone of inhibition i.e highest inhibitory activity. However, IZD of some gram negative strains ( i.e. P. aureoginosa) were more than gram positive strains. Among the fungal strains, Candida spp. and Pencillum notatum were more susceptible to both extracts at higher concentrations. However C. neoformans, A. fumigates and Rhizopus spp. C.Parapsilosis were completely resistant.

 The results of MIC and MBC depicted that extracts of E. ferox can be bactericidal in action and among standard strains tested, S. aureus ATCC 25923 was inhibited by least concentration of extracts used. Phyto-chemical screening of methanolic seed and leaf extracts revealed the presence of alkaloids, phenols, flavonoids, steroids and tannins while as steroids and Saponins were present in only seed extract. The separation of methanolic extracts by thin layer chromatography (TLC) yields different compounds with seed extract showed five compounds of Rf values 0.176, 0.41, 0.58, 0.63 and 0.84 while as leaf extract yields four compounds with Rf. value of 0.176, 0.35, 0.45 and 0.59.

The substances that can inhibit pathogens and have a little toxicity to host cells are considered good for developing antimicrobial drugs. Therefore, the antimicrobial activity of E. ferox extracts on these UTI organisms reveals that it can be safely used to treat these kinds of infections after proper drug formulation. The literature reveals that no prior work has been done on evaluating antimicrobial activity of E. ferox and it is now expected after results that it will be helpful in obtaining broad spectrum herbal formulation as well as new antimicrobial substances.