International Journal of Advances in Science and Technology,

Vol. 3, No.2, 2011

Bio-assay and Phytochemical Screening of Water

hyacinth of Nepalese Wetland
Bikash Baral* and Geeta Shrestha Vaidya
Nepal Academy of Science and Technology (NAST)

*Corresponding author: bikubaral@yahoo.com / ursbiku@gmail.com

Abstract

An experiment on the bioassay of the obnoxious water weed, water hyacinth, was conducted using the soxhlet
extraction (hot method) and cold percolation method differing in polarity i.e. methanol and aqueous. The
antimicrobial assay was performed using agar well diffusion method against different clinical bacteria and
phytopathogenic fungi. The extracts showed the different range of antimicrobial activities. Among them, the
methanolic hot extract showed the better result than the cold percolation method. Similarly, in aqueous extraction
process, the hot extraction showed the better result than the cold extraction method. Salmonella typhi (ZOI: 19 mm)
was the most inhibited bacteria by the hot methanolic extract followed by Shigella dysentriae (ZOI: 16 mm) and
Staphylococcus aureus (ZOI: 16 mm). The cold aqueous extraction did not show any effect against the different
bacteria. Comparatively, the extract showed the better result against the phytopathogenic fungal strains. Among the
extracts used, the hot aqueous extraction showed the greatest Zone of inhibition (ZOI: 20 mm) against F.
moniliforme at 200 mg/ml, followed by Sclerotium rolfsiii (ZOI: 16mm). Among the different organisms used, fungi
were found to be highly inhibited by hot aqueous extract of Water hyacinth. Chemical analysis indicated that the
major component in these extracts were alkaloid salts, reducing compounds, polyoses and saponins.

Key-words: bacteria, bio-assay, fungi, phytochemical screening, extracts, MIC, ZOI, MBC and MFC.

1. Introduction
New discoveries on extraction of new antimicrobial compounds from various kinds of sources such as microorganisms,
plants and animals have been achieved by implying various techniques. Discoveries of novel effective compounds on
folk medicines may be one of them (Tomoko et al. 2002). In contrary to the synthetic drugs, antimicrobials of plant
origin do not have side effects and possess massive therapeutic potential to heal many infectious diseases. Development
of the antimicrobials from higher plants appears rewarding, leading to the development of phytochemicals to act against
microbes (Iwu et al. 1999). Anti-microbial activity has been reported in several plant constituents such as phenols,
quinones, flavones, flavonoids, flavonols, tannins, terpenoids, essential oils and alkaloids etc (Cowan 1999, Harborne
& Williams 2000).

Since centuries back, plants have been continuously used for curing human diseases because of having attributes of
therapeutic values (Nostro et al. 2000). About 80% of the world population directly or indirectly relies on the use of
traditional medicine predominately based on plant material (WHO, 1993).

Water hyacinth, the fast growing perennial aquatic macrophyte (Reddy & Sutton, 1984) locally called ‘Jalkumbi’ is one
of the world’s most obnoxious waterweeds when not controlled. It is found to have a good antimicrobial activity
against different pathogenic bacteria and fungi. Many of the biologically active compounds have been extracted from
this weed. It is also known for its well ability to grow in severe polluted waters (So et al. 2003), perhaps a good
surviving strategy where the other weeds fail to grow.

Recently considerable attention has been given at harvesting the aquatic plant for practical uses to partially defray the
cost of removing plants from waterways and use as economical sources in many parts of the world. This study was
designed to evaluate the phytochemical screening and antimicrobial assay of pharmacological active substances of
water hyacinth.

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2. Materials and Methods

2.1. Sampling: Water hyacinth plants were collected from Fewa lake, Pokhara. The whole plant was washed several
times under running tap water. These were shade dried, grinded into uniform powder and used for analysis.

2.2. Preparation of plant extracts: The extraction of the powdered plant materials was carried out by soxhlet
extraction method and cold percolation method using methanol and aqueous as solvent. The solvent of the methanolic
extract was removed using rotary vacuum evaporator while that of aqueous extract using hot water bath. The
concentrated extracts were stored at 4oC. The different working solutions of extracts, (i.e., 5 %, 10 %, 15 % and 20 %)
were prepared in dimethyl sulfooxide (DMSO).

2.3. Phytochemical analysis: Standard phytochemical test was carried out on the plant samples using the method as
given by Harborne (1998) to demonstrate the presence of the different pharmacologically active compounds.

2.4. Preparation of standard culture inoculum: The bacterial strains used for antimicrobial assay were Proteus
mirabilis (ATCC 49132), Salmonella typhi, S. paratyphi, Enterococcus faecalis (ATCC 29212), Bacillus subtilis
(ATCC 6633), Acinetobacter spp, Klebsiella pneumoniae, Schigella dysentriae, Staphylococcus aureus (ATCC 25923),
Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). Similarly, the fungal strains employed
for the experiment were Fusarium oxysporum, F. moniliforme, F. proliferatum, F. erundiforme, Sclerotium rolfsii,
Exserohilum turticum and Curvularia sp. For bacterial inoculum, colonies were selected from 18 - 20 h old cultures.
Turbidity was adjusted to 0.5 McFarland standard (1.5 x 108 CFU/mL. Similarly, the standard culture inoculum of each
fungal strain was prepared in potato dextrose broth (PDB) and adjusted to a range of 1×106 - 5×106 spores/ml.

2.5. Antimicrobial susceptibility testing: Well diffusion method using Mueller-Hinton agar plates were used to
demonstrate the antimicrobial properties of the crude extracts. The prepared inoculums for each bacteria was seeded on
to Muller Hinton Agar with a scraping from a glycerol stock frozen at –20°C and incubated during 18 - 20 h at 37°C.
Wells of 6mm were punctured in the culture media using sterile cork borers. The antifungal activity was observed in
the Potato Dextrose Agar (PDA) medium. In each well, 50µl of each plant extract (5%, 10%, 15%, 20%) obtained was
dispensed with the help of micropipette. The plates were incubated at 37oC for 24 h for bacterial culture and at 27oC till
5 days for fungal culture. ZOI as indicated by the clear zone i.e. without growth of organism around the well was
measured. The tests were done in triplicates and the mean value was taken. The solvents used for preparation of
working solution was at the centre were used as the negative control. The control zones of the solvents, if observed,
were deducted from the zones of inhibition created by the crude extracts.

The antimicrobial tests of extracts showing ZOI were further done by two fold broth dilution method to determine
minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)/ minimum fungicidal
concentration (MFC). The MIC and MBC for bacteria were evaluated using NB while PDB was used to evaluate MIC
and MFC for fungi.

3. Results
The methanolic and aqueous extracts of water hyacinth was obtained by soxhlet extraction and cold percolation
method. The antimicrobial activity of the different concentration (5%, 10%, 15% and 20%) of the extracts was
determined against 11 human pathogenic bacteria and 7 phytopathogenic fungi. The yield of the extract was found to be
4.132 and 5.22% in cold and hot extraction using methanolic solvent while the yield was 3.289 and 3.773 % in aqueous
solvent respectively.

Compared to aqueous extract the methanolic extract was found far effective against the bacterial pathogens, while
mixture result was obtained on fungal strains. The soxhlet extract was found to be effective against the bacterial and
fungal pathogens than the cold extract except for P. mirabilis, B. subtilis, F. proliferatum and S. rolfsii. Among the
bacterial strains (Table 1), S. typhi was found to be more susceptible against the methanolic (soxhlet and cold) extract.

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In the fungal strains, aqueous (soxhlet and cold) extract was found to be more effective against F. moniliforme (Table
2). The cold aqueous extract was not found to have any activity against tested bacterial pathogens.

The extracts did not show any activity against few bacteria (Pseudomonas aeruginosa and Enterococcus faecalis) and
few fungi (Exserohilum turticum and Curvularia sp.). The MIC value could not be obtained due to the colored extract.
The MBC value of the methanolic hot and cold extract against S. typhi was 7.185 mgml-1. Similarly, the MFC value of
the aqueous soxhlet and cold extract against F. moniliforme was 1.953 mgml-1. Klebsiella pneumoniae and Salmonella
typhi were the most susceptible bacterium of all the tested bacteria, while Fusarium moniliforme was found to be the
most susceptible fungus.

The phytochemical screening of various solvent extracts revealed the presence of alkaloid salts, reducing compounds,
polyoses and saponins in the different extracting solvents (Table 3). The presence of more pharmacologically active
compounds in aqueous extract is presumed to be responsible for the high potency recorded than methanolic extracts.
Phytochemicals in plants have been proved to be responsible for their therapeutic effect and by extension, antimicrobial
activity.

Table 1. Antibacterial properties of the extracts (in mm.)

Organism Pathogens used Soxhlet extraction Cold percolation Soxhlet extraction Cold percolation
(methanol) (methanol) (aqueous) (aqueous)
Conc. (%) Conc. (%) Conc. (%) Conc. (%)
5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20
Proteus mirabilis - - - - 7 8 9 10 - - - - - - - -
Salmonella paratyphi 7 9 12 14 - - 7 8 - - - - - - - -
S. typhi 11 14 15 19 8 8 9 10 - - - - - - - -
Bacillus subtilis - - 7 8 - - 7 8 - - - - - - - -
Bacterial strains

Acinetobacter spp 9 10 10 12 - 7 7 9 - - - - - - - -
Klebsiella pneumoniae 7 7 8 9 - - 8 10 9 11 12 13 - - - -
Schigella dysentriae 9 13 14 16 - - - - - - - - - - - -
Staphylococcus aureus 10 13 14 16 - - 7 10 - - - - - - - -
Escherichia coli - - - - - - - - - 7 9 12 - - - -
Pseudomonas aeruginosa - - - - - - - - - - - - - - - -
Enterococcus faecalis - - - - - - - - - - - - - - - -
The values in the tables are the diameter of ZOI (in mm) obtained as a mean of three replicates

Table 2. Antifungal properties of the extracts (in mm.)

Organism Pathogens used Soxhlet extraction Cold percolation Soxhlet extraction Cold percolation
(methanol) (methanol) (aqueous) (aqueous)
Conc. (%) Conc. (%) Conc. (%) Conc. (%)
5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20
F. oxysporum - - - - - - - - - 7 9 13 - - - -
Fungal strains

F. moniliforme - 7 8 12 7 8 11 12 13 16 17 20 12 13 14 14
F. proliferatum 7 8 10 11 7 8 12 13 - - - - - - - -
F.erundiformae 7 9 11 12 - - - - 7 7 11 12 - - - -

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 Sclerotium rolfsiii 8 11 12 12 14 15 15 16 - - - - 7 9 9 12

Exserohilum turticum - - - - - - - - - - - - - 7 8 10
Curvularia sp - - - - - - - - - - - - 7 9 12 14
The values in the tables are the diameter of ZOI (in mm) obtained as a mean of three replicates

Table 3 . Qualitative phytochemical screening of methanolic and aqueous extracts of Eichhornia crassipes
Phytochemicals Hexane extract Methanolic extract Aqueous extract
Volatile salts -- -- --
Fatty acids -- -- --
Coumarins -- -- --
Flavon aglycones -- -- --
Alkaloids ++ ++ ++
Emodins -- -- --
Sterols -- -- --
Triterpenes -- -- --
Polyphenols -- -- --
Reducing compound -- ++ ++
Glycosides -- -- --
Quinines -- -- --
Anthocyanosides -- -- --
Anthracenosides -- -- --
Polyoses ++ ++ ++
Saponins -- ++ ++
'++': Presence; '—' : absence

4. Discussion and Conclusion

An important characteristic of plant extracts and their components is their hydrophobicity, which enable them to
partition the lipid of the bacterial cell membrane and mitochondria, disturbing the cell structures and rendering them
more permeable. Extensive leakage from bacterial cells or the exit of critical molecules and ions will lead to death (
Rastogi & Mehrotra, 2002).

The inhibition produced by the plant extracts against particular organism depends upon various extrinsic and intrinsic
parameters. Due to variable diffusability in agar medium, the antibacterial property may not demonstrate as ZOI
commensurate to its efficacy. Therefore MBC and MFC values have also been computed in this study. MBC is the
lowest concentration of antibacterial substance required to produce a sterile culture (Cheesbrough, 1993).

Plant extracts are potential sources of novel antimicrobial compounds especially against bacterial and fungal pathogens.
Plant substances continue to serve as viable source of drugs for the world population and several plant-based drugs are
in extensive clinical use. The plant extract was found to have a good potential against F. moniliforme, leading to best
controlling and regulating mechanism against this phytopathogen. This may be due to the presence of saponins because
saponins are active antifungal agents (Sodipo et al. 1991) having expectorant action, which is very useful in
management of upper respiratory tract inflammation (Finar 1989, Trease & Evans 1989).

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The variation in the effectiveness of the extract against different microorganisms depends upon the chemical
composition of the extracts and membrane permeability of the microbes for the chemicals and their metabolism. It has
been suggested that the antimicrobial activity is mainly due to the presence of essential oils, flavonoids and

triterpenoids and other natural polyphenolic compounds or free hydroxyl groups (Rojas et al. 1992). The preliminary
phytochemical screening was also performed to observe the presence of secondary metabolites on test plant.

Sclerotium rolfsii, a phytopathogen that mainly attacks stems, roots, leaves and fruits and usually restricted to plant
parts in contacts to the soil can widely be controlled by the application of water hyacinth. More effect of this water
hyacinth can be observed in Fusarium moniliforme. Exserohilum , an emerging human pathogen has not been found to
be inhibited by the application of the different concentration of the extracts. The tested plant extract did not show any
activity against F. oxysporum, Curvularia sp, Pseudomonas sp and Enterococcus faecalis. In comparison to other
phytopathogens, Fusarium species are found to be more susceptible against the extract of water hyacinth.

Alcohol extracts provide a more complete extraction, including less polar compounds, and many of these extracts have
been found to possess antimicrobial properties (Ali-Shtayeh &Abu Ghdeib 1999). Also the aqueous extract was less
potent than methanol extracts. This may probably be due to the solvent and/or method of extraction (Rios and Recio,
2005). In addition, Nostro et al. (2000) demonstrated the effect of different extractives on the activity of the bioactive
molecules on the test organisms, thus supporting the reason for greater potency in methanol extract than aqueous
extract. The methanol solvent is known with its ability to isolate more antimicrobials from plants including tannins,
polyphenols, terpenoids, saponins, xanthoxyllines, totarol, quassinoids, lactones, flavones and phenones , while the
aqueous extracts could contain only anthocyanins, starches, tannins, saponins, polypeptides and lectins (Cowan 1999).
Many alkaloids contain at least one nitrogen atom in an amine type structure that makes them pharmacologically
active.The antimicrobial effect of the extracts could be explained by disturbance of the permeability barrier of the
bacterial membrane structure (Cowan, 1999).

In summary, our data indicate that the extracts from Water hyacinth efficaciously inhibit Klebsiella pneumoniae,
Salmonella typhi, S. paratyphi, Acinetobacter sp., Fusarium moniliforme and Sclerotium rolfsii which make serious
problems worldwide. This indicates that this plant may be useful for developing alternative compounds to treat
infections caused by these antibiotic resistant pathogens. As suggested in a recent report (Betoni et al., 2006), these
biologically active compounds of plant origin may be used together with known drugs in the development of
pharmacological agents against different hazardous pathogens.

Of all the test organisms used, B. subtilis and S. aureus were gram positive bacteria. The extract showed no potency on
B. subtilis and S. aureus for aqueous extract, but showed some activity in methanol extract. This may be due to the
presence of oval centrally located endospore, in B. subtilis making it difficult for the bioactive molecules of the extract
to get into the cells.

The yields of methanolic and aqueous extracts of water hyacinth were quite adequate inhibiting the growth of the test
organisms. The degree of inhibition was found to be both organism(s) and concentration dependent. These findings
tend to lend credence to the traditional use of Eichhornia crassipes for the treatment of microbial infections.

Acknowledgement
We would like to thank all those helping hands, especially Bijaya Laxmi Maharjan, for helping through the entire
process of our research. A thank also goes to the NAST family for providing a well equipped research laboratory and
co-operating with us in our study.

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390, 2006.

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Photoplates

Methanolic hot extract against Aqueous hot extract against Fusarium

Salmonella typhi moniliforme

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Aqueous cold extract against Curvularia MBC of methanolic extract against

sp. Salmonella typhi

Authors Profile:

Bikash Baral is a Research Fellow working in Nepal Academy of

Science and Technology (NAST). He did his graduation in Botany
specializing in Plant Biotechnology and Plant Biochemistry. He has
published several articles regarding the medicinal and invasive
plants. Currently, he is pursuing his research in Molecular
biotechnology laboratory of NAST.

Dr. Geeta Shrestha Vaidya is a Senior Scientific officer

working actively in NAST. She has been working here as an
active Scientist and as a Chief of Biological Resources Unit of
Nepal Academy of Science and Technology (NAST). She has
published more than three dozens of papers in National and
International Journal.

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