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25. ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF VANILLA PLANIFOLIA GROWN IN SHER-E-BANGLA AGRICULTURAL UNIVERSITY

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This study was carried out in 2a biotech lab, Department of Horticulture, Sher-e-Bangla Agricultural University with an objective to investigate the antibacterial and antifungal potentials of the leaf extracts of Vanilla planifolia. The aim of the study is to assess the antimicrobial activity and to determine the zone of inhibition of extracts on some bacterial and fungal strains. The antimicrobial activity was determined in the extracts using agar disc diffusion method. The antibacterial and antifungal activities of extracts (50, 100, 200 mg/L) of Vanilla planifolia were tested against 4 bacterial (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis) and two fungal strains (Aspergillus niger, Aspergillus flavus). Zone of inhibition of extracts were observed. The results showed that the remarkable inhibition of the bacterial growth was shown against the tested organisms. Fungus strains showed low sensitivity than bacteria although Aspergillus niger showed moderate sensitivity to the extract in all the concentrations. The phytochemical analyses of the plants were carried out. The microbial activity of the Vanilla planifolia was due to the presence of various secondary metabolites. Hence, these plants can be used to discover bioactive natural products that may serve as leads in the development of new agricultural research activities.
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BANGLADESH RESEARCH PUBLICATIONS JOURNAL
ISSN: 1998-2003, Volume: 11, Issue: 1, Page: 34-39, March - April, 2015
Review Paper
ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF VANILLA PLANIFOLIA
GROWN IN SHER-E-BANGLA AGRICULTURAL UNIVERSITY
A.F.M. Jamal Uddin1*, A. Nusrat1, S. Parvin1, M.Z.K. Roni2 and U. Mayda3
A.F.M. Jamal Uddin, A. Nusrat, S. Parvin, M.Z.K. Roni and U. Mayda (2015). Antibacterial and Antifungal Activities of Vanilla
Planifolia Grown in Sher-E-Bangla Agricultural University. Bangladesh Res. Pub. J. 11(1): 34-39. Retrieve from
http://www.bdresearchpublications.com/admin/journal/upload/1410058/1410058.pdf
Abstract
This study was carried out in 2a biotech lab, Department of Horticulture, Sher-e-
Bangla Agricultural University with an objective to investigate the antibacterial and
antifungal potentials of the leaf extracts of Vanilla planifolia. The aim of the study is
to assess the antimicrobial activity and to determine the zone of inhibition of
extracts on some bacterial and fungal strains. The antimicrobial activity was
determined in the extracts using agar disc diffusion method. The antibacterial and
antifungal activities of extracts (50, 100, 200 mg/L) of Vanilla planifolia were tested
against 4 bacterial (Staphylococcus aureus, Escherichia coli, Pseudomonas
aeruginosa, Bacillus subtilis) and two fungal strains (Aspergillus niger, Aspergillus
flavus). Zone of inhibition of extracts were observed. The results showed that the
remarkable inhibition of the bacterial growth was shown against the tested
organisms. Fungus strains showed low sensitivity than bacteria although Aspergillus
niger showed moderate sensitivity to the extract in all the concentrations. The
phytochemical analyses of the plants were carried out. The microbial activity of the
Vanilla planifolia was due to the presence of various secondary metabolites.
Hence, these plants can be used to discover bioactive natural products that may
serve as leads in the development of new agricultural research activities.
Key words: Vanilla, Antibacterial, Antifungal.
Introduction
Plants being the most reliable source of curatives are used as folk medicines for centuries.
The presence of antibacterial substances in the higher plants is well established (Srinivasan
et al., 2001). Even 80% of the modern day man still focuses on plant based remedies, for
their better adaptability, lesser side effects and economical affordability with a huge
compliment to the cultural acceptance (Firas and Bayati, 2009). Infections caused by
pathogenic bacteria and fungi are increasingly recognized as an emerging threat to
agricultural crops as well as human health (Walsh et al., 1996; Wu, 1999). Numerous
naturally occurring antimicrobials are present in plant tissues (Smid and Gorris, 1999) where
they probably evolved as part of the defense mechanisms of the host against microbial
invasion. There are many plants that demonstrate antimicrobial activity (Beales, 2002). The
bioactive compounds in plants are produced as secondary metabolites. Examples
include alkaloids, proteins (Chakraborty & Brantner, 1999), which may be stage specific or
organ specific or tissue specific. In fact there are several studies which have revealed the
presence of such compounds with antimicrobial properties (Cowan, 1999). As compared
to synthetic antimicrobial agents, plant based antimicrobials are cost effective,
affordable and exhibit lesser side effects. Herbs are widely exploited in the traditional
medicine and their curative potentials are well documented (Dubey, et al., 2004).
Numerous orchid species are traditionally used in herbal medicine as a remedy for
microbial infections and number of other ailment. Vanilla planifolia is a commercial as well
as medicinal orchid belonging to Orchidaceae family. The extract of this plant is used in
treating hysteria, rheumatism and other low forms of fever. This flat-leaved vanilla is a
Mexican species from where vanilla flavor is derived due to its high vanillin content. The
*Corresponding Author Email: E-mail: jamal4@yahoo.com
1 Department of Horticulture, Sher-e-Bangla Agricultural University;
2 Department of Agricultural Botany, Sher-e-Bangla Agricultural University;
3 Department of Botany, Jahangirnagar University
Antibacterial and Antifungal Activities of Vanilla Planifolia
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35
pods of Vanilla plantifolia are used for the extraction of vanillin. Moreover it contains
alkaloids, flavonoids, glycosides, carbohydrates and other phytochemicals. The presence
of tannin, flavonoid, carbohydrate, and glucoside have been reported to inhibit bacterial
growth (Clark, 1981). This present study represent an attempt aimed at investigating the
antimicrobial and antifungal activity of methanol extract of Vanilla plantifolia leaf against
some pathogenic microbes (viz. Staphylococcus aureus, Escherichia coli, Pseudomonus
aeruginosa, Bacillus subtilis, Aspergillus flavus and Aspergillus niger ).
Materials and methods
This experiment was conducted in 2a biotech laboratory, Department of horticulture,
Sher-e-Bangla Agricultural Univesrity. Fresh vanilla plant leaves were collected from 2a
Roof top garden, academic vaban, Sher-e-Bangla Agriculture University.
Preparation of plant extracts
Preparation of powder: Collected leaves were dried under shade condition. These dried
leaf materials were mechanically made coarse powdered with grinder machine and
preserved in air tight container. These vanilla leaves powder were used for further
phytochemical and fluorescent analysis.
Extraction of plant material: The coarse powder material was subjected to Soxhlet
extraction separately and was added methanol. These extract were concentrated to
dryness in flash evaporator under reduced pressure and controlled temperature.
Methanolic extract was stored in a refrigerator in air tight container. These extract was use
for phytochemical screening, antibacterial and antifungal activity.
Qualitative phytochemical test: Qualitative phytochemical screening was done using
different chemical procedure. Carbohydrate, steroid, alkaloids, tannins, flavones, phenols,
glycoside and terpine were qualitatively analyzed.
Test organisms: The stored culture of Staphylococcus aureus, Escherichia coli,
Pseudomonas aeruginosa, Bacillus subtilis, and the fungal strains Aspergillus flavus,
Aspergillus niger were collected from Jahangirnagar University, Savar.
Antibacterial test: At first made bacterial media for test. Nutrient agar was mixed with
distilled water and then sterilized in autoclave. The sterilized media were poured into Petri
plate. The solidified plates were bored with 6mm dia cork borer. The plates then
incubated for 24 hours and observe antimicrobial activity.
Antifungal test: 39 gm potato dextrose agar (PDA) was dissolved in 1liter distilled water.
Then shake it and autoclave this PDA. Then its poured into Petri plate and bored with 6
mm dia cork borer.
Result and discussion
The results of fluorescence analysis of the leaf powder of vanilla plantifolia in different
chemical reagents in visible light have been shown in table 1.
Table 1: Analysis of fluorescence characters of leaf powder of Vanilla plantifolia in
different chemical reagents
Sl No. Chemical reagent Appearance
1 Powder color Green
2 5% NaOH Green
3 10% NaOH Dark green
4 Conc. H2SO4 Green
5 Acetic acid Green
6 Conc. HNO3 Light brown
7 50% H2SO4 Green
8 50% HCl Green
9 Conc. HCl Dark green
10 5% FeCl3 Green
Phytochemical characteristics of leaf extract of vanilla planifolia were summarized in
table 2. Plants are rich in a wide variety of secondary metabolites such as tannins,
Uddin et al.
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36
terpenoids, alkaloids, flavonoids, glycosides, etc., which have been found in vitro to have
antimicrobial properties (Dahanukar et al, 2000 and Cowan, 1999). Many plants and their
extracts used against microbial infections due to the presence of these secondary
metabolites. The results revealed the presence of medically active compounds in leaf
extracts of Vanilla planifolia.
Table 2. Results of phytochemical screening of leaf extracts of Vanilla planifolia
Sl. no Name of the compounds Status of the substancex
1 Carbohydrate test +
2 Alkaloids test +
3 Steroids test +
4 Tannins test +
5 Phenol test +
6 Flavonoid test +
7 Glycoside test +
8 Terpine test +
x + = Presence
From the table 2, it could be seen that carbohydrates, alkaloids, steroids, tannins, phenol,
flavonoid, glycoside, terpine were present in the extract of vanilla planifolia.
Phytochemical analysis conducted on the plant extracts revealed the presence of
constituents which are known to exhibit medicinal as well as physiological activities
(Sofowra et.al, 1993). Alkaloids have been associated with medicinal uses for centuries
and one of their common biological properties is their cytotoxicity (Nobori et al., 1994).
Several workers have reported the analgesic (Antherden et al., 1969 and Harborne et.al.,
1973), antispasmodic and antibacterial (Stray et al., 1998 and Okwu et al., 2004)
properties of alkaloids. Steroids have been reported to have antibacterial properties
(Raquel, 2007). Kull et al. (1953) and Tar-bet, Oura and Sternberg (1953) indicated that
certain steroids containing nitrogen in the form of amino groups attached to the steroid
nucleus are most effective when tested against micro-organisms. Tannins bind to proline
rich protein and interfere with protein synthesis. Tannins are secondary metabolites
responsible for antimicrobial properties in various plants (Chung, 1998). Tannins are very
important for controlling microbial growth. Tannins exhibited antibacterial activities
against different tested microorganisms. A. Doss et al. (2009) revealed that
Staphylococcus aureus was highly sensitive to tannins, isolated from the plant material
followed by Escherichia coli and Pseudomonas aeruginosa. Tannins have shown potential
antiviral (Lin et al., 2004), antibacterial (Akiyama et al., 2001; Funatogawa et al., 2004),
and antiparasitic effects (Bhagavathi et al, 1999; Yang et al., 2000; Tanimura et al., 2005).
The ability of tannin compounds to cause the bacterial colonies to disintegrate probably
results from their interference with the bacterial cell wall; thereby inhibiting the microbial
growth (Erasto et al., 2004; Viljoen et al., 2003).
The phenolic compounds are one of the largest and most ubiquitous groups of plant
metabolites (Singh et al, 2007). They possess biological properties such as antiapoptosis,
antiaging, anticarcinogen, anti-inflammation, antiatherosclerosis, cardiovascular
protection and improvement of endothelial function, as well as inhibition of angiogenesis
and cell proliferation activities (Han et al. 2007). Several studies have described the
antioxidant properties of medicinal plants which are rich in phenolic compounds. Natural
antioxidant mainly come from plants in the form of phenolic compounds such as
flavonoid, phenolic acid etc. Phenols exhibit antimicrobial activities (Robbins, 1980). The
presence of the phenolic compounds proved that the extract had antimicrobial and
antifungal effect ( Vasantha K. et al ,2012). Flavonoids are hydroxylated phenolic
substances known to be synthesized by plants in response to microbial infection and they
have been found to be antimicrobial substances against wide array of microorganisms in
vitro (Brown et al,1998 and Krings et al, 2001). Their activity is probably due to their ability
to complex with extracellular and soluble proteins and to complex with bacterial cell wall
(Marjorie, 1996). They also are effective antioxidant and show strong anticancer activities.
Flavonoids can function as direct antioxidants and free radical scavengers, and have the
capacity to modulate enzymatic activities and inhibit cell proliferation (Duthie and
Crozier, 2000). In plants, they appear to play a defensive role against invading pathogens,
Antibacterial and Antifungal Activities of Vanilla Planifolia
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37
including bacteria, fungi and viruses (Sohn et al., 2004). Damage to pathogenic cell
membranes is a common feature of the antibacterial activity of terpene (Yoshihiro et al,
2004). Terpines play a role in and are under investigation for Antibacterial, Antineoplastic
and other Pharmaceutical functions (Yamunadevi et al., 2011). On basis of this study it is
indicated that, the identified phytochemical compounds may be the bioactive
constituents. For these constituents methanol leaf extract of Vanilla planifolia may show
antimicrobial and antifungal effect on Staphylococcus aureus, Escherichia coli,
Pseudomonas aeruginosa, Bacillus subtilis and Aspergillus niger.
Table 3: Antimicrobial activity of leaf extract of Vanilla planifolia
Sl. No. Name of the organisms Zone of inhibition
(Methanol extract)
50 mg 100 mg 200 mg
1 Staphylococcus aureus 12 21 26
2 Escherichia coli 18 21 29
3 Pseudomonas aeruginosa 16 21 31
4 Bacillus subtilis 13 25 28
Table 4: Inhibition zone of methanol extracts of Vanilla planifolia against fungal
pathogens
Sl. No. Name of the organisms Zone of inhibition
Methanol extract
50 mg 100 mg 200 mg
1 Aspergillus flavus - - 13
2 Aspergillus niger 12 16 19
The leaf extract of Vanilla planifolia was tested for their antimicrobial activity against
Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and
the results were presented in Table 3. The presented values indicated the zone of inhibition
formed around the discs (mm). Staphylococcus aureus showed comparatively low
susceptibility to methanol leaf extract of Vanilla planifolia in all the concentrations.
Escherichia coli was found to be more susceptible towards the methanol extracts of leaf
of Vanilla planifolia in case of using 50 mg extract. Bacillus subtilis showed maximum zone
of inhibition when 100 mg extract was used. Pseudomonas aeruginosa showed maximum
zone of inhibition when 200 mg extract was used. This is may be due to the presence of
alkaloids, steroids, tannins, phenol, flavonoid, glycoside etc in the leaf extract of Vanilla
planifolia.
The leaf extract of Vanilla planifolia was tested for their antifungal activity against
Aspergillus flavus and Aspergillus niger in Table 4. The table showed that Aspergillus niger
was more susceptible than Aspergillus flavus to the leaf extract of Vanilla planifolia in all
the concentrations.
Conclusion
The present study conclusively demonstrates that the leaf extract of Vanilla planifolia is a
good source of various phytochemicals like alkaloids, flavonoids, carbohydrates,
glycosides, saponins, tannins, Terpenoids. The antibacterial activity of Vanilla planifolia
leaf extract was clearly shown by the present study against various bacteria
(Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis). But
the study revealed that fungas such as Aspergillus flavus and Aspergillus niger were not
that sensitive like the tested bacteria. All these preliminary reports warrant an in depth
analysis of the usefulness of Vanilla planifolia as miracle agent against various harmful
bacteria and fungus.
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Compounds of pharmacological interest (tannins) were isolated from the plant species, Solanum trilobatum Linn and assayed against the bacteria, Staphylococcus aureus, Streptococcus pyrogens, Salmonella typhi, Pseudomonas aeruginosa, Proteus vulgaris and Escherichia coli using agar diffusion method. Tannins exhibited antibacterial activities against all the tested microorganisms. S.aureus was the most resistant to tannins isolated from the plant material followed by Streptococcus pyrogens, Salmonella typhi, Escherichia coli, Proteus vulgari and Pseudomonas aeruginosa. Minimum inhibitory concentration of the tannins ranged between 1.0 and 2.0 mg/ml while the minimum bactericidal concentration ranged between 1.5 and 2.0 mg/ml.
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