antibacterial and antifungal activities of vanilla ...

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. Parv in1, 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-eBangla Agricultural Univ ersity with an objectiv e to investigate the antibacterial and antifungal potentials of the leaf extracts of Vanilla planifolia. The aim of the study is to assess the antimicrobial activ ity and to determine the zone of inhibition of extracts on some bacterial and fungal strains. The antimicrobial activ ity was determined in the extracts using agar disc diffusion method. The antibacterial and antifungal activ ities 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 sensitiv ity than bacteria although Aspergillus niger showed moderate sensitiv ity to the extract in all the concentrations. The phytochemical analyses of the plants were carried out. The microbial activ ity of the Vanilla planifolia was due to the presence of various secondary metabolites. Hence, these plants can be used to discov er bioactiv e natural products that may serv e as leads in the development of new agricultural research activ ities.

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 effectiv e, 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: [email protected] 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

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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 activ ity. 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. 1 2 3 4 5 6 7 8 9 10

Chemical reagent Powder color 5% NaOH 10% NaOH Conc. H2SO4 Acetic acid Conc. HNO3 50% H2SO4 50% HCl Conc. HCl 5% FeCl 3

Appearance Green Green Dark green Green Green Light brown Green Green Dark green 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, http://www.bdresearchpublications.com/journal/

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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 1 2 3 4 5 6 7 8 x + = Presence

Name of the compounds

Status of the substancex

Carbohydrate test Alkaloids test Steroids test Tannins test Phenol test Flavonoid test Glycoside test Terpine test

+ + + + + + + +

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 activ ities and inhibit cell proliferation (Duthie and Crozier, 2000). In plants, they appear to play a defensive role against invading pathogens, http://www.bdresearchpublications.com/journal/


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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. 1 2 3 4

Zone of inhibition (Methanol extract) 50 mg 100 mg 200 mg 12 21 26 18 21 29 16 21 31 13 25 28

Name of the organisms Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Bacillus subtilis

Table 4: Inhibition zone of methanol extracts of pathogens Sl. No. 1 2

Name of the organisms Aspergillus flavus Aspergillus niger

Vanilla planifolia against fungal

Zone of inhibition Methanol extract 50 mg 100 mg 200 mg 13 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 prelim inary reports warrant an in depth analysis of the usefulness of Vanilla planifolia as miracle agent against various harmful bacteria and fungus. References Akiyama H, F. Kazuyasu, O. Yamasaki, T. Oono and K. Iwatsuki .2001. Antibacterial action of several tannins against Staphylococcus aureus. J. Antimicrobial Chemotherapy,48 (48), 487-491 Annonimous. 2010. Clinical Laboratory Standard Institute. Performance Standard for antimicrobial susceptibility testing M. 30 (15), 100-520. http://www.bdresearchpublications.com/journal/

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