Phytochemical and Antibacterial studies of
Seed extracts of
Memecylon edule
Tamizhamudu Elavazhagan1*, Kantha D. Arunachalam2
1
Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur- 603203, Tamilnadu, India.
2
Center for Inter disciplinary Research, SRM University, Kattankulathur, Chennai, Tamilnadu, India.
Abstract
The present study was carried out to analyse the preliminary phytochemical screening for ten principle bioactive compounds in Petroleum ether, chloroform, ethyl acetate and ethanol extracts of seeds of Memecylonedule collected from Potheri forest, Kattankulathur in Tamilnadu. Chloroform and ethyl acetate extracts of seeds of Memecylon edule were also investigated against ten bacteria using disc diffusion method. Preliminary phytochemical screening revealed the presence of Alkaloids, Triterpenes, Flavonoids and saponins. Themaximum activity was observed in chloroform extract against
Bacillus subtilis with zone of inhibition (15mm) and minimum activity was observed in ethyl acetate extract against
Yersinia enterocolitica and Vibrio cholera at inhibition range was observed (10mm). The results suggest ethyl acetate and chloroform extracts of this plant with moderent antibacterial activity and secondary metabolites of this plant used for wound healing property and other forms of bacterial infections.
Key words: Preliminary phytochemical, Memecylon, Dyeing property, Disc diffusion, Bacillus, Melastomataceae INTRODUCTION
Memecylon edule (Melastomataceae)
The genus Memecylon belongs to the Melastomataceae family that grow as small shrubs or trees in tropical and subtropical regions of the world (Clausing G, et al., 2001). Among them in india Memecylon edule mostly found in Karnataka , Andhra Pradesh and Tamil Nadu (Ranjit Daniels RJ et al., 2007). M. edule is an iron wood tree and it’s used for decorative work, walking sticks, light axe handles, combs. M.edule has a dyeing property. The fruits of this plant are small, green and turning red then black as they ripen.
Tradional uses
Fruits of this plant are act as a cooling astringent and leaves are used for the treatment of anti leucorrhoeic, spasmolytic, hypoglycaemic and lotion prepared from leaves was used for ophthalmia and conjuctivities. A decoction prepared from leaves of Memecylon edule is used internally for gonorrhea. Roots of this plant are used for excessive menstrual discharge (Khare CP 2007). Leaves of M. edule have strong anti-inflammatory and analgesic activity (Nualkaew S et al 2009). Phytochemistry
A survey concerning the secondary metabolites of Memecylon edule showed the presence of flavonoids and triterpenes in crude ethyl acetate extract of leaves and no biological report is present in the literature (Nualkaew S et al ., 2009). An old phytochemical study of this whole Memecylon genus evidenced the presence of 13 fatty acids, 12 methyltetradeconate, glucose, amino acids, carotenoids, a phenolic glycoside and possibly undefined saponins (Stephen L et al., 1981).
The aim our work was to carry out the phytochemical investigation in Petroleum ether, chloroform, ethyl acetate and ethanol extracts of seeds of Memecylon edule. The chloroform and ethyl acetate extracts were also evaluated for antimicrobial activity (Hullatti K et al., 2004).
MATERIALS AND METHODS
Plant materials collection and authentification
The seeds of Memecylon edule were collected from the Potheri forest, Kattankulathur, Kancheepuram, District, Tamilnadu, India. The seed of this plant was authentified by Dr. D. Narasiman, Centre for floristic research, Department of Botany, Madras Christian College, Chennai, Tamilnadu, India.
Extraction
The air-dried seed powder (100 g) was successively extracted with hexane, chloroform, ethyl acetate and ethanol using a Soxhlet apparatus. The extraction procedure was carried out in the order of increasing polarity of the solvents. In all extracts, the solvent was removed under reduced pressure by rotary evaporator to yield a dry condensed residue.
The condensed extracts were used for preliminary screening of secondary metabolites such as alkaloids (Mayer’s, Wagner, Dragendorff’s), flavonoids (Alkaline reagent, Shinoda), phenolics (lead acetate, alkaline reagent test), coumarins (NaoH), Anthroquinones (Magnesium acetate test, conc.H2SO4), catechins (Ehrlisch test), fatty acids (KoH), triterpenes (liberman-burchard test), saponins (foam test), tannins (gelatin) were carried out (Harborne, 1976), (Dey PM et al., 1987) (Evans WC, et al., 1989)
Bacterial strains
Bacterial strains were used for testing included Staphylococcus aureus, Bacillus subtilis, Proteus vulgaris, Vibrio cholera, Klebsiella pneumoniae, Salmonella typhi, Pseudomonas aeruginosa, Escherichia coli, Yersinia enterocolitica, Streptococcus faecalis for ethyl acetate and chloroform extracts of seeds of Memecylon edule
Antibacterial assay
Antibacterial activity of the above mentioned ethyl acetate and chloroform extracts of seeds of Memecylon edule were tested against a set of ten selected strains of bacteria using disc diffusion method (Biol TJ et al., 2003) (Erdogrul T et al.,
2002) (Jantova S et al., 2000). All ten bacterial strains were incubated at 35˚ C for 24 h and the plates containing nutrient agar were allowed to dry (Ahmad I et al., 2001) (Nascimento GG et al., 2000). The Nutrient agar was inoculated with 200
μl of the prepared inoculum was spread into the sterile Petri plates. The infusions were assayed at one concentration: 50mg/ml were prepared for each extract. Blank discs of 6 mm diameter impregnated with DMSO were used as negative controls, and discs of ampicillin (10 mg) as positive controls for the antibacterial assay (Nair R., et al 2005). The disc was saturated with 20 μl of crude extract (50mg/ml) were allowed to dry and filter paper discs made from Whatmann filter paper no.1, which were carefully transferred onto the surface of seeded agar plate. The plates were incubated in overnight at 37˚ C (bacteria) and the plates were observed and the diameter of resulting zones of inhibition (mm) measured after 24 h. The effects of zone of inhibition of plant extracts (50mg/ml) were compared with standard ampicilin at a concentration of 10 mg/ml. Triplicates were performed and the experiment was repeated thrice and the average values of antibacterial activity of chloroform and ethyl acetate extracts of seeds were calculated.
RESULTS AND DISCUSSION Preliminary phytochemical
The results of preliminary phytochemical screening tests of the petroleum ether, chloroform, ethyl acetate and ethanolic extracts of seeds of Memecylon edule are as shown in Table 1. The results indicated the presence of triterpenes, flavonoids and saponins. Some extracts showed trace amount of positive result for triterpenes, phenolics, anthroquinones in chloroform extract.
Antibacterial activity
Regarding antibacterial activity the ethyl acetate extract did not show activity against Bacillus subtilis, Salmonella typhi, Streptococcus faecalis. The results of ethyl acetate extract of seed of Memecylon edule as shown in (Table 2). The solvents used for extraction (chloroform and ethyl acetate) were used for dissolving the extracts also and all the solvent controls did not show any activity. The ethyl acetate extract showed activity against seven bacteria and the highest antibacterial effect on Eschericia coli was found with zone of inhibition (14 mm) and lowest antibacterial effect in ethyl acetate extract on
Vibrio cholera and Yersinia enterocolitica (10mm). The ethyl acetate extract also showed activity against Staphylococcus aureus, Proteus vulgaris, Klebsiella pneumonia, Pseudomonas aeruginosa with zone of inhibition ranging from (11-13 mm). Results were summarized in (Fig 1).
Table 1. Phytochemical tests on the petroleum ether, chloroform, ethyl acetate and ethanolic extracts of seeds of Memecylon edule
S.no Secondary metabolites
Petroleum ether
Chloroform Ethyl acetate
Ethanol
1 Alkaloids - - - -
2 Flavanoids - - + -
3 Phenolics - ± - -
4 Coumarins - - - -
5 Anthroquinones - ± - -
6 Catachins - - - -
7 Fatty acids - - - -
8 Triterpenes - ± + -
9 Saponins - - - -
10 Tannins - - - +
Key: + = Present, - = Absent, ± Doughtful
Table 2. Antibacterial activity of ethyl acetate extract of seed of Memecylon edule
S.no Bacteria Seed Ethyl acetate
extract
(Diameter of zone of inhibition in mm)
Standard (Diameter of zone of
inhibition in mm)
1 Staphylococcus aureus 13 17
2 Bacillus subtilis - 15
3 Proteus vulgaris 13 16
4 Vibrio cholera 10 18
5 Klebsiella pneumoniae 11 15
6 Salmonella typhi - 17
7 Pseudomonas aeruginosa 11 14
8 Escherichia coli 14 25
9 Yersinia enterocolitica 10 16
Fig 1.
0 5 10 15 20 25 30
Seed Ethyl acetate extract Standard
Staphylococcus aureus
Bacillus subtilis
Proteus vulgaris
Vibrio cholerae
Klebsiella pneumoniae
Salmonella typhi
Pseudomonas aeruginosa
Escherichia coli
Yersinia enterocolitica
Streptococc faecalis
zone of
in
hibi
tio
n
(m
m
)
(Zone of inhibition are the mean values of triplicate readings; Extract, SEAE, Seed ethyl acetate extract (50mg/ml); Standard-Ampicillin (10mg/ml).
Table 3. Antibacterial activity of chloroform extract of seed of Memecylon edule
S.no Bacteria Seed Chloroform
extract (Diameter of zone of inhibition in mm)
Standard (Diameter of zone of
inhibition in mm)
1 Staphylococcus aureus 14 17
2 Bacillus subtilis 15 15
3 Proteus vulgaris - 16
4 Vibrio cholera - 18
5 Klebsiella pneumoniae 14 15
6 Salmonella typhi 13 17
7 Pseudomonas aeruginosa - 14
8 Escherichia coli 12 25
9 Yersinia enterocolitica - 16
Fig 2.
0 5 10 15 20 25 30
Seed chloroform extract Standard
Staphylococcus aureus
Bacillus subtilis
Proteus vulgaris
Vibrio cholerae
Klebsiella pneumoniae
Salmonella typhi
Pseudomonas aeruginosa
Escherichia coli
Yersinia enterocolitica
Streptococc faecalis
zon
e of
inhib
itio
n
(m
m
)
(Zone of inhibition are the mean values of triplicate readings; Extract, SCHE, Seed chloroform extract (50mg/ml); STD, Standard-Ampicillin (50mg/ml). DISCUSSION
Memecylon edule has not attracted much attention so far, but in this study preliminary phytochemical screening of the extracts of petroleum ether, chloroform, ethyl acetate and ethanol showed the presence of alkaloids, tannins, triterpenes, flavonoids and saponins in seeds of Memecylon edule. During the antibacterial test for both extracts in disc diffusion method result was observed that the positive control drug ampicillin showed the largest zone of inhibition compared to chloroform and ethyl acetate extracts on the ten bacterial strains investigated in this study. No zone of inhibition was observed in Proteus vulgaris, Vibrio cholera, Pseudomonas aeruginosa, Yersinia enterocolitica, Streptococcus faecalis.
However, the ethyl acetate extract showed activity against seven bacterial strains and no zone of inhibition for Bacillus subtilis Salmonella typhi and Streptococcus faecalis. The strongest antibacterial effect was found in chloroform extract of seed extract of M.edule against Bacillus subtilis and the weakest activity against Vibrio cholera and Yersinia enterocolitica.
Conclusion
This is the first report for phytochemical analysis and antibacterial study in seeds of Memecylon edule.
The results obtained from the antibacterial studies have shown that the chloroform and ethyl acetate extracts were less active than ampicillin.
Acknowledgement
This work was supported by SRM University. The authors thank Dr. D. Narasiman, Centre for floristic research, Department of Botany, Madras Christian College, Chennai, Tamilnadu, India for identification of plant sample. First author acknowledges the fellowship from SRM University, Kattankulathur and also thankful to all members in Department of Biotechnology, School of Bioengineering, SRM University for financial support and providing the necessary infrastructure to carry out this work.
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