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Preliminary phytochemical screening and antimicrobial activities of various fractions of Mallotus philippensis Muell.

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The phytochemical studies and biochemical screening of various fractions, that is, hexane, chloroform, ethyl acetate, butanol and aqueous (F1 to F5) obtained from the medicinal plant Mallotus phillipensis were studied. The presence of alkaloids, flavonoids, glycosides, phenols, quinines, saponins, tannins and terpenoids were confirmed by performing chemical tests using standard protocols. The infrared (IR) spectroscopic analysis revealed the presence of oxygenated and carbo-nitro functionalities, mainly in the polar fractions of the plant. All the fractions were tested for their antimicrobial activities against nine of the human pathogens including Bacillus subtilis, Staphylococcus aureus, Staphylococcus pneumonia, Pseudomonas aeruginosa, Proteus vulgaris, Salmonella typhi, Aspergillus flavus, Aspergillus niger and Candida albicans using standard procedures. Among all, ethyl acetate (F3) and butanol (F4) fraction exhibited strong antibacterial and antifungal activities. F3 revealed to be the most promising by showing 80% inhibition zone in case of P. vulgaris (80%), S. typhi (80%), while almost 70% against B. subtilis (68%) and S. pneumonia (67%). In fungicidal assay, F3 showed 75 and 73% inhibition against A. flavus and A. niger, respectively.
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Vol. 7(41), pp. 3066-3070, 3 November, 2013
DOI: 10.5897/JMPR2013.5182
ISSN 1996-0875 ©2013 Academic Journals
http://www.academicjournals.org/JMPR
Journal of Medicinal Plants Research
Full Length Research Paper
Preliminary phytochemical screening and antimicrobial
activities of various fractions of Mallotus philippensis
Muell.
Masood Afzal1, Zhao Wang2, Farman ali1, Zhongshu Song3, Russel Cox3 and Shafiullah
Khan1,2*
1Department of Chemistry, Gomal University Dera Ismail Khan, KPK, Pakistan.
2State Key Laboratory of Organic-Inorganic Composites, Beijing University of Chemical Technology, Beijing 100029,
China.
3School of Chemistry, Bristol University, Bristol BS8 1TS, United Kingdom.
Accepted 14 October, 2013
The phytochemical studies and biochemical screening of various fractions, that is, hexane, chloroform,
ethyl acetate, butanol and aqueous (F1 to F5) obtained from the medicinal plant Mallotus phillipensis
were studied. The presence of alkaloids, flavonoids, glycosides, phenols, quinines, saponins, tannins
and terpenoids were confirmed by performing chemical tests using standard protocols. The infrared
(IR) spectroscopic analysis revealed the presence of oxygenated and carbo-nitro functionalities, mainly
in the polar fractions of the plant. All the fractions were tested for their antimicrobial activities against
nine of the human pathogens including Bacillus subtilis, Staphylococcus aureus, Staphylococcus
pneumonia, Pseudomonas aeruginosa, Proteus vulgaris, Salmonella typhi, Aspergillus flavus,
Aspergillus niger and Candida albicans using standard procedures. Among all, ethyl acetate (F3) and
butanol (F4) fraction exhibited strong antibacterial and antifungal activities. F3 revealed to be the most
promising by showing 80% inhibition zone in case of P. vulgaris (80%), S. typhi (80%), while almost 70%
against B. subtilis (68%) and S. pneumonia (67%). In fungicidal assay, F3 showed 75 and 73% inhibition
against A. flavus and A. niger, respectively.
Key words: Mallotus phillipensis, phytochemical screening, infrared (IR) spectral analysis, antimicrobial
activities.
INTRODUCTION
The genus Mallotus belongs to the family Euphorbiaceae
which plays a major role in the ethno pharmacology of
several areas of the world where they are indigenous.
Plants of this genus has been used in folk medicine such
as topical antiseptic, anthelminthic and useful in
treatment of bronchitis, abdominal diseases, spleen
enlargement as well as to treat chronic hepatitis in
traditional Vietnamese medicine (Chi et al., 1997; Loi et
al., 2001). The genus as a whole is significantly
efficacious against helminth parasites infections (Singh et
al., 1997; Khunkitti et al., 2000; Asha et al., 2001),
especially its fruit used to cure worm constipation,
infestation as well as abdominal diseases (Pandey et al.,
1991). Mallotus phillipensis, locally known as Kamala, is
a woody plant of this genus, having wide geographical
range extending from North America, East Asia to
Northern Indo-Pak. Medicinally, the bark juice of this
plant is used in diarrhea and dysentery (Samy et al.,
1998). The fruits and roots are strong laxative,
anthelminthic, vulnerary, detergent, maturant, and
*Corresponding author. E-mail: s.khan@gu.edu.pk. Tel: +92 966-750359. Fax: +92 966-750250.
carminative; while leaves of this plant were shown to
strongly inhibit mouse skin tumor promotion (Reiko et al.,
2008). Moreover, “Kamala oil, obtained from this plant is
used as a constituent for tung oil (Tanaka et al., 1998).
The diverse medicinal importance of genus Mallotus has
prompted us to carry out investigation on M. phillipensis.
This study reports the phytochemical screening and
antimicrobial activities of M. phillipensis.
MATERIALS AND METHODS
Plant
The whole plant M. phillipensis was collected in June 2008 from
village Kuwari, district Mansehra, Khyber Pakhtunkhwa, Pakistan.
The plant was identified by Prof. Dr. Manzoor Ahmad, Botany
Department, Government Post Graduate Collage Abbottabad,
where a voucher specimen was deposited in the herbarium
(Accession No. C-0027).
Extraction and isolation
The shed-dried powdered material of M. phillipensis (2.5 kg) was
extracted with methanol (65 L) at room temperature for a period of
seven days (3 × 65 L). The resulting extract was filtered and
evaporated with the help of rotary evaporator to obtain greenish
gummy crude (81.6 g). This methanolic crude was then succes-
sively partitioned into n-hexane (F1, 14.2 g), chloroform (F2, 14.8 g),
ethyl acetate (F3, 18.8 g), n-butanol (F4, 14.1 g) and water soluble
fractions (F5, 15.7 g).
Phytochemical screening
Chemical tests were carried out on all the fractions (F1 to F5) of the
M. phillipensis using standard procedures to identify the phyto-
constituents as described by Sofowora (Pour et al., 2011), Trease
(Paulraj et al., 2011) and Evans and Harborne (Rajan et al., 2011).
The following are a brief description of the procedures.
Test for alkaloids
About 0.2 g of each extract was heated with 2% H2SO4 for two
minutes. It was filtered and few drops of Dragondorff r eagent were
added. Orange red precipitate confirmed the presence of alkaloids.
Test for tannins
About 0.5 g of each extract was mixed with water and heated on
water bath. It was filtered and few drops of ferric chloride were
added to the filtrate. Dark green solution colour confirmed the
presence of tannins.
Test for anthraquinones
About 0.5 g of each extract was boiled with 10% HCl for few
minutes in water bath and filtered. Then it was filtered and allowed
to cool. Equal volume of CHCl3 was added to the filtrate. Then
drops of 10% ammonia was added to the mixture and heated.
Rose-pink color indicated the presence of anthraquinones.
Afzal et al. 3067
Test for glycosides
All the fractions were hydrolyzed with HCl and neutralized with
NaOH solution. A few drops of Fehling solutions A and B were
added. Appearance of orange r ed precipitate indicated the
presence of reducing sugars.
Test for saponins
About 0.2 g of each extract was shaken with 5 ml of distilled water.
It was then heated to boil. Appearance of creamy miss of small
bubbles (Frothing) confirmed the presenc e of saponin.
Test for flavonoids
About 0.2 g of each extract was dissolved in dilute NaOH. After it
HCl was added to the mixture. A yellow s olution that turns colorless
confirmed the presence of flavonoids.
Test for phlobatanins
About 0.5 g of each extract was dissolved in distilled water and
filtered. The filtrate was boiled with 2% HCl solution. Red precipitate
confirmed the presence of phlobatanins.
Test for steroids
About 2 ml of acetic anhydride was added to 0.5 g of the extract of
each with 2 ml of H2SO4. The colour changed from violet to blue or
green in some samples confirmed the presence of steroids.
Test for terpenoids (Salkowski’s test)
About 0.2 g of each of the extract was mixed with 2 ml of chloroform
(CHCl3) and 3 ml of concentrated H2SO4 was carefully added from a
layer. A reddish brown coloration of the interface was formed to
indicate the positive results for the presence of terpenoids.
Test for reducing sugars (Fehling’s test)
A small portion of each of the extract was shaken with distilled
water and filtered. The f iltrate was boiled with drops of Fehling’s
solutions A and B for 2 min. An orange-red precipitate on boiling
with Fehling’s solution indicated the presence of reducing sugars.
IR Spectroscopy
Perkin Elmer Spectrum 100FT was used for IR spectroscopic
analysis. The fractions were scanned in accordance with ASTM
1252-98. A drop of each extract was applied on a sodium chloride
cell to obtain a thin layer. The cell was mounted on the Fourier
transform-infrared (FTIR) and scanned through the IR region. The
characteristic absorption peaks in IR spectra of all the fractions are
shown in Table 2.
Antibacterial activity
The antibacterial activity was checked by the agar–well diffusion
method (Kavanagh, 1963). In this method, one loop full of 24 h old
culture containing approximately 104 to 106 CFU was spread on
3068 J. Med. Plants Res.
Table 1. Preliminary phytochemical screening of t he various fractions of M.
phillipensis.
S/N
Class
F
1
F
2
F
3
F
4
F
5
01 Alkaloids - + + - -
02 Tannins - - ++ - -
03 Glycosides - + +++ + +
04 Anthraquinones - - ++ - -
05 Reducing sugars - + + + +++
06 Saponins - - ++ - -
07 Flavonoids + ++ +++ ++ ++
08 Phlobatanins - + + + +
09 Steroids - + + + +
10 Terpenoids - - +++ + +
- = Absent, + = Present, ++ = Present appreciable, +++ = Present very appreciable.
Table 2. IR Spectroscopic data of solvent extract of M. philippensis (absprotion in cm-1).
Functionality F1 F2 F3 F4 F5
O - H 3296.35 3381.21 3365.78 3290.59 3323.35
C = O 1924.28 1726.21 1683.86 1732.08 1730.15
C = C 1604.77 1614.42 1608.63 1606.70 1611.45
C - H 2922.25 2927.06 2925.04 2927.02 2925.07
C - O 1186.73 1071.48 1108.28 1043.57 1065.78
C – N 1355.85 1345.77 1233.54 1345.98 1356.63
the surface of Mueller-Hinton agar plates. Wells were dug in the
medium with the help of sterile metallic c ork borer. Stock solutions
of the test samples (F1 to F4) in the concentration of 1 mg/ml were
prepared in dimethyl sulfoxide (DMSO) and 100 μl dilutions were
added in their respective wells. The antibacterial activities of all the
fractions were compared with standard drug, streptomycin which
served as positive control.
Antifungal activity
The antifungal activity was determined by the agar well diffusion
method (Kavanagh, 1963). In this method, miconazole were used
as the standard drug. All the fractions (F1 to F4) were dissolved in
DMSO (50 mg/5 ml). Sterile Sabouraud’s dextrose agar medium (5
ml) was placed in a test tube and inoculated with the sample
solutions (400 µg/ml) k ept in slanting position at room temperature
overnight. The fungal culture was then inoculated on the slant. The
samples were incubated for 7 days at 29°C and growth inhibition
was observed.
RESULTS
The results of different experiments performed for the
phytochemical investigations revealed the presence of
flavonoids in all fractions (F1 to F5). Glycosides, reducing
sugars, phlobatanins and steroids were also present in all
fractions except F1. Terpenoids were present in F3, F4 and
F5, but absent in F1 and F2. The presence of alkaloids
was confirmed in F2 and F3 only. Tannins, anthraquinones
and saponins were found only in F3 (Table 1).
The IR spectra of all the fractions (F1 to F5) were
obtained to confirm the presence of various functionalities
(Egwaikidi et al., 2009). The IR spectra exhibited
characteristic absorption for OH group in the region
between 3296.35 and 3381.21 cm-1, the absorption
appeared between 2927.06 to 2922.25 cm-1
due to C-H
stretching indicating aliphatic groups, absorption in the
region 1924.35 and 1683.86 cm-1
clearly indicated the
presence of carbonyls in all the fractions. Absorption
bands for the olefinic functionalities appeared in between
1604.77 and 1614.42 cm-1, while the indication of carboxy
functional groups was found at 1186.73 and 1043.57 cm-
1. The absorption bands at 1355.85, 1345.77, 1233.54,
1345.98 and 1356.63 cm-1 showed the presence of C-N
bond stretching (Table 2).
DISCUSSION
Antimicrobial activity
Antibacterial activity of v arious fractions (F1 to F4) of M.
phillipensis were performed against six human patho-
gens including Bacillus subtilis, Staphylococcus aureus,
Staphylococcus pneumonia, Pseudomonas aeruginosa,
Afzal et al. 3069
Table 3. Antimicrobial activities of fractions (F1 to F4) of M. phillipensis.
Fraction Gram positive bacteria
a
Gram negative bacteria Fungi
BS SA SP PA PV ST AF AN CA
F1 6 5 5 4 5 4 6 5 4
F2 12 14 11 12 17 15 14 10 12
F3 22 20 20 18 22 24 24 22 20
F4 16 14 15 16 18 15 15 18 14
Positive controlb,c 32 34 30 32 34 30 32 30 34
a = Zone of inhibition in mm, b = Streptomycin (Standard drug in antibacterial assay, mg/ml), c = Miconazole (Standard drug in
antifungal assay, mg/ml). BS = Bacillus subtilis, SA = Staphylococcus aureus, SP = Staphylococcus pneumonia, PA =
Pseudomonas aeruginosa, PV = Proteus vulgaris, ST = Salmonella typhi, AF = Aspergillus flavus, AN = Aspergillus niger, CA =
Candida albicans.
Percentage
Figure 1. Percentage inhibition of fraction F3 and F4 in antimicrobial ass ays against all the
tested organisms. BS = Bacillus subtilis, SA = Staphylococcus aureus, SP =
Staphylococcus pneumonia, PA = Pseudomonas aeruginosa, PV = Proteus vulgaris, ST =
Salmonella typhi, AF = Aspergillus flavus, AN = Aspergillus niger, CA = Candida albicans.
Proteus vulgaris, Salmonella typhi. The results are shown
in Table 1. Fraction F3 displayed the highest activity
amongst all by inhibiting P. vulgaris (80%), S. typhi
(80%), B. subtilis (68 %) and S. pneumonia (67%), while
it showed minimum activity against P. aeruginosa (36%).
F4 revealed moderate inhibitory potential against all the
tested cultures as it showed 50% of the inhibition zone
against almost all the bacteria. F2 showed moderate to
weak inhibition, while F1 revealed minimum activity
against all the bacteria. These results were compared
with standard drug (Streptomycin) which was more
effective by showing maximum inhibition zones (Table 3).
Antifungal activity
The fungicidal activity of the various fractions (F1 to F4) of
M. phillipensis was evaluated against three fungi include-
ing Aspergillus flavus, Aspergillus niger and Candida
albicans (Table 1). The results indicated that both fraction
F3 and F4 displayed significant activity while fraction F1
and F2 showed low activity in killing all the three tested
fungi. The area of inhibition (in percent) for the F3 was
promising compared to the standard drug (Miconazole)
against A. flavus (75%), A. niger (73%), while moderate
inhibition was observed in case of C. albicans (59%). It
was further observed that the fraction F4 showed high
activity against A. niger (60%), while weak activity against
the rest of fungi. The fractions F2 remained less effective,
while F1 showed no activity in killing the tested fungi
(Figure 1).
Conclusion
Phytochemicals studies of M. phillipensis Muell. reveal
the presences of several secondary metabolites, e.g.
alkaloids, tannins, glycosides, saponins, flavonoids and
terpenoids. The chemical tests as well as the IR spectral
data were significant in identifying the presence of
3070 J. Med. Plants Res.
various functionalities in the fractions. Furthermore, ethyl
acetate and butanol soluble fractions (F3 and F4) showed
maximum inhibition zone in antimicrobial assay, hence
signifying the therapeutic effect which strongly supports
the conventional use of this plant against various
diseases. These activities may be due to the presence of
biologically active compounds present as alkaloids and
the glycosides (Cheng et al., 1998). The inhibition against
fungi may be attributed towards potent monoterpenes,
flavonoids (Kosalec et al., 2005; Pereira et al., 2007;
Lauro et al., 2008) and steroids, which shows more
activity in higher concentration against the growth of all
fungi (Lauro et al., 2008; Winkelhausen et al., 2005;
Subhisha et al., 2005). These results findings confirm the
medicinal perspective of the ethyl acetate and butanol
fractions against septicaemia, urinary tract and typhoid. It
is further suggested that more phytochemical investi-
gation should be carried out to isolate and characterize
the potent compounds.
ACKNOWLEDGEMENT
The authors greatly acknowledged Higher Education
Commission of Pakistan (HEC) for providing financial
support through HEC Indigenous 5000 Ph.D Fellowship
Scheme.
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Anti-microbial activity and phytochemical constituents of methanol extract of Plumeria rubra (flower and leaf) and Eucalyptus globules (leaf) was investigated. Phytochemical screening of the crude extract revealed the presence of tannins, phlobatannins, saponins, flavonoids, steroids, terpenoids, cardiac glycosides and reducing sugar in the plants investigated. Phlobatanins were found to be absent in the methanol extract of Plumeria rubra (flower) and Eucalyptus globulus (leaf) except the methanol extracts of Plumeria rubra (leaf). All the crude extract displayed higher inhibitory effects at the tested concentration (20 mg/ml) except on Corynebacterium pyogenese and Bacilhis anthracis of Plumeria rubra leaf and Streptococcus faecalis of Eucalyptus globules leaf. The infra-red (IR) spectra of the crude extract revealed the presence of different functional group ranging from hydroxyl group (OH) (3406 -3338.6 cm -1), C-Hstr., alkyl group (2926.6 cm -1), C=O stretching for carbonyls (2162.1 cm -1), C-O bending for alcohols, ethers, esters, carboxylic acid and anhydrides (1310.6 -1059.6 cm -1), C – H bending alkyl (1453.4 – 1376.2 cm -1) and C – H bending for methyl group (864.1 – 668.4 cm -1).