Phytochemical, antibacterial and antifungal activities of leaves and bark of Colebrookea oppositifolia

14 Pages·2017·1.29 MB·English

Pure Appl. Biol., 7(1): 138-151, March, 2018
http://dx.doi.org/10.19045/bspab.2018.70017
Research Article

Phytochemical, antibacterial and
antifungal activities of leaves and bark
of Colebrookea oppositifolia: an
ethnomedicinal plant

Muhammad Ajaib1*, Samia Abid1, Musfirah Anjum1, Qumqum

Noshad1, Muhammad Faheem Siddiqui2 and Muhammad Asif Iqbal3
1. Department of Botany, Mirpur University of Science and Technology (MUST), Mirpur-10250 (AJK)-
Pakistan
2. Department of Botany, University of Karachi, Karachi 75270-Pakistan
3. Department of Chemistry, University of Management and Technology (UMT), Lahore-Pakistan
*Corresponding author’s email: [email protected]
Citation
Muhammad Ajaib, Samia Abid, Musfirah Anjum, Qumqum Noshad, Muhammad Faheem Siddiqui and
Muhammad Asif Iqbal. Phytochemical, antibacterial and antifungal activities of leaves and bark of Colebrookea
oppositifolia: an ethnomedicinal plant. Pure and Applied Biology. Vol. 7, Issue 1, pp138-151.
http://dx.doi.org/10.19045/bspab.2018.70017
Received: 21/10/2017 Revised: 05/01/2018 Accepted: 05/01/2018 Online First: 17/01/2018
Abstract
The phytochemical, antibacterial, antifungal and MIC (minimum inhibitory concentration)
study of the leaves and bark of Colebrookea oppositifolia Smith was evaluated. Phytochemical
screening C. oppositifolia Sm. indicated the presence of alkaloids, flavonoids, glycosides,
steroids and saponins whereas terpenoids, tannins, and cardiac glycosides are showed only in
bark extract. Petroleum ether extract showed maximum effectiveness against P. aeruginosa
with bark extract (18±2.2 mm) and maximum antifungal potential exhibited by petroleum ether
extract of leaves (15 ±0.3 mm) against A. nigar. It was noticed that the extracts in distilled
water had not shown much potential. The MIC assay was carried out for further verification of
results obtained by the Zone of Inhibition against both bacterial and fungal strains. The lowest
MIC value is 0.09±0.3 at 1 mg/mL against E. coli exhibited by bark extract and 0.09±0.2 at 0.9
mg/mL against K. pneumoniae shown by leaf extract. Whereas, in fungi minimal MIC value
exhibited 0.11±0.3 at 0.9mg/mL by leaf extract against A. oryzae. Hence, it is recommended
further investigation to explore toxicological and clinical trials before using this plant as drug.
Keywords: Antibacterial; Antifungal;Colebrookea oppositifolia; Ethnomedicinal plant
Introduction properties by the identification of a critical
Products obtained from plants have been number of helpful compounds [1, 2].
used medicinal purposes since pre-historic Ethno-botanical plants frequently use as
time. For instance, certain conventional pharmaceuticals, Nutraceuticals, makeup
pharmaceuticals including numerous trees, and nourishment supplements [3]. With an
bushes, climbers and herbs have been expansion of resistant microorganisms,
utilized to remedy of certain diseases. conventional plants are being researched
Traditionally important plants have become for their antibacterial and therapeutic
sources for examining their bioactive qualities. The antibacterial and antifungal
impact of a plant is because of the
Published by Bolan Society for Pure and Applied Biology 138
Ajaib et al.
phytochemicals such as saponins, phenolic The plant Colebrookeaoppositifolia Sm.
compounds, tannins, terpernoids, heart commonly known as Bansa Siyah was
glycosides, alkaloids and flavonoids [4]. collected from the Kakra town, District
Numerous emphasis made on deciding the Mirpur AJK. Some plants of that species
antimicrobial action of plant concentrates also seen in Khoirata, Peer Gali and Kotli.
found in local societies in the form of oils, This plant belongs to the family Lamiaceae,
flavonoids, alkaloids, lactones etc.[5]. also called as Mint family. The plant
Foodborne diseases fall out 76 million species collected was mounted on
individuals every year in the United States, herbarium sheet and submitted in the
represent almost 325,000 hospitalizations Herbarium Department of Botany MUST,
and more than 5,000 passings. The diseases Bhimber campus. A voucher No. MUST.
caused by food are generally dangerous,
even fatal for example, microscopic
organisms, growths, infections and
parasites that enter the body essentially by
the ingestion of debased sustenance [6].
Because of the rapid resistance to
antibiotics by using allopathic medicines,
there is a need to grow new and inventive
antimicrobial compounds. As plants are
cheap, non-poisonous source, there is a
long convention of utilizing them as a part
of the treatment of irresistible illness in
Cameroonian people[7]. There are
numerous strategies for hindering or
inactivating microorganisms by utilizing
plant derived products as antimicrobials BOT. 880 to obtain for further reference.
[8]. Figure 1. Colebrookea oppositifolia
Colebrookea oppositifolia Smith (Figure1) Test organisms
is a genus of family Lamiaceae/Labiateae. Total six bacteria were selected, the three
C. oppositifolia is commonly known as strains were from gram
Bansa.It is medicinally important plant and positiveEnterococcus
it is utilized locally as sickness for faecalis,Staphylococcus aureus and
differentpurposes. The leaves paste is Bacillus subtilis and two strains of gram
utilized for wound healing and bruises negativeKlebsiellapneumonia,Escherichia
while the paste of its roots is utilized against coli and Pseudomonas aeruginosaand two
the epilepsy [9]. Juice assimilated by strains of fungal i.e. Aspergillus oryzae and
beating young leaves is utilized as Aspergillusnigerwere selected used for
anthelmintic [10]. The root and stem juice investigation of antimicrobial perspective
are used in eye diseases. It has intriguing of C. oppositifolia Smith. Four standard
antibacterial action and had exceptional antibiotics of bacterial strains
radical searching potential to be utilized as (azithromycin, methicillin, amoxicillin and
cell reinforcement [11]. Keeping in view ampicillin) and four standard antibiotics of
the significance of ethno botanical status of fungal strains (nystatin, tezole, fungivin,
C. oppositifoliaSmith and other members of and griseofulvin) were tested for comparing
family Lamiaceae the present investigation the potential of plant extract with them.
was carried out. Methodology adopted
Materials and methods The whole plant was washed with cold
Plant material water for removing of mud. After washing
the plant parts such as bark and leaves were
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Pure Appl. Biol., 7(1): 138-151, March, 2018
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separated and kept for drying under room Chloroform, Methanol and distilled water
temperature about 20-30 days. After drying and then evaporated to get final
the powder was made with the help of concentrated form of extracts.Thecorporeal
pestle and mortar and then exposed for analysis of herbal extracts considered its
maceration method [12]. texture, colour and appearance, and
Maceration of the plant specimen calculated yield before further study was
The shade dried plant material of C. accompanied.
oppositifolia was subjected for maceration Yields percentage (%) was calculated with
different solvents i.e. Petroleum ether, the following method:

Weight of plant extract
𝐸𝑥𝑡𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝑦𝑖𝑒𝑙𝑑 = ×100
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑝𝑙𝑎𝑛𝑡 𝑠𝑎𝑚𝑝𝑙𝑒

Qualitative study of phytoconstituents Phytochemical recognition key
The phytochemical investigation of plant − = Absent, + = Present in minimal
extracts were done by using typical process quantity, ++ = Existing in moderate
[13]. quantity whereas, +++ = Present in
Estimation of antimicrobial activity noticeableamount. Six bacterial strains
By using agar well diffusion method for the including three gram-positive
antimicrobial activity of leaf and bark (Staphylococcus aureus, Bacillus subtilis,
extracts of C. oppositifolia. For Enterococcus facealis) and three gram
antibacterial activity methodology of [14] negative (Escherichia coli, Pseudomonas
was used. For the bacterial strains i.e., P. aeruginosa, Klebsiella pneumoniae) were
aeruginosa,K. pneumonia, E.faecalis, S. used for the estimation of antimicrobial
aureus, B. subtilis and E.coli, the nutrient activity of bark and fruit of Cholebrookia
agar media was used. For antifungal oppositifolia Smith. All of these bacteria
activity the methodology of[15] was used. were pathogenic. Furthermore, strains of
Potato Dextrose Agar media was used for fungal i.e.,A.oryzaeandA. niger, selected
antifungal activity. for antifungal prospective.
Assessment of minimum inhibitory We also used some standard discs, to find
concentration (MIC) out the receptiveness of the microbial
Plant extracts of C. oppositifolia for MIC strains. Which is used as the test organisms
was investigated by broth-dilution method (Tables 3 & 4). According to these
adapted by [16]. standards, the specimens were categorized
Statistical Analysis as vulnerable, transitional, and resistant
Allthe statistical analysis was done utilizing prone. Leaf and bark of
Microsoft excel 2010. C.oppositifoliaproducediffrent Zone of
Results and discussion Inhibition in response of bacterial strains
To checked the antimicrobial activity of the was recorded in mm. Zone of Inhibition
plant material i.e. Colebrookea was produced by the p. ether extract of leaf
oppositifolia Smith, extracts were prepared. showed maximum results i.e. 18±0.6 mm
Macerated and their % yield was intended against B. subtilis. Whereas, bark extract in
before further analysis, to provide glance of p. ether showed maximum Zone of
the economical perspective of the Inhibition i.e. 18±0.8 mm against S.
completepractice.The phytochemical aureusand 18±2.2 mm against P.
screening the plant extracts of C. aeruginosa. The leaf of C.oppositifolia
oppositifolia Smith was also achieved. Smith had displayed activity under the
Results showed the presence and absence of range of 0±0 mm to 18±0.6mm. Bark had
secondary metabolites (Table 1 & 2). exhibited the potential under the range of
0±0 mm to 18±2.2mm.
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Ajaib et al.

Table 1. Phytochemical evaluations of Bark extracts of C. oppositifolia Smith
Presence / Absence of Phytochemical Constituents

Plant Cardiac
Solvent Alkaloids Steroids Tannins Flavonoids Glycosides Terpenoids Saponens
Part Glycosides
P. Ether ++ _ _ _ _ + + +++
Chloroform + + _ ++ _ +++ + ++

Methanol +++ ++ + _ + + _ _
Bark
Aqueous _ _ + + _ _ _ _

Table 2. Phytochemical evaluations of Leaves extract of C. oppositifolia Smith
Presence / Absence of Phytochemical Constituents
Plant Solvent Cardiac
Alkaloids Steroids Tannins Flavonoids Glycosides Terpenoids Saponens
Part Glycosides
P. Ether +++ + _ + + _ _ ++

Chloroform + ++ _ _ + _ _ +

Leaves Methanol ++ + _ _ ++ _ _ +

Aqueous _ _ _ + _ _ _ _

Table 3. Zone of Inhibition (mm) produced by the bacterial strainsagainst standard
antibiotic discs
Zone of inhibition (mm)
Antibiotic Conc
S.
standard disc (µg) E. coli P. aeruginosa K. pneumoniae E. faecalis B. subtilis
aureus
Methicillin 15 8±0.6 9±1.2 9±0.5 8±0.5 8±0.7 8±0.5
Amoxicillin 30 8±1.5 9±1.0 8±1.0 9±1.3 - 11±1.0
Azithromycin 16 14±2.6 14±1.3 10.5±0.7 11±0.5 10±1.5 12±1.5
Ampicillin 10 9±1.7 8±2.0 9±1.2 8±1.0 7±0.5 8±0.5
GeneralResponse Resistant Resistant Resistant Resistant Resistant Resistant
Table 4. Zone of Inhibition (mm) of fungal strains against the standard antifungal discs
Concentration Zone of inhibition (mm)
Antifungal standard disc
(µg/ml) A.niger A.oryzae
Nystatin 99.9 23±2.1 30±1.6
Tezole 99.9 23±0.6 67±2.6
Fungivin 99.9 16±3.5 40±1.8
General Response Intermediate Intermediate
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The extract of leaf had showed supreme muchalike potential with little difference.
potential in contrast to S. Aureusas The potential exhibited by both parts was
compared to the bark with the petroleum better as compared to standard discs.
ether and aqueous, as shown in (Figure 2). Theleast potency was showed by the
Whereas, chloroform extracts of leaf and methanolic extracts of both parts (Table5).
bark, mutually had displayed nearly
Zone of Inhibition (mm) against S. aureus
25
20
)
m
m
(
n 15
o Leaf
it
ib Bark
ih 10
n
I f Azithromycin
o
e 5 Ampicillin
n
o
Z
0
P.E Chl. MeOH Aq.
Solvents
Figure 2. Graphical representation of the Zone of Inhibition against S. aureus

Table 5. The Zone of Inhibition produced by both parts of C.oppositifolia against the
bacterial strains was documented in mm
Zone of Inhibition (mm)

Plant
Solvents S. aureus E. coli P. aeruginosa K. pneumoniae E. faecalis B. subtilis
Part
P. ether 15±1.6 14±0.5 14±1.2 14±1.0 16±2.0 18±0.6
Chloroform 13±0.8 12±0.5 12±0.5 14±0.7 14±1.5 12±0.3
Methanol 10±0.8 11±0.6 10±0.3 12±0.6 11±0.7 13±0.8
Leaf Aqueous 17±0.3 17±0.3 16±1.0 15±0.3 15±0.5 17±1
P. ether 18±0.8 15±0.3 18±2.2 17±2.0 13±0.4 15±0.3
Chloroform 11±0.8 10±0.3 10±0.3 13±0.5 12±0.4 11±0.6
Methanol 11±0.8 11±0.5 11±1.2 10±1.0 16±0.7 10±0.5
Bark Aqueous 16±0.5 17±0.3 15±2.8 11±0.6 15±0.6 17±0.3
Both leaf and bark extracts had showed The leaf extract had exhibited the record
marvelous potential against E. coli as well potential against P. aeruginosaas well.
with the extracts in distilled With the petroleum ether being the most
waterdisplaying the record potential in effective one. The aqueous extracts of
response of the specific bacterial strain bothparts of C. oppositifolia Smith had
(Figure 3). The p. ether extracts of both showed also satisfactory results against the
parts had showed also better results against particular bacterial strains (Figure 4).
E. coli.
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Ajaib et al.
Zone of Inhibition (mm) against E. coli
20
18
)
m
16
m
( n 14 Leaf
o 12
it
ib 10 Bark
ih
n 8
I f Azithromycin
o 6
e
n 4 Ampicillin
o
Z 2
0
P.E Chl. MeOH Aq.
Solvents
Figure 3. Graphical representation of the Zone of Inhibition against E. coli

Zone of Inhibition (mm) against P. aeruginosa

25
)
m
m 20
(
n
oit 15 Leaf
ib
ih 10 Bark
n
I fo e 5 Azithromycin
no Ampicillin
Z 0

P.E Chl. MeOH Aq.

Solvents

Figure 4. Graphical representation of the Zone of Inhibition against P. aeruginosa

Petroleum ether extract of bark (17±2.0 satisfactory results against E. faecalisi.e.
mm) and aqueous extract of leaf (15±0.3 17±1 mm and 17±0.3 mm. Whereas,
mm) exhibited maximum potential against chloroform bark extract and methanolic
K. pneumoniae. Petroleum ether leaf leaf extract shown minimal potential. The
extract and chloroform leaf extract had potential showed by both parts was better as
showed somewhat similar results against compared to antibiotic discs which were
particular strains of bacteria i.e., 14±1.0 used as standard (Figure 6).
mm and 14±0.7 mm respectively. The The leaf extract had showed incredible
activity displayed by both parts was greater, activity against B. subtilis, as well with the
as compared to antibiotic discs (Figure 5). extracts soaked in petroleum ether
The petroleum ether extract of leaf (16±2.0 represented the maximum potential i.e.,
mm) and methanolic extract of bark 18±0.7 against the particular bacterial
(16±0.7 mm) showed maximum potential strain. The aqueous extracts of both parts
against E. faecalis. Aqueous extract of both had expressed significant activity against B.
leaf and bark of C. oppositifolia Smith subtilis (Figure 7).
exhibited somewhat similar and

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Pure Appl. Biol., 7(1): 138-151, March, 2018
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Zone of Inhibition (mm) against K.pneumoniae

20
)
m
m 15
(
n Leaf
o
itib 10 Bark
ihn 5
I fo Azithromycin
e 0 Ampicillin
n
o P.E Chl. MeOH Aq.
Z
Solvents

Figure 5. Graphical representation of the Zone of Inhibition against K. pneumonia

Zone of Inhibition (mm) against E. faecalis

18
)
m 16
m
( 14
no 12
itib 10 Leaf
ih 8 Bark
nI fo 6 Azithromycin
e 4
no 2 Ampicillin
Z
0

P.E Chl. MeOH Aq.

Solvents

Figure 6. Graphical representation of the Zone of Inhibition against E. faecalis

Zone of Inhibition (mm) against B. subtilis

25
)
m
m 20
(
n
o 15 Leaf
it
ibih 10 Bark
n
I f 5 Azithromycin
o
en 0 Ampicillin
o
Z P.E Chl. MeOH Aq.

Solvents

Figure 7. Graphical representation of the Zone of Inhibition against B. subtilis
MIC Assay showed maximum activity against E. coli
Themethanolic extracts were only (Table 6).
employed for the MIC estimation of C. Methanolic extract inhibited growth of S.
oppositifolia Smith. The leaf extract aureus very effectively and MIC value was
repressedthe growth of S. 0.49±0.01 at conc. of 0.9 mg/mL and
aureousefficientlywhile the bark extract 0.10±0.5 at conc. of 0.8 mg/mL (Figure 8).
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Ajaib et al.
The bark extract contained the significant of C.oppositifoliaSmith were very effective
potential against the all microbes, so we can against E. coli, their MIC values are
say the bark extracts were more effective 0.10±0.33 and 0.09±0.3 at conc. of 0.9 and
than leaf extracts. The bark and leaf extracts 1 respectively (Figure 9).

Table 6. MIC values (mg/mL) exhibited by leaf and bark of C. oppositifolia against Gram-
positive and Gram-negative bacterial strains.
K.
S. aureus E. coli P. aeruginosa E. faecalis B. subtilis
Plant pneumoniae
Part Conc. MIC Conc. MIC Conc. MIC Conc. MIC Conc. MIC Conc. MIC
0.10± 0.13±0. 0.09±
Leaf 0.9 0.49±0.01 0.9 0.8 0.9 0.8 0.14±0.5 0.7 0.10±0.3
0.33 7 0.2
0.15±0. 0.10±
Bark 0.8 0.10±0.5 1 0.09±0.3 0.9 0.8 0.8 0.12±0.7 0.8 0.11±0.3
5 0.3

MIC values against S. aureus

0.4

0.35

0.3
)L
m 0.25
/
g
m 0.2
( Leaf
C 0.15
IM Bark
0.1

0.05

0

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Conc.

Figure 8. Graphical representation of the MIC values against S. aureus

MIC values against E. coli

0.6

0.5
)
Lm 0.4
/
gm 0.3
( Leaf
C 0.2
IM Bark
0.1

0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Conc.

Figure 9. Graphical representation of the MIC values against E. coli

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The bark extract displayedsupreme The bark extract showed maximum
potential in response of P. aeruginosa. The potential against E. faecalis then leaf
leaf extract were less effective than bark extract. The bark extracts were extra
extract (Figure 10). MIC values estimated effective as compared to leaf extracts in that
for both parts were 0.13±0.7 at 0.81 mg/mL case. MIC values estimated for both parts
and 0.15±0.5 at 0.9 mg/mL against E. faecalis were 0.14±0.5 at 8.0
correspondingly. mg/mL and 0.12±0.7 at 8.0 mg/mL
The K. pneumoniae was stated to be correspondingly (Figure 12).
maximum delicate against the bark extract. The B. subtilis was stated to be the
When all methanolic extracts of C. maximum resistant, against the methanolic
oppositifoliaSmith during analysis were extract of bark. Whereas, leaf extracts were
taken to consideration. The bark extract extrareal than bark extracts. MIC values
were more active than leaf extracts assessed for both parts of C.oppositifolia
(Figure11). Actual MIC values estimated Smith against B. subtilis were 0.10±00.3 at
for both parts were 0.09±0.2 at 0.9 mg/mL 0.7mg/mL and 0.11±0.3 at 0.8mg/mL
and 00.10±0.3 at 0.8 mg/mL subsequently (Figure 13).
correspondingly.
MIC values against P. aeruginosa

0.5

0.4
)
Lm
/g 0.3
m
( C 0.2 Leaf
IM Bark
0.1

0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Conc.

Figure 10. Graphical representation of the MIC values against P. aeruginosa

MIC values against K. pneumoniae

0.5

0.4
)L
m
/ 0.3
g
m

( C 0.2 Leaf
IM Bark
0.1

0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Conc.

Figure 11. Graphical representation of the MIC values against K. pneumoniae

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Ajaib et al.

MIC values against E. facealis

0.5

0.4
)L
m
/ 0.3
g
m
( 0.2 Leaf
C
IM Bark
0.1

0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Conc.

Figure 12. Graphical representation of the MIC values against E. faecalis

MIC values against B. subtilis

0.5

0.4
)
L
m
/ 0.3
g
m
( C 0.2 Leaf
IM Bark
0.1

0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Conc.

Figure 13. Graphical representation of the MIC values against B. subtilis
The antimycotic potential of the C. The A. oryzae strain had presented resistant
oppositifolia Smith was estimated using towards the plant extracts in comparison to
two fungal strains i.e. A.niger and A.oryzae. A. niger. The supreme potential against the
The extracts had shown variable potential respective fungal strain was showed by the
against these fungal strains with the distilled water extract of both parts. The
extreme potency being showed by the leaf extract macerated in methanol had showed
extracts soaked in distilled water. However, resistance compared to other extracts. The
petroleum ether, chloroform and potential exhibited by the bark extract of C.
methanolic extracts had showed minimal oppositifolia Smith in chloroform was
potential against these fungal strains (Table greater compared to leaf extract (Figure 15
7). & 16).
The A. niger was additional sensitive near The extracts macerated in the methanol
the plant extracts when equated to the A. were also utilized for the MIC estimation.
oryzae. The supreme potential against the The leaf extract had exhibited maximum
A. niger had showed by the aqueous extract potential against A. niger with the MIC
of leaf. The petroleum ether extract of leaf value 0.55±0.5 at 0.9mg/mL whereas, bark
had displayed less potential as compared to extract showed 0.50±0.5 MIC value at
the petroleum extract of bark against the A. 0.8mg/mL (Table 8).
niger(Figure 14; Table 8).
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