Antibacterial efficacy of Bacopa monnieri leaf extracts against pathogenic bacteria

Abstract

Background: Bacopa monnieri (Linn) Pennell (Scrophulariaceae) is widely distributed in tropical regions of Asia, and used in the treatment of cough or as an antiseptic. The traditional use of this plant suggests its possible antibacterial properties, but its efficacy has not been examined yet. Objective: Evaluate the antibacterial efficacy against pathogenic bacteria using the disk diffusion method. Materials and methods: Five different concentrations (500 μg, 1, 2, 5, 10, and 15 mg/mL) of crude leaf extracts of Bacopa monnieri (L.) Pennell were tested for antibacterial efficacy against seven Gram-positive and 11 Gram-negative bacteria. The sensitivity of plant fractions was tested using the disk diffusion method. Results: Maximum activity was revealed by ethyl acetate and methanol extracts, followed by aqueous, benzene, and petrol extracts. Phyto-chemical analysis of the plant leaf showed the presence of alkaloids, flavonoids, and saponins. Conclusion: This plant may be effective for treatment of different pathogenic diseases. Although large numbers of plant products are used to treat human diseases worldwide, information on the effectiveness of most plant species is either insufficient or lacking [1, 2]. India has great potential for discovery of novel plant-derived-drugs needed to combat various human diseases [3, 4]. Many medicinal plants are used in traditional phyto-therapy for centuries [5, 6]. Bacopa monnieri (Linn) Pennell (Scrophulariaceae) (B. monnieri) is growing widely in tropical regions of Asia, and used in the treatment of cough or as an antiseptic (see Appendix). The traditional use of this plant suggests possible antibacterial properties. However, its efficacy has not been examined fully yet. This study was aimed to evaluate the antibacterial efficacy of B. monnieri. Antibacterial screening of the crude leaf extracts was made using seven Gram-positive (G +ve) and 11 Gram negative (G -ve) bacteria.

Full text

Asian Biomedicine Vol. 4 No. 4 August 2010; 651-655
Antibacterial efficacy of Bacopa monnieri leaf extracts
against pathogenic bacteria
Abdul Viqar Khan
a
, Qamar Uddin Ahmed
b
, Indu Shukla
c
, Athar Ali Khan
a
a
Department of Botany,
b
Department of Microbiology, Faculty of Life Sciences, Aligarh Muslim
University, Aligarh-202002, State of Uttar Pradesh, India.
b
Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, International Islamic University Malaysia, 25200-Kuantan,
Pahang Darul Makmur, Malaysia
Background: Bacopa monnieri (Linn) Pennell (Scrophulariaceae) is widely distributed in tropical regions of
Asia, and used in the treatment of cough or as an antiseptic. The traditional use of this plant suggests its
possible antibacterial properties, but its efficacy has not been examined yet.
Objective: Evaluate the antibacterial efficacy against pathogenic bacteria using the disk diffusion method.
Materials and methods: Five different concentrations (500 μg, 1, 2, 5, 10, and 15 mg/mL) of crude leaf extracts of
Bacopa monnieri (L.) Pennell were tested for antibacterial efficacy against seven Gram-positive and 11 Gram-
negative bacteria. The sensitivity of plant fractions was tested using the disk diffusion method.
Results: Maximum activity was revealed by ethyl acetate and methanol extracts, followed by aqueous, benzene,
and petrol extracts. Phyto-chemical analysis of the plant leaf showed the presence of alkaloids, flavonoids, and
saponins.
Conclusion: This plant may be effective for treatment of different pathogenic diseases.
Keywords: Antibacterial efficacy, Bacopa monnieri (L.) Pennell, crude extracts, traditional use
Although large numbers of plant products are used
to treat human diseases worldwide, information on
the effectiveness of most plant species is either
insufficient or lacking [1, 2]. India has great potential
for discovery of novel plant-derived-drugs needed to
combat various human diseases [3, 4]. Many
medicinal plants are used in traditional phyto-therapy
for centuries [5, 6].
Bacopa monnieri (Linn) Pennell
(Scrophulariaceae) (B. monnieri) is growing widely
in tropical regions of Asia, and used in the treatment
of cough or as an antiseptic (see Appendix). The
traditional use of this plant suggests possible
antibacterial properties. However, its efficacy has not
been examined fully yet. This study was aimed to
evaluate the antibacterial efficacy of B. monnieri.
Antibacterial screening of the crude leaf extracts was
made using seven Gram-positive (G
+ve
) and 11 Gram
negative (G
-ve
) bacteria.
Materials and methods
Plant material
Fresh plant of B. monnieri was collected after
the September rainy season. The specimen were
collected in the wild, where they grow naturally, in
the marshy location at the Herbarium, Department of
Botany of Aligarh Muslim University, India. They were
authenticated by Dr. Athar Ali Khan (Taxonomist,
India).
Preparation of extracts and fractions
Plant extracts of B. monnieri were prepared
according to the following procedure [7, 8].
1) Freshly dried and healthy plant material was
pulverized into fine powdered form by an electric
grinder and was stored in a dessicator.
2) Five hundred gram plant powder was refluxed
with 95% methyl alcohol (MeOH) on a water bath
for 10 hours. Mother liquor (crude MeOH extract)
Correspondence to: Dr. Abdul Viqar Khan, Department of
Botany, Faculty of Life Sciences, Aligarh Muslim University,
Aligarh 202002, State of Pradesh, India. E-mail: viqarvicky@
gmail.com
Brief communication (Original)

652
A.V. Khan, et al.
was filtered, residual plant material was refluxed with
95% MeOH for 10 hours. The process was repeated
four times for maximum yield of MeOH extract. The
extract was evaporated to dryness at 35°C under
reduced pressure using a rotary evaporator.
3) Dried MeOH extract was refluxed with petroleum
ether (60-80°C) for five hours. After filtration, the
residual MeOH extract was refluxed with petroleum-
ether for five hours. It was then tiltered and the process
was repeated five times. Petrol was evaporated under
a reduced pressure to obtain petroleum ether-soluble
extract.
4) Petroleum ether insoluble fraction of MeOH
extract obtained in step 3, was refluxed with benzene
for five hours. Then, it was refluxed with benzene
for five hours and filtered. The process was repeated
five times. Benzene was evaporated under reduced
pressure to obtain benzene soluble extract.
5) Benzene insoluble fraction obtained in step 4 was
refluxed with ethyl acetate (EtOAc) for five hours.
Then, it was refluxed with EtOAc for five hours and
filtered. The whole process was repeated five times
to ensure a maximum yield. EtOAc was evaporated
under reduced pressure to obtain EtOAc-soluble
extract.
6) EtOAc-insoluble fraction obtained in step 5 was
refluxed with MeOH (95%) for five hours, filtered
and was repeatedly refluxed five times with methanol.
The MeOH-soluble fraction was evaporated under
reduced pressure to obtain MeOH extract, while
MeOH-insoluble residue was discarded.
Preparation of aqueous extract
Five hundred gram plant powder was poured with
double distilled water, and left for 72 hours at room
temperature. Then, the flask was refluxed over
hot water bath for 10 hours, and the mother liquor
was filtered, process was repeated for four times to
obtain the maximum yield. The filtrate obtained
was evaporated to complete dryness under reduced
pressure using a rotary evaporator. The aqueous
extract thus obtained was kept in a closed bottle at
low temperature until further use.
Dried plant extract were stored in sterilized screw-
capped bottles at -20
ο
C in a deep freezer.
Microorganisms test
The leaf extracts of B. monnieri were tested
for possible antibacterial activity in the disk assay
using seven G
+ve
bacteria (Staphylococcus aureus,
Staphylococcus aureus ATCC 25953, Staphylococcus
albus, Streptococcus haemolyticus Group-A,
Streptococcus haemolyticus Group-B, Streptococcus
faecalis, and Bacillus subtilis) and 11 G
-ve
bacteria
(Escherichia coli, Edwardsiella tarda, Klebsiella
pneumoniae, Proteus mirabilis, Proteus vulgaris,
Pseudomonas aeruginosa, Salmonella typhi, Shigella
boydii, Shigella dysenteriae, Shigella flexneri, and
Plesiomonas shigelloides). The bacterial strains were
obtained from the Bacterial Stock of the Department
of Microbiology, Jawaharlal Nehru Medical College,
India. The bacterial cultures were maintained at
4
ο
C on nutrient agar.
Antimicrobial assay
Agar plates were inoculated using a sterile swab
dipped into culture inoculums adjusted to 1.5x10
8
bacterial/mL. The agar was streaked in three directions
turning the plates by 60 degree for each streak. All
the extracts were sterilized by filtration thorough
membrane filter (diameter: 0.045 mm), and autoclaved
at 122
ο
C. The paper disk (Whatman filter paper)
impregnated with 1mg/mL, 2 mg/mL, 5 mg/mL,
10 mg/mL, and 15 mg/mL plant extracts were dried
and placed on the agar surface. The sensitivity disks
were placed to make complete contact with the surface
of the medium on the plate. Plates were kept at room
temperature for 30 minutes (pre-diffusion time).
Inoculated petri dishes were incubated at 37
ο
C
overnight and at the end of the period. Inhibition zones
formed on the medium were evaluated in millimeter,
and experiments were repeated three times.
Studied activity
Antibacterial activity was tested using the disc
diffusion method [9]. Diameters of petri dish and disk
were 9.0 cm and 0.6 cm, respectively.
Phyto-chemical analysis
Binary and tertiary solvent systems were used to
detect the presence of alkaloids, flavonoids, triterpenes,
anthraquinones, coumarins, saponins, and glycosides
in non-polar and polar extracts of plant on the thin
layer chromatography plates. Reagents used were
dragendorff reagent, mayers reagent, potassium
hydroxide, alcoholic ferric chloride, Vaniline + supfuric
acid, and liebermann-burchard reagent [10]. Solvent
system with different ratios for non-polar extracts
included toluene-acetone, toluene-chloroform-acetone
for semi-polar extracts, and n-butanol-glacial acetic
acid-water for polar extracts.

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August 2010
Antibacterial efficacy of B. minnieri leaf extracts
Statistical analysis
All values are expressed as mean ±standard error
of mean (SEM). Linear regression analyses were
made to determine correlation between two variables
using MS-DOS software (GraphPad InStat statistical
program).
Results
Antibacterial efficacy of B. monnieri leaf
extracts using different solvents (Petroleum ether,
Benzene, EtOAc, MeOH, and aqueous) against
seven G
+ive
and 11 G
-ve
bacteria is shown in Table 1.
Apparently, EtOAc and MeOH extracts exhibited
strong antibacterial activity against maximum number
of bacterial species (15 each). Both extracts were
sensitive against all G
+ve
bacterial strains. Aqueous
extract also demonstrated biological activity against
all G
+ve
bacteria strains, but it could only resist the
growth of five of the G
-ve
pathogens.
Table 1. Antibacterial efficacy of B. monnieri leaf extracts against 18 pathogenic bacteria.
Gram positive (G
+ve
) bacteria Gram negative (G
+ve)
bacteria
1 234 567 1234 56 7 891011
Petrol
500 μg -02-- 02-01----- - - ----
1 mg -02-- 02-06----- - - ----
2 mg 0305-- 06-07----- - - ----
5 mg 0508-- 09-10----- - - ----
10 mg 0710-- 12-12----- - - ----
15 mg 1016-- 14-16----- - - ----
Benzene
500 μg -02-04--04---04- - 03----
1 mg -03-05--04---04- - 04----
2 mg -06-07--07---06- - 07-05--
5 mg -11-09--10---09- - 10-07--
10 mg -15-13--14---12- - 15-10--
15 mg -17-15--16---15- - 19-12--
Ethylacetate
500 μg 04 04 04 05 01 03 04 - 04 04 - 04 - 01 03 02 02 03
1 mg 04 05 05 05 02 04 05 - 04 05 - 04 - 02 05 03 03 04
2 mg 08 08 07 08 04 06 09 - 08 07 - 06 - 04 09 06 06 07
5 mg 14 14 12 14 07 10 15 - 10 10 - 09 - 06 14 09 09 10
10 mg 17 16 17 19 09 12 19 - 13 13 - 14 - 09 17 11 11 12
15 mg 21 20 21 21 12 18 21 - 17 18 - 19 - 11 21 13 15 17
Methanol
500 μg 03 04 04 - - 02 04 05 - - 03 - 03 - - 02 02 04
1 mg 04 05 04 02 - 03 05 05 - 02 04 - 04 - - 02 05 05
2 mg 08 09 08 06 04 06 08 08 - 06 08 - 06 - 04 05 06 07
5 mg 14 15 12 09 07 10 14 14 - 09 11 - 09 - 06 09 09 12
10 mg 17 17 17 13 09 12 17 17 - 12 13 - 12 - 09 11 11 14
15 mg 21 21 20 19 12 16 20 20 - 16 18 - 16 - 11 15 14 16
Aqueous
500 μg ---02--- --01-01- - ----
1 mg -030203--- --02-02- - ----
2 mg 05 06 05 06 - 05 03 - 02 05 - 05 - - 04 - 05 -
5 mg 10 11 10 10 03 07 08 - 05 08 - 07 - - 06 - 07 -
10 mg 13 13 13 14 05 10 11 - 08 10 - 09 - - 08 - 10 -
15 mg 16 17 15 16 08 12 15 - 10 13 - 11 - - 11 - 12 -
Chloramphenicol
10 μg/disc 18 18 16 - - - 16 18 16 - 16 18 - 16 17 19 18 20
G
+ve
bacteria: 1. Staphylococcus aureus, 2. Staphylococcus aureus ATCC 25953, 3. Staphylococcus albus, 4. Streptococcus
haemolyticus Group-A, 5. Streptococcus haemolyticus Group-B, 6. Streptococcus faecalis, 7. Bacillus subtilis. G
-ve
bacteria:
1. Escherichia coli, 2. Edwardsiella tarda, 3. Klebsiella pneumoniae, 4. Proteus mirabilis, 5. Proteus vulgaris, 6. Pseudomonas
aeruginosa, 7. Salmonella typhi, 8. Shigella boydii, 9. Shigella dysenteriae, 10. Shigella flexneri, 11. Plesiomonas shigelloides.
a
Values are expressed as mean of replication of three values. (-): no inhibition.
Inhibition zone (mm)

654
A.V. Khan, et al.
Table 2 shows results by phyto-chemical analysis
of B. monnieri leaf. Interestingly, the plant leaf has
alkaloids, flavonoids, and saponins.
Discussion
The present result showed that the maximum
activity is revealed by B. monnieri EtOAc extracts
and MeOH, followed by aqueous, benzene, and petrol
extracts (Table 1). This suggests that this plant may
be of clinical benefit for uses in different pathogenic
diseases. Bioactive compounds in these extracts may
be used in the development of novel antibacterial
drugs.
Pathogenic bacteria are known to develop
resistance for antibiotics, thus search for new
antibiotics is a never-ending process [11]. Crude
extracts of B. monnieri, especially EtOAc and
MeOH extracts, may deserve further investigations
to develop a new antibiotic that may help in combating
several bacterial infirmities in tropical countries.
In conclusion, B. monnieri may be effective for
treatment in different pathogenic diseases. Our finding
may be useful for development of antibacterial drugs
from this plant. B. monnieri (L.) Pennell
Acknowledgement
Thanks are due to Department of Science and
Technology SERC Division, New Delhi for financial
support to the author Dr Abdul Viqar Khan (DST No:
304/2004:SR/FT/L-129/2004). The authors have no
conflict of interest to report.
Appendix
Bacopa monneiri (Linn) Pennell
(Scrophulariaceae)
B. monnieri is a perennial, creeping herb whose
habitat includes wetlands and muddy shores.
The leaves are succulent and relatively thick and
oblanceolate, and are arranged oppositely on the stem
(Fig. 1). It commonly grows in marshy areas
throughout India, Nepal, Sri Lanka, China, Taiwan,
Vietnam, and some southern states of USA.
Chemical constituents are Hersaponin, Brahmin,
Herpestatine, Herstatin, β-Sitosterol, D-Mannitol,
Stigmasterol, Betulic acid, Stigmastanol, Monnierin,
Bacoside A & B, A
3
-A and I, II, III, IV and V, Nicotine,
Bacogenin A
1
-A
4
, Triterpenoid agelycone, Monnierin,
Apigenine, Luteolin, Aaspartic acid, Glutamic acid, and
Serine [12].
Table 2. Phtotochemical analysis of B. monnieri leaf.
Plant part Alkaloids Anthraquinones Coumarins Flavonoids Saponins Polyphenols Cardiac glycoside
Leaf ++++ - - ++++ ++ - -
(-): negative, (+): small amount, (++): average, (++++): high
Fig. 1 Leaf of Bacopa monnieri. This plant is commonly named as Thyme leaved Gratiola, Brahmi.

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Antibacterial efficacy of B. minnieri leaf extracts
Leaf analysis shows that moisture is 88.4%,
protein 2.1%, fat 0.6%, carbohydrates 5.9%, crude
fiber1.05%, and ash (1.9g/100g) where calcium is
202.0, phosphorus 16.0, iron 7.8, ascorbic acid 63.0,
and nicotinic acid 0.3mg/100g [12].
Alcoholic extract (50% EtOH) of whole plant has
a number of biological activities in vivo. These
includes analgesic [13], cardio tonic [14], tranquilizer,
muscle relaxant and anti-stress [15], anticonvulsant
[16], anticancer [17], improvement in maize learning
in rats [18], and as an antioxidant [19].
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