Download Indian nutraceutical plant leaves as a potential source of natural

Science against microbial pathogens: communicating current research and technological advances
_______________________________________________________________________________
A. Méndez-Vilas (Ed.)
Indian nutraceutical plant leaves as a potential source of natural
antimicrobial agents
Sumitra Chanda* and Mital Kaneria
Phytochemical, Pharmacological and Microbiological Laboratory, Department of Biosciences, Saurashtra University,
Rajkot 360 005, Gujarat, India
* Author for correspondence, E-mail: [email protected]
The quest for plants with medicinal properties continues to receive attention as they are known for a range of biological
activities which range from antibiotics to antitumor. Medicinal plants represent one of the most important fields of
traditional medicine all over the world. In light of the recent emergence of bacteria which are resistant to multiple
antimicrobial drugs posing a challenge for the treatment of infections, the need to discover new antimicrobial substances
for use in combating such microorganisms become pertinent. Bioactive compounds are normally accumulated as
secondary metabolites in all plant cells but their concentration varies according to the plant parts. Leaf is one of the highest
accumulatory plant part of such compounds and generally it is preferred for therapeutic purpose. Some of the active
compounds that inhibit the disease causing microbial growth act either singly or in combination. They can inhibit the
growth of the microbes by many mechanisms of actions. It is believed that crude extracts from plants are more effective
than isolated compounds due to their synergistic effect. Here we report the antimicrobial activity of five different solvent
extracts of four Indian nutraceutical plants viz. Manilkara zapota (L.) var. Royen., Psidium guajava L., Punica granatum
L. and Syzygium cumini L. leaves against 14 pathogenic microorganisms. The antimicrobial activity was evaluated by agar
well diffusion method. P. guajava leaves showed best and promising antimicrobial activity, indicating the possibilities of
its potential use in the formula of natural remedies for the treatment of infections.
Keywords antimicrobial activity; solvent extracts; Manilkara zapota; Psidium guajava; Punica granatum; Syzygium
cumini
1. Introduction
Infectious diseases account for a high proportion of health problems and burden in India. Large amounts of antibiotics
are consumed in fighting infections, some of them saving lives, but every use adds antibiotic resistance in bacteria.
Antimicrobial resistance has in fact turned into a global health issue, which hampers the control of infectious diseases;
multi-drug resistant bacteria are thus posing a problem in treatment regimens. Multi-drug resistant organisms are also an
epidemiological concern as they may spread locally, regionally or globally through individual contacts, poor sanitation,
travel or the food chain. During the present millennium, overcoming resistance to antibiotics is one of the major issues
facing WHO [1]. Hence, WHO has declared antimicrobial resistance as the theme for the World Health Day, 2011 [2].
Antimicrobials have been used successfully for over six decades to treat infectious diseases; but antimicrobial resistance
threatens the continued effectiveness of antimicrobials and simultaneously the downward trend in the development of
new antibiotics has serious implications [3].
Resistant bacteria dramatically reduce the possibilities of treating infectious diseases effectively and increase the risk
of complications and fatal outcomes for patients with severe infections. The increasing prevalence of multi - drug
resistant bacteria and fungi, the recent appearance of strains with reduced susceptibility to antibiotics, the side effects
associated with antibiotics, the high costs of antimicrobial drugs and the re-emergence of diseases are the key factors
that obstruct effective management of bacterial infections in many developing countries. Unfortunately, the future does
not look bright in the war against infectious diseases, as multi drug resistant strains of microbes continue to increase at
an alarming rate. In fact, multi drug resistant strains are adapting faster than the introduction of new, more potent
antibiotics. Consequently, this has raised the specter of untreatable microbial infections and adds urgency to the search
for new infection-fighting strategies. This necessitates greater efforts to discover new potent antibiotics. A potential
post-antibiotic era is threatening present and future medical advances.
The development of antibiotic resistance can be natural or acquired. Bacteria usually become resistant to antibiotics
by one of the following mechanisms (Fig. 1). Natural resistance is achieved by spontaneous gene mutation and acquired
resistance is through the transfer of DNA fragments like transposons from one bacterium to another. Bacteria gain
antibiotic resistance due to any of the four reasons namely: (I) by alteration of drug binding or target sites; by alteration
of membrane permeability; (II) direct destruction or in ability of the drug to enter in the cell; (III) by developing the
ability to produce multi resistance pumps, due to this pump drug drained out of the cells before drug can damage or kill
the cell or (IV) replacement of a sensitive pathway and acquisition of a new enzyme to replace a sensitive one [4].
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A. Méndez-Vilas (Ed.)
Fig. 1 Mechanisms of antibiotic resistance.
In light of the recent emergence of bacteria which are resistant to multiple antimicrobial drugs causing a challenge
for the treatment of infections, the need to discover new antimicrobial substances for use in combating such
microorganisms becomes pertinent. Thus, there is a constant and urgent need to develop new antimicrobial drugs for the
treatment of infectious diseases from medicinal plants. Naturally occurring compounds have been and continue to be, an
important source of new leads for many pharmaceuticals and agrochemicals. In this context, there is a growing
popularity in nutraceutical products or functional foods.
1.1 Medicinal plants as antimicrobials
India is a varietal emporium of medicinal plants and is one of the richest countries in the world with regard to genetic
resources for medicinal plants. India is rich in medicinal plant diversity; all known types of agroclimatic, ecologic and
edaphic conditions are found within India. India is rich in all the three levels of biodiversity viz. species diversity,
genetic diversity and habitat diversity. The increased interest in medicinal plant cures is because, primarily plants as
medicines are safe, less rigorous and more affordable than synthetic alternatives. In fact, plants produce a diverse range
of bioactive molecules, making them a rich source of different types of medicines [5]. Many plants contain non-toxic
glycosides that can get hydrolyzed to release phenolics that are toxic to microbial pathogens [6]. Phytochemicals or the
bioactive compounds in medicinal plants could be used as a novel source in infectious disease management as an
alternative to synthetic drugs. Phytochemicals can be derived from any part of the plant like bark, leaves, roots, fruits,
seeds, fruit rind, flowers, stem, whole plant, etc. and antimicrobial activity of different parts of the plant is reported in
literature [7]. However, leaf is generally the preferred part for therapeutic purpose [8]. This may be because leaves
contain more number of secondary metabolites which may be responsible for its antimicrobial activity. They can inhibit
the growth of microbes in many ways such as by inhibiting protein synthesis, interfering with nucleic acid synthesis,
breaking the peptide bonds, acting as chelating agents, inhibiting metabolic pathway, interfering with cell wall synthesis
or by preventing utilization of available nutrients by the microorganisms. Some compounds also cause lyses of
microbial cells.
1.2 Nutraceuticals as a novel source of antimicrobials
The traditional and herbal medicines are known as essential resources of nutraceuticals which could provide a variety of
beneficial effects on human health. Nutraceuticals or functional foods are natural bioactive, chemical compounds that
have health promoting, disease preventing or medicinal properties. The secondary metabolites present in them give
them a specific medical benefit other than a purely nutritional. Nutraceuticals have thus dual role to play: as food and
therapeutic agent i.e. aids in prevention and/or treatment of disease and/or disorder. The other benefit is, being natural,
they have no side effects as other therapeutic agents. Nutraceuticals may range from single isolated nutrients, dietary
supplements, or secondary metabolites to genetically engineered designer foods [9]. There are many reports of
nutraceutical plant leaves that are effective as antimicrobials (Table 1).
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Petroleum ether,
acetone, methanol
Methanol, acetone,
N, Ndimethylformamide
Manilkara hexandra
(Roxb.) Dubard
(Sapotaceae)
Psidium guajava L.
(Myrtaceae)
Water, acetone,
ethanol
Water, ethanol,
methanol
Aqueous,
methanol, saponins
Tamarindus indica
Linn. (Fabaceae)
Thymus vulgaris L.
(Lamiaceae)
Ziziphus mauritiana
Lam. (Rhamnaceae)
Acetone, methanol,
water
Hexane, methanol
Crude EtOH/H2O
Ethanol
Ficus exasperata Vahl
(Moraceae)
Rosmarinus officinalis
L. (Lamiaceae)
Rumex dentatus L.
(Plygonaceae)
Rumex ecklonianus
Meissner
(Polygonaceae)
Methanol
[33]
[34]
Staphylococcus aureus, Pseudomonas aeruginosa, S. facecalis, Klebsiella pneumoniae, Escherichia coli,
Enterobacter faecalis, Enterobacter faecium, Proteus mirabilis
[32]
[31]
[30]
[29]
[28]
[27]
Pseudomonas aeruginosa
Staphylococcus aureus, S. Setubal, Bacillus subtilis, Micrococcus luteus, Eschericia coli, Pseudomonas
picketii, Bordetella bronchiseptica
Bacillus cereus, Staphylococcus epidermidis, S. aureus, Micrococcus kristinae, Streptococcus pyogenes,
Escherichia coli, Salmonella pooni, Serratia marcescens, Pseudomonas aeruginosa, Klebsiella
pneumoniae
Escherichia coli, Proteus mirabilis, Pseudomonas aerugenosa, Salmonella typhi, S. paratyphi, Shigella
flexnerri, Staphylococcus aureus, Bacillus subtilis, Streptococcus pyogenes, Aspergillus flavus, A.
fumigatus, A. niger, Candida albicans
Streptococcus mutans, S. salivarius, S. sobrinus, S. mitis, S. sanguinis, Enterococcus faecalis
Bacillus cereus, B. megaterium, B. subtilis, Corynebacterium rubrum, Micrococcus flavus, Staphylococcus
aureus, S. epidermides, S. subfava, Alcaligenes fecalis, Citrobacter freundii, Enterobacter aerogenes,
Escherichia coli, Klebsiella aerogenes, K. pneumoniae, Proteus mirabilis, P. morganii, P. vulgaris,
Pseudomonas aeruginosa, P. pseudoalcaligenes, P. putida, P. testosteroni, Salmonella typhimurium,
Candida albicans, C. glabrata, C. tropicalis, C. neoformans, Cryptococcus luteolus, Trichosporon
beigelii, Aspergillus candidus, A. flavus, A. niger, Mucor hiemalis wehmer
Staphylococcus sp.-13, S. aureus, S. epidermidis, S. subfava, Bacillus cereus, B. subtilis, B. megaterium,
M. flavus, Pseudomonas sp.-16, P. aeruginosa, P. testosterone, P. pseudoalcaligenes, E. coli-15, E. coli,
Enterobacter sp.-2, E. aerogenes, Klebsiella sp.-5, K. pneumoniae, Proteus sp.-2, P. mirabilis, P. vulgaris,
P. morganii, Providencia sp.-1, Citrobactor sp.-2, C. freundii, Alcaligenes fecalis, Salmonella
typhimurium, Candida sp.-5, C. albicans-2, C. glabrata, C. tropicalis, C. apicola, Cryptococcus
neoformans, C. luteolus, Trichosporan beigelii, Aspergillus flavus, A. candidus A. niger
[26]
[25]
Streptococcus mutans, S. sanguinis, S. sobrinus, S. ratti, S. criceti, S. anginosus, S. gordonii,
Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Prevotella intermedia,
Porphyromonas gingivalis, Escherichia coli, Staphylococcus aureus, S. epidermidis, S. pyogenes
Escherichia coli, Staphylococcus albus
[24]
[23]
Reference
Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis
Bacillus subtulis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherchia coli, Candida albicans,
Aspergillus niger
Petroleum ether,
70% ethanol
Water
Microorganisms
Extracts
Ficus carica Linn.
(Moraceae)
Plant name
Anacardium
occidentale Linn.
(Anacardiaceae)
Carissa edulis Vahl
(Apocynaceae)
Table 1 List of some nutraceutical plant leaves showing antimicrobial activity against some microorganisms causing infectious diseases.
Science against microbial pathogens: communicating current research and technological advances
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A. Méndez-Vilas (Ed.)
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A. Méndez-Vilas (Ed.)
2. Objective
The purpose of this work was to investigate the potential of four Indian nutraceutical plant leaves as natural
antimicrobial agents. Therefore leaves of four plants viz. Manilkara zapota L. var. Royen., Psidium guajava L., Punica
granatum L. and Syzygium cumini L. were selected and their antimicrobial potential was explored against some
common pathogenic bacteria and fungi.
3. Experimental methodology
3.1
Chemicals
Petroleum ether, toluene, ethyl acetate, acetone, dimethyl sulphoxide (DMSO), Nutrient broth, Sabouraud dextrose
broth, Muller Hinton agar No. 2 and Sabouraud dextrose agar, agar powder were obtained from Hi-media or Merck. All
reagents used were of analytical grade.
3.2
Collection of plant materials and preparation of plant extracts
Fresh leaves of four nutraceutical plants were collected in September, 2008, from Jam-jodhpur, Gujarat, India. The
ethnobotanical description of the four plants is given in table 2 [10]. The leaves were separated, washed thoroughly with
tap water, shade dried, homogenized to fine powder and stored in airtight bottles.
The dried powder of the leaves was extracted sequentially [11] by cold percolation method [12], using different
solvents depending upon their polarities like petroleum ether, toluene, ethyl acetate, acetone and water (Flow chart 1).
The extracts were concentrated under reduced pressure, using rotary vacuum evaporator (Equitron, India) to dryness.
The dried crude concentrated extracts were stored at 4 °C in a refrigerator in air tight bottles.
Plant powder
Extraction with Petroleum ether
Residue
Petroleum ether Extract
Extraction with Toluene
Residue
Toluene Extract
Extraction with Ethyl acetate
Residue
Ethyl acetate Extract
Extraction with Acetone
Residue
Acetone Extract
Extraction with Water
Residue
Aqueous Extract
Flow Chart. 1 Systematic representation of different solvent extracts of leaves by sequential extraction.
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Dadum
Jambu
Punica granatum L.
(PSN311)
Syzygium cumini L.
(PSN295)
4
Jamphal
Psidium guajava L.
(SU/BIO/510/Thakrar)
2
3
Chiku
Manilkara zapota (L.)
var. Royen.
(PSN429)
1
Vernacular
name
Plant name
(Voucher specimen No.)
No.
©FORMATEX 2011
Myrtaceae
Punicaceae
Myrtaceae
Sapotaceae
Family
Therapeutic uses
The bark is astringent, sweet, sour, acrid, refrigerant, carminative, diuretic, digestive, anthelmintic,
febrifuge, constipating, stomachic and antibacterial. The fruits and seeds are sweet, acrid, sour, tonic and
cooling. The bark is useful in diabetes, leucorrhoea, stomachaigia, fever, gastropathy, strangury and
dermatopathy. The tender leaves are used for vomiting. The leaves are used for strengthening the teeth and
gums. The fruits and seeds are used in diabetes, diarrhoea, pharyngitis, splenopathy, urethrorrhoea and ring
worm.
The root and stem bark are astringent, cooling and anthelmintic. Flowers are stypic. Fruits are sweet, sour,
astringent, cooling, tonic, aphrodisiac, laxative, diuretic. Seeds are astringent, stomachic, diuretic,
cardiotonic. Root and stem bark is used in tape worm infection, vomiting. Flowers are used in vomiting,
pitta, ophalmodynia, ulcers, pharyngodynia, hydrocele. Fruits are used in anaemia, hyperdipsia,
pharygodynia, opthalmodynia, pectoral diseases, splenopathy, bronchitis, otalgia. Fruit rind is used in
dysentery, diarrhoea and gastralgia. Seeds are used in vomiting, opthalmodynia, pitta, scabies,
hepatopathy, splenopathy.
The roots are astringent, haemostatic, constipating, antiemetic. Leaves are astringent, anodyne, febrifuge,
antispasmodic, tonic. Flowers are cooling, laxative, tonic. Fruits are sweet, astringent, sour, cooling,
aphrodisiac, laxative, tonic. Roots are used in haemorrhages, diarrhoea, dysentery, ulcers, gingivitis,
proctoptisis, vomiting. Leaves are used in wounds, ulcers, cholera, diarrhea, vomiting, nephritis, cachexia,
vata, epilepsy, odontalgia, gum boils. Flowers are used in bronchitis, opthalmodynia, colic, ulemorrhagia.
Fruits are used in pitta, dipsia, burning sensation, colic, ulemorrhagia, diarrhoea, dysentery, debility.
The seeds are aperients, diuretic, tonic and febrifuge. Bark is antibiotic, astringent and febrifuge. Fruits are
edible, sweet with rich fine flavour. Chicle from bark is used in dental surgery. Bark is used as tonic and
the decoction is given in diarrhoea and peludism.
Table 2 Ethnobotanical description of four studied nutraceutical plants.
Science against microbial pathogens: communicating current research and technological advances
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A. Méndez-Vilas (Ed.)
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3.3
Microbial strains and culture condition
The bacterial and fungal strains used to assess the antimicrobial properties included five Gram positive bacteria
[Bacillus megaterium ATCC9885 (BM), Bacillus subtilis ATCC6633 (BS), Corynebacterium rubrum ATCC14898
(CR), Staphylococcus aureus ATCC25923 (SA), Staphylococcus epidermidis ATCC12228 (SE)], five Gram negative
bacteria [Citrobacter freundii ATCC10787 (CF), Enterobacter aerogenes ATCC13048 (EA), Klebsiella pneumoniae
NCIM2719 (KP), Proteus mirabilis NCIM2241 (PM), Salmonella typhimurium ATCC23564 (ST)] and four fungi
[Candida albicans ATCC2091 (CA), Candida epicola NCIM3367 (CE), Candida glabrata NCIM3448 (CG), Candida
neoformans NCIM3542 (CN)]. The investigated bacterial and fungal strains were obtained from National Chemical
Laboratory (NCL), Pune, India. The organisms were maintained on nutrient agar and MGYP medium (Hi-media, India)
bacteria and fungi respectively, at 4 °C and sub-cultured before use. The microorganisms studied are clinically
important ones causing several infections, food born diseases, spoilages, skin infections and it is essential to overcome
them through some active therapeutic agents.
3.4 Antimicrobial assay
In vitro antimicrobial activity of the different solvent extracts of the four screened plants was studied against 14
pathogenic microbial strains by the agar well diffusion method [13, 14]. Muller Hinton No. 2 / Sabouraud dextrose agar
(Hi-media) was used for antibacterial and antifungal susceptibility test respectively. The extracts were prepared in
100% DMSO at a concentration of 20 mg/ml. The microbial activity was evaluated at a concentration 2.0 mg/well. The
Muller Hinton agar / Sabouraud dextrose agar was melted and cooled to 48-50 °C and a standardized inoculum (1.5 ×
108 CFU/ml, 0.5 McFarland) was then added aseptically to the molten agar and poured into sterile Petri dishes to give a
solid plate. Wells were prepared in the seeded agar plates. The test compound (100 µl) was introduced in the well (8.5
mm). The plates were incubated over night at 37 °C and 28 °C for 24 h and 48 h for bacteria and fungi respectively. The
antimicrobial spectrum of the extract was determined in terms of zone sizes around each well. DMSO was used as
negative control. The control zones were subtracted from the test zones and the resulting zone diameter is shown in Fig.
2. The experiment was performed three times to minimize the error.
3.5 Data analysis
The results are expressed as the mean value ± Standard Error of Mean (S.E.M.).
4. Results and discussion
In the present work, the four nutraceutical plant extracts showed considerable antibacterial activity; the various solvent
extracts showed 71% activity against Gram positive bacteria and 79% activity against Gram negative bacteria.
Generally, it is believed that Gram negative bacteria are more resistant to plant based antimicrobials than Gram positive
bacteria [15-17]. This is because the Gram negative bacteria have an effective permeability barrier, comprised of outer
membrane, which restricts the penetration of antimicrobial compounds, which extrude plant extracts across this barrier.
The single membrane of Gram positive bacteria is considerably more accessible to permeation by plant extracts in
region where these bacteria provide limited protection [7, 12, 18]. But contradicting this general belief, in the present
work, Gram negative bacteria were more susceptible than Gram positive bacteria. All the five different solvent extracts
of P. guajava showed activity against all the ten tested bacteria, while all the five different solvent extracts of P.
granatum showed activity against all the five tested Gram negative bacteria (Fig. 2). The most resistant bacteria was S.
aureus. The antifungal activity was comparatively less than that of antibacterial activity. It was only 61%. It appears
that fungi were more resistant than bacteria. Resistance of fungi to plant extracts can be explained by chitinous structure
of the cell wall, which does not allow easy penetration of bioactive substances [19]. There are many reports in literature
where plant extracts showed good antibacterial activity but poor antifungal activity [20-22]. All plants extracts showed
better antibacterial activity than antifungal activity. P. guajava showed best antimicrobial activity. Overall, a broad
spectrum antimicrobial activity was exhibited by four nutraceutical plants. Therefore, it can be stated that all the four
nutraceutical plant leaf extracts can be a good source of natural antimicrobics.
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A. Méndez-Vilas (Ed.)
Fig. 2 Antimicrobial activity of five different solvent extracts of leaves of Manilkara zapota, Psidium guajava, Punica granatum and
Syzygium cumini.
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Science against microbial pathogens: communicating current research and technological advances
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A. Méndez-Vilas (Ed.)
5. Concluding remarks
The broad spectrum activity exhibited by four nutraceutical plant extracts, establishes the scientific basis of their use as
ethnomedicine and use their potential for development of novel antimicrobial agents effective for treatment of microbial
infectious diseases. Its usefulness in the formulation of antiseptics and disinfectants is also recommended if the active
principles can be isolated and purified. On the basis of the present findings, we conclude that amongst four nutraceutical
plants, P. guajava showed best antimicrobial activity. It might turn out to be a good candidate in the search for effective
and efficient antimicrobial agents.
Acknowledgements The authors thank Prof. S.P. Singh, Head, Department of Biosciences, Saurashtra University, Rajkot, Gujarat,
India for providing excellent research facilities. One of the authors, Mr. Mital Kaneria, is thankful to University Grants Commission,
New Delhi, India for providing financial support as Junior Research Fellow.
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