chapter-iii antimicrobial studies -...
TRANSCRIPT
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CHAPTER-III
ANTIMICROBIAL STUDIES
Antimicrobial is an agent that either kills microorganisms or inhibits their
growth. They may be of various types on the basis of microorganisms on which they
primarily act against e.g. antibacterials are used against bacteria and antifungals are
used against fungi. These antimicrobials may be of plant origin, fungal or synthetic
chemicals. In past few decades the synthetic chemicals have been used to treat various
infections including epidermal infections. However, synthetic antimicrobial chemicals
are sometimes associated with adverse side effects such as hypersensitivity, allergic
reactions and immunity suppression (Cakir et al, 2005).
Therefore, there has been a growing interest in research concerning alternative
natural antimicrobial agents, including the extracts and essential oils from various
species of medicinal plants that are relatively less damaging to human health.
Keeping these things in mind an effort has been made in the present study to
screen some medicinal plants for their antibacterial activity against epidermal
infections.
A total of 75 extracts in three solvents (water, methanol and cowurine) and
essential oils from 14 plants have been screened for their antibacterial activity against
nine epidermal infection causing bacteria. Further Minimum Inhibitory Concentration
(MIC) i.e. the lowest concentration of the antimicrobial agent that will inhibit the
visible growth of microorganism after overnight incubation (Andrews, 2001) was
determined for the extracts and essential oils that showed inhibition against bacteria.
3.1 Materials and Methods
3.1.1 Selection of medicinally significant samples: Plants belonging to different
families were selected on the basis of traditional applications and other
pharmacological reports. The plant materials were collected from herbal gardens,
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surroundings and local market. Authentication of plant material was done by Wild
Life Institute of India Dehradun.
3.1.2 Chemicals: Cow urine sealed bottles were purchased from counter of Divya
Pharmaceuticals (Ramdev). Before evaluation of antimicrobial activity, cow urine
was tested for presence of other pathogens microscopically as well as in broth culture.
All other chemicals were of analytical grade.
3.1.3 Preparation of Plant Extracts: To determine the antimicrobial activity of
plants, their extracts were prepared in various solvents. Extraction is the separation of
medicinally active portions of plant tissues from the crude samples by using selective
solvents. During extraction the, solvent diffuses into the solid plant material and
solubilize compounds with similar polarity. (Ncube et al., 2008). The extracted plant
material contains complex mixture of many medicinal plant metabolites such as
alkaloids, glycosides, terpenoids, flavonoids and lignans (Handa et al., 2008). The
quality of the extract are influenced by parameters such as plant part used as starting
material, solvent used for extraction and the extraction procedure followed.
The samples were carefully washed under running tap water followed by
sterile water and shade dried for 4-5 days
The dried plant materials were ground to powder and stored in airtight
containers.
Three different solvents namely Water, Methanol and Cowurine were used for
extraction.
10g of powdered sample was separately soaked in conical flasks each
containing 100ml of water, methanol and cow urine for 24 hrs.
Conical flasks were allowed to stand for 30 mins in a water bath (at 100°C)
with occasional shaking followed by keeping all the flasks on rotary shaker at
200 rpm for 24h (Ogundiya, 2006).
Each preparation was filtered through a sterilized Whatman No. 1 filter paper
and finally concentrated to dryness under vacuum at 40°C using a rotary
evaporator.
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The dried extract, thus, obtained was sterilized by overnight UV-irradiation,
checked for sterility on nutrient agar plates and stored at 4°C in refrigerator for
further use. (Aneja, 2010).
The dried extracted plant material was weighed to determine the percent yield
of each extract using the following formula (Ali et al., 2001).
Fig 3.1 Preparation of Plant Extracts
Collection of plant material Drying and homogenizationof plant material into a fine powder
Addition of solvents (100ml Water, Methanol and Cow urine in 10g plant material)
Extraction with solvent
Drying of plant extract through Rotary evaporator
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Weight of extracted plant obtained (g) Percentage extract yield (w/w) = x 100 Weight of plant taken for extraction (g)
The dried extracts were reconstituted to 10% in dimethylsulphoxide (DMSO) for the
antibacterial analysis.
Table 3.1
List of Plants used in the study
S. No Scientific Name Vernacular Name
Family Plant Parts Used
1. Amomum subulatum Large cardamom Zingiberaceae Fruit
2. Azadirachta indica Neem Meliaceae Leaves
3. Calotropis procera Aak Asclepiadaceae Leaves
4. Capsicum annum Red Pepper Solanaceae Fruit
5. Cassia fistula Amaltas Fabaceae Leaves
6. Catharanthus roseus Sadabahar Apocyanaceae Leaves
7. Cinnamomum zeylanicum
Cinnamon Lauraceae Bark
8. Coriandrum sativun Coriander Apiaceae Seeds
9. Cuminum cyminum Cumin Apiaceae Seeds
10. Curcuma longa Turmeric Zingiberaceae Rhizome
11. Diospyros melanoxylon Tendu Ebenaceae Leaves
12. Elettaria cardamomum Small cardamom Zingiberaceae Fruit
13. Lawsonia inermis Henna Lytharceae Leaves
14. Murraya koenigii Curry leaves Rutaceae Leaves
15. Myristica fragrans Nutmeg Myristicaceae Fruit
16. Nicotiana tobaccum Tobacco Solanaceae Leaves
17. Piper nigrum Black Pepper Piperaceae Fruit
18. Psidium guajava Guava Myrtaceae Fruit
19. Rosa indica Rose Rosaceae Petals
20. Syzygium aromaticum Clove Myrtaceae Flower bud
21. Tagetes erecta Marigold Compositae Flower
22. Tectona grandis Teak Lamiaceae Leaves
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23. Terminalia chebula Haritkari Combretaceae Fruit
24. Trigonella foenum graecum
Fenugreek Fabaceae Seeds
25. Zingiber officinale Ginger Zingiberaceae Rhizome
3.1.4 Essential oil extraction
Essential oils can be obtained by expression, fermentation or extraction but the
method of steam distillation is most commonly used. Air-dried plant materials were
washed and dried at room temperature. They were pulverized into powdered form.
The powder of samples was subjected to hydrodistillation for 5 h in a Clevenger-type
apparatus to obtain the essential oils. Distillate (aqueous phase) was extracted with
dichloromethane (DCM). The organic phase was dried over anhydrous sodium
sulfate, filtered and the solvent was separated from the oil using the rotary evaporator
and preserved in sealed dark vials at 4 °C until further analysis. Three exotic essential
oils (T. vulgaris, O .europea and M. alternifolia) were procured from the commercial
suppliers.
TABLE 3.2
List of Essential Oils used in the study S.
NO
PLANTS COMMOM NAME
FAMILY PLANT PARTS USED
1. Azadirachta indica Neem Meliaceae Leaves
2. Cinnamomum
cecidodaphne
Sugandh kokila Lauraceae Fruits
3. Cinnamomum
zeylanicum
Cinnamon Lauraceae Bark
4. Coriandrum sativum Coriander Apiaceae Seeds
5. Cyperus scariosus Nagarmotha Cyperaceae Roots
6. Eucalyptus globules Eucalyptus Myrtaceae Leaves
7. Foeniculum vulgare Fennel Apiaceae Seeds
8. Melaleuca alternifolia Tea tree Myrtaceae Leaves
9. Nardostachys jatamansi Jatamansi Valerianaceae Roots
10. Olea europea Olive Oleaceae Fruits
11. Syzygium aromaticum Clove Myrtaceae Flower buds
12. Thymus vulgaris Thyme Labiatae Flowering tips and
Leaves
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13. Valerian officinalis Valerian Valerianaceae Roots
14. Zanthoxylum rhetsa Teppal Rutaceae Leaves
3.1.5 Selection of Bacterial strains
Nine epidermal infection causing bacteria were procured from Microbial type
culture collection (MTCC), Institute of microbial technology (IMTECH), Chandigarh
and National Dairy Research Institute (NDRI), Karnal. All bacterial cultures were
maintained in Nutrient agar slants except K. pneumoniae which was maintained in
Luria Bertani slants. All strains were stored at 4oC and periodically sub cultured. Five
Gram positive and four Gram negative bacterial strains used in this investigation are
given below in Table 3.3.
TABLE 3.3 List of Bacteria used in the Study
BACTERIAL STRAINS S. No.
Gram-positive Bacteria
MTCC/NCDC
CODE
GROWTH
MEDIUM
1. Staphylococcus epidermidis 435 NA
2. Staphylococcus epidermidis 3086 NA
3. Staphylococcus aureus 109 NA
4. Staphylococcus aureus 3160 NA
5. Staphylococcos hominis 4435 NA
Gram-negative Bacteria
6. Klebsiella pneumoniae 4030 LB
7. Proteus vulgaris 426 NA
8. Pseudomonas aeruginosa 424 NA
9. Pseudomonas aeruginosa 7453 NA NA: Nutrient Agar, LB: Luria Bertani.
3.1.6 Preparation of Mc Farland standard: Mc Farland standard is used to
standardize the approximate number of cells in a liquid suspension by comparing the
turbidity of the test organism suspension. It is a chemical mixture of 0.5 ml of 0.048
M BaCl2 (1.17% w/v BaCl2.. H2O) to 99.5 ml of 0.18 M H2SO4 (1% v/v), with
constant stirring, resulting in production of a fine precipitate of barium sulphate, the
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turbidity is visually comparable to a bacterial suspension of approximately 1.5 × 106
CFU/ml. The standard solution was stored in dark at room temperature and prepared
fresh after six months (Andrews, 2001).
3.1.7 Agar well diffusion assay: Antibacterial activity of all plant extracts and
essential oils was determined by agar well diffusion method. One hundred microlitre
(100μl) of the inoculum of tested organism (1.5 × 106 CFU /ml) was poured into semi
hot condition medium. The plates were allowed to solidify and used. The medium
seeded with organisms, were bored (8mm) at equidistant using sterile borer and 100
μl of the different sample preparations were added to respective wells. The plates
were allowed to stand for 1h at room temperature for diffusion of the extract and
essential oils into agar and incubated at 37°C for 24h (Okeke et al., 2001). Sterile
DMSO (10%) served as the negative control and ciprofloxacin served as the positive
control. The antimicrobial activity, indicated by an inhibition zone surrounding the
well containing the extract, was recorded if the zone was greater than 8mm (Aneja et
al., 2010). The experiments were performed in triplicates and the mean values of the
diameter of inhibition zones ± standard deviations were calculated.
3.1.8 Minimum Inhibitory Concentration: Minimum Inhibitory Concentration
(MIC) of the extracts that showed antibacterial activity was determined by
microdilution technique as described by the National Committee for Clinical
Laboratories standards (2000). The bacteria inoculums were prepared in 5 ml nutrient
broth and incubated at 370C. The final inoculums were of approximately 1.5 ×
106 CFU/ml. Controls with 0.5 ml of culture medium without the extract samples and
other without microorganisms were used in the tests. Tubes were incubated at 370C
for 24 h. The activity was measured as a function of turbidity at 660 nm. Lack of
turbidity was further confirmed by pouring suspension aliquot of 0.1 ml into pre-
sterilized Petri dishes with nutrient agar medium. The tests were conducted in
triplicate.
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3.2 Results and Discussion 3.2.1 Percent Extract Yield: Medicinal plants are the richest bio-resource of drugs.
They are the backbone of traditional systems of medicine, modern medicine,
nutraceuticals, food supplements, folk medicine, pharmaceutical intermediates and
chemical entities for synthetic drugs (Ncube et al., 2008).
Plant materials can be used in fresh or dried form for the extraction of
secondary plant components. But as many plants are used in the dry form by
traditional healers and due to differences in water content within different plant
tissues plants are usually air dried to a constant weight before the extraction.
Three solvents water, methanol and cow urine were used for the preparation of
extracts. After extraction, the extracted plant material was weighed to determine the
percentage yield of each extract. Percent yield of plant extracts varied from 15.7% to
41.4%. The aqueous extracts gave the highest yield percentage followed by cow urine
and methanol. Highest percent yield was given by aqueous extract of T. chebula
(41.4%) whereas lowest percent yield was given by methanol extract of M. fragrans
(15.7%) (Table 3.4).Whereas for essential oils percent yield varied from 0.76% to
1.34%. Maximum percent yield was obtained for E. globulus. The percent yield of
various essential oils is shown in Table 3.5. In the present investigation, the variation
of the extract yields among different solvents and plant materials may be associated to
the different chemical nature of the compounds present in these materials, as well as
the polarity of the extraction solvents. The yields of extractable components, in
addition to their chemical nature, are also strongly influenced by the concentration,
polarity and nature of the extraction solvent, as well as the extraction technique
employed. Therefore, an appropriate extraction system has to be employed to recover
optimum contents of extractable antioxidant components. Typically, high polarity
solvents such as methanol and ethanol are widely used to extract plants phenolic
antioxidant components due to their compatibility and efficacy towards solubilization
of such compounds (Shabbir et al., 2011).
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TABLE 3.4
Percent Extract Yield from Different
Plants in Three Solvents
Percent Yield (w/w) of material extracted
from 10g Plant material.
S.No Plant
Water Methanol Cow urine
1. Amomum subulatum 37.3 26.7 34.2
2. Azadirachta indica 28.4 23.4 25.5
3. Calotropis procera 34.9 27.8 35.6
4. Capsicum annum 32.6 22.4 28.9
5. Cassia fistula 30.0 24.8 28.9
6. Catharanthus roseus 28.3 22.4 24.5
7. Cinnamomum zeylanicum 39.8 28.7 37.6
8. Coriandrum sativun 25.6 19.7 21.2
9. Cuminum cyminum 35.4 26.5 32.2
10. Curcuma longa 33.4 25.6 30.0
11. Diospyros melanoxylon 28.7 23.4 25.5
12. Elettaria cardamomum 35.8 28.7 34.4
13. Lawsonia inermis 29.9 21.2 22.3
14. Murraya koenigii 24.3 18.7 20.9
15. Myristica fragrans 22.3 15.7 19.4
16. Nicotiana tobaccum 33.3 25.6 28.7
17. Piper nigrum 26.3 19.4 22.6
18. Psidium guajava 37.4 24.3 33.2
19. Rosa indica 28.3 21.4 20.6
20. Syzygium aromaticum 31.0 23.3 26.9
21. Tagetes erecta 37.4 27.5 32.5
22. Tectona grandis 23.5 16.3 20.5
23. Terminalia chebula 41.4 34.4 38.2
24. Trigonella foenum graecum 32.4 25.5 29.7
25. Zingiber officinale 38.7 27.8 33.5
46
TABLE 3.5
Percent Yield of Essential Oils from Different Plants.
3.2.2 Antibacterial Assay
Plant Extracts
The results of the antibacterial assay of plant extracts are shown in Table 3.6.
Out of the tested 75 extracts, all the three extracts of T. chebula showed antibacterial
activity against all the tested bacteria. Maximum diameter of zone of inhibition (36
mm) was reported for the methanol extract against S. aureus (3160). Our findings
were in accordance with Malckzadeh et al., 2001, Kim et al., 2006, Chatotopadhyay
et al., 2007, Bag et al., 2009 reports.
Among other samples, methanol extract of S. aromaticum and P. guajava also
showed inhibitory activity against all nine bacteria. For P. guajava the diameter of
S.No Plants Percent yield
(w/w)
1. Azadirachta indica 0.78
2. Cinnamomum cecidodaphne 0.63
3. Cinnamomum zeylanicum 1.68
4. Coriandrum sativum 1.13
5. Cyperus scariosus 0.81
6 Eucalyptus globules 1.34
7. Foeniculum vulgare 0.91
8. Melaleuca alternifolia -
9. Nardostachys jatamansi 0.87
10. Olea europea -
11. Syzygium aromaticum 0.91
12. Thymus vulgaris -
13. Valerian officinalis 0.76
14. Zanthoxylum rhetsa 0.87
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inhibition zones ranged from 14 mm to 22 mm. The inhibitory effects of aqueous and
alcoholic extracts of P. guajava on the growth of different bacteria have also been
reported by Gutierrez et al., 2009.
The zones of inhibition of S. aromaticum extracts ranged from 15.6 mm to 25
mm. Our results were in agreement to Sulieman et al., (2007) who reported the
inhibition of growth of pathogenic bacteria by S. aromaticum. All three extracts of R.
indica showed good antibacterial activity against all the bacteria except S. hominis
(4435). Mishra et al., 2001 also reported bactericidal effects of R. indica extracts on
pathogenic organisms. Extracts of C. fistula, C. cyminum, L. inermis also showed
potent antibacterial activities against most of the tested bacteria.
Minimum Inhibitory concentration (MICs) is considered as the gold
standard for determining the susceptibility of organism to antimicrobials (Andrews,
2001). The MIC for various extracts ranged from 0.93 mg/ml to 30 mg/ml. Lowest
MIC was reported for methanol extract of T. chebula against the two strains of S.
aureus, thereby confirming it as the best antibacterial agent of the present study. Our
studies have also confirmed that MIC values did not exhibit substantial variations
when compared to the trend of inhibition shown with the well diffusion method and
large inhibition zones correlated with lower MIC.
The present work summaries (Fig.3.2), methanol extracts exhibited 43.11%
(97/225) antibacterial activity. The most sensitive bacteria against various methanol
extracts were S. epidermidis (435), S. aureus (3160) and S. hominis i.e. 13.4% (13/97)
each. The most resistant bacteria observed against methanol samples was K.
pneumoniae i.e. 6.18% (6/97).
While water extracts possessed only 28.88% (65/225) antibacterial activity.
Among all bacterial strains. P. vulgaris was most sensitive bacteria. For water extracts
15.38% (10/65) samples exhibited activity against P. vulgaris while the most resistant
strain was K. pneumoniae [3.07%, (2/65)]. Our studies are in accordance to earlier
reports, where organic extracts exhibit more activity than water extracts.
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Although traditional healers use primarily water but plant extracts from
organic solvents have been found to give more consistent activity compared to water
extract (Parekh et al., 2006). Also water soluble flavonoids have no antimicrobial
significance and water soluble phenolics only important as antioxidant compounds
(Das et al., 2010). The higher activity of alcoholic extracts as compared to the
aqueous extracts can be attributed to the presence of higher amounts of polyphenols
as compared to aqueous extracts. All the identified components from plants
possessing activity against microorganisms are aromatic or saturated organic
compounds, they are most often obtained through initial ethanol or methanol
extraction (Cowan, 1999).
The least antibacterial prevalence was exhibited by cow urine [22.22%
(50/225)]. P. vulgaris was most sensitive bacteria for cow urine extract i.e. 18%
(9/50) of the respective samples. Like methanol and water extracts K. pneumoniae
was found most resistant for cow urine samples also i.e. 2.00% (1/50). Cow urine
extracts of only T. chebula possessed activity against all the nine strains studied. The
sensitivity of P. vulgaris against cow urine extracts is supported by Ahuja et al., 2012.
Our findings are supporting the sensitivity of S. aureus against cow urine extracts
(Upadhyay et al., 2010). As per Ayurvedic literature cow urine possess many
medicinal properties and is used in curing number of diseases like skin diseases,
kidney problems, epilepsy, anemia, constipation, respiratory diseases etc (Chauhan et
al., 2001 and Krishanamurthi et al., 2004). Due to its therapeutic values majority of
rural population in India use cow urine as a folklore remedy to get rid of various
diseases. But our findings do not support a good success rate. The reason of low
success rate may be the fact that distilled cow urine possesses low activity as
compared to fresh cow urine. The fresh cow urine is most acidic in nature, supporting
the claim of traditional practitioner.
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Table 3.6 Diameter of Zones of Inhibition (mm) and Minimum Inhibitory Concentration (mg/ml) of Plant Extracts Amomum subulatum
Solvent extracts (mg/ml)
SE (435) SE (3086)
SE (109)
SE (3160)
SH (4435)
KP (4030)
PV (426)
PA (424)
PA (7453)
Aqueous - - - - - - - - -
Methanol - - 14.6±0.57 (15)
16.6±0.57 (15)
- - - - -
Cowurine - - - - - - - -
Azadirachta indica
Aqueous - - - - - - - - -
Methanol 13.3±0.57 (15)
13.3±1.1 (30)
12±0 (30)
- - - -
Cowurine - - - - - - - -
Calotropis procera
Aqueous - - 11.6±0.57 (30)
09±1 (30)
- - 18±1 (15)
11.6±0.57 (30)
14.6±0.57 (15)
Methanol - - - - - - 10.6±1.15 (30)
- -
Cowurine - - 12±0 (30)
- - - 20±1 (30)
10.6±0.57 (30)
10.3±0.57 (15)
50
Capsicum annum
Solvent
extracts
(mg/ml)
Se
(435)
Se
(3086)
Sa
(109)
Sa
(3160)
Sh
(4435)
Kp (4030) Pv
(426)
Pa
(424)
Pa
(7453)
Aqueous - - - - - - - - -
Methanol 14.6±0.57 (15)
16±1 (7.5)
- - 17.8±0.63 (7.5)
- - - -
Cowurine - - - - - - - - -
Cassia fistula
Aqueous 11.3±0.57
(30)
- - - 10.3±0.57
(30)
- 13±1
(30)
12.6±0.57
(30)
15.6±0.57
(15)
Methanol 16±1
(15)
12.3±0.57
(30) - 14±1
(15)
12±1
(30) - 12.3±0.57
(30)
15.3±0.57
(30)
16±1
(7.5)
Cowurine 12.3±0.57
(30)
- - 11.6±0.57
(30)
11±0
(30)
- 15.3±0.57
(30)
- 14.6±0.57
(15)
Catharanthus roseus
Aqueous - - 23±1
(30)
- - - 10.6±0.57
(30)
- 13.6±0.57
(30)
Methanol - 21.3±0.57
(30)
- 19±1
(15)
- 11±0
(30)
- 11±0
(30)
Cowurine - - - - - - 10.3±0.57
(30)
- -
51
Cinnamomum zeylanicum
Solvent
extracts
(mg/ml)
SE (435) SE (3086) SA(109) SA (3160) SH (4435) KP (4030) PV (426) PA (424) PA (7453)
Aqueous - - - - - - - - -
Methanol 14.3±0.57
(15)
15.6±0.57
(15) 19±1
(7.5)
11.33±1.15
(30)
- - - - -
Cowurine - - - - - - - - -
Coriandrum sativum
Aqueous - - - - 15±0
(30)
- - - -
Methanol - - - - 17.3±0.57
(15)
- - - -
Cowurine - - - - 14.6±0.57
(30)
- - - -
Cuminum cyminum
Aqueous 23.6±0.57
(7.5)
- 18±1
(15)
14.3±0.57
(15)
15.3±0.57
(15)
- - 13.6±0.57
(30)
-
Methanol 18±0
(3.75)
- 15±1
(7.5)
16.6±0.57
(7.5)
18.6±0.57
(15)
- - 20.6±0.57
(7.5)
13±0
(30)
Cowurine - - 18.66±1.1
(15)
13±1
(15)
14.6±0.57
(15)
- - 17.3±0.57
(15)
-
52
Curcuma longa
Solvent
extracts
(mg/ml)
SE(435) SE(3086) SA(109) SA (3160) SH (4435) KP (4030) PV (426) PA (424) PA
(7453)
Aqueous - - - - - - 10.6±0.57
(30)
- -
Methanol - - - - - - 12.3±0.57
(30)
- -
Cowurine - - - - - - 15.3±0.57
(30)
- -
Diospyros melanoxylon
Aqueous 11±0
(30)
- - - - - 10±1
(30)
- -
Methanol 14±1
(15)
- - 10±0
(30)
- - 11±0
(30)
- -
Cowurine - - - - - - - - -
Eletarria cardamomum
Aqueous 19.5±1.25
(7.5)
- 19±1
(7.5)
- - - - - -
Methanol - - - 13±1
(30)
- - - - -
Cowurine 19.6±0.57
(7.5)
- 23±1
(3.75)
- - - - - -
53
Lawsonia inermis
Solvent
extracts
(mg/ml)
SE (435) SE (3086) SA (109) SA(3160) SH (4435) KP (4030) PV (426) PA (424) PA
(7453)
Aqueous 14±1
(7.5)
12.6±0.57
(30)
15.6±0.57
(7.5)
10.6±0.57
(30)
- - 16±1
(15)
11±0
(30)
9.6±0.57
(30)
Methanol - 15±1
(3.75)
10.3±0.57
(30)
12.3±0.57
(30)
- - 15.3±0.57
(7.5)
11.6±0.57
(30)
11.6±0.57
(30)
Cowurine 16.6±0.57
(15)
- 14±1
(15)
09.6±1.52
(30)
- - 10.6±0.57
(30)
10±1
(30)
9.6±0.57
(30)
Murraya koenigii
Aqueous - - - - - - - - -
Methanol 14.6±0.57
(15)
- 22.3±0.57
(3.75)
23.3±0.57
(3.75)
- - - - -
Cowurine - - - - - - - - -
Myristica fragrans
Aqueous - - - - - - - - -
Methanol - - - - - - - - -
Cowurine - - - - - - - - -
Nicotiana tobaccum
Aqueous - - - - 10.3±0.57
(30)
- - -
Methanol - - - - 11.3±0.57
(30)
- - - -
Cowurine - - - - 10.3±0.57
(30)
- - - -
54
Piper nigrum
Solvent
extracts
(mg/ml)
SE(435) SE (3086) SA(109) SA (3160) SH
(4435)
KP (4030) PV (426) PA (424) PA (7453)
Aqueous - - - - - - - - -
Methanol 19±1
(3.75)
18.33±0.57
(7.5) - - - 16.3±0.57
(15) 25±1
(3.75)
- 16.6±0.57
(7.5)
Cowurine - - - - - - - - -
Psidium guajava
Aqueous 16.3±0.57
(15)
21.33±0.57
(7.5) 17±1
(30)
18.33±0.57
(15)
- 12.3±0.57
(30)
18±0.57
(15)
Methanol 18±1
(7.5)
22±0
(3.75)
20.33±0.57
(15)
16±1
(7.5)
14±1
(15)
22±0
(7.5)
22±1
(3.75)
14.6±0.57
(15)
20±1
(7.5)
Cowurine - - - - - - - 09.6±0.57
(30)
14±0.57
(15)
Rosa indica
Aqueous 11±0
(30)
10.33±0.57
(30) 28±1
(1.87)
- - 13.33±0.57
(30) 20.33±0.57
(15)
13.66±0.57
(30)
20.66±0.57
(3.75)
Methanol 12±1
(30)
13±1
(15)
26.66±0.57
(1.87) 10.33±0.57
(30)
- 14±0
(15)
20±0
(15)
20.66±0.57
(15)
19.66±0.57
(3.75)
Cowurine 11±1
(30)
09.66±0.57
(30) 15±1
(3.75)
- - - 14.33±0.57
(30)
17.33±0.57
(30)
14.33±0.57
(15)
55
Syzygium aromaticum
Solvent
extracts
(mg/ml)
SE (435) SE (3086) SE(109) SE (3160) SH(4435) KP(4030) PV (426) PA (424) PA
(7453)
Aqueous - 13±1
(15)
18.5±1.25
(7.5)
- 18±1
(15)
- 12.3±0.57
(30)
21.3±1.1
(7.5)
19±0
(15)
Methanol 16.3±0.57
(1.87)
19±1
(7.5)
20±1
(3.75)
15.6±0.57
(15)
15.6±0.57
(15)
20.3±0.57
(7.5)
25±1
(3.75)
22.3±0.57
(3.75)
20.6±0.57
(7.5)
Cowurine - - 13.3±0.57
(30)
- 14±1
(15)
- 10.3±1.1
(30)
17.6±0.57
(30)
16.6±0.57
(15)
Tagetes erecta
Aqueous - - - - 10.33±0.57
(30)
10±0
(30)
- -
Methanol - - - - 11±0
(30)
10.66±0.57
(30)
11.33±0.57
(30)
25±1
(3.75)
12±1
(30)
Cowurine - - - - 09.66±0.57 (30) - 10±1
(30)
- -
Tectona grandis
Aqueous - - - - 10.3±0.57
(30)
- - - -
Methanol - - - - 11±0
(30)
- - - -
Cowurine - - - - 10±1
(30)
- - - -
56
Terminalia chebula Solvent extracts (mg/ml)
Se (435) Se (3086) Sa (109) Sa (3160) Sh (4435) Kp (4030) Pv (426) Pa (424) Pa (7453)
Aqueous 21±1 (3.75)
27±1 (3.75)
33±1 (1.87)
33±1 (1.87)
17±1 (7.5)
10.6±0.57 (30)
27±1 (3.75)
25±1 (3.75)
27.6±0.57 (1.87)
Methanol 22.3±0.57 (1.87)
26±1 (3.75)
35±0 (0.93)
36±1 (0.93)
23±1 (3.75)
19±1 (7.5)
26±1 (3.75)
29.3±0.57 (3.75)
25.6±0.57 (0.93)
Cowurine 18.3±0.57 (3.75)
27.3±0.57 (3.75)
31±1 (1.87)
30±0 (1.87)
14.6±0.57 (7.5)
11±1 (30)
26±1 (3.75)
24.6±0.57 (7.5)
29±1 (1.87)
Trigonella foenum graecum Aqueous - - - - - - - - -
Methanol - - 16.6±0.57 (7.5)
17±1 (7.5)
14.6±0.57 (15)
- - - -
Cowurine - - - - - - - - -
Zingiber officinale
Aqueous 14.3±0.57 (30)
- - 19±1 (15)
- - - - -
Methanol 15±1 (30)
- - - - - - - -
Cowurine
- - - - - - - -
SE (109): S. epidermidis (109), S.E (3086); S.epidermidis (3086), SA (109): S. aureus (109), SA (3160); S. aureus (3160), SH (4435): S. hominis (4435), KP (4030): K. pneumoniae (4030), PV (426): proteus vulgaris (426), PA (424): P. aeruginosa (424), PA (7453): P. aeruginosa (7453).
57
Fig 3.2 Antibacterial activity percentage of various extracts
Essential oils
The results of antibacterial assay of essential oils are shown in Table 3.7.
Among 14 essential oils studied 71.4% (10/14) exhibited 100% resistance against
studied strains. Most of the essential oils showed very good antibacterial activities
against the tested bacteria with zones of inhibition ranging between 10.6 mm to 46
mm and MIC ranging between 0.3 µl/ml to 100µl/ml. Most potent essential oils were
T. vulgaris, M. alternifolia, S. aromaticum, C. zeylanicum, E. globulus and V.
officinalis. Maximum zone of inhibition (46 mm) was reported for V. officinalis
against P. aeruginosa (7453) followed by T. vulgaris (45.6 mm) against S. hominis.
Good antibacterial activities were shown by A. indica, C. cecidodaphne, C. sativum,
C. scariosus, N. jatamansi and Z. rhetsa. Little activity was shown by F. vulgare
against the two strains of S. epidermidis where as no activity was shown by O.
europeae.
Our finding were in accordance to Penalver et al., 2005 who proposed thyme
essential oil can inhibit pathogenic bacterial strains such as E. coli, Salmonella
enteritidis, Salmonella choleraesuis and Salmonella typhimurium with the inhibition
directly correlated to the phenolic components carvacrol and thymol.
58
Tea tree oil also showed efficient antibacterial potential with zones of
inhibition ranging from 21.6 mm to 35 mm. This validates the use of tea tree oil
against dematological infections. Reports suggest that repeated use of formulations
containing tea tree essential oil (TTO) does not lead to dermatological problems, nor
affect the original protective bacterial flora of the skin (Carson and Riley, 1995).
Clove oil exhibited activity against all the bacteria in the range of 17.3 mm to
25.3 mm. According to Trongtokit et al., 2005, the essential oil extracted from the
dried flower buds of clove is used for acne, warts, scars and parasites. The clove oil is
also used as a topical application to relieve pain and to promote healing and also finds
use in the fragrance and flavouring industries (Chaieb et al., 2007).
Maximum inhibitory activity was reported by essential oil of valerian against
P. aeruginosa (7453) having a zone of inhibition of 46 mm, the findings are supported
by Wang et al., 2010 who reported broad spectrum antibacterial and antifungal
activities of valerian. Similarly Eucalyptus oil also possessed antibacterial activities
against all bacteria with zones of inhibition ranging between 25.3 mm to 35.3 mm.
According to Vratnica et al., 2011, Eucalptus oil exhibited good inhibitory activity
against food poisoning, spoilage and human pathogens. The present study reports
92.8% (13/14) of E.Os shows activity against gram positive, while only 85.7% were
active against gram negative. This is in accordance to earlier findings by Nevas et al.,
2004.
The major mode of infection transmission in hospital acquired infections is
thought to be through hand carrying of pathogens from staff to patient, and from
patient to patient (Boyce and Pittet, 2002), and a relationship between hand hygiene
and reduced transmission of infections has been reported (Reybrouck, 1986). Most
antiseptic agents can damage the skin, leading to a change in microbial flora, and an
increased shedding of the original protective bacterial flora of the hand leads to an
increased risk of transmission of pathogenic microorganisms (Larson, 2001). Our
present study is an effort to make validation of essential oils efficacy as well as to
search any new oils possessing efficient activity against epidermal infections. The
promising results illustrated here may promote further investigations in this area.
59
Table 3.7 Diameter of Zones of Inhibition (mm) and Minimum Inhibitory Concentration (µl/ml) of Essential Oils
S.
No.
Essential oils S. E
(435)
S. E
(3086)
S. A
(109
S. A
(3160)
S. H
(4435)
K.P
(4030)
P.V
(426)
P.A
(424)
P.A
(7453)
1. Azadirachta indica 14.33±0.57
(25)
11±0
(50)
16.66±0.57
(12.5)
17.33±0.57
(12.5)
13.66±0.57
(12.5)
- - 14±1
(25)
16.33±0.57
(25)
2. Cinnamomum
cecidodaphne
13.66±0.57
(50)
12.66±0.57
(50)
18.33±0.57
(12.5)
15.33±0.57
(100)
12.33±1.1
(50)
14±1
(50)
14.66±0.57
(50)
10.33±0.57
(50)
10.66±0.57
(50)
3. Cinnamomum
zeylanicum
34±0.57
(0.7)
31±1
(1.5)
39±1
(0.7)
21.66±0.57
(0.7)
22±0
(1.5)
25.33±0.57
(1.5)
29.33±0.57
(1.5)
31±1
(1.5)
34±1
(1.5)
4. Coriandrum sativum 19±1
(12.5)
19±1
(12.5)
15.66±0.57
(12.5)
11.33±0.57
(100)
17.66±0.57
(6.2)
14.33±0.57
(50)
20.33±1.52
(12.5)
15±1
(25)
16±0
(12.5)
5. Cyperus scariosus 12±1
(50)
11.33±1.1
(50)
19±1
(12.5)
12.66±0.57
(100)
- 19.33±0.57
(50)
18.33±0.57
(50)
23.66±0.57
(50)
14.33±0.57
(50)
6. Eucalyptus globules 31±1
(0.7)
32±1
(1.5)
35.33±0.57
(0.7)
31±0
(0.7)
24.33±0.57
(3.1)
25.33±0.57
(3.1)
25.33±0.57
(3.1)
29±0
(3.1)
26±0
(1.5)
7. Foeniculum vulgare 11±0
(50)
9.66±0.57
(50)
- - - - - - -
8. Melaleuca
alternifolia
25.66±0.57
(0.7)
24.66±0.57
(1.5)
35±0
(0.7)
31±1
(6.2)
21±1
(3.1)
23±0
(3.1)
23±1
(0.7)
21.66±0.57
(1.5)
25.33±0.57
(1.5)
9. Nardostachys
jatamansi
16.33±0.57
(50)
18.66±0.57
(50)
24±1
(6.2)
13±0
(50)
12±1
(50)
19.33±0.57
(25)
17.66±0.57
(25)
14.66±1.15
(50)
19.66±(50)
10. Olea europea - - - - - - - - -
11. Syzygium 24±1 23±1 25.33±0.57 17.33±0.57 21.33±0.57 21±1 20±1 18±1 20±1
60
aromaticum (1.5) (1.5) (3.1) (1.5) (1.5) (1.5) (1.5) (1.5) (3.1)
12. Thymus vulgaris 43.33±1.52
(0.7)
45±1
(1.5)
43±1
(0.3)
35±0
(0.3)
45.66±0.57
(0.7)
43±1
(0.3)
26±1
(0.7)
27.33±0.57
(1.5)
28±1
(1.5)
13. Valerian officinalis 33±1
(6.2)
42±0
(0.7)
43.66±1.52
(0.3)
31±1
(0.7)
40±0
(0.7)
36±1
(0.3)
31±1
(1.5)
39.66±0.57
(0.7)
46±1
(0.7)
14. Zanthoxylum rhetsa 14±1
(50)
18±1
(12.5)
19±1
(12.5)
14.66±0.57
(50)
13±1
(50)
13.33±0.57
(50)
20.66±0.57
(12.5)
15±0
(50)
19.33±0.57
SE (109): S. epidermidis (109), S.E (3086); S.epidermidis (3086), SA (109): S. aureus (109), SA (3160); S. aureus (3160), SH (4435): S. hominis (4435), KP
(4030): K. pneumoniae (4030), PV (426): p. vulgaris (426), PA (424): P. aeruginosa (424), PA (7453): P. aeruginosa (7453).
61
3.3 Conclusions
Of the 75 plant extracts screened for antibacterial activity, three extracts of T.
chebula exhibited best antibacterial activity against all nine bacterial strains.
Lowest MIC was also reported for its methanol extract thereby considering it
as the most potent antibacterial agent of the present study.
P. guajava, S. aromaticum and R. indica extracts also possessed broad
spectrum antibacterial activities.
Of the 14 essential oils 10 possessed antibacterial activities against all the
bacteria. Best activities were shown by T. vulgaris, M. alternifolia, S.
aromaticum, C. zeylanicum, E. globulus and V. officinalis.
Although P. vulgaris was sensitive bacteria against all three types of extracts
along with S. epidermidis, S. aureus and S. hominis in case of methanol extract
only. But on parameters of zones of inhibition gram-positive bacteria were
more sensitive to plant extracts and essential oils than gram-negative bacteria.
These observations are likely to be the result of the differences in cell wall
structure between gram-positive and gram-negative bacteria, with gram-
negative outer membrane acting as a barrier to many environmental
substances.
62
A B
C D A: Zone of inhibition of T. chebula against S. aureus (3160) B: Zone of inhibition of S. aromticum against S. hominis C: Zone of inhibition of P. guajava against P. vulgaris D: Zone of inhibition of R. indica against S. aureus (3160) Aq: Aqueous, Me: Methanol, Cu: Cow urine, C: Control, Ab: Antibiotic.
63
A
B C
D E A: Zone of inhibition of C. zeylanicum against S. epidermidis
B: Zone of inhibition of E. globulus against S. aureus
C: Zone of inhibition of S. aromaticum against S. aureus
D: Zone of inhibition of V. officinalis against S. hominis
E: Zone of inhibition of T. vulgaris against K. pneumoniae