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CERTIFICATE OF ORIGIN

This is to certify that Mr. Bharat Singh Chauhan, student of B-Tech (Biotech), has
undergone a training programme at Amity Institute of Biotechnology, Amity
University, Noida, Uttar Pradesh.

The project report and data submitted by him, is authentic and genuine to my
knowledge. This Summer Internship report has the requisite standard for the partial
fulfilment for the award of Degree B-Tech (Biotech). To the best of my knowledge
no part of this report has been reproduced from any other report and the contents are
based on original research.

Signature

DECLARATION

I hereby declare that the present work on Analysis of Phytoconstituents and


Antimicrobial Activity of Essential oil against selected Bacterial and fungal
Pathogenic Isolates is a record of original work done by me under guidance of

Dr.

Praveen Dahiya during 1st March 2013 to 23rd May 2013, at Amity Institute of
biotechnology and is submitted in partial fulfilment of the requirements for the award
of the degree in B.Tech (Biotechnology).

I also declare that no part of this project has previously been submitted to any
university or any examining body for acquiring any diploma or degree.

DATE: 30-05-2013
PLACE: NOIDA

ACKNOWLEDGEMENT

This project report itself is an acknowledgement to the intensity drive and technical
competence of many individual who have contributed to it
I am very much indebted to Prof. K. C. Upadhyaya, Amity Institute of Biotechnology
Amity University, Noida for his continued guidance and invaluable encouragement
throughout the project. I am very much thankful to Dr. P.K. Paul, Dy. Director &
Head, AIB for providing me this opportunity to increase my knowledge in
Biotechnology.
I bestow my heartful sense of gratitude to Dr. Praveen Dahiya, faculty Guide for
giving me an opportunity to work on this project and for excellent guidance and
tremendous support. Without her encouragement and help this project would not have
materialized.
I would also like to thank my colleagues and friends for their constant support and
help in the successful completion of my project.
I am grateful to my teachers and to all those people who provided me the best
information directly or indirectly throughout my project work. I owe my soulful
thanks to my parents and God for their blessings.

ABSTRACT

The three essential oils taken are labelled as BS-01, BS-02 and BS-03. BS-01 belongs
to plant family Pinaceae. BS-02 belongs to Melaleuca and BS-03 belongs to poaceae
family. In the present investigation the oils has been evaluated for phytochemical
constituents, sensory evaluation, Antibacterial activity, total phenolics, chemical
characterisation and TLC bio autography assay. The essential oil BS-01 contains few
phytochemicals

including

reducing

sugars,

cardiac

glycosides.

However,

anthraquinone, flavanoids, tannins and phlobatanins were not observed. Similar results
were observed for BS-02 and BS-03 where phytochemical screening reveals major
constituents present in the essential oils.

Antibacterial activity of the oils was assessed on bacterial isolates. Antifungal activity
of the oil was also assessed on fungal isolates of Aspergillus niger. Thin layer
chromatography and bio autography assay demonstrated well defined growth
inhibition zones against clinical isolates. This established a good support to the use of
this Essential oil in herbal medicine and a base for the development of novel potent
drugs and phytomedicine.

Contents

Page No.

Introduction

Review Literature

Materials and Methods

13

Results and Discussion

21

Summary and conclusion

32

References

34

TABLE OF CONTENTS

INTRODUCTION

The spread of drug resistance pathogen is one of the most serious threats to successful
treatment of microbial diseases. Down the ages, essential oils and other extracts of
plants have evoked interest as sources of natural products. They have been screened
for their potential uses as alternative remedies for the treatment of many infectious
diseases (Bora et al.,, 2000). World health organisation noted that majority of the
worlds population depends on traditional medicine for primary healthcare. Medicinal
and aromatic plants which are widely used as medicine and constitute a major source
of natural organic compounds (Avato et al.,., 2006).

Essential oils (also called as volatile oils) are aromatic oily liquids obtained from plant
materials. They are naturally synthesized by plants for different reasons according to
their needs. Some synthesize it to help fend off parasites and insects, some to attract
pollinators and some are simply vital for the physiology of the plant. This is why they
are called Essential- because they carry the very essence and undoubtly the most
important part of plant. They can be obtained by expression, fermentation or
extraction but the method of steam distillation is most commonly used for commercial
production. An estimated 3000 essential oil are known, of which 300 are
commercially important in fragrance market (Kowalska et al., 2007).

Essential oils have been shown to possess Antibacterial, antifungal, antiviral


insecticidal and antioxidant properties (Burt et al.,, 2004, kordali et al.,, 2005). Some
oils have been used in cancer treatment (Sylvestre et al.,, 2006). Some oils have been
used as muscle relaxant, emollient, soothing agent, stimulant. Some other oils have
been used in food preservation, aroma therapy (Buttner et al.,, 1996) and fragrance
industry. Technically essential oils are not true oil as they contain no lipid content but
are instead made up of a variety of complex, volatile compounds so they are rich
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source of biologically active compounds. There has been a increased interest in


looking at antimicrobial properties of extracts from aromatic plants particularly
essential oils. Therefore, it is reasonable to expect a variety of plants compounds in
these oils with specific as well as general antimicrobial activity (Neeta et al.,, 2007)
and antibiotic potential.
For this project we have picked three essential oils. The essential oil is obtained from
steam distillation of plant parts. Although, these oils have been reported for their
medicinal activity, however very less scientific evidence supports their potential.
Hence, to explore the medicinal potential of

each oil we determined their

Antibacterial activities and phytochemical constituents individually using various


experiments.
In search of broad spectrum Antibacterial activity from essential oils, we performed
agar well diffusion test on the selected oil. Nowadays, multiple drug resistance has
developed due to the indiscriminate use of commercial antimicrobial drugs commonly
used in the treatment of infectious disease. In addition to this problem antibiotics are
sometimes associated with adverse effects on the hosts including hypersensitivity,
immune suppression and allergic reactions. This situation forced scientists to search
for new antimicrobial substances. Given the alarming incidence of anti biotic
resistance in bacteria of medical importance, there is a constant need for new and
effective therapeutic agents. Therefore, there is a need to develop alternative
antimicrobial drugs for the treatment of infectious diseases from essential oils.

The objectives for the study includes:


1.
2.
3.
4.

Sensory evaluation and phytochemical screening


Chemical characterisation and total phenolic content of oil
Antibacterial and antifungal activity
Thin layer chromatography and bioautography

REVIEW LITERATURE
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BS-01
Family: Pinaceae

Fig 1: BS-01
BS-01 belonging to Pinaceae family is the most widespread conifer tree in the
Romanian forests. It can be used to avoid the erosion of the soil, and is of great
economical importance, as construction wood, resonance material for musical
instruments, paper manufacturing, and even in phytotherapy (some parts as shoots and
needles). The content and the chemical composition of volatile oil isolated from
different species of the family Pinaceae depends on the geographic origin the type of
the plant material (needles, twigs, cones), the isolation and determination techniques
used for analysis. The volatile oil quality is also strongly affected by soil and air
pollution.

BS-01 contains methyl salicylate.

It has a beautiful fragrance.

It contains

monoterpenes. Monoterpenes are Antibacterial, antifungal, and antiparasitical. BS-01


also contains sesquiterpenes, and alpha-pinenes (Dahanukar et al., 2000). Put alphapinenes and sesquiterpenes together and you have powerful oil for the brain. Alphapinenes plus sesquiterpenes plus methylsalicilate stimulate the hypothalamus to
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secrete cortisone in the body from the adrenal glands. Cortisone is the hormone that
regulates the inflammation associated with arthritis or osteoporosis. It also has a
hormone-like action in stimulating the thymus gland. BS-01 is a natural tonic for the
nervous system and the solar plexus. It grounds and balances. BS-01 is absolutely
fabulous for Bi-polar problems.

BS-02
Family: Melaleuca

Fig 2 : BS-02
BS-02 is

a volatile

oil obtained

by

distillation

from

the

leaves

of

the myrtaceous tree Melaleuca leucadendra, and probably otherMelaleuca species.


The trees yielding the oil are found throughout the Malay archipelago, the Malay
Peninsula and over the hotter parts of the Australian continent; but the greater portion
of the oil is produced from Sulawesi. The name cajuput is derived from its
Indonesian name, kayu putih or "white wood".

The oil is prepared from leaves collected on a hot dry day, macerated in water, and
distilled after fermenting for a night. This oil is extremely pungent, and has the odour
of a mixture of turpentine and camphor. It consists mainly of cineol (Brug, 1947) from
which cajuputene, having a hyacinth-like odour, can be obtained by distillation
with phosphorus pentoxide. The drug is a typical volatile oil, and is used internally in
doses of 2 to 3 minims, for the same purposes as, say, clove oil. It is frequently
employed externally as a counterirritant. It is an ingredient in some liniments for sore
muscles such as Tiger Balm and Indonesian traditional medicine Minyak Telon.
It is also used as an ingredient in inhalants/decongestants and topical
pain/inflammation remedies such as Olbas Oil. It is noted for its antiseptic properties,
as is its cousin, Tea tree oil. Like Tea tree oil it may have use against resistant staph
infections

BS-02 oil is produced by steam distillation of fresh leaves and twigs of the cajuput
tree (Melaleuca leucadendra) and the paperback tree (Melaleuca quinquenervia). BS02 is used to treat colds, headaches, toothache, and tumors; to loosen phlegm so it can
be coughed up (as an expectorant); and as a tonic. Some people apply BS-02 to
the skin for mites (scabies) and a fungal infection of the skin (tinea versicolor).
BS-02 is also used either alone or in combination with other ingredients in
commercially available antiseptic lotions to treat joint pain (rheumatism) and other
pains.

Some

people

inhale

BS-02

oil

as

an

expectorant.

In dentistry, BS-02 is used to relieve gum pain after a tooth is removed or lost.
In food and beverages, BS-02 oil is used as flavouring in very small amounts.

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BS-03
Family: Poaceae

Fig 3: BS-03
BS-03 is extracted through the process of steam distillation. The oil is composed of
75% geraniol and 20% geranyl acetate. The scent of jamrosa can be described as a
rose-grassy scent but it is less stronger than that of the palmarosa. Jamrosa blends well
with palmarosa, geranium, rose geranium, ginger grass and juniper berry. It is an
antiseptic, bactericidal, digestive stimulant and febrifuge. The BS-03s are used in the
food and flavouring industries. These oils are replete with valuable medicinal
properties and other health giving properties.
Scientifically known as Cymbopogon khasans, it refers to a grass hybrid, which is
generally of a pale yellowish colour. The origin of jamrosa grass is in India and it is
widely grown in areas like Chhattisgarh, Maharashtra, Madhya Pradesh and also in the
southern parts of the country. The BS-03 is extracted from the highly aromatic
jamrosa grass. This medium sized grass is of hybrid origin, being a cross between the
palmarosa and citronella. It is a plant which generally grows in the wild. The high
quantities of ocimene and geraniol present in jamrosa imparts it with the scents of
mangoes and roses and this is how it differs from the palmarosa. Recently, the jamrosa
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products have seen a rise in demand and commercial cultivation of the grass on a
larger scale is required. The Regional Research Laboratory (RRL) in Jammu has
developed a new variety of the jamrosa which is called CN-5.

Uses
The strong fibers of the oil are also used to make idols and statues containing plaster
of Paris. This grass provides highly useful products and is greatly favoured by the
industry. Its long and strong fibers are useful in the making of ropes. Jamrosa supplies
the raw materials for industries based on fiber. Traditionally, jamrosa has been used as
a perfumery compound as it possesses the scent of roses. Geraniol & Geranyl Acetate
are two useful products derived from the BS-03. The spent grass of jamrosa is used in
the making of cattle feed and organic manure. Handmade papers are also made from
plant products and also for making dhoop, agarbatti/ masala agarbatti and raw
agarbatti. The spent water of the plant is used to make natural insect repellent.
The spent water of the plant is highly pure in quality and has many chemical agents
which can be dissolved easily in water. It is considered good for health reasons.
Jamrosa is also used for creating fragrances like rose fragrances. These grasses are
ecologically important as they have the ability to prevent soil erosion, recharge the
ground water and to detoxify environmental contamination.

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MATERIALS AND METHODS

The experiments were carried out at Amity Institute of Biotechnology, Amity


University, Sec- 125, Noida(U.P.)

Essential Oil
The essential oils (three) were purchased from Belleza Aromatica, an essential oil
importer in New Delhi. These oils were selected based on literature survey and their
use in traditional medicine. As per the certification obtained from the manufacturer,
the oils were prepared by steam distillation. Essential oil was dissolved in methanol
(0.3 ml oil/ 2 ml methanol). The oils was transferred into sterile vials and stored at
-4 0 C.
Essential oil

Family name

Plant part used

BS-01

Pinaceae

Needles and Twigs

BS-02

Melaleuca

fresh

leaves

and

twigs
BS-03

Poaceae

Roots

Sensory Evaluation
Sensory evaluation of oils is considered a critical test of quality. The first steps of
essential oil testing usually begins with sensory evaluation, since this prevents
wasting precious time and money on more expensive analytical procedures and can
identify inferior oils quickly. The viscosity, colour, clarity and odour of an essential
oil can help to identify poor quality oil right at the outset, provided you know

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precisely what you look for. Sensory evaluation of edible oils is governed by three
factors, namely, medium in which the oil is dispersed, temperature of oil during
testing and panel training for unambiguous understanding of descriptors (Kowalska et
al.,. 2007). The information on the sensory profile of oil could facilitate other useful
studies that would help draw a picture of what makes the oil unique from other oil.
Although sensory analysis is one of the most fifteen sensitive methods available, it is
not practical for routine analysis and generally lacks reproducivity.
The scoring by taste or odour panellist may vary greatly from laboratory to laboratory.
Chemical and physical methods developed therefore attempt to improve
reproducivity, sensitivity, and quantitativeness (Joy et al.,.2008).
The essential oil was evaluated on colour, clarity and odour. The colour and clarity of
the oil were evaluated visually. Odour grading was based upon a five point intensity
scale:
1 = no precipitation; 2= weak; 3= medium; 4= strong and 5= extreme.
Chemical characterisation of oil :
Many instrumental and chemical methods have been proposed over the years to
evaluate the quality and stability of essential oils. GC-MS (Mittman et al.,., 2000) is
mostly used to provide a chromatographic profile of the constituents of essential oils.
Every individual component of the essential oil can be identified by the time at which
the peak eluted on the trace. The data produced can then be compared to an
established profile or fingerprint for that particular essential oil to finally determine
the purity of the oil. Analysis by volatiles by gas chromatography is closely related to
flavour evaluation and is therefore the most suitable method for comparison with the
results of sensory panel tests. The oxidative stability of oil can be determined by %
free fatty acids and acid value.
Percentage Free Fatty Acids: free fatty acids, as oleic acids percentages in oil
sample, were determined using an alkali titration method. 50gms of oil were dissolved
in 50 ml in neutralised methylated spirits. 2 drops of phenolphthalein indicator were
added to the sample and this was the heated on a hot sample only enough to produce a
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well emulsified sample on shaking. The sample was titrated with 0.1M sodium
hydroxide until a faint end point was raised and which remains for only 30 seconds.
The volume of sodium hydroxide used in the titration determined the percentage of
free fatty acids in the sample by using the conversion table. The results is expressed in
terms of oleic acids. Free fatty acid s were calculate using the formula (Brunmark,
1989):
% free fatty acids= Vol. NaOH(ml )X normality NaOH X 28.2(oleic acid) X 100
Weight of Sample(g)
Acid value: each oil sample (1.0 g) was weighed and neutralised with 50 ml of fat
solvent (Benzene). 2 drops phenolphthalein indicator were added and titrated to pink
end point(which persisted for 15minutes) with 0.1N potassium hydroxide solution().
Acid value was calculated using the formula(Xiao Mei WEI et al., 1967):
Acid value = V x 0.00561 x1000
Weight of oil sample
Total Phenolic content :
The essential oil were analysed for total phenolic content. This phenolic component is
responsible for antioxidant activity. Total phenolic content (TPC) were determined
according to folins ciocalteus methods (Kragujevac J. 2011). The essential oils were
diluted to a suitable concentration for analysis.
A 0.5 ml of essential oil sample, 1ml of N folins ciocalteus reagent and 1ml of 20%
Na2CO3 were mixed. The mixture was incubated for 2 hours with continuous shaking
at ambient temperature. After two hours of incubation, the optical density was
measured at 765nm. Different concentrations of galic acid (10-90g/ml) were
determined to be a calibration curve. Y is absorbance; X is concentration of galic acid;
the results was shown as g galic acid equivalents (GAE)/ 5 mg essential oil.
Phytochemical analysis
Phytochemical tests for flavonoids, steroids, tannins, saponins, reducing sugars,
cardiac glycosides, anthroquinones, triterpenoids and phlobatannins, Flavonosides
15

were performed on the hexane, acetone, methanol and ethanol extracts of all the ten
plant materials (Cameron et al.,. 1943).
Flavonoids: Sodium hydroxide test- 0.5ml of sample was dissolved in 3-4 drops of
1% HCL and 5 drops of 20% NaOH was added. Yellow colour indicates the presence
of flavonoids.
Steroids: Salkowski reaction- To 0.5ml of sample same amount of CHCl 3 and
concentrated H2SO4 were added and shaken well. Appearance of red colour
chloroform layer and greenish yellow acid layer indicates the presence of steroids.
Tannins: Ferric test- 0.5 ml of sample was added with 3 drops of 5% FeCl 3 solution.
Appearance of dark green or deep blue colour indicates the presence of tannins.
Saponins: 0.5ml of each sample was shaken with 5ml of distilled water and heated to
boil. Frothing indicates the presence of saponins.
Reducing sugars: Fehlings test- Equal volumes of each sample and Fehlings
reagent were added and the solutions were heated in boiling water bath for 5-10
minutes. First a yellow then brick red precipitate indicates the presence of reducing
sugars.
Cardiac glycosides: Keller-Killiani test- 0.5ml of sample were mixed with 0.5ml of
glacial acetic acid and then treated with one drop of 5% ethanolic FeCl 3 solution.
0.5ml of concentrated H2SO4 was carefully poured down the sides of the test tubes.
Appearance of brownish ring between the two layers indicates the presence of cardiac
glycosides.
Anthraquinones: 0.5ml of sample was boiled with 10% HCl for few minutes in
water bath. After cooling, equal volumes of CHCl3 was added. Then few drops of
10% NH3 was added and heated. Appearance of rose pink colour indicates the
presence of anthraquinones.

Phlobatanins: 0.5ml of sample added to 1.25ml of HCl, Red precipitate indicates of


phlobatanins.
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Polysaccharides: 0.5ml of sample added to few drops of iodine. Change in colour


indicates the presence of polysaccharides.
Coumarins and Lactones: 0.5ml of sample added to 1% NaOH solution (2 drops)
and then heated in boiling water bath to get clear solution. Add 4 drops of HCl,
presence of cloudy solution indicates the presences of coumarins and lactones.

Antimicrobial activity
Stock preparation
1. Culture slants of multi-drug resistant strains of Staphyllococcus aureus,
Klebsiella, Enterococcus, Acinetobacter, Pseudomonas,

E.coli and fungal

strains including Aspergillus niger, Aspergillus sp. were obtained.


2. The suitable media was prepared and 3ml of media was added to each culture
slant under a laminar flow hood.
3. The slants were tapped to dissolve the culture cells in the media added.
4. Equal amounts of 50% glycerol and nutrient broth were added in eppendorfs
and were stored in the freezer for future use.
Agar well diffusion method

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1. Cultures of all the bacterial and fungal strains were prepared by adding 100l
of stock solution to 50ml of nutrient media and were incubated at 37C
overnight.
2. Eleven agar plates were prepared out of which every plate were swabbed with
each bacterium.
3. Four wells were made in each plate using cork screw borer.
4. 50l, methanol and oil sample were pipetted in the four different wells of one
plate swabbed with each bacterium. This was done for all the three oil samples.
5. These plates were then incubated at 37C for 24 hours and then the formation
of zones of inhibition were observed and calculated.

oil

BS-01
B3-03

BS-02

Oil

oil

Thin layer chromatography and bioautography:


Thin-layer chromatography (TLC) is without doubt one of the most versatile and
widely used separation methods in chromatography. TLC has remained relatively
inexpensive and one can easily see why it is still popular today. TLC is a simple,
quick, and inexpensive procedure that gives the chemist a quick answer as to how
many components are in a mixture. TLC is also used to support the identity of a
compound in a mixture when the Rf of a compound is compared with the Rf of a
known compound (preferably both run on the same TLC plate).
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Biological activities of the separated compounds in medicinal plants extracts using


TLC assays can be tested. These can be anti-oxidant, Antibacterial and antifungal TLC
assays. For Antibacterial TLC assay, a method known as TLC direct bioautography
can be used. This is whereby after running TLC, a particular bacteria or fungi, that is
known as a plant pathogen, is inoculated onto the TLC plate with the separated
compounds. The TLC plates with the microorganisms are then incubated for two days
at 37C, in a moist atmosphere. The Antibacterial compounds are then identified by
observing where the growth of the microorganism is inhibited or reduced and then
further analysis using these compounds can be carried out.
TLC Autobiography
1. Silica gel powder is mixed with distilled water to form a thick mixture and
spread on the glass

plates evenly.

2. The plates are desiccated for an hour at 60C. Thus silica plates are prepared.
3. The methanol extracts (5 l) of oils were spotted one centimetre above the rim
of the plates with the help of capillary tubes. Twin copies of each plate were
prepared.
4. Running solvent was prepared taking Chloroform: ethyl acetate: Acetic Acid
(50:50:1).
5. The plates along with their replicas were kept in the running solvent and were
allowed to run about 6-8 cm from spot.
6. The plates were allowed to dry and the copies of each plate were separately
kept for autobiography.
7. The other set of plates were sprayed with staining solution
8. Number of spots of varying colours was observed and their Rf values were
calculated.
9. The agar media inoculated with culture were poured over the plates separately
kept for autobiography to form a thin layer.

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10. When the agar solidified, the plates were kept in a trough surrounded with
water soaked cotton to maintain the moisture. The trough was then incubated at
37C for 24hours.
11. After 24 hours the plates were sprayed with TTC (triphenyl tetrazolium
chloride (2%)) and incubated for 2 hours.
12. The plates were then observed for clear zones within the pink colour showing
bacterial growth.
13.

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RESULTS AND DISCUSSION

Sensory Evaluation:
The results of the sensory evaluation carried out on the essential oil BS-01, BS-02 and
BS-03 is shown in Table 1.

Table 1: Sensory evaluation of essential oils

S.NO

Characteristics

BS-01

BS-02

BS-03

.
1.

Colour

transparent

Pale yellow

Transparent
and

slightly

2.

Clarity

Clear

Slightly clear

yellow
Clear

3.

Odour

Herbal

Spicy

Pungent

4.

Odour Intensity

Medium

Medium

High

CHEMICAL CHARACTERISATION OF OIL


Table 2: Table for chemical indices
Chemical indices

BS-01

BS-02

BS-03

PERCENTAGE

2.37

4.99

2.73

0.20

0.42

0.23

FREE

FATTY

ACID
ACID VALUE

Phytochemical Analysis
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Preliminary phytochemical screening of essential oils showed that the essential oil
BS-01 contain few phtyochemicals including steroids, reducing sugar and cardiac
glycosidase. However, flavanoids, anthraquinone, tannins, saponins phlobatanins were
not observed.
Similarly results were reported for BS-02 and BS-03 and results were recorded in
table 3.
Table 3: Phytochemical analysis of essential oils
Phytoconstituents

BS-01

BS-02

BS-03

Flavanoids

Steroids

Tannins

Saponins

Reducing Sugar

Cardiac Glycoside

Anthraquinone

Phlobatanins

Polysaccharides

and -

Coumarins
lactones

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Fig 4: Test for Steroids

Fig 5: Test for reducing sugars

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Fig 6: Test for coumarins and lactones

Antimicrobial activity
The essential oils were screened for potential antimicrobial activity against MDR
bacteria by agar well diffusion method. E. coli, Pseudomonas aeruginosa, klebsiella
sp. isolates were used. All the three oils showed susceptibility against all the isolates.
Furthermore, the highest activity was observed for MDR clinical isolates in essential
oil BS-01 was E. Coli with wide inhibition diameter (30 mm) followed by klebsiella
sp.
Similarly, the highest activity was observed for MDR clinical isolates in essential oil
BS-02 was S. aureus with wide inhibition diameter (30 mm) followed by
Acinetobactor.
Similarly, the highest activity was observed for MDR clinical isolates in essential oil
BS-03 was Acinetobactor with a wide diameter of (30 mm) followed by S. aureus.

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Table 5: Antibacterial activity of Essential oils


Bacterial cultures

BS-01

BS-02

BS-03

(mm)

(mm)

(mm)

Culture 1

14

20

18

Culture 2

12

18

14

Culture 4

10

30

15

Culture 6

16

25

30

Culture 9

15

26

12

Culture 11

16

35

25

Culture 12

30

18

Culture 13

18

25

Culture 14

05

15

Culture 15

26

12

Culture 16

21

16

Culture 1, 15, 16 (klebsiella sp.) Culture 2, 12 (E. coli)


Culture 4, 9, 11, 13(S. aureus) Culture 6 (Acinetobactor)
Culture 14(Pseudomonas aeruginosa)

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Fig 7: Antibacterial activity shown by Essential oil against culture 4

Fig 8: Antibacterial activity shown by Essential oil against culture 12

26

Fig 9: Antibacterial activity shown by Essential oil against culture 14

Fig 10: Antibacterial activity shown by Essential oil against culture 4

27

Fig 11: Antibacterial activity shown by Essential oil against culture 2

Fig 12: Antibacterial activity shown by Essential oil against culture 11

Antifungal Activity

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The essential oils were screened for potential antifungal activity against fungal strains
by agar well diffusion method. Aspergillus sp. and Aspergillus niger isolates were
used. BS-02 showed highest susceptibility towards Aspergillus niger whereas BS-03
does not show activity against any of the two fungal strains.
Table 6: Antifungal study of essential oils
Fungal culture

BS-01

BS-02

BS-03

Culture F1

(mm)
0.5

(mm)
2.0

(mm)
-

Culture F2

2.0

30

Fig 13: Antifungal activity shown by Essential oil against culture F1

29

Fig 14: Antifungal activity shown by Essential oil against culture F2

Thin Layer Chromatography And Bioautography


Bioautographic assay is usually used to screen for antimicrobial activity by separating
components on to the surface of chromatographic plates and overlaying the TLC plate
with molten bacterial agar. TLC bioautography was carried out only on the selected
oils with good results in agar well diffusion technique experiments.
Following observation infer that the essential oils with positive results in agar well
diffusion technique contained various phytochemical constituents responsible for the
antibacterial potential of the respective essential oil.
UV fluorescent compounds were absent in all the samples. The R f value for BS-01 is
0.083; for BS-02 is 0.33 and for BS-03 is 0.25.
The length of the solvent 6 cm
Reference Plate

Bioautography
30

Spot No.

1.
2.
3.

Distance from Spot Rf Value

Distance

(cm)

clear area from

0.5
2.0
1.5

spot(cm)
-

0.083
0.33
0.25

of

Rf Value

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SUMMARY AND CONCLUSION


The research was conducted on three essential oils. Before the start of experiments a
solution of the oils was prepared in methanol (0.3ml oil in 2 ml methanol)
The whole training comprised of basically four experiments :- phytochemical analysis
and sensory evaluation , antibacterial assay and minimum inhibitory concentration,
Total phenolics and chemical characterization and TLC autobiography.
In sensory evaluation, the oils were assessed for four physical characteristics- colour,
intensity, odour and clarity by five different people and the average was recorded.
In phytochemical analysis the oil solutions were subjected to various tests and it was
found that the essential oil BS-01 contains few phytochemicals including reducing
sugars, cardiac glycosidase. However, anthraquinone, saponins, steroids, flavanoids,
tannins were not observed. Similar results were reported for essential oil BS-02 and
BS-03.
The second set of experiments was conducted for antimicrobial and antifungal activity
of the three oils using different bacterial and fungal cultures. The activities were
observed on the basis of availability and growth pattern by agar diffusion method. For
this nutrient agar plate was prepared and nutrient broth were inoculated with different
bacterial cultures. Then after swabbing on nutrient agar plate with overnight grown
cultures, wells were created using sterilised cork borer. Then the fixed amount of
different oil samples were poured in wells.
After 24 to 48 hours the plates were checked for zone of inhibition. Zone of inhibition
were measured using a scale and were analysed. In chemical characterisation the
percentage free fatty acids and the acid value of the essential oil was calculated .
The last experiment was of thin layer chromatography and autobiography in which the
phytoconstituents of the essential oil was separated on a silica gel plate by using a
suitable solvent medium of chloroform : benzene : diethyl ether (2:2:0.5) v/v.

32

After drying the plates at room temperature they were covered in nutrient agar
inoculated with four different bacterial strains. After incubation of 24 hours in high
humidity the agar covered TLC plates were sprayed with 2,3,5- triphenyl tetrazolium
chloride and further incubated for 4 hours. Post incubation the agar plates were found
to be pink colour showing the growth of bacterial colonies. The absence of pink colour
or clear regions on the TLC plates denoted antibacterial action which correspondent to
the phytoconstituents band responsible for that activity.
These experiments helped in proving that the three oils contained phytoconstituents
with potent antibacterial activity. These constituents can be further isolated by gas
chromatography and mass spectrometry and high performance liquid chromatography.
This opens future prospects of these plant constituents to be used as potential drugs for
the treatment of infectious diseases. These can be further screened for their antioxidant
potential which could prove useful for the treatment of cancer and anti-inflammatory,
antirheumatic disease; thus expanding new horizons for alternate and complimentary
medicines.

33

REFERENCES

1. James A. Duke. 1983. Handbook of Energy Crops.


2. Bora KS, Sharma A. Phytochemical and pharmacological potential of
Medicago sativa: A review. Pharm Biol. 2010.
3. Avato, P. Antimicrobial activity of saponins from Medicago sp.: structureactivity relationship. Phytotherapy Research. 2006. 20: 454457.
4. Kowalska I. et al.,. Flavanoids from barrel medic (Medicago tranculata) aerial
parts. J Agric Food Chem. 2007; 55(7):2645-52.
5. Burt SA . Antimicrobial activity of native and naturalized plants of Minnesota
and Wisconsin. Journal of Medicinal Plants Research. 2008, 2(5), 98-110.
6. Kundan Singh Bora et al.,. In Vitro Antioxidant and Free Radical Scavenging
Potential of Medicago sativa Linn. Journal of Pharmacy Research 2010, 3(6),
1206-1210.
7. Brunmark A, Cadenas E (1989). "Redox and addition chemistry of quinoid
compounds and its biological implications"
8. Kragujevac J. Sci. 33 (2011) 63-72.
9. David V. Huhman and Lloyd W. Sumner. Metabolic profiling of saponins in
Medicago sativa and Medicago truncatula using HPLC coupled to an
electrospray ion-trap mass spectrometer. Phytochemistry. 2002, 347-360.
10. Burg S. L .Journal of Microscopical Science s3-88, 163-164.
11. Neeta Shrivastava,Tejas Patel.Clerodendrum and Heathcare: An Overview.
Medicinal and Aromatic Plant Science and Biotechnology.2007 Global Science
Books.
12. S.M.Vidya et al.,.Evaluation of hepatoprotective activity of clerodendron
serratum L.Indian Journal of Pharmacology 2000; 32: S81-S118
13. Narayanan N. et al.,. Antinociceptive, anti-inflammatory and antipyretic effects
of ethanol extract of Clerodendron serratum roots in experimental animals.J
Ethnopharmacol. 1999; 65(3):237-41.

34

14. Xiao Mei WEI, Qi Xiou ZHU, Jin Chun CHEN, Dong Liang CHENG.Two
New Iridoid Glucosides from Clerodendrum serratum.Chinese Chemical
Letters.2000, 415416.
15. Garg, V. P. and Verma, S. C. L. (1967), Chemical examination of Clerodendron
serratum.

Isolation

and

characterization

ofD-mannitol.

Journal

of

Pharmaceutical Sciences, 56: 639640.


16. S.A. Dahanukar et al.,. Pharmacology of medicinal plants and natural
products.Indian Journal of Pharmacology 2000; 32: S81-S118
17. Santosh S. Bhujbal et al.,.Protective Effects of Icosahydropicenic Acid Isolated
from the Roots of Clerodendrum serratum (L) Moon on Experimental Allergic
Asthma.Journal of Complementary and Integrative Medicine.2010.
18. N.Narayanan et al.,.Comparative Antibacterial activities of clerodendron
serratum and Premna herbacea roots.Indian Journal of Pharm Sciences.2004.
19. SS Bhujbal et al.,.Antioxidant Effects of Roots of Clerodendrum serratum
Linn.Journal of Pharmacognosy research.2009, 1:5; 294-298.
20. SS Bhujbal et al.,. In-vitro and In-vivo antiasthamati studies of clerodendron
sp.in guinea pigs.IntJ pharm res and development.2010.2:4
21. Polunin. O. and Stainton. A. Flowers of the Himalayas.
22. Obertreis . Antiphlogistic effects of Urtica dioica folia extract in comparison to
caffeic malic acid. Arzneimittelforschung. 1996; 46:52-6.
23. Hirano M. Effect of stinging nettle root extracts and their steroidal components
on the Na+, K+-ATPase of the benign prostatic hyperplasia. Planta Med. 1994;
60:30-3.
24. Mittman et al.,. Randomized, double-blind study of freeze-dried Urtica dioica
in the treatment of allergic rhinitis. Planta Med. 1990; 56:44-7.
25. P.M. Njogu. Phytochemical investigation and antimicrobial activity of
girardinia diversifolia (link) Friis, M.Pharm. (Pharmaceutical Analysis) thesis.
2007, University of Nairobi.
26. Riehemann K, Plant extracts from stinging nettle (Urtica dioica), an

antirheumatic remedy, inhibit the pro-inflammatory transcription factor NFkappaB. FEBS Lett. 1999 ; 442(1):89-94.
35