Journal of Pharmacognosy and Phytochemistry 2019; 8(4): 3338-3343
E-ISSN: 2278-4136
P-ISSN: 2349-8234
JPP 2019; 8(4): 3338-3343 Phytochemical screening and qualitative analysis
Received: 04-05-2019
Accepted: 06-06-2019 of Cymbopogon citratus
Praveen Kumar Gupta
Department of Biotechnology, Praveen Kumar Gupta, BS Rithu, Shruthi A, Anushree Vinayak Lokur
R.V College of Engineering, and Raksha M
Bangalore, Karnataka, India
BS Rithu Abstract
Department of Biotechnology, Cymbopogon citratus, commonly known as Lemongrass is a small herbaceous plant of Poaceae family. It
R.V College of Engineering, is used as traditional medicine for treatment of several diseases such as fever, sore throats, cough,
Bangalore, Karnataka, India laryngitis, bronchitis, oral candiasis, body ache, head ache, digestive problems etc. This study deals with
the preliminary phytochemical screening of ethyl acetate extract of lemon grass leaves followed by
Shruthi A qualitative analysis by Thin Layer Chromatography. Antimicrobial activity of the extract was also been
Department of Biotechnology, tested to highlight the medicinal values. Phytochemical analysis revealed the presence of several
R.V College of Engineering, bioactive compounds such as flavonoids, phenols, tannins, alkaloids etc. And TLC analysis further
Bangalore, Karnataka, India
confirmed the presence of those secondary metabolites. The ethyl acetate extract showed significant
Anushree Vinayak Lokur
antibacterial activity against Bacillus subtilis. The study revealed the medicinal potential of lemongrass.
Department of Biotechnology,
R.V College of Engineering, Keywords: Phytochemical screening, qualitative analysis, Cymbopogon citratus
Bangalore, Karnataka, India
1. Introduction
Raksha M Plants are important source of medicinal agents as they possess numerous active constituents
Department of Biotechnology,
R.V College of Engineering,
of immense therapeutic value [1]. Since ancient times, plants and herbs have been given a
Bangalore, Karnataka, India unique place in all the civilizations throughout the world [2]. Plant-based drugs are used
worldwide for the treatment of various diseases because of their easy availability and less toxic
effect to recipient compared to that of synthetic drugs [1]. The use of herbal drugs increasing
rapidly and it represents a substantial part world drug market [3]. More than 75% of the world
population depends upon medicinal plants for their basic health needs. Plant-based medicine
has become a popular alternative for synthetic medicine because it does not cause any adverse
effect [4].
1.1 Secondary metabolites
Plants synthesize a vast variety of chemical compounds classified as primary and secondary
metabolites. Primary metabolites are involved directly in growth and development whereas
secondary metabolites have several medicinal importance. There are wide range of secondary
metabolites such as alkaloids, flavonoids, saponins, tannins, terpenoids, cardiac glycosides etc.
Each of these have specific functions and health benefits. Hence they are used as raw materials
for pharmaceutical and cosmetic industries [5]. India has tremendous variety of plant sources
and is origin of traditional system of medicines [6].
1.2 Lemongrass
Cymbopogon citratus, commonly known as Lemongrass belongs to the poaceae family and
genus Cymbopogon is a tall, monocotyledonous aromatic perennial plant with slender
sharpedge green leaves with pointed apex. The origin of the plant is tropical Asia.
Taxonomic details of the lemongrass:
Kingdom : Plantae
Division : Magnoliophyta
Class : Liliopsida
Order : Poales
Family : Poaceae
Genus : Cymbopogon
Species : citrates
Correspondence
Praveen Kumar Gupta
Department of Biotechnology, The name lemongrass is due to typical lemon-like odour of the essential oil present in the
R.V College of Engineering, shoot. The Cymbopogon genus members also known as aromatic grasses since they produce
Bangalore, Karnataka, India volatile oils [4].
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1.3 Medicinal Properties of Lemongrass 2.3 Extraction Using Soxhlet Unit
Leaves of the Cymbopogon citratus plant used for food, The extraction process using the Soxhlet unit was followed as
cosmetic as well as pharmaceutical applications. Lemongrass per the description given by Williams et al. 2007 [10]. The
is one of the important medicinal plant and it has various processed sample powder was subjected to the extraction in
applications in traditional medicines. Also it can be used for the Soxhlet unit using the ethyl acetate as solvent.
treatment of HIV complications, especially secondary Here 20grams of powder sample was taken for the process of
bacterial infections [7]. extraction with 230ml of ethyl acetate as solvent. The boiling
Lemon grass has been traditionally used to treat various temperate of the Soxhlet unit was maintained to 67-70 degree
medical conditions due to various secondary metabolites Celsius. The flask containing the extraction solvent was
present in it. It has been also used to treat fever, cough, heated to reflux. The process of extraction was carried out for
elephantiasis flu, leprosy, malaria and other digestive 3 days (15-20 cycles). After the process of extraction, the
problems. Antimicrobial activity of lemongrass against sample was collected from the distillation flask and subjected
various bacteria, fungi, protozoa has also been reported. The to the process of filtration using Whatman filter paper and
scientific investigations and information on the therapeutic followed by the evaporation using the water bath to allow the
potentials of lemongrass is limited. The lack of scientific solvent to get evaporated and to obtain the concentrated
knowledge has restricted the use of lemongrass for clinical sample which can be used for the further experimental
applications [4]. This study focusses on qualitative analysis of process [11].
the phytochemistry of lemongrass (Cymbopogon citratus),
and determination of the antimicrobial effect of the leaf 2.4 Phytochemical Screening
extracts on some selected microbial isolates. Phytochemicals are the chemical compounds which are
produced by the plants. They are produced as a result of
2. Methodology primary and secondary metabolism in plants. These
phytochemicals are usually considered as the research
compounds because of the biological activity of the
compounds are still under the scientific and experimental
study towards the health effects.
Thereby the phytochemical analysis of lemon grass extract
was carried out using the standard protocol method [12].
2.4.1 Test for alkaloids
Mayer’s test: 1o 1 ml of extract add 1 ml of conc. Hcl
followed by few drops of Mayer’s reagent, formation of white
or green precipitate indicate the presence of alkaloids.
2.4.2 Test for phenols
Ferric chloride test: To 1ml of extract add 1ml of 5 % ferric
chloride solution, formation of reddish-brown precipitate
Methodology Flow
indicates the presence of phenols.
2.1 Sample Collection
2.4.3 Test for flavonoids
The fresh leaves of lemon grass were collected from an farm
Lead acetate test: To 1 ml of the extract add 1 ml of 10%
land at Hoskote, Bengaluru Karnataka, on 23rd February
lead acetate solution, formation of yellow precipitate indicates
2019.
the presence of flavonoids.
2.2 Sample Preparation 2.4.4 Test for tannins
The collected sample was washed with running tap water to Braymer’s test: To 0.5 ml of extract add 1 ml of distilled
remove the dust particles and other contaminants present on water followed by 1 ml of 5 % ferric chloride solution,
the sample. The clean sample was once again washed with formation of blue-green colour indicates the presence of
distilled water to avoid any cross contamination of the sample tannins.
and the sample was air dried under the sun for 8-10 days at
27-30 degree Celsius and then subjected to electrical grinder 2.4.5 Test for saponins
to obtain fine powder. The powder was then subjected to Foam test: To 1 ml of extract add 1ml of distilled water and
sieving to obtain fine powder so that the extraction can be shake vigorously, formation of the foam indicates the
carried out at larger surface area [8, 9]. presence of saponins.
2.4.6 Test for cardiac glycosides
Keller- killiani test: To 1 ml of sample add 2 ml of glacial
acetic acid followed by 2ml of glacial acetic acid, add 1 ml of
5% ferric chloride solution along with 1 ml of dilute Hcl,
formation of brown ring at the interface indicate the presence
of cardiac glycosides.
2.4.7 Test for terpenoids
Ferric chloride test: To 1 ml of extract add 2 ml of water
followed by 1ml of 10% ferric chloride solution, formation of
Fig 1: Sample processing intense colour indicates the presence of terpenoids.
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Journal of Pharmacognosy and Phytochemistry
2.4.8 Test for quinones Solvent systems used
To 1 ml of extract add 0.5 ml of con Hcl, formation of yellow Solvent system Ratio
precipitate indicates the presence of quinones. Hexane: ethyl acetate 18: 2
Ethyl acetate: chloroform: distilled water 5: 3: 1
2.4.9 Test for coumarins n-butanol: ethyl acetate: distilled water 5: 10:15
To 1ml of extract add 1.5 ml of 10% NaOH, formation of
Chloroform: distilled water 6: 4
yellow colour indicates the presence of coumarins.
Methanol: distilled water 6: 3
2.5 Qualitative Analysis Using TLC
2.6 Anti-Microbial Activity
The process of TLC was carried out in order to isolate the
The potency of the plant extract in inhibiting the growth of
components that were present in the plant extract. Different
the microbe was studied by carrying out the disc diffusion
solvent systems with different ratio was prepared to carry out
method.
the thin layer chromatography studies and to identify the best
solvent system that is capable of showing better resolution [13,
14, 15, 16] 2.6.1 Anti-Bacterial Activity: The ability of the plant extract
.
to inhibit the growth of the bacteria was studied by
The precoated silica TLC plates were taken and cut into the
performing the disc diffusion method, the study was carried
required dimension. The sample was loaded on the TLC plate
out using the Bacillus subtilis. To prepare 150 ml of LB
using the capillary tube. The sample loaded TLC plate were
media, 3grams of LB & 1.8 grams of agar was dissolved in
then placed in mobile phase and allowed the capillary action
150 ml of distilled water and then autoclaved at 121 degree
of the solvent to take place. The TLC plates were removed
Celsius at 100kPa above atm pressure for 15mins [17].
once the solvent reaches 3/4th of the TLC plate. The TLC
The sample stock of 100mg/ml was made and from that the
plate was then air dried using the hot air oven for one min and
20ul, 25ul & 30ul of sample was taken to observe the anti-
then observed under the UV-TLC reader.
microbial activity
Rf = Distance moved by sample/distance moved by solvent
3. Result & Discussion
(Rf: retention factor)
3.1 Results of Phytochemical Analysis
Table 1: Results of phytochemical screening
TEST Inference Ethyl acetate extract
Test for flavonoids Formation of white precipitate +ve
Test for phenol Formation of dark blue/intense color +ve
Test for saponins Formation of persistence foam. -ve
Test for tannins Formation of blue greenish color. +ve
Test for cardiac glycosides Brown ring at the interface. +ve
Test for alkaloids Presence of green color or white precipitate. +ve
Test for terpenoids Formation of intense color. -ve
Test for quinones Yellow precipitate -ve
Test for coumarins Formation of yellow color. +ve
The results of this study showed the presence of the phytochemicals namely (flavonoids, phenols, tannins, alkaloids, cardiac
glycosides & coumarins).
Fig 2: Results of flavonoids Fig 3: Results of phenols
Fig 4: Results of tannins Fig 5: Results of saponins
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Journal of Pharmacognosy and Phytochemistry
Fig 6: Results of cardiac glycosides Fig 7: Results of alkaloids
Fig 9: Results of Terpenoids Fig 10: Results of quinones test
Fig 11: Results of coumarins test
(c is negative control& 1,2,3 test tubes are three trials of the test)
3.2 Results of thin Layer Chromatography combination of various solvents and calculating the Rf value
The qualitative analysis ethyl acetate extract was carried out to identify the respective phytochemical by comparing with
by performing the thin layer chromatography using the the standard chart.
Table 2: Results of TLC
Solvent system Ratio Rf Inference
Methanol: distilled water 18:2 0.81 Flavonoids
Ethyl acetate: chloroform: distilled water 5: 3:1 0.86 Flavonoids
n-butanol: ethyl acetate: distilled water 5:10:15 0.88 Tannins
Chloroform: distilled water 6: 4 0.92 Tannins
0.92 Tannins
0.61 Alkaloids
Hexane: ethyl acetate 6:3 0.56 Alkaloids
0.49 Terpenoids
0.35 Terpenoids
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Journal of Pharmacognosy and Phytochemistry
Fig 12: TLC plates observed under the UV light
From the study we can conclude that the solvent (hexane: 3.3 Anti-Bacterial Activity
ethyl acetate) showed the better resolution in separation of the The microbe was inoculated on the LB media and kept for
phytochemicals, almost 6 phytochemicals was inferred by incubation at 28 degree Celsius for 2 days and observed that
comparing the standard. there was zone of inhibition, from the above figure we can
interpret that the ethyl acetate extract has inhibition on the
growth of the bacteria.
Table 3: Zone of inhibition
S. No Zone of Inhibiotion at 3rd Day Zone of Inhibition at 6th Day Zone of Inhibition at 9th Day
1 20ul: 0.8cm 20ul: 0.8cm 20ul: 0.8cm
25ul:1cm 25ul:1cm 25ul:1cm
30ul:1.1cm 30ul:1.1cm 30ul:1.1cm
2 20ul:0.9cm 20ul:0.9cm 20ul:0.9cm
25ul:1.1cm 25ul:1.1cm 25ul:1.1cm
30ul:1.3cm 30ul:1.3cm 30ul:1.3cm
3 20ul:0.8 cm 20ul:0.8 cm 20ul:0.8 cm
25ul:1.2cm 25ul:1.2cm 25ul:1.2cm
30ul:1.5cm 30ul:1.5cm 30ul:1.5cm
From this study we can observe that the 30ul (3mg/30ul) was 5. Conflict of Interest
the optimal concentration in inhibition the growth of the The authors listed in this paper have no conflict of interest
microbe, where as minimal zone of inhibition was observed known best from our side. There was also no problem related
even in 20ul &25ul of sample. to funding. All authors have contributed equally with their
valuable comments which made the manuscript to this form.
6. Acknowledgment
The authors listed in this paper wish to express their
appreciation to the RSST trust Bangalore for their continuous
support and encouragement. As a corresponding author, I also
express my sincere thanks to all other authors whose valuable
contribution and important comments made this manuscript to
this form.
Fig 13: Zone of inhibition observed (c is control, 1 is 20ul, 2 is 25ul 7. References
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