Phytochemical Investigation and Antimicrobial Study of Leaf Extract of Plantago lanceolata

Abstract

Leaves of Plantago lanceolata were traditionally used to treat wounds, burns, inflammations, fevers, diabetes and cancer. The present study was carried out on the phytochemical investigation and antimicrobial activities of the leaves extract of Plantago lanceolata since the plant was used for wound healing in Ethiopia. The powdered leaves of Plantago lanceolata herb was sequentially extracted with organic solvents: petroleum ether, chloroform/methanol (1:1) and methanol respectively. The crude extracts was subjected to phytochemical screening and revealed the presence of steroids, alkaloids, flavonoids, saponins, glycosides, phenols, tannins and terpenoids compounds that might be responsible for the claimed activities by local people. The petroleum ether extract was purified over silica gel preparative thin layer chromatography and yielded an isolated compound PL-5. The structure of this compound was elucidated using different spectroscopic techniques such as FT-IR, 1H-NMR, 13C-NMR and DEPT-135 spectral data and by comparing the data with literature reports. The crude extracts, isolated pure compound and n-hexane extracted oil were tested against four bacterial species (Gram negative bacteria: Escherichia coli and Salmonela thyphei; Gram positive bacteria: Staphylococcus aureus, Streptococcus agalactiae) and two fungal species (Aspergillus niger and Fusarium solani) using paper disc diffusion method. All crude extracts, isolated pure compounds and extracted oil were active against all the tested bacterial. Additionally, petroleum ether and chloroform/methanol (1:1) crude extracts and n-hexane extracted oil were active against the two fungal species and hence the present work supported the medicinal use of Plantago lanceolata.

Full text

Phytochemical Investigation and Antimicrobial Study of Leaf Extract of
Plantago lanceolata
Shuma Fayera1*, Neelaiah Babu G2, Aman Dekebo3 and Yiheyis Bogale2
1Department of Chemistry, Debark University, Gonder, Ethiopia
2Department of Chemistry, Haramaya University, Ethiopia
3Program of Applied Chemistry, Adama Science and Technology University, Ethiopia
*Corresponding author: Shuma Fayera, Department of Chemistry, Debark University, Gonder, Ethiopia, Tel: 251930875097; E-mail: shumafayera@gmail.com
Received: January 05, 2018; Accepted: January 17, 2018; Published: January 25, 2018
Copyright: © 2018 Fayera S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Leaves of Plantago lanceolata were traditionally used to treat wounds, burns, inflammations, fevers, diabetes and
cancer. The present study was carried out on the phytochemical investigation and antimicrobial activities of the
leaves extract of Plantago lanceolata since the plant was used for wound healing in Ethiopia. The powdered leaves
of Plantago lanceolata herb was sequentially extracted with organic solvents: petroleum ether, chloroform/methanol
(1:1) and methanol respectively. The crude extracts was subjected to phytochemical screening and revealed the
presence of steroids, alkaloids, flavonoids, saponins, glycosides, phenols, tannins and terpenoids compounds that
might be responsible for the claimed activities by local people. The petroleum ether extract was purified over silica
gel preparative thin layer chromatography and yielded an isolated compound PL-5. The structure of this compound
was elucidated using different spectroscopic techniques such as FT-IR, 1H-NMR, 13C-NMR and DEPT-135 spectral
data and by comparing the data with literature reports. The crude extracts, isolated pure compound and n-hexane
extracted oil were tested against four bacterial species (Gram negative bacteria: Escherichia coli and Salmonela
thyphei; Gram positive bacteria: Staphylococcus aureus, Streptococcus agalactiae) and two fungal species
(Aspergillus niger and Fusarium solani) using paper disc diffusion method. All crude extracts, isolated pure
compounds and extracted oil were active against all the tested bacterial. Additionally, petroleum ether and
chloroform/methanol (1:1) crude extracts and n-hexane extracted oil were active against the two fungal species and
hence the present work supported the medicinal use of Plantago lanceolata.
Keywords: Antimicrobial activities; Phytochemical; Screening;
Plantago lanceolata
Introduction
Medicinal plants are now more focused than ever because they have
the capability of producing many benets to society indeed to
mankind, especially in the line of medicine and pharmaceutical. Plant
parts such as leaves, roots and bark are used for the therapeutic
purposes and as well serve as precursors for the synthesis of useful
drugs due to their ethnomedical importance in nature. e medicinal
potentials of these plants could be traceable to the bioactive
phytochemical constituents that are responsible for the physiological
action on the human body [1]. Substances derived from plants have
recently being of great interest due to their versatility. ese substances
in the plant which enhance their usefulness globally are classied as
phytochemicals [2].
Majority of the people living in the developing world are struggling
to increase the standard of living and to improve the health care
delivery in the face of increasing poverty and growing population.
According to WHO survey, 80% of populations living in the
developing countries rely exclusively on traditional medicine for their
primary health care needs of which most involve the use of plant
extracts [3].
Plantago lanceolata
belonging to the Plantaginaceae
family has also taken place in many medicinal uses as a wound healing
remedy. is herbaceous plant was traditionally used in North Africa
to treat wounds, burns, abscesses, inammations, hemorrhoids and
fevers [4]. Previous reports have indicated that the plant was also
eective against diarrhea, dysentery, an anesthetic, antiviral, anti-
inammatory, astringent, anti-helmintic, analgesic, analeptic,
antihistaminic, anti-rheumatic, antitumor, anti-ulcer, diuretic,
expectorant and hypotensive in traditional medicine [5,6]. Among
many popular medicinal plants,
Plantago lanceolata
has accessed a
scientic value, as it has been taken place in many traditional medicine
uses as a wound healing remedy for centuries [7]. In this study, better
extraction methods of
Plantago lanceolata
leaves were investigated. For
eective extraction and better yield of extract of
Plantago lanceolata
oil, organic solvent extraction methods was used and certain
parameters such as extraction time and temperature was manipulated.
Materials and Methods
Sample collection
e leaves of
Plantago lanceolata
L., herb was collected from
Haramaya University main campus, Ethiopia, in December 2016
(Figure 1).
e bacterial and fungal test microorganisms used in this study
were two Gram positive bacterium:
Staphylococcus aureus
and
Streptococcus agalactiae
and two Gram negative bacterium:
Escherichia coli
and
Salmonela thyphei
and the fungi:
Aspergillus
niger
and
Fusarium solani
were obtained from Plant Pathology
Laboratory of the School of Plant Science, Haramaya University.
N
a
t
u
r
a
l
P
r
o
d
u
c
t
s
C
h
e
m
i
s
t
r
y
&
R
e
s
e
a
r
c
h
ISSN: 2329-6836
Natural Products Chemistry &
Research
Fayera et al., Nat Prod Chem Res 2018, 6:2
DOI: 10.4172/2329-6836.1000311
Research Article Open Access
Nat Prod Chem Res, an open access journal
ISSN: 2329-6836
Volume 6 • Issue 2 • 1000311

Figure 1: Image of
Plantago lanceolata
herb taken by Shuma.
Sample preparation and crude extraction
e collected leaf was washed repeatedly and dried in an open air
protected from direct exposure to sun light. Air dried leaves of
Plantago lanceolata
L., was grinded by analytical mill and packed in
polyethylene bags. A 210 g of the powdered of
P. lanceolata
L., leaves
was then extracted with petroleum ether for 8 h in soxhlet apparatus
(60 g of each) and soaked with 300 mL of petroleum ether and heated
with heating mantle at 45°C for 8 h and ltered with lter paper
(Figure 2). e ltrate was collected and concentrated at 40°C under
reduced pressure using a Rotary evaporator. Aer air drying at room
temperature, the defatted marc was subjected soxhlet extractor with
MeOH: CHCl3 (1:1) for 8 hrs. en ltered using what man No.1 lter
paper and concentrated using Rotary evaporator at 40°C under
reduced pressure. e marc collected aer CHCl3/MeOH extraction
was dried at room temperature for further extraction with MeOH by
the same procedure. is procedure was repeated twice until sucient
crude extract was collected and the crude extract was kept at 4°C until
analysis.
Yield of extract %=Weight of crude extract/Weight of samples × 100.
Phytochemicals screening on leaves of
Plantago lanceolata
ere was phytochemical screening of methanol crude extract
report on the leaves of the
Plantago lanceolata
[8]. In this study, the
preliminary phytochemical screening was carried out on the plant
extract (petroleum ether crude extract, CHCl3/MeOH (1:1) crude
extract and MeOH crude extract), following the standard procedures
were described [9,10].
1. Detection of saponins:
Froth test: All extracts (0.25 g each) were diluted with distilled water
to 10 mL and shaken in a graduated cylinder for 15 min vigorously.
e foam formation indicated the presence of saponins.
2. Detection of quinones: 0.2 g of each of the Pet.ether, CHCl3/
MeOH (1:1) and MeOH crude extracts were treated separately with
alcoholic potassium hydroxide solution. en, the formation of blue
color from red indicated the presence of quinones.
3. Detection of carbohydrates: Pet.ether, CHCl3/MeOH (1:1) and
MeOH crude extracts were dissolved individually in 5 ml distilled
water and ltered. e ltrates were used to test for the presence of
carbohydrates.
Figure 2: Scheme for Successive Soxhlet extractions of
Plantago
lanceolata
leaves.
Molisch’s solution test: Molish’s solution was added to the crude
plant extracts dissolved in distilled water then 2 mL of H2SO4
concentrated was added and poured carefully along the side of the test
tube, a violet ring appeared at the interphase of the test tube indicated
the presence of carbohydrate.
4. Detection of avonoids:
Lead acetate test: Pet.ether, CHCl3/MeOH (1:1) and MeOH crude
extracts (0.2 g) were treated with few drops of lead acetate solution.
Formation of yellow color precipitate indicates the presence of
avonoids.
5. Detection of alkaloids: 0.2 g of Pet.ether, CHCl3/MeOH (1:1) and
MeOH crude extracts were dissolved separately in 5 mL diluted
Hydrochloric acid and ltered.
Wagner’s test: 2 mL Filtrates were treated with Wagner’s reagent
(Iodine in Potassium Iodide). Formation of brown/reddish precipitate
indicates the presence of alkaloids.
6. Detection of tannins: 0.2 g of Pet.ether, CHCl3/MeOH (1:1) and
MeOH crude extracts was dissolved in 10 mL distilled water and
ltered. 3-4 drops of 1% aqueous Iron chloride (FeCl3) solution was
added to the ltrate. e appearance of intense green, purple, blue or
black color indicated the presence of tannins in the test samples.
7. Detection of phenols: Ferric Chloride Test: Pet.ether, CHCl3/
MeOH (1:1) and MeOH crude extracts (0.25 g) were treated with 3-4
drops of ferric chloride solution. Formation of bluish black color
indicated the presence of phenols.
8. Detection of terpenoids: 0.2 g of the organic extract was dissolved
in 2 mL of CHCl3 and evaporated to dryness. 2 mL of conc. H2SO4
Citation: Fayera S, Neelaiah Babu G, Dekebo A, Bogale Y (2018) Phytochemical Investigation and Antimicrobial Study of Leaf Extract of
Plantago lanceolata. Nat Prod Chem Res 6: 311. doi:10.4172/2329-6836.1000311
Page 2 of 8
Nat Prod Chem Res, an open access journal
ISSN: 2329-6836
Volume 6 • Issue 2 • 1000311

was then added and heated for about 2 min. Development of a grayish
color was indicated the presence of terpenoids.
9. Detection of steroids:
Salkowski’s test: e formation of red color in the lower chloroform
layer when 0.2 g of organic extracts (Pet.ether, CHCl3/MeOH (1:1) and
MeOH crude extracts) dissolved in 2 mL of chloroform and 2 mL
concentrated sulphuric acid added to it, indicates the presence of
steroids.
10. Detection of glycosides:
Keller-Kiliani Test: 0.2 g extracts of pet. Ether, CHCl3/MeOH (1:1)
and MeOH was added to glacial acetic acid, one drop of 5% FeCl3 and
conc.H2SO4. Reddish brown color was appeared at junction of the two
liquid layers and upper layer appears bluish green indicated the
presence of glycosides.
Structural elucidation
Characterizations of the isolated compounds were governed by
spectroscopic techniques through the overdue conditions. NMR
spectra were recorded on Brucker Avance DMX 400 FT-NMR
spectrometer operating at 400 MHz for 1H and 100 MHz for 13C at
room temperature by using CDCl3. Tetra methylsilane (TMS) was used
to refer as standard. IR spectra were recorded between 400-4000 cm-1
in KBr pellets.
Isolation of compounds
Preparative thin layer chromatography and solvent system:
Preparative in-layer chromatography (PTLC), glass plates
admeasuring 20 × 20 cm were coated with silica gel G (1-7 mm). ey
were dried at room temperature for 5-6 hours and activated in oven at
110°C for almost the same period. Aer dried the band subjected to
isolate through a chromatographic chamber using the overdue solvent
system. Approximately 5-10 mg of sample (fractions) was dissolved in
1-2 ml Pet. Ether and applied over the activated silica layer as a narrow
streak across the plate. e solvent from the streak was allowed to
evaporate. e plate was then developed in toluene:ethylacetate (40:
10) binary mobile phase in a rectangular tank. Aer the development,
the plate was removed from the tank and the solvents were allowed to
evaporate at room temperature. e compound from the scraped silica
was recovered by adding appropriate volume of Pet. Ether followed by
shaking and ltration. e solute was thus extracted four times and the
extracts were combined.
A mixture of toluene and ethyl acetate in the ratio of (4:1) was used
as solvent systems to isolate six bands with Rf values of 0.17, 0.50, 0.58,
0.70, 0.78 and 1.00 and the purity of (Rf=0.78, 1.00) was checked on
analytical TLC.
Coding system for isolated pure compound
e coding system of the isolated pure compound was based on the
rst two letters from the scientic name of the plant; P stands for the
genus name
Plantago
, L stands for the place of specimen
lanceolata
and followed by the number that indicating the location of the
compounds starting from the highest Rf value to the lowest. TLC
examination of the crude extracts revealed the presence of at least six
spots visualized under UV lamp at 254 nm and 356 nm. PL-1, PL-2
and PL-5 indicate the rst, second and h compounds respectively.
Antimicrobial Assay
Preparation of inoculums: e test bacterial strains were transferred
from the stock cultures and streaked on Mueller Hinton plates and
incubated for 24 hrs at 37°C. Well separated bacterial colonies were
then used as inoculums. Bacteria was transferred using bacteriological
loop to autoclaved MHA that was cooled to about 45°C in water bath
and mixed by gently swirling the asks. e medium was then poured
to sterile Petridishes, allowed to solidify and used for the Biotest [11].
For test fungi, mycelial plugs from stock cultures were transferred to
PDA plates and incubated for 3 days. en spores of
A. niger
was
harvested by washing the surface of the colony using 10 mL sterile
distilled water and was transferred to 250 mL autoclaved PDA which
was cooled to about 45°C in water bath. Likewise, mycelium of
F.
solani
was washed with 10 mL sterile distilled water, macerated in
blander and the mycelia suspension was transferred to 250 mL
autoclaved PDA cooled to about 45°C in water bath. e medium
containing spore or mycelia suspension was poured to sterile a plates
allowed to solidify and used for disk diusion bioassay [11].
Testing for antifungal and antibacterial activity: A lter paper disc
of about 6 mm in diameter was placed in beaker sterilized in an oven
at 180°C for 1 hrs. 10 and 20 µL of the samples were pipetted to the
discs in three replications. e paper discs impregnated with the
extract solutions were then transferred using sterile forceps to PDA
seeded with spore or mycelia suspension of test fungi as described
under inoculums preparation above. e Petri dishes were incubated
at 24°C for 3 days. e antifungal activity was evaluated by measuring
of the inhibition zone against the tested organisms. e entire test was
performed in triplicate. Similar procedures to that of antifungal test
were followed. Sterilized paper discs were transferred to MHA plates
seeded with at 37°C bacteria for 24 hrs. All the tests were performed in
triplicate. e leaves crude extracts, fractions, n-hexane extracted oil
and pure compounds were taken to test the sensitivity towards four
bacteria.
Results and Discussion
Determination of extraction yield
e extraction yield is a measure of the solvent eciency to extract
specic components from the original material. e percentage yield of
crude extract in respective solvent crude extract was shown in Table 1.
It could be calculated according to the formula as follows [12].
Percentage yield=Weight of crude extract/Weight of samples × 100
Crude extract Yield in (%)
Petroleum ether 7.97
CHCl3/MeOH 19.16
MeOH 27.18
Table 1: Yield of Petroleum ether, CHCl3/MeOH and MeOH crude
extracts by Soxhlet extractor.
Phytochemical constituent
In the present study, the qualitative analyses of
P. lanceolata
leaf
extracts were carried out for dried leaf samples. e preliminary
phytochemical screenings on leaves (petroleum ether, CHCl3/MeOH
Citation: Fayera S, Neelaiah Babu G, Dekebo A, Bogale Y (2018) Phytochemical Investigation and Antimicrobial Study of Leaf Extract of
Plantago lanceolata. Nat Prod Chem Res 6: 311. doi:10.4172/2329-6836.1000311
Page 3 of 8
Nat Prod Chem Res, an open access journal
ISSN: 2329-6836
Volume 6 • Issue 2 • 1000311

and MeOH) extracts indicated the presence of saponins, tannins,
alkaloids, avonoids, terpenoids, and phenolic compounds were
conrmed in leaves of extracts of
P. lanceolata
. e phytochemical
screening test results of Pet.ether, CHCl3/MeOH and MeOH crude
extracts of
P. lanceolata
leaves is presented in the Table 2 below.
No Constituents Pet. Ether CHCl3/MeOH MeOH
1 Steroids + + +
2 Alkaloids + + -
3 Carbohydrates - - -
4 Flavonoids - + +
5 Saponins - + ++
6 Glycosides - + +
7 Phenols - - +
8 Tannins - + ++
9 Terpenoids + + -
10 Quinones - - -
+ve=the presence of phytochemical constituents.
-ve=the absence of phytochemical constituents.
+=High presence of phytochemical constituents.
Table 2: Phytochemical screening results of
P. lanceolata
leaves.
e phytochemical screening test of Pet.ether crude extract showed
that the presence of steroids, alkaloids, terpenoids and the absence of
glycosides, carbohydrates, avonoids, saponins, phenols, tannins and
quinones. e CHCl3/MeOH crude extract has steroids, alkaloids,
avonoids, saponins, glycosides, tannins, and terpenoids and has no
carbohydrates, phenols and quinones. e methanol extract has
constituted steroids, avonoids, saponins, glycosides, phenols, and
tannins and has no constituted alkaloids, carbohydrates, terpenoids
and quinones (Table 2).
Structural elucidation of the compound PL-5
Compound PL-5 was isolated as a purple reddish amorphous solid
with an Rf value of 0.78 which was obtained from petroleum ether
extract isolated by PTLC in toluene/ethyl acetate (40:10). Structural
elucidation of the compounds was based on the spectroscopic data
obtained from FT-IR, NMR (1H-NMR, 13C-NMR and DEPT-135).
e IR spectrum of compound PL-5 revealed a medium absorption
band at 3433 cm-1 due to the presence of a stretching vibration of -O-
H group. e strong absorption band at 2922 and 2853 cm-1 is due to
aliphatic CH3 and CH2 stretching vibration frequencies respectively.
e absorption band at 1754 cm-1 indicates the presence of carboxyl
groups whereas the absorption band at 1464 cm-1 and 693 cm-1
indicates the -CH2 bending for alkanes and -CH bending for
unsubstituted alkene.
e 1H-NMR spectrum of PL-5 showed a downeld shied at 5.314
ppm, due to the presence of olenic proton (H-20). e spectrum
showed the presence of methyl proton at δH 0.81-1.28 (3H each;
Me-23, Me-28, Me-29, Me-30 and Me-31) and methylene proton at δH
1.208-4.58 (H-1,…4, H-7, H-8, H-10,…12, H-15, H-16, H-19, H-22,
H-25, H-26 and H-27) ppm respectively. e signals at δH 1.50-2.27
(H-6, H-9, H-14, H-17 and H-18) ppm are due to the methine proton
whereas 2.77 ppm indicates the presence of oxygenated protons
(Figure 3).
Figure 3: Isolation of PL-5 (toluene:ethylacetate [40:10]) using
PTLC.
13C-NMR spectrum of compound PL-5 showed well resolved
aliphatic, olenic and carbonyl carbons of ester functional groups. 13C-
NMR of the compound revealed that the compound has 31 carbon
atoms. By comparing the 13C-NMR and DEPT-135 NMR spectra there
existed four quaternary carbon atoms with one oxygenated and one
olenic carbon at 173 and 142 ppm whereas the other quaternary
carbons at δ 29.47 and 77.21 pm. e DEPT-135 NMR also displayed
down ward peaks at δC, 24.49, 24.79, 25.03, 29.16, 29.28, 29.35, 29.69,
31.93, 34.42, 36.63, 37.29, 37.35, 37.42, 39.36, 39.85 and 61.19 ppm
which showed the presence of sixteen methylene groups. In addition to
that DEPT-135 NMR shows ve methyl groups at 14.12, 16.36, 19.70,
19.74 and 22.62 ppm and six methine carbons at 22.68, 22.72, 27.97,
32.67, 32.80 and 118.15 ppm as upward peaks out of others. Based on
the above information the compound PL-5 was proposed to have the
following structure.
Antimicrobial assay
Antibacterial and antifungal activity test: e antimicrobial
activities of crude extracts, isolated pure compounds and leaves oil
were assayed by the presence or absence of inhibition zones diameter
compared with some standard antibiotics
in vitro
against four bacteria
(Table 3) and two fungi (Table 4). 10 µL and 20 µL of the crude extract
and isolated compound (PL-5) were used for the study. e standard
samples (chloramphenicol for bacteria and tilt for fungi) showed the
greatest inhibition. e negative control solvent (petroleum ether) did
not show any inhibition zone against tested fungi and bacteria (Figures
4 and 5).
All crude extracts have higher inhibition eect against the tested
bacteria in dose 20 µL when compared with dose 10 µL. PL-5 have
lower inhibition eect against the tested bacteria in dose 20 µL
compared with other all crude extract, which might be due to naturally
occurring combinations of these components, might have synergistic
eects [13]. Inhibition zones of the tested samples were to a little extent
higher in gram negative bacterial (
E. coli
and
S.
thyphei
) than in gram
positive bacterial (
S. aureus
and
S. agalactiae
), hence, the tested
Citation: Fayera S, Neelaiah Babu G, Dekebo A, Bogale Y (2018) Phytochemical Investigation and Antimicrobial Study of Leaf Extract of
Plantago lanceolata. Nat Prod Chem Res 6: 311. doi:10.4172/2329-6836.1000311
Page 4 of 8
Nat Prod Chem Res, an open access journal
ISSN: 2329-6836
Volume 6 • Issue 2 • 1000311

samples have a stronger antibacterial activity towards gram negative
than gram positive. e commercial standard drug (Chloramphenicol)
showed the greatest inhibition eect against both tested bacteria in
both doses (10 and 20 µL) compared with the tested samples and the
negative control (solvent). e crude extracts and pure components are
known to be active against a wide variety of microorganisms, including
Gram-negative and Gram-positive bacteria. erefore, leaves of
Plantago lanceolata
extracts are valuable and could be a future target
for replacing synthetic antibacterial agents (Figure 6).
Figure 4: (E)-Butyl 2-(4-(2-(2-hydroxy-2-methylcyclohexyl)
ethyl)-7-methyloctahydro-1H-inden-1-yl)-5-methylhept-4-enoate.
Sample Dose (µl)
Types of bacteria with mean inhibition zone diameter (mm)
Gram-negative bacteria Gram positive bacteria
E. coli S. thyphei S. aureus S. agalactiae
1
10 11 ± 0.10 5 ± 0.20 8 ± 0.10 11.2 ± 0.03
20 24 ± 0.30 24.5 ± 0.11 23 ± 0.20 17 ± 0.06
2
10 10 ± 0.01 2 ± 0.40 10 ± 0.16 11 ± 0.10
20 23 ± 0.10 24 ± 0.15 23 ± 0.3 19 ± 0.10
3
10 10 ± 0.08 3 ± 0.50 10 ± 0.10 9 ± 0.10
20 20.3 ± 0.10 24.5 ± 0.15 19.7 ± 0.30 16 ± 0.13
4
10 10 ± 0.61 NI 10 ± 0.24 7 ± 0.10
20 10 ± 0.12 11 ± 0.10 11 ± 0.30 9 ± 0.20
5
10 7 ± 0.40 2 ± 0.40 3 ± 0.50 7 ± 0.35
20 10 ± 0.20 9 ± 0.20 9 ± 0.10 9 ± 0.10
6
10 NI NI NI NI
20 NI NI NI NI
7
10 33.5 ± 0.03 25 ± 0.25 28 ± 0.09 31 ± 0.10
20 43 ± 0.09 43.5 ± 0.05 37.7 ± 0.14 32.2 ± 0.20
Value represents mean of zone inhibition of three replication in mm ± SD; NI: Stands for No Inhibition
Where
1 Pet. Ether crude extract
2 CHCl3/MeOH crude extract
3 MeOH crude extract
4 PL-5
5 Fr-6
6 Negative standards (Pet. Ether)
7 Positive standards (Chloramphenicol)
Table 3: Zone of bacterial growth inhibition (mm) crude extracts, Fr-6 and PL-5 of
Plantago lanceolata
.
Citation: Fayera S, Neelaiah Babu G, Dekebo A, Bogale Y (2018) Phytochemical Investigation and Antimicrobial Study of Leaf Extract of
Plantago lanceolata. Nat Prod Chem Res 6: 311. doi:10.4172/2329-6836.1000311
Page 5 of 8
Nat Prod Chem Res, an open access journal
ISSN: 2329-6836
Volume 6 • Issue 2 • 1000311

Figure 5: Photograph showing the antibacterial activity of
Plantago lanceolata
leaves extracts.
Sample Dose (µl)
Types of bacteria with mean inhibition zone diameter (mm)
A. niger F. solani
1 10 18.2 ± 0.10 19.7 ± 0.23
20 22 ± 0.10 20 ± 0.20
2 10 7.5 ± 0.09 8 ± 0.09
20 10 ± 0.20 8 ± 0.10
3 10 NI 6.8 ± 0.08
20 NI 8 ± 0.10
4 10 NI NI
20 NI NI
5 10 NI NI
20 NI 8.2 ± 0.06
6 10 NI NI
20 NI NI
7 10 32 ± 0.10 44 ± 0.40
20 36 ± 0.11 50 ± 0.10
Value represents mean of zone inhibition of three replication in mm ± SD; NI; Stands for No Inhibition
Where
1 Pet. Ether crude extract
2 CHCl3/MeOH crude extract
5 Fr-6
6 Negative standards (Pet. Ether)
Citation: Fayera S, Neelaiah Babu G, Dekebo A, Bogale Y (2018) Phytochemical Investigation and Antimicrobial Study of Leaf Extract of
Plantago lanceolata. Nat Prod Chem Res 6: 311. doi:10.4172/2329-6836.1000311
Page 6 of 8
Nat Prod Chem Res, an open access journal
ISSN: 2329-6836
Volume 6 • Issue 2 • 1000311

3 MeOH crude extract
4 PL-5
7 Positive standards (tilt)
Table 4: Zone of fungal growth inhibition (mm) crude extracts, Fr-6 and PL-5 of
Plantago lanceolata.
Figure 6: Photograph showing the antifungal activity of
Plantago
lanceolata
leaves extracts.
e highest activity was determined in positive control (tilt) against
F. solani
50 ± 0.10 mm the antifungal activities of other crude extracts
were followed by petroleum ether, CHCl3/MeOH, MeOH and Fr-6
respectively. e lowest activity was determined in a MeOH against
F.
solani
6.8 ± 0.08 mm. In general the susceptibility of
A. niger
and
F.
solani
to petroleum ether crude extract and CHCl3/MeOH crude
extract demonstrate higher antifungal activities in terms of growth
inhibition on the tested organisms make it a good candidate for
investigation of in control of new multi-drug resistant on the tested
fungi.
Conclusion
In this study the leaves
P. lanceolata
were extracted using petroleum
ether, MeOH:CHCl3 (1:1) and MeOH. From the study, the leaves
extract was found to contain steroids, alkaloids, avonoids, tannins,
saponins, glycosides, phenols and terpenoids. e presence of these
important phytochemicals in the plant is a scientic justication of the
plant use in the medicinal treatment against various diseases aecting
humans. Additionally, crude extracts, isolated pure compound of the
leaves were tested for the antimicrobial activities against four bacteria
(Gram negative bacterium;
E. coli
and
S. thyphei
and the Gram-
positive bacterium;
S. aureus
and
S. agalatiae
) and two fungi (
A. niger
and
F. solani
). Antimicrobial activity tests showed some antimicrobial
potency of the extracts when it was compared with standard
antibiotics. Aer repeated successive solvent extraction and
preparative thin layer chromatography dierent compounds were
isolated from the plant leaf. Based on TLC analysis the plant contains
several chemical constituents which were not isolated in this study
because of nancial and time constraints. e isolated pure compound
coded as PL-5 was characterized using FT-IR, 1H-NMR, 13C-NMR and
DEPT-135 analysis. Based on those spectral data, PL-5 was identied
as one of non-aromatic steroid compound, (Tables 5-7) and Figures of
Spectral data of compound PL-5.
Observed
frequency (cm-1)
Possible
frequency range
(cm-1)
Assignments
3433 3200-3600 -O-H stretching
2922 2850-3000 Asymmetric -C-H3 stretching of
alkanes
2853 2850-2950 Symmetric -C-H2 stretching of
alkanes
1754 1665-1760 Carboxyl compound
1464 1350-1470 -C-H2 bending for alkanes
693 665-1000 -C-H bending (unsubstituted alkene)
Table 5: Typical Infra-Red (IR) absorption frequencies of the
compound PL-5.
Chemical
shift
of PL-5
Multiplicity,
No of Hydrogen
Literature
value
Remark
0.78-0.96 Multiplet, 15H 0.80-1.28 CH3-
1.02-1.208 Multiplet, 18H 1.00-1.44 Cycloalkanes proton
1.50-1.58 Multiplet, 12H 1.50 -CH2-
1.60-1.96 Singlet, 4H 1.40-1.80 -CH-
2.27-2.28 Triplet, 1H (J=15.2 Hz) 0.50-2.00 -CH-
2.77-2.80 Singlet, 1H 2.25-3.50 Hydroxyl proton
4.43-4.58 Doublet, 1H (J=7.2 Hz) 4.20-5.00 -OCH2-
5.02-5.314 Multiplet, 1H 5.00-5.3 Olefinic proton
Table 6: 1H-NMR spectral data of PL-5 in CDCl3.
Carbon position 13C-NMR δ (ppm) DEPT-135 δ (ppm) Remark
Me-23 14.12 14.12 -CH3
Me-30 16.36 16.37 -CH3
Me-28 19.70 19.71 -CH3
Me-29 19.74 19.75 -CH3
Me-31 22.62 22.63 -CH3
C-9 22.68 22.69 -CH-
C-14 22.72 22.72 -CH-
Citation: Fayera S, Neelaiah Babu G, Dekebo A, Bogale Y (2018) Phytochemical Investigation and Antimicrobial Study of Leaf Extract of
Plantago lanceolata. Nat Prod Chem Res 6: 311. doi:10.4172/2329-6836.1000311
Page 7 of 8
Nat Prod Chem Res, an open access journal
ISSN: 2329-6836
Volume 6 • Issue 2 • 1000311

C-27 24.49 24.47 -CH2
C-11 24.79 24.80 -CH2
C-3 25.03 25.03 -CH2
C-6 27.97 27.98 -CH-
C-15 29.16 29.15 -CH2
C-19 29.28 29.27 -CH2
C-2 29.35 29.35 -CH2
C-13 29.47 - Quaterna
ry
C-8 29.69 29.65 -CH2
C-1 31.93 31.92 -CH2
C-18 32.67 32.66 -CH-
C-17 32.80 32.79 -CH-
C-16 34.42 34.40 -CH2
C-22 36.63 36.63 -CH2
C-26 37.29 37.30 -CH2
C-10 37.35 37.36 -CH2
C-7 37.42 37.44 -CH2
C-12 39.36 39.37 -CH2
C-4 39.85 39.86 -CH2
C-25 61.19 61.20 -OCH2
C-5 77.21 - Quaterna
ry
C-20 118.15 118.16 -CH-
C-21 142.60 - Quaterna
ry
C-24 173.94 - Quaterna
ry
Table 7: 13C-NMR and DEPT-135 spectral data of PL-5 in CDCl3.
References
1. Fagbohun ED, Lawal OU, Ore ME (2012) e proximate, mineral and
phytochemical analysis of the leaves of Ocimum gratissimum L.,
Melanthera scandens A. and Leea guineensis L. and their medicinal value.
2. Nsi EW, Dyegh AH (2004) Preliminary phytochemical screening and
antimicrobial activity of terminalia aricennoides. Intern J Sci Technol 2:
42-44.
3. Sandhya B, omas S, Isabel W, Shenbagarathai R (2006) Ethnomedicinal
plants used by the Valaiyan community of Piranmalai hills (Reserved
forest), Tamilnadu, India-A pilot study. African J Trad, Complem Altern
Med 3: 101-114.
4. Veale DJH, Furman KI, Oliver DW (1992) South African traditional
herbal medicines used during pregnancy and childbirth. J
Ethnopharmacol 36: 185-191.
5. Basri DF, Tan LS, Shaei Z, Zin NM (2012) In vitro antibacterial activity
of galls of Quercus infectoria Olivier against oral pathogens. Evidence-
Based Complementary and Alternative Medicine.
6. Kobeasy I, Abdel-Fatah M, El-Salam SM, Mohamed ZE (2011)
Biochemical studies on Plantago major L. and Cyamopsis tetragonoloba
L. Intern J Biodiver Conserv 3: 83-91.
7. Samuelsen AB (2000) e traditional uses, chemical constituents and
biological activities of Plantago major L. J Ethnopharmacol 71: 1-21.
8. Asressu KH (2013) Antimicrobial activity and phytochemical screening
of crude extract of medicinal plants grown in Eastern Ethiopia. Int J
Pharm Biol Sci 4: 326-333.
9. Tiwari P, Kumar B, Kaur M, Kaur G, Kaur H (2011) Phytochemical
screening and extraction: A Review. Intern Pharmac Sci 1: 98-106.
10. Santhi R, Lakshmi G, Priyadharshini AM, Anandaraj L (2011)
Phytochemical Screening of Nerium oleander Leaves and Momordica
charantia Leaves. Intern Res J Pharm 2: 131-135.
11. Ogunniran KO (2009) Antibacterial eects of extracts of Ocimum
gratissimum and piper guineense on Escherichia coli and Staphylococcus
aureus. African J Food Sci 3: 77-81.
12. Zhang SQ, Bi HM, Liu CJ (2007) Extraction of bio-active components
from Rhodiola sachalinensis under ultrahigh hydrostatic pressure. Separ
Puric Technol 57: 277-282.
13. Delaquis PJ, Stanich K, Girard B, Mazza G (2002) Antimicrobial activity
of individual and mixed fractions of dill, cilantro, coriander and
eucalyptus essential oils. Intern J Food Microbiol 74: 101-109.
Citation: Fayera S, Neelaiah Babu G, Dekebo A, Bogale Y (2018) Phytochemical Investigation and Antimicrobial Study of Leaf Extract of
Plantago lanceolata. Nat Prod Chem Res 6: 311. doi:10.4172/2329-6836.1000311
Page 8 of 8
Nat Prod Chem Res, an open access journal
ISSN: 2329-6836
Volume 6 • Issue 2 • 1000311
Acknowledgement
My sincere gratitude is given to Ethiopian Ministry of Education for
financial support.