Journal of Pharmacognosy and Phytochemistry 2018; 7(3): 603-616
E-ISSN: 2278-4136
P-ISSN: 2349-8234
JPP 2018; 7(3): 603-616
Received: 12-03-2018
Accepted: 16-04-2018
Siraj khan
Department of Plant Sciences,
Abdul wali khan university
Mardan, Pakistan
Gul Jan
Department of Plant Sciences,
Abdul wali khan university
Mardan, Pakistan
Hameeda bibi
Department of Plant Sciences,
Abdul wali khan university
Mardan, Pakistan
Jan Sher
Department of Plant Sciences,
Abdul wali khan university
Mardan, Pakistan
Shakir Ullah
Department of Plant Sciences,
Abdul wali khan university
Mardan, Pakistan
Syed Abidullah
Department of Plant Sciences,
Abdul wali khan university
Mardan, Pakistan
Correspondence
Siraj khan
Department of Plant Sciences,
Abdul wali khan university
Mardan, Pakistan
Phytochemical screening and antimicrobial
activity of the Cichorium intybus (Family-
asteraceae) and Medicago sativa (Family-
fabaceae) Peshawar, Pakistan
Siraj Khan, Gul Jan, Hameeda Bibi, Jan Sher, Shakir Ullah and Syed
Abidullah
Abstract
The plants (Cichorium intybus L and Medicago sativa L) are two important plants of Pakistan. In the
present study attention is being paid to Cichorium intybus ethanolic extracts ((18µl/disc) inhibited the
growth of K. pneumoniae (the Gram negative bacterium) and made the 22.5±0.5 mm zone of inhibition
followed by 22±0.1 mm zone of inhibition against the Gram positive bacterium, B. subtilis and 19±0.1
mm zone of inhibition against the fungus, C. albican. Similarly, 15±0.1, 22±0.2 and 19±0.1 mm zones of
inhibition were made by methanolic extract of Medicago sativa against Gram positive bacterium, (B.
subtilis), Gram negative bacterium (E.coli) and fungus, C. albican. In other words, the order of inhibitory
potential was Cichorium intybus > Medicago sativa. Ethanolic extract was more effective followed by
methanolic, ethyl acetate, hexane and aqueous extracts. Gram positive bacteria were more resistant
followed by Gram negative bacteria and fungi. Antimicrobial assay were analysed for the extraction and
its major components. Preliminary phytochemical analysis of the extracts indicated the presence of
steroids, glycosides, saponins and tannins.
Keywords: phytochemistry; biological chemistry; antimicrobial activity; Cichorium intybus l; Medicago
sativa l; Pakistan
1. Introduction
The helping hand of our health is nature because it provides all necessary material for survival.
Plants having medicinal characteristics are nature’s gift to us to put together disease free life
and play fundamental role to protect our health (Begum and Vimalnath, 2009) [5]. Pakistan is a
developing country and upto great extent, depends on plant assets for agricultural, food,
fodder, shelter and herbal medicines (Shinwari, 2010). In emergent countries like Pakistan,
extracts of the plants are still the chief source of conventional handling. Recent study have
determined that about 60-80% of the total world population, still use extracts of the plant as a
conventional, medicinal and antimicrobial agents. Cichorium intybus L. belonge to family
Asteraceae, local name is Kasni, and Engish name is Chicory. It is distributed in Europe, New
Zealand, Pakistan and India (Ahmad et al. 2009) [1]. In Pakistan, it is found in areas of Hazara,
Swat, Kurram, Gilgit and Kashmir (Qureshi et al. 2008). It is cultivated as annual herb or may
be found as weed in Trifolium field (Ahmad et al. 2009) [1]. Phytochemical work showed that
the plant material contains vitamins i.e. ascorbic acid, thiamine, retinol, riboflavin (Gilani and
Janbaz, 1996). Medicinaly, it is used for eye diseases, hepatitis, liver diseases, cough antidote,
headache, throat disease (Ahmad et al. 2009) [1]. The local name of Medicago sativa is Alfalfa,
also known as Lucerne (Gaweł. 2012) [10]. This plant belongs to family Leguminosae; and is
considered as a green food of the millennium (Liang et al. 2011). This plant is medicinally
valuable because of its phytochemical substances create specific physiological actions in living
organisms. Carbohydrates, starch, basic proteins as well as L-canaverine are nearly all
important bioactive material of the plant. Besides this, Medicago sativas also has pectin
substances, tannins, amines, saponins, triterpene glycosides, coumarin derivatives, purines
base, carotenoids, plant sterols, flavones, cumestrol (phytoestrogens), phenolic compounds and
isoflavonoids (Gaweł, 2012) [10]. Medicinally, M.sativa is used in central nervous system
disorders. M.sativa is also used as a anti-inflammatory, antidiabetic and antioxidant (Kundan
and Anupam, 2011). In Pakistan, this plant is distributed in Hazara, Chitral, Hunza, Peshawar,
Rawalpindi, Attock, Jhelum, and Lahore and in the provinces of Sind and Baluchistan
(Qureshi et al. 2008). Phyotochemical analysis of Oxalis corniculata
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shows the occurrence of flavanoids, phenol, tannins,
phytosterol, glycoseides volatile oil and fatty acids. The
leaves have vitexine-2-O-beta-D-glucopyrunoside iso-
vitexine and flavonoids. It is high source of important fatty
acids like oleic, linolenic palmitic acid, stearic acids and
linoleic acid. It is high source of carotene, vitamin C and
contains a high level of oxalates (Badwaik et al. 2011) [3].
This plant is a good appetizer, removes vata, kapha and piles,
cures dysentery and diarrhoea, astringent for skin diseases and
quarten fevers. Small leaves are externally used to eliminate
opacities and warts of cornea. The leaves are refrigerant and
anti-inflammatory (Kirtikar and Basu, 1975) [25].
2. Material and Methods
2.1 Chemical constituents
Plant Cichorium intybus and Medicago sativa Have vitamins
i.e. ascorbic acid, thiamine, retinol, riboflavin, niacin
carotenoids, inulin, sesquiterpenes i.e. cichorin, esculetin,
esculin, lactucin and lactucopicrin (Gilani and Janbaz, 1996).
Medicago sativas also has pectin substances, tannins, amines,
saponins, triterpene glycosides, coumarin derivatives, purines
base, carotenoids, plant sterols, flavones, cumestrol
(phytoestrogens), phenolic compounds and isoflavonoids
(Gaweł, 2012) [10].
Laboratory organization translational medicine in Pakistan
National Research centre (NRC) shared laboratories. The
Institute established the Centre for pharmaceutical
development (Research and Development) which will
implement a comprehensive pharmaceutical development of a
full cycle: synthesis and screening of new molecules,
development, analysis and implementation of new drugs and
medical products (diagnostic systems) into production. Also
pharmaceutical development of innovative medicines.
2.2 Plants materials
The Two Plant species Cichorium intybus and Madicago
sativa were collected from northern Pakistan. The collected
plant species were identified by taxonomist and ethnobotanist
Dr. Hamayun (Associat Professer botany depertment) voucher
specimen was deposited at the herbarium of Abdul wali khan
university Mardan (AWKUM), Pakistan.
2.3 Extraction
The plants were dipped in tap water and kept at room
temperature in shade, at Pakistan Council of Scientific and
Industrial Research Centre Peshawar (PCSIR). The dried
whole plants of the Cichorium intybus and Madicago sativa
were grinded with ordinary grinder. Three hundred grams of
powder of whole plant of Cichorium intybus and Madicago
sativa and were dipped in ethanol in flask for one week. The
extract was filtered by using Rotary evaporator and
concentrated below reduced pressure. The extract was
completely dried out to condense into liquid form. Extracts
such as methanol, ethyl acetate, hexane and aqueous extracts
were prepared by using separating funnel. The extracts were
kept at 4 °C in fridge.
2.4 Plant extracts and stock solution
Extracts of medicinal plants such as ethanol, methanol, ethyl
acetate, hexane and aqueous were assessed for antimicrobial
activity. The plant extracts were adjusted to 1 milligram/6
microliters in dimethyl sulfoxide (DMSO) and diluted. The
solutions of stock were prepared. (Each 6 microliters of the
solution contains 1 milligram of the extract)
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2.5 Microbes
The test microbes were 3 Gram positive bacteria Bacillus
subtilis, Styphyloccus aureus, Bacillus atrophoeus and 3
Gram negative bacteria Salmonella typhi, Klebsella
pneumoniae and Escherichia coli and as well as fungal
strains, Candida albicans, Rhizopus sp and Aspergillus sp.
2.6 Media preparation
In this research, nutrient broth medium was used for
standardization shaking, incubation and inoculation of
selected microbes. Nutrient agar medium was added in one
litter D.W used for culturing and growth of selected
microbes.1.3g of nutrient broth and 2.8g of nutrient agar were
prepared in distilled water and transferred into flasks. Nutrient
broth (about 20 ml) was transferred into the test tubes. The
medial test tubes as well as flasks were plugged up by the
cotton and then kept in autoclave at 121 ºC under 15 pounds
pressure for fifteen minutes for sterilization. The Nutrient
medium (agar) was transferred into uncontaminated Petri
plates in a biological safety cabinet. A germ free atmosphere
was sustained for the period of pouring to keep away from
impurity. The media were allowed to solidify in Petri plates
for one hour and then such Petri plates were kept in inverted
position (to keep away from vanishing of water from the
medium within such plates) in incubator for 24 hrs at 37 ºC.
For the shaking incubation of microorganisms, about 20 ml
per flask of nutrient broth was used. Whereas, for the
standardization of culturing of microorganisms, nutrient broth
was used in test tubes.
3. Phytochemical screening
The plant extracts were screened Have vitamins i.e. ascorbic
acid, thiamine, retinol, riboflavin, niacin carotenoids, inulin,
sesquiterpenes i.e. cichorin, esculetin, esculin, lactucin and
lactucopicrin, pectin substances, tannins, amines, saponins,
triterpene glycosides, coumarin derivatives, purines base,
carotenoids, plant sterols, flavones, cumestrol
(phytoestrogens), phenolic compounds and isoflavonoids,
flavanoids, phenol, tannins, phytosterol, glycoseides volatile
oil and fatty acids.
4. Antimicrobial activities determination
4.1 Disc diffusion method
Antimicrobial activity of Cichorium intybus and Madicago
sativa were resoluted by the disk diffusion process. For the
antifungal activity, Candida albicans, Rhizopus sp. and
Aspergillus sp. was adjusted to the 10cfu/milliliter
concentration. Candida albicans, Rhizopus sp. and
Aspergillus sp. were cultured in sterilized solution of normal
saline (0.9%) and inoculated onto agar plates (Saboured
dextrose agar medium). upto 0.5 turbidity Mcfarland
principles and immunized onto agar plates (nutrient agar).
Whattman No. 1 (sterilized filter paper) discs were soaked
with extracts of plants in 1, 2 and 3 milligram and in the
concentrations of 6, 12 and 18 microliter of extract were
applied on the discs. Fungal and bacterial cultures were then
left for 18 hrs at 37 ºC.
4.2 Antimicrobial bioassay
The subsequent method was used for the determination of
antimicrobial activity.
4.2.1 Preparation and pouring of media
The required amount of 1.3 gram/100 ml of nutrient broth and
2.8gram/100 ml of nutrient agar were taken in flasks and
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dissolved in distilled water. All the equipment’s and media 60mm against Candida. albicans, Aspergillus sp. and
used in the experiment were sterilized at 121 ºC at 15 psi for Rhizopus sp. respectively. (Table#1 to Table# 3. Graph#1).
1h than sterilized media, were transferred into plates in
biological safety cabinet. The media was allowed to solidify Table 1: Antibacterial activity of Azithromycin against Gram
in Petri plates for about one hour and then such Petri plates positive bacteria
were kept in inverted position in incubator for 24 hrs at 37 ºC. Azithromycin Inhibition zone diameter
Microbes
Concentration (mm) (mean± standard deviation)
4.3.2 Streaking and incubation S. aureus 50µg/6µl 48±0.0
In biological safety cabinet, the microbe’s stock cultures B. subtilis 50µg/6µl 50±0.0
were freshened by streaking with germ-free inoculating loop B. atropoeus 50µg/6µl 65±0.0
on the agar plate. These cultures were heated for 24 hrs at 37
ºC in incubator. Table 2: Antibacterial activity of Ciprofloxacin against Gram
negative bacteria
4.4.3 Inoculation and shaking Microbes Ciprofloxacin Inhibition zone diameter
Uncontaminated agar (nutrient broth) streaked cultures were Concentration (mm) (mean± standard deviation)
inoculated. For the shaking incubation of microorganisms. E.coli 50µg/6µl 35±0.0
About 20 ml per flask of nutrient broth were used. K. pneumoniae 50µg/6µl 45±0.0
S.typhi 50µg/6µl 40±0.0
4.5.4 Turbidity and Standardization
For standardization, the microbes cultures were diluted (from Table 3: Antifungal activity of Clotrimazole against three strains
flasks) in test tube containing uncontaminated nutrient broth. Clotrimazole Inhibition zone diameter (mm)
With the help of spreader, fifty micro liters of microbial Microbes
Concentration (mean± standard deviation)
standardized cultures were spread on agar (nutrient agar). For Candida albicans 50µg/6µl 25±0.0
15 minutes these plates were kept in a refrigerator. Aspergillus sp. 50µg/6µl 65±0.0
Rhizopus sp. 50µg/6µl 60±0.0
4.6.5 Applying test
The plates containing microbial standardized inoculums were
again kept in biological safety cabinet after absorption.
Whattman No. 1 (sterilized filter paper) disc soaked with
extracts of plants in 1, 2 and 3 milligram discs in
concentrations of 6, 12 and 18 micro liter were applied on the
discs. Fungal cultures and bacterial cultures were kept for 18
hrs at 37 ºC. Antibiotics (Coltrimazole Ciprofloxacin,
Azithromycin,) were applied at the doze of 6 micro liter/disc
as a positive control against fungi, Gram negative and Gram
positive bacteria, respectively.

4.7.6 Measurement of zone of inhibition

By measuring the zones of inhibition in each extract
antimicrobial activity was recorded properly. By taking the
mean of inhibitory zones, the results were compiled.
5. Statistical analysis
Standard error means were found with the help of SPSS.V.16.
6. Results and Discussion
The standard antibiotic Ciprofloxacin was used against three
Gram negative bacteria (Escherichia coli, Klebsella
pneumonia and Salmonella typhi) and Azithromycin against
three Gram positive bacterial strain (Styphyloccus aureus,
Bacillus subtilis and Bacillus atropoeus) while Coltrimazole
was used against fungal strains (Candida albicans,
Aspergillus sp. and Rhizopus sp.) in fifty microgram per six
microliter concentration. Azithromycin demonstrated (50mm,
48mm and 65mm zone of inhibition) against Bacillus subtilis,
Staphylococcus aureus and Bacillus atropheous.
Ciprofloxacin showed (40mm, 35mm and 45mm against
Salmonella typhi, Escherichia coli and Klebsella pneumoniae
respectively While Coltrimazole showed 25mm, 65mm and
Graph 1: Antimicrobial activities of standard antibiotics against
different bacteria and fungi
6.1 Cichorium intybus
Five different solvents (ethanol, methanol, hexane, ethyl
acetate, and aqueous) were used for the antimicrobial activity
of Cichorium intybus (whole plant).
6.2.1 Ethanolic extract of Cichorium intybus
The results of the antimicrobial activities measured in term of
diameter of zone of inhibition in mm. The antimicrobial
activity of the ethanolic extract of Cichorium intybus is
showed (Table 4.2.1. Graph 4.2.1 and Fig. 6). Ethanolic
extract of Cichorium intybus showed the maximum inhibitory
activity against the Gram positive bacterium, B. subtilis
(22±0.1 mm zone of inhibition), against the Gram negative
bacterium, K. pneumoniae (22.5±0.5 mm zone of inhibition)
and fungus, C. albicans (19±0.1 mm zone of inhibition).
While the controls results were 50±0.0 mm, 40±0.0 mm and
25±0.0 mm, respectively.

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Table 4: Antimicrobial activity of the ethanolic extract of Cichorium intybus

Microbes Extract Concentration (mg/µl) Inhibition zone diameter (mm) (mean± standard deviation)
6 mg/µl 12.5±1.8
S. aureus 12 mg/µl 15±0.1
18 mg/µl 16.5±0.5
6 mg/µl 14.5±0.5
B. subtilis 12 mg/µl 19.5±1.3
18 mg/µl 22±0.1
6 mg/µl 12±0.0
B. atropoeus 12 mg/µl 14.5±0.5
18 mg/µl 15.5±0.5
6 mg/µl 10.5±1.3
E. coli 12 mg/µl 14.5±0.5
18 mg/µl 17±0.1
6 mg/µl 11±0.1
S. typhi 12 mg/µl 13.5±0.5
18 mg/µl 15±0.2
6 mg/µl 14±0.0
K. pneumoniae 12 mg/µl 19±0.1
18 mg/µl 22.5±0.5
6 mg/µl 11.5±0.5
C. albicans 12 mg/µl 16.5±0.5
18 mg/µl 19±0.1
6 mg/µl 12±0.1
Rhizopus sp. 12 mg/µl 17±0.1
18 mg/µl 20±0.1
6 mg/µl 11±0.1
Aspergillus sp. 12 mg/µl 15±0.1
18 mg/µl 18±0.2

Graph 2: Antimicrobial activity of the ethanolic extract of Cichorium intybus

Fig 1: Ethanolic extract of Cichorium intybus against K.pneumoniae Fig 2: Use of ciprofloxacin (standard) against K. pneumoniae
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6.3.2 Methanolic extract of cichorium intybus
The antimicrobial activity of the methanolic extract of
Cichorium intybus is showed (Table 4.2.2 and Graph 4.2.2.)
Methanolic extract of Cichorium intybus showed the
maximum inhibitory activity against the Gram positive
bacterium, B. subtilis (15.5±0.5 mm zone of inhibition),
against the Gram negative bacterium, E. coli (17±0.1 mm
zone of inhibition) and fungus, C. albicans (14±0.5 mm zone
of inhibition). While the controls resuls were 50±0.0 mm,
35±0.0 mm and 25±0.0 mm, respectively.

Table 5: Antimicrobial activity of the methanolic extract of Cichorium intybus

Microbes Extract Concentration (mg/µl) Inhibition zone diameter (mm) (mean± standard deviation)
6 mg/µl 9.5±0.5
S. aureus 12 mg/µl 11±0.1
18 mg/µl 12±0.2
6 mg/µl 12±0.2
B. subtilis 12 mg/µl 14±0.2
18 mg/µl 15.5±0.5
6 mg/µl 11.5±0.5
B. atropoeus 12 mg/µl 14.5±0.5
18 mg/µl 16±0.1
6 mg/µl 13±0.1
E. coli 12 mg/µl 14.5±0.5
18 mg/µl 17±0.1
6 mg/µl 12.5±0.1
S. typhi 12 mg/µl 13±0.1
18 mg/µl 14±0.1
6 mg/µl 11±0.5
K. pneumoniae 12 mg/µl 12.5±0.5
18 mg/µl 13±0.1
6 mg/µl 9±0.1
C. albicans 12 mg/µl 12±0.0
18 mg/µl 14.5±0.5
6 mg/µl 10±0.1
Rhizopus sp. 12 mg/µl 14±0.2
18 mg/µl 17±0.2
6 mg/µl 11±1.7
Aspergillus sp. 12 mg/µl 15±0.1
18 mg/µl 18±0.2

Graph 3: Antimicrobial activity of the methanolic extract of Cichorium intybus

6.4.3 Ethyl acetate extract of Cichorium intybus
The antimicrobial activity of the ethyl acetate extract of
Cichorium intybus is showed in (Table 4.2.3. and Graph
4.2.3). Ethyl acetate extract of Cichorium intybus showed the
maximum inhibitory activity against the Gram positive
bacterium, S. aureus (13±0.1 mm zone of inhibition), against
the Gram negative bacterium, E. coli (18±0.1 mm zone of
inhibition) and fungus, C. albicans (18±0.1 mm zone of
inhibition). While the controls results were 48±0.0 mm,
35±0.0 mm and 25±0.0 mm, respectively.

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Table 6: Antimicrobial activity of the ethyl acetate extract of Cichorium intybus

Microbes Extract Concentration (mg/µl) Inhibition zone diameter (mm) (mean± standard deviation)
6 mg/µl 11±0.1
S. aureus 12 mg/µl 12.5±0.5
18 mg/µl 13±0.1
6 mg/µl 10.5±0.5
B. subtilis 12 mg/µl 13±0.1
18 mg/µl 14±0.1
6 mg/µl 10±0.0
B. atropoeus 12 mg/µl 14±0.1
18 mg/µl 19±0.1
6 mg/µl 11±0.1
E. coli 12 mg/µl 14.5±0.5
18 mg/µl 18±0.1
6 mg/µl 8±0.1
S. typhi 12 mg/µl 11.5±0.5
18 mg/µl 14±0.1
6 mg/µl 8±0.1
K. pneumoniae 12 mg/µl 11.5±0.5
18 mg/µl 13±0.1
6 mg/µl 12±0.1
C. albicans 12 mg/µl 17±0.0
18 mg/µl 18±0.1
6 mg/µl 11±0.1
Rhizopus sp. 12 mg/µl 13.5±0.5
18 mg/µl 15±0.1
6 mg/µl 9±0.1
Aspergillus sp. 12 mg/µl 14±0.1
18 mg/µl 17±0.1

Graph 4: Antimicrobial activity of the ethyl acetate extract of Cichorium intybus

6.5.4 Hexane extract of Cichorium intybus
The antimicrobial activity of the hexane extract of Cichorium
intybus is showed in (Table 4.2.4. and Graph 4.2.4). Hexane
extract of Cichorium intybus showed the maximum inhibitory
activity against the Gram positive bacterium, B. subtilis
(17±0.3 mm zone of inhibition), against the Gram negative
bacterium, E. coli (17±0.1 mm zone of inhibition) and fungus,
C. albicans (19±0.1 mm zone of inhibition). While the
controls results were 50±0.0 mm, 35±0.0 mm and 25±0.0 mm
respectively.

Table 7: Antimicrobial activity of the hexane extract of Cichorium intybus

Microbes Extract Concentration (mg/µl) Inhibition zone diameter (mm) (mean± standard deviation)
6 mg/µl 9±0.2
S. aureus 12 mg/µl 13±0.2
18 mg/µl 15±0.0
6 mg/µl 11±0.1
B. subtilis 12 mg/µl 14.5±0.5
18 mg/µl 17±0.3
B. atropoeus 6 mg/µl 10±0.0
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Journal of Pharmacognosy and Phytochemistry

12 mg/µl 12.5±0.5
18 mg/µl 14±0.2
6 mg/µl 10.5±0.5
E.coli 12 mg/µl 14.5±0.5
18 mg/µl 17±0.1
6 mg/µl 8.5±0.5
S. typhi 12 mg/µl 12.5±0.5
18 mg/µl 15±0.1
6 mg/µl 11±0.0
K. pneumoniae 12 mg/µl 13.5±0.5
18 mg/µl 14±0.1
6 mg/µl 12±0.1
C. albicans 12 mg/µl 16±0.1
18 mg/µl 19±0.1
6 mg/µl 11±0.1
Rhizopus sp. 12 mg/µl 14±0.0
18 mg/µl 18±0.0
6 mg/µl 10.5±0.5
Aspergillus sp. 12 mg/µl 14.5±0.5
18 mg/µl 16±0.0

Graph 5: Antimicrobial activity of the hexane extract of Cichorium intybus

6.6.5 Aqueous extract of Cichorium intybus
The antimicrobial activity of the aqueous extract of
Cichorium intybus is showed in (Table 4.2.5. and Graph
4.2.4). Aqueous extract of Cichorium intybus showed the
maximum inhibitory activity against the Gram positive
bacterium, S. aureus (11±0.1 mm zone of inhibition), against
the Gram negative bacterium, E. coli (14±0.0 mm zone of
inhibition) and fungus, C. albicans (9.5±0.1 mm zone of
inhibition). While the controls results were 48±0.0 mm,
35±0.0 mm and 25±0.0 mm, respectively.

Table 8: Antimicrobial activity of the aqueous extract of Cichorium intybus

Microbes Extract Concentration (mg/µl) Inhibition zone diameter (mm) (mean± standard deviation)
6 mg/µl 7±0.0
S. aureus 12 mg/µl 9±0.1
18 mg/µl 11±0.1
6 mg/µl 8±0.1
B. subtilis 12 mg/µl 10±0.1
18 mg/µl 12±0.1
6 mg/µl 12.5±0.5
B. atropoeus 12 mg/µl 13.5±0.5
18 mg/µl 14.5±0.5
6 mg/µl 10.5±0.5
E. coli 12 mg/µl 12±0.0
18 mg/µl 14±0.0
6 mg/µl 7.5±0.5
S. typhi 12 mg/µl 10±0.1
18 mg/µl 12±0.1
6 mg/µl 12.5±0.5
K. pneumoniae
12 mg/µl 13±0.1
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18 mg/µl 14±0.1
6 mg/µl 8±0.1
C. albicans 12 mg/µl 9±0.0
18 mg/µl 9.5±0.5
6 mg/µl 12±0.1
Rhizopus sp. 12 mg/µl 15±0.1
18 mg/µl 17±0.1
6 mg/µl 11±0.1
Aspergillus sp. 12 mg/µl 14.5±0.5
18 mg/µl 17±0.1

Graph 6: Antimicrobial activity of the aqueous extract of Cichorium intybus

7. Medicago sativa Ethanolic extract of Medicago sativa showed the maximum

Five different solvent were used such as (ethanol, methanol,
hexane, ethyl acetate, and aqueous). Different solvents were
tested against different species of bacteria and fungi.
7.1 Ethanolic extract of Medicago sativa
The antimicrobial activity of the ethanolic extract of
Medicago sativa is showed in (Table 4.3.1. and Graph 4.3.1).
inhibitory activity against the Gram positive bacterium, B.
subtilis (21±0.1 mm zone of inhibition), against the Gram
negative bacterium, E. coli (15±0.1 mm zone of inhibition)
and fungus, C. albicans (15.5±0.1 mm zone of inhibition).
While the controls results were 50±0.0 mm, 35±0.0 mm and
25±0.0 mm, respectively.

Table 9: Antimicrobial activity of the ethanolic extract of Medicago sativa

Microbes Extract Concentration (mg/µl) Inhibition zone diameter (mm) (mean± standard deviation)
6 mg/µl 9.5±0.5
S. aureus 12 mg/µl 12.5±0.5
18 mg/µl 15±0.1
6 mg/µl 10.5±0.5
B. subtilis 12 mg/µl 15.5±0.5
18 mg/µl 21±0.5
6 mg/µl 11±0.1
B. atropoeus 12 mg/µl 15±0.0
18 mg/µl 19±0.1
6 mg/µl 11±0.1
E. coli 12 mg/µl 13.5±0.5
18 mg/µl 15±0.1
6 mg/µl 11.5±0.5
S. typhi 12 mg/µl 14.5±0.5
18 mg/µl 17±0.1
6 mg/µl 13±0.0
K. pneumoniae 12 mg/µl 15.5±0.5
18 mg/µl 19±0.1
6 mg/µl 11.5±0.5
C. albicans 12 mg/µl 14.5±0.5
18 mg/µl 15.5±0.5
6 mg/µl 10±0.0
Rhizopus sp.
12 mg/µl 14±0.1

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18 mg/µl 18±0.0
6 mg/µl 15±0.0
Aspergillus sp. 12 mg/µl 18±0.1
18 mg/µl 19±0.2

Graph 7: Antimicrobial activity of the ethanolic extract of Medicago sativa

7.2 Methanol Extract of Medicago sativa
The antimicrobial activity of the methanolic extract of
Medicago sativa is showed in (Table 4.3.2. Graph 4.3.2. And
Fig.8). Methanolic extract of Medicago sativa showed the
maximum inhibitory activity against the Gram positive
bacterium, B. subtilis (15±0.1 mm zone of inhibition), against
the Gram negative bacterium, E. coli (22±0.2 mm zone of
inhibition) and fungus, C. albicans (19±0.1 mm zone of
inhibition). While the controls results were 50±0.0 mm,
35±0.0 mm and 25±0.0 mm, respectively.

Table 10: Antimicrobial activity of the methanolic extract of Medicago sativa

Microbes Extract Concentration (mg/µl) Inhibition zone diameter (mm) (mean± standard deviation)
6 mg/µl 10±1.7
S. aureus 12 mg/µl 11.5±1.5
18 mg/µl 12±0.0
6 mg/µl 12.5±0.5
B. subtilis 12 mg/µl 14.5±0.5
18 mg/µl 15±0.1
6 mg/µl 15.5±1.5
B. atropoeus 12 mg/µl 17.5±2.5
18 mg/µl 21±0.0
6 mg/µl 11.5±1.5
E. coli 12 mg/µl 15.5±0.5
18 mg/µl 22±0.2
6 mg/µl 9±0.1
S. typhi 12 mg/µl 12±0.0
18 mg/µl 15±0.2
6 mg/µl 14.5±0.5
K. pneumoniae 12 mg/µl 15±0.0
18 mg/µl 17±0.2
6 mg/µl 13±0.1
C. albicans 12 mg/µl 17±0.2
18 mg/µl 19±0.1
6 mg/µl 14±0.2
Rhizopus sp. 12 mg/µl 18±0.2
18 mg/µl 19±0.0
6 mg/µl 10±0.1
Aspergillus sp. 12 mg/µl 14±0.1
18 mg/µl 20±0.0

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Graph 7: Antimicrobial activity of the methanolic extract of Medicago sativa

Fig 3: Ethanolic extract of Medicago sativa against C.albicans Fig 4: Use of Ciprofloxacin (standard) against C.albicans

7.3 Ethyl acetate extract of Medicago sativa
The antimicrobial activity of the ethyl acetate extract of
Medicago sativa is showed in (Table 4.3.3. and Graph 4.3.3).
Ethyl acetate extract of Medicago sativa showed the
maximum inhibitory activity against the Gram positive
bacterium, B. subtilis (20±0.2 mm zone of inhibition), against
the Gram negative bacterium, E. coli (13±0.1 mm zone of
inhibition) and fungus, C. albicans (14±0.0 mm zone of
inhibition). While the controls results were 50±0.0 mm,
35±0.0 mm and 25±0.0 mm, respectively.

Table 11: Antimicrobial activity of the ethyl acetate extract of Medicago sativa
Microbes Extract Concentration (mg/µl) Inhibition zone diameter (mm) (mean± standard deviation)
6 mg/µl 12.5±1.5
S. aureus 12 mg/µl 15.5±0.5
18 mg/µl 17.5±1.5
6 mg/µl 12.5±0.5
B. subtilis 12 mg/µl 15±0.1
18 mg/µl 20±0.2
6 mg/µl 12.5±0.5
B. atropoeus 12 mg/µl 15.5±1.5
18 mg/µl 17±0.0
6 mg/µl 11±0.1
E. coli 12 mg/µl 12±0.0
18 mg/µl 13±0.1
6 mg/µl 7±0.0
S. typhi 12 mg/µl 10±0.2
18 mg/µl 12±0.0
6 mg/µl 10.5±0.5
K. pneumoniae 12 mg/µl 13±0.2
18 mg/µl 15±0.1
6 mg/µl 10±0.2
C. albicans
12 mg/µl 12±0.2
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18 mg/µl 14±0.0
6 mg/µl 0±0.0
Rhizopus sp. 12 mg/µl 0±0.0
18 mg/µl 0±0.0
6 mg/µl 7±0.1
Aspergillus sp. 12 mg/µl 12.5±0.5
18 mg/µl 14±0.0

Graph 8: Antimicrobial activity of the ethyl acetate extract of Medicago sativa

7.4 Hexane extract of Medicago sativa
The antimicrobial activity of the hexan extract of Medicago
sativa is showed in (Table 4.3.4. and Graph 4.3.4). Hexan
extract of Medicago sativa showed the maximum inhibitory
activity against the Gram positive bacterium, B. subtilis
(17±0.0 mm zone of inhibition), against the Gram negative
bacterium, E. coli (18.5±0.5 mm zone of inhibition) and
fungus, C. albicans (17±0.2 mm zone of inhibition). While
the controls results were 50±0.0 mm, 35±0.0 mm and 25±0.0
mm, respectively.

Table 12: Antimicrobial activity of the hexane extract of Medicago sativa

Microbes Extract Concentration (mg/µl) Inhibition zone diameter (mm) (mean± standard deviation)
6 mg/µl 10.5±0.5
S. aureus 12 mg/µl 12.5±0.5
18 mg/µl 13.5±0.5
6 mg/µl 13.5±0.5
B. subtilis 12 mg/µl 15.5±0.5
18 mg/µl 17±0.0
6 mg/µl 11.5±0.5
B. atropoeus 12 mg/µl 14±0.0
18 mg/µl 18±0.0
6 mg/µl 12.5±1.5
E. coli 12 mg/µl 15.5±2.5
18 mg/µl 18.5±0.5
6 mg/µl 7.5±1.5
S. typhi 12 mg/µl 10.5±1.5
18 mg/µl 12.5±0.5
6 mg/µl 8±0.0
K. pneumoniae 12 mg/µl 9.5±0.5
18 mg/µl 10±0.0
6 mg/µl 12±0.2
C. albicans 12 mg/µl 15±0.1
18 mg/µl 17±0.2
6 mg/µl 11.5±0.5
Rhizopus sp. 12 mg/µl 12.5±0.5
18 mg/µl 14±0.0
6 mg/µl 11.5±1.5
12 mg/µl 16.5±1.5
Aspergillus s
18 mg/µl 18.5±0.5

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Graph 9: Antimicrobial activity of the hexane extract of Medicago sativa

7.5 Aqueous Extract of Medicago sativa
The antimicrobial activity of the aqueous extract of Medicago
sativa is showed in (Table 4.3.5. and Graph 4.3.5). Aqueous
extract of Medicago sativa showed the maximum inhibitory
activity against the Gram positive bacterium, S. aureus
(10±0.1 mm zone of inhibition), against the Gram negative
bacterium, E. coli (10.5±0.5 mm zone of inhibition) and
fungus, C. albicans (13.5±0.5 mm zone of inhibition). While
the controls results were 48±0.0 mm, 35±0.0 mm and 25±0.0
mm, respectively.

Table 13: Antimicrobial activity of the aqueous extract of Medicago sativa

Microbes Extract Concentration (mg/µl) Inhibition zone diameter (mm) (mean± standard deviation)
6 mg/µl 6±0.1
S. aureus 12 mg/µl 9±0.0
18 mg/µl 10±0.1
6 mg/µl 5±0.0
B. subtilis 12 mg/µl 6±0.0
18 mg/µl 6.5±0.5
6 mg/µl 9±0.1
B. atropoeus 12 mg/µl 12±0.0
18 mg/µl 15.5±0.5
6 mg/µl 7±0.1
E. coli 12 mg/µl 9±0.1
18 mg/µl 10.5±0.5
6 mg/µl 5.5±0.5
S. typhi 12 mg/µl 7.5±0.5
18 mg/µl 8±0.0
6 mg/µl 8±0.1
K. pneumoniae 12 mg/µl 9.5±0.5
18 mg/µl 10±0.0
6 mg/µl 5±0.1
C. albicans 12 mg/µl 9.5±0.5
18 mg/µl 13.5±0.5
6 mg/µl 13.5±0.5
Rhizopus sp. 12 mg/µl 15.5±0.5
18 mg/µl 16.5±0.5
6 mg/µl 14±0.1
Aspergillus sp. 12 mg/µl 16±0.1
18 mg/µl 17.5±0.5

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Graph 11: Antimicrobial activity of the aqueous extract of Medicago sativa
8. Discussion
Out of three studied plants during the present studies
Cichorium intybus L. showed the highest inhibitory activity
followed by Medicago sativa, and Oxalis corniculata.
Ethanolic extract showed the best efficacy followed by
methanolic, ethyl acetate, hexane and aqueous extracts. The
inhibition ratio was more against fungi as compared Gram
negative bacteria, and Gram positive bacteria respectively.
Ethanolic extracts of whole plant of Cichorium intybus
showed the highest inhibitory activity against K. pneumoniae
(22.5±0.5 mm zone of inhibition) a Gram negative bacterium.
Similarly, against the Gram positive bacteria the most
significant results were in the case of ethanolic extract against
B. subtilis (22±0.1 mm zone of inhibition). In addition to this,
the growth inhibition was more in the case of ethanolic extract
of Cichorium intybus against the fungus, C. albicans (19±0.1
mm zone of inhibition). Previously, Rehman et al. (2014)
worked on the antibacterial activities of the ethanolic extract
of Cichorium intybus and found the significant results against
Gram positive Gram negative bacteria and fungi. Similar
finding were reported by Khakzadihe et al. (2014) [24].
Nandagopal and Kumari (2007). Reported that Cichorium
intybus has inulin, esculin, volatile compound, coumarins,
flavonoids and vitamins in considerable quantity. These
compounds are highly antimicrobial agents. Our findings are
agreement with these studies. Methanolic extract of Medicago
sativa showed the maximum inhibitory activity against E. coli
(22±0.21 mm zone of inhibition) a Gram negative bacterium.
Similarly, against the Gram positive bacterium, the most
significant results were in the case of methanolic extract
against B. subtilis (15±0.1 mm zone of inhibition) In addition
to this, the growth inhibition was more in the case of
methanolic extract of Medicago sativa against C. albican
(19±0.1 mm zone of inhibition). Previously Doss et al. (2011)
worked on the antimicrobial activities of the methanolic
extract of Medicago sativa and found the significant results
against Gram positive and Gram negative bacteria and fungi.
Similar finding were reported by Baloch et al. (2013) [4].
Desoukey (2015) [7] reported that Medicago sativa contains
flavonoids, tannins, alkaloids, saponin and glycosides
compounds in considerable quantity. The strong antimicrobial
agents. Our finding are in agreement with Desoukey (2015)
[7]
. Our result are supported by the previous raport. When
ethanolic extract of Oxalis corniculata was tested the
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maximum inhibitory activity against the fungus C. albican
was found (17.5±2.5 mm zone of inhibition). Similarly,
against the Gram positive and Gram negative bacteria,
significant results were in the case of ethanolic extract against
S. aureus (19±0.5 mm zone of inhibition) and E. coli (15±0.1
mm zone of inhibition) Feresin et al. (2001) [9] worked on the
antimicrobial activities of the ethanolic extract of Oxalis
corniculata and found the encouraging results against the
Gram positive and Gram negative bacteria and fungi. Similar
finding were reported by Handali et al. (2011) [14].
Raghavendra et al. (2005) reported that Oxalis corniculata
contains carbohydrates and glycosides, phytosterols, phenolic
compounds, flavonoids, proteins aminoacids and volatile oils
in enough quantity the presence of antimicrobial compounds
in these plants justifies our finding. As a whole ethanol
extract were found as more effective because ethanol, as a
dehydrating agent, causes the cell membrane damage, rapid
denaturalization of proteins subsequently metabolism
interference and cell lysis (Larson and Morton, 1991). The
studied plants have varying degree of inhibitory potential
which must be explored with respect to biochemical point of
view.
9. Conclusion
The conclusion is generalized as:
 The order of effectiveness of extracts (plant-wise) is
Cichorium intybus > Medicago sativa > Oxalis
corniculata.
 The order of effectiveness of extracts (solvent -wise) is
ethanol > methanol > ethyl acetate > hexane > aqueous
extract
 The order of inhibition according to the different groups
of organisms was fungi > Gram negative bacteria> Gram
positive bacteria.
 The most resistant Gram positive bacterium was Bacillus
atropoeus, Gram negative bacterium was Salmonella
typhi and fungus was Rhizophus sp.
 The most sensitive Gram positive bacterium was Bacillus
subtilise, Gram negative bacterium was Escherichia coli
and fungus was Candida albicans.
10. Acknowledgments
The authors are very thankful to my labefellow and my great
supervisor for sharing their indentions knowledge during
Journal of Pharmacognosy and Phytochemistry

current study. We are thankful to herbarium of Pakistan (ISL), extract of Oxalis corniculata L. with antibacterial effects
Abdulwali khan university, Mardan, Pakistan for their role in of common antibiotics in Staphylococcus aureous and E.
plant identification and for submission of plant specimens for coli infections. J Med. Plants. 2010; 9:33.
future referenc 16. Hulina N. Wild marigold-Tagetes minuta L. New weed
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