Sagar Bashyal1, Avijit Guha2
of Biotechnology, IILM College of Engineering and Technology, U.P, India
Objective: To analyze the presence of
different phytochemicals and to investigate the antimicrobial activity of Trachyspermum ammi seeds.
Methanol, acetone, chloroform and distilled water (aqueous) extracts
were prepared using a Soxhlet apparatus, agar well diffusion method was used
for the antimicrobial test.
Results: The results revealed that
flavonoids and saponins presence was found in methanol, acetone, chloroform and
distilled water extracts. Alkaloids and phenols presence was seen in methanol
and aqueous extracts. Glycosides and carbohydrates in methanol, chloroform, and
aqueous extracts. Further, Proteins, terpenoids, and tannins presence were
found in methanol, chloroform, and aqueous extracts respectively. Ciprofloxacin
was taken as a control against E. coli.
The maximum zone of inhibition was found in the methanolic extract (13.5 mm). Acetone,
chloroform, and water extracts showed 9mm, 10.5mm, 11mm respectively, while
Ciprofloxacin (control) showed 17.5mm of the zone of inhibition.
results conclude that different extracts of Trachyspermum ammi seeds exert biological properties due to the presence of various
chemical constituents. Thus, it can be used to obtain novel antibacterial
compounds for the treatment of infectious diseases in the future.
KEYWORDS: Trachyspermum ammi, Phytochemicals, Solvent
extraction, Antimicrobial activity,
Since prehistoric times,
medicinal plants, also known as medicinal herbs have been discovered and used
in traditional medicine practices. Naturally found plants synthesize many
chemical compounds for botanical functions, including defense against insects, fungi,
diseases, and herbivorous mammals. There is a continuous need for the development of
new effective antimicrobial drugs because of the emergence of new infectious
diseases and drug resistance 1,2. In the present scenario, herbal drugs and
their formulations has become an alternative to the synthetic drugs 3. The
plant-derived natural products are the products of secondary metabolism; the
compounds which are not essential for existence in laboratory conditions, but
are certainly responsible for self-defense coordination in natural conditions 4. Ajwain, Trachyspermum ammi, (L.)
Sprague ex-belonging to the family Apiaceae is also known as Ajowan caraway,
Oomam in Tamil, bishop weeds or Carom. Trachyspermum ammi is mostly found
throughout India and is cultivated in Rajasthan and Gujarat. Trachyspermum ammi
is a seed which is native of Egypt and is cultivated in Iraq, Afghanistan, Pakistan,
and India. In India, the seeds are cultivated in Gujarat, Rajasthan, Madhya
Pradesh, Uttar Pradesh, Maharashtra, Bihar and West Bengal 5. The oil obtained
from the seeds exhibits fungicidal 6 antimicrobial 7 and anti-aggregatory
effects on humans 8. It is an important remedial agent for flatulence, atonic
dyspepsia and diarrhea 9. An essential oil obtained after the
hydrodistillation of the fruits of the plant consists thymol, gamma-terpinene,
and p-cymene as well as more than 20 trace compounds (predominately terpenoids)
10. Trachyspermum ammi has been shown to possess antimicrobial,11 hypolipidemic,12
digestive stimulant,13 antispasmodic, Broncho-dilating,14 antihypertensive,
hepatoprotective, diuretic,15 abortifacient,16 anti-lithiasis,
galactogogic,17 antiplatelet-aggregator,18 anti-inflammatory,19
antitussive,20 anti-filarial,21 gastroprotective,22 nematicidal,23
anthelmintic,24 detoxification of aflatoxins,25 and ameliorative effects.26
Therapeutic uses of Trachyspermum ammi fruits include; stomachic, expectorant
and carminative,27 antiseptic and amoebiasis, antimicrobial. The current
study was aimed to carry out the phytoconstituents testing and to analyze
antibacterial activity against E. coli using the extracts prepared in the
Tracheobionta, Vascular plants
Division: Magnoliophyta, Flowering plants
Plant Material Collection and Authentication:
Ajwain (Trachyspermum ammi) was obtained from the local market and field of
Greater Noida, India. The seeds were verified by Associate Professor Dr. Avijit
Guha in the Department of Biotechnology, IILM College of Engineering and
Technology. The seeds were dried using an oven and powdered using an electric
grinder. The study of plant morphology was done using a simple determination
technique, the shape, size, color, odor.
of crude extracts:
About 3 gm of coarse powder
sample in each 4-conical flask (200ml) was Soxhlet with distilled water (50
ml), methanol and water (7:3, v/v), chloroform and acetone (70%) for 48 hours
in the successive mode using a Soxhlet apparatus.
The extract obtained was further
concentrated using a rotary evaporator (Rotavap, Heidolph Labortechnik VV 2000)
with the water bath set at 55°C. The dried extracts obtained was
weighed and percentage extracted was calculated which was then transferred to airtight
jars and stored at 4°C in the refrigerator for future use. The crude extracts obtained was taken for further investigation of
phytochemicals, and antimicrobial evaluation.
The Petri dishes and pipettes
packed into metal canisters were appropriately sterilized in the hot air oven
at 170°C for 1 h at each occasion. Laminar air flow was
cleaned with 70% ethanol before starting the culturing of microbes.
of Test Organisms:
The E. coli sample was maintained weekly by sub-culturing on agar
slants. Before starting the experiment, the cells were activated by successive
sub-culturing and incubation.
The phytochemical tests were carried out for four
different extracts as mentioned above using the standard method 29-32.
To 0.5 ml of plant extracts the
Dragendorff’s reagent was added. A reddish-brown precipitate confirms that test
About 0.5 mg of plant extracts
was shaken with 2.5 ml of water, filtered and the filtrate was concentrated. To
this 1.25 ml of Benedict’s solution was added and boiled for 5 minutes. Brick
red precipitate indicated the presence of carbohydrates.
A pinch of the dried plant extracts was added to 3
ml of distilled water. The mixture was shaken vigorously for a few minutes. Foam
formation indicated the presence of saponin.
To 0.5 ml of plant extracts few
drops of sodium hydroxide solution were added. A yellow coloration which turns
to colorless by the addition of a few drops of dilute acetic acid indicated the
presence of flavonoids.
To 0.5 ml of plant extracts, 4%
NaOH solution and a few drops of 1% CuSO4 solution were added. The violet color
appears, indicating the presence of protein.
To 0.5 ml of plant extracts, few
drops of 0.1% ferric chloride solution were added. Formation of brownish green
or a blue-black coloration indicating the presence of tannins.
steroids and terpenoids:
0.5 ml of each extract was
treated in chloroform with a few drops of concentrated sulphuric acid, shaken
well and allow to stand for some time. After few minutes red color on the lower
layer indicates the presence of sterols and the formation of a yellow colored
lower layer indicates the presence of terpenoids.
To 1 ml of plant extract, 1 ml of
benzene and 0.5 ml of dilute ammonia solution was added. A reddish pink color
indicated the presence of glycosides.
OF ANTIMICROBIAL ACTIVITY:
Test microorganisms and control:
extracts of the seeds of Trachyspermum
ammi were tested against E. coli.
The sample of E. coli was obtained
from the sample taken from clinical sites. The isolated culture in the nutrient
agar medium was sub-cultured in a nutrient broth, which was further kept at
37°C for 24 hours.
Ciprofloxacin was taken as the control for E.
coli cells. And the zone of inhibition was compared with the control.
diffusion method was used to determine the antimicrobial activity. E. coli suspension was seeded on two
MHA (Muller Hinton Agar) plates which were maintained in the sterilized
condition. In each of these plates, two wells were punched using the sterilized
corn borer. Using a micropipette 70 µl of methanol extract and control was
loaded in the first plate (well 1 and 2) and again, the same concentration of
acetone, chloroform, and aqueous extract was loaded in the second plate in
respective numbered wells. Plates were incubated for 24 hours at 37°C.
antimicrobial activity was analyzed using the diameter measurement method of
inhibition zone formed around well. The effects were compared with that of the
standard antibiotic Ciprofloxacin.
test of three different extracts prepared using a Soxhlet apparatus (fig. 1) is
shown in Table 1. Flavonoids and saponins presence was found in methanol,
acetone, chloroform and distilled water (aqueous) extracts. Alkaloids and
phenols presence was seen in methanol and aqueous extract. Alkaloids show a
potent antioxidant property.
antioxidant is an important property by which living organisms can neutralize
the toxic and cell-damaging the molecules called free radicals, which are produced
during various metabolic reactions of the body. 33
Glycosides and carbohydrates presence were seen in methanol, chloroform, and
aqueous extracts. Further, Proteins, terpenoids, and tannins presence were
found in methanol, chloroform, and aqueous extracts respectively. Plant
terpenoids are used extensively for their aromatic qualities and play a role in
traditional herbal remedies. 34
Fig. 1. Soxhlet apparatus
Table 1. Preliminary phytoconstituents screening of
different extracts of Trachyspermum ammi.
sign indicates the presence and ‘– ‘sign indicates absence.
After incubation for 24 hours
from the time of loading of extracts, inhibition zones were measured. From this
process, we came to know that different forms of extracts have different
anti-microbial potential. The controlled region showed inhibition zone of
17.5mm, the methanolic, acetone, chloroform and aqueous extracts showed
inhibition zone of 13.5mm, 19mm,10.5mm, and 11mm (Table 2, fig. 2.). A maximum
zone of inhibition was found in the methanolic extract.
Table 2: Antimicrobial activity of four different extracts of Trachyspermum ammi on E. coli
Zone of Inhibition (mm)
Fig. 2. A
chart showing different inhibition zone for four different extracts.
The study revealed that the seeds
of Trachyspermum ammi have potent
antimicrobial activity and can be used for pharmacological evaluation, drug
discovery, and treatment of various infectious diseases. We found that the
seeds contain alkaloids, carbohydrates, glycosides, flavonoids, proteins,
terpenoids, tannins, phenols which have the high medicinal purpose. The high
zone of inhibition was seen in the methanolic extract which signifies the high
antimicrobial action than other three extracts. This medicinal plant needs a
scientific exploration of the hidden curative and therapeutic potential.
The authors are thankful for the
Head, Department of Biotechnology, IILM College of Engineering and Technology,
Greater Noida to provide necessary laboratory facilities to conduct this
The authors declare that no conflict of interest occurred
during the work.
Sagar Bashyal carried out the
experiment, wrote the manuscript along with the support and supervision of
Associate Professor Dr. Avijit Guha. Both authors conceived the original idea.
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