Sagar Bashyal1, Avijit Guha2
of Biotechnology, IILM College of Engineering and Technology, U.P, India
Objective: To identify medicinally
important phytochemicals and evaluate the antimicrobial potential of Trachyspermum ammi seeds.
Methods: Four different extracts
(methanol, acetone, chloroform, and water) were prepared using a Soxhlet
apparatus, antimicrobial activity was tested using Agar well diffusion
Results: The results revealed the
presence of flavonoids and saponins in all the extracts prepared. Similarly, alkaloids
and phenols presence were obtained 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. At 70 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.
Conclusion: 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 methods 29-32.
About 1ml of aqueous extract was
taken and stirred properly with the addition of 1 ml of the Dragendorff’s
reagent A reddish-brown precipitate confirms that test as positive.
About 1 mg of seed extracts was
shaken with 2.5 ml of water, filtered and the filtrate was concentrated. 1 ml
of Benedict’s solution was added and boiled for 5 minutes. Brick red
precipitate indicated the presence of carbohydrates.
About 0.5 mg of the dried seed
extracts was added to 3 ml of distilled water and concentrated. The mixture was
shaken vigorously for a few minutes. Foam formation indicated the presence of
About 3 ml of aqueous extract was
added with few drops of sodium hydroxide solution. 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 seed extracts
solution, 4% NaOH solution and a few drops of 1% CuSO4 solution were added. The
violet color indicated the presence of protein.
To 3 ml of seed extracts, few
drops of 0.1% ferric chloride solution were added and allowed to stand for few
minutes. Formation of brownish green or a blue-black coloration indicating the
presence of tannins.
steroids and terpenoids:
1 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.
About 3 ml of seed aqueous extract was shaken
vigorously, 1 ml of benzene and 1 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 medium for isolation and
sub-culture was sterilized using wet heat sterilization method. 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 for incubation. Ciprofloxacin was taken
as the control for E. coli cells. And
the zone formation was compared with the control along with the measurement of
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 all extracts. Alkaloids
and phenols presence was seen in methanol and aqueous extracts. 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 for providing 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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