Sagar diseases, and herbivorous mammals. There is a

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Sagar diseases, and herbivorous mammals. There is a

Sagar Bashyal1, Avijit Guha2

Department
of Biotechnology, IILM College of Engineering and Technology, U.P, India

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ABSTRACT:

 

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
technique.

 

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, Seed extracts, Phytochemicals, Antimicrobial
activity.

 

INTRODUCTION:

 

Since from past ages, medicinal
plants, also known as medicinal herbs have been discovered and used in
traditional medicine practices. The plants that are found naturally are capable
to synthesize various chemical compounds for botanical functions that provide
defense capability against number of 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. The seeds of Trachyspermum ammi
is native of Egypt and is cultivated in different regions of Iraq, Afghanistan,
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 source which acts as a remedial agent
for flatulence (gas problem), atonic dyspepsia (indigestion) 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 found to possess the following properties:

·        
Antimicrobial
11

·        
 Hypolipidemic: used for treatment of high
level of fats. 12

·        
 Digestive stimulant 13

·        
 Antispasmodic: used for smooth muscle
relaxation.

·        
 Broncho-dilating: provide relief from acute
bronchoconstriction. 14

·        
 Antihypertensive

·        
 Hepatoprotective

·        
 Diuretic: increases the production of urine 15,

·        
 Abortifacient 16

·        
 Anti-lithiasis

·        
 Galactogogic 17

·        
 Antiplatelet-aggregator 18

·        
 Anti-inflammatory 19

·        
 Antitussive: suppress coughing 20

·        
 Anti-filarial 21

·        
 Gastroprotective 22

·        
 Nematicidal 23

·        
 Anthelmintic 24

·        
 Detoxification of aflatoxins 25

·        
 Ameliorative effects 26

 

Therapeutic
uses of Trachyspermum ammi fruits
include; stomachic, expectorant and carminative, 27 antiseptic and
amoebiasis, antimicrobial. The current study aimed to carry out the
phytoconstituents testing and to analyze antibacterial activity against E. coli using the extracts prepared in
the laboratory.

 

 

Taxonomic
classification 28   

 

Kingdom:        
Plantae, Plant

Subkingdom:  
Tracheobionta, Vascular plants

Superdivision:  Spermatophyta,
Seed plants

Division:           Magnoliophyta, Flowering plants

Class:              
 Magnoliopsida, Dicotyledons

Order:             
 Apiales

Family:             Apiaceae

Genus:              Trachyspermum

Species:             Ammi

 

MATERIALS
AND METHODS:

 

 

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.

 

Preparation
of crude extracts:

 

About 3 gm of coarse seed 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.

 

Sterilization
of Materials:

 

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.

 

Maintenance
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.

 

PHYTOCONSTITUENTS
ANALYSIS:

 

The phytochemical tests were carried out for four
different extracts as mentioned above using the standard methods 29-32.

 

Test for
alkaloids:

 

Dragendorff’s
test

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.

 

Test for
carbohydrates:

 

Benedict’s
test

1 ml of Benedict’s solution was
added to the concentrated and filtrate aqueous extract obtained by mixing 1 mg
of seed extract in 2.5 ml of water and boiled for 5 minutes. The presence of
carbohydrates was identified by formation of a brick red precipitate.

 

Test for
saponins:

 

Froth
test

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. Saponin presence was identified by the
formation of foam across the surface for a few minutes.

 

 

Test for
flavonoids:

 

Alkaline
reagent test

About 3 ml of aqueous seed extract
was added with a few drops of sodium hydroxide solution. Flavonoids presence
was identified by the formation of yellow color which in addition of dilute
acetic acid disappears.

 

Test of
proteins:

 

Biuret test

About l ml of the seed extracts
solution was taken and 4% NaOH solution and 1% CuSO4 solution were added. Proteins
presence was identified by the formation of violet color accordingly.

 

Test of
tannins:

 

Ferric
chloride test

To 3 ml of seed extracts was
taken and a few drops of 0.1% ferric chloride solution were added and allowed
to stand for a few minutes. Tannins presence was identified by formation of
brownish green or blue-black color.

 

Tests for
steroids and terpenoids:

 

Salkowski
test

1 ml of each aqueous extract was
treated in chloroform with a few drops of concentrated H2SO4,
shaken well and left to stand for a few minutes. After few minutes steroids
presence was identified by formation of red color at the lower layer and
terpenoids by the formation of a yellow colored lower layer.

 

Tests for
glycosides:

 

Borntrager’s
test

About 3 ml of seed aqueous extract was shaken
vigorously. Further, 1 ml of benzene and 1 ml of dilute ammonia solution was
added. Glycosides presence was identified by formation of reddish pink color.

 

 

EVALUATION
OF ANTIMICROBIAL ACTIVITY:

 

Test microorganisms and control:

 

The
extracts of the seeds of Trachyspermum
ammi were tested against E. coli.
Clinical site was the major source used for the isolation of E. coli cells. The medium for isolation
and sub-culture was sterilized using the wet heat sterilization method. The
isolated culture in the nutrient agar medium was sub-cultured in a nutrient
broth and was taken for the incubation, which was kept at temperature of 37°C
for 24 hours for.
Ciprofloxacin was taken as the control for E.
coli cells. And the zone formation was compared with the control along with
the measurement of the length (mm).

 

Antimicrobial assay:

 

Agar well
diffusion method was used to determine the antimicrobial activity. E. coli suspension was seeded on two
Muller Hinton Agar (MHA) plates which were maintained in the sterilized
condition. Using the sterilized corn borer, two wells were punched in each
plate. Using a micropipette 70 µl of water extract and control was loaded in the
first plate (well 1 and 2) and again, the same concentration of acetone,
chloroform, and methanol extract was loaded in the second plate in respective
numbered wells. Then, plates were incubated for 24 hours at temperature of 37°C.

 

The
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.

 

RESULT
& DISCUSSION:

 

Phytoconstituents screening:

 

Phytochemical
test of four 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.
An
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.

 

S.No.

Phytochemicals

Methanol

Acetone

Chloroform

Water

1.

Alkaloids

+

   

    

2.

Carbohydrates

+

+

+

3.

Saponins

+

+

+

+

4.

Flavonoids

+

+

+

+

5.

Proteins

+

+

6.

Tannins

+

7.

Steroids

   

   
 –

8.

Terpenoids

+

9.

Glycosides

+

+

  +

‘+’
sign indicates the presence and ‘– ‘sign indicates absence.

 

 

 

 

Antimicrobial activity:

 

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, 9mm,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

 

 

Solvent Extract

Zone of Inhibition (mm)

Methanolic

13.5

Acetone

9

Chloroform

10.5

Aqueous

11

Control
(Ciprofloxacin)

17.5

 

Fig. 2. A
chart showing different inhibition zone for four different extracts.

 

 

 

 

CONCLUSION:

 

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.

 

ACKNOWLEDGEMENT:

 

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
research work.

 

CONFLICT
OF INTEREST:

 

The authors declare that no conflict of interest occurred
during the work.

 

AUTHORS
CONTRIBUTION:

 

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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