Sagar Trachyspermum ammi seeds. Methods: Four different extracts

Sagar Bashyal1, Avijit Guha2

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

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

[email protected]

 

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 of the phytochemical
analysis revealed the presence of flavonoids, saponins alkaloids, phenols, glycosides,
carbohydrates, proteins, terpenoids, and tannins. 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.

 

Conclusion: The
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.

 

INTRODUCTION:

 

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
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 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 method 29-32.

 

Test for
alkaloids:

 

Dragendorff’s
test

To 0.5 ml of plant extracts the
Dragendorff’s reagent was added. A reddish-brown precipitate confirms that test
as positive.

 

Test of
carbohydrates:

 

Benedict’s
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.

 

Test of
saponins:

 

Froth
test

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.

 

 

Test of
flavonoids:

 

Alkaline
reagent test

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.

 

Test of
proteins:

 

Biuret test

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.

 

Test of
tannins:

 

Ferric
chloride test

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.

 

Tests for
steroids and terpenoids:

 

Salkowski
test

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.

 

Tests for
glycosides:

 

Borntrager’s
test

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.

 

 

DETERMINATION
OF ANTIMICROBIAL ACTIVITY:

 

Test microorganisms and control:

 

The
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.

 

Antimicrobial assay:

 

Agar well
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.

 

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 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.
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, 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

 

 

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 to provide 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.

 

REFERENCES:

 

1)     
Richard E.
Lenski, “Bacterial evolution and the cost of antibiotic resistance” International
Microbiology 1 1998;1:1265-270.

 

2)     
Raghunath
D, “Emerging antibiotic resistance in bacteria with special reference to
India”. J Biosci 2008;33:593-603.

 

3)     
Patel, I., & Talathi, A. (2016). USE OF TRADITIONAL INDIAN HERBS
FOR THE FORMULATION OF SHAMPOO AND THEIR COMPARATIVE ANALYSIS. International
Journal of Pharmacy and Pharmaceutical Sciences, 8(3), 28-32.

 

4)     
Adhikari, P., & Paul, S. (2018). HISTORY OF INDIAN TRADITIONAL
MEDICINE: A MEDICAL INHERITANCE. Asian Journal of Pharmaceutical and Clinical
Research, 11(1), 421-426.

  

5)     
Ayurvedic
Pharmacopoeia of India. Government of India, Ministry of Health and Family
Welfare Department of Ayush. Part 1. 1999-2011;1:170–1.

 

6)     
Singh, I,
Singh VP. Antifungal properties of aqueous and organic extracts of seed plants
against Aspergillus flavus and A. niger. Phytomorphology 2000;20:151–7.

 

7)     
Sivropoulou
A, Papanikolaou E, Nilolaou C, Kokkini S, Lanaras T, Arsenakis M. Antimicrobial
and cytotoxic activities of origanum essential oils. J Agric Food Chem 1996;44(5):1202–1205.

 

8)     
Srivastava
KC. Extract of a spice Omum (Trachyspermum ammi) shows antiaggregatory effects
and alters arachidonic acid metabolism in human platelets. Prostaglandins
Leukot Essent Fatty Acids 1988;33:1–6.

 

9)     
Bentely
R, Trimen H. Medicinal Plants. New Delhi: Asiatic Publishing House; 1999. pp.
107–15.

 

10)  Singh, Gurdip; Maurya, Sumitra;
Catalan, C.; de Lampasona, M. P. (June 2004). “Chemical Constituents,
Antifungal and Antioxidative Effects of Ajwain Essential Oil and Its Acetone
Extract”. Journal of Agricultural and Food Chemistry 52(11):3292–3296.

 

11)  Bonjar GH. Anti-yeast activity of
some plants used in traditional herbal-medicine of Iran. J Biol
Sci 2004;4:212–5.

 

12)  Kumari KS, Prameela M. Effect of
incorporating Carum copticum seeds in a high fat diet for albino rats. Med
Sci Res 1992;20:219–20.

 

13)  Vasudevan K, Vembar S,
Veeraraghavan K, Haranath PS. Influence of intragastric perfusion of aqueous
spice extracts on acid secretion in anesthetized albino rats. Indian J
Gastroenterol 2000;19:53–6.

 

14)  Gilani AH, Jabeen Q, Ghayur MN,
Janbaz KH, Akhtar MS. Studies on the antihypertensive, antispasmodic,
bronchodilator and hepatoprotective activities of the Carum copticum seed
extract. Journal of Ethnopharmacol 2005;98:127–35. 

 

15) 
 Ahsan
SK, Shah AH, Tanira MO, Ahmad MS, Tariq M, Ageel AM. Studies on some herbal
drugs used against kidneystones in Saudi
folk medicine. Fitoterapia 1990;61:435–8.

 

16) 
Nath D, Sethi N,
Srivastav S, Jain AK, Srivastava R. Survey on indigenous medicinal plants used
for abortion in some districts of Uttar Pradesh. Fitoterapia 1997;68:223–5.

 

17) 
Kaur H.
Estrogenic activity of some herbal galactogogue constituents. Indian J
Anim Nutr 1998;15:232–4.

 

18) 
Srivastava KC.
Extract of a spice-omum (Trachyspermum ammi)-shows antiaggregatory effects and
alters arachidonic acid metabolism in human platelets. Prostaglandins
Leukot Essent Fatty Acids. 1988;33:16.

 

19) 
Thangam C,
Dhananjayan R. Antiinflammatory Potential of The Seeds of Carum Copticum Linn. Indian J
Pharmacol 2003;35:388–91.

 

20) 
Boskabady
MH, Jandaghi P, Kiani S, Hasanzadeh L. Antitussive effect of Carum copticum in
guinea pigs. J Ethnopharmacol 2005;97:79–82. 

 

21) 
Mathew N,
Bhattacharya SM, Perumal V, Muthuswamy K. Antifilarial Lead Molecules Isolated
from Trachyspermum ammi. Molecules 2008;13:2156–68. 

 

22) 
Ramaswamy
S, Sengottuvelu S, Sherief S Haja, Jaikumar S, Saravanan R, Prasadkumar C, et
al. Gastroprotective Activity of Ethanolic Extract of Trachyspermum
Ammi Fruit. Int J Pharm Biosci 2010;1:1–15. 

 

23)  Pelczar MJ, Chan EC, Krieg
NR. Control of microorganism by physical agents, in microbiology. New
York: Mcgraw Hill International; 1988. pp. 469–509.

 

24)  Priestley CM, Williamson EM,
Wafford KA, Sattelle DB. Thymol, a constituent of thyme essential oil, is a
positive allosteric modulator of human GABAA receptors and a homooligomeric
GABA receptor from Drosophila melanogaster. Br J Pharmacol 2003;40:1363–72. 

 

25) 
Velazhahan
R, Vijayanandraj S, Vijayasamundeeswari A, Paranidharan V, Samiyappan R,
Iwamoto T, et al. Detoxification of aflatoxins by seed extracts of the
medicinal plant, Trachyspermum ammi (L.) Sprague ex Turrill
Structural analysis and biological toxicity of degradation product of aflatoxin
G1. Food Control 2010;21:719–25.

 

26) 
 Anilakumar
KR, Saritha V, Khanum F, Bawa AS. Ameliorative effect of ajwain extract on hexachlorocyclohexane-induced
lipid peroxidation in rat liver. Food Chem Toxicol 2009;47:279–82.

 

27) 
 Chialva
F, Monguzzi F, Manitto P, Akgül A. Essential oil constituents
of Trachyspermum copticum (L.) Link fruits. J Essent Oil
Res 1993;5:105–6.

 

28) 
Choudhury
S, Riyazuddin A, Kanjilal PB, Leclercq PA. Composition of the seed oil
of Trachyspermum ammi (L.) Sprague from Northeast India. J
Essent Oil Res 1998;10:588–90.

 

29) 
Mameta
S., Jyoti S. Phytochemical screening of Acorus Calamus and Lantana Camara. Int
Res J Pharm 2012;3(5):324-326.

 

30)  Sanjay Parihar, Kartik D. Virani,
E. A. Pithawala, M. D. Shukla, S. K. Lahiri, N. K. Jain and H. A. Modi.
Phytochemical screening, total phenolic content, antibacterial and antioxidant
activity of wild edible mushroom Pleurotus ostreatus. Int. Res. J. Pharm
2015;6(1):65-69.

 

31) 
Mumtaz,
F., Shahid Massod R., Zubair A., Iftikhar A. and Musaddique H. Qualitative
phytochemical analysis of some selected medicinal plants in local area of
Faisalabad Pakistan. Journal of Pharmacy and Alternative Medicine 2014;3(3):17-
23.

 

32) 
Reddy S.,
Ammani,Ch., RoseMary K., Nikhil Rajesh T., Aravind G. and Bala Sekaran Ch.
Phytochemical and GC-MS analysis of Commiphora caudata (Wt.) Eng. Bark,
Indian Journal of Advances in Plant Research 2014;1(5):24-29.

 

33)  Swargiary, A., Nath,
P., Basumatary, B., & Brahma, D. (2017). PHYTOCHEMICAL, ANTIOXIDANT, AND
TRACE ELEMENT ANALYSIS OF ANTHELMINTIC PLANTS OF NORTH-EAST INDIA.
International Journal of Pharmacy and Pharmaceutical Sciences, 9(9), 228-232.

 

34)  Sakthi, A. (2016).
PRELIMINARY PHYTOCHEMICAL SCREENING AND IN-VITRO FREE RADICAL SCAVENGING
ACTIVITY OF ROOT EXTRACTS OF GLYCYRRHIZA GLABRA L. Asian Journal of
Pharmaceutical and Clinical Research, 9(6), 85-90.