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, 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 laboratory.
Taxonomic classification 28
Kingdom: Plantae, Plant
Subkingdom: Tracheobionta, Vascular plants
Superdivision: Spermatophyta, Seed plants
Division: Magnoliophyta, Flowering plants
Class: Magnoliopsida, Dicotyledons
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.
The phytochemical tests were carried out for four different extracts as mentioned above using the standard methods 29-32.
Test for alkaloids:
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 of carbohydrates:
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.
Test of saponins:
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 saponin.
Test of flavonoids:
Alkaline reagent test
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.
Test of proteins:
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.
Test of tannins:
Ferric chloride test
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.
Tests for 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.
Tests for glycosides:
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.
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 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 the length(mm).
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:
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 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.
‘+’ 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 research work.
CONFLICT OF INTEREST:
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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