ABSTRACT: Objective: To analyze the presence of different phytochemicals and to determine the antimicrobial activity of Trachyspermum ammi seeds. Methods: 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.
Conclusion: The results conclude that different extracts of Trachyspermum ammi seeds exert biological properties due to the presence of secondary metabolites in the form of flavonoids and phenols. 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 fungi, insects, 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 present scenario, herbal drugs and their formulations will 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 5, bishop weeds 6 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 Iran, Iraq, Afghanistan, Pakistan, and India. In India, the seeds are cultivated in Gujarat, Rajasthan, Madhya Pradesh, Uttar Pradesh, Maharashtra, Bihar and West Bengal 7.
The oil obtained from the seeds exhibits fungicidal 8 antimicrobial 9 and anti-aggregatory effects on humans 10. It is an important remedial agent for flatulence, atonic dyspepsia and diarrhea 11. Hydrodistillation of ajwain fruits yields an essential oil consisting primarily of thymol, gamma-terpinene, and p-cymene as well as more than 20 trace compounds (predominately terpenoids) 12. Trachyspermum ammi has been shown to possess antimicrobial,13 hypolipidemic,14 digestive stimulant,15 antispasmodic, Broncho-dilating,16 antihypertensive, hepatoprotective, diuretic,17 abortifacient,18 anti-lithiasis, galactogogic,19 antiplatelet-aggregator,20 anti-inflammatory,21 antitussive,22 anti-filarial,23 gastroprotective,24 nematicidal,25 anthelmintic,26 detoxification of aflatoxins,27 and ameliorative effects.28 Therapeutic uses of Trachyspermum ammi fruits include; stomachic, expectorant and carminative,29 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 30 Kingdom: Plantae, PlantSubkingdom: Tracheobionta, Vascular plantsSuperdivision: Spermatophyta, Seed plantsDivision: Magnoliophyta, Flowering plantsClass: Magnoliopsida, DicotyledonsOrder: ApialesFamily: ApiaceaeGenus: TrachyspermumSpecies: 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 thus obtained were used for further investigation for of phytochemical screening, 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 31-34.
Test for alkaloids: Dragendorff’s testTo 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 testAbout 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 testA pinch of the dried plant extracts was added to 3 ml of distilled water. The mixture was shaken vigorously for a few minutes. Formation of a foam indicated the presence of saponin. Test of flavonoids: Alkaline reagent testTo 0.
5 ml of plant extracts few drops of sodium hydroxide solution was 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 testTo 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 testTo 0.5 ml of plant extracts, few drops of 0.1% ferric chloride solution was added.
Formation of brownish green or a blue-black coloration indicating the presence of tannins. Tests for steroids and terpenoids: Salkowski test0.5 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.