EVALUATION the presence of secondary metabolites in the

EVALUATION OF TRACHYSPERMUM AMMI SEEDS FOR ANTIMICROBIAL ACTIVITY AND PHYTOCHEMICAL ANALYSIS. Sagar Bashyal1, Avijit Guha2Department of Biotechnology, IILM College of Engineering and Technology, U.

P, [email protected]

in  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.

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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 the 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 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. An essential oil obtained after the hydrodistillation of fruits of plant consist 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 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 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. Foam formation 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.  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. 35 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.

36 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. AUTHOR 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.

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