Sagar Bashyal1, Avijit Guha2Department of Biotechnology, IILM College of Engineering and Technology, U.P, [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 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.

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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 act 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 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, 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 methods 29-32. Test for alkaloids: Dragendorff’s testAbout 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 test1 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 brick red precipitate. Test for saponins: Froth testAbout 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 few minutes.  Test for flavonoids: Alkaline reagent testAbout 3 ml of aqueous seed extract was added with few drops of sodium hydroxide solution.

Flavonoids presence was identified by the formation of yellow color which on addition of dilute acetic acid disappears. Test of proteins: Biuret testAbout l ml of 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 testTo 3 ml of seed extracts was taken and few drops of 0.1% ferric chloride solution were added and allowed to stand for few minutes. Tannins presence was identified by formation of brownish green or blue-black color.

 Tests for steroids and terpenoids: Salkowski test1 ml of each aqueous extract was treated in chloroform with a few drops of concentrated H2SO4, shaken well and left to stand for 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.  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).

 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 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, 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 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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Asian Journal of Pharmaceutical and Clinical Research, 9(6), 85-90.