Study on Phytochemical Screening and Antimicrobial Activity of Adhatoda Vasica

and chest, or breast have been traditionally treated using Adhatoda vasica nees. The present study aimed to evaluate Adhatoda vasica 's phytochemical analysis and antimicrobial activity. The preliminary phytochemical screening of alkaloids, steroids, saponins, phenols, and terpenoid yielded positive results. The antibacterial activity of ethanol and leaf extracts of Adhatoda vasica was investigated in this study. Based on the data presented herein, the largest zone of inhibition was found to be against Pseudomonas aeruginosa . The antifungal activity of Aspergillus

Medicinal plants, which are the basis of traditional medicine, have been the subject of much pharmacological research in recent decades. Since ancient times, higher plants have played a controlling role in preserving human health as suppliers of therapeutic chemicals. Over 50% of all modern clinical drugs are of natural product origin, and natural products play an important role in drug development programs in the pharmaceutical industry [1]. Adhatoda vasica have been useful for years in daily life to treated diseases all over the world. Adhatoda vasica belonging to the Acantharean family is found in India [2]; the leaves are of dark green colors and pale yellow below. The flowers are typical, white, arrange in a pedunculated spike [3]. Adhatoda vasica native range is Asia and is a well-known plant in Ayurveda and Unani systems of medicinal plant. Various parts of this plant have been used to treat several ailments as herbal remedies for many porpoises, e.g., cold, cough, whooping cough, chronic bronchitis, fever, jaundice asthma as a sedative expectorant, diarrhea, and dysentery, as well as inflammatory rheumatic diseases. The roots, leaves, and flowers of the plant are used to remove volatile oils and alkaloids, which have great medicinal importance [3]. The local practitioners use many medicinal plants for treatment of infectious diseases like pneumonia. In many cases, pneumonia is treated at home. It frequently cleared up in 2 to 3 weeks. But older adults, babies, and immune-compromised patients may develop significant complications. Literature survey revealed that the leaves of Adhatoda vasica contain many secondary metabolites and phytochemicals [4]. Several plants have been reported to have antimicrobial activity [5]. Pneumonia has bacterial, viral, fungal, and other primary causes [6]. Medicinal plants are considered a potential source of new compounds of therapeutic value and important sources of new compounds in drug development [7]. These plants make many chemical compounds to protect themselves against fungi, and bacteria, this act in the same way on the human body as allopathic drugs [8,9]. The different health benefits of Adhatoda vasicaare are shown in Figure  1.

Materials and Methods
The leaves of the plant Adhatoda vasica were collected from the open fields of Kareli baug, Vadodara District, Gujarat, India. The fresh leaves were recognized and validated in the college of Agriculture of Parul University. The fresh leaves were dried for 10 days in sunlight. The powdered sample was stored in a bottle at room temperature before analysis.

Method
Antimicrobial studies of Adhatoda vasica have been performed in the Micro care lab, Surat. Zinc acetate dehydrate (99% purity), and sodium hydroxide (Pellet 99%) was used as introductory materials supplied by Sigma-Aldrich chemicals.

Preparation of Plant Extract
The powder sample of Adhatoda vasica and ethanol was extracted by using the Soxhlet apparatus. First, the extract of selected plant material in solvents such as ethanol was prepared. Then, the 30 gms of dried powder were extracted with 250 ml solvent using the Soxhlet apparatus to complete eight cycles. The ethanol extracts were lyophilized and stored in 4 O C. The resulting extract was sent to NABL Accredited micro care lab of Surat.  [12] Phytochemical Screening Tests 1. Test for alkaloids In this regard, 1ml of extract sample and 1 ml of Mayer's reagent were added. As a result, the formation of whitish-yellow or cream-colored precipitate exhibits the presence of alkaloids.

Test for steroids
1 ml of extract sample, 2 ml of acetic anhydride, and 2 ml of concentrated sulphuric acid were added. The formation of violet to blue or green color shows the presence of steroids.
3. Test for terpenoids 1 ml of extract, 2 ml of chloroform, and 3-5 drops of sulphuric acid were added. Theformation of the reddish-brown ring indicates the presence of terpenoids.

Test for flavonoids
1 ml of extract sample, few drops of dilute ammonium solution, and few drops of concentrated hydrochloric acid were added. The yellowish color indicates the presence of flavonoids.

Test for saponins
1 ml of extract and 5 ml of distilled water were added and mixed strenuously. The formation of froth desired the presence of saponins.

Test for phenols
1ml of extract sample and 1 ml of lead acetate solution were used. The formation of a precipitate shows the presence of phenols.

Test for amino acids
1ml of extract sample and 3 to 4 drops of ninhydrin solution were added and then boiled in a water bath for 10 minutes. The formation of purple or blue color indicates the presence of amino acids.

Test for reducing sugars
1ml of extract and equal quantities of Fehling solution A and B were added and then heated. The formation of brick-red precipitate reveals the presence of reducing sugars. The IC50 esteem is determined utilizing the direct connection between the inhibitory likelihood and fixation logarithm as per the strategy. The IC50 esteem is communicated as the mean ± standard deviation of three autonomous investigations.
Serial weakening was set up in essential and optional screening.

2.
The control tube containing no anti-microbial was promptly sub-cultured [before inoculation] by spreading a loopful over a fourth of the plate of the medium, reasonable for the development of the test microorganisms, followed by short term incubation at 37 0C for hatching 3.
The MIC of the control life form was perused to check the exactness of the medication fixations.

4.
The least focus repressing development of the creature was recorded as the MIC.

5.
Before hatching, the measure of development from the control tube [which addresses the first inoculum] 6.
Strategies used for essential and optional screening: Essential screening: Centralizations of combined medications were taken (1000 miniature/ml, 500 miniature/ml, and 250 miniature/ml). The combination medications found in this essential screening were also tested in the second arrangement of weakening against all microorganisms. Optional screening: The medications discovered dynamically in essential screening were likewise weakened.

Results
The preliminary phytochemical screening was carried out on the ethanolic crude leaves extracts of Adhatoda vasica (Table 1). Phytochemical results revealed the presence of various bioactive auxiliary metabolites in leaf ethanol concentrates of Adhatoda vasica. The most extreme auxiliary metabolites were seen in ethanolic extracts. Table 1 and 2 show preliminary analysis and antibacterial and antifungal activity of leaf extract of Adhatoda vasica. Table 3 shows minimal bactericidal concentration and minimal fungicidal concentration of standard drugs on specific microorganisms   Table 3 Minimal

Bactericidal Concentration and Minimal Fungicidal Concentration of Standard Drugs on Specific Microorganisms
Observations Gentamycin showed growth inhibition at 0.05 ug/ml on E.coli, followed by chloramphenicol (inhibition halo at 50 ug/ml on S. aureus), ciprofloxacin (inhibition at 25 ug/ml on E. coli and P. aeruginosa), nystatin (inhibition at 100 ug/ml on A.niger), and greseofulvin (inhibition at 100 ug/ml on A. niger and A. clavus).
Adhatoda vasica also showed the same inhibitory activity at the same concentration on P. aeruginosa, S. aureus, S.pyogenus, A.niger, E.coli C. albicans. Thus, Adhatoda vasica extract might be the same as ampicillin, chloramphenicol, ciprofloxacin, nystatin, and griseofulvin. However, further cytotoxicity studies are needed to confirm these findings. The presence of qualitative phytochemicals in A. vasica leaf extracts (ethanol) has been investigated [2]. The findings showed that the pure ethanol extracts of A. vasica leaves provided the greatest antibacterial activity against the microorganisms examined by using the agar-well diffusion method. Ample evidence exhibited that the ethanolic extract of the plant has a high antibacterial effect. The antibacterial activity of ether and aqueous extracts has been found to be poor [3]. In addition to petroleum ether and aqueous extract, the ethanolic extract should be tested for antifungal activity. Additional research on ethanolic extract could lead to the development of a good antibacterial agent. The extracts were tested for the content of phenols, tannins, alkaloids, anthraquinones, saponins, flavonoids, amino acids, and reducing sugars by using the agar well diffusion method [3]. The pure cultures of organisms, including gramnegative (Klebsiella pneumoniae and Escherichia coli) and gram-positive (Staphylococcus aureus and Enterococcus faecalis) were sub-cultured in MacConkey agar (HI Media, India) nutrient broth based on previously used method [3]. Methanol as negative and Terramycin (30µg/l) as positive controls were also tested. The antimicrobial potential of leaf extraction of Adhatoda vasica leaves (Family Acanthaceae) alone has a significant contribution. The effect of a specific antimicrobial agent on the production of growth inhibition zone manifests itself as a clear area around the source of the agent [4]. It can be employed as an eco-safe, biodegradable alternative in the prevention and treatment of bacterial infections, where leaves extracts exhibited activity comparable to that of a typical antibacterial activity (i.e., ceftriaxone) against tested microbial isolates [4]. Antifungal activity of methanolic and leaf extracts of J. adhatoda, solvent against four fungi including Aspergillus niger,A. flavus, Fusarium oxysporum and Penicillium digitatumwas has been previously evaluated, where Potato Dextrose Agar Medium was applied as the medium for the evaluation of the antifungal activity by the disc diffusion method [5].
In a comparative investigation, aqueous extracts of A. vasica were found to be more effective against gram-negative Klebsiella pneumoniae than commercially available vasicine. The MIC and MBC values of water and ethanol extracts have been found to be promising against Klebsiella pneumoniae in plankton and biofilm forms. However, there was an unidentified activated compound in water and ethanol extract of Adhatoda vasica, showing more efficiency towards gram negative Klebsiella pneumoniae biofilm intervention than vasicine even at low MIC concentration, that are in need of further studies to know the compound in detail [5,6].
The traditional use of the Justicia adhatoda appeared to be promising for treating infectious diseases, particularly bacterial and fungal infections. A study evaluated the antibacterial, antifungal, and phytochemical features of Justicia Adhatoda leaf extracts and then assessed its bioactive components. Physicochemical and Proximate analysis exhibited a 15.20% moisture content (leaves), and the total ash content, insoluble acid ash, water-soluble ash, and foreign matter values were found to be 11.40%, 1.50%, 3.85%, and 1.68%, respectively. The antibacterial and anti-fungal properties of four crude extracts of Justicia Adhatoda (petroleum ether, ethyl acetate, chloroform, methanol extracts) were studied by the aforementioned study, where all assessed organisms were slightly affected by all extracts except Pseudomonas aeruginosa and Proteus vulgaris. Among standard antibiotics used, ciprofloxacin (10mcg/disc) and ketoconazole (10mcg/disc) showed good inhibitory effects [6,7].
The A. vasica is an excellent medicinal plant for producing ZnO-NPs through fast Zn ion reduction. Since bacterial strains are resistant to traditional antibiotics, ZnO-NPs showed a significant potential antibacterial activity against bacterial cell development; thus, they may be employed in the manufacture of apoptotic agents and the synthesis of novel antibiotics [18].

Conclusion
The current study aimed to determine the phytochemical content of Adhatoda vasica leaf extracts and to evaluate their antibacterial activity. The results indicated the existence of eight phytochemical components. The leaves of Adhatoda vasica have considerable antibacterial action against both gram-negative and gram-positive microorganisms. Antibacterial and antifungal efficacy of Adhatoda vasica against microorganisms is superior because the leaf extracts showed strong effectiveness against tested microbial isolates. Its effectiveness was comparable to that of a typical antibacterial drug (ceftriaxone). Thus, it may be employed as an eco-friendly, biodegradable alternative to prevent and treat bacterial infections.