Introduction

Nature gives several things against several diseases since last many decades. (Samuelsson et al., 2017) Adhatoda Vasica is a perpetual plant having a place with family Acanthaceae, generally known as Vasaka. (Claeson UP, et al., 2000) It is a small evergreen, perpetual bush dispersed all through India. It has been utilized for the treatment of different infections and clutters, especially for the respiratory tract illnesses. It is recognized with an upsetting smell and bitter taste. (Maurya S, et al., 2010) Vasicine and vasicinone alkaloids are the significant chemical constituents of the plant and are liable for its solid respiratory energizer activity. (Rastogi RP, et al., 1999) The drug for the most part contains fresh or dried leaves however the flowers, foods grown from the ground are likewise broadly utilized for different illnesses. Different measurement types of leaf-like powder, fresh juice, decoction, alcoholic and aqueous extract, and so on., are portrayed to be utilized for different diseases. (Anonymous ,1985)

A.Vasica was especially utilized as a home-grown medication for treating respiratory grievance including cold, hack, incessant bronchitis, asthma and as antispasmodic. (Singh B, et al., 2013) It has been accounted for antibacterial, injury mending, hypoglycemic, abortifacient, antitussive, anti-inflammatory and hepatoprotective activities. (Ahmad S, et al., 2009) Most part contains phytochemical likes pyrroquinazoline alkaloids, flavonoids, triterpenes, (Atta-Ur-Rahman, et al., 1997) steroids, tannins, saponins and glycosides. (Yusuf M, 2016) Therefore, influencing the bacterial pathogenic potential through majority detecting restraint is one of the new methodologies that are utilized to battle the microbial resistance. (Bhardwaj K, et al., 2013)     In laboratory experiments on cats and dogs, A. Vasica increases bile activity when animals are given a dose of 5mg / kg. in dogs, the amount of excreted bile increases by 40-100%. Animals also showed increased bilirubin excretion. (Gangwar et al. 2014)

In recent time medicine in surgery, cancer chemotherapy, and limb transplantation is credited to the use of antibiotics. (Livermore et al., 2002) Antibiotics working today will not work tomorrow New drugs must be examined, with fewer resistance (Sarkar et al., 2003) As a resistance to the spread of old antibiotics, new immune agents are rapidly developed. Yet, records of the rapid emergence and emergence of new antimicrobial agents suggest that a new family of antimicrobial agents will have a shorter life span. (Coates et al., 2002) For that much chemical contained the Adhatoda Vasica is effective against serval disease. Combination of plant extracts and antibiotics were used to increase sensitivity of plant extract and lower side effect of allopathy tablets.

Common Name
English : Malabar nut, Adulsa
Hindi : Adosa, Adalsa, Vasaka
Gujarati :  Ardusi, Adusi
Marathi : Vasuka

Plant Anatomy (Flora of Gujarat state, 1978)
Kingdom :   Plantae
Division  :  Angiosperm
Class        : Dicotyledonae
Series       :  Bicarpellatae 
Order       :  Personales
Family     :  Acanthaceae
Genus      :  Adhatoda
Species    : Vasica

Figures 1: Adhatoda Vasica Plant Click here to View Figure

Materials and Methods

Material Use for Study              

Adhatoda Vasica Plant

Antibacterial Antibiotics :   Amoxicilline,   Ciprofloxacin, Ceftazidime, Erythromycin
Antifungal Antibiotics :   Amphotericin-B, Fluconazole
Bacteria  :   Bacillus subtilis (MTCC 441), Escherichia coli (MTCC 1687), Pseudomonas aeruginosa (MTCC 1688), Staphylococcus aureus (MTCC 737)
Fungi :  Aspergillus Niger (MTCC 1344), Candida albicans (MTCC 81)

Method

Leaves of the Adhatoda Vasica was conglomerate from local area of north Gujarat in June 2018. Identification of the plant was done by Dr. N. K. Patel, head of the botany department, Sheath M. N. Science college, Patan.  The first stages of the study of medicinal plants are to prepare plant samples, which is to store biomolecules in plants before extracted. The leaves were thoroughly wash with tap water and then washed with deionized water to remove particles of dust and sand. The leaves were dried in the dark at room temperature for several days and then powder with mortar. And it is extracted by using Soxhlet extraction.

Qualitative Phytochemical Analysis

Following methods were used for analysis of different type phytochemicals.

Test for Alkaloids

Plant Extractions were dissolved in dil. HCl and filtered

Dragendroff’s Test

Plant extract   +   Potassium Bismuth Iodide solution (Dragendroff’s Reagent)   →   Orange Precipitate

Hagers’s Test

Plant extract   +   Picric Acid (Hagers’s Reagent)   →    Yellow Precipitate

Mayers’s Test

Plant extract   +   Potassium Mercuric Iodide solution (Mayers’s Reagent)   →   Cream precipitate

Wagner’s Test

Reaction of plant extract   +   Iodine in Potassium Iodide (Wagner’s Reagent)   →   Red-brown precipitate

Test for Flavonoids

Test with Alkaline

Plant extract   +   Sodium Hydroxide   →   intense yellow color

which become colorless on further adding Dil. acid

Test with Lead Acetate

Treat plant extract   +   few drops of Lead Acetate   →   Yellow color precipitate

Shinoda Test

Dried powder of plant   +   5 ml of 95% CH₃OH   +   Few drops of conc. HCl along   +   0.5 g magnesium   →   turnings Pink precipitate

Test for Glycosides

Keller-Kiiani Test

Plant extract   +   gla. CH₃COOH    +   few drops 5% FeCl_{3   +}   concentrated sulfuric acid   →    formation of   blue color in acetic layer.

Legal Test

Dissolve plant extract in pyridine   +   sodium nitroprusside solution   +   made alkaline   →    Pink or red Color produce

Test for Phenolics

FeCl₃ Test

Plant extract   +   aqueous FeCl_{3   →}    blue color

Test for Saponins

Foam Test

Plant extract in test tube with a little quantity of water   →    foam produced persisted for 10 minutes.

Test for Tannins

Gelatin Test

Plant extract   +   1% Gelatin Solution containing 10% sodium chloride   →    White precipitate

FeCl₃ Test

Plant extract   +   FeCl_{3   →}    Blue-black precipitate

Test for Terpenoids

Salkowski Test

Extract   +   Con. H₂SO_{4   →}    formation of yellow colored at lower layer

Preparation of Combination

Activities of various extract and their combination were resolute by the zone of ​​the inhibition method. It was investigated by agar diffusion method using agar cup method. Purified extract was diluted in dimethyl sulfoxide and purified antibiotics were used by purification; and stand at 4⁰ C.  For compare slandered results we used standard antibiotic. Antimicrobial activity of all extracts and their combination were tested against for some bacteria and fungi which was prepared in Acetone (CH₃COCH₃), Methanol (CH₃OH) and ethanol (CH₃CH₂OH) solvents. Muller-Hinton agar plates were seeded with indicator bacterial and fungal strains, and incubated for 24 hours at 37⁰ C. The sensitivity of microbial species to plant extracts was determined by measuring the resistance zone size on the agar surface.

Result and Discussion

Table 1: Qualitative Phytochemical Analysis

(+) shows the Positive result and (-) show Negative Result of the Test

Antimicrobial Analysis Adhatoda Vasica, Antibiotics and Combination both for 25 μl.

*Growth of the Bacteria and Fungi shown in table number 2 to table number 9 are in Millimeter (mm)

Table 2: Antibacterial activity of Adhatoda Vasica

In the above table three different solvent were used for biological study. The study for each solvent were done with four different concentration. Table shows methanolic extract of in 1000 µg/ml concentration gives highest growth Which is used to decide dosage for human body.

Table 3: Antibacterial activity of Adhatoda Vasica with Amoxicilline

Amoxicilline which is an antibacterial antibiotic is combined with the plant. The study shows the combination with 1000 µg/ml in MeOH extract gives highest zone of inhibition.

Table 4: Antibacterial activity of Adhatoda Vasica with Ciprofloxacin

 The mixture of Plant extract and ciprofloxacin in 1000 µg/ml gives good values in zone of inhibition of bacteria.

Table 5: Antibacterial activity of Adhatoda Vasica with Ceftazidime

Ceftazidime which is also an antibacterial antibiotic also gives effective results in methanol when it mixed with plant 

Table 6: Antibacterial activity of Adhatoda Vasica with Erythromycin

Combination of plant extract and an antibacterial antibiotics Erythromycin gives good activity in higher concentration.

Table 7: Antifungal activity Adhatoda Vasica

In this table antifungal activity of Adhatoda Vasica extract was studied in Acetone, Methanol and Ethanol solvent. Biological study in two fungi, Aspergillus Niger and Candida Albicans fungi shows increasing of zone inhibition in higher concentration.  Methanolic extract shows higher   activity then other two solvent.

Table 8: Antifungal activity of Adhatoda Vasica with Amphotericin B

An antifungal antibiotic Amphotericin B when combined with Adhatoda Vasica extract gives very good activity in 1000 µg/ml concentration.

Table 9: Antifungal activity of Adhatoda Vasica with Fluconazole

Fluconazole which is also an antifungal antibiotic also gives effective activity in higher concentration of the solvent.

Figure 2: Activity in Adhatoda Vasica extract (a) Antibacterial (b) antifungal Click here to View Figure

Conclusion

Plant drugs from Ayurvedic framework are being investigated globally. The consequences of the essential examination demonstrated that alongside its traditional helpful cases and some experimentally demonstrated pharmacological exercises, Adhatoda Vasica additionally has the possibility to be created as a powerful plant with combination of antibiotics. Combination of A. Vasica and antibiotics indicated pronounced antimicrobial activities with wonderful restraints. This work gives logical confirmations to medicinal uses of A. Vasica with commitment of a portion of the distinguished and tried phytoconstituents in the got organic impacts. From the study we find that the combination of plant extract with antibiotics gives very effective activity then only plant and antibiotics. It also suggests that methanolic extract with higher concentration gives best results than others.

Acknowledgement

The authors are thankful to the Faculty member of Chemistry Department of Sheth M. N Science College, Patan. We are also thankful to the Microbiology Department of Dr. Indu Dayal Meshri College of Science and Technology, Patan for their kind support during the biological Study. We are also thankful to Dr. N. K. Patel, Head of Botany Department, Sheth M. N. Science College, Patan for identification of plant

Conflict of Interest Statement

We, the authors of the submitted manuscript declare that the work and data present in the manuscript entitled – qualitative analysis and biological study is genuine research carried out by us. The work finally belongs to the institutes. We have not misused the data previously published and have not manipulated the original work.

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