INTRODUCTION:

A great source of antibacterial agents are medicinal plants. Plants are utilised medicinally and are a source of many powerful and powerful medicines in different nations. A broad range of plant components are utilised as raw medicines for extracts and have therapeutic effects. Medicinal plants are considered key sources for the potential therapeutical benefits of novel chemical substances. A number of phytochemicals were identified to provide a source for the secondary metabolites of plants, which may be directed to novel medication synthesis.

In the contemporary primary health system of developing nations traditional medicine should be allowed to play an even larger role. In contrast to contemporary synthetic medications the natural remedies are considered to be more suitable for the human body^1-3. The most essential element is to profit from the traditional medical system in order to provide the rural people with proper health care. Nature is a significant source of therapeutic products for many years. An astonishing number of contemporary medicines, based on their usage in traditional medicine, were either isolated or derived from natural sources. The plants have long since been traditionally utilised, and current scientific investigations have demonstrated a good link between traditional or folkloric plant use and the quest for pharmcologically active components in plants has been further strengthened. Taking into account the enormous potential of plants as sources for antibacterial medicines. Anthocephalus cadamba is a wide and straight umbrella-shaped tree. The branches are classified in levels. The tree may reach 45 m in height and 100-160 cm in diameter and occasionally has a little buttock up to 2 m in height^4-7.

Bark on young trees is grey, smooth and extremely light, but tough and split in elderly trees. The branches extend and droop at the tip horizontally. The leaves are bright, green, simple sessile, elliptical oval. In young fertilised trees, the leaves are considerably bigger, submissive at the basis and accumulated at the peak. Fruits with their top portions comprising 4 hollow or solid structures are abundant and slightly meaty^8-12. The fruit comes in a tiny body package which forms an orange fleshy infructescence with around 8000 seeds. It is packed in tight cooperation.The grains are somewhat trigonal or irregular, not winged. Miq, of the Rubiaceae family in Indian subcontinent including Bangladesh, is widely known as Kadam. A number of components of the plant are utilised in tumour, inflammation, fever, haematological disorders, skin conditions, leprosy and hypoglycemic drugs as traditional medicine^11-13. So far, scientific bioactivity determination investigations have shown the antipalarial, antihepatotoxic and anti-inflammatory properties of A. cadamba¹⁴ as well as anti-diabetic actions¹⁵. However, no scientific data has been available on A. cadamba leaves' antioxidant, cytotoxic and antibacterial activities. This work aims at determining A. cadamba leaves phytochemicals quantitatively and at analysing its antioxidant, cytotoxicity, inhibiting amylase and anti-bacterial capabilities²⁶.

Sample collection:

The leaves are collected from AKRG college of Pharmacy, Nallajerla, W.G District and are authenticated by botanist.

Preparation of Extract:

Anthocephalus cadamba (Roxb.) Miq's leaves are shaded dried, then ground in a powdered shape using a dry grind. After extraction the solvent was removed by distillation and then the concentrated extract obtained was dried by means of a lowering pressure using a rotary evaporator not exceeding 40°C, and moderated heating was provided on water b (180g) with a pumped plant floor, continuously removed with petrol ether (40-60°C), which was then removed by distillation.

An about 1 gramme yellowish extract has been obtained. After completion of the extraction, the solvent was removed by distilling and then concentrated extract was dried under a lower pressure at temperatures not exceeding 40°C and then given moderate temperature heating in the water bath. The deforestated drug was removed by extraction with ethanol (95 percent) until completion of extraction. The resulting ethanol extract was dark yellow with a weight of about 42.8 gramme. In Petridis, ethanol extract was preserved and stored in a cold area in a desiccator.

Aqueous Extraction:

Cold maceration technique aqueous extract. Approximately 50g polished material combined with 300 ml distilled water, stored at room temperature for 7 days. The extract was filtered from water by the Whommann filter paper No.1 and the residual water content (40°C) with heating mantle was evaporated.

Experimental work:

The extracts were vacuum-concentrated to get the appropriate waste (7.4gm). The following chemical tests were performed and given in Table 1. TLC residue with ethanolic extract exhibited 4 spots on the solvent system 1:1 chloroform:methanol. Chloroform extract was collected by column chrome-coded over silica gel (100-200 mesh) with eluitous polarity and 250-ml fractions of petroleum ether, benzene, chloroform and methanol. Each fraction was monitored on TLC after concentration.

Table 1. Chemical tests for the ethanolic extract of leaves of Anthocephalusc adamba

Name of the test

Anthocephalus cadamba ethanolic extract

Steroids:

A)Salkowski Test

B)Liebermann Buchard’s Test

(+) ve

Triterpenes:

A) Salkowski Test:

B)Liebermann- Buchard’s Test

(-) ve

Alkaloids:

A)Mayer’s Test

B)Dragendroff’s Test

C)Wagner’s Test

D)Hager’s Test

(-) ve

Carbohydrates

A)Fehling’s Test

B)Molisch’s Test

D)Barfoed’s Test

C)Benedict’s Test

(+) ve

Flavonoids:

A)Shinoda Test

B)Ferric chloride Test

C)Lead Acetate Test

D)Zinc- Hcl reduction Test

(+) ve

Tannins:

A)Ferric Chloride Test

B)Gelatin Test

(+) ve

Glycosides:

A)Baljet Test

B)Legal Test

C)Keller- Killiani Test

D)Kedde's test

(+) ve

(-)ve

1. Antibacterial activity of the Anthocephalus cadamba leaf extract:

Antimicrobial activity of every natural sample is individually evaluated using the agar diffusion technique, which uses a 24-hour culture of fungal strains and test bacteria. The technique and the agar cup plate are commonly employed to conduct these research. But only the later approach has been selected in the current investigations. By measuring the diameter of the inhibition areas in mm, the impact of the extract and the conventional medication has been studied.

Method used for the experiment:

Cup-Plate Agar Diffusion Method.

Extract and dosage used in the experiment:

Ethanolic extract of the whole plant of Anthocephalus cadamba was used in two dose levels of 100mg/ml and 300mg/ml.

Test organisms:

Gram (+) ve bacteria: Staphylococcus aureus, Bacillus subtilis,.

Gram (-) ve bacteria: Escherichia coli

Composition of Nutrient Agar Medium:

Component	Quantity
Peptone	5g
Sodium chloride	5g
Meat extract	5g
Agar-agar	20g
Distilled water	Upto 1000ml
P^H	7.22-7.4

Procedure:

In 1000ml of hot distilled water 28g of powdered nutritional agar medium (Hi-Media) was suspended. For 20minutes, in an autoclave at 120°C (15 lb/sq.in) the produced nutritional agar medium was sterilised. In the above described test organisms, 25ml of the prepared nutrient agar medium were inoculated with the 24-hour stock cultivation and transferred to sterile Petri dish of 15cm diameter. The media in the plates may be adjusted to harden the laminar air flow unit for approximately 30 minutes at room temperature. Every plate was equally far from 4 cups of 6mm diameter. Residual extract test solutions were developed at 100mg/ml and 300mg/ml concentrations. The sterile pipettes placed 50μl of each concentration in the cups. One cup for every plate was used as standard for controlling antibiotic chloramphenicol (1mg/ml). The Petri dishes so produced were incubated at 30BC for 24 hours and afterwards investigated in millimetres by the measurement of inhibitory zones, with a table of results.

RESULTS:

The ethanolic extract of Anthocephalus cadamba laeves showed moderate activity against Gram (+)ve and Gram (-)ve organisms staphylococcus aureus, streptococcus anginosus, Bacillus subtilis, Micrococcus luteus, Lactobacillus acidophilus, streptococcus mutans, Proteus vulgaris, Erwinia, Enterobacteraerogens, E.coli, Pseudomonas aeuriginosa , Klebsiella pneumonia the increase in the extract concentration will results in increase in the anti bacterial activity.

Table 2. Antibacterial activity of ethanolic extract of the leaves of Anthocephalus cadamba

S. No	Name of the extract	Diameter of zone of inhibition
		Gram +Ve bacteria		Gram +Ve bacteria
		S. A	B.S	E.C
1	Ethanolic extract (50mg/ml)	10	18	10
2	Ethanolic extract (100mg/ml)	12	19	12
3	Ethanolic extract (150mg/ml)	14	21	15
4	Ethanolic extract (200mg/ml)	15	22	17
5	Ethanolic extract (300mg/ml)	18	24	18
6	Standard	27	50	20
7	Control	-	-	-

Zone of inhibition in millimeters, cup diameter: 6mm

S.A: Staphylococcus aureus;

B.S: Bacillus subtilis; E.C: Escherichia coli

2. Anti-fungal activity of the leaf extracts of Anthocephalus cadamba:

The antifungal activity of an ethanolic extract of Anthocephalus cadamba leaves was tested using the Agar cup plate technique. In two dosage levels of 100mg/ml and 300mg/ml, chloroform extracts of the entire plant Anthocephalus cadamba were used.

Test organisms:

Aspergillus niger, Rhizopus stolonifer, Mucor recemosus

Composition of PDA medium

Component	Quantity
Potatoes(peeled)	200g
Dextrose	20g
Agar-Agar	20g
Distilled water	Upto 1000ml

Procedure:

200g of peeled potatoes were chopped into small pieces and cooked for 30 minutes in 500ml of water. The pieces were crushed during the boiling process, and the pulp was filtered out after cooling using muslin cloth. Dextrose was added to the filtrate in the appropriate amounts, and the volume was increased to 1000ml. It was sterilised in an autoclave at 121o(15lbs/sq.in) for 20 minutes after boiling it on a water bath, then adding agar-agar and stirring constantly for 5 minutes. It was then distributed in 20ml amounts into boiling tubes and sterilised in an autoclave at 121o(15lbs/sq.in) for 20 minutes. The medium was injected with organisms that had been cultured in aseptic conditions for two days and then transferred to sterile Petri plates with a diameter of 15cm. For around 30minutes, the medium in the plates was allowed to set at room temperature. 25mL of nutritional PDA medium (Hi-media) was produced and inoculated with 5 microliters of aqueous suspension of the above-mentioned test organisms, which were generated from 48hour cultures, into sterile Petri plates. For around 10 minutes, the medium in the plates was allowed to set at room temperature. In each plate, four 5mm diameter cups were created at equal intervals. Stock solutions of the test residual extract were produced at 100mg/ml and 300mg/ml concentrations. Using sterile pipettes, 50l of each of the aforementioned stock concentrations were pipetted into the cups.One cup was used for control and standard in each plate. The reference standard is fluconozole (10mg/ml). The Petri plates were then incubated at 37°C for 48hours before being evaluated by measuring the inhibitory zones in millimetres.

RESULTS:

Chloroform extract of the entire plant Anthocephalus cadamba exhibits no antifungal effect on the examined species at doses of 100mg/ml and 300mg/ml.

3. Anti oxidant activity of leaf extract of Anthocephalus cadamba:

Introduction:

Antioxidants are chemicals that can protect your cells from free radical damage. Free radicals are molecules created when your body breaks down food or when you are exposed to cigarette smoke or radiation in the environment. Free radicals may harm cells and are linked to heart disease, cancer, and other illnesses.

Method of assay:

(a) Hydrogen peroxide scavenging assay:

Chemicals: Hydrogen peroxide,phosphate buffer (pH 7.4), Gallic acid and extract.

Apparatus: Spectrophotometer and p^H meter.

Preparation of standard solution:

Required quantity of Gallic acid was dissolved in to give (10, 20, 30, 40, 50)μg/ml

Preparation of Hydrogen peroxide solution:

Required quantity of Hydrogen peroxide is dissolved in phosphate buffer to give 100mM solution with Ph 7.4.

Preparation of sample solution:

Required quantity of sample was dissolved in Phosphate buffer to give (100, 200, 400, 600, 800, 1000)μg/ml.

Procedure:

Take 2mL of hydrogen peroxide solution and add 1mL of normal Gallic acid at different concentrations to it.

2mL of hydrogen peroxide solution is obtained, and 1 mL of various quantities of extract is added to it.Incubate the above-prepared solutions for 10 minutes.

Phosphate buffer was used as a blank and the absorbance was measured at 230nm.

Hydrogen peroxide scavenging activity:-

Although hydrogen peroxide is not a particularly reactive substance, it can be harmful to cells when it produces the hydroxyl radical. As a result, removing the hydrogen peroxide radical is critical for food system safety. The capacity of extraction to scavenge was tested in the experiment. Table 3 shows the free radical hydrogen peroxide, which is displayed against a standard using a bar chart. As can be seen in the graph, all of the samples' activity increased in a concentration-dependent way. In the higher dosages, the comparison standard Gallic acid exhibited extremely high radical scavenging activity (97.85percent).

RESULTS:

Anthocephalus cadamba methanol extract has a substantial effect on hydrogen peroxide radical scavenging activity.

Table 3: Hydrogen peroxide scavenging activity

S. No.	Concentration (μg/ml)	Absorbance	% Scavenging activity
	Control	1.256	-
Gallic acid
1	50	0.758	37.58
2	100	0.386	67.21
3	150	0.285	82.57
4	200	0.986	91.65
5	250	0.025	97.85
Leaf extract
1	50	0.955	24.89
2	100	0.205	83.05
3	150	0.198	88.89
4	200	0.165	88.98
5	250	0.135	91.85

(b) Nitric oxide scavenging activity:

Damage to cellular organelles and enzymes, as well as an increase in lipid peroxidation and the development of insulin resistance, can result from abnormally high amounts of free radicals and a concurrent reduction in antioxidant defence systems. These oxidative stress side effects might hasten the onset of complications.Under aerobic conditions, nitric oxide (NO) is an unstable species. Through intermediates such as NO₂, N₂O₄, and N₃O₄, it interacts with O₂ to create the stable products nitrates and nitrite. is a pleiotropic inhibitor of physiological processes such smooth muscle relaxation, neuronal signalling, platelet aggregation inhibition, and cell mediated toxicity regulation. It is a diffusible free radical that functions as an effector molecule in a variety of biological systems, including neuronal messenger, vasodilation, antibacterial, and anticancer properties.Under aerobic conditions, nitric oxide is an extremely unstable species. Through intermediates such as NO₂, N₂O₄, and N₃O₄, it interacts with O₂ to create the stable products nitrates and nitrite. The Griess reagent is used to calculate it. The quantity of nitrous acid will decrease in the presence of the test chemical, which is a scavenger. In comparison to the standard of Ascorbic acid, the higher dosages demonstrated valuable high radical scavenging activity (93.85%). Table 4 shows the results, as well as a comparison plot with the standard.

Chemicals required:

Sodium nitroprusside, phosphate buffersaline, sulfanlic acid, napthaline diamne dihydrochloride.

Preparation of reagents:

Sulfanilic acid reagent: 20% of glacial acetic acid was prepared by dissolving 20ml of glacial acetc acid in 100ml of distilled water , 0.33ml of sulfanilic acid is taken and is make up to 100ml wth 20% glacial acetic acid.

Napthyl Ethylene Diamine Diamine Dihydro Chloride (0.1%w/v): 0.1gm of napthyl ethylene damine dihydrochloride is dissolved n 100ml of distlledwater.

Sodium nitroprusside (10mm): Molecular weight is 297.95gm n 1000ml = 1ml.

29.795gm in 1000ml = 0.1ml, 2.9795gm in 1000ml = 0.01ml, 0.29795gm in 1000ml = 0.001ml, 0.2979gm in 100ml = 10mm.

Preparation of Aqueous Extract:

100gms of crude powder is combined with 2000ml (1:20) of chilled water in a stainless vessel, then boiled for 2 hours on a gas burner. The filterate is then put into a borosilicate 500ml beaker and evapourated on a hot plate until it reaches the concentrated quantity (do not be in viscous state).

Procedure:

The inhibition of ntric oxide radicals was measured using napthyl ethylene d amine d hydrochloride (0.1 percent w/v). Aqueous ethanolic extract of 500-1000 g/ml and a reaction mixture comprising 2ml of 10mm sodium nitroprusside, 0.5ml saline phosphate buffer, and 0.5ml of standard solution (taken as ascorbic acid or quercetin) are incubated at 25°C for 150 minutes. Following incubation, 0-5ml of the aforementioned reaction mixture was mixed with sulfanlc acd reagent (0-33percent n 20% glacial acetic acid) and left to stand for 5 minutes to complete the diazotzation process.

After that, 1mL napthyl ethylene diamine hydrochloride was added, stirred, and let to stand for 30 minutes at 25 degrees Celsius. The concentration of nitrile was measured at 540nm and compared to the absorbance of a standard nitrile solution as a control.

Ascorbic acid and quercetin were used as standards, and the blank was used as a buffer and to build up solvents.

% Scavenging activity = {(Acontrol - Atest or Astd)}*100

Where, A control = Obsorbance of control

Table 4: Nitric oxide scavenging activity

S. No.	Concentration (μg/ml)	Absorbance	% Scavenging activity
	Control	1.6661	-
Ascorbic acid
1	50	0.168	89.83
2	100	0.136	91.33
3	150	0.132	91.45
4	200	0.113	91.08
5	250	0.102	93.59
Leaf extract
1	50	0.662	59.12
2	100	0.628	62.95
3	150	0.611	63.25
4	200	0.539	67.85
5	250	0.509	68.12

DISCUSSION:

Many components of plant products have been found to be specifically targeted against resistant pathogenic bacteria, and there has recently been a lot of interest in using plant material as an alternate approach to manage pathogenic microorganisms. Multidrug-resistant strains of numerous diseases have emerged, posing a major danger and complicating treatment. Furthermore, the present cost of most chemotherapeutic drugs is unaffordable for the general people, particularly in developing nations such as India. As a result, efforts must be directed toward the creation of a therapy that is both effective and non-toxic. The present work was a pioneer to explore the antimicrobial, anti fungal and anti oxidant properties of ethanolic extract of Anthocephalus cadamba.

SUMMARY AND CONCLUSION:

The results revealed that the ethanolic leaf extract had significant antibacterial activity, which was higher than the aqueous extract but lower than the standard. It also has hydrogen peroxide scavenging and nitric oxide scavenging properties. It was determined that this research would lead to the discovery of a valuable chemical that might be utilised to develop new, different, and more powerful natural antibacterial medicines. More research is needed to discover the biologically active chemicals and assess their efficacy against pathogenic bacteria linked to a variety of human illnesses.

REFERENCES:

1. Gontijo DC, Nunes LG, Farias LM, Duarte MG, Carvalho AF, Fietto LG, Leite JP. Assessment of the phenolic content, mutagenicity and genotoxicity of ethanolic extracts of stem bark and leaves from Strychnospseudoquina A. St.-hil. Drug ChemToxicol 2018; 10: 1-7.

2. Nostro A, Germanò MP, D'angelo V, Marino A, Cannatelli MA. Extraction methods and bioautography for evaluation of medicinal plant antimicrobial activity. Lett Appl Microbiol 2000; 30: 379-384.

3. Choudhary R, Tandon RV. Consumption of functional food and our health concerns. Pak J Physiol 2009; 5: 76-83.

4. Shahrbandy K, Hosseinzadeh R. In vitro antioxidant activity of Polygoniumlyrcanicum, Centaureaedepressa, Sambusebulus, Menthe spicata and Phytolaceae Americana. Pak J BiolSci 2007; 10: 637-640.

5. Saeed N, Khan MR, Shabbir M. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilisleptophylla L. BMC Complement Altern Med 2012; 12: 221-232.

6. Song JM, Lee KH, Seong BL. Antiviral effect of catechins in green tea on influenza vírus. Antivir Res 2005; 68: 66–74.

7. Cushnie TT, Lamb AJ. Antimicrobial activity of flavonoids. Int. J. Antimicrob Agents 2005; 26: 343–356.

8. Da Silva MA, Cardoso CA, Vilegas W, Dos Santos LC. High-performance liquid chromatographic quantification of flavonoids in Eriocaulaceae species and their antimicrobial activity. Molecules 2009 ; 14: 4644-54.

9. Ferguson PJ, Kurowska E, Freeman DJ, Chambers A.F, Koropatnick DJ. A flavonoid fraction from cranberry extract inhibits proliferation of human tumor cell lines. J Nutr 2004; 134: 1529–1535.

10. Russo A, Cardile V, Sanchez F, Troncoso N, Vanella A, Garbarino JA. Chilean propolis: antioxidant activity and antiproliferative action in human tumor cell lines. Life Sci 2004; 76: 545–558.

11. Alam MA, Akter R, Subhan N, Rahman MM, Majumder MM, Nahar L, Sarker SD. Antidiarrhoeal property of the hydroethanolic extract of the flowering tops of Anthocephalus cadamba. Brazilian J Pharmacog 2008; 18: 155–9.

12. Schwikkard S, van Heerden FR. Antimalarial activity of plant metabolites. Nat Prod Rep 2002; 19: 675–692.

13. Kapil A, Koul IB and Suri OP. Antihepatotoxic effects of chlorogenic acid from Anthocephaluscadamba. Phytother Res 1995; 9: 189–193.

14. Kodangula SC, Borthakur A and Kodangala SP. Anti-inflammatory effect of the methanol extract from Anthocephalus cadamba stem bark in animal modelsa. Int J Plant Biol 2010; 1: 30–2.

15. Acharyya S, Dash GK, Mondal S and Dash SK. Studies on Glucose Lowering Efficacy of the Anthocephalus cadamba (Roxb.)Miq. roots. Int J Pharma Bio Sci 2010; 1: 1–9.

16. Verza SG, Kreinecker MT, Reis V, Henriques AT, Ortega GG. Evaluation of analytical variables of the Folin-Ciocalteu method for the quantitation of the total tannins content using a Psidiumguajava L. leaves aqueous extract as a model. Química Nova 2007; 30: 815-20.

17. Krishna Kondragunta. V, Karuppuraj. V, Perumal. K. Antioxidant activity and Folic acid content in indigenous isolates of Ganodermalucidum. Asian J. Pharm. Ana. 2016; 6(4): 213-215.

18. P. Muthukumaran, P. Padmapriya, S. Salomi, R. Umamaheshwari, P. Kalaiarasan, C. Malarvizhi. In Vitro Anti Microbial Activity of Leaf Powder. Asian J. Pharm. Res. 1(4): Oct. - Dec. 2011; Page 108-110.

19. MuthusamySenthil Kumar, SrinivasanBalachandran, ShibaniChaudhury. Influence of Incubation Temperatures on Total Phenolic, Flavonoids Content and Free Radical Scavenging Activity of Callus from Heliotropium indicum L. Asian J. Pharm. Res. 2(4): Oct. - Dec. 2012; Page 148-152.

20. Ch. Madhu, J. Swapna, K. Neelima, Monic V. Shah. A Comparative Evaluation of the Antioxidant Activity of Some Medicinal Plants Popularly Used in India. Asian J. Res. Pharm. Sci. 2(3): July-Sept. 2012; Page 98-100.

21. Konda Ravi Kumar, K.N.S. Karthik, P. Reshma Begum, Ch. M.M. PrasadaRao.Synthesis, characterization and biological evaluation of benzothiazole derivatives as potential antimicrobial and analgesic agents. Asian J. Res. Pharm. Sci. 2017; 7(2):115-119.

22. Patel D.S. Shah P. B., Managoli N. B. Evaluation of In-vitro Anti-oxidant and Free Radical Scavenging activities of Withania somnifera and Aloe vera. Asian J. Pharm. Tech. 2(4): Oct. - Dec. 2012; Page 143-147.

23. Pranita A. Argade, Mangesh A. Bhutkar, Chandrakant S. Magdum. Albizzialebbeck extract mediated synthesis of Zinc Oxide Nanoparticles and study of its In-vitro Anti-diabetic and Anti-oxidant activity. Asian J. Pharm. Tech. 2019; 9(2):93-98.

24. Balakrishnan N, Panda A B, Raj N R, Shrivastava A, Prathani R. The Evaluation of Nitric Oxide Scavenging Activity of AcalyphaIndica Linn Root. Asian J. Research Chem. 2(2): April.-June, 2009 page 148-150.

25. Ankush Garg, Radhika Maheshwari, Pooja Chawla, Shubhini A. Saraf. Free Radical Scavenging Activity of Novel 5-Substituted Arylidene-3-Substituted-Benzyl-Thiazolidine-2, 4-Diones. Asian J. Research Chem. 3(3): July- Sept. 2010; Page 528-530.

26. Shlini P., Siddalinga Murthy K.R. Extraction of Phenolics, Proteins and Antioxidant Activity from Defatted Tamarind Kernel Powder. Asian J. Research Chem. 4(6): June, 2011; Page 936-941.

Received on 17.08.2021 Modified on 09.11.2021

Asian J. Pharm. Res. 2022; 12(4):275-280.

DOI: 10.52711/2231-5691.2022.00044