Research Articles

2022  |  Vol: 7(3)  |  Issue: 3(May-June) | https://doi.org/10.31024/apj.2022.7.3.4

Evaluation of antioxidant and antimicrobial activity of Plumbago auriculata stem extract

Sangeeta Saraswat, Suresh Kumar Dev*

Pacific College of Pharmacy, Pacific Academy of Higher Education and University, Udaipur, Rajasthan, India

*Address for Corresponding Author

Dr. Suresh K. Dev

Pacific College of Pharmacy, Pacific Academy of Higher Education and University, Udaipur, Rajasthan, India

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Abstract

Objectives: The present study was designed to assess the preliminary phytochemical, antioxidant and antimicrobial activity of Plumbago auriculata Linn methanol extract (PAME) stem part. Material and Methods: The preliminary, phytochemical analysis, antioxidant, isolation of phenolic compound and antimicrobial activity of PAME was determined. Antioxidant activity was estimated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay method. While antimicrobial activity was determined by agar plate diffusion method using Staphylococcus aureus, Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa bacterial and fungal strains. The isolation of phenolic compound was done by TLC method, and was characterized by UV, FTIR, and NMR spectra peaks. Results: The outcomes demonstrated that PAME found as most potential due to presence of higher phenolic content (TPC) 8.34±0.68 mg of equivalent gallic acid, measured by the Folin-ciocaltechu method. The flavonoid content (TFC) was found to be 12.7±0.81 quercetin equivalent measured by aluminium chloride colorimetric method. DPPH Free Radical Scavenging Activity showed by IC50 value was found to be 121µg/mL for the methanol extract. The antimicrobial activity was also confirmed that the PAME led to a higher inhibition zone against bacterial and fungal strains. Spectral results of UV, FTIR and NMR indicated that isolated compound was rutin. The Rutin content in the PAME was found to be 3.55 ±1.55%, w/w. Conclusion:This study suggests that the PAME from stem has strong antioxidant, TPC and TFC potential components and it could be a significant source of natural antioxidant and antimicrobial agent for topical formulation. 

Keywords: Plumbago auriculatalam, antioxidant, antimicrobial, total phenolic content, total flavonoid content

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Introduction

Plants and herbal extracts are commonly used to treat a number of serious illnesses due to their well-known therapeutic capabilities. Ayurvedic, Unani, and Siddha medicine are founded on these. Since the beginning of time, much work has been done in this field, but more work is still needed to effectively utilise these invaluable resources for the sake of humanity. Since ancient times, people have used plants as a sort of medicine. Numerous plant species have been used for ages by humans as remedies for a wide range of illnesses due to their well-known ability to produce a variety of secondary metabolites. For a variety of reasons, including growth regulation, inter and intra-specific interactions, and defence against pathogens and predators, secondary metabolites are biosynthesized in plants. Many of these natural compounds have been shown to exhibit intriguing biological and pharmacological properties, and they are employed as chemotherapeutic agents or as the basis for the creation of contemporary medications (Abubakar et al., 2010). Novel medicinal molecules have been inspired by plants since plant-derived medications have greatly improved human health and wellbeing. They play a dual role in the creation of novel medications: (a) they might serve as the foundation for a drug, a model for natural drug development, or (b) a phytomedicine for illness therapy (Iwu et al., 1999; Ciocan et al., 2007). There is no denying that modern medicine has made tremendous advances, but many illnesses and diseases still lack a treatment. Herbal plants are essential to human life. Since the dawn of time, people have used plants' flowers, leaves, stems, berries, barks, and roots as antioxidants, antimalarial, antibacterial, analgesic, and other medicinal remedies. Commonly found in medicinally active plants are phenolic acids, flavonoids, stilbenes, tannins, coumarins, lignans, and lignins, which have a variety of biological effects, including antioxidant and antibacterial activity (Abera et al., 2015).

In many medical settings, herbal remedies are used. Since ancient times, the traditional medical system has demonstrated that a wide range of medicinal plants contain powerful medications that can lessen or eradicate illnesses. This made it necessary to create safer medications to address inflammatory conditions, skin conditions, microbial and fungal infections, diabetes, liver ailments, and gastrointestinal disorders. For this reason, distinct health care programmes in different nations complement one another in terms of the isolated active principles needed to be administered in modern pharmaceuticals and herbal medicines. Secondary metabolites or substances found in medicinal plants, including flavonoids, tannins, terpenoids, alkaloids, and others, may be what give them their analgesic, antifungal, and antibacterial properties. Some human pathogenic fungi and bacteria have been discovered to be more resistant to bioactive components like flavonoids' antifungal and antibacterial action (Oladeji et al., 2016). A rise in the hunt for antimicrobial agents from natural sources is a result of antimicrobial multiple drug resistance to widely used commercial medications. More research is required in this field since plant-derived antimicrobial compounds are a resource with immense medical potential that is virtually untapped (Abubakar et al., 2010).

Plumbago auriculata is a perennial herb or small shrub with a stem that is upright, trailing, or climbing, with branches that are widely spaced. The leaves are simple, alternate, elliptic to obovate, 3- 41.5-2 cm, with an acute to attenuate base and an acute, apiculate apex. The petiole is up to 1 cm long. Compact inflorescence measuring 3-5 cm. 1 centimetre calyx. violet to blue corolla tube 3 cm long, 1 cm long, widely obovate lobes. 5 free stamens, 3 cm long filaments, and an additional 3 mm. Ovary superior, 1 celled, filiform, 4 cm by 2 cm in size. Fruit a membranous, oblong capsule. One seeded, rectangular, 7mm long and somewhat flattened, dark brown or black, up to 8mm long, tapering to an apex encased in the persistent calyx, dehiscent.

The powdered root of this plant is used as a snuff for headaches and to ward off unpleasant nightmares. It has also traditionally been used to treat a variety of illnesses, including warts, broken bones, wounds, and wart therapy. To prevent lightning, a stick from the plant is tucked into the thatch roof of cottages. In East Africa, P. auriculata flowers and leaves are used to dye fabrics a variety of colours, including beige, lemon, yellow (when coupled with alum), and gold (combined with chrome). Root sap, which is grey-blue, is applied as tattoo ink (Burkill et al., 1997). The root juice or extract of this plant is traditionally consumed by the Pawara Bhil and Pardhi tribes in the Satpuda Forest region of the Dhule and Jalgaon district of Maharashtra before each meal for a week (Jain et al., 2010). In order to explore the antioxidant and antibacterial properties of Plumbago auriculata stem extract, this plant with therapeutic potential was chosen.

Material and methods

Selection procurement of plant materials

Plumbago auriculata lam, crude stem was collected herbal drug shop, Hathipol, Udaipur, Rajasthan. The stem part of the plant was selected for investigating antioxidant and antimicrobial activity.

Preparation of extract

To remove the appropriate components from the plant materials for further separation and characterization, extraction is required. Processes including pre-washing, drying of plant materials, or freeze drying, and grinding to obtain a uniform sample are carried out before extraction. The bioactive component was extracted from natural materials using several solvent systems. Several techniques were utilized for extraction, including sonification, continuous hot percolation (Soxhlet extraction), maceration, microwave assistance, etc (Sasidharan et al., 2011). Plumbago auriculata stem component was the plant part for which the extraction was carried out. To preserve homogeneity, the plant components were sieved 20 times after being ground into a coarse powder and dried in the shade for two weeks. To remove fatty components, the coarsely dried powder was first treated with petroleum ether (60–80 °C) for 24 hours. Methanol was then used again, along with the Soxhlet apparatus, at the predetermined temperatures. Whatman filter paper was used to capture the extracts, which were then concentrated or dried in a rotatory vacuum evaporator. In order to determine the % yield, the extracts were kept in a desiccator (Singh et al., 2017; Sharma et al., 2013).

Phytochemical Evaluation Parameters

Qualitative chemical tests

All of the chosen medicines were concentrated after being extracted in methanol or a mixture of methanol and water (70:30). Then, other qualitative tests were performed on these extracts to determine their various contents, including alkaloids, glycosides, volatile oils, flavonoids, tannins, etc (Bajaj et al., 20212; Sharma et al., 2013).

a. Tests for Glycosides

Liebermann’s Test: the aqueous plant crude extract was treated with 2.0 ml of acetic acid and 2 ml of chloroform. A few drops of concentrated H2SO4 were then added when the liquid had reached room temperature. Green hues indicated the amount of aglycone and the steroidal component of glycosides.

Keller-Kiliani Test: The 10 ml aqueous plant extract and 1 ml of concentrated conc.H2SO4 were combined with a 4.0 ml solution of glacial acetic acid and a drop of a 2.0% Fecl3 combination. Between the layers, a brown ring formed, revealing the presence of cardiac steroidal glycosides.

Salkowski’s Test: A 2 ml of concentrated H2SO4 was added to the aqueous plant crude extract. A reddish-brown hue developed, indicating that the glycoside's steroidal aglycone component was present (Gul et al., 2017).

b. Test for Alkaloids

Mayer’s test: To a 10 ml of plant extract, two drops of Mayer's reagent were applied along the test tube's sides. Alkaloids are present, as evidenced by the white creamy precipitate.

Wagner’s test: A few drops of the Wagner's reagent are added to a few millilitres of plant extract along the test tube's sidewalls. Alkaloids are present, as shown by a reddish-brown precipitate (Gupta et al., 2013; Gupta et al., 2015).

c. Test for Amino acids

100 mg of the extract are diluted in 10 ml of distilled water, then run through a Whatman filter. The filtrate from No. 1 filter paper is tested for amino acids.

Ninhydrin test: 2 ml of aqueous filtrate is combined with two drops of ninhydrin solution (10 mg of ninhydrin in 200 ml of acetone). Amino acids were present, as evidenced by the purple tint.

d. Test for Carbohydrates

Molish’s test: Two drops of an alcoholic solution of naphthol are added to 2 ml of plant sample extract. A few drops of concentrated sulfuric acid are progressively added along the test tube's sides after the mixture has been well shaken. Carbohydrates are detected by a violet ring.

Benedict’s test: Benedict's reagent is mixed with 0.5 cc of aqueous plant filtrate. For two minutes, the mixture is boiled in a bath of boiling water. A distinctively coloured precipitate suggested that sugar was present.

e. Test for Fixed oils and Fats

Spot test: The extract is pressed between two filter sheets in a tiny amount. The presence of fixed oils was indicated by an oil stain on paper.

Saponification test: A tiny amount of extract is mixed with a few drops of 0.5 N alcoholic potassium hydroxide solution and one drop of phenolphthalein. For two hours, the mixture is heated in a water bath. Fixed oils and fats were revealed by the production of soap or by the partial neutralisation of alkali.

f. Test for Glycosides

Using strong hydrochloric acid for two hours in a water bath, the extract (50mg) is hydrolyzed. After filtering, the hydrolysate is put through the following tests:

Borntrager’s test: 3 ml of chloroform are added to 2 ml of filtered hydrolysate and agitated for 15 minutes. After removing the chloroform layer, 10% ammomia solution is applied. Glycosides were denoted by the pink colour.

Legal’s test: In pyridine, the extract (50 mg) is dissolved. Added the Sod nitroprusside solution and used 10% NaOH to make the solution alkaline. Pink hue indicated glycoside presence. g. Check for tannins and phenolic chemicals.

Ferric Chloride test: A 50 mg dose of the extract is dissolved in 5 ml of distilled water. This is then mixed with a few drops of a neutral 5% ferric chloride solution. Phenolic substance was identified by a dark green colour (Tiwari et al., 2016; Mourya et al., 2017).

Gelatin test: 50 mg of the extract are diluted in 5 ml of distilled water before being mixed with 2 ml of a 1% solution of gelatin containing 10% NaCl. Phenolic chemicals were present as white precipitate (Banu et al., 2015)

h. Test for Tannins: All of them are filtered, warmed aqueous extracts. Filtrate (5ml) and a 1ml solution of 5% ferric chloride was given time to react. The presence of tannins is indicated by the hue of deep blue or dark green.

i. Test for Saponins

Extract (1 ml) was shaken in a graduated cylinder for 15 minutes after being diluted with distilled water to a volume of 20 ml. Stable foam development indicated the presence of saponins.

A solution of 1% lead acetate was used to treat an extract (1ml). The presence of saponins is shown by the production of white precipitate (Devmurari et al., 2010).

j. Test for Flavonoids

Ferric chloride test: A few drops of neutral ferric chloride solution were added to the extract's alcoholic solution. The presence of flavonoids was revealed by the emergence of green colour.

Shinoda Test: Five millilitres of 95% ethanol, a few drops of concentrated hydrochloric acid, and 0.5 grammes of magnesium turnings were added to the dry extract. Pink was spotted. Pink colour revealed the presence of flavonoids.

Zinc-hydrochloric acid-reduction test: Zinc dust and a few drops of hydrochloric acid were added to the test solution, and the resultant magenta red colour confirmed the presence of flavonoids.

Alkaline reagent test: The test solution's intensity of yellow hue rose after being treated with sodium hydroxide solution and it turned colourless after a few drops of diluted acetic acid were added.

Lead acetate solution test: Yellow precipitate results from the test solution when a few drops of lead acetate solution (10%) are added.

k. Test for Proteins

Biuret test: 2 ml of Biuret reagent were added to 2 ml of extract. It was properly shook and cooked in a water bath. Proteins could be detected by the emergence of red or violet colour. In a different procedure, 4% NaOH and a few drops of 1% CuSO4 solution were added to the extract (3ml). Proteins were present because of the emergence of pink or violate.

 l. Test for Steroids

Salkowski reaction: Chloroform and conc. H₂SO₄ were added to 2 ml of extract. It had a good shake. The chloroform layer is red, while the acid layer is fluorescing in a greenish yellow colour

Liebermann’s reaction: With 3 ml of acetic anhydride, add 3 ml of extract. It was heated to ambient temperature and then cooled. Few drops of concentrated H₂SO₄, A blue hue is visible 80 (Jamadar et al., 2017).

Estimation of phytoconstituents by thin layer chromatography (TLC)

The thin layer chromatography was carried out using conventional techniques. PAME sample concentrations (2 mg/ml) were dissolved in the appropriate solvents in small amounts. Utilized were solvent systems created by adjusting the concentration of mobile phases. The plates were seen using a UV-TLC viewer immediately following drying (Figure 3). Different places' Rf values were determined, and the results were compared to the reference values (Svendsen et al., 1983; Sharma et al., 1998; Medicsaric et al., 2004).

Estimation of Rutin Content

A conventional technique was used to determine the rutin content with certain changes. Rutin (10 mg) was dissolved in 10 ml of methanol (80%), after which 10, 20, 30, 40, and 50 mg/ml serial dilutions were made. Separately, 100 ml of methanol (80%) was used to dissolve around 100 mg of the test extracts. A UV spectrophotometer was used to assess the sample's absorbance at 260 nm while using distilled water as a control. The Rutin standard curve served as the foundation for the estimation. Results were presented as % weighted Rutin content.

In-vitro antioxidant activity by DPPH method

Measurement of the polyherbal combination's and individual extracts' capacity to scavenge free radicals using the standardized 1, 1-diphenyl-2-picryl-hydrazil (DPPH) technique with some modifications in Srivastava et al. In methanol, a solution of DPPH (0.1 mM) was made. Higher DPPH concentrations revealed an absorbance that was beyond the limits of spectrophotometric measurement precision (Ayres, 1949; Sloane et al., 1977). The concentration of DPPH was so chosen such that it obeyed beer’s lambert laws. From the stock solution (10mg/ml) of ascorbic and methanol extract test sample (PAME), serial dilution of 2, 4, 6 and 8 mg/ml were prepared. To 50μl of test sample of different concentration, 1ml of DPPH solution (0.1 mM) was added in each test tube and final volume in each test tube was made up to 3 ml with methanol. The reaction mixture was shaken vigorously and allowed to stand at room temperature for about 30 minutes. A UV-visible spectrophotometer was used to measure the mixture's absorbance at 517 nm with methanol serving as the reference. Each sample was measured three times, with the mean representing the outcomes. In this manner, the ascorbic acid served as a typical antioxidant. The following percentage of DPPH free radical scavenging activity (FRSA) was calculated:

% (FRSA) = (Absorbance of control-Absorbance of test sample) Absorbance of controlx100

The operations were carried out in a dark chamber because it was discovered that the absorbance of DPPH decreased with exposure to light due to changes in its magnetic property (Figure 3). To stop changes in the magnetic characteristics of the DPPH, especially during working and storage, standardized preventive techniques were devised. Within 24 hours, a freshly produced DPPH solution in methanol that had been kept at ambient laboratory temperature (24–25^oC) and in the dark was used (Otohinoyi et al., 2014).

Evaluation of antimicrobial activity

Agar well diffusion method

Gram-positive (Staphylococcus aureus, Bacillus subtilis) and gram-negative (Escherichia coli and Pseudomonas aeruginosa) strains were used to examine the effects of each test medication utilizing the cup plate method.

Culture of test microbe

Nutrient broth medium (NBM) was made using agar-agar and 8% nutrient broth in double-distilled water for the growth of the bacterial strains. It was autoclaved for 25–30 minutes at 15 Ibs psi. 15 ml of NBM were poured into petri dishes in an aseptic manner to create the agar test plates, which were then left to stabilize at room temperature. Regular sub-culturing was used to maintain bacterial cell cultures in peptone saline solution, and the cultures were incubated at 37^oC for 24hrs) (Yadav et al., 2014).

Preparation of agar plates and sampling of the test drugs

In order to evenly distribute the inoculum, agar plates were infected by streaking bacterial strains across the entire sterile surface for 1-2 times. The agar plates were then drilled with 9 mm diameter wells after being allowed to dry at ambient temperature. Using dimethyl sulphoxide (DMSO) as a diluting solvent, test drug (PAME) concentrations of 50, 100, 150, and 200 mg/ml and standard medication Ofloxacin (10 mg/ml) were created. Using a sterile micropipette, the standard and test medicines (100 l) were applied to the wells. The plates were then kept in an incubator for 24 hours at 37 degrees Celsius. Using a calibrated digital Vernier calliper, the zone of inhibition was quantified. Each bacterial strain had a triple run of the technique

Determination of minimum inhibitory concentration (MIC)

Serial dilutions of extract (50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39 and 0.19 mg/ml) and standard drug ofloxacin (10, 5.0, 2.5, 1.25, 0.62, 0.31, 0.15, 0.07, 0.03 and 0.01μg/ml) were prepared. Each test tube containing 100μl of 105 CFU/ml of test strain (Staphylococcus aureus) in tubes with an equal volume of nutritional broth, three bacteria (Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa) were injected. The tubes were incubated aerobically for 24-48 hours at 37 °C. For every strain, 3 control tubes were kept (media control, organism control and extract control). The MIC was defined as the lowest concentration of extract that, when compared to the control tubes, did not result in any turbidity or discernible growth after 24 hours (Jahan et al., 2013). Each of the four chosen microorganisms has its own MIC established.

Results and Discussion

The yield of the extract from the stem section of Plumbago auriculata was determined to be 9.31%. All extracts were dark brown in colour, and the methanolic extract of the stem portion of Plumbago auriculata was semisolid.

Preparation of plant extracts

Extraction was done by the procedure mentioned and all selected extracts were further evaluated for solubility as freely soluble in methanol and ethanol and insoluble in n-hexane, ethyl acetate, chloroform and water.

Phytochemical investigation

The methanolic extract of Plumbago auriculata stem portion (PAME) was used for phytochemical screening tests, and it was discovered that PAME extract contained Glycosides, Alkaloids, Amino acids, Carbohydrates, Glycosides, Phenolic chemicals, Tannins, Saponins, Flavonoids, and Proteins. The bioactive substances offer semi-qualitative data on the extract's active ingredients. A phytochemical analysis of PAME revealed that the methanolic extract included a variety of phytoconstituents (Table 1). As a result, PAME underwent additional in-vivo research.

Table 1. Results of phytochemical screening test

S. No.	Experiment	Observations
		Pet. Ether Extract	Ethyl Acetate	Methanol
Test for Carbohydrates
1.	Molisch’s Test	-	-	+
2.	Fehling’s Test	-	-	+
3.	Benedict’s Test	-	-	+
4.	Bareford’s Test	-	-	+
Test for Alkaloids
1.	Mayer’s Test	-	+	+
2.	Hager’s Test	-	+	+
3.	Wagner’s Test	-	+	+
4.	Dragendroff’s Test	-	+	+
Test for Terpenoids
1.	Salkowski Test	-	-	+
2.	Libermann-Burchard’s Test	-	-	+
Test for Flavonoids
1.	Lead Acetate Test	-	+	+
2.	Alkaline Reagent Test	-	+	+
3.	Shinoda Test	-	+	+
Test for Tannins and Phenolic Compounds
1.	FeCl3 Test	-	+	+
2.	Lead Acetate Test	-	+	+
3.	Gelatine Test	-	+	+
4.	Dilute Iodine Solution Test	-	+	+
Test for Saponins
1.	Froth Test	+	-	-
Test for Protein and Amino acids
1.	Ninhydrin Test	-	-	+
2.	Biuret’s Test	-	-	+
3.	Million’s Test	-	-	+
Test for Glycosides
1.	Legal’s Test	-	-	+
2.	Keller Killani Test	-	-	+
3.	Borntrager’s Test	-	-	+

+ = Components present

Preparation of standard curve of Rutin

The flavonoids are useful in tissue remodelling and have high activity against microbial viability. As a result, the flavonoid concentration was calculated using the aluminium chloride colorimetric method, using rutin flavonoid as the reference. Rutin was dissolved in methanol to create the standard curve, which was then serially diluted in the concentration range of 1 to 50 g/ml (Figure 1).

Figure 1: Standard curve of Rutin

Rutin content of macerated methanolic water extract

The flavonoids are useful in tissue remodelling and have high activity against microbial viability. As a result, the flavonoid concentration was calculated using the aluminium chloride colorimetric method, using rutin flavonoid as the reference. Rutin was dissolved in methanol to create the standard curve, which was then serially diluted in the concentration range of 1 to 50 g/m.

Estimation of phytoconstituents by thin layer chromatography (TLC)

Alkaloids, tannins, and phenols/flavonoids were found in the samples PAME, according to the Rf values. The criteria and values were compared, and a conclusion was reached. Using industry-standard techniques, thin layer chromatography (TLC) was carried out (Harborne, 1998). Methanol was used to dissolve all of the PAME (2 mg/ml) samples that were chosen. Used were solvent systems that were created by adjusting the concentration of the mobile phases. The plates were seen using a UV-TLC viewer immediately following drying (Fig. 2). The computed Rf values for the various sites were then compared to the reference values from studies (Svendsen et al., 1983; Sharma et al., 1998; Medi-Ari et al., 2004).

Table 2. Determination of R_f values of different spots

Alkaloids (Cyclohexane: Chloroform: Diethyl amine 5:4:1)
Sample extract	No. of spots	Rf value	Inference
PAME	2	0.37	Plumbogin
		0.23	Napthaquinone derivatives
Tannins (Chloroform: Ethyl acetate: Acetic acid 50:50:10)
PAME	1	0.60	Cinnamic acid derivatives
Phenol/Flavonoid (Toluene: Ethyl acetate: Formic acid 36:12:5)
PAME	2	0.67	6-Hydroxy flavones
		0.70	6-Hydroxy flavones derivatives

Figure 2. TLC Images of extracts PAME and Standard

Antioxidant evaluation of PAME Extract

PAME extracts were used for phytochemical screening; however the amount of each extract in the composition was chosen based on its unique in-vitro efficacy, or percentage free radical scavenging activity. Methanol extracts were discovered to have effective antioxidant properties.

Free radical scavenging activity of extract

The results of the free radical scavenging activity of PAME tested by the DPPH method are depicted in Table 3, Fig 3. The comparative antioxidant activity among the test samples clearly revealed the effect of PAME, as compared to the standard Ascorbic acid.

Table 3. Antioxidant effects of PAME and Ascorbic acid

Sample	% Inhibition at different concentration
	2mg/ml	4mg/ml	6mg/ml	8mg/ml
AA	62.8	66.5	81.2	90.9
PAME	19.5	51.77	53.8	72.2

Ascorbic acid, PAME: Plumbago auriculata methanolic extract. All values are represented as mean ± SEM, n = 3 for each group

Figure 3. Graphical Representation of DPPH radical Scavenging activity of extract

Screening of extracts by in-vitro antimicrobial assays

Determination of zone of inhibition of PAME extract by agar well diffusion technique was used. The antimicrobial spectrum showed that all the test samples were found to be effective against both gram positive and gram-negative strains in a concentration dependent manner. The sample PAME was found to be effective (Maximum ZI among all test samples) against S. aureus, S. bacillus, E. coli and P. aureginosa at maximum concentration level (200 mg/ml). The results were showed antimicrobial efficacy of PAME in compare to standard. Ofloxacin shows its maximum antimicrobial efficacy as standard drug as compared to PAME under consideration (Fig. 4, 5 and 6). The antimicrobial activities of the selected plant extracts were consistent with the ethno pharmacological relevance. Antimicrobial efficacies of PAME against bacterial strains are shown in table 4.

Table 4. Antimicrobial efficacy of PAME against bacterial strains

Strains	Test drug	Zone of inhibition (in mm), mean ± SEM				Ofloxacin
		50 mg/ml	100 mg/ml	150 mg/ml	200 mg/ml	(10µg/ml)
S. aureus	PAME	16.50±0.412****	21.50±0.415****	25.10±0.415****	27.00±0.624***	30.0±0.618
S. bacillus	PAME	17.29±0.655****	22.69±0.421****	24.75±0.656****	26.00±0.521****	32.4±0.530
E. coli	PAME	16.10±0.480****	19.20±0.532****	21.00±0.547****	23.55±0.510****	31.50±0.577
P. aureginosa	PAME	17.30±0.538****	20.15±0.400****	23.50±0.522****	24.50±0.444****	32.8±0.500

All values are represented as mean ± SEM, n = 3 for each group, Data were analysed by two-way ANOVA, for each bacterial strain, followed by Dunnett's multiple comparisons test Multiple Comparisons Test, ****p< 0.01, Asterisk (*) denotes significant difference as compared to test drug PAME

Figure 4: Antimicrobial spectrum of plants extract PAME and against S. aureus, S. bacillus, E. Coli, P. aureginosa

Figure 5: Antimicrobial activity of ofloxacin against different microbial strains at 10µg/ml

Figure 6. In-vitro culture plates (Agar cup plate method) of standard drug (ofloxacin) and Methanolic extracts (PAME) showing zone of inhibition (ZI) against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa at 50, 100, 150 and 200mg/ml concentrations.

Determination of MIC of plant extract by Broth dilution method

The antibacterial effectiveness of test medicines is assessed using MICs using the broth dilution method. With this technique, the effect of lowering test medication concentrations on the inhibition of microbial growth is measured. These analyses can be used to establish the proper concentrations needed to create the desired effect. The test drug's MICs are considerably lower than the concentration found in the final dose form. Both MICs for gram-positive and gram-negative bacteria were visible in the methanolic test extracts. It was discovered that the PAME had the lowest MIC against S. bacillus. The MIC values for E. coli and P. aureginosa against S. aureus were identical to those for PAME (Fig. 7). The phenolic and flavonoid content of the chosen herbs probably killed the microbes by preventing the formation of their cell walls or by reducing their permeability. These raised the likelihood that all essential cellular components, including the membrane, would cease to function, which would cause mutation, cellular damage, and ultimately cell death.

Table 5. Minimum Inhibitory Concentration of different methnolic extract of Plumbago auriculata

Extracts	S. aureus	S. bacillus	E. coli	Pseudomonas
PAME	3019µg/ml	3021µg/ml	1459µg/ml	6152µg/ml

Figure 7. Minimum Inhibitory Concentration of different methanolic extract of Plumbago auriculata

Conclusion

The phytoconstituents selected for healing wounds were chosen after conducting a literature search. According to the outcomes of the selected phytochemical screening, triterpenoids, tannins, glycosides, flavonoids, polyphenols, carbohydrates, alkaloids, and other classes of compounds with significant antioxidant potentials were discovered in the Plumbago auriculata methanolic extract (PAME). It is generally known that flavonoids and polyphenols are potent sources of antioxidants and can activate the body's defence mechanisms. They show protective benefits against a variety of infectious bacterial and viral infections, and it was shown that vicinyl dihydroxyl groups in rutin and its metabolites impact phenols' capacity to block the creation of starting radical species that is catalysed by iron and copper. It was probably going to neutralise radicals by preventing glucose. Gallic acid (GA), a type of polyphenol (3, 4, 5-trihydroxybenzoic acid), has been found to have both pro- and strong antioxidant properties. The presence of alkaloid, tannins, polyphenols, and flavonoid components in the Plumbago auriculata methanolic extract (PAME) was discovered by (TLC) thin layer chromatography. Free radicals are continuously produced in living systems and cause significant harm to tissues and biological molecules, which results in a number of diseases. The usage of natural antioxidants through food supplements and conventional medicine may be an alternative to the many synthetic medications that can prevent oxidative damage due to their negative side effects. This has given scientists fresh insight into how to treat or attenuate oxidative stress-induced pathogenesis for a variety of diseases using either novel chemical alone or in combination with natural sources possessing antioxidant properties. The plant extract was (PAME) exhibits strong antioxidant effectiveness. The chosen herbs' physiologically active compounds, which are responsible for the broad-spectrum antibacterial action, were what gave Plumbago auriculata methanolic stem extract its powerful antimicrobial potency.

Source of Support: Nil

Conflict of Interest: None declared

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