In-vitro evaluation of Antioxidant and Antimicrobial Activity of Sankarjata (Uraria picta) Leaves Ethanolic Extract

Indications: boiling cough; chills & fever. The leaves of are also known to treat gonorrhoea¹ Finally, roots and pods cure infantile rectal prolapse. These are oral pain relief capsules given to children. It helps in urinary tract ailments, tumours, inflammation, damage caused by smoking and breathlessness. Paste in water, use to treat snake bite². It is ayurvedic medicine for general body weakness. It is an antioxidant, analgesic and anti-inflammatory drug. In vitro studies of U. picta are laboratory investigations performed outside of living organisms, usually in a controlled environment like a petri dish or test tube.

1. Antioxidant Activity³
2. Antimicrobial Activity⁴
3. Antimicrobial Resistance Modulation

Objectives of Studying Antioxidant Activity of U. picta:

1. Quantification of Antioxidant Capacity:

To measure the antioxidant potential of U. picta extracts using in vitro assays which are DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging, ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay.

2. Analysis of Phytochemicals:

To isolate and quantify bioactive phytochemicals including flavonoids, phenolics, and tannins responsible for the antioxidant potential of the plant.

3. Comparison of Solvent Extracts:

To measure and compare the antioxidant activities for extracts of U. picta prepared with solvents of different polarities (water, ethanol, methanol) among others.

4. Mechanistic Insights:

To investigate the mechanisms through which U. picta mitigates oxidative stress, including free radical scavenging and metal ion chelation.

5. Correlation with Traditional Uses:

To correlate the observed antioxidant activity with the plant's traditional medicinal applications, such as wound healing and anti-inflammatory treatments.

6. Exploration of Synergistic Effects:

To study potential synergistic interactions between U. picta extracts and standard antioxidants to enhance oxidative stress mitigation.

7. Baseline for Further Research:

To provide a foundation for the development of U. picta-based antioxidant formulations for pharmaceutical, nutraceutical, or cosmetic applications.

Objectives of Antimicrobial Studies of U. picta:

A test of antimicrobial activity:

The aim was to evaluate the antimicrobial effectiveness of U. picta extracts against a wide range of microorganisms, including gram-positive, gram-negative bacteria as well as fungi.

Calculation of MIC: 

To determine the minimum concentrations of U. picta extracts, which will inhibit the growth of microorganisms using standard in vitro assays.

Comparison Across Solvent Extracts:

To compare the antimicrobial activity of extracts prepared using different solvents (e.g., aqueous, methanolic, ethanolic) and identify the most potent extract.

Screening Against Multidrug-Resistant (MDR) Strains:

To evaluate the effectiveness of U. picta extracts against multidrug-resistant pathogens to explore its potential in combating antimicrobial resistance (AMR).

Investigation of Mechanisms of Action:

To explore the possible mechanisms by which U. picta extracts exert antimicrobial effects, such as disruption of microbial cell walls, inhibition of protein synthesis, or interference with metabolic pathways.

Phytochemical Correlation:

To correlate the antimicrobial activity with specific phytochemicals, eg: alkaloids, flavonoids, and saponins from Uraria picta.

Synergistic Potential with Antibiotics:

To examine whether U. picta extracts enhance the efficacy of standard antibiotics, potentially serving as adjuvants in antimicrobial therapies.

Toxicity Assessment:

To assess the cytotoxicity of the extracts to ensure their safety for potential therapeutic applications.

Baseline for Drug Development:

To provide foundational data for developing U. picta-derived antimicrobial agents for pharmaceutical use.

Validation of Traditional Usage:

To scientifically validate the traditional use of U. picta as an antimicrobial agent in folk medicine.

Objectives of Antimicrobial Resistance Modulation Studies on U. picta:

1. Evaluation of Resistance Modulation Activity:

To investigate the ability of U. picta extracts to enhance the efficacy of conventional antibiotics against multidrug-resistant (MDR) pathogens.

2. Assessment of Synergistic Effects:

To determine whether combining U. picta extracts with antibiotics produces synergistic effects, reducing the minimum inhibitory concentration (MIC) on antibiotics.

3. Screening opposed to Resistant Strains:

To assay the modulation potential of U. picta extracts against various MDR bacterial strains, including Extended-Spectrum Beta-Lactamase (ESBL) producers, Methicillin-Resistant Staphylococcus aureus (MRSA), and Carbapenem-Resistant Enterobacteriaceae (CRE).

4. Mechanistic Exploration:

To explore the molecular mechanisms underlying the resistance-modulating effects of U. picta, such as inhibition of efflux pumps, disruption of biofilms, or interference with resistance gene expression.

5. Phytochemical Correlation:

To identify bioactive compounds in U. picta responsible for antimicrobial resistance modulation and establish their role in synergizing with antibiotics.

6. Reduction of Resistance Development:

To assess whether U. picta extracts can delay or reduce the development of resistance in pathogens when used in combination with antibiotics.

7. Broad-Spectrum Modulation Potential:

To evaluate the efficacy of U. picta extracts in modulating resistance across a diverse range of bacterial species and classes of antibiotics.

8. Safety and Cytotoxicity Studies:

To ensure that the extracts are non-toxic to human cells at effective concentrations used for resistance modulation.

9. Validation of Traditional Knowledge:

To validate traditional uses of U. picta in enhancing the effectiveness of herbal or synthetic antimicrobial therapies.

10. Foundation for Adjuvant Development:

To provide data supporting the development of U. picta-based adjuvants for use in combination therapies against MDR infections.

Fig:1 U. picta Plant

Future Prospects of Study of U. picta:

Because of its therapeutic qualities, U. picta, often referred to as "Prishnaparni" in Ayurveda, is a medicinal plant with a great deal of room for further study and use. U. picta may have bright futures in the following important areas:

1. Potential for Pharmaceuticals:

Bioactive substances found in U. picta, such as flavonoids, saponins, alkaloids, and tannins, have demonstrated a range of medicinal benefits. The plant has long been utilised for its hepatoprotective, antioxidant, anti-inflammatory, and antibacterial properties.
Research is still being conducted to assess its potential for medication development, namely in natural plant-based treatments for infections, inflammation, and liver function.

2. Cosmetic and Nutraceutical Markets:

• U. picta extracts may help skincare products lower inflammation and oxidative damage because of their antioxidant qualities. Additionally, it may be used in dietary supplements for immunity, wellbeing, and overall health.
• Given the growing demand for natural and organic products, the nutraceutical industry may be interested in the plant's bioactive components.

3. Genetic and Biotechnological Research:

• By investigating the genetic underpinnings of its therapeutic qualities, active compounds' quality and yield may be improved. Techniques for tissue culture and genetic manipulation may improve U. picta commercial viability.
• Biotechnological advances could help optimize its growth and conservation, as some populations of this plant face threats from overharvesting.

4. Applications in Agriculture⁵ and the Environment:

• Because U. picta is a hardy plant that can thrive in a variety of soil types, it is a good choice for improving soil health and reclaiming land, especially in marginal or degraded soils.
• It may improve soil fertility by fixing nitrogen, which would make it beneficial for sustainable farming methods.

MATERIALS AND METHODS:

Preparation of Plant Extract:

Drying and Powdering:

Collect fresh U. picta leaves (or other parts), wash them thoroughly, and dry them in the shade until they are completely dry. Once dry, grind leaves to form a fine powder used by grinder.

Extraction:

1. Weigh about 50 grams of the powdered plant material.
2. Soak the powder in 250 ml of ethanol, methanol, or water (depending on the compound you want to extract) in a conical flask.
3. Seal the flask and allow it to stand for 24-48 hours, shaking it occasionally.
4. After the extraction period, filtration is done by using Whatman filter paper or a similar filter.
5. Then filtrate is obtained, which will be used for the phytochemical tests.

Fig 2: U. picta plant extract

Phytochemical Analysis:

• Phytochemical Screening: Qualitative and quantitative tests for alkaloids, flavonoids, tannins, saponins, and other secondary metabolites.
• Chromatographic procedures:
  • Thin Layer Chromatography (TLC)
  • High-Performance Liquid Chromatography (HPLC)

Antioxidant Activity

• Assays:
  • DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical scavenging assay
  • ABTS (2,2′-azino bis (3-ethylbenzothiazoline-6-sulfonic acid)) assay
  • Ferric Reducing Antioxidant Power (FRAP) assay
• These tests measure plant's ability to neutralize free radicals.

Antimicrobial Studies

• Agar Well Diffusion Method: To test against bacterial and fungal strains.
• Minimum Inhibitory Concentration (MIC): Using broth dilution methods.
• Zone of Inhibition: Quantification of antimicrobial activity by measuring the inhibited zone around the sample.

Anti-oxidant assay of U. picta:

1. DPPH Free Radical Scavenging Assay⁶

This assay evaluates the extract's ability to donate hydrogen to neutralize the DPPH radical.

• Principle: The DPPH radical exhibits a violet colour. Upon reduction by an antioxidant, it turns yellow.
• Protocol:
  1. Prepare a DPPH solution in methanol (e.g., 0.1 mM).
  2. Mix the plant extract with DPPH solution at various concentrations.
  3. Allow the sample to stand inside chamber for approximately half an hour at room temperature absence from the light.
  4. Measure absorbance at 517 nm using a UV-Vis spectrophotometer.
  5. Measure % inhibition using this formula:

Inhibition %=Abs(control) - Abs(sample)?Abs(control) ×100

Antimicrobial Studies with U. picta:

1. 50442660183178960. Testing Methods

A. Agar Well Diffusion Method⁷

1. 3271573590719. The diffusion of plant extract into agar inhibits microbial growth around wells.
   125903990432.  

• • Prepare agar plates and inoculate with test organisms using a spread plate method.
  • Make wells (6–8 mm diameter) in the agar.
  • Put the extract of the plants into different concentrations in separate wells.
  • Incubate at 37°C for bacteria (24 hours) or at 28°C for fungi (48 hours).
  • Measure zone of inhibition (clear area around the well in mm).

1. 204141490. Interpretation

1. 449207. of Inhibition (mm): Indicates antimicrobial potency in agar-based methods.

1. 1001. and MBC (Minimum Bactericidal Concentration)⁸:
   1001. Lowest concentration inhibiting visible growth.

1. 1000. Lowest concentration killing 99.9% of organisms.

• • Inhibition: Calculated for biofilm or microbial viability.

1. 204141490. AND DISCUSSION:

Table 1: (+) = Present (-) = Absent

Fig 3:

Fig 3:

Principle:

DPPH is a deep violet-coloured compound having absorption maximum at 517 nm. The interaction of DPPH withAn antioxidant, it is reduced, leading to a colour change from violet to pale yellow. The decrease in absorbance at 517 nm is used to estimate the antioxidant activity.

Materials:

• DPPH solution (usually prepared in methanol or ethanol)
• Test sample (antioxidant or extract to be tested)
• Methanol or ethanol (solvent)
• UV-Vis spectrophotometer or microplate reader
• Control and blank solutions

Procedure:

1. Prepare the DPPH solution: Dissolve DPPH in methanol or ethanol to obtain a working solution (e.g., 0.1 mM). Store it in a dark bottle to avoid light degradation.
2. Prepare sample solutions: Dissolve the test sample in a suitable solvent at various concentrations.
3. Mix solutions: Mix a fixed volume of the DPPH solution (e.g., 1 mL) with the test sample (e.g., 0.5 mL).
4. Incubation: Allow reaction mixture to stand at room temperature in a dark place for known duration Usually, 30 minutes.
5. Quantify the absorbance: Take the absorbance of the reaction mixture at 517nm with a

Spectrophotometer. Control and blank:

• • Control: Mix DPPH solution with the solvent only (no sample).
  • Blank: Mix the sample solution with solvent (no DPPH).

Calculation of Radical Scavenging Activity:

The percentage of DPPH radical scavenging activity is calculated using the formula:

Inhibition %=Abs(control) - Abs(sample)?Abs(control) ×100

Where:

• Abs control? = Absorbance of the control
• Abs sample? = Absorbance of the test sample

Antioxidant Potentiality of U. Picta Using DPPH.

Table 2:

Fig 4: Sample And Control

Minimum Inhibitory Concentration (MIC):

The MIC study is one of the standard methods for evaluating the antimicrobial Attributes associated with an extract from plant sources, including Utricularia picta; MIC refers to the weakest concentration of the extract whereby it prevents visible microbial growth. Below is a comprehensive guideline on how to perform the MIC study on Utricularia picta.

Preparation:

Materials:

1. Plant extract: Utricularia picta extract (e.g., methanolic, ethanolic, aqueous, etc.).
2. Microorganisms: Bacterial or fungal strains (e.g., Escherichia coli, Staphylococcus aureus, Candida albicans).
3. Growth medium:
   • Nutrient broth or Mueller-Hinton broth for bacteria.
   • Sabouraud dextrose broth for fungi.
4. Solvent: DMSO, ethanol, or water to dissolve the plant extract.
5. Sterile microplates: 96-well microtiter plates.
6. Positive control: Known antimicrobial agent (e.g., ampicillin, ciprofloxacin).
7. Negative control: Solvent or medium alone.
8. Indicator dye: Resazurin or tetrazolium salts (optional, for colorimetric detection).
9. Incubator: Maintain the optimal growth temperature (e.g., 37°C for bacteria, 25–30°C for fungi).

Procedure:

1. Extraction Methods from Plants

• First, prepare a stock solution of the plant extract using the chosen solvent; for example, dissolve the plant extract in DMSO at: 10 mg/mL.
• Sterilize the extract using a 0.22 μm syringe filter, which reduces the chance of contamination during experiments.

2. Microbial Growth Preparation

• Incubate the test microorganism in the broth until it reaches a required optical density (e.g., OD600=0.1, approx. 1×108CFU/mL).Dilute the inoculum to achieve a final”: concentration of 5×10⁵ CFU/mL.

3. Serial Dilution of the Extract:

• Serial title of two from the plant extract in the microplate:
• Add 100 μL of sterile growth medium to all wells.
• Add 100 μL of plant extract into the first well, mix very well.
• Transfer 100 μL from the first well to the next, repeat until the desired dilutions occur (e.g., 10 mg/mL to 0.078 mg/mL).
• Leave one row as the solvent control (no extract).

4. Inoculation:

• To all wells, add 100 μL of prepared inoculum from the isolated microbial.
• And this gives a final volume for each well of 200 μL.

5. Controls:

• Positive control: Add a known antibiotic or antifungal agent in a separate row.
• Negative control: Include wells with the solvent or medium but without the extract.

6. Incubation:

• Incubate the microplate for 18–24 hours at the appropriate temperature (e.g., 37°C for bacteria, 25–30°C for fungi).

7. Detection of MIC:

• Visual inspection: Check for turbidity in each well (growth indicates a lack of inhibition).
• Indicator dye (optional):

Add 20 microliters of resazurin solution (0.02%) in each well and keep it for additional 2 hours.

Colour changes from blue to pink indicate microbial growth.

• The MIC is defined as the minimal concentration of plant extract that does not exhibit visible growth or any colour change.

Data Interpretation:

• MIC value: The concentration at which microbial growth is completely inhibited.
• Lower MIC values indicate stronger antimicrobial activity.
• Compare the MIC of the extract with the positive control to evaluate its relative efficacy.

Determination of minimum inhibitory concentration (MIC) of leaf extract of U. picta with respect to different bacteria

The leaf extract MIC of U. picta was determined by the agar dilution method (NCCLS, 2003) by adding the extract individually at concentration of (control), 500, 1000,2000 and 5000 mg/ml. Nutrient agar medium was poured in 80 mm Petri dishes at 20 ml volume. At first, the organisms were grown in peptone water, adjusted to a McFarland standard value of 0.5, and were spot inoculated on nutrient agar plates such that each inoculum contained around 5x 10 5 CFU/ml. The inoculated plates were incubated at 37 C for 24 hours and further incubated for 72 hours if needed. The lowest concentration of either an antibiotic or non-antibiotic that completely inhibited the growth of organism was defined as the MIC according to NCCLS, 2003.

Minimum inhibitory concentration (MIC mg/ml) of aqueous extract of leaf of U. picta against Gram positive bacteria.

Minimum inhibitory concentration (MIC mg/ml) of aqueous extract of leaf of U. picta against Gram negative bacteria,

Table 4