Unveiling the Bioactive Potential of Lantana camara in Relation to its Anti-oxidant and Antimycotic Properties

Introduction

Infectious diseases are a major cause of morbidity and a increase in mortality rates, especially in developing countries. Nowadays, an extensive range of antimicrobial agents are available, but resistance to them has rapidly evolved.¹ Infections, including the fungal infections have rapidly increased.² Weeds in agricultural land impact economic crops like rice, cassava, and corn. It is necessary to manage these weeds due to high resistance to microbial attack.¹

Lantana camara Linn. is prominently known as “Sleeper weed or Wild sage”. It is a short perennial shrub, two meters in height, with opposite, ovate leaves having scaly or rough surfaces and flowers that are red, pink, white, yellow, and violet in colour.²  In 1809, L. camara was introduced by the British as an ornamental plant in the Kolkata botanical garden.² In Asian countries, leaves of L. camara are employed for tetanus, cuts, rheumatism, eczema, tumors, and catarrhal infections.³ It demonstrates various medicinal activities due to the presence of phytochemicals.⁴ The biologically active components include quercetin, isorhamnetin, palmitic acid, and dodecanoic acid.⁵

The polyphenols recognize antioxidant activity due to their redox property.⁶ They inhibit free radical correlated injuries by direct scavenging or inhibiting enzymes involved in free radical production. The antioxidant activity of L. camara was determined on DPPH (1,1-diphenyl-2-picrylhydrazyl), OH (hydroxyl), NO-(nitric oxide) radicals.⁷ Antioxidant potential was determined by iron chelating, enzymatic activity (peroxidase, polyphenol oxidase), FRAP (ferric reducing antioxidant power) and ABTS (2,2-azino-bis-3-ethylbenzotiazolin-6-sulphonic acid).⁸

To determine antioxidant activity, various tests are used that accelerate oxidative conditions with high temperature and oxygen, which enhances lipid oxidation and risk of degradation.⁹ At ambient temperature, DPPH method is used to measure antioxidant activity.¹⁰  When DPPH free radical reacts with L. camara, the unpaired valence electron on 'N' bridge of DPPH is reduced by ‘H' atom of L. camara, measured by spectrophotometry.¹¹ For comparison of the antioxidant activity of L. camara and ascorbic acid, it is essential to reduce the initial concentration of DPPH free radical by 50%.¹²

L. camara plant extract possesses antifungal activity. It disrupts fungi by breaking cell walls and altering the membrane’s integrity.¹³ It demonstrates effective antifungal activity against various pathogenic fungi, as mentioned in Table 1.

Literature revealed that L. camara plant extract has potent antifungal activity on isolated fungi. The present study explored the anti-oxidant property by using the DPPH method, preliminary phytochemicals screening and antifungal activity against Candida albicans on L. camara gathered from a botanical garden in Davanagere.

Materials and Methods

Chemicals

1,1-diphenyl-2-picrylhydrazyl radical (DPPH) reagents were purchased from Sisco Research Laboratories Pvt. Ltd, Mumbai, while methanol, Wagner’s reagent, Mayer’s reagent, Molisch's reagent, Benedict’s reagent, Millon’s reagent, dilute hydrochloric acid, magnesium tunings, ethanol, 5% w/v ferric chloride solution, 10% lead acetate, ethyl acetate, concentrated sulphuric acid, acetone were purchased from SD - Fine Chem Limited, Mumbai. All the chemicals procured were of ARG grade.

A UV-visible spectrophotometer (UV-19001 SHIMA- DZU CORP 80866) and a weighing balance (scale TEC®) were used during the research.

Plant material

The L. camara plant was gathered from the GM Institute of Pharmaceutical Sciences and Research Institute's garden in Davanagere, Karnataka (Figure 1). A Professor of plant taxonomy at the Faculty of Department of Botany, GM University, Davanagere, Karnataka, identified the plant. Plant leaves were rinsed with distilled water and air-dried at room temperature. Subsequently, the dried components were finely ground using an electric mill, and the resulting powders were stored in dry containers for the extraction process.¹⁹

Extraction yield

The optimal method for extracting 150 g of dried L. camara leaves involved maceration using an equimolar solution of methanol (250 mL) and water (250 mL), followed by thorough mixing.²⁰ Later the mixture was placed in a dark place at room temperature for 10 days and stirred 2-3 times a day. After 10 days, the leaf mixture was taken, filtered, and placed in a water bath to reduce half of its concentration. Then it was placed in a hot air oven at 60°C to obtain a solid form of extract. This method not only facilitates a substantial yield of the crude extract but also results in elevated levels of total phenolics, total flavonoids, and major active compounds. Furthermore, it demonstrates the highest antioxidant activity among the extraction methods studied. This underscores the effectiveness of the maceration process with methanol and water (1:1) in obtaining a comprehensive and potent extract from L. camara leaves.

Phytochemical screening

Identifying phytoconstituents is crucial for predicting the potential pharmacological activity of plants. Conventional phytochemical tests are highlighted as a cost-effective and resource-efficient alternative for preliminary phytochemical screening. These tests are considered economic, easy to perform, and require fewer resources.

Test for Alkaloids:  0.01 g of crude extract dissolved in ethanol and divided into two parts.

• Wagner’s test: A few drops of Wagner’s reagent were added to one part, forming a reddish-brown precipitate 
• Mayer’s test: A few drops of Mayer’s reagent were added to another part to form a creamy white precipitate.

Test for Carbohydrates: 0.01 g of plant crude extract dissolved in 5 mL of distilled water and filtered.

• Molisch’s test: To filtrate, Molisch's reagent and sulphuric acid are added slowly along the sides of the test tube forming a reddish violet ring at the junction of two layers. 
• Benedict’s test: A few drops of Benedict’s reagent were added to the filtrate and boiled, forming a brick-red or orange precipitate.

Test for proteins and amino acids: 0.01 g of crude extract dissolved in water.

• Millon’s test: 5-6 drops of Millon's reagent are added forming a white precipitate.
• Ninhydrin test: 5-8 drops of 2% Ninhydrin reagent are added forming blue colour.

Test for Flavonoids

• Ferric chloride test: 0.01 g of crude extract dissolved in 5% ferric chloride solution, results in appearance of green, purple or brown colour.
• Shinoda test: 0.01 g of crude extract dissolved in 5 mL of ethanol, 10 drops of dilute HCl are added followed by a few pieces of magnesium tunings. The pink or reddish-brown colour appears.

Test for Tannins and phenols: 0.01 g of crude extract dissolved in water.

• Lead acetate test: 10% lead acetate solution added to plant extract solution to form a white precipitate.
• Ferric chloride test: 5% ferric chloride solution added to plant extract, which appears green in colour.

Test for steroids or phytosterols

Liebermann Burchard test: Crude extract was mixed with a few drops of ethyl acetate, boiled and cooled. Concentrated sulfuric acid was carefully added along the sides of the test tube, and the formation of a brown ring at the interface between the two layers was observed. The green colouration of the upper layer indicates a positive test for steroids.

Test for saponins

Foam formation test: Water is mixed with the plant extract solution, shaken, and then observed for froth formation, which should remain stable for 15 minutes.

Test for resins

Turbidity test: 0.01 g of crude extract is dissolved in acetone and 5-7 mL of distilled water is added, and the turbidity is observed.

Determination of free radical scavenging activity by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay

The DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay is a widely used method for determining the antioxidant properties of compounds, including plant extracts.

• 0.5 mM DPPH solution in methanol was prepared.
• A stock solution of L. camara was prepared by dissolving 3 g of solid extract in 100 mL of methanol.
• From the stock solution, various concentrations, 30 μg/mL, 60 μg/mL, 90 μg/mL, 120 μg/mL, 150 μg/ mL of sample were prepared using methanol.
• The DPPH solution was mixed with the plant extract at different ratios. The reaction mixture was allowed to incubate in the dark at room temperature for a specified period (30 minutes).
• After the incubation period, the absorbance of each reaction mixture was measured at the appropriate wavelength of 517 nm using a UV-visible spectrophotometer. Methanol was used as a blank.²¹
• The percentage of DPPH radical scavenging activity was calculated using the following formula:

Radical scavenging activity = "absorbance of control absorbance of sample" / "absorbance of control" × 100

The control was the DPPH solution without any plant extract or standard. A graph of scavenging activity percentage against the concentration of the plant extract was plotted. The IC₅₀ value (concentration at which 50% of the DPPH radicals are scavenged) was determined.

Determination of antifungal activity

Antifungal activity is the capacity of a substance to hinder the growth or development of fungi. The assessment of antifungal activity involves laboratory methods like well diffusion assays. The method enables researchers and practitioners to evaluate the effectiveness of antifungal agents against specific fungal strains. The details of the well diffusion method are tabulated in Table 2.

Test organism: Candida albicans

Test compound as standard: Ketoconazole

Inoculum

Candida albicans cell suspension was prepared and grown on media and cultures were incubated for 24 hours at 24-30°C. The cell suspensions of all the cultures were adjusted to 1-2x 10⁶ spores/mL.

Test compound

Sample - 500 µg/mL

Standard - Ketoconazole (100 μg/mL)

Procedure

Determination of Antifungal activity

Candida albicans were inoculated on media (90 mm).

Test compounds: Sample (25 µL), Standard Ketoconazole (50 µL) Candida albicans were added to the 5 mm wells on the agar plates.

The treated plates with Candida albicans were incubated in the aerobic chamber at 24-30°C for 24 hours. The treated plates were observed for the zone of inhibition around the wells.

Results

Radical scavenging activity: DPPH method

The methanolic leaf extract demonstrated antioxidant activity as determined by % RSA (Radical Scavenging Activity) and it varied with different concentrations as shown in Figure 2. The % RSA value increased with increase in concentration, 47.50% for high concentration (150 µg/mL) which was the highest % RSA value and 26.11% for lower concentration (30 µg/mL). IC₅₀ (inhibitory concentration) indicates the efficacy of the extract, the lowest IC₅₀ shows higher potency (IC₅₀ 1.8 has 26.11% radical scavenging activity) and the highest IC₅₀ shows the lowest potency (IC₅₀ - 24.1 has 47.50% radical scavenging activity) as shown in Figure 2.

Sample

Preliminary phytochemical screening

In this study, the preliminary phytochemical screening of the methanolic extract of leaves of Ketoconazole 100 μg/mL was carried out. The results in Table 3 revealed the presence of alkaloids, flavonoids, tannins, steroids, triterpenoids, and saponins in the leaf extract of L. camara.

Antifungal activity

The results of inhibitory activity of methanolic leaf extract of L. camara against the organism (Candida albicans) at 50 µl/ml. With standard (ketoconazole) at 10 µl/ml. In this test the inhibitory activity is determined by zone of inhibition (90 mm inoculated media with 5 mm well on an agar plate) as shown in Table 4 with pictorial representation included in Table 4.

Discussions

The IC₅₀ value is an essential metric for assessing the efficacy of antioxidants, indicating the concentration needed to block 50% of DPPH radicals. A lower IC₅₀ value signifies enhanced antioxidant capacity, as a less quantity of extract is necessary to counteract free radicals. This investigation recorded the lowest IC₅₀ at 1.8, associated with a 26.11% radical scavenging activity, signifying modest antioxidant effectiveness at reduced doses.

The maximum IC₅₀ value was 24.1, corresponding to a 47.50% radical scavenging activity. This indicates a greater proportion of free radical inhibition; however, the elevated IC₅₀ implies that a considerably higher concentration of the extract is necessary to attain this effect, signifying reduced efficacy relative to lower IC₅₀ values.

The findings indicate that the methanolic extract of L. camara has antioxidant characteristics, with its effectiveness being significantly concentration dependent. The existence of bioactive substances, including flavonoids, phenolic acids, and other phytochemicals, probably enhances this effect. The range in IC₅₀ values signifies the complexity of the extract, suggesting that more purification or fractionation may be required to separate the most effective antioxidant components.₂₂

The antioxidant capacity of the methanolic leaf extract underlines its potential therapeutic implications, especially in addressing disorders associated with oxidative stress. Subsequent research may concentrate on refining the extraction technique and investigating the synergistic interactions of various phytochemicals to augment its antioxidant effectiveness.

Preliminary phytochemical screening

From results it is revealed the presence of alkaloids, f lavonoids, tannins, steroids, triterpenoids, and saponins from the leaf extract of L. camara. Alkaloids are present only in the leaves of L. camara and also the concentrated methanolic leaf extract had a higher number of flavonoids.²³

Alkaloids

It is the defensive mechanism against herbivores and pathogens. It can be toxic and bitter.

Phenolic and flavonoid contents

The methanolic leaf extract of plant L. camara showed higher phenolic and flavonoid content. It has been considered that the phenolic and flavonoid compounds have highly potent antioxidants as they possess the ability to absorb and neutralize free radicals as well as reactive oxygen species

Phenols in this plant showed anti-oxidant, disinfectant, and preservative activities. Flavonoids present in this plant possessed anti-oxidant, anti-inflammatory properties, for preventing cardiovascular diseases, cancer, and maintaining skin health.

Antifungal activity

The antifungal efficacy of the methanolic extract of L. camara against C. albicans, using ketoconazole as the reference antifungal drug. Ketoconazole, at a concentration of 100 µg/mL, showed a zone of inhibition of 18 mm, demonstrating its significant antifungal efficacy against C. albicans.

The methanolic extract of L. camara, evaluated at a concentration of 500 µg/mL, demonstrated a zone of inhibition measuring 9 mm. This zone, although considerably smaller than that produced by ketoconazole, indicates that the plant extract exhibits modest antifungal activity. The diminished efficacy relative to ketoconazole may be ascribed to various reasons, including the intricate composition of plant extracts, the concentration of active constituents, and their bioavailability.

The antifungal action of L. camara extract may be attributed to phytochemicals such as flavonoids, phenolic acids, and alkaloids, which are recognized for their antimicrobial characteristics.²² Nonetheless, the comparatively diminished inhibitory zone comparison to the standard indicates that, although the plant extract shows promise, it may want elevated concentration or additional refinement to attain efficiency akin to synthetic antifungal drugs such as ketoconazole.

Conclusion

In the present study, the methanolic leaf extract of L. camara was examined for phytoconstituents, antioxidant and antifungal activity. The L. camara contained more extractable bioactive metabolites. The presence of the phenolic group in the extract indicates a positive correlation between phenolic content and free radical scavenging activity. The DPPH assay method allows an easy and rapid way to evaluate the free radical scavenging activity. The methanolic leaf extract of L. camara was subjected to antifungal activity by an agar well diffusion method against Candida albicans, a pathogenic fungi, and about 9 mm of zone of inhibition was observed. However, more exploration is necessary to isolate and identify the dependable antioxidant and antifungal molecules within the crude extract.

Conflicts of Interest

Nil