Phytochemical Screening and Antimicrobial Activity of Leaf, Stem, and Roots in Sida Cordifolia Plant Extract

The global rise of antimicrobial resistance (AMR) has emerged as one of the most critical health threats of the 21^st century. As conventional antibiotics become increasingly ineffective, there is a pressing need to explore alternative sources of antimicrobial agents that are both effective and safe. Medicinal plants, which have been used for centuries in traditional healing systems across the world, represent a promising avenue for novel drug discovery. Among these, Sida cordifolia L., a member of the Malvaceae family, stands out for its broad spectrum of ethno- pharmacological applications and rich phytochemical profile. Commonly known as “Bala” in the Ayurvedic system of medicine, Sida cordifolia has traditionally been utilized to treat respiratory conditions, inflammatory disorders, neurological ailments, and infectious diseases (Kumar et al., 2012; Nadkarni, 1976). The plant is native to tropical and subtropical regions, particularly abundant in India, Sri Lanka, and Africa. Every part of the plant leaves, stems, and roots has been incorporated into therapeutic preparations in systems like Ayurveda, Unani, and Siddha. In traditional formulations, Sida cordifolia has been used for its adaptogenic, anti-inflammatory, analgesic, diuretic, and rejuvenative properties (Bensky & Gamble, 1993). Despite its traditional importance, there remains a considerable gap in scientific literature regarding a comparative phytochemical and antimicrobial analysis of the individual plant parts. Most studies focus on crude extracts or specific chemical classes without offering a holistic understanding of the plant’s full medicinal potential. Therefore, there is significant scope for evaluating the phytoconstituents and bioactivities of the leaf, root, and stem independently (Singh et al., 2010). Phytochemicals are naturally occurring secondary metabolites that plants produce for self-defense against pests, pathogens, and environmental stress. These compounds, which include flavonoids, alkaloids, terpenoids, phenolics, tannins, and saponins, have been extensively studied for their therapeutic properties (Newman & Cragg, 2020). In the case of Sida cordifolia, previous studies have reported the presence of biologically active constituents like ephedrine-like alkaloids, β-sitosterol, stigmasterol, quercetin, and various glycosides (Devi et al., 2009; Sharma et al., 2011). However, few investigations have systematically compared the phytochemical content of different plant parts and correlated these findings with antimicrobial potency. The antimicrobial activity of plant-derived compounds is largely attributed to their ability to disrupt microbial cell membranes, interfere with enzyme activity, chelate essential metal ions, or modulate virulence factors (Hemaiswarya et al., 2008). The emergence of multidrug-resistant (MDR) strains such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and fungal pathogens like Candida albicans and Aspergillus niger has spurred greater interest in natural antimicrobial agents (Bharti et al., 2013). In this context, a systematic evaluation of Sida cordifolia’s antimicrobial potential across its anatomical parts may yield valuable insights for developing plant-based antimicrobial therapeutics

II. Literature Review

A. National Status

Multiple studies have investigated the phytochemical composition and antimicrobial properties of Sida cordifolia, guiding its use in ethnomedicine and modern research. Kumar et al. (2014) identified key phytochemicals such as alkaloids, flavonoids, tannins, and glycosides, and demonstrated strong antibacterial and anthelmintic activity. Prabhakar et al. (2007) confirmed antimicrobial effects of methanol and chloroform extracts, including antifungal action. Rachel (2008) compared Sida acuta extracts and highlighted ethanol’s superior extraction efficiency, influencing solvent choices. Kumari and Raja (2019) attributed significant antibacterial activity to flavonoids and phenolics. Sharma et al. (2009) provided benchmark MIC values for methanolic extracts, while Nirmal et al. (2007) linked antifungal activity to tannins and alkaloids. Raut and Karuppayil (2014) found ethanol extracts more effective against foodborne pathogens. Patel et al. (2010) emphasized the leaf’s phytochemical richness, making it a key target in screening. Rathi et al. (2011) identified bioactive compounds with pharmacological relevance, and Bharti et al. (2013) validated the presence of flavonoids, tannins, and terpenoids. Collectively, these findings informed solvent selection, microbial targets, and the phytochemical focus of the user’s research, supporting S. cordifolia’s potential as a broad-spectrum antimicrobial agent.

B. International Status

Recent international studies have further emphasized the pharmacological and antimicrobial potential of Sida cordifolia. Singh et al. (2022) revealed the immunomodulatory role of its polysaccharides, suggesting indirect antimicrobial mechanisms. Sharma and Gupta (2021) highlighted bioactives like vasicinone and ephedrine, emphasizing the plant’s anti-inflammatory and antibacterial capabilities. Ribeiro et al. (2020) demonstrated that S. cordifolia essential oil showed potent activity against S. aureus, indicating membrane-disruptive effects and justifying oil-based extractions. Datta and Subban (2003) confirmed the presence of steroids, triterpenoids, and alkaloids, guiding the selection of key phytochemical tests. Yuan et al. (2010) and Sagnia et al. (2014) supported ethanol extraction for isolating alkaloids and terpenoids from roots and stems, influencing extraction and screening strategies. Singh and Ali (2011) reported broad-spectrum antimicrobial effects against K. pneumoniae, S. typhi, and P. vulgaris, validating aqueous and ethanolic solvents. Dos Santos et al. (2012) found S. cordifolia effective against S. aureus and S. pyogenes, supporting ethanol use and Gram-positive testing. Further research by Sagnia et al. (2014) confirmed bactericidal action against E. coli and S. epidermidis, reinforcing the focus on alkaloids and terpenoids. Together, these findings shaped the user’s methodological framework and substantiated the plant’s antimicrobial versatility.

III. METHODOLOGY

1. Collection and Preparation of Plant Material:

Fresh leaves, stems, and roots of Sida cordifolia will be collected from a previously designated location. Proper identification and authentication will be performed by a botanist. Voucher specimens will be deposited in a recognized herbarium for further reference.

Cleaning and Drying: Parts of the plants will be extensively cleaned with distilled water to eliminate debris. The cleaned parts will be shade-dried at room temperature or air-dried in a well-ventilated place. Alternatively, the process can be hastened by using a plant dryer.

Grinding: Powdered plant material will be ground to a fine powder using a mechanical grinder or mortar and pestle.

2. Extraction Procedure:

Solvent Extraction: Solvents such as methanol, ethanol, and water will be used for extraction to maximize the recovery of different phytochemical compounds.

Maceration /Soxhlet Extraction:  Appropriate extraction methods such as maceration or Soxhlet extraction will be used. The extracts will then be filtered and concentrated with a rotary evaporator.

Extract Preparation: Extracts will be prepared at different concentrations for further analysis.

3. Preliminary Phytochemical Screening:

Standard qualitative tests will be conducted in duplicate to detect major secondary metabolites in crude plant extracts.

Alkaloids

Dragendorff’s Test: Add Dragendorff’s reagent to the extract. Orange/reddish-brown precipitate → Alkaloids present.

Mayer’s Test: Add Mayer’s reagent. Cream/ white precipitate → Alkaloids present.

Flavonoids

Shinoda Test: Add ethanol, magnesium, and HCl. Pink/red/orange color → Flavonoids present.

Lead Acetate Test: Add 10% lead acetate. Yellow /brown precipitate → Flavonoids present.

Aluminum Chloride Test: Add 1% AlCl? in methanol. Yellow fluorescence → Flavonoids present.

Terpenoids

Salkowski Test: Mix extract with chloroform, add H?SO?. Reddish-brown interface → Terpenoids present.

Liebermann– Burchard Test: Add acetic anhydride and H?SO?. Blue-green/red color → Terpenoids/steroids present.

Saponins

Frothing Test: Shake aqueous extract. Persistent 1 cm foam → Saponins present.

Tannins

Ferric Chloride Test: Add FeCl?. Blue-black (hydrolyzable) or green-black (condensed) → Tannins present.

Lead Acetate Test: Add 10% lead acetate. White/ yellow precipitate → Tannins present.

Steroids

Liebermann– Burchard Test: Same as terpenoids test. Bluish-green color → Steroids present.

Salkowski Test: Same as terpenoids test. Reddish-brown interface → Steroids present.

Cardiac Glycosides

Keller–Kiliani Test: Add glacial acetic acid (with FeCl?) and H?SO?. Brown ring at interface, violet ring below → Cardiac glycosides present.

4. Assessment of Antimicrobial Activity

4.1 Selection of Test Microorganisms

A panel of pathogens will be used:

• Gram-positive: Staphylococcus aureus, Bacillus subtilis
• Gram-negative: Escherichia coli, Pseudomonas aeruginosa
• Fungi: Candida albicans, Aspergillus niger, Aspergillus fumigatus

Strains will be sourced from MTCC/ATCC and maintained on nutrient or Sabouraud’s dextrose agar.

4.2 Inoculum Preparation

• Bacterial inocula will be standardized to 0.5 McFarland (~10? CFU/mL).
• Fungal inocula will be prepared from 5–7 day-old cultures.

5.  Antimicrobial Susceptibility Testing

5.1 Disc Diffusion Assay

• Mueller-Hinton agar plates will be swab-inoculated with microbial suspensions.
• Sterile discs (6 mm) will be loaded with 10–20 µL of plant extract (100 mg/mL).
• Plates will be incubated at 37°C (bacteria, 24 h) or 28°C (fungi, 48 h).
• Zones of inhibition (mm) will be measured; tests run in triplicate.
• Positive controls (e.g., ampicillin, fluconazole) and negative controls (DMSO/water) included.

5.2 Broth Microdilution (MIC)

• Serial dilutions of extracts will be prepared in broth (Mueller-Hinton/ Sabouraud).
• Wells inoculated with 100 µL microbial suspension.
• MIC: Lowest concentration showing no visible growth (visually or via OD???).
• Optionally, MBC/MFC will be determined by subculturing from clear wells.

IV. RESULTS AND DISCUSSION

1. Phytochemical Screening:

1. The methanolic extracts of Sida cordifolia leaf, stem, and root were subjected to preliminary phytochemical screening, revealing a rich diversity of bioactive constituents across different plant parts. Among the three, the leaf extract demonstrated the most abundant presence of phytochemicals, indicating it as the most pharmacologically active part of the plant.
2. Alkaloids, known for their antimicrobial, analgesic, and anti-inflammatory properties, were detected in high amounts in the leaf (+++), and in moderate quantities in both the stem and root (++). Flavonoids, which possess strong antioxidant and anti-inflammatory activities, were found in very high concentrations in both the leaf and root extracts (++++), while the stem showed only moderate levels (++). Similarly, phenolic compounds, another class of potent antioxidants, followed a comparable pattern very high in the leaf and root, and moderate in the stem supporting their potential role in oxidative stress modulation.
3. Tannins, which contribute to astringent and antimicrobial activities, were present in high concentrations (+++) in the leaf and root, and moderately (++) in the stem. Saponins, associated with immune-modulatory and anti-hyper lipidemic properties, were moderately present (++) in the leaf and root, but only in trace amounts (+) in the stem. Interestingly, steroids, known for their anti-inflammatory and hormonal effects, were moderately present (++) in the stem, but only in trace amounts (+) in the leaf and root. Glycosides, which may contribute to cardiovascular and diuretic activity, were found in moderate amounts (++) uniformly across all three plant parts.

Table 1: The methanolic extracts of Sida cordifolia (leaf, stem, and root) revealed the presence of various bioactive compounds:

Legend: + (trace), ++ (moderate), +++ (high), ++++ (very high)

2. Antimicrobial Activity:

1. The antimicrobial activity of methanolic extracts of Sida cordifolia (leaf, stem, and root) was evaluated using the agar well diffusion method, and the results were expressed in terms of the diameter of the zone of inhibition (in mm) against selected bacterial and fungal pathogens.
2. Among the three plant parts tested, the leaf extract exhibited the strongest antimicrobial activity across all tested microorganisms. Against Staphylococcus aureus, a major Gram-positive pathogen, the leaf extract showed the largest inhibition zone of 21 mm, indicating high antibacterial potency. This was followed by the root extract (19 mm) and stem extract (14 mm), showing comparatively weaker but noticeable activity.
3. Similarly, against Bacillus subtilis, another Gram-positive bacterium, the leaf extract showed a zone of inhibition of 19 mm, while the root and stem extracts measured 16 mm and 12 mm respectively. In the case of Gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa, the leaf extract again demonstrated higher activity with inhibition zones of 20 mm and 17 mm, respectively. The root extracts showed moderate activity (18 mm and 15 mm), whereas the stem extract had the least effect (13 mm and 11 mm).
4. For fungal pathogens, the leaf extract continued to show superior efficacy. It produced 18 mm and 16 mm inhibition zones against Candida albicans and Aspergillus niger, respectively. The root extracts followed with 17 mm and 15 mm, while the stem extracts had lower antifungal activity (12 mm and 11 mm).

Table 1: The antimicrobial activity was tested via agar well diffusion and measured by the diameter (in mm) of the zone of inhibition.

Overview of the Graph Structure

• X-axis: Lists the tested microorganisms
  (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger)
• Y-axis: Zone of inhibition in millimeters (mm) (ranging approximately from 0 to 25 mm)
• Bars: Each group contains three bars representing the leaf (usually tallest), stem (shortest), and root (moderate height) extracts.

Detailed Interpretation by Microorganism

1. Staphylococcus aureus (Gram-positive bacterium)

• Leaf extract shows the highest inhibition zone (21 mm), suggesting strong antibacterial activity.
• Root extract is also effective (19 mm), while stem extract is less active (14 mm).
• Interpretation: High sensitivity of S. aureus to compounds in the leaf and root (likely due to flavonoids and alkaloids).

2. Bacillus subtilis (Gram-positive bacterium)

• Leaf: 19 mm, Root: 16 mm, Stem: 12 mm
• Again, the leaf shows dominant activity. The effectiveness pattern is similar to S. aureus.
• Indicates broad Gram-positive antibacterial potential of leaf extract.

3. Escherichia coli (Gram-negative bacterium)

• Leaf: 20 mm, Root: 18 mm, Stem: 13 mm
• Leaf and root extracts demonstrate notable inhibition, indicating the presence of compounds capable of penetrating the outer membrane of Gram-negative bacteria.

4. Pseudomonas aeruginosa (Gram-negative bacterium)

• Leaf: 17 mm, Root: 15 mm, Stem: 11 mm
• Activity is slightly reduced compared to E. coli, but the pattern remains consistent (Leaf > Root > Stem).

5. Candida albicans (Fungus)

• Leaf: 18 mm, Root: 17 mm, Stem: 12 mm
• The extracts are effective against fungal strains as well, with good antifungal activity in leaf and root, likely due to phenolic compounds and flavonoids.

6. Aspergillus niger (Fungus)

• Leaf: 16 mm, Root: 15 mm, Stem: 11 mm
• Follows the same trend as Candida albicans, though with slightly lower inhibition zones.

General Observations from the Graph

• The leaf extract consistently produces the highest zones of inhibition across all microbes.
• The root extract shows moderate antimicrobial activity, typically just below the leaf.
• The stem extract exhibits the least activity, possibly due to lower concentrations of bioactive compounds.
• Gram-positive bacteria (S. aureus, B. subtilis) are slightly more sensitive than Gram-negative ones (E. coli, P. aeruginosa), which is a typical pattern in plant extract studies due to cell wall differences.
• The extract also shows promising antifungal activity, especially against Candida albicans.

Fig.: Graph: Zone of Inhibition (mm)