Isolation and Evaluation of Anti-Prevotella Activity of Moringa Oleifera Leaf Extract by HPLC

Periodontal diseases, encompassing a range of inflammatory conditions affecting the supporting structures of the teeth, remain a major global public health concern.[1, 2] Among the diverse microbial populations implicated in the pathogenesis of periodontitis, Prevotella species particularly Prevotelladenticolaplay a central role.[3] These Gram-negative, anaerobic bacteria contribute significantly to biofilm formation and chronic oral infections due to their virulence, tissue-destructive enzymes, and ability to evade host immunity.[4] The persistence and resistance of Prevotella within polymicrobial biofilms pose serious therapeutic challenges, especially as antibiotic resistance becomes increasingly prevalent.[5, 6] The limitations of current antibiotic therapies, including the emergence of resistant strains, adverse drug reactions, and disruption of normal flora, necessitate the exploration of alternative antimicrobial agents.[7, 8] Natural products, especially plant-derived phytochemicals, have emerged as promising candidates due to their broad-spectrum activity, lower resistance potential, and favorable safety profiles.[9] One such plant of significant interest in ethnomedicine is Moringaoleifera, commonly referred to as the “drumstick tree” or “miracle tree.”[10, 11] Moringaoleifera, native to the Indian subcontinent and widely cultivated in tropical and subtropical regions, has long been used in traditional medicine for its antimicrobial, anti-inflammatory, antioxidant, and immunomodulatory properties (figure 1). Its leaves are particularly rich in bioactive compounds, including phenolic acids, flavonoids, alkaloids, and tannins.[12, 13] Several studies have confirmed the efficacy of Moringaoleifera extracts against a broad range of bacterial pathogens, both Gram-positive and Gram-negative. However, limited research exists on its specific effect against Prevotella species, particularly in the context of oral health and biofilm inhibition.[14]

Figure 1:Moringaoleifera^[15]

High-Performance Liquid Chromatography (HPLC) has emerged as a powerful analytical technique for the identification and quantification of plant secondary metabolites.[16, 17] It allows the detection of specific phenolic and flavonoid compounds responsible for bioactivity, offering insights into the phytochemical profile and potential mechanisms of antimicrobial action.[18] The precise profiling of Moringa extracts by HPLC can, therefore, serve as a bridge between ethnobotanical knowledge and pharmacological validation.[19, 20] This study aims to isolate and evaluate the anti-Prevotella activity of Moringaoleifera leaf extract through a combination of microbiological and analytical methods. Specifically, the research involves: (1) extraction of phytochemicals from Moringaoleifera leaves; (2) phytochemical screening to identify key secondary metabolites; (3) HPLC-based analysis at two wavelengths (280 nm and 320 nm) to quantify individual bioactive compounds; (4) isolation and confirmation of Prevotella species from clinical sources; (5) assessment of antimicrobial activity through agar well diffusion; (6) determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC); and (7) evaluation of anti-biofilm activity using a crystal violet assay.  In summary, the present research is a multidisciplinary investigation that integrates microbiology, phytochemistry, and analytical chemistry to evaluate the potential of Moringaoleifera leaf extract as a natural therapeutic agent against Prevotella species. It not only addresses the growing need for alternative antimicrobial strategies but also contributes to the scientific validation of traditional medicinal plants. By combining classical phytochemical screening with advanced HPLC analysis and modern microbiological assays, this study provides a comprehensive evaluation of the antimicrobial and anti-biofilm capabilities of Moringaoleifera, with a specific focus on oral anaerobic pathogens. The outcomes of this study are expected to offer new insights into plant-based interventions for oral microbial infections and support the development of novel bioactive compounds for dental therapeutics. Furthermore, this work sets the foundation for future in vivo and formulation studies aimed at harnessing the full potential of Moringaoleifera in managing resistant anaerobic infections.

MATERIALS AND METHODS

Materials

Fresh leaves of Moringaoleifera were collected from a botanical garden, authenticated by a plant taxonomist, and shade-dried for seven days. The dried leaves were ground into a fine powder using a mechanical grinder and stored in an airtight container until extraction. The chemicals and reagents used included methanol (≥99.9%, HPLC grade, Sigma-Aldrich), ethanol (≥99.8%, Merck), dimethyl sulfoxide (DMSO, Sigma-Aldrich), Mueller-Hinton Agar (MHA, HiMedia), Brain-Heart Infusion (BHI) broth (HiMedia), phosphate-buffered saline (PBS, pH 7.4, Sigma-Aldrich), crystal violet (0.1%, Sigma-Aldrich), sodium chloride (NaCl, Merck), chlorhexidine (0.2% solution, Sigma-Aldrich), and standard antibiotics such as metronidazole and ciprofloxacin (Sigma-Aldrich).

1. Plant Extraction

The powdered Moringaoleifera leaf material (100 g) was subjected to Soxhlet method. The extract was filtered through Whatman No. 1 filter paper and the filtrate was concentrated using a rotary evaporator at 40°C under reduced pressure (figure 2).

[a]                                                                [b]

Figure 2:[a] Soxhletextraction of powdered Moringaoleifera leaf [b] Moringaoleifera leaf Extract

2. Analysis of Compounds in Moringaoleifera Extracts by HPLC

The phytochemical profiling of Moringaoleifera extract was performed using High-Performance Liquid Chromatography (HPLC) to identify and quantify major phenolic and flavonoid constituents.

Sample Preparation

An accurately weighed amount (10 mg) of dried methanolic extract was dissolved in 10 mL of methanol (HPLC grade) and filtered through a 0.45 µm syringe filter prior to injection.

HPLC Instrumentation and Conditions

The analysis was performed using a Shimadzu LC-20AD HPLC system equipped with a photodiode array (PDA) detector. Separation was achieved using a C18 reverse-phase column (250 mm × 4.6 mm, 5 µm particle size). The mobile phase consisted of solvent A: water with 0.1% formic acid and solvent B: acetonitrile, with the following gradient program: 0–5 min (5% B), 5–15 min (5–25% B), 15–30 min (25–50% B), followed by a re-equilibration phase. The flow rate was 1.0 mL/min and the injection volume was 20 µL. The detection wavelengths were set at 280 nm for phenolic acids and 320 nm for flavonoids. Total run time was 35 minutes.

Standard Preparation and Quantification

Standard solutions of gallic acid, chlorogenic acid, ferulic acid, quercetin, and rutin were prepared in methanol at concentrations ranging from 10–100 µg/mL. Calibration curves were constructed for each standard based on peak area. The compounds in the extract were identified by comparing retention times and UV spectra with those of the standards. Quantification was carried out based on the area under the peak in relation to the calibration curves.

3. Phytochemical Screening of Moringaoleifera Extracts

Qualitative phytochemical screening was performed to identify the presence of bioactive compounds such as alkaloids, flavonoids, tannins, saponins, and phenolics. Alkaloids were detected using Dragendorff’s test, where the formation of an orange precipitate indicated a positive result. The presence of flavonoids was confirmed using the Shinoda test, resulting in pink coloration. Tannins were identified using the ferric chloride test, producing a green-black precipitate. The foam test was used to detect saponins, with stable froth formation indicating their presence. Phenolics were detected using the ferric chloride test, where a bluish-black coloration confirmed their presence.

4. Isolation and Verification of Prevotella

Prevotella species were isolated from human subgingival plaque samples using Wilkins-Chalgren anaerobic agar supplemented with 5% sheep blood. Plaque samples were collected using sterile cotton swabs, which were then inoculated into Brain Heart Infusion (BHI) broth and incubated anaerobically at 37°C for 48 hours. The culture was streaked on Wilkins-Chalgren agar and incubated anaerobically at 37°C for 72 hours. Colonies were identified based on Gram staining, which confirmed the presence of Gram-negative rods, and biochemical tests, including indole-negative, catalase-negative, and bile-sensitive reactions. Molecular identification was further confirmed using 16S rRNA sequencing.

5. Preparation of Bacterial Suspension

A standardized bacterial suspension was prepared in phosphate-buffered saline (PBS, pH 7.4) to match the 0.5 McFarland turbidity standard, which corresponds to approximately 1.5 × 10? CFU/ml.

6. Antimicrobial Evaluation of the Extracts

The antimicrobial activity of Moringaoleifera leaf extract against Prevotella was evaluated using the agar well diffusion method. Mueller-Hinton Agar plates were inoculated with Prevotella suspension using a sterile cotton swab. Wells of 6 mm diameter were punched into the agar using a sterile cork borer. Each well was loaded with 50 µL of extract prepared at a concentration of 10 mg/mL in DMSO. A well containing metronidazole (10 µg/mL) was used as a positive control, while a well containing DMSO served as a negative control. The plates were incubated anaerobically at 37°C for 48 hours, after which the zones of inhibition were measured.

7. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

The MIC and MBC of Moringaoleifera leaf extract against Prevotella were determined using the broth microdilution method in 96-well plates. Serial two-fold dilutions of the extract were prepared in Brain Heart Infusion (BHI) broth, ranging from 1.25 to 1000 µg/mL. Each well was inoculated with 100 µL of Prevotella suspension adjusted to 1.5 × 10? CFU/mL. The plates were incubated anaerobically at 37°C for 24 hours. MIC was determined as the lowest concentration of extract that inhibited visible bacterial growth. MBC was determined by plating samples from MIC wells onto Mueller-Hinton Agar and incubating them at 37°C for 48 hours. The lowest concentration at which no bacterial growth was observed was recorded as the MBC.

8. Anti-Biofilm Assay of Moringaoleifera Extracts

The ability of Moringaoleifera extract to inhibit Prevotella biofilm formation was assessed using the crystal violet staining method. A 96-well plate was inoculated with 100 µL of bacterial suspension and 100 µL of extract at MIC and sub-MIC concentrations. The plate was incubated anaerobically at 37°C for 24 hours. After incubation, wells were washed with phosphate-buffered saline (PBS) and stained with 0.1% crystal violet for 15 minutes. Excess stain was removed by washing with distilled water. The bound crystal violet was solubilized using 200 µL of ethanol, and biofilm biomass was quantified by measuring absorbance at 595 nm using a microplate reader.

9. Statistical Analysis

All experiments were performed in triplicate, and results were expressed as mean ± standard deviation (SD). Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test, with p < 0.05 considered statistically significant.

RESULTS

1. HPLC Analysis of Moringaoleifera Extracts

The High-Performance Liquid Chromatography (HPLC) analysis of the methanolic extract of Moringaoleifera leaves revealed the presence of several phenolic and flavonoid compounds. The chromatographic separation was performed at two wavelengths: 280 nm (for phenolic acids) and 320 nm (for flavonoids) (figure 3).

Figure 3:HPLC chromatographic profiles of the constituents in Moringaoleifera leaf extract were obtained at 280 nm (A) and 320 nm (B)

At 280 nm, major peaks corresponded to gallic acid, chlorogenic acid, and ferulic acid, while at 320 nm, prominent peaks were attributed to quercetin and rutin. The retention times (Rt) and quantified concentrations were compared with respective standard curves.

Table 1 shows the retention times and concentrations of the identified bioactive constituents.

Table 1: Identified Phenolic and Flavonoid Compounds in Moringaoleifera Extract by HPLC

2. Phytochemical Analysis

Qualitative phytochemical screening revealed the presence of multiple bioactive constituents. The extract tested positive for alkaloids, flavonoids, tannins, saponins, and phenolics, suggesting a complex mixture of phytochemicals that may contribute to antimicrobial effects.

Table 2 presents the qualitative results of phytochemical screening.

Table 2: Qualitative Phytochemical Constituents of Moringaoleifera Extract

Table 2: Presence of key secondary metabolites was confirmed in the methanolic extract. (+++: strongly present, ++: moderately present, +: slightly present)

3. Identification of Prevotella

The bacteria isolated from subgingival plaque samples were grown anaerobically on Wilkins-Chalgren agar. Colonies appeared pigmented, black, and mucoidcharacteristic of Prevotella species. Gram staining confirmed Gram-negative rods. Biochemical identification yielded negative catalase and indole reactions. The identity was further confirmed by 16S rRNA gene sequencing, which showed a 99.4% sequence homology to Prevotelladenticola(figure 4).

Figure 4:The 16S rRNA sequence trace data for the isolates Prevotelladenticola HJX050

The results confirmed the successful isolation of Prevotella species, which was used for subsequent antimicrobial evaluations.

4. Antimicrobial Evaluation of Moringaoleifera Extracts

The agar well diffusion method revealed significant antimicrobial activity of Moringaoleiferamethanolic leaf extract against Prevotella. The inhibition zones were measured and compared with the standard antibiotic (metronidazole). A dose-dependent response was observed (figure 5).

Figure 5: (A) After 7 days, Prevotella colonies appearing black-pigmented, spherical, smooth, and shiny, and (B) Prevotella appearing as gram-negative coccobacilli under a light microscope

M. oleifera L. showed dose-dependent antimicrobial activity at all tested concentrations, with the lowest and highest mean values of inhibition zone estimated.

Table 3 displays the diameter of inhibition zones formed by different concentrations of the extract.

Table 3: Antibacterial Activity of Moringaoleifera Extract AgainstPrevotella

Table 3displaysThe antimicrobial activity increased with rising extract concentration. Metronidazole showed the highest inhibition zone, followed closely by the 40 mg/ml extract.

5. MIC and MBC of Moringaoleifera Extracts

The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined using broth microdilution and subculturing methods.

The extract demonstrated a MIC of 12.5 µg/mL and an MBC of 25 µg/mL, suggesting potent bactericidal activity against Prevotella.

Table 4 summarizes the MIC and MBC values.

Table 4: MIC and MBC of Moringaoleifera Extract AgainstPrevotella

Table 4: The extract displayed low MIC and MBC values, indicating strong antimicrobial efficacy. Metronidazole remained more potent, but the extract showed comparable potential.

6. Anti-Biofilm Assay of Moringaoleifera Extracts  : The anti-biofilm activity of the Moringa extract was evaluated using a crystal violet assay. The extract significantly inhibited biofilm formation at MIC and sub-MIC concentrations (figure 6).At MIC (12.5 µg/mL), the biofilm formation was reduced by 72.8%, while at half-MIC (6.25 µg/mL), the inhibition was 53.6%.

Table 5 presents the quantitative results of the biofilm inhibition assay.

Table 5: Anti-Biofilm Activity of Moringaoleifera Extract

Table 5: The extract exhibited dose-dependent inhibition of biofilm formation, with near equivalence to metronidazole at MIC concentration.

Figure 6:Anti-Biofilm Activity of Moringaoleifera Extract