Preliminary Analysis of Bioactive Compounds and Antibacterial Properties in Methanolic Extract of Achillea fragrantissima

Introduction

Medicinal plants remain a primary source of drugs in both modern and traditional medicine worldwide ^1,2. For centuries, plants have provided valuable natural products essential for maintaining the health of both animals and humans. Natural products derived from higher plants present a promising source of antimicrobial agents, potentially offering novel mechanisms for treating infectious diseases ^3,4. Medicinal plants are rich in active compounds that can provide effective herbal alternatives for treating common bacterial infections. These plants are a valuable reservoir of diverse drugs and bioactive substances. Therefore, they should be extensively researched to better understand their properties, safety, and effectiveness. The genus Achillea, part of the Asteraceae family, is commonly found across various Middle Eastern countries. Several studies have demonstrated the diverse pharmacological effects of its hydro-distilled volatile oils, which are effective in treating various diseases, both when applied topically and taken orally⁵.

Researchers have explored the antibacterial properties of various plants against both Gram-negative and Gram-positive bacterial strains, but there are limited reports on their activity against drug-resistant bacteria. Our study was designed to assess, in vitro, the antibacterial properties of the methanolic extract of A. fragrantissima against bacterial pathogens.

Materials and Methods

Preparation of Plant Materials

Leaves of A. fragrantissima were harvested during the spring season Gathered in the Hail province of Saudi Arabia. Plant identification was carried out by Dr. Naila Alkafei from the University of Hafer Albatin, KSA. The collected leaves were left to dry naturally in a shaded area at room temperature for a period of two weeks. After drying, the plant material was finely ground into powder form for extraction purposes⁶.

Extraction Procedure

Only the dried leaves were used for the extraction process. These were separately ground into a fine powder. A total of 30 grams of this powder was mixed with 250 mL of methanol at room temperature. Extraction was enhanced using ultrasonic treatment, applied in four 20-minute cycles spread over a 24-hour period in a water bath. Following extraction, the mixture was filtered, and both the filtrate and residue were preserved for subsequent analyses.

GC–MS Analysis

This method was employed to identify the major bioactive compounds in the methanolic leaf extract. The analysis was performed using a Gas Chromatography–Mass Spectrometry system equipped with AS 3000 autosampler and an Ion Quantification System detector. A non-polar fused silica capillary column was utilized, with helium gas as the mobile phase at a constant flow rate of 1.2 milliliters per minute. For the analysis, 2 milliliters of the methanol-diluted extract were injected to enable partial separation of its chemical constituents. Spectral data were collected via mass spectrometry and analyzed using Xcalibur software. The resulting mass spectra were compared with reference data from the NIST and MAINLIB libraries for compound identification ⁷.

Liquid Chromatography–Mass Spectrometry (LC–MS) Analysis

This method was employed to analyze and identify the bioactive constituents present in the methanolic extracts of the selected plant leaves. Chromatographic separation was performed using a Shimadzu ExionLC system, with the mobile phase comprising 0.1% formic acid in water and acetonitrile. A Gas & Liquid column (100 × 2.1 mm, 3 µm particle size) was used for separation, with a flow rate maintained at 0.35 mL/min. The gradient elution profile was set as follows: 5% solvent B from 0 to 5 minutes, a linear increase from 5% to 95% B between 5 and 30 minutes, and a return to 5% B from 30 to 40 minutes. Mass spectral data were acquired using a SCIEX X500R QTOF system, using an electrospray ionization source, the analysis was carried out in both positive and negative ionization modes. The compounds were identified by cross-referencing the acquired mass spectra with entries in the NIST spectral library database.

In Vitro Antibacterial Activity

The plant extracts were tested for their antibacterial potential against a panel of bacterial strains, including S. aureus ATCC 512477 (B-1), S. epidermidis ATCC 12228 (B-2), E. faecalis ATCC 29212 (B-3), E.coli ATCC 25922 (B-4),, K. pneumoniae ATCC 700603 (B-5), S. choleraesuis ATCC 10708 (B-6), P. aeruginosa ATCC 27853 (B-7),  and P. mirabilis ATCC 299 (B-8),. Prior to testing, 24-hour cultures were prepared from the respective stock strains. Antibacterial susceptibility testing was conducted following the standardized method described in ⁸. Mueller-Hinton agar plates were prepared for the assays. Plant extracts were assessed for anti-bacterial properties via the agar well diffusion technique, whereas the disc diffusion approach was utilized for the reference antibiotic, ciprofloxacin (5 µg/disc). To ensure uniform distribution of the bacterial inoculum, sterile cotton swabs were dipped into standardized bacterial suspensions (based on CFU/mL) and uniformly distributed over the surface of Mueller-Hinton agar by gently rotating the Petri dishes. After allowing the plates to dry for around 10 minutes, wells were made in the agar using a sterile stainless-steel borer. Following sample application, the plates were incubated at 37°C for 24 hours. Antibacterial activity was evaluated by measuring the diameters of the inhibition zones formed around the wells. The size of each zone indicated the effectiveness of the extract, with larger zones representing stronger antibacterial effects. The results are summarized in Table 3.

Results and Discussion

According to the results obtained from the fractionation process, a number of bioactive compounds were successfully isolated using both the GC-MS and LC-MS. These separated compounds were identified as the key contributors to the antibacterial activity observed in this plant. Their presence points to the plant’s promise as a source of natural antimicrobial agents, supporting its traditional use and encouraging further pharmacological investigation. GC-MS analysis was done on a methanolic extract of A. fragrantissima leaves to find out what its main components and come up with an answer for the antibacterial activity that was seen in vitro. Table 1 & figure 1 shows the major chemical compositions of A. fragrantissima leaves methanolic extract via GC-MS. Thujone⁹, α-Resorcylic acid¹⁰, (+)-α-Funebrene¹¹, Dihydroxanthin¹², and Strophanthidin¹³ were among the bioactive compounds identified in the extracts through GC-MS analysis and are believed to contribute to the observed antibacterial activity of the plant’s extract in methanol. 

Figure 1: GC-MS chromatogram of A. fragrantissima leaves extract in methanol.Click here to View Figure

Table 1: Chemical compositions of A. fragrantissima leaves methanolic extract via GC-MS

*Retention time.

The methanolic extracts of physiologically active A. fragrantissima leaves were analyzed using LC-MS to discover any chemical components that may be present. Many chemicals were discovered utilizing both positive and negative ionization techniques (Table 2). Some of these chemicals may have been responsible for the observed antibacterial activity. 2 -Oxovaleric acid ¹⁴, chorismic acid ¹⁵, quercetagetin ¹⁶, calcipotriol ¹⁷, niacinamide ¹⁸, butylated hydroxyanisole ¹⁹, xanthurenic acid ²⁰, and dihydromyricetin ²¹ were discovered to have significant antibacterial action.

Table 2: LC-MS examination of the methanolic extract of A. fragrantissima leaves

*Retention time.

As presented in Table 3, the methanolic extract of A. fragrantissima leaves exhibited the highest antibacterial activity against S.aureus, E. coli, P. mirabilis, and S.choleraesuis. However, its efficacy was significantly lower compared to the standard antibiotic ciprofloxacin.

Table 3: Results of In vitro antibacterial activity 

*Each value is the mean of 6 batches with standard deviation. All the values are compared to the standard ciprofloxacin disc by performing Tukey Kramer test (post hoc). All the test values are significantly lesser than the standard ciprofloxacin disc at p < 0.05. Std: Standard deviation

Conclusions

The methanolic extract of A. fragrantissima leaves grown in Hail, KSA has a range of components that exhibit antibacterial properties against S. aureus, E. coli, P. mirabilis, and Salmonella choleraesuis. Our findings, together with earlier research, confirm the plant’s antibacterial qualities in the face of rising antibiotic resistance. It can serve as an antibacterial complement for the creation of novel medicinal medicines. Further research is needed to confirm the plant’s potential as an antibacterial agent in topical or therapeutic applications, as well as to assess the effects of its active components in vivo.

Acknowledgment

The author would like to thank, (Insert university name and Dept. name) for their guidance and support to complete this article.

Conflicts of Interest

There are no conflicts of interest declared by the authors.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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