Unraveling the antimicrobial efficacy and chemical fingerprinting of medicinal plants against the WHO’s prioritized pathogens

Abstract

Background

The global spread of drug-resistant organisms has necessitated the search for alternative treatments against bacterial and candidal resistant pathogens. Plants have long been used as traditional medicines to ameliorate various diseases, and their antimicrobial properties are still being explored. The aim of the present study is to assess the antimicrobial activity of extracts from Alstonia scholaris, Orthosiphon aristatus, Sphaeranthus amaranthoides, Crateva magna and Garcinia travancorica against bacteria and Candida pathogens.

Results

Out of 60 different sequential extracts tested, several showed moderate to good antimicrobial activity. Among them, ethyl acetate extract of G. travancorica exhibited significant activity against Lactobacillus acidophilus (17 mm) followed by Staphylococcus aureus (16 mm), Escherichia coli (13 mm), Proteus mirabilis (12 mm), Staphylococcus epidermis, Candida krusei (11 mm), Candida glabrata (10 mm) and the chloroform extract from O. aristatus showed good activity against S. epidermis, L. acidophilus (13 mm), S. aureus, Escherichia fergusonii, C. krusei (12 mm), C. glabrata, E. coli (11 mm) and Klebsiella pneumoniae (10 mm), respectively. In addition, GC–MS analysis revealed the presence of nine major compounds in G. travancorica and ten compounds in O. aristatus which were responsible for the significant antimicrobial activity.

Conclusions

These findings highlight the potential of G. travancorica and O. aristatus as sources for developing new antimicrobial agents against the World Health Organization’s (WHO) prioritized pathogens. Further research on these plants could lead to the discovery and synthesis of novel therapeutic agents with enhanced antimicrobial properties.

Graphical abstract

Background

The spread of antibiotic resistance has been a dire worriment for humanity and is responsible for over 5.3 million deaths annually across the globe (Ogbole et al. 2018). Antibiotic resistance is emerging as a major threat to humankind, as new resistance mechanisms are being developed periodically by microorganisms (Costa et al. 2016). With the emergence of new drugs, bacteria tend to acquire various mechanisms of resistance to survive. Researchers have put in a lot of efforts on keeping bacterial proteins as drug target; however, mechanisms such as translation control, exploiting trans translational pathway and controlling the regulatory RNAs have emanated to enhance bacteria’s resistance towards antibiotics (Aswathanarayan and Vittal et al. 2013). Antibiotic resistance, particularly in microorganisms have occurred in epidemic waves beginning with the emergence of strains that were resistant to penicillin and progressing to the present pandemic of community-associated Methicillin-Resistant Staphylococcus aureus (MRSA) (Timpau et al. 2023). Organisms belonging to genera of Escherichia, Klebsiella, Proteus, Salmonella, Staphylococcus, and Candida are the causative agent for infections ranging from bacteremia, pneumonia, meningitis, cellulitis, urinary tract infections, stomach ulcers, candidemia and many more and are infamously known for their expeditious nature to be highly resistant to antibiotics. According to the recent report by the World Health Organization (WHO), traditional medicinal plants are widely utilized as a primary source of healthcare by 70–80% of the global population (Muhammad et al. 2011). These plants have been relied upon for their broad spectrum of biological properties across different cultures, where they hold a prominent place in ethnomedicine (Moglad et al. 2020). The effectiveness of medicinal plants in treating various ailments, including antimicrobial effects against a range of pathogens can be attributed to the presence of active chemical constituents’ alkaloids, flavonoids, phenols, and tannins, terpenoids, steroids, resins and other metabolites (Archana and Bose 2022). Globally, researchers have explored the potential of these natural metabolites in combating pathogenic microorganisms due to their promising therapeutic properties and offers a vast repertoire of potential compounds with varying mechanisms of action, which can be harnessed for the development of novel antimicrobial agents (Pratheeshkumar et al. 2015). Therefore, this knowledge contributes to the development of alternative treatments for infectious diseases and addresses the growing concern of antimicrobial resistance. Many natural products derived from medicinal plants, which have been traditionally employed in folk remedies, have been extensively studied and possess strong scientific evidence supporting their antimicrobial properties. Therefore, investigation of neglected wild plants as potential alternative sources for biomedical applications has gained significant attention in recent times, emphasizing the importance of exploring untapped resources. In line with this context, our study focuses on evaluating the antimicrobial activities of traditional medicinal plant extracts against Candida and bacterial pathogens that could serve as alternative therapeutic options for the management of various infections caused by drug-resistant strains. Further, it also offers exciting prospects for the development of novel pharmacological agents and reinforces the importance of traditional medicine in modern healthcare.

Methods

Plant materials

The fresh aerial parts of the selected plants were collected in the summer season from different parts of Tamilnadu (TN), India such as Tirunelveli, Thirukkurungudi and South Veeravanallur between March–April 2023. The plants were identified and authenticated as Alstonia scholaris (L.) R.Br., Orthosiphon aristatus (Blume) Miq., Sphaeranthus amaranthoides Burm.f., Crateva magna DC., and Garcinia travancorica Bedd., (Fig. 1) by one of the authors Dr S. Mutheeswaran. The ethnobotanical information on the selected plants belonging to five families are depicted in Table 1 (Baliga 2010, 2012; Shang et al. 2010; Khyade et al. 2014; Jagetia and Baliga 2005; Adam et al. 2009; Houghton et al. 2005; Rethinam et al. 2021; Thanigaivel et al. 2019; Othman et al. 2011; Galani et al. 2010; Mostafa et al. 2018; Kiruba et al. 2011; Magalhães et al. 2023; Patel 2023; Espirito Santo et al. 2020). The plants voucher specimens (SM-0017–21) were deposited at Xavier Research Foundation herbarium in St. Xavier's College, Palayamkottai, TN, India for further reference.

Fig. 1

Photographs of selected plants in their natural habitat, a Alstonia scholaris (L.) R.Br., b Orthosiphon aristatus (Blume) Miq., c Crateva magna Burm.f., d Sphaeranthus amaranthoides DC., and 1e. Garcinia travancorica Bedd.,

Table 1 Ethnobotanical data on plants used in this study

Preparation of extracts

The collected plant materials were shade dried at room temperature and then ground into either coarse/fine powder using an electric blender. For each sample, 25 g of powdered plant materials were soaked sequentially in 100 mL of hexane, chloroform, ethyl acetate and methanol. The mixtures were subjected to 30 min of ultra-sonication and kept at room temperature for 48 h with intermittent shaking. The extracts were filtered and the solvents were removed under vacuum in a rotavapor at 35 °C. The extracts were further dried at room temperature and kept at 4 °C until they were screened for their antimicrobial activity (Gishen et al. 2020).

Test microbes

Microbial strains used in the present investigation are as follows: Gram-positive bacteria such as Staphylococcus epidermis (ATCC 49134), Staphylococcus aureus (ATCC11632), Lactobacillus acidophilus (MTCC 10307); Gram-negative bacteria such as Escherichia coli (ATCC 35218), Escherichia fergusonii (ATCC 35469), Salmonella para-typhi (MTCC 735), Proteus mirabilis (MTCC 7002), Klebsiella pneumoniae (MTCC 70063) and yeast such as Candida glabrata (MTCC 3019), and Candida krusei (MTCC 9215). The test ATCC and MTCC strains were procured from HiMedia Laboratories, and the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH) Chandigarh, India, respectively.

Inoculum preparation and antimicrobial susceptibility assay

Candida and bacterial inoculums were first grown in nutrient broth and incubated in a shaker incubator at 37ºC overnight. Prior to the experiment, the turbidity of the overnight cultures was adjusted to an optical density equivalent to 0.5 McFarland standard, which corresponds to approximately 10⁷ CFU/mL for bacteria and Candida. For the antimicrobial assay, 100μL of the adjusted test cultures were evenly spread on sterile Muller-Hinton agar (MHA) plates, ensuring confluent growth of the organisms. The plates were then allowed to dry for 5 min. Followed by 25µL of the plant extracts with concentrations of 1, 2, and 5 mg/disc were loaded onto sterile discs. The discs were dried and then placed on the surface of the pre-inoculated agar MHA plates using sterile forceps. Imipenem 10 µg/disc and cefotaxime/clavulanic acid 30/10 µg/disc were used as positive controls; 5% DMSO was used as a solvent control was included. The antimicrobial potency of the plant extracts was determined after incubation by measuring the zone of growth inhibition (in mm) surrounding the discs on the agar plates against the tested pathogens (Saravana Kumar et al. 2022).

Chemical fingerprinting using GC–MS

The bioactive compounds present in the active crude extract were analysed using GC–MS (Agilent 8890) with an HP-5MS column (30 m x 250 μm × 0.25 μm). The samples were dissolved in methanol, and 20μL of the solution was injected with an anterior column pressure of approximately 11.367 psi. The analysis involved programming the oven temperature to start at 75ºC and hold for 3 min, followed by a gradual increase to 350ºC at a rate of 5ºC per minute. The injection port temperature was set to 330ºC, while the transfer and ion source temperatures were maintained at 310ºC. Helium was used as the carrier gas, flowing at a rate of 1 mL/min. The instrument was calibrated to scan a mass range of m/z 35–500. To identify the compounds, their mass spectral fragmentation patterns were compared to the NIST98-MS and the Wiley KnowItAll 2020 Mass Spectral Library through spectral analysis.

Results

Antimicrobial efficacy of plant extracts

The antimicrobial activities of 60 different plants’ sequential extracts viz., hexane, chloroform, ethyl acetate and methanol from Alstonia scholaris, Orthosiphon aristatus, Sphaeranthus amaranthoides, Crateva magna and Garcinia travancorica were evaluated against 3 Gram-positive bacteria, 5 Gram-negative bacteria and 2 Candida human pathogens. In the line, the antimicrobial activities of the plant extracts showed zones of inhibition varied from good to weak. Specifically, for hexane extract the zones of inhibition ranged from 0–10 mm, 0–11 mm, and 0–13 mm. For the chloroform extract, the range was 0–10 mm, 0–12 mm, and 0–13 mm. The ethyl acetate extract exhibited zones of inhibition ranging from 0–14 mm, 0–15 mm, and 0–17 mm. The methanol extract showed zones of inhibition ranging from 0–10 mm, 0–12 mm, and 0–13 mm at the tested concentrations of 1 mg/disc, 2 mg/disc, and 5 mg/disc, respectively and the detailed results are depicted in Fig. 2 and Additional file 1: Table S1. However, the antibiograms of the extracts were observed to be different depending on the pathogens and their origin. Briefly, 80% of the chloroform extract tested exhibited considerable action against S. epidermis, L. acidophilus, C. glabrata, and C. krusei; 70% against S. aureus, E. coli, E. fergusonii, and 20% against P. mirabilis and K. pneumoniae. Followed by ethyl acetate extract demonstrated 80% activity against L. acidophilus, P. mirabilis, and C. krusei; 70% activity against E. fergusonii; 40% activity against E. coli and C. glabrata; and 20% activity against S. epidermis, S. aureus, S. para-typhi, and K. pneumoniae. 80% of the methanol extract inhibited E. coli, 70% inhibited S. para-typhi, P. mirabilis, and K. pneumoniae, 40% inhibited L. acidophilus, and 20% inhibited S. epidermis and S. aureus. None of the methanol is active against Candida strains. On the other hand, modest activities were observed in hexane extracts, with 80% activity against C. krusei and 20% activity against S. aureus, E. fergusonii, and C. glabrata (Fig. 3a, b). These results clearly demonstrated that the chloroform and ethyl acetate crude extracts exhibited higher and broad spectrum of inhibitory effects compared to the methanol extracts. Remarkably, among plant extracts screened, the ethyl acetate extract of G. travancorica exhibited significant activity against L. acidophilus (17 mm) followed by S. aureus (16 mm), E. coli (13 mm), P. mirabilis (12 mm), S. epidermis (11 mm), C. krusei (11 mm), C. glabrata (10 mm), and no activity were observed against E. fergusonii, S. para-typhi, K. pneumoniae. On the other hand, the chloroform extract from O. aristatus showed good activity against S. epidermis (13 mm), L. acidophilus (13 mm), S. aureus (12 mm), E. fergusonii (12 mm), C. krusei (12 mm) C. glabrata (11 mm), E. coli (11 mm), K. pneumoniae (10 mm), and no inhibition was observed for S. para-typhi, P. mirabilis. Furthermore, based on the broad spectrum of antimicrobial activity, the chemical analyses of the active ethyl acetate extract of G. travancorica revealed the presences of 6-methyloctadecane, β-D-glucopyranose, 1,4-dimethoxy-2,3-dimethylbenzene, bromoenol lactone, 1,3,5-benzenetriol, 3,7,11,15-tetramethyl-2-hexadecen-1-ol/ phytol, 2-hexadecen-1-ol,3,7,11,15-tetramethyl-,acetate, [R-[R*,R and trans-Geranylgeraniol (Fig. 4a, Table 2). The chloroform extract of O. aristatus indicates the presence of 6-methyloctadecane, 7-methoxy-2-methylquinolin-4-ol, caryophyllene, 2,4-di-tert-butylphenol, 3-trifluoroacetoxydodecane, 2-methyl-7-nonadecene, 9-eicosyne, pentadecanoic acid, 14-methyl, methyl ester, cis,cis,cis-7,10,13-hexadecatrienal and phytol (Fig. 4b, Table 3). These antimicrobial activities may be due to presence of several bioactive compounds in the ethyl acetate and chloroform extracts G. travancorica and O. aristatus, respectively.

Fig. 2

Heatmap showing the antimicrobial activity of the various plant extracts. 2a-c Antimicrobial activity of hexane extract; 2d-f Antimicrobial activity of chloroform extract; 2 g-i Antimicrobial activity of ethyl acetate extract; 2j-l Antimicrobial activity of methanol extract of Alstonia scholaris, Orthosiphon aristatus, Sphaeranthus amaranthoides, Crateva magna and Garcinia travancorica (1 mg, 2 mg and 5 mg/disc, respectively)

Fig. 3

a Parallel plot showing the cumulative percentage of antimicrobial activities of hexane, chloroform, ethyl acetate and methanol extracts (5 mg/disc) of Alstonia scholaris, Orthosiphon aristatus, Sphaeranthus amaranthoides, Crateva magna and Garcinia travancorica versus bacterial and candidal pathogens, respectively. b Chord plot showing the percentage of antimicrobial activity of different extracts of Alstonia scholaris, Orthosiphon aristatus, Sphaeranthus amaranthoides, Crateva magna and Garcinia travancorica against the tested bacterial and candidal pathogens

Fig. 4

Chromatogram showing compounds in the active extracts, a Orthosiphon aristatus and b Garcinia travancorica

Table 2 GC–MS analysis of chloroform extract from Orthosiphon aristatus

Table 3 GC–MS analysis of ethyl acetate extract from Garcinia travancorica

Discussion

Natural products have been reported to possess significant antimicrobial activities owing to their ability to interact with cell membrane, intercellular organs, enzymes and to inhibit cell cycle progression. However, the evidence on the distribution of microbial pathogens and therapy recommendations in patients remains insufficient for hospitalized and immunocompromised patients. There are also discouraging scenario persists due to poor adherence to the treatments and increased drug resistance among the bacterial and candidal pathogens are associated with nosocomial bloodstream infections, urinary tract infections, candidiasis, diabetic and immunocompromised patients (Teferi et al. 2023; Rossi et al. 2022). Therefore, proper screening and surveillance reports are required to guide antimicrobial stewardship efforts. The present investigation was conducted on sequential hexane, chloroform, ethyl acetate, and methanol extracts of Alstonia scholaris, Orthosiphon aristatus, Sphaeranthus amaranthoides, Crateva magna, and Garcinia travancorica aimed to delineate the inhibitory effect against a panel of clinically prioritized microbial pathogens. The obtained results revealed that the zones of inhibition varied significantly between extracts and concentrations. Extracts of hexane, chloroform, ethyl acetate, and methanol showed different antimicrobial properties against species of Candida implicated in nosocomial infections as well as Gram-positive and Gram-negative bacteria. Among the noteworthy findings were the stronger inhibitory effects of ethyl acetate and chloroform extracts in comparison to methanol which supported findings reported by Odongo et al. (2023) and Komape et al. (2017). In addition, these results are also in accordance with the Perera et al. (2022) indicating that the polar extracts exhibited higher antimicrobial activity compared to the non-polar extracts in treating bacterial and candidal infections. Moreover, it is possible that the methanol crude extracts contain lower concentrations of antimicrobial constituents, which may explain why larger quantities of decoctions are typically consumed over a longer duration to achieve therapeutic efficacy (Al-Tohamy et al. 2018). These observations highlight the importance of extraction methods and solvent choice in obtaining plant extracts with potent antimicrobial properties. The standout performance of the ethyl acetate extract from G. travancorica prompts a closer examination of its chemical composition. The identification of specific compounds, including 6-methyloctadecane, β-D-glucopyranose, 1,4-dimethoxy-2,3-dimethylbenzene, bromoenol lactone, 1,3,5-benzenetriol, 3,7,11,15-tetramethyl-2-hexadecen-1-ol/phytol, 2-hexadecen-1-ol,3,7,11,15-tetramethyl-,acetate, [R-[R*,R, and trans-Geranylgeraniol, sheds light on potential bioactive contributors to the observed antimicrobial effects. Similarly, the antimicrobial activity of essential oil from the leaves of G. travancorica has been reported to possess significant activity against Gram-Positive bacteria Micrococcus mucilaginosus and Gram-Negative bacteria Pseudomonas aeruginosa (Ramasubbu et al. 2020). In addition, the chloroform extract from O. aristatus revealed a complex composition featuring compounds such as 6-methyloctadecane, 7-methoxy-2-methylquinolin-4-ol, caryophyllene, 2,4-di-tert-butylphenol, 3-trifluoroacetoxydodecane, 2-methyl-7-nonadecene, 9-eicosyne, pentadecanoic acid, 14-methyl, methyl ester, cis,cis,cis-7,10,13-hexadecatrienal, and phytol. This result is in agreement with the previous report of Widyaningrum et al. (2022) in which the polar extract showed activity against Propionibacterium acnes when compared to non-polar ethanol and aqueous extracts. These antimicrobial activities may be due to the presence of several bioactive compounds were previously showed significant activity against microbial pathogens (Shahid et al. 2022; Tung et al. 2022).

Conclusions

Despite the significant advancements in modern medicine, the utilization of plants in healthcare remains crucial in several parts of the world. Plants continue to be a focus of research for drug development due to their accessibility and the ability to select them based on their traditional medicinal uses. In this study, five medicinal plants obtained from ethnobotanical sources demonstrated noteworthy antimicrobial activity. Interestingly, G. travancorica demonstrated significant activity against L. acidophilus, S. aureus, E. coli, P. mirabilis, S. epidermis, C. krusei, and C. glabrata, while the chloroform extract from O. aristatus displayed notable activity against S. epidermis, L. acidophilus, S. aureus, E. fergusonii, C. krusei, C. glabrata, E. coli, and K. pneumoniae with the zones of inhibition ranging from 10-17 mm. Moreover, the identification of major compounds in G. travancorica and O. aristatus through GC–MS analysis shed light on their significant antimicrobial properties as valuable resources for the development of novel antimicrobial medications. These findings validate the value of ethnomedicine as a reliable source for discovering novel anti-infective agents.