Introduction

Antibiotic therapy is among the primary approaches used in modern medicine to battle infectious diseases. The “golden period” of antibiotics lasted from the 1930s to the 1960s, during which time many antibiotics were developed (Nathan and Cars 2014; Narayanan et al. 2021a). Regrettably, this period finally ended when investigators could not keep up with the pace of antimicrobial agents in the face of potential bacterial strains (Schanté et al. 2011). Long-term inabilities to address or discover new medications, as well as indiscriminate medication use, are risk factors for the emergence of multidrug resistance (Narayanan et al. 2021b). The frequent use of specific antibiotics to treat specific pathogens that cause disease for several years results in drug resistance in pathogenic microbes, particularly bacteria (Narayanan et al. 2021c). Antimicrobial resistance (AMR) is a major global danger to human, animal, and environmental health. This is because of the resurgence, expanded, and perseverance of multidrug resistant (MDR) bacteria, also known as “superbugs” (Davies and Davies 2010). The global use of antibiotics in cattle has recently shown hotspots of antibiotic usage across countries, which will have environmental and socio—economic implications in the coming years (Narayanan et al. 2021d). Antibiotics are widely used in the food animals such as cows, poultry, and pigs, and it is predicted that by 2030, such use will increase by up to 67% in the world’s most populous countries (Van Boeckel et al. 2015). In 2019, WHO recognized 32 antibiotics in drug development that discuss the WHO list of priority pathogens, only six of which were deemed innovative and furthermore, a major issue is a lack of access to high-quality antimicrobials (Narayanan et al. 2021e). Antibiotic shortfalls are influencing countries at all stages of development, particularly health care systems. Numbers of multidrug resistance bacteria such as Mycobacterium tuberculosis (MDR) (Singh and Chibale 2021), Clostridium difficile (MDR) (Chow et al. 2017), Enterococci sp. (vancomycin-resistant) (Roberts et al. 2009), Staphylococcus aureus (methicillin) (Lee et al. 2018), Neisseria gonorrhoea (cefixime, ceftriaxone) (Unemo and Shafer 2011), Enterobacteriaceae (carbapenem-resistant) (Van Duin et al. 2013) have been reported regularly. Numerous researchers have found that chemotherapy indications, agent selection, and antibiotic treatment duration are all improper in 30–50% of cases (Aslam et al. 2018). According to WHO, AMR is one of the top ten worldwide public health vulnerabilities. To achieve the Sustainable Development ambition, vital multi-sectoral battle is essential. Hence, in this study two pre-recognized medicinal plants namely Evolvulus alsinoides and Mucuna pruriens have been selected. The E. alsinoides is a perennial herb, deep root system, with spreading branches, blue color flowers, and found in all over India. Furthermore, the E. alsinoides have been used as an efficient neuroprotective agent to treat memory loss, insomnia, lung disease, respiratory problems, and syphilis (Yadav et al. 2019a, b). It can also treat epilepsy, leukoderma, cuts, and ulcers (Singh 2008). The M. pruriens is a perennial wild leguminous climbing plant, enriched with protein (23–35%), present in tropical and sub-tropical regions of the world, especially from southern China and eastern India called as velvet bean (Lampariello et al. 2012). The various medicinal properties such as anti-neoplastic, anti-diabetic, anti-epileptic, aphrodisiac, anti-venom, anti-microbial, and anti-helminthic activities of this plant have been investigated by numbers of researchers (Lampariello et al. 2012; Pathania et al. 2020; Sathiyanarayanan and Arulmozhi 2007). This is the first report deals about the antimicrobial potentials of solvent extracts of E. alsinoides and M. pruriens against multidrug resistant bacteria and evaluate the possible predominant bioactive components in these plant extract through GC–MS analysis. Since, finding novel bioactive components to treat the multidrug resistant bacterial strain is timely need. Hence, this research was designed to analyse the phytochemical profile of this various solvent leaf extract of this plant, in addition to this analyse the antibacterial activity of these extracts against multidrug resistant pathogenic bacteria such as Pseudomonas aeruginosa 1, P. aeruginosa 2, P. aeruginosa 3, P. aeruginosa 4, Staphylococcus aureus, and Klebsiella pneumoniae.

Materials and methods

Plant samples collection

The fresh and disease free leaves of Evolvulus alsinoides and Mucuna pruriens were collected from Kolli hills of Namakkal district of Tamil Nadu, India. Dr. D. Natarajan, Department of Biotechnology, Periyar University, Salem, Tamil Nadu, India, identified and recognized the collected plant samples. The collected leaf samples were rinsed with clean tap water and dehydrated under shadow conditions until suitable for pulverizing process. The well-dried plant leaf samples were pulverized individually, and grounded samples were filtered through standard flour filter (Narayanan et al. 2021a) and refrigerated for further extraction.

Brief profile about test multidrug resistant bacteria culture

The pre-isolated (Mohanasundari et al. 2021) multidrug resistant (MDR) bacteria such as Pseudomonas aeruginosa 1, P. aeruginosa 2, P. aeruginosa 3, P. aeruginosa 4, Staphylococcus aureus, and Klebsiella pneumoniae were used in this study to evaluate their sensitivities to solvent extracts derived from E. alsinoides and M. pruriens leaf samples. These tests MDR bacteria were reported from various clinical samples such as pus, wound, tissue, and so on (Mohanasundari et al. 2021).

Extract preparation

The standard hot plate extraction method (Narayanan et al. 2021a) was applied to extract the phytochemical ingredients present in the powdered leaf samples of E. alsinoides and M. pruriens. In brief, about 15 g of (each) powdered leaf samples of E. alsinoides and M. pruriens were dissolved in 100 mL (each) of various solvents such as benzene, ethanol, and methanol in 250-mL conical flasks. These individual reaction mixers were placed over the water bath for 20 min at 55 °C, the extracts were filtered then, and using Whatman (No. 1) filter paper and filtrates were stored at 4 °C for study.

Qualitative phytochemicals analysis

The benzene, ethanol, and methanol extracts derived from the leaf of E. alsinoides and M. pruriens were subjected to qualitative phytochemicals analyses such as alkaloids, saponins, protein, glycosides, phlobatannins, amino acid, coumains, steroids, carbohydrate, starch, phenol, anthraquinone, flavonoids, and tannin. The standard qualitative phytochemicals analyses methods (Narayanan et al. 2021b) were for followed to assess presence of these phytochemicals on various extracts of these test plants.

Quantitative phytochemicals analysis

The phytochemicals include alkaloids, protein, glycosides, phlobatannins, amino acid, steroids, carbohydrate, phenol, flavonoids, tannin, coumarins, and anthraquinone were focused on assessing their quantity in individual extracts based on the results obtained from the qualitative phytochemicals analysis. Typical methods were followed for this quantification analysis (Ezeonu and Ejikeme 2016; Gul et al. 2017).

In-vitro antibacterial activity analysis

The antibacterial potential of various extracts of E. alsinoides and M. pruriens against pre-isolated multidrug resistant (MDR) bacteria such as P. aeruginosa 1, P. aeruginosa 2, P. aeruginosa 3, P. aeruginosa 4, S. aureus, and K. pneumoniae through typical agar diffusion method. Initially, 100 µL of each solvent extract was tested with these MDR strains through the agar well diffusion method (Narayanan et al. 2021a). Subsequently, each solvent extracts of both test plants were blended in two proportions: I. 25 µL of E. alsinoides with 25 µL of M. pruriens. II. 50 µL of E. alsinoides with 50 µL of M. pruriens. These proportions were poured on well perforated on Muller Hinton Agar plates and 30 µg mL−1 combinations of penicillin and gentamicin were used as a positive control. The culture inoculated and extracts loaded plates were incubated at 37 °C for 24 h and zone of inhibition was measured then and compared with control and determine the sensitivity of test MDR bacterial pathogens.

Gas chromatography–mass spectrometry (GC–MS) analysis

The in-vitro antibacterial assay revealed that the methanol extract of both plants (E. alsinoides and M. pruriens) were effective against most of these MDR strains. Thus, the methanol extracts were subjected to GC–MS (7000D Triple Quadrupole GC/MS, Agilent) analysis to determine the predominant bioactive components present in these extracts by following standard operating protocol (Kanthal et al. 2014). The initial temperature for the column was set as 35 °C for 3 min and programmed to increase the temperature by 8 °C min−1 up to 280 °C. About 1 μL of the sample was injected into the sample port with the sample flow rate of 1 mL min−1 with helium gas (carrier gas). Subsequently, the MS spectrum was operated at 70 eV to analyze the column and components were further investigated by Flame Ionization Detector (FID). The obtained peaks corresponding chemical components were studied by comparing with the NIST database.

Results and discussion

Qualitative and quantitative analyses of phytochemicals

The qualitative phytochemicals analysis results revealed that the methanol extract of E. alsinoides and M. pruriens contains more numbers of phytochemicals followed by ethanol and benzene extracts (Table 1). The methanol extract of E. alsinoides contains alkaloid, protein, glycosides, amino acid, carbohydrate, phenol, anthraquinone, flavonoids, and tannin. Similarly, the methanol extract of M. pruriens contains alkaloids, phlobatannins, carbohydrate, phenol, flavonoids, and tannin. These results confirmed that the E. alsinoides and M. pruriens have more phytochemicals, which dissolved in the methanol solvent. Most of these major phytochemicals were previously recognized as pharmaceutically valuable components able to cure various health issues. In the quantitative analysis, the obtained results revealed that the methanol extract of E. alsinoides contains significant quantities of alkaloid 38.0 mg g−1, phlobatannins 7.3 mg g−1, carbohydrate 1.52 mg g−1, phenol 89.0 mg g−1, flavonoids 141.0 mg g−1, and tannin 98.2 mg g−1 (Table 2). The methanol extract of M. pruriens contains alkaloids 1.87 mg g−1, glycosides 4.0 mg g−1, amino acid 0.87 mg g−1, carbohydrate 48.1 mg g−1, phenol 4.6 mg g−1, flavonoids 26.4 mg g−1, tannin 12.0 mg g−1, coumarins 0.98 mg g−1, and anthraquinone 1.89 mg g−1 (Table 2). Remaining phytochemicals were not in a quantifiable quantity in both plant methanol extracts, furthermore these quantities were quite higher than the remaining extracts. The obtained phytochemicals possess excellent pharmaceutical properties, for example several components of alkaloid have been documented as outstanding medicinal potential such as quinine (antimalarial activity), homoharringtonine (anticancer), vincamine (vasodilator), morphine (analgesic), piperine (antihyperglycemic), ephedrine (antiasthma), cheleythrine (antibacterial), and so on (Cushnie et al. 2014; Prasat et al. 2014; Russo et al. 2013). Phlobatannins possess good wound healing, anti-inflammatory, analgesic and antioxidant properties (Wadood et al. 2013). The phenol contains significant anti-inflammatory, antioxidant, and anti-carcinogenic, or anti-mutagenic effects (Huang et al. 2009). Flavonoids, like phenol, are a class of multi-potential pharmaceutical agents because they have anticancer, antioxidant, immune system booster, antimicrobial, antiinflammatory, and cardioprotective properties, among other things (Tungmunnithum et al. 2018). The tannin compound has been used to cure hemorrhoids, tonsillitis, skin eruptions, and pharyngitis (Pizzi 2019). The other most important phytochemicals found in this plant extract coumarins are most significant phytochemicals applied to cure several critical health complications such as neuroprotective, antiadipogenic, anticancer, and antimicrobial antihyperglycemic, antihypertensive, anticonvulsant, antitubercular, anticoagulant, antioxidant, and anti-inflammatory (Venugopala et al. 2013). The anthraquinone also possess excellent anticancer, insecticidal, antibacterial, antiviral, antiparasitic, fungicidal, diuretic, immunomodulatory properties and so on (Yadav et al. 2019a, b).

Table 1 Qualitative phytochemicals profile of various solvents extracts of E. alsinoides and M. pruriens
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Table 2 Quantitative phytochemicals analysis of various solvents extracts of E. alsinoides and M. pruriens
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Antibacterial potential of plant extract against MDR

The individual extract antibacterial activity results showed that the methanol extract of M. pruriens has a significant level of antibacterial activity at 100 µL dosage against P. aeruginosa 1 (11 mm), P. aeruginosa 2 (10 mm), P. aeruginosa 3 (10 mm), P. aeruginosa 4 (9 mm), S. aureus (11 mm), and K. pneumoniae (10 mm) than methanol extract of E. alsinoides (Table 3). However, in the combined plant extract study also the 100 µL (50 µL of E. alsinoides + 50 µL of M. pruriens) of methanol extract showed a significant level of antibacterial activity against P. aeruginosa 1 (13 mm), P. aeruginosa 2 (12 mm), P. aeruginosa 3 (13 mm), P. aeruginosa 4 (10 mm), S. aureus (23 mm), and K. pneumoniae (13 mm) and it followed by ethanol extract combined activity (Table 4). This antibacterial efficiency was determined by the size of the zone of inhibition around the treated wells. The obtained zones of inhibitions results for combined methanol extract activity were almost close to the positive control. These results were partially correlates with the findings of Atef et al. (2019). They reported that the methanol extract of Moringa oleifera L.(leaves) and Matricaria recutita L. (flowers) have excellent antibacterial potential MDR (P. aeruginosa, Staphylococcus sp., E. coli, Klebsiella sp., and P. mirabilis) at the dosage of 7.8–62.5 mg mL−1 (Atef et al. 2019). The obtained results suggested that the phytochemicals present on the methanol extract of both plants were effective against MDR when it in combined form than the individual. Thus it suggests that bioactive components present in these extracts were combined randomly and showed their efficiency against MDR (Khan et al. 2009). The bioactive components present in these extracts may inhibit the growth of MDR by interrupting several essential mechanisms such as cell wall biosynthesis, protein synthesis, nucleic acid synthesis, and destruction of the bacterial membrane (Khameneh et al. 2019). Another report states that the extracts derived from A. nilotica, S. aromaticum and C. zeylanicum showed excellent antibacterial activity against MDR bacterial strains such as E. coli, K. pneumoniae, S. mutans, S. aureus, E. faecalis, S. bovis, P. aeruginosa, and S. typhimurium (Khan et al. 2009). This result suggests that certain plant extracts have pharmaceutically valuable bioactive components that can be very effective to MDR bacterial strains (Mehta et al. 2013).

Table 3 Antibacterial activity of E. alsinoides and M. pruriens leaf extracts (individual) in various solvents against MDR bacteria
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Table 4 Antibacterial activity of E. alsinoides and M. pruriens leaf extracts (combined) in various solvents against MDR bacteria
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GC–MS analysis

Based on the results obtained from the antibacterial activity assay, the methanol extracts of E. alsinoides and M. pruriens have the reasonable potential to act against the multidrug resistant bacterial strains. Thus, the predominant bioactive components present in the methanol extracts of both plants were investigated through GC–MS analysis. The methanol extract of E. alsinoides contains about 11 predominant peaks obtained at various retention times and respective bioactive chemical components. Their structure and molecular formulas were found and mentioned in Table 5 (Fig. 1). Those predominant components were identified as propanoic acid, 2,3-dihydroxy-(RT 9.331), butanoic acid, 3-hydroxy-(RT 13.308), (6Z)-nonen-1-ol (RT 15.108), n-hexadecanoic acid (palmitic acid) (RT 16.152), phytol (RT 17.352), 9,12-octadecadienoic acid (17.530), 2-methyl-Z,Z-3,13-octadecadienol (RT 17.563), octadecanoic acid (stearic acid) (RT 17.730), stigmasterol (RT 21.263), octadecane (RT 21.385), and squalene (RT 21.952) (Table 5; Fig. 1). Nevertheless, the methanol extract of M. pruriens contains only six major peaks with various retention times (Fig. 2). based on the NIST database, the obtained peaks were matched with the pharmaceutically valuable bioactive components such as butanal, 2-methyl-(RT 11.486), n-hexadecanoic acid(palmitic acid) (RT 16.208), 9,12-octadecadienoic acid (Z,Z)-(RT 17.630), octadecanoic acid (RT 17.785), hexadecanoic acid, 2-hydroxy-1-(palmitoglycerol) (RT 20.218), and Z,E-7,11-hexadecadien-1-yl acetate (RT 21.363) (Table 6). Various concentrations and various proportions of these bioactive components can be act as a potential agent against MDR.

Table 5 GC–MS analysis: predominant bioactive compounds present in methanol extract of E. alsinoides
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Fig. 1
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GC–MS analysis of methanol extract of E. alsinoides

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Fig. 2
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GC–MS analysis of methanol extract of M. pruriens

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Table 6 GC–MS analysis: predominant bioactive compounds present in methanol extract of M. pruriens
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Similarly, 11 bioactive components have been reported in methanolic whole plant extract of Withania somnifera and that crude extract has been documented as excellent antibacterial activity against urinary infections causing bacteria (Mishra and Patnaik 2020). Some of the individuals identified bioactive components researchers have reported biomedical activities. For example, propanoic acid has excellent inhibition activity on numbers of bacteria and molds (Abd-Elaal et al. 2018). The byproducts or derivatives or blend with any suitable adjuvants can act as excellent anticancer activity in the form of butyric acid or butyrate. These statements strongly suggest that the methanolic extract of both plant species contained significant volume of pharmaceutically valuable bioactive components. Palmito-glycerol has been reported as excellent antimicrobial activities against various pathogenic microbes (Wang et al. 2019).

Conclusion

The methanol extracts of E. alsinoides and M. pruriens contain significant quantity of pharmaceutically valuable phytochemicals than other solvent xtracts such as ethanol and benzene. The combined proportions (50 µL of E. alsinoides + 50 µL of M. pruriens) of methanol extract showed reasonable antimicrobial activity against pre-identified MDR strains such as such P. aeruginosa 1, P. aeruginosa 2, P. aeruginosa 3, P. aeruginosa 4, S. aureus, and K. pneumoniae and it followed by ethanol extract showed considerable antibacterial activity. No significant antibacterial activity was found in benzene extracts. Under the GC–MS analysis, about 11 and 6 predominant peaks were found for methanol extracts of E. alsinoides and M. pruriens. These RT peaks were corresponding to valuable pharmaceutical bioactive components. Thus, these results conclude that the crude methanol extracts of these plants contain pharmaceutically valuable bioactive molecules and can be considerable for pharmaceutical drug formulation preparation against MDR after completing compound purification and in-vivo studies.