Potential antimicrobial compounds in flower extract of Plumeria alba

2.1 Preparation of extract

Healthy flowers of P. alba were collected from Lahore, Pakistan during September 2019 and stored in paper bags to carry to the laboratory. After washing, petals were separated from the flowers and kept in trays to evaporate water from their surface. Flowers were dried under shade and thoroughly crushed in a pestle and mortar. Flower extract was prepared in 95% methanol by gentle heating at 40 °C for 60 min in a closed container. The solvent was filtered and then evaporated on a rotary evaporator at 40 °C under reduced pressure and the remaining gummy material (methanolic extract) was used for antimicrobial bioassays.

2.2 Evaluation of antibacterial activity

Five bacterial species namely Bacillus sp., Salmonella sp., Pseudomonas sp., Escherichia coli and Staphyllococcus sp. were procured from First Culture Bank of Pakistan (FCBP). LBA growth medium (beef extract 10 g + typton 10 g + NaCl 5 g + agar 20 g + water 1000 mL) was autoclaved at 121 °C for 30 min. For preparation of different concentrations of the methanolic extract, procedure of Kanwal et al. (2009) was followed with little modifications. To 75 mL of LBA media, appropriate quantity of the methanolic extract was added and mixed well to prepare 5, 10, 20, 50, 100, 500 and 1000 ppm concentrations. This volume was divided into three equal portions and poured in sterilized Petri plates aseptically to serve as replicates. Agar discs (5 mm) containing bacterial inocula were placed in the centers of the plates and incubated at 37 °C for 24 h, and radial growth of bacteria in each plate was recorded thereafter (Balouiri et al., 2016).

2.3 Evaluation of antifungal activity

Five fungal species namely Trichoderma koningii, T. viride, T. hamatum, T. harzianum and T. reesei were obtained from FCBP. A 2% malt extract agar (20 g malt extract + 20 g agar + 1000 mL water) was autoclaved at 121 °C for 30 min. To avoid bacterial growth, streptomycene capsule was added after autoclaving during the cooling stage. A volume of 75 mL of the autoclaved growth media was poured in 250-mL flasks and appropriate quantities of the flower extract were added to prepare 5, 10, 20, 50, 100, 500 and 1000 ppm concentrations. Control was without the flower extract. Media was poured in autoclaved Petri plates, each containing 25 mL of the media. Media was allowed to solidify and thereafter the plates were inoculated in the center (5 mm disc) with the test fungal species. Experiment was carried out in a completely randomized design using three replications. After 7-day incubation at 27 °C, radial growth of the fungi was measured (Kanwal et al., 2010).

Percentage decreases in the fungal growths due to different concentrations were calculated using the following equation (Khan et al., 2021): $$\text{Growth inhibition}(\%)=\frac{\text{Growth in control - Growth in extract}}{\text{Growth in control}}\times \text{100}$$

2.4 GC–MS analysis

The gas chromatograph (GC) machine model was 7890B and that of mass spectroscopy (MS) was 5977A, used for the identification of different compounds from the sample while both were branded by Agilent Technologies. The column used was DB 5 MS (30 m × 0.25 µm × 0.25 μm). Injection volume was 1 µL and the carrier gas was helium. Oven ramping; initial temperature was 80 °C and then raised 10 °C per minute up to 300 °C. Inlet temperature was 280 °C. MS conditions were as mode: scan 50–500 m/z, the source temperature was 230 °C and quadrupole temperature was 150 °C. Chemical compounds were identified by comparison of their spectra with library and arranged in the ascending order of their retention times and retention indices. The relative abundance was reported by using their peak areas (Ferdosi et al., 2021).

2.5 Statistical analysis

All the experimental data regarding the effect of flower extract on growth of different bacterial and fungal species were subjected to two-way ANOVA using the software Statistix 8.1. The significant differences among the treatment means were determined by LSD test at P ≤ 0.05.

3.1 Antibacterial activity

The effect of bacterial species (B), extract concentration (C) and B × C was significant for bacterial growth (Table 1). The highest growth was recorded in negative control treatments of all the five bacterial species. Flower extract of P. alba significantly reduced growth of all the bacterial species over control. However, the effect was variable against different bacterial species (Fig. 1A). There was 36–90%, 8–84%, 19–90%, 23–93% and 12–92% reduction in growth of E. coli, Salmonella sp., Pseudomonas sp., Bacillus sp., and Staphylococcus sp., respectively, over corresponding control treatments (Fig. 1B).

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Fig. 1

Antibacterial activity of methanolic flower extract of Plumeria alba. Different letters indicate significant differences (P ≤ 0.05) among the treatments as determined by LSD Test.

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In general, the extract was effective against all the five bacterial species, however, different bacterial species showed variable growth responses to the extract. Similar variable antibacterial effects of different products such as extracts and essential oil of this plant have been reported earlier in other parts of the world. Zaheer et al. (2010) studied the antibacterial activity of essential oils of P. alba flower and reported that the oil was more toxic to Gram positive than Gram-negative bacteria. The oil was more effective against Staphylococcus aureus and Bacillus subtilis than against Escherichia coli and Salmonella typhi. Radha et al. (2008) reported that methanolic extract (MIC 200 μg/mL) of P. alba flowers and its triterpene rich fraction (MIC 200 μg/mL) showed pronounced antibacterial activity against S. aureus, E. coli, S. typhi and Pseudomonas aeuroginosa. Gold nanoparticles of P. alba flower extract showed more antibacterial activity than the standard antibiotics. Moreover, the nanoparticles were more effective against E. coli than against Streptobacillus sp. (Nagaraj et al., 2012).

3.2 Antifungal activity

The effect of fungal species (F), extract concentration (C) and their interaction (F × C) was significant (P ≤ 0.001) for fungal growth (Table 2). All the concentrations of the applied extract significantly suppressed growth of all the five test fungal species. The effectiveness of the extract was concentration dependant as there was a gradual decrease in fungal growth with increase in extract concentration. Growth response to the applied extract also varied with the change in fungal species (Fig. 2A). Different concentrations of the extract reduced growth of T. viride, T. harzianum, T. hamatum, T. reesei and T. koningii by 31–90%, 24–86%, 7–77%, 11–97% and 9–76%, respectively, over the respective control treatments (Fig. 2B).

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Fig. 2

Antifungal activity of methanolic flower extract of Plumeria alba against Trichoderma species. Different letters indicate significant differences (P ≤ 0.05) among the treatments as determined by LSD Test.

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Like bacteria, the methanolic flower extract also significantly reduced growth of all the fungal species and the susceptibility of the different fungi was variable to the applied extract. Earlier, petroleum ether extract of P. alba showed pronounced fungistatic activity against Penicillium digitatum, Aspergillus flavus and Rhizopus arrhizus but not against Aspergillus fumigatus and A. niger (Sibi et al., 2012). Similarly, Kumari et al. (2012) reported that essential oil of P. alba flowers was highly toxic to the growth of Candida albicans, Aspergillus niger, and Penicillium chrysogenum while Ralstonia entropha and Phaenorochaete chrysporium showed less susceptibility to the applied oil. Alkaloids are the major compounds in flowers of P. alba followed by flavonoids and glycosides in addition to phenols, tannins and terpenoids (Sibi et al., 2012), which could be responsible for antifungal activity (Yadava and Tiwari, 2007; Kanwal et al., 2010; Javed et al., 2021). Radha et al. (2008) reported antifungal activity of methanolic flower extract of P. alba against A. niger, P. chrysogenum and Microsporum gypseum, and correlated the activity with triterpenes present in the extract. Extracts of other parts of this plant also known to exhibit antifungal activity against various fungal species, such as ethyl acetate bark extract showed marked activity against C. albicans (Murniana et al., 2011).

3.3 GC–MS analysis

GC–MS chromatogram presented in Fig. 3 demonstrated the presence of 21 natural compounds in methanolic flower extract of P. alba whose details are given in Table 3. Principal components of this extract included benzofuran, 2,3-dihydro- (27.95%), 9-octadecyne (14.29%), and cyclononanone (14.59%). Moderately abundant compounds included 9,12,15-octadecatrienoic acid, methyl ester, (Z,Z,Z)- (10.28%), benzyl alcohol (5.57%), 1-decanol, 2-hexyl- (5.44%), and 2,6-octadien-1-ol, 3,7-dimethyl-, acetate, (Z)- (3.76%). Compounds such as 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- (2.48%), heptadecane (2.43%), 1,3-propanediamine, N-methyl- (1.86%), 1,6,10-dodecatrien-3-ol, 3,7,11-trimethyl-, [S-(Z)]- (1.35%), hexadecanoic acid, 15-methyl-, methyl ester (1.19%), 4-methyl-1,5-heptadiene (1.07%), and phenol (1.04%) were identified as less abundant ones. Four compounds namely hydroquinone (1.01%), catechol (0.99%), 3,3′-dimethoxybenzil (0.99%), 3,5-dimethylanisole (0.95%), 3,7,11-tridecatrienenitrile, 4,8,12-trimethyl- (0.94%), 2-cyclopenten-1-one, 2-hydroxy- (087%), and 1,4-cyclohexanedimethanamine (0.86%), with peak area less than 1% were ranked as the least abundant. The most abundant compound in this study was benzofuran, 2,3-dihydro-. Earlier, Idan et al. (2015) also reported this compound from methanolic fruit extract of Citrullus colocynthis. Compounds possessing benzofuran nucleus (bicyclic ring system) are known for their broad-spectrum bioactivities including antimicrobial activities (Kamal et al., 2011). Recently, Zaher et al. (2022) identified a benzofuran glucoside in ethyl acetate extract as a new and the potent antimicrobial agent inhibiting growth of two Gram-positive and one Gram-negative bacteria, and a fungal species. Some of the uother identified compounds are also known to possess antimicrobial potential. 2-Cyclopenten-1-one, 2-hydroxy- is a diterpene with good antimicrobial activity (Soonthornchareonnon et al., 2012; Revathi et al., 2015). Phenol is known to have antibacterial activity (Bennett, 1959). Phenolic compounds inhibit bacterial growth by damaging the bacterial membrane, inhibiting the virulence factors like toxins and enzymes, and by suppressing the biofilm formation (Miklasińska-Majdanik et al., 2018). Benzyl alcohol, an aromatic alcohol, is a naturally occurring substance present in many fruits and in many essential oils, and is known for its antimicrobial activity (Lucchini et al., 1990). Catechol is a phenolic allelochemical produced in plants. It showed antibacterial activity against Corynebacterium xerosis, Pseudomonas putida, P. pyocyanea, and antifungal activity against Penicillium italicum, Colletotrichum circinans and Fusarium oxysporum (Farkas and Kiraly, 1962; Kocaçalişkan et al., 2006). This compound is also effective against harmful intestinal bacterial species namely Escherichia coli, Clostridium perfringes, C. difficile (Jeong et al., 2009). Hydroquinone showed strong antibacterial activity against Staphylococcus aureus by destroying cell wall and membrane, and increasing permeability leading to leakage of intracellular substances, and by influencing gene expression (Ma et al., 2019).

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Fig. 3

GC–MS chromatogram of methanolic flower extract of Plumeria alba.

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