Phytochemical Analysis And Antibacterial Activity Of Lygodium Microphyllum Extract

Lygodium microphyllum

Lygodium microphyllum (Lygodiaceae) is one of Malaysian’s medicinal ferns which has important contributions for prevention and treatment of various human ailments. Alternatively known in Malay as “ribu-ribu kecil, selada, kapai alus’’, L.microphyllum is a large and distinctive ground-to-crown climbing fern species. It has long climbing, stem-like fronds and wiry brown rhizomes. Fronds spread along the ground, overgrown shrubs, and also climb up and overgrow trees and other structures by twining around them (Figure 01 ). The rhizomes can accumulate to form dense mats on the ground. The leaflets (pinnules) are of two types (a) non-reproductive leaflets are unlobed along their margins, and oblong to lanceolate (pointed), and (b) reproductive leaflets (have tiny lobes along their margins covering the sporangia (Lott, Volin, Pemberton, & Austin, 2003).

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Phytochemicals

Phytochemicals or secondary metabolites from plants are produced to strengthen the plants in order for them to adapt to their environment, and also to defend themselves from predators and pathogens. Each species of the plant kingdom contains a great number of secondary metabolites. The presence of plant secondary metabolites has however fluctuated due to the environmental factors surrounding the plants of the same species including biotic (invaded by pathogens causing stress to plants) and abiotic (water stress, salinity stress and temperature stress) factors. Consequently, the plants of the same species may have different concentrations of a certain secondary metabolite, thereby affecting the effectiveness of certain pharmacology activity due to the lack of corresponding bioactive compound ( Verma & Shukla, 2015).

Exploration of the L. microphyllum for the development of new compounds is beneficial for the production of target drugs and is a good solution to overcome problems caused by antibiotic-resistant gram-negative bacteria. Secondary metabolites such as phenolics, alkaloids, tannins and terpenoids which are synthesized by ferns to defend themselves from abiotic and biotic stresses have been responsible to provide a lot of therapeutic effects for clinical use. Therefore, the secondary metabolites have remained as the essential components and are always being explored when developing new drugs.

Antibacterial activity

In the 21st century, the spread of drug-resistant bacteria is a growing worldwide problem. Infectious diseases are difficult to treat because of the antibacterial resistance due to intensive use and misuse of available antibiotics. Common mechanisms of resistant bacteria to survive doses of antibiotics that would previously exhibit bactericidal effects are due to the production of enzymes which inactivate or modify antibiotic, modify the target site and changes in the bacterial cell membrane, thereby preventing the uptake of antibiotic ( Wilson et al., 2002).

Lygodium microphyllum , a fern that has been reported to have therapeutic effects on diarrhoea or dysentery problem; and controlling high fever and typhoid fever ( Benniamin, 2011) will be tested to find the potential constituents that promote antibacterial activity. So far, there is no comparative study specifically done on this fern species.

Extraction and fractionation of crude methanol L. microphyllum extract

Dried powder leaves (200 g) was macerated with 2 L of methanol solvent (1:10, w/v) ( Perumal & Mahmud, 2013) at room temperature (27± 3 °C) for three days. The extract was filtered and the residue solvent eliminated by concentrating it using a rotary evaporator (Rotavapor® R-200, Buchi, Switzerland) under reduced pressure of 40 °C until dryness, which was then oven-dried (40 °C) to afford 58.13 g of dried extract. The crude extract was stored in a refrigerator at 4 °C until further use.

Crude methanol extract was fractionated via liquid-liquid extraction using a separatory funnel. The extracted organic solvents from polar to non-polar solvents were used (hexane, chloroform, ethyl acetate and n-butanol). The extract was dissolved in distilled water first, and then mix with hexane (1: 3, v/v). The mixture was then gently shaken and allowed to settle down overnight until two layers of solution were formed. The hexane layer was identified (supernatant layer), decanted and replaced with fresh hexane. This process was repeated until the hexane layer becomes colourless. The pool of decanted solution was concentrated using a rotary evaporator and dried in the fume cupboard to obtain hexane fraction. The residue (aqueous layer) was further mixed with other extracting solvents and the respective chloroform, ethyl acetate and n- butanol fractions were obtained. Lastly, the water fraction was obtained by concentrating using rotary evaporator and oven-dried (Hot-air drying 80 °C) the aqueous residue ( Ngoc et al., 2015). These fractions were stored in refrigerator at 4 °C prior to further use.

Quantitaive and qualitative phytochemical analysis

The phytochemical compounds of phenolic and flavonoids of crude methanol L. microphyllum extract and its fractions were determined and quantified by standard procedures.

The qualitative analysis of secondary metabolites present in the crude methanol of L. microphyllum extract and its fractions (hexane, chloroform, ethyl acetate, n-butanol and water) were conducted and analyzed following the standard procedures of Edeoga, Okwu, and Mbaebie ( 2005), Chanda, Parekh, and Karathia ( 2006), Onwukaeme, Ikuegbvweha, and Asonye, (2007), Parekh and Chanda (2007) and Kumar et al. (2007) with some modifications.

The stock concentration (1 mg/mL) of all the extracts was prepared and dissolved in methanol prior to use.

Determination of antibacterial activity

Antibacterial activities of crude methanol L. microphyllum extract and its fractions (hexane, chloroform, ethyl acetate, n-butanol and water) were determined by using broth micro-dilution assay according to the protocols described by Wiegand, Hilpert, and Hancock ( 2008), following the guideline of Clinical and Laboratory Standard Institute ( CLSI, 2015). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of L. microphyllum extracts were obtained against resistant clinical isolates of enteric bacteria and the American Type Culture Collection (ATCC) standard bacterial strain.

Quantitative phytochemical analysis

Total phenolic content (TPC) values of the samples (crude methanol L. microphyllum extract and its fractions) were determined by using an equation obtained from the standard gallic acid calibration curve and expressed as mg gallic acid equivalents (GAE)/g samples. The order of the decreasing TPC was as follows : ethyl acetate fraction (11.59 ± 0.59) > chloroform fraction (6.53 ± 0.10) > hexane fraction (5.62 ± 0.09) > crude methanol extract (4.51 ± 0.47) > n-butanol fraction (2.44 ± 0.23) > water fraction (1.15 ± 0.15) (Table 01 ). The TPC value of the ethyl acetate fraction was 2.6 times higher than the crude methanol extract.

Total flavonoid contents (TFC) of the samples (crude methanol L. microphyllum extract and its fractions) were calculated from the equation obtained from the standard quercetin calibration curve and expressed as mg quercetin equivalents (QE)/g samples. The highest content of total flavonoids was found in hexane fraction (60.27 ± 2.03) followed by chloroform fraction (58.37 ± 1.46), ethyl acetate fraction (16.22 ± 1.94), crude methanol extract (15.66 ± 0.23), n-butanol fraction (5.32 ± 0.34) and water fraction (3.29 ± 0.15). The TFC value of hexane fraction was recorded 3.8 higher than the crude methanol extract.

The crude methanol L. microphyllum extract which was fractionated in less polar solvent (n-butanol fraction) and the polar solvent (water fraction) showed the lowest phenolic and flavonoid contents compared to the intermediate polar and non-polar solvents.

In these assays, ethyl acetate fraction (intermediate polarity solvent) and hexane fraction (non-polar solvent) exhibited higher value of TPC and TFC respectively, compared to crude methanol. This may be due to the presence of complex mixtures of secondary metabolites in the crude methanol that interfered and inhibited these activities. As was previously noted by Tiwari, Kumar, Mandeep, Kaur, and Kaur ( 2011), the crude extracts are believed to have complex mixtures of various classes of bioactive compounds and need to subject to further separation to obtain an active portion for bioassay studies.

Qualitative phytochemical analysis

The qualitative analysis of the crude methanol L. microphyllum extract and its fractions showed the presence of secondary metabolite compounds namely alkaloid, cardiac glycosides, flavonoids, quinones, saponin and steroid (Table 02 ). All extracts however showed the absence of anthraquinones. Tannins were only present in the intermediate polarity ethyl acetate fraction. Meanwhile, terpenoid was found in the crude methanol L. microphyllum extract and less polar butanol and the polar water fractions. Terpenoids constitute the largest chemical group found in ferns including mainly triterpenoids, diterpenoids, and sesquiterpenoids having various biological interests. Occasionally terpenoids will be found in the aqueous phase, but they are more often obtained by treatment with less polar solvents ( Tiwari et al., 2011).

The phytochemicals present in the crude methanol L. microphyllum extract and its fractions also play important roles for the evident antibacterial activity; since, phenolic and polyphenols (such as quinones, flavonoids and tannins), terpenoids and alkaloids possess the beneficial mechanism of actions to combat bacteria such as binds to adhesins, complex with cell wall, inactivates enzymes, substrate deprivation and membrane disruption which are responsible to exhibit antibacterial activity ( Tiwari et al., 2011). These findings from quantitative and qualitative phytochemical analysis showed that intermediate polarity ethyl acetate fraction (which exhibited higher value of TPC and present of tannin) and non-polar hexane fraction (which exhibited higher value of TFC) have the potentials to promote antibacterial activity.

Antibacterial activities of crude methanol L. microphyllum extract and its fractions

The results from the in vitro antibacterial susceptibility test revealed that the crude methanol extract of L. microphyllum in non-polar solvent (hexane fraction) and intermediate polar solvent (ethyl acetate fraction) showed strong inhibitory effects (MIC and MBC values of 6.25 mg/mL and 12.5 mg/mL, respectively) compared to the crude methanol extract in less polar and polar (n-butanol and water fractions) solvent systems against all tested gram-negative bacteria including E. coli as standard strain. This could have resulted from the presence of phenolic, flavonoid, alkaloid, glycoside, quinones, saponin and tannins (only presence in ethyl acetate) compounds that are known to possess antibacterial activity ( Cowan, 1996). Thus, all the resistant clinical isolates which consist of enteric bacteria were susceptible to hexane and ethyl acetate fractions.

Crude methanol L. microphyllum extract and its butanol and water fractions (extracted in less polar and the polar solvent system, respectively) displayed weak inhibitory effects against all bacterial isolates with the MIC values varied from 12.5–50 mg/mL (Table 03 ). The crude methanol extract, butanol fraction and water fraction are rich in terpenoid compound which is listed as a useful antibacterial compound. However, these extracts exhibited poor inhibitory effects compared to other fractions. Thus, this clearly showed that the antibacterial activity of the extract did not depend solely on the presence of a certain type of bioactive secondary metabolite. The expression of additive, synergistic or antagonistic effects of various bioactive compounds present in the crude methanol extract, butanol and water fractions may affect the bacterial growth inhibitory activity.

Tetracycline, an inhibitor of bacterial protein synthesis was found to possess the strongest inhibitory activity against all tested gram-negative bacteria.

Standard isolate Escherichia coli (ATCC 25922) was used in order to verify the accuracy of the susceptibility results as recommended by Wiegand et al. (2008). Results are acceptable when the test values of MIC for control strains are within the suggested MICs range of antibiotics used. The MIC for routinely used tetracycline for quality control strain of Escherichia coli (ATCC 25922) was based on CLSI ( 2015) whereby the test value for this strain must be within 0.5 x 10 ^-3 to 2.0 x 10 ^-3 mg/mL. Hence, the MIC value of the studied Escherichia coli (ATCC 25922) was 1.0 x 10 ^-3 mg/mL. Therefore, the results from this finding are acceptable. Furthermore, the susceptibility results of 5 isolates of clinically resistant enteric bacteria when tetracycline was used in this study were also acceptable since the MIC values of these bacterial isolates conformed with the suggested range for MIC determination of Enterobacteriaceae which is less than or equal to 4.0 x 10 ^-3 mg/mL.

Findings from MBC determinations suggested that the hexane, chloroform, ethyl acetate and butanol fractions showed bactericidal effect; whilst the crude methanol of L.microphyllum extract and water fraction showed a bacteriostatic effect against tested enteric bacteria (Table 04 ).

The presence of secondary metabolite compounds in L.microphyllum were associated with the antibacterial effect. However, the antibacterial activity did not only depend on a single compound but a combination of different bioactive compounds may produce better expression of bacterial inhibition activity.

Thus, the present study confirmed the antibacterial activity of L.microphylum extracts, particularly hexane and ethyl acetate fractions which distinctly showed antibacterial activities against tested clinically resistant enteric bacteria.