New Pharmacophore from the Stem Bark Fractions of Acacia decurrens ( Willd ) , an Invasive South Africa Tree

The tolerance of Acacia decurrens, an invasive species, was exploited pharmacologically in this study. Phytochemical screening revealed important secondary metabolites. Importantly, the assay shows that ethyl acetate and methanol fractions are sources of phytochemicals compared to the hexane and chloroform fractions. A bioassay-guided in vitro assay of the extracts led to the eventual isolation of four bioactive compounds by column chromatography, identification, and characterisation with the aid of GCMS, UV-Vis, FTIR, and NMR. The antimicrobial screening by disc diffusion assay revealed 22.2%, 44.4%, 66.7%, and 77.8% microbial inhibition by 2-methyl-octahydro-indene-4-carboxylic acid (AD1), 6-methyldecahydro-1H-phenanthren-9one (AD2), 8-hydroxytetradecahydro-chrysene-1-carb aldehyde (AD3), and 8,9-dihydroxy-7-(2-hydroxy-ethyl)-9,9a-hexahydro1H,3H-2-thia-5a-aza cyclopenta[b]anthracen-6-one (AD4), respectively. Compounds AD3 and AD4 are the most potent antibacterial compounds against Gram-positive bacteria with MIC 12.5–6.25 μg/ml. Antioxidant study of the compounds assayed with DPPH and ABTS revealed that compound (AD4) is the most efficient DPPH radical scavenger with IC50 30.07 ± 0.31 and ABTS scavenging activity of 4363.2 ± 452.4 μmol of TE/gDW.This provides scientific information on four pharmacophores with phyto-antioxidants and antimicrobial potential, despite the classification of A. decurrens as a Category 2 invasive plant by the National Water Act.


Introduction
Invasive species are flora introduced into a foreign environment from their native host region and with significant consequences on the available resources in the host region [1].The chances of an invasive species establishing dominance in a new environment are minuscule, but research has proven that once they do, the economic damage can be enormous [2].Literature has shown that invasive species are responsible for threat on indigenous species, although some other biotic and abiotic factors might contribute [3].
Acacia (wattle) is native shrubs and trees to Australia, from the subfamily of Mimosoideae of the family Fabaceae, due to its prolific nature being found in all terrestrial habitats [4].They are classified as a Category 2 invasive plant by the Act on Alien and Invasive Species Regulations of South Africa [5]; such exempted flora does require a permit to be introduced to the environment [6].
Indigenous wildlife may not have the potentials of combating or competing against invasive species that has no predators on the food chain within the new environment [7]; however, this phytotolerance can be exploited for its pharmacological activity against infections of the region.The plant parts have various applications in its native country Australia: the flowers are edible and the seed pods are used for the production of green dye, being grown for firewood or as a fast-growing windbreak or shelter tree.The gum from the trunk is used as a low-quality gum arabic [8].

Experimental
2.1.Chemicals.The reagents and chemicals used in this study are from Sigma-Aldrich Chemicals Co., St. Louis, MO, USA, and are of analytical grade.

Collection and Extraction of A. decurrens Stem Bark.
The fresh stem bark of A. decurrens was harvested around October 2016 near Vaal Dam Road, Heidelberg (26.5033 ∘ S, 28.4397 ∘ E), South Africa, diced, and dried at an ambient temperature at a relatively low humidity.Authentication of the of A. decurrens stem bark was carried out by the South Africa National Biodiversity Institute, Pretoria, and voucher specimen number: 1200-1, and was deposited at Pretoria National Botanical Garden.The extraction was performed by serial maceration using 4 L of each solvent: hexane, chloroform, ethyl acetate, and methanol with slight agitation at 111 revs/min for seven days.The solvents were removed using a rotary evaporator and the yield of the extracts was determined.

Qualitative and Quantitative Phytochemical Screening.
The fractions were analyzed for the presence of secondary metabolites using standard procedures.The total phenolic content (TPC) was determined, and the results were expressed in mg of gallic acid per g of the sample [12].The total flavonoids (TFC) were measured and expressed as mg of rutin equivalents per g of the sample [13].The tannin content (TC) measured and expressed as mg of tannin per g of the sample [14], and also the alkaloid, saponins, and terpenoids contents were determined and expressed in percentage [15][16][17].

Fractionation and Isolation of Compounds.
The fractionation of the crude extracts was conducted on a Si-gel column of 640 mm by 60 mm with various solvent systems.The obtained bioactive fractions of the extracts were further subjected to subfractionation on a Si-gel column of 330 mm by 30 mm for isolation.Figure 1 is a summary of the fractionation, isolation, and purification of bioactive compounds from the extracts.

GC-MS Analysis of Compounds.
The first step involves the solubilization of compounds AD1 and AD2 in chloroform and AD3 and AD4 in acetone prior to introduction into the GC injection port.The retention time and molecular ion determination were carried out on Clarus 500 PerkinElmer Gas Chromatograph equipped with an Elite-5 (100% dimethylpolysiloxane) column coupled to a mass spectrometer detector.The initial column temperature is set at 110 ∘ C and held for 2 min and the oven temperature increased at a rate of 5 ∘ C/min, to 230 ∘ C, and held for 9 min.The helium (He) flow rate was maintained at 1 ml/min, while keeping the injection port temperature at 250 ∘ C. The injection of the compounds was by split mode method of 10 : 1 with a mass scan range of 45-450 (/).

Spectroscopic Analysis of Compounds.
Spectroscopic studies were carried out on the compounds by scanning the UV-Vis region using PerkinElmer Spectrophotometer and identification of functional groups by using VERTEX 80 FTIR Spectrophotometer and the 1D and 2D; the characterization was carried out on Agilent VnmrJ3 Spectrometer operating at 400 MHz.

Inoculums and Inoculation
Procedure.Stock culture test organisms maintained at 4 ∘ C on slants of nutrient agar were obtained from the Biotechnology Department, Vaal University of Technology, South Africa.The growth method was employed in the standardisation of the inoculum density to achieve a concentration of 1.5 × 10 8 CFU/ml.The agar plates were cultured from colonies in a Mueller-Hinton broth and incubated at 37 ∘ C to 0.5 McFarland standard [18].Furthermore, the inoculum suspension was used within a quarter of an hour to avoid any change in the size or loss of viability [19].A uniform streaking of the dry Mueller-Hinton agar plate surface was conducted twice with standardised bacterial inoculum suspension.

Preparation of Compound Impregnated Discs.
Sensitivity discs of 6 mm diameter were immersed in stock solutions of each compound prepared by dissolving 50,000 g of the compound in 5 ml of dimethyl sulfoxide [20].The disc was sterilised by autoclaving at 121 ∘ C for 15 minutes and impregnated with 10 mg/ml of each compound.Dimethyl sulfoxide-loaded discs (negative controls), ampicillin (positive controls), and compound impregnated discs were dried in an incubator at 45 ∘ C for 24 h before the application on the bacterial lawn [21].The disc diffusion method was carried out to investigate the in vitro sensitivity of Micrococcus luteus (ATCC 26883), Staphylococcus aureus (ATCC 25923), Escherichia coli (NCTC 11954), Salmonella typhi (ATCC 29692), Klebsiella pneumonia (BAA 1706), Shigella sonnei (ATCC 25931), Staphylococcus epidermis (ATCC 12228), Listeria monocytogenes (ATCC(R) BAA-751TM), and Enterococcus faecalis (ATCC 22735) against the compounds.The analysis was performed in triplicate.the minimum inhibitory concentration by broth microdilution method [22].The 12 wells of each row were filled with 0.5 ml sterilised Mueller-Hinton agar.To wells 3-12 sequentially, an additional 0.5 ml of a mixture of agar and compounds was serially diluted to create a concentration of 100-0.3906g/mL.The first well served as growth control and well 2 as an antibiotic control (ampicillin) with MIC of 3.125 g/mL.The incubation of the deep wells was for 24 h at 37 ∘ C and turbidity was measured after 24 h at 600 nm using a PerkinElmer UV-Vis Spectrophotometer to determine the MIC.These tests were performed in triplicate.

Total Antioxidant Activity by ABTS •+ Decolourization
Assay.In the method of Siddhuraju and Manian [24], the total antioxidant activity of the compounds was measured by the decolourization of ABTS •+ .Exactly 7 mM of 2,2  -azinobis(3-ethylbenzothiazoline-6-sulfonic acid and 2.45 mM of potassium persulfate were prepared in distilled water, and the mixture was allowed to stand in the dark at ambient temperature for 24 h to generate the ABTS •+ .After incubation for 10 h at 28 ∘ C in the dark, the bluish green ABTS •+ colour was produced.However, the ABTS cation radical solution was diluted with distilled H 2 O to an absorbance of 1.00 at 734 nm.The 3 mL of the generated ABTS •+ solution was mixed with 30 L of the compounds and Trolox (control).The antioxidant potential was expressed as the concentration of Trolox (mol/g), having the equivalent antioxidant activity of the compounds on dry weight basis.
The absorbance was read at 734 nm exactly after 30 min.

Influence of Menstruum on the Recovery Yield of Compounds.
The fractions obtained were different in colours and nature and the percentage yields were recorded.The methanolic fraction has a higher yield compared to the yield of chloroform, ethyl acetate, and hexane (Table 1).This result implies that most of the secondary metabolites are hydrophilic in nature and consistent with the literature report which confirms methanol as the most suitable solvent for extraction of the polar secondary metabolite [25].There has been a report that polarity significantly affects the recovery yields of the secondary metabolites from the plant [26].

Qualitative and Quantitative Phytochemical Screening.
The phytochemical screening of the fractions reveals the presence of terpenoids, phenols, tannins, flavonoids, saponins, and alkaloids.All the menstruum contains terpenoids and tannins; however, the saponins were found only in the methanolic fraction (Table 2).
The methanolic and ethyl acetate fractions contain about 85.7% of the tested secondary metabolites with a high degree of precipitation, while hexane menstruum contains moderately 57.1%.The secondary metabolites identified have established pharmacological activities consequentially [27,28], indicating that the stem bark contains health-enhancing metabolites, other than growing invasive classification [29].
The alkaloid contents of the chloroform are higher than observed in hexane fraction (Figure 2(a)).However, the % steroids found in the hexane fraction is greater than chloroform, ethyl acetate, and methanol fractions ( According to the literature, alkaloids, saponins, and steroids are potent antimicrobial compounds because of their ability to prevent the growth of a microorganism, activating  perturbations of the membrane [30] and interfering with gene expression pathways and metabolic processes [31]. The total phenolic contents of the chloroform fraction are higher than those observed in ethyl acetate and methanol fractions, calculated from the calibration curve ( 2 = 0.9903) (Figure 3(a)).The total flavonoid from the calibration curve ( 2 = 0.9986) is slightly higher in the methanol than ethyl acetate fraction (Figure 3(b)).However, the calibration curve ( 2 = 0.9921) shows that the tannins are richer in solvents with polarity index 4.4-5.1,an indication of a highly polar class of tannins (Figure 3(c)).Reports have shown that phenols, flavonoids, and tannins have redox properties, which allow them to act as phytoantioxidants [32].A significant rise in the level of the tannin and flavonoids extracted was observed as polarity changes with solvent.The observation above is because the tannins and flavonoids are water-soluble polyphenol class of compounds [33], and simple skeleton phenolic rings are connected by a propionic chain [34].The HSQC spectrum shows a correlation between the protons at 2.15 (2H, m, 2-H) and 1.74 (1H, m, 10-H) and the methylene carbon at 32.74 (C-7) and 31.93 (C-6) ppm (see Supporting File 7).COSY data reveals a correlation between the protons at 2.15 (2H, m, 2-H) and 1.74 (1H, m, 10-H) ppm to the protons at 1.41 (1H, m, 6-H) and 1.24 (2H, m, 2-H) ppm (see Supporting File 8).

Characterization of 2-Methyl-octahydro-indene-4-carboxylic acid (AD1
The HMBC shows that the proton at 11.312 (1H, s, OH) ppm is two bonds away from the carbon at 178.14 (C-1) ppm and three bonds away from the carbon at 37.09 ppm.Another notable correlation is between methine proton at 2.15 (2H, m, 2-H) ppm and the carbons at 30.87 (C-10) and 26.89 (C-3) ppm which are two bonds away (see Supporting File 9).The above spectroscopic data confirms structure as AD (Scheme 1) with a molecular formula of C 11 H 18 O 2 .
The IR stretching vibrations at 2954.72(w), 2920.47(s), and 2851.24(s) are the sp 3 C-H of alkane, and C=O of a ketone at 1723(s), 1640(m), and 1650(m) is sp 2 C=C of an alkene.The bending vibrations of alkane sp 2 and sp 3 C-H are at 1462.67(m) cm −1 and 1377.32(m)cm −1 , respectively (see Supporting File 11).The HSQC shows a correlation between the proton at  3.47 (2H, d, CH 2 ) ppm and the carbon peak at 39.01 (C-14) ppm (see Supporting File 14).The 1H-1H COSY spectrum shows that the methylene proton at 3.47 (2H, d, CH 2 ) and the methine proton at 1.71 (1H, m, CH) ppm are in the same chemical environment (see Supporting File 15).
The GCMS analysis revealed a retention time of 15.98 min (see Supporting File 17) and a molecular ion peak at 218 (see Supporting File 18) which is in agreement with the molecular formula of C 15 H 22 O.The molecular structure of 6-methyldecahydro-1H-phenanthren-9-one based on the spectroscopic information above is deduced to be AD (Scheme 2).The IR spectrum of 8-hydroxytetradecahydro-chrysene-1-carbaldehyde reveals the presence of -OH stretching vibration of alcohol at 3274 cm −1 , C-H of alkane 2968, 2945, 2919, and 2853 cm −1 the =C-H of aldehyde at 2869 cm −1 , C=O of aldehyde at 1735 cm −1 , and two peaks of C-O of alcohol at 1190 and 1097 cm −1 .The bending vibration at 1464 cm −1 is the C-H of alkane and 1386 cm −1 of C-H of alkane (see Supporting File 22).
The COSY spectrum indicates that the olefinic protons at 5.12 (1H, t, 12-H) and 5.20 (1H, d, 19-H) ppm show correlation to the methine protons at 1.98 and 1.94 ppm.There is a correlation between the oxymethine proton at 3.20 (1H, m, 3-H) ppm and the protons at 1.53 and 1.52 ppm (see Supporting
The COSY spectrum indicates that the aromatic protons at 6.04 (1H, s, 2-H) and 6.04(1H, s, 13-H) ppm are three bonds away from the thiomethylene protons at 3.73 (1H, dd, 9-H) and 3.70 (2H, s, 15-H) ppm (see Supporting File   strains, including six Gram-positive and three Gram-negative strains.The compounds AD( -) have little inhibitory activity against Gram-negative strains, but the results on the six Gram-positive strains were significant.Compounds AD , AD , and AD were the most potent antimicrobial compounds inhibiting 44.4%, 66.7%, and 77.8% of the tested microorganisms, respectively (Table 3 and Figure 4).On the other hand, compound AD has the lowest bacterial inhibition at 22.2% compared to the positive control and the other compounds.
Compound AD indicated the highest antimicrobial activity due to the presence of functional groups such as the thiol, hydroxyl, and amide.However, E. coli and S. sonnei were predominantly resistance, due to the production of lactamases as expected of Gram-negative organisms [35]; similar strength was observed for compounds AD2 and AD3.
For -lactams to reach the binding proteins located in the inner membrane of the organism specific functional group is required.In this study, the bacterial cell production of lactamases was in insufficient concentration to tip the kinetics in favour of the destruction of compounds AD , AD , and AD .Compound AD clearly was ineffective due to the absence of the primary functional groups by decreasing penetration or actively extruding the antimicrobial compound [36].The breaching of the intact cell walls and membranes of the peptidoglycan and cytoplasmic layer of M. luteus, E. faecalis, and L. monocytogenes is also due to the type of functional groups on compounds AD2, AD , and AD .They induce cells to lyse and cause breakage of walls and membranes.M. luteus and L. monocytogenes were sensitive to all tested compounds despite the fact that these organisms survive oligotrophic environments for extended periods of time [37], confirming the possible use of the compounds for the treatment of opportunistic infection caused by M. luteus and L. monocytogenes in immunosuppressed patients.
The ascorbic acid used as a control in this study readily forms dehydroascorbate which does not possess radical scavenging potentials, but it easily reverts to ascorbate radical by donating an electron to the radical [40].The radical anions on the oligonucleotides DNA are always prone to the attack of polyhydroxy compounds such as quercetin, and this explains the observed activities of compounds AD and AD .The comparable scavenging activity of compounds AD due to the presence of the polyhydroxy groups in the neighbourhood of the aromatic ring is enhanced by the inductive effect of neighbouring groups, consequently, having the ease at which the protons are donated compared to

Conclusion
The findings from this study confirm ethyl acetate and methanol as the most efficient solvent for extracting potent phytoantioxidant and antimicrobial metabolites.

Figure 1 :
Figure 1: A brief summary of the general fractionation, isolation, and purification of bioactive compounds from the extracts.
Figure 2(c)).High alkaloid and steroid contents in solvents of polarity index of 0.1-4.1 are an indication that they are primarily nonpolar, unlike the saponins which are richer in ethyl acetate compared to the methanol fraction (Figure 2(b)).

3. 7 .
Antimicrobial Potential of Compounds AD1-AD4.The inhibition effect of the compounds is tested on nine bacterial

Figure 4 :
Figure 4: Antibacterial activity of the bioactive compounds.
). Microorganism sensitive compounds were screened to determine

Table 1 :
Nature of the extracts and recovery yield/g of A. decurrens stem bark.

Table 2 :
Qualitative screening of the stem bark of A. decurrens of extracts.

Table 3 :
Sensitivity of the bioactive compounds against the test organisms.

Table 4 :
Minimum inhibitory concentration (g/ml) of the bioactive compounds.

Table 5 :
[41]cal scavenging activity of the compounds from the stem bark of A. decurrens.-carbaldehyde(AD) being a tetracyclic steroid has an equally likely activity due to the long carbon chains, imposing a lipophilic property which plays a significant role in the radical scavenging activity[41].2-Methyl-octahydroindene-4-carboxylic acid (AD ) and 6-methyldecahydro-1H-phenanthren-9-one (AD ) are weak phytoantioxidant compounds due to the absence of electron donating and polyhydroxy groups on the structure.