Anti-adhesion potential of non-polar compounds and extracts from Ficus natalensis

Abstract

Four triterpenoids, ergosta-4,6,8(14),22-tetraene-3-one 1, stigma-4-ene-3-one 2, 3β-hydroxy-21β-H-hop-22(29)-ene 3, sitosterol and a quinone, tectoquinone 4, were isolated from the leaf, stem bark and fruit extracts of Ficus natalensis subsp. natalensis, Moraceae, a medicinal fig found in Africa. The pure compounds 1–4 and crude extracts were tested for their antibacterial activity against five Gram-negative and seven Gram-positive strains and for their potential anti-biofilm activity. Antimicrobial susceptibility was observed with all pure compounds tested at 250 µg against the majority of Gram-negative and Gram-positive strains. The dichloromethane-soluble fruit extract was active against sensitive and resistant Staphylococcus aureus strains, Enterococcus faecalis and Staphylococcus xylosus. Compounds 2, 3 and 4 demonstrated broad-spectrum antibiotic effects against eight of the twelve bacterial strains tested. In the anti-biofilm assay, exposure to ethyl acetate, methanol and aqueous methanol leaf, stem bark and fruit extracts decreased adhesion with a biofilm reduction of ≥100% for all three tested organisms: Escherichia coli, Pseudomonas aeruginosa and S. aureus. The methanol leaf extract demonstrated the most potent anti-adhesion potential against E. coli (218% biofilm reduction). The greatest ability to decrease adhesion was observed with compounds 2, 3 and 5 against P. aeruginosa at the lowest concentration tested (100 µg ml⁻¹).

Keywords
Ficus natalensis subsp. natalensis; Fig; Bacterial adhesion; Biofilm reduction

Introduction

Adhesion of microorganisms to diverse surfaces and to other microbes is essential for biofilm development and establishment of infections. When bacteria colonise and adhere to surfaces, they are associated with severe, recurrent infections and become 10–1000 times more resistant to antimicrobial agents (Borges et al., 2015). Pharmaceuticals or pharmaceutical extracts capable of preventing bacterial adhesion to surfaces are therefore desirable antibacterial agents. One source of these are medicinal plants used in traditional medicine, since many plant-based remedies are known to have therapeutic antimicrobial activity against clinical pathogens and an increasing number have even been identified as having anti-adhesion properties (Borges et al., 2016).

Ficus natalensis Hochst. subsp. natalensis, Moraceae, is a rock splitter tree which grows up to almost 20 m in height. It is geographically situated in South Africa, Zimbabwe, Zambia, Malawi, Mozambique and Kenya. The different parts of the tree have various applications varying from cultural, decorative, and commercial applications to folk medicine, is a major source of bark cloth and a potential natural dye (Burrows and Burrows, 2003). The root, bark, leaves and latex are used in several African countries to treat a variety of medical ailments including malaria, influenza, whooping cough, dysentery and guinea worm (Hutchings et al., 1996; Burrows and Burrows, 2003).

In previous studies on the plant, antibacterial activity has been reported for the methanol extract of the root (Rabe and van Staden, 1997). Apart from this, Olaokun et al. (2013) also reported the anti-diabetic potential of the acetone extract of the leaves.

Materials and methods

The fruits, leaves and stem bark from Ficus natalensis Hochst. subsp. natalensis, Moraceae, were collected in Durban, KwaZulu-Natal, South Africa in December 2012. The plant was identified at the Ward herbarium of the University of KwaZulu-Natal, where a voucher specimen was deposited under the collector number G.V. Awolola & H. Baijnath 3. The ground leaves, stem bark and fruits were extracted twice sequentially with organic solvents of increasing polarity, hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH). Details of the extraction are given in the supplementary data.

The crude extracts were subjected to vacuum column chromatography (CC) on silica gel using a Hex-DCM-EtOAc-MeOH gradient elution, to afford five major fractions (A–E) by TLC. Compound 3 (250.6 mg), identified as 3β-hydroxy-21β-H-hop-22(29)-ene crystallised out of fraction A of the DCM and Hex crude extract of the leaves. Fraction B of the same crude extracts contained 1 ergosta-4,6,8(14),22-tetraene-3-one (140.5 mg). Sitosterol (80.6 mg) was isolated from fraction B of the EtOAc fraction of the leaves. Compound 4, tectoquinone (70.8 mg) was obtained by purification of fraction B of the Hex and DCM crude extracts of the stem bark. Stigma-4-ene-3-one (2) (70.4 mg) was obtained from fraction C of the same extracts. All compounds were isolated and purified by silica gel CC using various ratios of Hex:EtOAc and identified by their ¹H and ¹³C NMR data and verified by data in the literature, 1 (Fangkrathok et al., 2013), 2 (Liu et al., 2014), 3 (Sousa et al., 2012), sitosterol (Rasoanaivo et al., 2014) and 4 (Cheng et al., 2008).

Antibacterial susceptibility testing (AST) was carried out in duplicate on 12 bacterial indicator strains (given in the supplementary material) using the disc diffusion method according to standard procedures (CLSI, 2007). Bacterial adhesion on three bacterial strains: Escherichia coli ATCC 35218, Staphylococcus aureus ATCC 43300 and Pseudomonas aeruginosa ATCC 27853 based on the results from the AST according to the crystal violet microtitre plate assay were carried out in triplicate (Basson et al., 2008). Details of the assays are given in the supplementary data.

Results and discussion

The fractionation and purification of the leaf, stem bark and fruit extracts led to the isolation and identification of five non-polar compounds. To the best of our knowledge, this is the first report of secondary metabolites from F. natalensis subsp. natalensis.

The crude extracts of the different organs of F. natalensis were tested along with compounds 1–4 against the bacterial strains to determine whether they had any antibacterial activity to substantiate the reported use of the plant as an antibacterial agent (Rabe and van Staden, 1997). There has been extensive antibacterial studies on the ubiquitous sitosterol and therefore its antibacterial properties was not tested again.

The DCM extract of the fruit was the most active of all extracts demonstrating activity against several of the Gram-positive strains. The antibacterial activity of all extracts are reported in the supplementary material. Rabe and van Staden (1997) have previously observed MIC of 4 mg ml⁻¹ for S. aureus and Staphylococcus epidermidis and 8 mg ml⁻¹ for Bacillus subtilis, using methanolic root extracts of F. natalensis.

Varying antibacterial activity was demonstrated by the isolated compounds, which ranged from resistant to susceptible against both Gram-negative and Gram-positive organisms (Table 1). The antibacterial activity was dose-dependent, being more active at 250 µg than at 100 µg. Compounds 1–4 showed good activity against β-lactam resistant E. coli ATCC 35218 and the extended spectrum β-lactamase producing Klebsiella pneumoniae ATCC 700603 but was only moderately active against P. aeruginosa. Amongst the Gram-positive bacteria, the best activity was seen by the methicillin-resistant S. aureus (MRSA) strain ATCC 43300 by all four compounds following 250 µg exposure (Table 1). B. subtilis and Enterococcus faecalis strains demonstrated varying levels of susceptibility to all four compounds. All compounds, 1–4, showed broad-spectrum antibiotic effects with as much as 10 of the 12 indicator bacterial strains being susceptible to them. Tectoquinone 4 showed high zones of inhibition of 25 mm and 23 mm against Staphylococcus sciuri and K. pneumoniae, respectively and stigma-4-ene-3-one 2 at 250 µg demonstrated a high zone of inhibition against Staphylococcus xylosus of 23 mm.

Table 1
Antibacterial activity of isolated compounds (zones of inhibition in mm) from Ficus natalensis subsp. natalensis.

Compound

Zones of inhibition (mm)

Gram-negative bacteria

E. coli ATCC 25922

E . coli ATCC 35218

P . aeruginosa ATCC 27853

P . aeruginosa ATCC 35032

K. pneumoniae ATCC 700603

100 µg

250 µg

100 µg

250 µg

100 µg

250 µg

100 µg

250 µg

100 µg

250 µg

1

10R

20S

9R

15S

7R

15S

9R

15S

9R

19S

2

10R

18S

9R

16S

0R

15S

10R

14I

9R

17S

3

0R

16S

0R

15S

0R

141

0R

18S

7R

16S

5

9R

18S

10R

20S

8R

13I

10R

15S

0R

23S

AMP10

22S

0R

0R

0R

0R

TE20

28S

23S

15S

14R

12R

Compound

Zones of inhibition (mm)

Gram-positive bacteria

B. subtilis ATCC 6633

E. faecalis ATCC 29212

E. faecalis ATCC 51299

S. aureus ATCC 29213

S. aureus ATCC 43300

S. sciuri ATCC 29062

S. xylosus ATCC 35033

100 µg

250 µg

100 µg

250 µg

100 µg

250 µg

100 µg

250 µg

100 µg

250 µg

100 µg

250 µg

100 µg

250 µg

1

0R

20S

0R

13I

0R

13I

9R

16S

9R

15S

9R

15S

11I

16S

2

15S

18S

0R

14I

0R

16S

10R

18S

8R

22S

10R

19S

0R

23S

3

0R

0R

0R

17S

0R

16S

7R

20S

7R

19S

0R

19S

0R

17S

5

0R

20S

0R

16S

0R

111

9R

17S

0R

19S

9R

25S

16S

18S

AMP10

40S

nt

25S

25S

20S

34S

0R

TE20

36S

nt

0R

28S

36S

25S

26S

• R, resistant; S, susceptible; I, intermediate; nt, not tested; AMP10, ampicillin (10 µg); TE20, tetracycline (20 µg).

Biofilms in body tissues can be formed by bacteria such as Acinetobacter baumannii, E. coli, P. aeruginosa, and S. aureus (Borges et al., 2016). Decreased adhesion was observed for all EtOAc, MeOH and aq/MeOH leaf, stem bark and fruit extracts against three resistant bacterial strains, E. coli ATCC 35218, P. aeruginosa ATCC 27853 and S. aureus ATCC 43300 (see supplementary data).

The MeOH leaf extract displayed the greatest anti-adhesion potential against E. coli, while majority of the extracts, with the exception of the DCM and Hex extract (leaves and fruit) showed reduced biofilm formation by P. aeruginosa. For S. aureus, the aq/MeOH leaf, EtOAc stem bark and fruit and MeOH stem bark extracts reduced biofilm formation. All the polar extracts decreased adhesion suggesting interference by the extracts on the ability of these microorganisms to adhere to polystyrene surfaces. There are varying reports on anti-adhesion effects of polar extracts against different bacterial strains in the literature, which corroborate our findings. The anti-adhesion effects of polar extracts have been reported for E. coli and Bacillus cereus (Bräunlich et al., 2013), oral bacteria Streptococcus mutans and Streptococcus sobrinus (Rahim and Khan, 2006), both S. aureus and MRSA (Chusri et al., 2012) and P. aeruginosa (Sarkar et al., 2014).

The activity of the isolated compounds with regard to bacterial adhesion was variable. All four compounds increased adhesion of E. coli ATCC 35218 at all concentrations (Table 2). Stigmast-4-en-one (2) decreased adhesion of P. aeruginosa ATCC 27853 at all concentrations, with 100 µg ml⁻¹ being the most effective. Ergost-4,6,8(14),22-tetraen-3-one (1) increased adhesion at 100 µg ml⁻¹, but decreased adhesion at inhibitory and supra-inhibitory concentrations. For 3 and 4, decreased adhesion was only observed at sub-inhibitory (100 µg ml⁻¹) concentrations, while increased adhesion was observed at inhibitory and supra-inhibitory concentrations. Increased adhesion of S. aureus ATCC 43300 was observed with all four compounds at all concentrations tested (Table 2).

Table 2
Percentage biofilm reduction following exposure to 100-500 µg ml^-1 of four compounds isolated from F. natalensis subsp. Natalensis.

Compound	Percent biofilm reduction (%)a
	E. coli ATCC 35218				P. aeruginosa ATCC 27853				S. aureus ATCC 43300
	100	250 µg ml-1	500		100	250 µg ml-1	500		100	250 µg ml-1	500
1	-315.62	-1456.10	-1176.99		-64.04	28.16	18.05		-445.34	-338.40	-437.78
2	-783.27	-1168.21	-1151.57		59.65	8.59	19.45		-1.81	-330.97	-381.66
3	-149.72	-1346.58	-1071.16		39.07	-26.45	-38.54		-54.09	-302.98	-474.39
5	-593.81	-1220.89	-1189.00		54.69	-6.49	-48.49		-51.30	-395.19	-416.02

• ^a
  Biofilm reduction calculated according to Pitts et al. (2003). Negative values are indicative of an increase in attachment/biofilm formation.

The variable activity observed with the isolated compounds was in accordance with the effects of oleanolic acid and β-amyrin acetate from Vernonia auriculifera against biofilm formation by seven different bacterial strains (Kiplimo et al., 2011). Strain-specific effects have been observed with the triterpenes ursolic and betulinic acids, from the liverwort Lepidozia chordulifera which inhibited biofilm formation and elastolytic activity of P. aeruginosa ATCC 27853, but increased adhesion of S. aureus ATCC 6538 (Gilabert et al., 2015). The increased adhesion being observed with some of the isolated compounds may be due to promotion of microbial adhesion by providing a suitable conditioning film for enhancement of cell attachment (Selim et al., 2014).

The crude extracts did not demonstrate a significant antibacterial effect. It is, however, quite possible that the compounds isolated from F. natalensis subsp. natalensis in this work could be responsible for the antibacterial activity experienced by those who use the plant for medicinal purposes since most of them have demonstrated promising antibacterial activity. Decreased adhesion with >100% biofilm reduction was demonstrated by all the crude polar extracts against resistant bacterial strains. The isolated compounds exhibited strain-specific anti-adhesion potential, with biofilm reduction against P. aeruginosa, but not E. coli or S. aureus.

Ethical disclosures

Protection of human and animal subjects

The authors declare that no experiments were performed on humans or animals for this study.

Confidentiality of data

The authors declare that they have followed the protocols of their work center on the publication of patient data.

Right to privacy and informed consent

The authors declare that no patient data appear in this article.

Acknowledgements

The authors are thankful to the Research Office, University of KwaZulu-Natal (UKZN), Westville Campus, Durban, South Africa for a doctoral research grant. GVA was awarded a PhD study fellowship (study leave) by the University of Ilorin, Kwara State, Nigeria.

Appendix A Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi: 10.1016/j.bjp.2017.07.004.

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Publication Dates

• Publication in this collection
  Sep-Oct 2017

History

• Received
  14 Apr 2017
• Accepted
  5 July 2017