A new antibacterial lupane ester from the seeds of Acokanthera oppositifolia Lam.

Abstract As a part of ongoing investigation of Acokanthera oppositifolia (Lam.) Codd., four compounds were isolated from its seeds, a new compound; lup-20(29)-en-3β-O-(3′-β-hydroxy) palmitate (1), three known compounds; a triterpene; lupeol (2), a cardiac glycoside; acovenoside A (3) and a sterol; β-sitosterol (4). Their structures were investigated using 1D & 2D- 1H and 13CNMR spectroscopy. Antimicrobial potential of the compounds was evaluated against 10 microorganisms responsible for endocarditis. The minimum inhibitory concentration (MIC) of the compounds was determined using broth microdilution method. The new compound (1) evidenced significant antibacterial activity especially aganist Pseudomonas aeruginosa with (MIC 7.81 μg/ml). Lupeol (2) exhibited remarkable antimicrobial activity against Methicillin-resistant Staphylococcus aureus, Aspergillus fumigates and Candida albicans (MIC 3.9, 0.24 and 3.9 μg/ml, respectively). On the other hand, acovenoside A (3) inhibited the growth of Escherichia coli (MIC 0.98 μg/ml). We herein present the potential of A. oppositifolia as a cardioprotective agent against the microorganisms responsible for endocarditis.

including dental work, surgery, urinary infections, infected cuts and bad throat infections (Zadik et al. 2008). Many microorganisms can cause infective endocarditis (IE) (Morris 2006). Staphylococcus aureus followed by Streptococci of the viridans group and coagulase-negative Staphylococcii together with Enterococci are the most common organisms responsible for IE (Murdoch et al. 2009). Viridians alpha-hemolytic Streptococci that are present in the mouth are the most frequently isolated micro-organisms, especially when the infection is acquired in a community setting. On the other hand, Staphylococcus can cause IE when it enters blood stream, these infections are frequently acquired in a health care setting and through procedures that involve breaking in the integrity of skin, for example in case of surgery, catheterisation or secondary to intravenous injection of recreational drugs (Elad et al. 2011).
In addition to bacteria, some fungi, such as Candida albicans, are associated with endocarditis especially in I.V drug users and immunocompromised patients. Other fungi demonstrated to cause endocarditis are Histoplasma capsulatum and Aspergillus fumigates (Lamas & Eykyn 2003).
Resistance to methicillin is now widely described in the community setting. The prevalence of healthcare-associated methicillin resistance strains of S. aureus (MRSA) continues to increase, and therefore, the development of new drugs or alternative therapies are urgently necessary. Human beings have used plants for the treatment of diverse ailments for thousands of years, and therefore, considerable effort is taken to discover plant-derived antibacterial agents that can defeat MRSA, especially those strains which have developed resistance to most existing antibiotics, including the last line of defence, vancomycin (Chung et al. 2011).
Acokanthera oppositifolia is evergreen shrubs or small trees used as the source of an arrow poison (Van Wyk et al. 2002). Numerous cardenolides were detected in the wood, leaves, seeds and fruits of A. oppositifolia (Van Wyk et al. 2000), among which acovenosides A, B and C, were the most important members as they have cardio-vascular effect (De Villers 1962). Pentacyclic triterpenes have been also isolated from various parts of Acokanthera (Karawya et al. 1974). The leaves of A. oppositifolia were reported as potential source of antibacterial and anticancer agents (Chaurasia & Sharma 2015). Pentacyclic triterpeoids, a biologically diverse plant-derived natural products, have been reported to show anti staphylococcus activities (Patocka 2003). Antifungal activity of a cardinolide genin has been reported against soil born and post harvest fungi (Abbassy et al. 2012).
The main objective of the present study is to isolate the major phytochemicals of the seeds of A. oppostifolia and to evaluate their possible antimicrobial (antibacterial and antifungal) potentiality especially on microorganisms that can cause infective endocarditis.

Results and discussion
The core principle in the treatment of endocarditis is optimal care of arrhythmia and of heart failure. As most viral infections cannot be treated with directed therapy, symptomatic treatment is the suitable strategy of therapy for those forms of myocarditis. For symptomatic patients, digoxin and diuretics provide clinical improvement (Nicholson et al. 2010). Antibiotics remain the mainstay of treatment for IE. Empiric antibiotic therapy is chosen based on the most likely infecting organisms. The emergence of methicillin-resistant S. aureus (MRSA) and penicillin-resistant streptococci has led to a change in empiric treatment (Weinstein & Brusch 1996). For this reason, we are investigating our plant for isolation of new antimicrobial agents.
The new compound (1) was isolated as white microcrystalline powder, its negative HR-EIMS showed a molecular ion peak at m/z 679.6187[M-H] − , corresponding to a molecular formula of C 46 H 80 O 3 . The HR mass of the compound suggested the presence of a lup-20(29)-3-Ol nucleus (C 30 H 50 O) esterified with a fatty acid (C 16 H 32 O 3 ). The 1 H NMR and 13 C NMR spectroscopic data of compound 1 (Table 1S and 2S & Figure 1S and 2S) indicated a lupane nucleus (Just et al. 1997;El-Gamal 2008;yan et al. 2010;Farimani et al. 2012;Nguyen & Nguyen 2013). The spectroscopic data of compound 1 was identical to that of compound 2 in every aspect except for the downfield shift of CH-3 (δ H 4.53 and δ C 81.4 ppm) indicating an esterification at C3-OH (Lakshmi et al. 2014). The esterification at C-3 has been further confirmed from the correlations in HMBC between the C=O (at δ C 172.8 ppm) and H-3 at δ H 4.53 ppm ( Figure 3S and 4S). The carbon at δ C 41.6 was assigned as C-2′ of the fatty acid by the direct correlation of this carbon with 2H at δ H 2.4-2.5 ppm in HSQC, the 2H at δ H 2.4-2.5 have a long range coupling with C=O in HMBC ( Figure 3S and 4S), so, the 2H were assigned as CH 2 -1′ of the fatty acid moiety. A signal at δ H 4.00 and δ C 68.2 ppm indicated an oxy carbon, the proton of this carbon showed a long range correlation with a C2` (δ C 41.6 ppm) in HMBC, and consequently, the oxy carbon was assigned as CH-3′. This indicated the presence of an OH group at C-3′ of the fatty acid (Waard et al. 1993). The obtained data showed similarity of the HR-MS of compound 1 and its NMR data with the published literature on lup-20(29)en-3-hexadecanoate (palmitate) (Razdan et al. 1996), with the presence of an extra OH group on the fatty acid. 1 H NMR and 13 C NMR assignments of the fatty acid moiety suggested that it is a 3-hydroxy palmitic acid (Waard et al., 1993). In the NOESy spectrum ( Figure 5S), crosspeaks between H-23 and H-3 indicated that the H-3 was axial and that the OH group at C-3 was equatorial. The configuration of the hydroxyl group at C-3′ of palmitic acid was assigned to be β, from the NOESy spectrum, on the basis of the presence of cross-peaks between H-5, H-23 and H-3`, this indicated the axial position of the H-3′ on the α-face and an equatorial orientation for the hydroxyl group on the β-face. This was opposite to the α-OH of the lupane fatty acid ester isolated from the leaves of Maclura pomifera (Lee et al. 1997). From the above data, compound 1 was identified as lup-20(29)-en-3β-O-(3′-β-hydroxy) palmitate and it is a new natural product.
Plants are known to produce a variety of compounds to protect themselves against a wide range of microorganisms including plant pathogens and environmental organisms, this is an indication of the successful defense mechanism developed. Therefore, plants and their secondary metabolites could be promising sources to provide structurally diverse bioactive compounds as potential therapeutic agents, including antimicrobial agents (Chung et al. 2011). Pentacyclic triterpenoids have shown anti-staphylococcal activities, although individual compounds showed weaker activity than common antibiotics produced from bacteria and fungi, but synergistically these compounds may use different mechanism of action or pathways to exert their antimicrobial effects (Chung et al. 2011). Therefore, the use of current antibiotics can be improved through combination with plant-derived antibacterial agents as a therapeutic option in the treatment of the resistant S. aureus infections.
In the present study, lup-20(29)-en-3β-O-(3′-β-hydroxy) palmitate was a good candidate against Pseudomonas aeruginosa (MIC 7.81 μg/ml) (Table 4S and 5S). Pseudomonas spp. are Gram-negative rod bacteria commonly found in soil, ground water, plants and animals (Mena & Gerba 2009). Pseudomonal infection causes a necrotising inflammation. P. aeruginosa is an opportunistic pathogen that can cause a wide range of infections, especially in immunocompromised people and people with severe burns, diabetes mellitus or cystic fibrosis (Mena & Gerba 2009). P. aeruginosa is relatively resistant to many antibiotics except, the broad-spectrum antibiotics such as ciprofloxacin and gentamicin. P. aeruginosa may infect heart valves in intravenous drug abusers, also prosthetic heart valves. The organism establishes itself on the endocardium by direct invasion from the blood stream. Thromboembolism from pseudomonal endocarditis may cause brain abscess, cerebritis and mycotic aneurysms (Mena & Gerba 2009). Lupeol and acovenoside A exhibited the same inhibitory effect against most of the tested microorganisms, especially against S. aureus which is the most common organism responsible for IE (Murdoch 2009). The cardiac glycoside, acovenoside A prevailed growth inhibitory effect against Escherichia coli with low MIC (0.98 μg/ml) (Table 4S and 5S) in double concentration compared to gentamicin (MIC 0.49 μg/ml) (Table 4S and 5S).

Conclusion
As a conclusion we herein present the potential of A. oppositifolia, which possesses a cardioprotective activity against endocarditis and their major compounds; lup-20(29)-en-3β-O-(3′-β-hydroxy) palmitate, lupeol and acovenoside A are good candidates as antimicrobial agents against microorganisms responsible for infective endocarditis.

Disclosure statement
No potential conflict of interest was reported by the authors.