In vitro susceptibility of Burkholderia pseudomallei to antimicrobial peptides

International Journal of Antimicrobial Agents 34 (2009) 309–314

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In vitro susceptibility of Burkholderia pseudomallei to antimicrobial peptides Sakawrat Kanthawong a,b , Kamran Nazmi c , Surasakdi Wongratanacheewin a,b , Jan G.M. Bolscher c , Vanaporn Wuthiekanun d , Suwimol Taweechaisupapong b,e,∗ a

Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Melioidosis Research Center, Khon Kaen University, Khon Kaen 40002, Thailand c Department of Oral Biochemistry, Academic Centre for Dentistry, Amsterdam, University of Amsterdam and VU University Amsterdam, 1081 BT Amsterdam, The Netherlands d Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand e Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand b

a r t i c l e

i n f o

Article history: Received 12 February 2009 Accepted 26 May 2009 Keywords: Burkholderia pseudomallei Antimicrobial peptides Histatins LL-37 Lactoferrin

a b s t r a c t Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics, resulting in high mortality rates of 19% in Australia and even 50% in Thailand. Antimicrobial peptides (AMPs) possess potent broad-spectrum bactericidal activities and are regarded as promising therapeutic alternatives in the fight against resistant microorganisms. Moreover, these peptides may also affect inflammation, immune activation and wound healing. In this study, the in vitro activities of 10 AMPs, including histatin 5 and histatin variants, human cathelicidin peptide LL-37 and lactoferrin peptides, against 24 isolates of B. pseudomallei were investigated. The results showed that the antibacterial activities of the individual peptides depended on peptide dose and bacterial isolate. Among the 10 peptides tested, LL-37 exhibited the most effective killing activity. The smooth type A lipopolysaccharide (LPS) phenotype B. pseudomallei appeared to be more susceptible than those expressing the smooth type B LPS and the rough type LPS. Four isolates of B. pseudomallei shown to be resistant to ceftazidime and trimethoprim/sulfamethoxazole were also highly susceptible to LL-37. These data indicate that LL-37 possesses antimicrobial activity against all isolates independent of the LPS phenotype and is therefore a promising peptide to combat B. pseudomallei infections. © 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

1. Introduction Burkholderia pseudomallei is the causative agent of melioidosis, a severe disease endemic in areas of Southeast Asia, Northern Australia, the Indian subcontinent, Iran, and Central and South America [1,2]. It is an environmental saprophyte that can be isolated from soil and water in these endemic areas and is known as a potential biological warfare agent. Infection is thought to be acquired either through a wound in the skin or through inhalation of aerosolised B. pseudomallei. Melioidosis may arise many years after exposure, commonly in association with compromised immunity. Overall mortality is 50% in Northeast Thailand (35% in children) [1] and 19% in Australia [2]. Recurrent infection is the most important complication in survivors despite 20 weeks of antimicrobial treatment and occurs in 10% of Thai patients who survive the primary episode [3]. Burkholderia pseudomallei is intrinsically resistant to many antibiotics, including penicillin, first- and second-generation

∗ Corresponding author. Present address: Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand. Fax: +66 43 202 862. E-mail address: suvi [email protected] (S. Taweechaisupapong).

cephalosporins, macrolides, rifamycins, colistin and aminoglycosides, but is usually susceptible to amoxicillin/clavulanic acid (AMC), chloramphenicol, doxycycline, trimethoprim/sulfamethoxazole (SXT), ureidopenicillins, ceftazidime and carbapenems [1,2]. Ceftazidime, the carbapenem antibiotics or AMC are used for the initial parenteral phase of therapy, followed by a prolonged course of oral antimicrobial therapy with either SXT with or without doxycycline or AMC [4]. However, ceftazidime- and/or AMC-resistant B. pseudomallei have emerged, ultimately leading to treatment failure [5–7]. The carbapenems have been reported to have good bactericidal activities against B. pseudomallei and have been used effectively to treat patients with septicaemic melioidosis [8,9]. However, increased use of carbapenems may again give rise to resistance as has been seen with ceftazidime and AMC. Owing to the difficulty in eradication of the organism following infection, prolonged antibiotic therapy is needed and a high relapse rate is observed if therapy is not completed [10]. Furthermore, recurrence of infection is common despite adequate antimicrobial therapy [2]. It has been shown that B. pseudomallei isolated from relapse cases and persistent infections were resistant to antibiotics [6,11]. With the increasing development of antibiotic resistance, it is necessary to search for novel anti-infective agents against B. pseudomallei.

0924-8579/$ – see front matter © 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2009.05.012

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S. Kanthawong et al. / International Journal of Antimicrobial Agents 34 (2009) 309–314 Table 1 Sequences and characteristics of the peptides investigated. Peptide

Sequence

Mol. wt.

Chargea

Histatin 5 dh5 dhvar2 dhvar3 dhvar4 dhvar5 bLFcin17-30 bLFampin265-284 bLFampin268-284 LL-37

DSHAKRHHGYKRKFHEKHHSHRGY KRKFHEKHHSHRGY KRLFKELLFSLRKY KRLFKKLKFSLRKY KRLFKKLLFSLRKY LLLFLLKKRKKRKY FKCRRWQWRMKKLG DLIWKLLSKAQEKFGKNKSR WKLLSKAQEKFGKNKSR LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

3036 1847 1841 1855 1840 1847 1922 2389 2047 4494

5+ 4+ 4+ 7+ 6+ 7+ 6+ 4+ 5+ 6+

a

Net positive charge at neutral pH.

Antimicrobial peptides (AMPs) have received special attention as a possible alternative means to combat antibiotic-resistant bacterial strains. They are attractive candidates for clinical development because of their selectivity, their speed of action and because bacteria may not easily develop resistance against them. In the last few decades, an increasing number of AMPs have been isolated and characterised from virtually all classes of organisms where they play an important role in the innate defence against microbial and viral infections. In addition to their antimicrobial role, these peptides may also serve as important effector molecules in inflammation, immune activation and wound healing [12–14]. The AMPs discovered so far have been divided into several groups based on their length, secondary and tertiary structure, and presence or absence of disulfide bridges [15]. Most of them share common features such as small size, cationic charge and an amphipathic nature. Several cationic peptides bind to the negatively charged residues of lipopolysaccharide (LPS) of the outer membrane by electrostatic interactions involving the negatively charged phosphoryl groups and by hydrophobic interactions involving the acyl chains of lipid A, thus destabilising the microbial membrane and leading to cell death for Gram-negative organisms [16,17]. However, different modes of action are proposed for several peptides, including inhibition of the synthesis of specific membrane proteins [18] or stress proteins [19], arrest of DNA synthesis [20], interaction with DNA [21] and production of hydrogen peroxide [22]. The AMPs LL-37, histatin 5 and histatin variants, and the lactoferrin peptides have been proven to be active against fungi as well as Gram-positive and -negative bacteria [23–27]. To identify the most effective peptides against B. pseudomallei, we compared the in vitro antimicrobial activities of histatin 5 and its variants, LL-37 and the lactoferrin peptides against 24 isolates of B. pseudomallei. Possible association between the LPS type and susceptibility of B. pseudomallei to these AMPs was also investigated. The results reported in this study indicate that, of the tested peptides, LL-37 exhibited the most effective killing activity and possessed antimicrobial activity against all isolates independent of the LPS phenotype. Therefore, it is a promising peptide to combat B. pseudomallei infections.

2. Materials and methods 2.1. Synthetic peptides Histatin 5 and its variants (dh5, dhvar2, dhvar3, dhvar4 and dhvar5), the lactoferrin peptides LFcin17-30, LFampin268-284 and LFampin265-284, corresponding to the amino acids 17–30, 268–284 and 265–284 of bovine lactoferrin (bLF), respectively, and the human cathelicidin peptide LL-37 were synthesised by fluoren9-ylmethoxycarbonyl (Fmoc) chemistry with a MilliGen 9050 peptide synthesizer (MilliGen-Biosearch, Bedford, MA) as described previously [23,26,27]. All peptides were ≥95% pure as determined by reverse-phase high-performance liquid chromatography

(RP-HPLC) (JASCO Corp., Tokyo, Japan). The authenticity of the peptides was confirmed by ion-trap mass spectrometry with an LCQ Deca XP (Thermo Finnigan, San José, CA). Amino acid sequences and characteristics of the 10 peptides investigated are shown in Table 1. 2.2. Bacteria and growth conditions Twenty-four isolates of B. pseudomallei were used in this study (Table 2), 13 of which were isolated from patients admitted to Srinagarind Hospital (Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand) and 11 were isolated from soil collected from the northeastern endemic region of Thailand. Individual isolates were identified by biochemical tests, antibiotic susceptibility profile and immunoreactivity with polyclonal and monoclonal antibodies as described elsewhere [28–30]. The B. pseudomallei isolates were divided into three groups based on their LPS phenotype (smooth type A, smooth type B and rough type) as described previously [31]. The media used in this study were brain–heart infusion (BHI) broth (Difco, Becton Dickinson Microbiology Systems, Franklin Lakes, NJ) and nutrient agar. Burkholderia pseudomallei isolates were cultured aerobically in BHI broth at 37 ◦ C overnight and, to yield a mid logarithmic growth phase, were subcultured at 37 ◦ C in a 200 rpm shaker-incubator for 1.5 h. 2.3. Antibacterial assay To determine the effective concentration of peptides against B. pseudomallei, various concentrations of histatin 5 and LFampin268284 were tested against B. pseudomallei isolate A2, which is a virulent strain with a 50% lethal dose of 19 in BALB/c mice [32]. Bacterial cells were washed three times and re-suspended to ca. 1 × 107 colony-forming units (CFU)/mL in 1 mM potassium phosphate buffer (PPB) (pH 7.0). The bacterial suspension was then added to an equal volume of the AMP to reach a final concentration Table 2 Source and lipopolysaccharide (LPS) phenotype of clinical and environmental Burkholderia pseudomallei isolates in this study. LPS smooth type A

LPS smooth type A

LPS smooth type B

LPS rough type

Isolate

Source

Isolate

Source

Isolate

Source

Isolate

Source

267 279 354 377 409 429 466 591 705 745 844

Soil, NE Soil, NE Soil, NE Soil, NE Soil, NE Soil, NE Soil, NE Soil, NE Soil, NE Soil, NE Soil, NE

A2 316c FL202 979b 2-173 365a

Blood Blood Fluid Sputum Urine Urine

A1 A20 G12 U2704

Blood Blood Pus Urine

316a U882b G207

Blood Pus Sputum

NE, northeast region of Thailand.

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of 10–100 ␮M. Bacterial suspension in PPB without peptide served as a control. Following incubation at 37 ◦ C for 60 min, the incubation mixture was serially diluted in sterile normal saline and plated in triplicate on nutrient agar. Colonies were counted after 20 h incubation at 37 ◦ C. The percentage killing or inhibiting effects of each AMP were calculated using the formula [1 − (CFU sample/CFU control)] × 100%. To compare the in vitro activities of the 10 peptides against the 24 isolates of B. pseudomallei, a final concentration of 100 ␮M of peptide was chosen based on the maximum killing activity in the previous initial tests. The antibacterial assay was performed as described above. 2.4. Killing kinetic assay of the most effective antimicrobial peptide Killing kinetics was determined using a culture of B. pseudomallei isolate A2 re-suspended in 1 mM PPB to give ca. 1 × 105 CFU/mL. Peptide was added to the bacterial suspension to a final concentration of 200 ␮M, which was the minimum bactericidal concentration (MBC), and incubated in a 200 rpm shaker-incubator at 37 ◦ C. At the indicated times (0, 1, 2, 4, 6, 8, 10, 12 and 24 h), samples were taken, serially diluted, plated in triplicate on nutrient agar and incubated for 24 h to allow colony counting. A bactericidal effect was defined as a ≥3 log10 reduction in CFU/mL compared with the initial inoculum. 3. Results 3.1. Antibacterial effect of the peptides To obtain initial information on the susceptibility of B. pseudomallei to AMPs, a test was performed with different concentrations of histatin 5 and LFampin268-284. Both peptides effected a clear dose-dependent killing activity against B. pseudomallei isolate A2 (Fig. 1). They exhibited 50% killing activities at a peptide concentration of 75 ␮M and 50 ␮M, respectively. Based on this result, the antibacterial activities of 10 peptides were determined at a concentration of 100 ␮M against 24 isolates of B. pseudomallei of different origin and distinct LPS types (smooth types A and B and rough type). The antimicrobial activities of the 10 peptides were largely isolate dependent. Of note, only LL-37 caused killing towards virtually all isolates (Fig. 2); 80–100% killing was observed for all soil isolates

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and 75% to almost 100% killing was observed for the clinical isolates with the exception of the 2 of 13 isolates that exhibited killing of ca. 50% and 60% (isolates 2-173 and A20, respectively). Isolate-dependent killing of the other peptides can best be illustrated on the basis of selected examples for the soil isolates (Fig. 2A). (i) The overall most sensitive soil isolate 377 turned out to be mainly susceptible to the dhvar series of peptides but not or much less susceptible to the parental histatin 5 or its active domain dh5, whilst soil isolate 466 appeared to be susceptible to dhvar4 and much less to the other variants. (ii) With respect to the lactoferrin peptides, isolate 377 was sensitive to LFcin17-30 and LFampin265284 but not to LFampin268-284, which is only three amino acids smaller. The opposite was found for soil isolate 354, which was more susceptible to LFampin268-284 than to LFampin265-284 or LFcin17-30. However, another soil isolate, 745, appeared equally sensitive to both LFampins and absolutely insensitive to LFcin1730. Similar examples can be given for the clinical isolates (Fig. 2B). (i) The most sensitive clinical isolates were A2 and FL202. Isolate FL202 expressed high sensitivity to histatin 5 and dh5 whilst having a lower sensitivity to dhvar2 and again increasing sensitivities to dhvar3, dhvar4 and dhvar5. Isolate 979b appeared to be most susceptible to dhvar3 and dhvar4, with less sensitivity to the others. (ii) Isolate FL202 also appeared to be very sensitive to the lactoferrin peptides LFcin17-30 and LFampin265-284 but less to LFampin268284. Similar to the soil bacteria, another isolate (316c) also showed the opposite effect, with highest sensitivity to LFampin268-284 and much less to the other two lactoferrin peptides. If the tested B. pseudomallei isolates are compared with respect to their LPS types, e.g. smooth types A and B and rough type, it is clear that B. pseudomallei with LPS of the smooth type A was more susceptible to all the peptides tested than those with smooth type B or rough type LPS (Fig. 3). The average percentage killing activities of LL-37 against the smooth types A and B and the rough type B. pseudomallei were ca. 94%, 77% and 83%, respectively. For histatin 5 and its variants, the average percentage killing activities against the smooth types A and B and the rough type B. pseudomallei were 41–55%, 24–42% and 16–36%, respectively, whilst the average percentage killing activities of lactoferrin peptides were 37–45%, 21–31% and 16–33%, respectively. Four of the tested B. pseudomallei isolates (316c, 365a, 979b and FL202) of smooth type A LPS tested in our laboratory and by others [33,34] were resistant to the classic antibiotics ceftazidime and SXT, at present the treatment of choice for severe melioidosis. The minimum inhibitory concentrations of ceftazidime and SXT towards these isolates (Table 3) are clearly beyond the breakpoints for resistance to ceftazidime and SXT in B. pseudomallei, adapted from data compiled by the National Committee for Clinical Laboratory Standards for similar organisms to be used for susceptibility testing [35]. To emphasise the antimicrobial activity of LL-37 against the antibiotic-resistant B. pseudomallei, the killing activities of all the peptides are given in Fig. 4. It is interesting and promising that although resistant to ceftazidime and SXT, these isolates were highly susceptible to LL-37, whereas the other peptides again showed isolate selectivity to these four isolates.

Table 3 Minimum inhibitory concentrations (MICs) of ceftazidime and trimethoprim/sulfamethoxazole (SXT) towards Burkholderia pseudomallei isolates. Fig. 1. Dose–response of the bactericidal activity of histatin 5 and LFampin268-284 against Burkholderia pseudomallei isolate A2. A bacterial suspension of ca. 1 × 107 colony-forming units (CFU)/mL was incubated with each antimicrobial peptide at final concentrations of 10–100 ␮M at 37 ◦ C for 1 h. Subsequently, the incubation mixture was serially diluted, plated in triplicate on nutrient agar and incubated at 37 ◦ C. After 18 h, colonies were counted and the percentage killing was calculated using the formula [1 − (CFU sample/CFU control)] × 100%.

Isolate

316c 365a 979b FL202

MIC (␮g/mL) Ceftazidime

SXT (1:19)

64 32 256 1

2/38 0.5/9.5 1/19 4/76

312

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Fig. 2. Bactericidal activities of 10 antimicrobial peptides (AMPs) against 24 isolates of Burkholderia pseudomallei. A bacterial suspension of ca. 1 × 107 colony-forming units (CFU)/mL of 11 isolates from environmental samples (A) and 13 isolates from clinical samples (B) were incubated with 100 ␮M AMP and processed as described in Section 2.3. Percentage killing was calculated using the formula [1 − (CFU sample/CFU control)] × 100% (mean value of triplicate incubations).

3.2. Killing kinetics of clinical Burkholderia pseudomallei isolate A2 by LL-37 Using B. pseudomallei isolate A2, the killing kinetics of the most effective antimicrobial peptide, LL-37, was further investigated at 200 ␮M, which was found to be the MBC (data not shown). The bactericidal endpoint was reached after 6 h of incubation (Fig. 5). There was a rapid decrease in the number of CFU/mL in the first 2 h, with slower killing after that point. Control incubations without peptide showed an increase in CFU/mL to >106 within 2 h and a further increase to 107 CFU/mL in the next 24 h. Other peptides showed initial reduction in CFU/mL, however they never reached a bactericidal endpoint and B. pseudomallei grew to the control level after 4–8 h (data not shown).

may have its own optimal activity against individual isolates, whilst the entire peptide is active against virtually all isolates (Fig. 2). Indeed, partially overlapping truncated peptides from LL-37 exhibit different levels of antimicrobial activity against different bacterial species [38], including Burkholderia thailandensis (S. Kanthawong et al., unpublished data). For the lactoferrin peptides, the mechanism of antibacterial action has been only partially clarified. The peptide binds to LPS in Gram-negative bacteria and to teichoic acid in Gram-positive

4. Discussion The results from this study show that although individual AMPs demonstrated selective antibacterial activity towards distinct isolates, the human cathelicidin peptide LL-37 possessed strong killing activity towards all B. pseudomallei isolates, as also observed in previous reports for LL-37 against other bacteria [36]. Although the exact mechanism by which the peptides kill bacteria is not clearly understood, it has been shown that LL-37 performs its bactericidal action by electrostatic binding of its cationic molecules to the outer surface of the bacterial cell. Insertion of the peptide into the cell membrane results in leakage of the cell cytoplasm into the extracellular space causing death of the bacterial cell [37]. The reason why LL-37, in contrast to the other peptides, is active against all tested B. pseudomallei isolates may be found in the fact that this peptide is much longer, intrinsically having a variety of domains differing in charge distribution and amphipathic conformation. Each domain

Fig. 3. Bactericidal activities of 10 antimicrobial peptides against different lipopolysaccharide (LPS) phenotypes of Burkholderia pseudomallei. Results represent the mean bactericidal activities of 17 isolates of B. pseudomallei expressing LPS of smooth type A, 4 isolates with smooth type B and 3 isolates with rough type LPS.

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Fig. 4. Antibacterial activity of the 10 antimicrobial peptides (AMPs) against ceftazidime- and SXT-resistant isolates Burkholderia pseudomallei. A bacterial suspension of ca. 1 × 107 colony-forming units (CFU)/mL was incubated with 100 ␮M AMP and processed as described Section 2.3. Percentage killing was calculated using the formula [1 − (CFU sample/CFU control)] × 100% (mean value of triplicate incubations).

Fig. 5. Killing kinetics of LL-37 against Burkholderia pseudomallei isolate A2. A bacterial suspension of ca. 1 × 105 colony-forming units (CFU)/mL was incubated with 200 ␮M of peptide incubated in a 200 rpm shaker-incubator at 37 ◦ C. At the indicated times (0, 1, 2, 4, 6, 8, 10, 12 and 24 h), samples were taken, serially diluted, plated in triplicate on nutrient agar and incubated at 37 ◦ C for 24 h. Colonies were counted and a bactericidal effect was defined as a ≥ 3 log10 reduction in CFU/mL compared with the initial inoculum.

bacteria [39]. From there it is assumed that it is brought to the cytoplasmic membrane where it exerts its effect by disintegrating the cytoplasmic membrane. This action can be explained by the conformational flexibility of the lactoferricin portion in the lactoferrin molecule that allows it to form an amphipathic structure in solution [40]. In this study, we found that the antibacterial activities of bLFampin265-284 and bLFampin268-284 were different from that of bLFcin17-30. Our results are in accordance with several previous studies [26,41]. This probably reflects the fact that these peptides, although sharing amphipathic and cationic features, are strikingly different with respect to amino acid composition and length as well as structure. In addition, besides peptide properties, other bacteria-associated features may also play an important role in the peptide-mediated killing. For the killing kinetic assay of LL-37 against B. pseudomallei isolate A2, there was a rapid decrease in the number of CFU/mL in the first 2 h (a reduction of >95% of the vital bacteria). The remaining <5% bacteria were killed at a slower rate, possibly owing to interference of the huge amount of bacterial debris with the peptide binding to vital bacteria or to scavenging of the peptide by this debris. The possible association between LPS type and susceptibility of B. pseudomallei to the AMPs was also investigated, as LPS is the major structural component of the outer membrane of Gram-negative bacteria and shields them from a variety of host defence factors, including AMPs. The results revealed that B. pseudomallei smooth type A LPS appeared to be the most susceptible to all

peptides tested compared with the smooth type B and the rough type B. pseudomallei. We recently reported the LPS heterogeneity among B. pseudomallei and that the two less abundant LPS patterns (smooth type B and rough type) were found more in isolates obtained from patients with relapsed melioidosis than from those with primary infection [31]. Since the modification of LPS structure has been reported to contribute to AMP resistance in several pathogens, i.e. Pseudomonas aeruginosa and Salmonella spp. [42,43], the higher resistance of the smooth type B and rough type B. pseudomallei to cationic peptides observed in this study may be related to modification of the LPS structure and the ability of these two LPS-containing B. pseudomallei to evade host immunity and cause relapse melioidosis. In contrast to the other peptides, LL-37 was capable of killing the smooth type B and rough type B. pseudomallei almost as well as the smooth type A, strongly affirming the potency of this peptide. Since 1989, ceftazidime became the treatment of choice for severe melioidosis because it was associated with 50% lower overall mortality than conventional treatment [44]. The antimicrobial combination SXT is commonly used in combination with ceftazidime or meropenem, or on its own as maintenance monotherapy because of its low cost, good efficacy and lower relapse rate compared with AMC [3]. However, the emergence of ceftazidime- and/or SXT-resistant clinical isolates and the wide distribution of B. pseudomallei in Southeast Asia increase the risk of malicious use of resistant strains. In the present study, it is very interesting that four B. pseudomallei isolates (316c, 365a, 979b and FL202) that were resistant to ceftazidime and SXT were highly susceptible to LL-37. In addition to the potential for direct bactericidal activity, LL37 may have diverse and complementary abilities to modulate the innate immune response to B. pseudomallei. LL-37 has been indicated to stimulate angiogenesis [45], to provoke cell migration [45] and to accelerate wound closure of the airway epithelium [46]. It is also able to neutralise LPS [37,47] and in doing so protects against endotoxic shock [48,49]. Furthermore, LL-37 provokes cytokine release, e.g. interleukin (IL)-8 and IL-18 secretion [14]. This latter role has been shown to play an essential part in the protective immune response to the severe form of melioidosis [50]. Because IL-18 is involved both in Th1 and Th2 functions, the ability of LL-37 to induce the secretion of this pro-inflammatory mediator indicates the role of this peptide in both innate and adaptive immunity. This in vitro study provides evidence that LL-37 has strong killing activity against B. pseudomallei, however the mechanism is still unknown. Thus, further investigations are needed to study the mechanisms of action and, in relation to its suggested

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