The stem bark of C. leptophloeos was collected at Parque Nacional do Catimbau, Pernambuco – Brazil. The authors confirm that the named authority Instituto Chico Mendes de Conservação da Biodiversidade granted permission (SISBIO 16806) for our described field searches. The botanical identification and the deposition of plant specimens were performed at the Herbarium of the Institute of Agricultural Research of Pernambuco (IPA-PE) (IPA n° 84037).

The dried bark (25 g) of C. leptophloeos was obtained by saturation in order of increasing polarity: submitted to Cyclohexane (CLCHE), Chloroform (CLCLE), Ethyl Acetate (CLAEE), Methanolic (CLMEE), and Aqueous (CLAQE) (250 mL) by agitation at 180 rotations per minute (rpm). After 24 h, the extract was filtered (Whatman^® number 2) and concentrated at 45°C under vacuum in a rotary evaporator (Concentrator 5301, Eppendorf^®). The powder produced was kept at –20°C for future use. For phytochemical and antimicrobial analysis, the extracts were dissolved in your respective solvents at the concentration of 100 mg/mL for all biological assays.

For hemolytic in vitro assay, whole blood (5 ml) was obtained from healthy, non-smoking volunteers by venipuncture, after obtaining written informed consent (National Ethics Committee reference number 30667014.5.0000.5208). Human erythrocytes from citrated blood were immediately isolated by centrifugation at 1500 rpm for 10 min at 4°C. After plasma removal, the erythrocytes were washed three times with phosphate-buffered saline (PBS; pH 7.4) and then re-suspended using the same buffer, and a 1% erythrocyte suspension was prepared. The hemolytic activity of C. leptophloeos extracts was tested under in vitro conditions. Each tube received 1.1 mL of erythrocyte suspension and 0.4 mL of extract with different concentrations ranging from 50 to 500 μg/mL. The negative control was solvent only and the positive control received 0.4 mL of Quillaja saponin (0.0025%). After 60 min of incubation at room temperature, cells were centrifuged at 1500 rpm for 10 min and the supernatant was used to measure the absorbance of the liberated hemoglobin at 540 nm length. The average value was calculated from triplicate assays (Sulaiman and Gopalakrishnan, 2013).

Each experiment was performed in biological duplicates and technical triplicates and results are presented as means and ± standard deviation (SD). Statistical analysis was performed by Student’s t-test and ANOVA tests. Differences were considered significant at p < 0.05. The concentration needed for 50% inhibition (IC₅₀) was estimated graphically by linear regression analysis.

The yields of the assessed extracts presented values varying between 9.8 and 12.3%, being 9.8% for CLCHE, 10.2% for CLECL, 10.5% for CLAEE, 11.8% for CLMEE, and 12.3% for CLAQE. The qualitative phytochemical analysis of C. leptophloeos by HPLC detected the presence of three particular compounds: Gallic acid (GA), Chlorogenic acid (CGA), and Protocatechuic acid (PCA), as showed in Figure 1. The qualitative phytochemical analysis by TLC of C. leptophloeos extracts detected the presence of Phenolic compounds, Flavonoids and Reducing sugars in all extracts (Table 2). The estimation of total phenolic content revealed that CLAQE (33.64 ± 0.5 mg of GAE/g) and CLMEE (20.3 ± 0.78 mg of GAE/g) exhibited the highest phenolic content (p < 0.05). The other extracts showed phenolic content values ranging from 13.8 ± 0.53 to 12.54 ± 0.55 mg of GAE/g, showed in Table 2. The fraction F16 from CLCLE studied shown the presence of hinokinin in our analysis and the spectrum ¹H NMR (CDCl₃, 200 MHz) of F16 from CLCLE showed two multiplets (δH 2.45 e 2.48) in high field, being referent to sp³ hydrogens connected to neighboring carbons of chiral carbon (C-7 and C-7’). In δH 2.85 was observed a doublet of doublet (dd, J = 4.7, 14.5 Hz,1H) of 1 H connected directly to chiral carbon C-8. In δH 3.00 the doublet of doublet (dd, J = 4.7, 14.5 Hz,1H) of hydrogen 1 connected directly to the chiral carbon (C-8’). In δH 3.85 (dd, J = 7.0, 9.2 Hz,1H) and 4.15 (dd, J = 6.2, 9.0 Hz,1H) two doublets of doublets regarding the hydrogens linked to the carbon C-9. In δH 5.9471 a multiplet in 4 H linked to the carbon C-10 and C-10’, simultaneously. Finally, a 6H a multiplet in δH 6.5 assigned to aromatic hydrogens were identified. The spectrum elucidation is show in Figure 2. Hinokinin is one of the constituents of secondary metabolites of C. leptophloeos described for the first time in this species.

The antibacterial activity of C. leptophloeos extracts was recorded against various microorganisms and is presented in Tables 3–5. Overall, all plant extracts exhibit a range of inhibitory potentials with broad spectrum, as they inhibited all bacteria and yeasts species tested. The better antimicrobial results observed were provided by CLMEE, in which MIC ranged from 0.097 to 50.0 mg/mL (p-value < 0.05) (Tables 3 and 4). CLMEE presented better antimicrobial activities against Gram-positive bacteria with best results for B. subtilis (MIC = 3.125 mg/mL), E. faecalis (MIC = 25 mg/mL) and M. luteus (MIC = 0.097 mg/mL and MMC = 12.5 mg/mL). Against S. aureus strain, the best bacteriostatic action was the CLCLE (MIC = 1.125 mg/mL) and, for this reason, we use F16 fraction purified C. leptophloeos hinokinin to evaluate the action of this lignan against MRSA strains (Table 5). C. leptophloeos also showed antifungal activity, such as Aspergillus sp. (MIC values ranged from 3.125 to 6.25 mg/mL) and C. albicans (MIC values ranged from 6.25 to 12.5 mg/mL, p-value < 0.05) showed in Table 3. The antimicrobial activities against Gram-negative bacteria were showed in Table 4. CLCHE showed better bacteriostatic effect against P. aeruginosa (MIC = 6.25 mg/mL) and K. pneumoniae (MIC and MMC = MMC 12.5 mg/mL), CLAEE showed the better activity against E. coli (MIC and MMC = 12.5 mg/mL), and CLCLE was tested against Mycobacterium, indicating M. smegmatis (MIC = 12.5 mg/mL) to be more susceptible to CLCLE than M. tuberculosis (MIC = 52 mg/mL), Table 4. The MIC values of hinokinin (obtained from the purification F16 from CLCLE) ranged from 0.0485 to 3.125 mg/mL (p-value = 0.002) for the different S. aureus clinical isolates tested, and showed a bactericidal activity against MRSA isolated from blood (MMC 0.40 mg/mL) and postoperative secretion (MMC = 3.125 mg/mL) showed in Table 5.

The concentration from CLAQE, CLMEE, CLAEE, CLCLEE, and CLCHE extracts demonstrated a HC₅₀ (concentration required for 50% of hemolysis) of 313 ± 0.5 μg/mL; 304.9 μg/mL ± 0.8; 287.49 μg/mL ± 3.0; 239.5 μg/mL ± 1.4, and 177.21 μg/mL ± 0.45, respectively (p-value = 0.001).

Species from Commiphora genus present bioactive compounds widely known and used as therapy for several pathologies in folk culture. Herein, we performed a thorough phytochemical characterization of C. leptophloeos, showing its notable amounts of phenolic compounds, namely Gallic acid (GA), Chlorogenic acid (CGA), and Protocatechuic acid (PCA). GA is endowed with pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial and antiproliferative activity (Kroes et al., 1992; Vishnu Prasad et al., 2010). On the other hand, CGA has been recently pointed as modulator of glucose and lipid metabolism in vivo, upon unbalanced metabolic conditions such as diabetes (Lou et al., 2011; Sato et al., 2011; Zhao et al., 2012; Hwang et al., 2014). Additionally, PCA, the major metabolite of anthocyanin, provides beneficial activities to human’s health such as reduced risk of cardiovascular diseases (Wang et al., 2010), anti-inflammatory, antioxidant and free radical scavenging activities (Li, 2011), as well as estrogenic and antiestrogenic activity (Kakkar and Bais, 2014).

Our phytochemical characterization showed the presence of other bioactive compounds in C. leptophloeos, such as hinokinin, an important class of lignans, which has been recently investigated in order to establish its biological activities (REFERENCIA). Lignans are important components of food and medicines biosynthetically deriving from the radical coupling of two molecules of coniferyl alcohol at C-8/C-8′ positions. In the past years, the biological activities of several lignans have been studied in depth (Aehle et al., 2011; Zhang et al., 2014) and among them, the hinokinin (Figure 2) is emerging as a new interesting compound with pharmacological potential. Hinokinin was isolated for the first time in 1933 by Yoshiki and Ishiguro from an ether extract of Hinoki wood – Chamaecyparis obtuse - as a colorless crystalline compound and over the years it has been gradually characterized and described by other researchers (Vanoeveren et al., 1994; Timple et al., 2013; Desai et al., 2014).

Commiphora leptophloeos shows antibacterial activity against several human Gram-positive pathogens such as B. subtilis, E. faecalis, M. luteus, and S. aureus (Table 3). The inhibition of bacterial growth in vitro by the extracts of C. leptophloeos could be due to the presence of some active compounds including flavonoids, phenolic acids and tannins, described in Table 2, known to be effective antimicrobial agents against a wide array of microorganisms. These active compounds may act alone or in combination to inhibit bacterial growth. Our results against M. luteus strain showed a MIC ranging from 0.097 to 12.5 mg/mL, and the CLMEE as the extract showing the best bacteriostatic and bactericidal action (MMC = 12.5 mg/mL), particularly when compared to other studies with Commiphora genus (Latha et al., 2005) and when compared with the MIC value obtained by the aminoglycoside Kanamycin (MIC = 6.8 mg/mL). M. luteus is a natural constituent of mammalian skin microflora and considered a nosocomial contaminant mainly in immunodeficient individuals, causing meningitis (Fosse et al., 1985) and endocarditis (Miltiadous and Elisaf, 2011) in severe infections. Immunodeficiency is a risk factor able to intensify selection and dissemination of multidrug resistance strains. C. leptophloeos extracts are also effective against Aspergillus sp. and C. albicans, the most prevalent fungal species of the human microbiota, often associated to several complications in immunosuppressed individuals (Nobile and Johnson, 2015).

Gram-negative pathogens are particularly alarming, due to their resistance to nearly all drugs firstly considered for treatment. The reason for this relies on their several pathways to β-lactam resistance through β-lactamases enzymes production, therefore interfering with the mechanism of action of β-lactam antibiotics. The same premise has become more frequent in Gram-positive infections (e.g., Staphylococcus). P. aeruginosa is a ubiquitous opportunistic pathogen, having outer membrane cell structure conferring pronounced resistance to xenobiotics (Gaspar et al., 2013). Previous studies had already reported the action of the aqueous extract of C. leptophloeos inhibition of biofilms of P. aeruginosa (Trentin et al., 2013) and S. epidermidis (Trentin et al., 2011). According to these aforementioned studies, CLCHE presented ample bacteriostatic activity against P. aeruginosa, with MIC of 6.25 mg/mL (Table 4), this greater potential of action is due the presence of tannins in CLCHE, as noted in our phytochemical profile (Table 2). E. coli is a bacterium usually found in intestinal microflora, nevertheless, some can cause debilitating and sometimes fatal human diseases (Riley, 2014). Pathogenic strains are divided into intestinal pathogens causing diarrhea and extraintestinal mainly related to urinary tract infections pyelonephritis, cystitis, and urosepsis (Croxen et al., 2013). Only two fractions, CLCHE and the CLMEE, showed bactericidal action (MMC ranging 12.5 to 25 mg/mL), suggesting that this action may be due to the presence of high amounts of phenolic acids (Figure 1), such as GA, which might contribute to the inhibition E. coli strain.

Klebsiella pneumoniae is a type of Gram-negative bacteria that may cause different types of infections including pneumonia, meningitis, bloodstream infections. Generation of extended- spectrum β- lactamases is one of the major mechanisms by which clinical K. pneumoniae develop resistance to antibiotics (Cai et al., 2016). Herein we identified that the CLCHE and CLAEE fractions effectively act as antibacterial agent in the Gram-negative tested strains (Table 4). The antibacterial activities of C. leptophloeos extracts were also detected against M. smegmatis and M. tuberculosis strains as showed in Table 4. A bacteriostatic effect against Mycobacterium species by plants secondary metabolites may be due to pathogen’s thick outer membrane that is highly hydrophobic and possibly provided a permeability to the extract (Yamori et al., 1992; Firmani and Riley, 2002).

Oxacillin and methicillin resistant S. aureus (MRSA) are resistant to all β-lactam agents including cephalosporin’s and carbapenems, causing global commitment in stopping its spread responsible for approximately 40% of S. aureus infections in global Intensive Care Units (ICU) (Frieden, 2013; Cuny et al., 2015). In our study, were evaluated four MRSA clinical isolated strains reported as Methicillin Resistance (Oxacillin Resistance) confirmed with CFX inhibition disc tests at where cefoxitin is used as a surrogate for mecA-mediated oxacillin resistance (CLSI, 2014). C. leptophloeos hinokinin showed the highest antibacterial activity against MRSA isolated of blood, with a bacteriostatic activity (MIC) of 0.39 mg/mL and bactericidal (MMC) of 0.40 mg/mL, both with very similar values (Table 5). Our results point towards a promising antimicrobial potential against S. aureus resistant, especially when compared to others studies (Saeed and Sabir, 2004; Abdallah et al., 2009; Abdulgader et al., 2015), once we obtained more pronounced antibacterial results, corroborating the antimicrobial potential of hinokinin.

Regarding hemolytic activity of C. leptophloeos extracts, the values obtained for hemolysis were superior to the ones regarding its antimicrobial activity, showing its safety. Even though a phytochemical study on another Commiphora genus (Hanus et al., 2005) has shown the presence of possibly toxic compounds, the toxicity assay performed in vitro for Amburana hemolytic properties did not show the same results. Regardless, we recognize the importance of applying other methods to assess toxicity from these extracts over other cellular components. Hemolytic assays were performed to assess cell safety in future pharmacological preparations without causing any harm. In the present study our trials with Commiphora showed lower hemolytic activity when comparing to other species from the Caatinga biome (Silva et al., 2011; Tam et al., 2015).