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HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC
JAB-15; No. of Pages 12 journal of applied biomedicine xxx (2014) xxx–xxx
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Original Research Article
HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC Aline Augusti Boligon a,*, Mariana Piana a, Thaís Felli Kubiça b, Débora Nunes Mario c, Tanise Vendruscolo Dalmolin d, Pauline Cordenonsi Bonez d, Rudi Weiblen b, Luciane Lovato b, Sydney Hartz Alves c, Marli M.A. Campos d, Margareth Linde Athayde a a
Phytochemical Laboratory, Department of Pharmacy Industrial, Federal University of Santa Maria (UFSM), 97105-900 Santa Maria, RS, Brazil b Virology Laboratory, Department of Veterinary Preventive Medicine, Federal University of Santa Maria (UFSM), 97105-900 Santa Maria, RS, Brazil c Mycological Research Laboratory, Department of Microbiology and Parasitology, Federal University of Santa Maria (UFSM), 97105-900 Santa Maria, RS, Brazil d Mycobacterial Research Laboratory, Department of Clinical and Toxicological Analysis, Federal University of Santa Maria (UFSM), Santa Maria, RS, Brazil
article info
abstract
Article history:
Antimicrobial, antimycobacterial and antiviral activities of crude extracts, fractions and
Received 29 November 2013
subfractions of Tabernaemontana catharinensis were evaluated. Best antimicrobial results
Received in revised form
occurred with the dichloromethane (DCM) and butanolic (NB) fractions against Staphylococ-
15 January 2014
cus aureus, Micrococcus sp., Enterococcus faecalis and Bacillus subtilis (minimal inhibitory
Accepted 20 January 2014
concentration, MIC = 31.25–1000 mg/mL). Considering the Gram-negative bacteria, only NB
Available online xxx
fraction was effective against Proteus mirabilis and Aeromonas sp. (MIC = 62.5 mg/mL and 250 mg/mL, respectively). In addition, the fungi Candida albicans, Candida glabrata, Cryptococcus
Keywords:
neoformans, Saccharomyces cerevisiae, Aspergillus flavus and Aspergillus fumigatus were particu-
Apocynaceae
larly vulnerable for DCM fraction (MIC = 31.25–1000 mg/mL). The fractions and subfractions
Cobrina
were effective against Mycobacterium smegmatis (MIC = 19.53–156.25 mg/mL). DCM (selectivity
HSV-1
index – SI = 77.92), ethyl acetate (EA) (SI = 40.27) and NB (SI = 28.97) fractions from the leaves
HPLC
exhibited a potential antiviral activity toward Herpes Simplex Virus type 1 whereas DCM2
Mycobacterium
subfraction from leaves (SI = 12.28) and alkaloidal fraction (AF) (10.71) maintained this good activity. Steroids, terpenoids and phenolics compounds were identified by high-performance liquid chromatography with diode array detection (HPLC/DAD) and may be partially responsible for the antimicrobial and antiherpes activities observed. The results obtained in this study showed that T. catharinensis has antimicrobial and anti-herpetic activities and that these properties are reported for the first time for this species. # 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
* Corresponding author. Tel.: +55 32209618. E-mail address: [email protected] (A.A. Boligon). 1214-021X/$ – see front matter # 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.jab.2014.01.004 Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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Introduction Compounds with antimicrobial activity have been investigated and the searches for new anti-infections agents showed that plant extracts, essential oils or isolated compounds such as alkaloids, flavonoids, diterpenes, triterpenes and sesquiterpene lactones are potential sources of bioactive compounds (Rios and Recio, 2005; Boligon et al., 2013a). Remedies derived from plants are still being used worldwide in the traditional medicine for the treatment of a wide range of diseases, including fungal, bacterial, mycobacterial and virus infections (Khan et al., 2005; Mohamad et al., 2011; Boligon et al., 2012a, 2013a). In this new millennium, research of chemical entities from plant species and other natural resources for activity against Mycobacterium species had intensified with the development of easier, faster and safer screening techniques. A good number of extracts and pure compounds isolated from these resources have been reported to exhibit considerable in vitro inhibitory activity against Mycobacterium tuberculosis and its related species (Copp and Pearce, 2007; Negi et al., 2010). Successful inhibition of M. tuberculosis by ethnobotanically selected plants in many countries has been published (Rastogi et al., 1998; Gautam et al., 2007; Askun et al., 2009; Boligon et al., 2012a; Pallant et al., 2012). Tabernaemontana catharinensis A. DC (syn. Peschiera catharinensis A. DC. Miers) belongs to family Apocynaceae, a family consisting of 415 genera and 4555 species. Recently, about 99 species from Tabernaemontana genus were reported and 44 species occur in America. Tabernaemontana species are well known for indole alkaloids and several pentacyclic triterpenoids with approximately 240 structurally different bases already described. These types of compounds are responsible for many pharmacological activities such as: antileishmanial; trypanocidal, antioxidant, antibacterial; antitumoral; hypoglycaemic, analgesic and cardiotonic activities (Van Beek et al., 1984; Pereira et al., 2008; Lim et al., 2009). T. catharinensis is popularly known as 'jasmim' (jasmine), 'leiteira de dois irmãos' (milkweed), and 'casca de cobra' (snake skin) and it occurs in Argentine, Uruguay, Paraguay and Southern Brazil. In folk medicine, it is used as an antidote for snakebites, to relieve toothache, as a vermifuge to eliminate warts, and as an anti-inflammatory (Leeuwenberg, 1994; Almeida et al., 2004; Pereira et al., 2004). Studies conducted by Batina et al. (1997, 2000) demonstrated that substances isolated and aqueous extracts of the root of T. catharinensis can inhibit the lethal and myotoxic activity of Crotalus durissus terrificus venom (South American Rattlesnake, cascavel). Substances with trypanocidal and antitumoral activities were also isolated and described from this plant (Pereira et al., 1999; Almeida et al., 2004). In addition, the aqueous extract of T. catharinensis was shown to have a potent cytotoxic action on human tumor lines such as SK-BR-3, MCF-7 and C-8161 in vitro. Furthermore, essential oil of T. catharinensis leaves has compounds with antioxidant and anti-inflammatory activities (Almeida et al., 2004; Boligon et al., 2013a). The present study aimed to identify and quantify triterpenes, steroids and phenolics compounds by HPLC/DAD in T. catharinensis leaves and stem bark, as well as evaluating the antimicrobial, antimycobacterial and antiherpetic properties of the fractions
and subfractions of T. catharinensis by a bioguided assay coupled with a high-performance liquid chromatography with diode array detection (HPLC–DAD) analysis.
Materials and methods Chemicals apparatus and general procedures Methanol was of HPLC grade. All extraction reagents, such as dichloromethane, ethyl acetate and n-butanol, were reagent grade. Gallic acid, chlorogenic acid, caffeic acid, rutin, quercetin and kaempferol were acquired from Merck (Darmstadt, Germany). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT), crystal violet solution, acyclovir, penicillin G, streptomycin, ampicillin, cefoperazone, imipenem, fluconazole, amphotericin B, bsitosterol, stigmasterol, lupeol, amyrin and ursolic acid were purchased from Sigma (Sigma Chemical Co., St. Louis, USA). Sabouraud dextrose agar medium, Muller Hinton (MH) agar medium, Tripticase soy broth (TSB), Löwenstein–Jensen medium, Middlebrook 7H9 broth, OADC (oleic acid–albumin–dextrose–catalase) were from Difco (Detroit, MI, USA). Silica Gel Merck 70–230 mesh was used for column chromatography and silica gel Merck GF254nm was used for thin layer chromatography. High-performance liquid chromatography with diode array detection (HPLC–DAD) was performed with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with Shimadzu LC-20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector and LC solution 1.22 SP1 software.
Plant collection Stem bark and leaves of T. catharinensis were collected in Bossoroca (Rio Grande do Sul State of Brazil) in September of 2009 (coordinates 288650 9300 S and 558010 2700 W). A dried voucher specimen is preserved in the herbarium of the Department of Biology at Federal University of Santa Maria by register number SMBD 12355. The parts of the plant were dried at room temperature and powdered in a knife mill. The powders of stem bark (1051.23 g) and leaves (1580.02 g) were macerated separately at room temperature with ethanol 70% for a week with daily shake-up. After filtration, the crude extract (CE) was evaporated under reduced pressure to remove the ethanol. Each extract was suspended in water and partitioned successively with dichloromethane, ethyl acetate and n-butanol, to obtain the dichloromethane fraction (DCM), ethyl acetate fraction (EA) and butanolic fraction (NB). The yield of the extract and fractions was calculated (see Table 1).
Phytochemical screening Compounds in the crude extract and fractions were separated on silica TLC plates (10 cm 10 cm) using a mobile phase of ethyl acetate: 2-propanol: ammonia (17:2:1, v/v/v). Developed TLC plates were assessed for the presence of various phytochemical groups, by utilization of visible and ultraviolet (UV) light, in addition to various spray reagents (Pallant et al., 2012)
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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Table 1 – Yield of crude extract and fractions and contents of total alkaloids of T. catharinensis. Crude extract and fractions
Stem bark Yield (%)
NB EA DCM CE *
Leaves
Alkaloids SE* (mg/g)
8.6 5.0 8.7 9.7
a
5.52 0.29 5.86 1.02 a 2.20 0.17 b 1.09 0.08 c
Yield (%)
Alkaloids SE* (mg/g)
8.5 4.1 9.7 10.3
0.41 0.09 a 3.02 0.05 b 7.34 0.11 c 0.29 0.03 a
SE: standard error; averages followed by different letters in each column differ by Tukey test at p < 0.001.
which were selected to visualize specific classes of compounds (alkaloids, coumarins, essential oils, flavonoids, phenols and sterols/steroids).
Estimation of alkaloids precipitable with Dragendorff's reagent To evaluate the amount of alkaloids precipitable with Dragendorff's reagent in the crude extracts and fractions of T. catharinensis we used a spectrophotometric method described for Boligon et al. (2012b). This method is based on the formation of yellow bismuth complex in nitric acid medium with thiourea that corresponds to the amounts of alkaloids present in plant. Briefly, 2 g of the crude extract was solubilized in ethanol and hydrochloric acid 1% until concentration of 60 mg/mL and pH was maintained at 2–2.5. The 5 mL of the extract solution was added 2 mL Dragendorff's reagent and the precipitate formed was centrifuged (2.400 rpm/30 min). The precipitate was washed with ethanol then treated with 2 mL disodium sulfide solution. The brownish black precipitate formed was then centrifuged and dissolved in 2 mL concentrated nitric acid. This solution was diluted to 10 mL in a standard flask with distilled water. To 1 mL of this solution, 5 mL of thiourea solution (3%) was added. Absorbance was measured at 435 nm against the blank containing nitric acid and thiourea. The equation obtained for the calibration curve of bismuth nitrate pentahydrate solution in the range of 0.01– 0.09 mg/mL was Y = 2.2783x + 0.0361 (r = 0.9997). The experiments were conducted in triplicate.
Alkaloids extraction Powdered drug (2 g) of T. catharinensis leaves was suspended in hydrochloric acid 2N and washed with ethyl ether three times. The aqueous fraction resulting from the partition was alkalized with ammonium hydroxide and again washed with ethyl ether three times. The ethereal fraction was dried resulting in alkaloidal fraction (AF). The residue from the extraction of alkaloids was dissolved in ethanol and investigated by TLC technique. A quantity of 10 mL was applied in plates and eluted with toluene:ethyl acetate:diethylamine (70:20:10) and chloroform:diethylamine (85:15). The plates were observed under UV light (254 and 365 nm) and sprayed with Dragendorff's reagent.
Bioassay-guided fractionation DCM fraction of T. catharinensis leaves (2.5 g) was submitted to a column chromatography on silica gel column (225 g), eluted
with hexane/dichloromethane (1:0–0:1, v/v). Thirty subfractions (50 mL each subfraction) were collected and their composition monitored by thin-layer chromatography (TLC), and grouped on the basis of similarity of the chromatographic profile to furnish two new subfractions, as DCM1 (subfractions 1–12, 45 mg) and DCM2 (subfractions 13–30, 109 mg).
HPLC analysis The phenolic acids analysis of the CE extracts, DCM, EA and NB fractions of T. catharinensis were carried out under gradient conditions using C18 column (4.6 mm 250 mm) packed with 5 mm diameter particles. The mobile phase was solvent A = water:acetic acid (98:2, v/v) and solvent B = acetonitrile. The gradient program was started with 95% of A and 5% of B until 2 min and changed to obtain 25%, 40%, 50%, 60%, 70% and 80% B at 10, 20, 30, 40, 50 and 80 min, respectively, following the method described by Boligon et al. (2012b) with slight modifications. The flow rate was 0.7 mL/min and the injection volume was 40 mL. Detection was performed with three wavelengths, 271 nm for gallic acid, 327 nm for caffeic and chlorogenic acids, and 365 for quercetin, rutin and kaempferol. The mobile phase was filtered through a membrane filter 0.45 mm and then degassed by an ultrasonic sound before use. The fractions and standard solutions (quercetin, rutin, kaempferol, gallic, caffeic and chlorogenic acids) were prepared in the HPLC mobile phase. Standard calibration curves were constructed in the concentration range of 0.012– 0.200 mg/mL. The chromatographic peaks were confirmed by comparing their retention times with those of reference standards and by DAD spectra (200–500 nm); the quantification was performed by peak integration using the external standard method. The subfractions DCM1 and DCM2 from the leaves were analyzed by HPLC–DAD. Chromatographic analyses were carried out in isocratic conditions using C8 column (4.6 mm 150 mm) packed with 5 mm diameter particles, the mobile phase was methanol:water (95:5, v/v) containing 1.0% acetic acid (Boligon et al., 2010). The mobile phase was filtered through a 0.45 mm membrane filter and then degassed by an ultrasonic bath prior to use. Stock solutions of b-sitosterol, stigmasterol, lupeol, amyrin and ursolic acid standard reference were prepared in the HPLC mobile phase at a concentration range of 0.010–0.200 mg/mL. The subfractions DCM1 and DCM2 were also dissolved in the mobile phase. Quantification was carried out by the integration of the peak using external standard method. The flow rate was 1.0 mL/min, injection volume was 40 mL and detection was done at 210 nm. The chromatographic peaks were confirmed by comparing their
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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retention times and UV spectra with those of the reference standards. All chromatographic operations were performed at room temperature and in triplicate. The limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation of the responses and the slope using three independent analytical curves. LOD and LOQ were calculated as 3.3 and 10s/S, respectively, where s is the standard deviation of the response and S is the slope of the calibration curve (Boligon et al., 2013a).
Antimicrobial assay The crude extract and fractions were individually evaluated against Staphylococcus aureus ATCC 29213, Micrococcus sp. ATCC 7468, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis ATCC 7002, Enterococcus faecalis ATCC 51299, Bacillus subtilis ATCC 6633, Saccharomyces cerevisiae ATCC 2601, Candida albicans ATCC 28967, Candida tropicalis (clinical isolate obtained from patients treated at the University Hospital of Santa Maria – HUSM/Brazil), Candida parapsilosis ATCC 90018, Cryptococcus neoformans ATCC 2857, Cryptococcus gattii ATCC 56990, Escherichia coli, Prototheca zopfii, Candida dubliniensis, Candida glabrata, Aeromonas sp., Malassezia pachydermatis, Aspergillus flavus, Aspergillus fumigatus and Fusarium solani (clinical isolate obtained from patients treated at the HUSM). The protocols used in the experiments were M27-A3 for yeasts (CLSI, 2008a,b), M38-A2 (CLSI, 2008a,b) for mold and M07-A9 (CLSI, 2012) for bacteria. The experiments were repeated twice and the results were determined as an average value. Six different dilutions of each fraction (1000, 500, 250, 125, 62.5 and 31.25 mg/mL) were prepared in DMSO. Filamentous fungi were sown on PDA (potato dextrose agar) for 7 days at 30 8C, the inoculum was obtained from a suspension of conidia in sterile saline is made by spectrophotometric reading (0.4–5 104 cells/mL). Bacterial strains were cultured overnight at 37 8C in Mueller–Hinton agar. The suspension of cells was prepared in sterile saline and made reading spectrophotometer (1–2 108 cells/mL). Yeasts were grown on Sabouraud dextrose agar for 48 h at 35 8C. The suspension of cells was prepared in sterile and made reading spectrophotometer (1–5 103 cells/mL) saline. The first column of the plate was reserved for negative control wells (without inoculants) and the last column, for the positive growth control wells (without antimicrobial agents). The MIC was considered as the lowest concentration of the extract or fraction inhibiting the total growth of microorganisms. MIC was detected by lack of visual turbidity (matching the negative growth control). Standard antibiotics (ampicillin, imipenem, and cefoperazone) were used to control the sensitivity of the tested bacteria, whereas fluconazole and amphotericin B were used as control against the tested fungi and the algae.
Determination of percent activity (A%) and microorganisms susceptible index (MSI) These parameters were determined according the equations listed below (Boligon et al., 2013a). A% ¼
100 number of susceptible strains to a specific extract or fraction Total of tested strains
MSI ¼
100 number of extracts of fractions effective against each strain total samples tested
Antimycobacterial assay Antimycobacterial activity was tested against Mycobacterium smegmatis mc2 155 (ATTC700084), M. tuberculosis H37Rv (ATTC25618) and M. avium LR541CDC. The stored mycobacterium were seeded onto Löwenstein–Jensen medium and incubated during 3–5 days. From this culture a portion was removed and placed into Middlebrook 7H9 broth, supplemented with 10% OADC and 0.2% glycerol (MD7H9) and then homogenized in ultrasonic bath for 1 min. The concentration of bacteria in this medium was determined by optical density on spectrophotometer (0.08–0.1 of absorbance at 625 nm) of 0.5 McFarland scale and then diluted with MD7H9 up to 105 CFU/mL for M. smegmatis, to reach the inoculum. The M. tuberculosis and M. avium were used at a concentration of 0.5 McFarland (not diluted) because they are slow-growing. Plant extracts, fractions and isolated alkaloids were dissolved in DMSO, at a concentration of 50 mg/mL and then diluted in MD7H9 until the desired concentrations, beginning the series with 2500 mg/mL. The activity tests were performed using the broth microdilution method, document M7-A6 (NCCLS, 2003) and are presented as minimum inhibitory concentrations (MICs) of extracts and fractions. 100 mL of mycobacterial culture was placed in each well of a microtitre plate, as well as the extracts and fractions at correspondent concentrations. Analysis was done in triplicate; controls were made to the medium and to the mycobacterium, and one blank for each concentration of samples. The plates were incubated during 48 h at 37 8C. The Alamar Blue (Boligon et al., 2012a) or MTT (Sankar et al., 2008) dye was used to check the growth of microorganisms. The Alamar Blue was diluted in a ratio of 1/10 with Tween 80, diluted to 1/9 with sterile water, supplemented with 0.025 mL in each well, considering the MIC into the pit where there was no change in color from purple to pink. MTT solution at 0.5 mg/mL was prepared by the dilution with absolute ethanol up to 1 mg/mL, and then by dilution half to half with a solution of 10% Tween 80. 25 mL of the final solution was added to each plate well, and the well where the drug prevented the color change from yellow to purple was considered the MIC.
Cells and viruses HEp-2 cells were cultivated in minimum essential medium (MEM – GIBCO Invitrogen Corporation, Grand Island, NY, USA) with 10% fetal bovine serum (SFB – GIBCO Invitrogen Corporation, Grand Island, NY, USA) and penicillin, streptomycin and amphotericin B, in the concentrations 100 U/mL, 100 mg/mL and 2.5 mg/mL, respectively. The cells were prepared in 96 well plates for both cytotoxicity and antiviral assays and they were maintained at 37 8C in a 5% CO2 incubator. The strain KOS of Herpes Simplex Virus type 1 (HSV-1) was kindly provided by Dr. Paulo Roehe from the Universidade Federal do Rio Grande do Sul (UFRGS). The viruses stocks were prepared as described in Boligon et al. (2013a) and kept at 70 8C. The virus titration
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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was performed according to the methodology of Reed and Muench (1938).
Cytotoxicity evaluation The cytotoxicity and antiviral tests were performed through the colorimetric assay MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide] according to Boligon et al. (2013a). Shortly, minimum essential medium (MEM) with 10% bovine fetal serum and increasing concentrations of the samples from 1.95 to 250 mg/mL were added to the HEp-2 cells (2 104 cells/well), in 96 well plates, in a total of six repetitions for each concentration. After 72 h of incubation at 37 8C, 5% CO2, the compound MTT (1 mg/mL) was added to the cells. The reagent was removed after 4 h of incubation and 100 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals. The supernatant was transferred to a new plate and readings were taken in an ELISA Spectra Count reader at the wavelength of 540 nm. The viable cells percentage for each compound was calculated by the formula: absorbance of the compound/absorbance of the cell control 100%. The CC50 (50% cytotoxic concentration) was obtained from the concentration–effect curves after linear regression as described in Freitas et al. (2009). The maximum non-toxic concentration (MNCC) of the crude extract, fractions and subfractions that did not cause cytotoxicity was used for the antiviral tests.
Antiviral evaluation HEp-2 cells monolayers were prepared in 96 well plates 24 h before performing the tests. The maintenance media on the preformed monolayer was then removed and replaced by a new media containing 100 mL/well of the virus suspension containing 104 TCID50/mL. The control cells media were replaced by 200 mL/well of MEM media, without virus. After that, 100 mL/well of the MNCC of the samples was added to the wells. The plates were then incubated for 72 h at 37 8C in a 5% CO2 incubator. The readings were taken as described above, for the cytotoxicity assays. Acyclovir (10 mg/mL) was used as positive control for the HSV-1 inhibition. The viral inhibition for each compound was calculated according to the formula: (absorbance of the compound absorbance of the viral control)/(absorbance of the cell control absorbance of the viral control) 100%. The IC50 (50% inhibitory concentration) was obtained from the concentration–effect curves after linear regression as described in Freitas et al. (2009). The selectivity index (IS) was calculated from the formula CC50/IC50.
Data analysis CC50 and IC50 values were obtained from linear regression analysis of concentration–effect curves. Results of the HPLC– DAD quantification were considered statistically significant by Tukey test at alpha 0.001. Data of biofilms assay were analyzed using one-way analysis of variance (ANOVA) followed by the Duncan's multiple range test when appropriate. Results were expressed as the mean standard deviation (SD) and evaluated at the significance level 2alpha = 0.0001.
Results and discussion The yields of crude extract, and dichloromethane, ethyl acetate and n-butanol-soluble fractions obtained from leaves and stem bark are given in Table 1. The highest yield was obtained from crude extract of the leaves (10.3%). The quantification of alkaloids for leaves followed the order: DCM fraction > EA fraction > NB fraction > CE; and for stem bark: EA fraction > NB fraction > DCM fraction > CE (Table 1). When comparing the results obtained, we may observe that the dichloromethane fraction of leaves showed the higher content of total alkaloids (7.34 0.11 mg/g). Purified fraction of alkaloids (AF) was obtained from the leaves of T. catharinensis and showed positive results to alkaloids according to Wagner et al. (2006) and Gonçalves et al. (2011) and the thin layer chromatography revealed the presence of indolic alkaloids classes (Table 2). These results are in agreement with previously published that reported on the presence of indolic alkaloids in large numbers in the species (Pereira et al., 2004, 2008). HPLC profile of crude extract and fractions from T. catharinensis was acquired (Figs. 1 and 2). Stem bark and leaves of T. catharinensis contain other minor compounds in addition to gallic acid (retention time – tR 14.7 min, peak 1), chlorogenic acid (tR = 22.9 min, peak 2), caffeic acid (tR = 33.1 min, peak 3), rutin (tR = 45.2 min, peak 4), quercetin (tR = 56.2 min, peak 5) and kaempferol (tR = 68.5 min, peak 6). Results published previously about T. catharinensis and other Tabernaemontana species support these findings, given that some authors reported the presence of alkaloids, triterpenoids and steroids (Pereira et al., 2008), flavonoids, phenylpropanoids, phenolic acids (Pallant and Steenkamp, 2008) and essential oils (Boligon et al., 2013b). In this work, the fractions with the highest amount of phenolic acids and flavonoids were NB (22.12% for stem bark and 20.45% for leaves) and EA (21.98% for leaves and 19.54 for stem bark); results of quantification are shown in Table 3. The respective standard solutions calibrations curves were: Y = 53985x + 1020.6 (r = 0.9987) for gallic acid; Y = 87603x + 4519.1 (r = 0.9995) for chlorogenic acid; Y = 81846x + 1093.4 (r = 0.9999) for caffeic acid; Y = 96030x + 7627.3 (r = 0.9979) for rutin; Y = 50833x + 4741.7 (r = 0.9899) for quercetin and Y = 87603x + 4519.5 (r = 0.9993) for kaempferol. Antimicrobial activities of CE extracts, DCM, EA and NB fractions of the T. catharinensis were tested (see Table 4). Regarding the Gram-positive bacteria, very good results were
Table 2 – Substances characterized by TLC technique in alkaloid extract. Mobile phase
Characteristic
Rf
Toluene:ethyl acetate:diethylamine (70:20:10)
254 nm: orange stain 365 nm: blue fluorescent stain
0.52 0.31
Chloroform:diethylamine (85:15)
254 nm: orange stain 365 nm: blue fluorescent stain
0.74 0.19
According Wagner et al. (2006).
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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Fig. 1 – High performance liquid chromatography phenolics profile of CE (a), DCM (b), EA (c) and NB (d) of T. catharinensis stem bark. Gallic acid (peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3), rutin (peak 4), quercetin (peak 5) and kaempferol (peak 6). Chromatographic conditions described in the experimental section.
Fig. 2 – High performance liquid chromatography phenolics profile of CE (a), DCM (b), EA (c) and NB (d) of T. catharinensis leaves. Gallic acid (peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3), rutin (peak 4), quercetin (peak 5) and kaempferol (peak 6).
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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Table 3 – Compounds determined by HPLC in Tabernaemontana catharinensis (%); LOD and LOQ variations for compounds (mg/mL). T. catharinensis
CE
DCM
EA
NB
LOD
LOQ
Stem bark Gallic acid Chlorogenic acid Caffeic acid Rutin Quercetin Kaempferol
1.72 0.03 a 1.24 0.01 b 2.35 0.06 c 1.18 0.09 b 0.80 0.13 d 0.39 0.08 e
2.46 0.04 a 2.30 0.05 a 2.21 0.12 a – – 0.43 0.03 b
4.05 0.06 a 3.74 0.09 b 4.31 0.13 c 0.99 0.17 d 5.38 0.10 e 1.07 0.02 d
4.26 0.14 a 4.49 0.05 a 2.83 0.09 b 6.30 0.13 c 2.91 0.05 b 1.33 0.08 d
0.022 0.019 0.027 0.034 0.030 0.024
0.067 0.057 0.082 0.103 0.091 0.072
Leaves Gallic acid Chlorogenic acid Caffeic acid Rutin Quercetin Kaempferol
0.87 0.01 a 1.09 0.02 b 1.71 0.02 c 1.43 0.05 c 0.45 0.01 d 0.49 0.03 d
2.39 0.03 a 1.67 0.01 b 3.63 0.05 c – – 0.83 0.02 d
4.72 0.01 a 3.29 0.04 b 4.68 0.05 a 3.31 0.02 b 4.01 0.03 c 1.97 0.01 d
3.89 0.02 a 4.52 0.01 b 3.93 0.02 a 4.67 0.03 b 2.36 0.05 c 1.08 0.01 d
0.022 0.019 0.027 0.034 0.030 0.024
0.067 0.057 0.082 0.103 0.091 0.072
Results are expressed as mean standard deviation (SD) of three determinations. Different letters in each column represent significant differences using analysis of variance followed by Tukey test ( p values < 0.001 were considered as significant). LOD, limit of detection; LOQ, limit of quantification.
obtained against S. aureus, Micrococcus sp., E. faecalis and B. subtilis for DCM and NB fractions, showing the minimal inhibitory concentration (MIC) ranging from 31.25 to 1000 mg/ mL. Considering the Gram-negative bacteria, only NB fraction of leaves was effective against P. mirabilis (MIC = 62.5 mg/mL)
and NB fractions of leaves and stem bark showed MIC of 250 mg/mL for Aeromonas sp. In addition, only S. cerevisiae was particularly vulnerable (MICs = 31.25 and 62.5 mg/mL), followed by C. neoformans and A. fumigatus (MIC = 250–500 mg/mL). The set of data indicated that DCM fractions of the leaves and stem
Table 4 – Minimum inhibitory concentration–MIC (mg/mL) values for crude extracts and fractions of Tabernaemontana catharinensis leaves and stem bark. Microorganisms
S. buxifolia Stem bark
Leaves
CE
DCM
EA
NB
CE
Gram-positive S. aureus Micrococcus sp. E. faecalis B. subtilis
>1000 500 >1000 >1000
1000 250 500 1000
>1000 >1000 >1000 >1000
>1000 500 1000 >1000
>1000 >1000 >1000 >1000
Gram-negative Aeromonas sp. K. pneumoniae P. mirabilis E. coli P. aeruginosa
>1000 >1000 >1000 >1000 >1000
>1000 >1000 >1000 >1000 >1000
>1000 >1000 >1000 >1000 >1000
250 >1000 >1000 >1000 >1000
>1000 >1000 >1000 >1000 >1000
Fungi C. albicans C. tropicalis C. glabrata C. parapsilosis C. dubliniensis C. neoformans C. gattii S. cerevisiae M. pachydermatis A. flavus A. fumigatus F. solani P. zopfii
>1000 >1000 >1000 >1000 >1000 >1000 >1000 1000 >1000 >1000 >1000 >1000 >1000
1000 >1000 500 >1000 >1000 500 >1000 62.5 >1000 1000 250 >1000 >1000
>1000 >1000 >1000 >1000 >1000 1000 >1000 500 >1000 >1000 >1000 >1000 >1000
>1000 >1000 >1000 >1000 >1000 250 >1000 500 >1000 >1000 250 >1000 >1000
>1000 >1000 >1000 >1000 >1000 >1000 >1000 500 >1000 >1000 >1000 >1000 >1000
Control EA
NB
mg/mL
>1000 >1000 >1000 >1000
>1000 31.25 250 >1000
Amp. 2.0 Amp. 8.0 Imi. 1.0 Imi. 0.6
>1000 >1000 >1000 >1000 >1000
>1000 >1000 >1000 >1000 >1000
250 >1000 62.5 >1000 >1000
Amp. 2.0 Imi. 1.0 Imi. 2.0 Amp. 8.0 Cpz. 16.0
1000 >1000 1000 >1000 >1000 250 >1000 31.25 >1000 500 500 >1000 >1000
>1000 >1000 >1000 >1000 >1000 >1000 >1000 500 >1000 >1000 >1000 >1000 >1000
>1000 >1000 >1000 >1000 >1000 >1000 >1000 500 >1000 >1000 1000 >1000 >1000
Flu. 16.0 Flu. 32.0 Flu. 32.0 Flu. 8.0 Flu. 16.0 Flu. 8.0 Flu. 32.0 Flu. 2.0 Flu. 16.0 Flu. 8.0 Flu. 2.0 Amph. 2.0 Amph. 0.5
DCM 500 31.25 62.5 500
CE, crude extract; DCM, dichloromethane fraction; EA, ethyl acetate fraction and NB, n-butanolic fraction. Controls: Amp, ampicillin; Cpz, cefoperazone; Imi, imipenem; Flu, fluconazole; Amph, amphotericin B.
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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acids and flavonoids (Table 3), in which additionally we emphasize the presence of rutin, quercetin and kaempferol, these compounds possess antimicrobial activity well described in the literature (Oliveira et al., 2007; Boligon et al., 2013a). They may act as antibacterial agents by means of at least three or more mechanisms: inhibition of nucleic acid synthesis, disruption of bacterial membranes and/or inhibition of energy metabolism (Cowan, 1999). Some compounds present in various plants are already identified in T. catharinensis, such as betulinic acid, ursolic acid, amyrin, lupeol, sitosterol and stigmasterol, and showed notable antifungal and antibacterial capacity (Jain et al., 2001; Patra and Thatoi, 2011). These results agree with Pallant et al. (2012), which describe good activity for the crude extract and alkaloidal fraction of T. elegans, particularly against mycobacterium (M. smegmatis and M. tuberculosis) and Gram-positive bacteria (B. subtilis, E. faecalis and S. aureus). DCM fraction of T. catharinensis leaves was the most active fraction in the antimicrobial assay (Table 4), and hence was submitted to chromatographic separation processes. Successive column chromatographic procedures with DCM fraction resulted in obtained two subfractions (DCM1 and DCM2). These subfractions were analyzed by HPLC/DAD and presented triterpenoids/steroids mixtures (Fig. 3). In DCM1 subfractions were identified and they quantified the mixture of lupeol (retention time, tR = 3.7 min), amyrin (tR = 5.1 min) and ursolic acid (tR = 6.9 min); and DCM2 the mixture of bsitosterol (tR = 7.3 min) and stigmasterol (tR = 9.0 min) (Fig. 4).
bark were the most effective ones in the antimicrobial assay; good results were obtained against the fungi and the Grampositive bacteria. Besides the determination of MIC, other methods could be used to express the antimicrobial and antifungal effectiveness of the fractions. The percent activity (A%) and the microorganisms susceptibility index (MSI) may help to choose the better plant or the better fraction to be thoughtfully examined (Boligon et al., 2013a). Percent activity indicates DCM as the effective fractions (45.45%), whereas MSI indicated S. cerevisiae as the most sensitive microorganism (MSI = 100%), showing sensibility for the CE, EA, NB and principally, to DCM fractions followed by Micrococcus sp. (MSI = 62.5%); E. faecalis, C. neoformans and A. fumigatus (MSI = 50.0%); S. aureus, B. subtilis, Aeromonas sp., C. albicans, C. glabrata and A. flavus (MSI = 25.0%); and P. mirabilis (MSI = 12.5%) with intermediate sensibility; and K. pneumoniae, E. coli, P. aeruginosa, C. tropicalis, C. parapsilosis, C. dubliniensis, C. gattii, M. pachydermatis, F. solani and P. zopfii as resistant microorganisms (MSI = 0%). Antimicrobial activity of T. catharinensis DCM fractions can be mainly attributed to the synergistic action of terpenes, alkaloids and phenols compounds previously described for this species (Pallant and Steenkamp, 2008; Pereira et al., 2008), since these classes of secondary metabolites have antifungal and antibacterial activity recognized (Harborne and Williams, 2000; Tabopda et al., 2009; Chen et al., 2008; Pallant et al., 2012). Furthermore, the antimicrobial properties shown by the NB fraction may be attributed to an interaction between phenolic
29
30
CH3
20
H
20
17
17
28 1
1
5
5
7
7
HO
HO
23
24
1
2 CH3
29 28 21
CH3
22
20
27 24
20 17
17
COOH
25 26
1 5
1
7
HO
5 24
7
HO
23
3
4 = ∆5 5 = ∆5,22
Fig. 3 – Chemical structures of lupeol (1), b-amyrin (2), ursolic acid (3), b-sitosterol (4) and stigmasterol (5). Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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Fig. 4 – High performance liquid chromatography profile of DCM1 (a) and DCM2 (b) subfractions from the T. catharinensis leaves. Lupeol (peak 1), amyrin (peak 2), ursolic acid (peak 3), b-sitosterol (peak 4) and stigmasterol (peak 5). Chromatographic conditions described in the experimental section.
showed activity, but weak. In relation to T. catharinensis leaves, all tested fractions and subfractions were active against M. smegmatis with MIC ranged from 19.53 to 156.25 mg/mL. The lowest MIC against M. smegmatis was observed for DCM fraction (MIC = 19.53 mg/mL), followed by AF and DCM1 (MIC = 39.06 mg/mL) and DCM2 (MIC = 78.12 mg/mL). However, only DCM and AF fractions of leaves showed activity against M. tuberculosis and M. avium, and presented MIC 312.50 and 625.00 mg/mL, respectively (see Table 6). Tosun et al. (2004) considered inactive the plant extracts that could not prevent growth of Mycobacterium up to concentration of 200.00 mg/mL. In our study, we consider all fractions and subfractions tested promising (except CE leaves). These results support the hypothesis that the presence of triterpenes/sterols and alkaloids, are primarily responsible for the antimicrobial activity of T. catharinensis; these compounds possess antimycobacterial activity well described (Cantrell et al., 2001; Pallant et al., 2012). Isolated alkaloids from various Tabernaemontana species, conoduramine, coronaridine, and voacamine, possess strong antibacterial activity against Grampositive bacteria (Van Beek et al., 1984). Furthermore, the alkaloids ibogamine and voacangine have proven antimycobacterial activity (Rastogi et al., 1998). These compounds, however, have been previously identified in T. catharinensis (Pereira et al., 2008), suggesting that the pharmacophore responsible for the antimycobacterial properties may be related to the alkaloids present in this species. Pallant et al. (2012) describe the antimycobacterial activity for crude extract
For quantification of the compounds in mixtures, calibration curves were constructed with reference standards, lupeol: y = 23157x + 1178.7 (r = 0.9995), amyrin: y = 16511x + 1438.3 (r = 0.9998), ursolic acid: y = 41118x + 1389.5 (r = 0.9983), bsitosterol: y = 24085x + 1210.9 (r = 0.9989) and stigmasterol: y = 19108x + 1750.3 (r = 0.9997). Table 5 shows the quantification of compounds in the subfractions. DCM1 is rich in triterpenes (lupeol, amyrin and ursolic acid) that represent 75.12% and DCM2 is comprised 85.78% of steroids (b-sitosterol and stigmasterol). Several researchers have utilized nonpathogenic, fastgrowing Mycobacterium species in rapid and easy screens for antimycobacterial activity in plant extract and pure plantderived compounds (Kuete et al., 2008; Boligon et al., 2012a). In one study realized by McGaw et al. (2008) it was concluded that M. smegmatis was better predictor of activity against pathogenic M. tuberculosis. However, more research is needed to validate the use of non-pathogenic species such as M. smegmatis as models for detecting activity of plant derived extracts against pathogenic M. tuberculosis. The results obtained in antimycobacterial assay performed with CE, fractions (DCM, EA and NB), subfractions (DCM1 and DCM2) and alkaloidal fraction (AF) of the T. catharinensis are summarized in Table 6. Fractions of the stem bark showed activity against M. smegmatis being DCM more active fraction (MIC = 39.06 mg/mL), followed by CE, EA and NB (MIC = 156.25 mg/mL); for M. tuberculosis the order of activity was CE and NB > DCM > EA; and for the M. avium only DCM
Table 5 – Subfractions composition of T. catharinensis leaves. Compounds
Lupeol Amyrin Ursolic acid b-Sitosterol Stigmasterol
DCM1
DCM2
LOD
LOQ
Quantities (%)
Quantities (%)
(mg/mL)
(mg/mL)
25.09 0.13 a 16.85 0.07 b 33.18 0.02 c – –
– – – 26.03 0.15 a 59.75 0.09 d
0.017 0.023 0.025 0.014 0.030
0.052 0.069 0.074 0.042 0.091
Results are expressed as means standard deviations of three determinations. Different letters represent significant differences using analysis of variance followed by Tukey test ( p values < 0.001 were considered as significant). LOD, limit of detection (mg/mL); LOQ, limit of quantification (mg/mL).
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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Table 6 – Minimal inhibition concentration (mg/mL) of fractions and isolated of Tabernaemontana catharinensis against M. smegmatis, M. tuberculosis and M. avium. Extract/fractions M. smegmatis M. tuberculosis M. avium Stem bark CE DCM EA NB Leaves CE DCM EA NB AF DCM1 DCM2
156.25 39.06 156.25 156.25
312.50 625.00 >1250.00 312.50
>1250.00 625.00 >1250.00 >1250.00
2500.00 19.53 156.25 156.25 39.06 39.06 78.12
ND 312.50 >1250.00 >1250.00 312.50 >1250.00 >2500.00
ND 625.00 >1250.00 >1250.00 625.00 >1250.00 >2500.00
AF, alkaloidal fraction; ND, not determined.
and alkaloidal fraction of T. elegans against M. smegmatis and M. tuberculosis showing a MIC = 32 mg/mL for alkaloidal fraction, and additive antibacterial effects were observed when the alkaloidal fraction was combined with antibiotics. Essential oils and extracts from a great diversity of plants have been examined concerning its ability to directly inactivate viruses or to interfere with the replication of these microorganisms (Chiang et al., 2002; Knockaert et al., 2002; Astani et al., 2011; Scala et al., 2011). The natural products are considered an important source of potential antiviral candidates due to the abundance of these products, as well as their chemical diversity and low toxicity (Chiang et al., 2002; Mukhtar et al., 2008). The antiviral activity of the crude extracts, fractions and subfractions of the T. catharinensis leaves and stem bark was tested against HSV-1. The results for the cytotoxicity and anti-HSV assay are shown in Table 7.
Table 7 – Cytotoxicity and anti-HSV-1 activity of Tabernaemontana catharinensis. Sample
Cytotoxicity CC50
Anti-HSV-1 activity IC50
SI
Stem bark CE DCM EA NB
62.82 4.75 4.21 1.42 26.85 3.18 119.38 15.6
NI 2.62 1.89 2.88 1.21 NI
– 1.61 9.32 –
Leaves CE DCM EA NB AF
59.53 4.56 46.75 5.29 89.03 9.21 119.35 9.71 116.20 5.50
NI 0.6 0.03 2.21 1.24 4.12 2.45 10.86 4.75
– 77.92 40.27 28.97 10.71
NI 48.34 4.17 1.50 0.30
– 12.28 >186.66
DCM subfractions (leaves) DCM1 49.69 4.91 593.88 14.24 DCM2 Acyclovir (10 mg/mL) >280.00
CE, crude extract; DCM, dichloromethane fraction; EA, ethyl acetate fraction and NB, n-butanolic fraction. CC50, 50% cytotoxic concentration (mg/mL). IC50, 50% inhibitory concentration (mg/mL). SI, selectivity index (=CC50/IC50); NI, no inhibitory activity.
CE of both bark and leaves of the T. catharinensis did not show any antiviral activity; however, the EA fraction of the stem bark and all the fractions and the DCM2 subfraction of the leaves of this plant exhibited values of IC50 under 48.34 mg/mL toward HSV-1 (Table 7). The IC50 values obtained with this plant are lower than the value of 100 mg/mL recommended by Cos et al. (2006) in order to consider a product as a potential candidate to antiviral. Indeed, the IC50 values varied from 0.6 to 10.86 mg/mL with most of them being lower than 4.5 mg/mL (Table 7). The best antiviral activity was obtained from the DCM fraction of the leaves of the T. catharinensis (Table 7). As already mentioned, the DCM fraction was subfractionated in DCM1, which did not show antiviral activity; and DCM2 that was effective against HSV-1 although showing less efficiency than the fraction as a whole (Table 7). A high content of b-sitosterol and stigmasterol was identified in this sub-fraction (85.78%), which could suggest a major role for those compounds in the anti-HSV1 activity. Nonetheless, the considerable higher activity of the fraction as a whole indicates that there is some important synergy among the compounds that is lost when it is fractioned (Fig. 4 and Table 5). Conversely, the presence of antiviral activity in all the fractions from the T. catharinensis and the absence of such action in the crude extract suggest that some of the compounds could have an antagonistic behavior. Actually, the phenomena of synergy and/or antagonism among compounds have been described for natural products (Wagner and Ulrich-Merzenich, 2009). The antiviral activity of isolated chemical compounds of extracts and essential oils from diverse natural products was already demonstrated (Chiang et al., 2002; Knockaert et al., 2002; Astani et al., 2011; Scala et al., 2011). Since all the fractions from the leaves showed antiviral activity, it is possible that in each case a combination of different chemical compounds was responsible for the activity. The presence of high amounts of polyphenolic compounds in some plant extracts was considered the main reason for its antiviral activity (Serkedjieva and Ivancheva, 1999). The EA and NB fractions of the T. catharinensis were rich in polyphenolic compounds (Figs. 1 and 2, Table 3) and it can be at least partially responsible for the high SI values observed in those cases (Table 7). On the other hand, the indole alkaloids present in T. catharinensis identified by Pereira et al. (2008) may be the reason of the antiviral activity of the alkaloidal fraction (AF). Scala et al. (2011) described the interference of indole-3,4-diones alkaloids with HSV-1 replication and reasoned that it could be due to the structural features of this compound that are similar to certain inhibitors of cyclin-dependent kinases (CDKs), which avoid viral replication in vitro (Knockaert et al., 2002). In addition, a recent survey by Boligon et al. (2013b) reported the presence of b-caryophyllene and bcaryophyllene oxide (56.87 and 2.51%, respectively) in the essential oil of T. catharinensis leaves. These sesquiterpenes are present in many essential oils, and the b-caryophyllene exhibited anti-HSV 1 properties that were attributed to its oxide-derived form (Astani et al., 2011). Then, considering all that were described above, the antiviral activity demonstrated by the diverse fractions and sub-fraction of the T. catharinensis cannot be related to any
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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particular compound; instead, such activity may be due to a combination of different compounds at each fraction and subfraction. Further studies to demonstrate the antiviral mechanisms of the fractions and sub-fraction that demonstrated the best anti-HSV1 activity are worthwhile.
Conclusion The fractions and subfractions of T. catharinensis demonstrated effective antiviral, antimicrobial and antimycobacterial properties, in vitro. High levels of steroids, alkaloids and phenolic compounds in the fractions suggest that these compounds are responsible for the antiviral and antimicrobial activities described here. Results obtained in the present study highlights the importance of the research of the natural products as a source for new antiviral and antimicrobial therapy.
Conflict of interest The authors have no conflicts of interest.
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Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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Original Research Article
HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC Aline Augusti Boligon a,*, Mariana Piana a, Thaís Felli Kubiça b, Débora Nunes Mario c, Tanise Vendruscolo Dalmolin d, Pauline Cordenonsi Bonez d, Rudi Weiblen b, Luciane Lovato b, Sydney Hartz Alves c, Marli M.A. Campos d, Margareth Linde Athayde a a
Phytochemical Laboratory, Department of Pharmacy Industrial, Federal University of Santa Maria (UFSM), 97105-900 Santa Maria, RS, Brazil b Virology Laboratory, Department of Veterinary Preventive Medicine, Federal University of Santa Maria (UFSM), 97105-900 Santa Maria, RS, Brazil c Mycological Research Laboratory, Department of Microbiology and Parasitology, Federal University of Santa Maria (UFSM), 97105-900 Santa Maria, RS, Brazil d Mycobacterial Research Laboratory, Department of Clinical and Toxicological Analysis, Federal University of Santa Maria (UFSM), Santa Maria, RS, Brazil
article info
abstract
Article history:
Antimicrobial, antimycobacterial and antiviral activities of crude extracts, fractions and
Received 29 November 2013
subfractions of Tabernaemontana catharinensis were evaluated. Best antimicrobial results
Received in revised form
occurred with the dichloromethane (DCM) and butanolic (NB) fractions against Staphylococ-
15 January 2014
cus aureus, Micrococcus sp., Enterococcus faecalis and Bacillus subtilis (minimal inhibitory
Accepted 20 January 2014
concentration, MIC = 31.25–1000 mg/mL). Considering the Gram-negative bacteria, only NB
Available online xxx
fraction was effective against Proteus mirabilis and Aeromonas sp. (MIC = 62.5 mg/mL and 250 mg/mL, respectively). In addition, the fungi Candida albicans, Candida glabrata, Cryptococcus
Keywords:
neoformans, Saccharomyces cerevisiae, Aspergillus flavus and Aspergillus fumigatus were particu-
Apocynaceae
larly vulnerable for DCM fraction (MIC = 31.25–1000 mg/mL). The fractions and subfractions
Cobrina
were effective against Mycobacterium smegmatis (MIC = 19.53–156.25 mg/mL). DCM (selectivity
HSV-1
index – SI = 77.92), ethyl acetate (EA) (SI = 40.27) and NB (SI = 28.97) fractions from the leaves
HPLC
exhibited a potential antiviral activity toward Herpes Simplex Virus type 1 whereas DCM2
Mycobacterium
subfraction from leaves (SI = 12.28) and alkaloidal fraction (AF) (10.71) maintained this good activity. Steroids, terpenoids and phenolics compounds were identified by high-performance liquid chromatography with diode array detection (HPLC/DAD) and may be partially responsible for the antimicrobial and antiherpes activities observed. The results obtained in this study showed that T. catharinensis has antimicrobial and anti-herpetic activities and that these properties are reported for the first time for this species. # 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
* Corresponding author. Tel.: +55 32209618. E-mail address: [email protected] (A.A. Boligon). 1214-021X/$ – see front matter # 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.jab.2014.01.004 Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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Introduction Compounds with antimicrobial activity have been investigated and the searches for new anti-infections agents showed that plant extracts, essential oils or isolated compounds such as alkaloids, flavonoids, diterpenes, triterpenes and sesquiterpene lactones are potential sources of bioactive compounds (Rios and Recio, 2005; Boligon et al., 2013a). Remedies derived from plants are still being used worldwide in the traditional medicine for the treatment of a wide range of diseases, including fungal, bacterial, mycobacterial and virus infections (Khan et al., 2005; Mohamad et al., 2011; Boligon et al., 2012a, 2013a). In this new millennium, research of chemical entities from plant species and other natural resources for activity against Mycobacterium species had intensified with the development of easier, faster and safer screening techniques. A good number of extracts and pure compounds isolated from these resources have been reported to exhibit considerable in vitro inhibitory activity against Mycobacterium tuberculosis and its related species (Copp and Pearce, 2007; Negi et al., 2010). Successful inhibition of M. tuberculosis by ethnobotanically selected plants in many countries has been published (Rastogi et al., 1998; Gautam et al., 2007; Askun et al., 2009; Boligon et al., 2012a; Pallant et al., 2012). Tabernaemontana catharinensis A. DC (syn. Peschiera catharinensis A. DC. Miers) belongs to family Apocynaceae, a family consisting of 415 genera and 4555 species. Recently, about 99 species from Tabernaemontana genus were reported and 44 species occur in America. Tabernaemontana species are well known for indole alkaloids and several pentacyclic triterpenoids with approximately 240 structurally different bases already described. These types of compounds are responsible for many pharmacological activities such as: antileishmanial; trypanocidal, antioxidant, antibacterial; antitumoral; hypoglycaemic, analgesic and cardiotonic activities (Van Beek et al., 1984; Pereira et al., 2008; Lim et al., 2009). T. catharinensis is popularly known as 'jasmim' (jasmine), 'leiteira de dois irmãos' (milkweed), and 'casca de cobra' (snake skin) and it occurs in Argentine, Uruguay, Paraguay and Southern Brazil. In folk medicine, it is used as an antidote for snakebites, to relieve toothache, as a vermifuge to eliminate warts, and as an anti-inflammatory (Leeuwenberg, 1994; Almeida et al., 2004; Pereira et al., 2004). Studies conducted by Batina et al. (1997, 2000) demonstrated that substances isolated and aqueous extracts of the root of T. catharinensis can inhibit the lethal and myotoxic activity of Crotalus durissus terrificus venom (South American Rattlesnake, cascavel). Substances with trypanocidal and antitumoral activities were also isolated and described from this plant (Pereira et al., 1999; Almeida et al., 2004). In addition, the aqueous extract of T. catharinensis was shown to have a potent cytotoxic action on human tumor lines such as SK-BR-3, MCF-7 and C-8161 in vitro. Furthermore, essential oil of T. catharinensis leaves has compounds with antioxidant and anti-inflammatory activities (Almeida et al., 2004; Boligon et al., 2013a). The present study aimed to identify and quantify triterpenes, steroids and phenolics compounds by HPLC/DAD in T. catharinensis leaves and stem bark, as well as evaluating the antimicrobial, antimycobacterial and antiherpetic properties of the fractions
and subfractions of T. catharinensis by a bioguided assay coupled with a high-performance liquid chromatography with diode array detection (HPLC–DAD) analysis.
Materials and methods Chemicals apparatus and general procedures Methanol was of HPLC grade. All extraction reagents, such as dichloromethane, ethyl acetate and n-butanol, were reagent grade. Gallic acid, chlorogenic acid, caffeic acid, rutin, quercetin and kaempferol were acquired from Merck (Darmstadt, Germany). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT), crystal violet solution, acyclovir, penicillin G, streptomycin, ampicillin, cefoperazone, imipenem, fluconazole, amphotericin B, bsitosterol, stigmasterol, lupeol, amyrin and ursolic acid were purchased from Sigma (Sigma Chemical Co., St. Louis, USA). Sabouraud dextrose agar medium, Muller Hinton (MH) agar medium, Tripticase soy broth (TSB), Löwenstein–Jensen medium, Middlebrook 7H9 broth, OADC (oleic acid–albumin–dextrose–catalase) were from Difco (Detroit, MI, USA). Silica Gel Merck 70–230 mesh was used for column chromatography and silica gel Merck GF254nm was used for thin layer chromatography. High-performance liquid chromatography with diode array detection (HPLC–DAD) was performed with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with Shimadzu LC-20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector and LC solution 1.22 SP1 software.
Plant collection Stem bark and leaves of T. catharinensis were collected in Bossoroca (Rio Grande do Sul State of Brazil) in September of 2009 (coordinates 288650 9300 S and 558010 2700 W). A dried voucher specimen is preserved in the herbarium of the Department of Biology at Federal University of Santa Maria by register number SMBD 12355. The parts of the plant were dried at room temperature and powdered in a knife mill. The powders of stem bark (1051.23 g) and leaves (1580.02 g) were macerated separately at room temperature with ethanol 70% for a week with daily shake-up. After filtration, the crude extract (CE) was evaporated under reduced pressure to remove the ethanol. Each extract was suspended in water and partitioned successively with dichloromethane, ethyl acetate and n-butanol, to obtain the dichloromethane fraction (DCM), ethyl acetate fraction (EA) and butanolic fraction (NB). The yield of the extract and fractions was calculated (see Table 1).
Phytochemical screening Compounds in the crude extract and fractions were separated on silica TLC plates (10 cm 10 cm) using a mobile phase of ethyl acetate: 2-propanol: ammonia (17:2:1, v/v/v). Developed TLC plates were assessed for the presence of various phytochemical groups, by utilization of visible and ultraviolet (UV) light, in addition to various spray reagents (Pallant et al., 2012)
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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Table 1 – Yield of crude extract and fractions and contents of total alkaloids of T. catharinensis. Crude extract and fractions
Stem bark Yield (%)
NB EA DCM CE *
Leaves
Alkaloids SE* (mg/g)
8.6 5.0 8.7 9.7
a
5.52 0.29 5.86 1.02 a 2.20 0.17 b 1.09 0.08 c
Yield (%)
Alkaloids SE* (mg/g)
8.5 4.1 9.7 10.3
0.41 0.09 a 3.02 0.05 b 7.34 0.11 c 0.29 0.03 a
SE: standard error; averages followed by different letters in each column differ by Tukey test at p < 0.001.
which were selected to visualize specific classes of compounds (alkaloids, coumarins, essential oils, flavonoids, phenols and sterols/steroids).
Estimation of alkaloids precipitable with Dragendorff's reagent To evaluate the amount of alkaloids precipitable with Dragendorff's reagent in the crude extracts and fractions of T. catharinensis we used a spectrophotometric method described for Boligon et al. (2012b). This method is based on the formation of yellow bismuth complex in nitric acid medium with thiourea that corresponds to the amounts of alkaloids present in plant. Briefly, 2 g of the crude extract was solubilized in ethanol and hydrochloric acid 1% until concentration of 60 mg/mL and pH was maintained at 2–2.5. The 5 mL of the extract solution was added 2 mL Dragendorff's reagent and the precipitate formed was centrifuged (2.400 rpm/30 min). The precipitate was washed with ethanol then treated with 2 mL disodium sulfide solution. The brownish black precipitate formed was then centrifuged and dissolved in 2 mL concentrated nitric acid. This solution was diluted to 10 mL in a standard flask with distilled water. To 1 mL of this solution, 5 mL of thiourea solution (3%) was added. Absorbance was measured at 435 nm against the blank containing nitric acid and thiourea. The equation obtained for the calibration curve of bismuth nitrate pentahydrate solution in the range of 0.01– 0.09 mg/mL was Y = 2.2783x + 0.0361 (r = 0.9997). The experiments were conducted in triplicate.
Alkaloids extraction Powdered drug (2 g) of T. catharinensis leaves was suspended in hydrochloric acid 2N and washed with ethyl ether three times. The aqueous fraction resulting from the partition was alkalized with ammonium hydroxide and again washed with ethyl ether three times. The ethereal fraction was dried resulting in alkaloidal fraction (AF). The residue from the extraction of alkaloids was dissolved in ethanol and investigated by TLC technique. A quantity of 10 mL was applied in plates and eluted with toluene:ethyl acetate:diethylamine (70:20:10) and chloroform:diethylamine (85:15). The plates were observed under UV light (254 and 365 nm) and sprayed with Dragendorff's reagent.
Bioassay-guided fractionation DCM fraction of T. catharinensis leaves (2.5 g) was submitted to a column chromatography on silica gel column (225 g), eluted
with hexane/dichloromethane (1:0–0:1, v/v). Thirty subfractions (50 mL each subfraction) were collected and their composition monitored by thin-layer chromatography (TLC), and grouped on the basis of similarity of the chromatographic profile to furnish two new subfractions, as DCM1 (subfractions 1–12, 45 mg) and DCM2 (subfractions 13–30, 109 mg).
HPLC analysis The phenolic acids analysis of the CE extracts, DCM, EA and NB fractions of T. catharinensis were carried out under gradient conditions using C18 column (4.6 mm 250 mm) packed with 5 mm diameter particles. The mobile phase was solvent A = water:acetic acid (98:2, v/v) and solvent B = acetonitrile. The gradient program was started with 95% of A and 5% of B until 2 min and changed to obtain 25%, 40%, 50%, 60%, 70% and 80% B at 10, 20, 30, 40, 50 and 80 min, respectively, following the method described by Boligon et al. (2012b) with slight modifications. The flow rate was 0.7 mL/min and the injection volume was 40 mL. Detection was performed with three wavelengths, 271 nm for gallic acid, 327 nm for caffeic and chlorogenic acids, and 365 for quercetin, rutin and kaempferol. The mobile phase was filtered through a membrane filter 0.45 mm and then degassed by an ultrasonic sound before use. The fractions and standard solutions (quercetin, rutin, kaempferol, gallic, caffeic and chlorogenic acids) were prepared in the HPLC mobile phase. Standard calibration curves were constructed in the concentration range of 0.012– 0.200 mg/mL. The chromatographic peaks were confirmed by comparing their retention times with those of reference standards and by DAD spectra (200–500 nm); the quantification was performed by peak integration using the external standard method. The subfractions DCM1 and DCM2 from the leaves were analyzed by HPLC–DAD. Chromatographic analyses were carried out in isocratic conditions using C8 column (4.6 mm 150 mm) packed with 5 mm diameter particles, the mobile phase was methanol:water (95:5, v/v) containing 1.0% acetic acid (Boligon et al., 2010). The mobile phase was filtered through a 0.45 mm membrane filter and then degassed by an ultrasonic bath prior to use. Stock solutions of b-sitosterol, stigmasterol, lupeol, amyrin and ursolic acid standard reference were prepared in the HPLC mobile phase at a concentration range of 0.010–0.200 mg/mL. The subfractions DCM1 and DCM2 were also dissolved in the mobile phase. Quantification was carried out by the integration of the peak using external standard method. The flow rate was 1.0 mL/min, injection volume was 40 mL and detection was done at 210 nm. The chromatographic peaks were confirmed by comparing their
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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retention times and UV spectra with those of the reference standards. All chromatographic operations were performed at room temperature and in triplicate. The limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation of the responses and the slope using three independent analytical curves. LOD and LOQ were calculated as 3.3 and 10s/S, respectively, where s is the standard deviation of the response and S is the slope of the calibration curve (Boligon et al., 2013a).
Antimicrobial assay The crude extract and fractions were individually evaluated against Staphylococcus aureus ATCC 29213, Micrococcus sp. ATCC 7468, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis ATCC 7002, Enterococcus faecalis ATCC 51299, Bacillus subtilis ATCC 6633, Saccharomyces cerevisiae ATCC 2601, Candida albicans ATCC 28967, Candida tropicalis (clinical isolate obtained from patients treated at the University Hospital of Santa Maria – HUSM/Brazil), Candida parapsilosis ATCC 90018, Cryptococcus neoformans ATCC 2857, Cryptococcus gattii ATCC 56990, Escherichia coli, Prototheca zopfii, Candida dubliniensis, Candida glabrata, Aeromonas sp., Malassezia pachydermatis, Aspergillus flavus, Aspergillus fumigatus and Fusarium solani (clinical isolate obtained from patients treated at the HUSM). The protocols used in the experiments were M27-A3 for yeasts (CLSI, 2008a,b), M38-A2 (CLSI, 2008a,b) for mold and M07-A9 (CLSI, 2012) for bacteria. The experiments were repeated twice and the results were determined as an average value. Six different dilutions of each fraction (1000, 500, 250, 125, 62.5 and 31.25 mg/mL) were prepared in DMSO. Filamentous fungi were sown on PDA (potato dextrose agar) for 7 days at 30 8C, the inoculum was obtained from a suspension of conidia in sterile saline is made by spectrophotometric reading (0.4–5 104 cells/mL). Bacterial strains were cultured overnight at 37 8C in Mueller–Hinton agar. The suspension of cells was prepared in sterile saline and made reading spectrophotometer (1–2 108 cells/mL). Yeasts were grown on Sabouraud dextrose agar for 48 h at 35 8C. The suspension of cells was prepared in sterile and made reading spectrophotometer (1–5 103 cells/mL) saline. The first column of the plate was reserved for negative control wells (without inoculants) and the last column, for the positive growth control wells (without antimicrobial agents). The MIC was considered as the lowest concentration of the extract or fraction inhibiting the total growth of microorganisms. MIC was detected by lack of visual turbidity (matching the negative growth control). Standard antibiotics (ampicillin, imipenem, and cefoperazone) were used to control the sensitivity of the tested bacteria, whereas fluconazole and amphotericin B were used as control against the tested fungi and the algae.
Determination of percent activity (A%) and microorganisms susceptible index (MSI) These parameters were determined according the equations listed below (Boligon et al., 2013a). A% ¼
100 number of susceptible strains to a specific extract or fraction Total of tested strains
MSI ¼
100 number of extracts of fractions effective against each strain total samples tested
Antimycobacterial assay Antimycobacterial activity was tested against Mycobacterium smegmatis mc2 155 (ATTC700084), M. tuberculosis H37Rv (ATTC25618) and M. avium LR541CDC. The stored mycobacterium were seeded onto Löwenstein–Jensen medium and incubated during 3–5 days. From this culture a portion was removed and placed into Middlebrook 7H9 broth, supplemented with 10% OADC and 0.2% glycerol (MD7H9) and then homogenized in ultrasonic bath for 1 min. The concentration of bacteria in this medium was determined by optical density on spectrophotometer (0.08–0.1 of absorbance at 625 nm) of 0.5 McFarland scale and then diluted with MD7H9 up to 105 CFU/mL for M. smegmatis, to reach the inoculum. The M. tuberculosis and M. avium were used at a concentration of 0.5 McFarland (not diluted) because they are slow-growing. Plant extracts, fractions and isolated alkaloids were dissolved in DMSO, at a concentration of 50 mg/mL and then diluted in MD7H9 until the desired concentrations, beginning the series with 2500 mg/mL. The activity tests were performed using the broth microdilution method, document M7-A6 (NCCLS, 2003) and are presented as minimum inhibitory concentrations (MICs) of extracts and fractions. 100 mL of mycobacterial culture was placed in each well of a microtitre plate, as well as the extracts and fractions at correspondent concentrations. Analysis was done in triplicate; controls were made to the medium and to the mycobacterium, and one blank for each concentration of samples. The plates were incubated during 48 h at 37 8C. The Alamar Blue (Boligon et al., 2012a) or MTT (Sankar et al., 2008) dye was used to check the growth of microorganisms. The Alamar Blue was diluted in a ratio of 1/10 with Tween 80, diluted to 1/9 with sterile water, supplemented with 0.025 mL in each well, considering the MIC into the pit where there was no change in color from purple to pink. MTT solution at 0.5 mg/mL was prepared by the dilution with absolute ethanol up to 1 mg/mL, and then by dilution half to half with a solution of 10% Tween 80. 25 mL of the final solution was added to each plate well, and the well where the drug prevented the color change from yellow to purple was considered the MIC.
Cells and viruses HEp-2 cells were cultivated in minimum essential medium (MEM – GIBCO Invitrogen Corporation, Grand Island, NY, USA) with 10% fetal bovine serum (SFB – GIBCO Invitrogen Corporation, Grand Island, NY, USA) and penicillin, streptomycin and amphotericin B, in the concentrations 100 U/mL, 100 mg/mL and 2.5 mg/mL, respectively. The cells were prepared in 96 well plates for both cytotoxicity and antiviral assays and they were maintained at 37 8C in a 5% CO2 incubator. The strain KOS of Herpes Simplex Virus type 1 (HSV-1) was kindly provided by Dr. Paulo Roehe from the Universidade Federal do Rio Grande do Sul (UFRGS). The viruses stocks were prepared as described in Boligon et al. (2013a) and kept at 70 8C. The virus titration
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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was performed according to the methodology of Reed and Muench (1938).
Cytotoxicity evaluation The cytotoxicity and antiviral tests were performed through the colorimetric assay MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide] according to Boligon et al. (2013a). Shortly, minimum essential medium (MEM) with 10% bovine fetal serum and increasing concentrations of the samples from 1.95 to 250 mg/mL were added to the HEp-2 cells (2 104 cells/well), in 96 well plates, in a total of six repetitions for each concentration. After 72 h of incubation at 37 8C, 5% CO2, the compound MTT (1 mg/mL) was added to the cells. The reagent was removed after 4 h of incubation and 100 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals. The supernatant was transferred to a new plate and readings were taken in an ELISA Spectra Count reader at the wavelength of 540 nm. The viable cells percentage for each compound was calculated by the formula: absorbance of the compound/absorbance of the cell control 100%. The CC50 (50% cytotoxic concentration) was obtained from the concentration–effect curves after linear regression as described in Freitas et al. (2009). The maximum non-toxic concentration (MNCC) of the crude extract, fractions and subfractions that did not cause cytotoxicity was used for the antiviral tests.
Antiviral evaluation HEp-2 cells monolayers were prepared in 96 well plates 24 h before performing the tests. The maintenance media on the preformed monolayer was then removed and replaced by a new media containing 100 mL/well of the virus suspension containing 104 TCID50/mL. The control cells media were replaced by 200 mL/well of MEM media, without virus. After that, 100 mL/well of the MNCC of the samples was added to the wells. The plates were then incubated for 72 h at 37 8C in a 5% CO2 incubator. The readings were taken as described above, for the cytotoxicity assays. Acyclovir (10 mg/mL) was used as positive control for the HSV-1 inhibition. The viral inhibition for each compound was calculated according to the formula: (absorbance of the compound absorbance of the viral control)/(absorbance of the cell control absorbance of the viral control) 100%. The IC50 (50% inhibitory concentration) was obtained from the concentration–effect curves after linear regression as described in Freitas et al. (2009). The selectivity index (IS) was calculated from the formula CC50/IC50.
Data analysis CC50 and IC50 values were obtained from linear regression analysis of concentration–effect curves. Results of the HPLC– DAD quantification were considered statistically significant by Tukey test at alpha 0.001. Data of biofilms assay were analyzed using one-way analysis of variance (ANOVA) followed by the Duncan's multiple range test when appropriate. Results were expressed as the mean standard deviation (SD) and evaluated at the significance level 2alpha = 0.0001.
Results and discussion The yields of crude extract, and dichloromethane, ethyl acetate and n-butanol-soluble fractions obtained from leaves and stem bark are given in Table 1. The highest yield was obtained from crude extract of the leaves (10.3%). The quantification of alkaloids for leaves followed the order: DCM fraction > EA fraction > NB fraction > CE; and for stem bark: EA fraction > NB fraction > DCM fraction > CE (Table 1). When comparing the results obtained, we may observe that the dichloromethane fraction of leaves showed the higher content of total alkaloids (7.34 0.11 mg/g). Purified fraction of alkaloids (AF) was obtained from the leaves of T. catharinensis and showed positive results to alkaloids according to Wagner et al. (2006) and Gonçalves et al. (2011) and the thin layer chromatography revealed the presence of indolic alkaloids classes (Table 2). These results are in agreement with previously published that reported on the presence of indolic alkaloids in large numbers in the species (Pereira et al., 2004, 2008). HPLC profile of crude extract and fractions from T. catharinensis was acquired (Figs. 1 and 2). Stem bark and leaves of T. catharinensis contain other minor compounds in addition to gallic acid (retention time – tR 14.7 min, peak 1), chlorogenic acid (tR = 22.9 min, peak 2), caffeic acid (tR = 33.1 min, peak 3), rutin (tR = 45.2 min, peak 4), quercetin (tR = 56.2 min, peak 5) and kaempferol (tR = 68.5 min, peak 6). Results published previously about T. catharinensis and other Tabernaemontana species support these findings, given that some authors reported the presence of alkaloids, triterpenoids and steroids (Pereira et al., 2008), flavonoids, phenylpropanoids, phenolic acids (Pallant and Steenkamp, 2008) and essential oils (Boligon et al., 2013b). In this work, the fractions with the highest amount of phenolic acids and flavonoids were NB (22.12% for stem bark and 20.45% for leaves) and EA (21.98% for leaves and 19.54 for stem bark); results of quantification are shown in Table 3. The respective standard solutions calibrations curves were: Y = 53985x + 1020.6 (r = 0.9987) for gallic acid; Y = 87603x + 4519.1 (r = 0.9995) for chlorogenic acid; Y = 81846x + 1093.4 (r = 0.9999) for caffeic acid; Y = 96030x + 7627.3 (r = 0.9979) for rutin; Y = 50833x + 4741.7 (r = 0.9899) for quercetin and Y = 87603x + 4519.5 (r = 0.9993) for kaempferol. Antimicrobial activities of CE extracts, DCM, EA and NB fractions of the T. catharinensis were tested (see Table 4). Regarding the Gram-positive bacteria, very good results were
Table 2 – Substances characterized by TLC technique in alkaloid extract. Mobile phase
Characteristic
Rf
Toluene:ethyl acetate:diethylamine (70:20:10)
254 nm: orange stain 365 nm: blue fluorescent stain
0.52 0.31
Chloroform:diethylamine (85:15)
254 nm: orange stain 365 nm: blue fluorescent stain
0.74 0.19
According Wagner et al. (2006).
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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Fig. 1 – High performance liquid chromatography phenolics profile of CE (a), DCM (b), EA (c) and NB (d) of T. catharinensis stem bark. Gallic acid (peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3), rutin (peak 4), quercetin (peak 5) and kaempferol (peak 6). Chromatographic conditions described in the experimental section.
Fig. 2 – High performance liquid chromatography phenolics profile of CE (a), DCM (b), EA (c) and NB (d) of T. catharinensis leaves. Gallic acid (peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3), rutin (peak 4), quercetin (peak 5) and kaempferol (peak 6).
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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Table 3 – Compounds determined by HPLC in Tabernaemontana catharinensis (%); LOD and LOQ variations for compounds (mg/mL). T. catharinensis
CE
DCM
EA
NB
LOD
LOQ
Stem bark Gallic acid Chlorogenic acid Caffeic acid Rutin Quercetin Kaempferol
1.72 0.03 a 1.24 0.01 b 2.35 0.06 c 1.18 0.09 b 0.80 0.13 d 0.39 0.08 e
2.46 0.04 a 2.30 0.05 a 2.21 0.12 a – – 0.43 0.03 b
4.05 0.06 a 3.74 0.09 b 4.31 0.13 c 0.99 0.17 d 5.38 0.10 e 1.07 0.02 d
4.26 0.14 a 4.49 0.05 a 2.83 0.09 b 6.30 0.13 c 2.91 0.05 b 1.33 0.08 d
0.022 0.019 0.027 0.034 0.030 0.024
0.067 0.057 0.082 0.103 0.091 0.072
Leaves Gallic acid Chlorogenic acid Caffeic acid Rutin Quercetin Kaempferol
0.87 0.01 a 1.09 0.02 b 1.71 0.02 c 1.43 0.05 c 0.45 0.01 d 0.49 0.03 d
2.39 0.03 a 1.67 0.01 b 3.63 0.05 c – – 0.83 0.02 d
4.72 0.01 a 3.29 0.04 b 4.68 0.05 a 3.31 0.02 b 4.01 0.03 c 1.97 0.01 d
3.89 0.02 a 4.52 0.01 b 3.93 0.02 a 4.67 0.03 b 2.36 0.05 c 1.08 0.01 d
0.022 0.019 0.027 0.034 0.030 0.024
0.067 0.057 0.082 0.103 0.091 0.072
Results are expressed as mean standard deviation (SD) of three determinations. Different letters in each column represent significant differences using analysis of variance followed by Tukey test ( p values < 0.001 were considered as significant). LOD, limit of detection; LOQ, limit of quantification.
obtained against S. aureus, Micrococcus sp., E. faecalis and B. subtilis for DCM and NB fractions, showing the minimal inhibitory concentration (MIC) ranging from 31.25 to 1000 mg/ mL. Considering the Gram-negative bacteria, only NB fraction of leaves was effective against P. mirabilis (MIC = 62.5 mg/mL)
and NB fractions of leaves and stem bark showed MIC of 250 mg/mL for Aeromonas sp. In addition, only S. cerevisiae was particularly vulnerable (MICs = 31.25 and 62.5 mg/mL), followed by C. neoformans and A. fumigatus (MIC = 250–500 mg/mL). The set of data indicated that DCM fractions of the leaves and stem
Table 4 – Minimum inhibitory concentration–MIC (mg/mL) values for crude extracts and fractions of Tabernaemontana catharinensis leaves and stem bark. Microorganisms
S. buxifolia Stem bark
Leaves
CE
DCM
EA
NB
CE
Gram-positive S. aureus Micrococcus sp. E. faecalis B. subtilis
>1000 500 >1000 >1000
1000 250 500 1000
>1000 >1000 >1000 >1000
>1000 500 1000 >1000
>1000 >1000 >1000 >1000
Gram-negative Aeromonas sp. K. pneumoniae P. mirabilis E. coli P. aeruginosa
>1000 >1000 >1000 >1000 >1000
>1000 >1000 >1000 >1000 >1000
>1000 >1000 >1000 >1000 >1000
250 >1000 >1000 >1000 >1000
>1000 >1000 >1000 >1000 >1000
Fungi C. albicans C. tropicalis C. glabrata C. parapsilosis C. dubliniensis C. neoformans C. gattii S. cerevisiae M. pachydermatis A. flavus A. fumigatus F. solani P. zopfii
>1000 >1000 >1000 >1000 >1000 >1000 >1000 1000 >1000 >1000 >1000 >1000 >1000
1000 >1000 500 >1000 >1000 500 >1000 62.5 >1000 1000 250 >1000 >1000
>1000 >1000 >1000 >1000 >1000 1000 >1000 500 >1000 >1000 >1000 >1000 >1000
>1000 >1000 >1000 >1000 >1000 250 >1000 500 >1000 >1000 250 >1000 >1000
>1000 >1000 >1000 >1000 >1000 >1000 >1000 500 >1000 >1000 >1000 >1000 >1000
Control EA
NB
mg/mL
>1000 >1000 >1000 >1000
>1000 31.25 250 >1000
Amp. 2.0 Amp. 8.0 Imi. 1.0 Imi. 0.6
>1000 >1000 >1000 >1000 >1000
>1000 >1000 >1000 >1000 >1000
250 >1000 62.5 >1000 >1000
Amp. 2.0 Imi. 1.0 Imi. 2.0 Amp. 8.0 Cpz. 16.0
1000 >1000 1000 >1000 >1000 250 >1000 31.25 >1000 500 500 >1000 >1000
>1000 >1000 >1000 >1000 >1000 >1000 >1000 500 >1000 >1000 >1000 >1000 >1000
>1000 >1000 >1000 >1000 >1000 >1000 >1000 500 >1000 >1000 1000 >1000 >1000
Flu. 16.0 Flu. 32.0 Flu. 32.0 Flu. 8.0 Flu. 16.0 Flu. 8.0 Flu. 32.0 Flu. 2.0 Flu. 16.0 Flu. 8.0 Flu. 2.0 Amph. 2.0 Amph. 0.5
DCM 500 31.25 62.5 500
CE, crude extract; DCM, dichloromethane fraction; EA, ethyl acetate fraction and NB, n-butanolic fraction. Controls: Amp, ampicillin; Cpz, cefoperazone; Imi, imipenem; Flu, fluconazole; Amph, amphotericin B.
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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acids and flavonoids (Table 3), in which additionally we emphasize the presence of rutin, quercetin and kaempferol, these compounds possess antimicrobial activity well described in the literature (Oliveira et al., 2007; Boligon et al., 2013a). They may act as antibacterial agents by means of at least three or more mechanisms: inhibition of nucleic acid synthesis, disruption of bacterial membranes and/or inhibition of energy metabolism (Cowan, 1999). Some compounds present in various plants are already identified in T. catharinensis, such as betulinic acid, ursolic acid, amyrin, lupeol, sitosterol and stigmasterol, and showed notable antifungal and antibacterial capacity (Jain et al., 2001; Patra and Thatoi, 2011). These results agree with Pallant et al. (2012), which describe good activity for the crude extract and alkaloidal fraction of T. elegans, particularly against mycobacterium (M. smegmatis and M. tuberculosis) and Gram-positive bacteria (B. subtilis, E. faecalis and S. aureus). DCM fraction of T. catharinensis leaves was the most active fraction in the antimicrobial assay (Table 4), and hence was submitted to chromatographic separation processes. Successive column chromatographic procedures with DCM fraction resulted in obtained two subfractions (DCM1 and DCM2). These subfractions were analyzed by HPLC/DAD and presented triterpenoids/steroids mixtures (Fig. 3). In DCM1 subfractions were identified and they quantified the mixture of lupeol (retention time, tR = 3.7 min), amyrin (tR = 5.1 min) and ursolic acid (tR = 6.9 min); and DCM2 the mixture of bsitosterol (tR = 7.3 min) and stigmasterol (tR = 9.0 min) (Fig. 4).
bark were the most effective ones in the antimicrobial assay; good results were obtained against the fungi and the Grampositive bacteria. Besides the determination of MIC, other methods could be used to express the antimicrobial and antifungal effectiveness of the fractions. The percent activity (A%) and the microorganisms susceptibility index (MSI) may help to choose the better plant or the better fraction to be thoughtfully examined (Boligon et al., 2013a). Percent activity indicates DCM as the effective fractions (45.45%), whereas MSI indicated S. cerevisiae as the most sensitive microorganism (MSI = 100%), showing sensibility for the CE, EA, NB and principally, to DCM fractions followed by Micrococcus sp. (MSI = 62.5%); E. faecalis, C. neoformans and A. fumigatus (MSI = 50.0%); S. aureus, B. subtilis, Aeromonas sp., C. albicans, C. glabrata and A. flavus (MSI = 25.0%); and P. mirabilis (MSI = 12.5%) with intermediate sensibility; and K. pneumoniae, E. coli, P. aeruginosa, C. tropicalis, C. parapsilosis, C. dubliniensis, C. gattii, M. pachydermatis, F. solani and P. zopfii as resistant microorganisms (MSI = 0%). Antimicrobial activity of T. catharinensis DCM fractions can be mainly attributed to the synergistic action of terpenes, alkaloids and phenols compounds previously described for this species (Pallant and Steenkamp, 2008; Pereira et al., 2008), since these classes of secondary metabolites have antifungal and antibacterial activity recognized (Harborne and Williams, 2000; Tabopda et al., 2009; Chen et al., 2008; Pallant et al., 2012). Furthermore, the antimicrobial properties shown by the NB fraction may be attributed to an interaction between phenolic
29
30
CH3
20
H
20
17
17
28 1
1
5
5
7
7
HO
HO
23
24
1
2 CH3
29 28 21
CH3
22
20
27 24
20 17
17
COOH
25 26
1 5
1
7
HO
5 24
7
HO
23
3
4 = ∆5 5 = ∆5,22
Fig. 3 – Chemical structures of lupeol (1), b-amyrin (2), ursolic acid (3), b-sitosterol (4) and stigmasterol (5). Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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Fig. 4 – High performance liquid chromatography profile of DCM1 (a) and DCM2 (b) subfractions from the T. catharinensis leaves. Lupeol (peak 1), amyrin (peak 2), ursolic acid (peak 3), b-sitosterol (peak 4) and stigmasterol (peak 5). Chromatographic conditions described in the experimental section.
showed activity, but weak. In relation to T. catharinensis leaves, all tested fractions and subfractions were active against M. smegmatis with MIC ranged from 19.53 to 156.25 mg/mL. The lowest MIC against M. smegmatis was observed for DCM fraction (MIC = 19.53 mg/mL), followed by AF and DCM1 (MIC = 39.06 mg/mL) and DCM2 (MIC = 78.12 mg/mL). However, only DCM and AF fractions of leaves showed activity against M. tuberculosis and M. avium, and presented MIC 312.50 and 625.00 mg/mL, respectively (see Table 6). Tosun et al. (2004) considered inactive the plant extracts that could not prevent growth of Mycobacterium up to concentration of 200.00 mg/mL. In our study, we consider all fractions and subfractions tested promising (except CE leaves). These results support the hypothesis that the presence of triterpenes/sterols and alkaloids, are primarily responsible for the antimicrobial activity of T. catharinensis; these compounds possess antimycobacterial activity well described (Cantrell et al., 2001; Pallant et al., 2012). Isolated alkaloids from various Tabernaemontana species, conoduramine, coronaridine, and voacamine, possess strong antibacterial activity against Grampositive bacteria (Van Beek et al., 1984). Furthermore, the alkaloids ibogamine and voacangine have proven antimycobacterial activity (Rastogi et al., 1998). These compounds, however, have been previously identified in T. catharinensis (Pereira et al., 2008), suggesting that the pharmacophore responsible for the antimycobacterial properties may be related to the alkaloids present in this species. Pallant et al. (2012) describe the antimycobacterial activity for crude extract
For quantification of the compounds in mixtures, calibration curves were constructed with reference standards, lupeol: y = 23157x + 1178.7 (r = 0.9995), amyrin: y = 16511x + 1438.3 (r = 0.9998), ursolic acid: y = 41118x + 1389.5 (r = 0.9983), bsitosterol: y = 24085x + 1210.9 (r = 0.9989) and stigmasterol: y = 19108x + 1750.3 (r = 0.9997). Table 5 shows the quantification of compounds in the subfractions. DCM1 is rich in triterpenes (lupeol, amyrin and ursolic acid) that represent 75.12% and DCM2 is comprised 85.78% of steroids (b-sitosterol and stigmasterol). Several researchers have utilized nonpathogenic, fastgrowing Mycobacterium species in rapid and easy screens for antimycobacterial activity in plant extract and pure plantderived compounds (Kuete et al., 2008; Boligon et al., 2012a). In one study realized by McGaw et al. (2008) it was concluded that M. smegmatis was better predictor of activity against pathogenic M. tuberculosis. However, more research is needed to validate the use of non-pathogenic species such as M. smegmatis as models for detecting activity of plant derived extracts against pathogenic M. tuberculosis. The results obtained in antimycobacterial assay performed with CE, fractions (DCM, EA and NB), subfractions (DCM1 and DCM2) and alkaloidal fraction (AF) of the T. catharinensis are summarized in Table 6. Fractions of the stem bark showed activity against M. smegmatis being DCM more active fraction (MIC = 39.06 mg/mL), followed by CE, EA and NB (MIC = 156.25 mg/mL); for M. tuberculosis the order of activity was CE and NB > DCM > EA; and for the M. avium only DCM
Table 5 – Subfractions composition of T. catharinensis leaves. Compounds
Lupeol Amyrin Ursolic acid b-Sitosterol Stigmasterol
DCM1
DCM2
LOD
LOQ
Quantities (%)
Quantities (%)
(mg/mL)
(mg/mL)
25.09 0.13 a 16.85 0.07 b 33.18 0.02 c – –
– – – 26.03 0.15 a 59.75 0.09 d
0.017 0.023 0.025 0.014 0.030
0.052 0.069 0.074 0.042 0.091
Results are expressed as means standard deviations of three determinations. Different letters represent significant differences using analysis of variance followed by Tukey test ( p values < 0.001 were considered as significant). LOD, limit of detection (mg/mL); LOQ, limit of quantification (mg/mL).
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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Table 6 – Minimal inhibition concentration (mg/mL) of fractions and isolated of Tabernaemontana catharinensis against M. smegmatis, M. tuberculosis and M. avium. Extract/fractions M. smegmatis M. tuberculosis M. avium Stem bark CE DCM EA NB Leaves CE DCM EA NB AF DCM1 DCM2
156.25 39.06 156.25 156.25
312.50 625.00 >1250.00 312.50
>1250.00 625.00 >1250.00 >1250.00
2500.00 19.53 156.25 156.25 39.06 39.06 78.12
ND 312.50 >1250.00 >1250.00 312.50 >1250.00 >2500.00
ND 625.00 >1250.00 >1250.00 625.00 >1250.00 >2500.00
AF, alkaloidal fraction; ND, not determined.
and alkaloidal fraction of T. elegans against M. smegmatis and M. tuberculosis showing a MIC = 32 mg/mL for alkaloidal fraction, and additive antibacterial effects were observed when the alkaloidal fraction was combined with antibiotics. Essential oils and extracts from a great diversity of plants have been examined concerning its ability to directly inactivate viruses or to interfere with the replication of these microorganisms (Chiang et al., 2002; Knockaert et al., 2002; Astani et al., 2011; Scala et al., 2011). The natural products are considered an important source of potential antiviral candidates due to the abundance of these products, as well as their chemical diversity and low toxicity (Chiang et al., 2002; Mukhtar et al., 2008). The antiviral activity of the crude extracts, fractions and subfractions of the T. catharinensis leaves and stem bark was tested against HSV-1. The results for the cytotoxicity and anti-HSV assay are shown in Table 7.
Table 7 – Cytotoxicity and anti-HSV-1 activity of Tabernaemontana catharinensis. Sample
Cytotoxicity CC50
Anti-HSV-1 activity IC50
SI
Stem bark CE DCM EA NB
62.82 4.75 4.21 1.42 26.85 3.18 119.38 15.6
NI 2.62 1.89 2.88 1.21 NI
– 1.61 9.32 –
Leaves CE DCM EA NB AF
59.53 4.56 46.75 5.29 89.03 9.21 119.35 9.71 116.20 5.50
NI 0.6 0.03 2.21 1.24 4.12 2.45 10.86 4.75
– 77.92 40.27 28.97 10.71
NI 48.34 4.17 1.50 0.30
– 12.28 >186.66
DCM subfractions (leaves) DCM1 49.69 4.91 593.88 14.24 DCM2 Acyclovir (10 mg/mL) >280.00
CE, crude extract; DCM, dichloromethane fraction; EA, ethyl acetate fraction and NB, n-butanolic fraction. CC50, 50% cytotoxic concentration (mg/mL). IC50, 50% inhibitory concentration (mg/mL). SI, selectivity index (=CC50/IC50); NI, no inhibitory activity.
CE of both bark and leaves of the T. catharinensis did not show any antiviral activity; however, the EA fraction of the stem bark and all the fractions and the DCM2 subfraction of the leaves of this plant exhibited values of IC50 under 48.34 mg/mL toward HSV-1 (Table 7). The IC50 values obtained with this plant are lower than the value of 100 mg/mL recommended by Cos et al. (2006) in order to consider a product as a potential candidate to antiviral. Indeed, the IC50 values varied from 0.6 to 10.86 mg/mL with most of them being lower than 4.5 mg/mL (Table 7). The best antiviral activity was obtained from the DCM fraction of the leaves of the T. catharinensis (Table 7). As already mentioned, the DCM fraction was subfractionated in DCM1, which did not show antiviral activity; and DCM2 that was effective against HSV-1 although showing less efficiency than the fraction as a whole (Table 7). A high content of b-sitosterol and stigmasterol was identified in this sub-fraction (85.78%), which could suggest a major role for those compounds in the anti-HSV1 activity. Nonetheless, the considerable higher activity of the fraction as a whole indicates that there is some important synergy among the compounds that is lost when it is fractioned (Fig. 4 and Table 5). Conversely, the presence of antiviral activity in all the fractions from the T. catharinensis and the absence of such action in the crude extract suggest that some of the compounds could have an antagonistic behavior. Actually, the phenomena of synergy and/or antagonism among compounds have been described for natural products (Wagner and Ulrich-Merzenich, 2009). The antiviral activity of isolated chemical compounds of extracts and essential oils from diverse natural products was already demonstrated (Chiang et al., 2002; Knockaert et al., 2002; Astani et al., 2011; Scala et al., 2011). Since all the fractions from the leaves showed antiviral activity, it is possible that in each case a combination of different chemical compounds was responsible for the activity. The presence of high amounts of polyphenolic compounds in some plant extracts was considered the main reason for its antiviral activity (Serkedjieva and Ivancheva, 1999). The EA and NB fractions of the T. catharinensis were rich in polyphenolic compounds (Figs. 1 and 2, Table 3) and it can be at least partially responsible for the high SI values observed in those cases (Table 7). On the other hand, the indole alkaloids present in T. catharinensis identified by Pereira et al. (2008) may be the reason of the antiviral activity of the alkaloidal fraction (AF). Scala et al. (2011) described the interference of indole-3,4-diones alkaloids with HSV-1 replication and reasoned that it could be due to the structural features of this compound that are similar to certain inhibitors of cyclin-dependent kinases (CDKs), which avoid viral replication in vitro (Knockaert et al., 2002). In addition, a recent survey by Boligon et al. (2013b) reported the presence of b-caryophyllene and bcaryophyllene oxide (56.87 and 2.51%, respectively) in the essential oil of T. catharinensis leaves. These sesquiterpenes are present in many essential oils, and the b-caryophyllene exhibited anti-HSV 1 properties that were attributed to its oxide-derived form (Astani et al., 2011). Then, considering all that were described above, the antiviral activity demonstrated by the diverse fractions and sub-fraction of the T. catharinensis cannot be related to any
Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004
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particular compound; instead, such activity may be due to a combination of different compounds at each fraction and subfraction. Further studies to demonstrate the antiviral mechanisms of the fractions and sub-fraction that demonstrated the best anti-HSV1 activity are worthwhile.
Conclusion The fractions and subfractions of T. catharinensis demonstrated effective antiviral, antimicrobial and antimycobacterial properties, in vitro. High levels of steroids, alkaloids and phenolic compounds in the fractions suggest that these compounds are responsible for the antiviral and antimicrobial activities described here. Results obtained in the present study highlights the importance of the research of the natural products as a source for new antiviral and antimicrobial therapy.
Conflict of interest The authors have no conflicts of interest.
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Please cite this article in press as: Boligon, A.A., et al., HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.01.004