Latex from Tabernaemontana catharinensis (A. DC)—Apocynaceae: An alternative for the sustainable production of biologically active compounds

Industrial Crops & Products 129 (2019) 74–84

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Latex from Tabernaemontana catharinensis (A. DC)—Apocynaceae: An alternative for the sustainable production of biologically active compounds

T

Carolina da Silva Menecuccia, Kathiele Luiza Mucellinib, Márcia Machado de Oliveirab, Bruna Higashia, Rafaela Takako Ribeiro de Almeidac, Carla Portod, Eduardo Jorge Pilauc, ⁎ José Eduardo Gonçalvesd, Regina Aparecida Correia Gonçalvesa,e, Arildo José Braz de Oliveiraa,e, a

Graduate Program in Pharmaceutical Sciences, State University of Maringá, Ave. Colombo 5790, 87.020-900, Maringá, Brazil Department of Basic Sciences of Health, State University of Maringá, Ave. Colombo 5790, 87.020-900, Maringá, Brazil c Department of Chemistry, State University of Maringá, Ave. Colombo 5790, 87.020-900, Maringá, Brazil d Program of Master in Clean Technologies, Program of Master in Science, Technology and Food Safety and Cesumar Institute of Science, Technology and Innovation – ICETI. University Center of Maringa, Ave. Guedner, 1610, 87.050-900, Maringa, Brazil e Department of Pharmacy, State University of Maringá, Ave. Colombo 5790, 87.020-900, Maringá, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Tabernaemontana catharinensis Laticiferous plant Proteases Non-timber forest products Extractivism

Lattices from the plant species Tabernaemontana catharinensis have been traditionally used as an antidote for snakebites, for the relief of toothaches, as a vermifuge for the elimination of warts, and as an anti-inflammatory compound. It is accepted that the antidote activity is the result of the proteolytic fraction of the water-soluble latex of the plant. Thus, the present study aimed to assess the proteolytic activity of T. catharinensis latex. The insoluble material from the latex of T. catharinensis was removed by centrifugation. The resulting water-soluble fraction of the latex was subjected to colorimetric and spectroscopic analysis. It was also assayed for proteolytic activity using azocasein as a substrate. The water-soluble fraction of the latex consisted mainly of carbohydrates, proteins, and amino acids. The soluble fraction exhibited strong proteolytic activity compared to trypsin. The latex was subjected to extraction with hexane to obtain a fraction rich in n-alkanes that was analysed by gas chromatography-mass spectrometry (MS) followed by acid-based alkaloid extraction, resulting in two alkaloidal extracts whose compounds were identified by off-line electrospray ionisation (+)-MS/MS analysis. The results suggest that latex from T. catharinensis is a rich source of sugars, amino acids, n-alkanes, alkaloids, and proteins.

1. Introduction Latex is widely found among plant species, with 12,000–35,000 species described as exuding the substance (Kekwick, 2001; Pickard, 2007; Agrawal and Konno, 2009). The occurrence of latex in plants is also widespread, with more than 40 families of plants characterised as establishing lactiferous structures, including Apocynaceae Juss., Asteraceae Brecht & Presl., Caricaceae Dumort, EuphorbiaceaeJuss., Moraceae Gaudich, and Papaveraceae Juss. (Lewinsohn, 1991; Yariswamy et al., 2013). Tabernaemontana catharinensis A. DC (syn. Peschiera catharinensis A. DC. Miers) belongs to the Apocynaceae family, which consists of 415 genera and 4555 species. Recently, 99 species from the Tabernaemontana genus were reported, while 44 species occur in the Americas (Leeuwenberg, 1994). T. catharinensis is popularly known as 'jasmim' (jasmine), 'leiteira de

⁎

dois irmãos' (milkweed), and 'casca de cobra' (snake skin) and occurs in Argentina, Uruguay, Paraguay, and southern Brazil. In traditional medicine, it is used as an antidote for snakebites, for the relief of toothaches, as a vermifuge for the elimination of warts, and as an antiinflammatory compound (Leeuwenberg, 1994; Almeida et al., 2004; Pereira et al., 2004). Studies conducted by Batina et al. (2000) demonstrated that isolated substances and aqueous extracts of the root of T. catharinensis could inhibit the lethal and myotoxic activity of Crotalus durissus terrificus venom (South American rattlesnake, Cascavel). Substances with trypanocidal and antitumoural activities have also been isolated from this plant (Pereira et al., 1999; Almeida et al., 2004). In addition, the aqueous extract of T. catharinensis has been shown to possess potent cytotoxic action against human tumour cell lines such as SK-BR-3, MCF-7, and C-8161in vitro. Furthermore, the essential oil of T. catharinensis leaves contains compounds with antioxidant and anti-inflammatory activity (Almeida et al., 2004).

Corresponding author at: Graduate Program in Pharmaceutical Sciences, State University of Maringá, Ave. Colombo 5790, 87.020-900, Maringá, Brazil. E-mail address: [email protected] (A.J.B. de Oliveira).

https://doi.org/10.1016/j.indcrop.2018.11.036 Received 19 December 2017; Received in revised form 30 October 2018; Accepted 13 November 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

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2.3. Preparation of crude acetone precipitate

Latex is a colloidal suspension that contains an extraordinary array of secondary metabolites and proteins (Agrawal and Konno, 2009). T. catharinensis contains specialised cells called laticifers dispersed throughout most of its tissues, which secrete latex. Its chemical composition may be related to the biological effects described above. The chemical profile and biological effects of the latex of T. catharinensis have not yet been reported, however. The objective of the present study was to characterise the main chemical constituents and evaluate the potential applications of the water-soluble fraction of T. catharinensis latex.

CLTc was resolubilised in distilled water and submitted to protein precipitation (Fig. 1A) by slowly adding three volumes of chilled acetone to one volume of crude extract. The precipitate was then separated by centrifugation at 5000 ×g for 25 min, dissolved in a minimum volume of water, and reprecipitated by the addition of chilled acetone. The supernatant and precipitate were obtained following centrifugation (5000 ×g for 25 min). All the centrifugation steps were performed at 5 °C. The supernatant was referred to as the soluble latex supernatant of T. catharinensis (SLSTc). The precipitate was resolubilised in distilled water, freeze dried, and used in subsequent experiments. This fraction, containing almost all of the soluble latex protein, is referred to as crude latex protein (CLPTc) and was used for total carbohydrate, amino acid, and protein determination assays.

2. Materials and methods 2.1. Reagents Azocasein, trypsin, and bovine serum albumin were purchased from Sigma-Aldrich. All other chemicals were of analytical grade and used without further purification.

2.4. n-Alkanes and total monoterpenoid indole alkaloid extraction To extract n-alkanes and total monoterpenoid indole alkaloids, the CLTc fraction was processed according to Fig. 1B.The freeze-dried water-soluble fraction of latex (4 g) was resuspended in 40 mL of distilled water and extracted three times with hexane under constant agitation, dried in anhydride Na2SO4, and filtered. The solvent was removed by rotary evaporation, and this extract was referred to as the hexane extract of T. catharinensis latex (HETCL; yield 0.2%). HETCL was characterised by nuclear magnetic resonance (NMR) spectroscopy analysis and submitted to gas chromatography coupled mass spectrometry (GC/MS) analysis. The water-soluble fraction of latex that resulted following the hexane extraction was submitted to an acid-based extraction to obtain total monoterpenoid indole alkaloid extracts (TMIAs) (Fig. 1B). The latex solution was acidified up to pH 2.0 with 1.0 mol/L HCl and maintained at 5 °C overnight. Then, the acid solution was filtered, extracted three times with 80.0 mL of CH2Cl2, dried in anhydride Na2SO4, and filtered. The solvent was removed by rotary evaporation under reduced pressure, and this extract was referred to as acid monoterpenoid indole alkaloids (MIA-A; yield 0.23%). The resulting acid water solution was then alkalinised to pH 9.0 with 1.0 M NaOH, and extracted three times with 80 mL of CH2Cl2, dried, and filtered. The

2.2. Collection of Tabernaemontana catharinensis latex Tabernaemontana catharinensis (A. DC) – Apocynaceae latex samples were obtained in 2014 from uncultivated plants from the campus of the State University of Maringá (lat: -23.4253 long. -51.9386 err: ± 19250 WGS84), Maringá, in the state of Paraná, Brazil. The plant material was identified and deposited as voucher code 29012 at the State University of Maringá Herbarium (Biology Department). The acquisition and fractionation of the water-soluble fraction of T. catharinensis latex was processed according to Fig. 1A. The latex was collected by incisions in the stem of the plant with sharp blades and diluted in distilled water (1:1 [v/v]). The sample was centrifuged (5000 ×g) at 10 °C for 25 min to separate the pellet, which was discarded. The supernatant was frozen or freeze-dried for further experiments. The water-soluble fraction, after lyophilisation, was referred to as the crude latex of T. catharinensis (CLTc), and was used for total carbohydrate, amino acid, and protein determination assays. The CLTc underwent other fractionation procedures.

Fig. 1. (A) Methodology for obtaining the crude latex protein of T. catharinensis (SLPTc); (B) Methodology for obtaining the n-alkane and MIA fractions from T. catharinensis latex. 75

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Table 1 Analysis of carbohydrates, proteins, amino acids and proteolytic activity of CLTc and SLPTc from T. catharinensis latex. Components

Media ± DP (%)

Total carbohydrates Reducing sugars Total proteins Amino acids

CLTc

CLPTc

26.3 ± 2.09 n.d.a 52.5 ± 1.02 21.2 ± 1.79

4,3 ± 0.78 n.d.a 85.6 ± 6.65 10.1 ± 3.31

Proteolytic activity

CLTc

CLPTc

SLSTc

Trypsin (Control)

Total activity (units) Specific activity (units/mg)

2.56 ± 0.37 0.57 ± 0.06

3.03 ± 0.02 1.22 ± 0.01

0.95 ± 0.05 0.20 ± 0.01

2.42 ± 0.75 0.48 ± 0.03

a

n.d.: not detected.

2.7. Analysis of total monoterpenoid indole alkaloids by off-line ESI/MS

solvent was removed by rotary evaporation under reduced pressure, and this extract was referred to as basic monoterpenoid indole alkaloids (MIA-B; yield 0.4%). Both extracts were dissolved in methanol for offline electrospray ionisation (ESI) MS analysis.

The samples of MIA-A and MIA-B were initially submitted to off-line ESI-MS analysis; alkaloid extracts were reconstituted in methanol (500 μL) and 100 μL, and this solution was diluted in 2 mL of acetonitrile. Following this, the solutions were filtered through a 0.2 μm pore membrane and introduced into the mass spectrometer using a syringe pump. Spectra were obtained using a quadrupole-time of flight mass spectrometer (Bruker, Impact II), which was operated in full-scan mode with MS data collected between m/z 50–1200 in positive ionisation mode. The nebuliser gas was set to a flow rate of 5 L/min at 0.4 bar with a temperature of 180 °C. The capillary and end plate offset were set to 4500 and −500 V, respectively. For ESI(+)-MS/MS, the energy for collision induced dissociations was optimised for each component. Diagnostic ions in different fractions were identified by comparison of their ESI(+)-MS/MS dissociation patterns with previously identified compounds.

2.5. Biochemical analysis Analysis of the total water-soluble carbohydrates, free amino acids, and proteins was performed by colorimetric techniques. Analysis was performed in triplicate and the mean values, standard deviations, and coefficients of variation were calculated. Total carbohydrate content was determined by the phenol-sulphuric acid method (Dubois et al., 1956). Free reducing sugar content was determined by p-hydroxybenzoic acid hydrazide assay (Lever, 1972). Amino acid content was determined by ninhydrin assay (Magné and Larher,1992). Protein content was measured according to Lowry's modified method using bovine serum albumin as the standard protein (Hartree, 1972).

2.8. NMR analysis

2.5.1. Monosaccharide composition The monosaccharide components of the CLTc and their ratios were determined by hydrolysis with 2 M trifluoroacetic acid for 8 h at 100 °C, followed by conversion to alditol acetates by successive NaBH4 reductions, and acetylation with Ac2O-pyridine (1:1 [v/v], 1 mL) at room temperature for 14 h. The resulting alditol acetates, pyridine, and excess acetic anhydride were removed with toluene and the sample was dried. The resulting alditol acetates were then extracted with dichloromethane and analysed by GC/MS using a Gas Chromatography Agilent 7890B coupled with Mass Spectrometry Agilent 5977 A MSD, with He as a carrier gas. A HP5-MS UI-Agilent fused silica capillary (30 × 0.25 mm × 0.25 mm; Agilent Technologies) was maintained at 80 °C during a 2 min injection, then programmed to increase 10 °C/min to 180 °C,remain at this temperature for 2 min, increase to 240 °Cat a rate of 5 °C/min, and remain at this temperature for 5 min.

The CLTc, SLSTc, and CLPTc (20 mg) samples were dissolved in 99.9% deuterium oxide (D2O), maintained at 45 °C for 24 h to achieve hydrogen exchange, and lyophilised. The dried waste was solubilised in 700 μL of D2O and the HETCL sample was dissolved in CDCl3. One- and two-dimensional NMR spectra were recorded at 298 K using a Bruker Spectrometer (Advance III HD model) operating at 500 MHz for the 1H nucleus and 125 MHz for the 13C nucleus using the standard pulse sequences of the Bruker software. The chemical shifts (δ) were expressed in parts per million.3-(Trimethylsilyl)propionic-2,2,3,3d4 acid sodium salt and tetramethylsilane were used as internal references (δ, 0 ppm). 2.9. Enzyme assays

2.6. Analysis of n-alkanes by GC/MS

CLTc, CLPTc, SLSTc, and trypsin (5 mg/mL, positive control) were used in the enzyme assays. Proteolytic activity was assayed using azocasein as a substrate (Charney and Tomarelli, 1947). In the first step, 500 μL of 2.5% (w/v) azocasein solution diluted in sodium bicarbonate buffer (0.5% [w/v]), pH 8.3 at 37 °C was added to all vials. Then, 500 μL of sodium bicarbonate buffer was added to a blank tube, as well as 300 μl of the samples and trypsin, mixed, and incubated at 37 °C until thermal equilibrium was achieved. Thereafter, 200 μL of CLTc, CLPTc, SLSTc, or trypsin was added to the respective tubes, gently mixed, and incubated at 37 °C for 30 min. In the second step, a 200 μL aliquot from all of the reactions was added to the other tubes and 800 μL of 5.0% (v/v) trichloroacetic acid solution, diluted in distilled water, was added. Next, the solutions were mixed and filtered using a syringe with a 0.45 μm filter. Aliquots of 200 μL were removed from the filtrates and transferred to other tubes, before 600 μL of 500 mmol/L sodium hydroxide solution was added.

HETCL was analysed by GC/MS using a Gas Chromatography Agilent 7890B coupled with Mass Spectrometry Agilent 5977 A MSD and a HP5-MS UI-Agilent fused silica capillary (30 × 0.25 mm × 0.25 mm; Agilent Technologies).The carrier gas was He and was used at a flow rate of 1 mL/min. Temperature of the split injector (split ratio 1:50) was 190 °C. The initial temperature of the oven was 150 °C, which was increased to 250 °C at 10 °C/min and maintained for 5 min, followed by an increase to 300 °C at 10 °C/min and maintained for 10 min. The mass spectrometer was operated in the electron impact mode, with electron energy of 70 eV for the positive mode. Ion source temperature was 200 °C. Mass spectrometric detection was conducted in scan mode from m/z 100 to 800 at 2.2 scans per second. Control, data acquisition, and processing were performed using the Agilent MSD Chemstation (Agilent Technologies) and the NIST MS Search 2.0 spectrum database. 76

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Fig. 2. Representative 1H NMR spectral profile of (A) crude latex of T. catharinensis (CLTc) (amplified spectra and full spectrum) and (B) crude latex protein of T. catharinensis (CLPTc).

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Fig. 3. Representative

13

C NMR spectral profile of (A) crude latex of T. catharinensis (CLTc) and (B) crude latex protein of T. catharinensis (SLPTc).

78

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Fig. 4. (A) GC/MS chromatogram obtained from n-alkanes isolatedfrom the latex of T. catharinensis by hexane extraction and (B) mass spectrum of the linear n-alkane C25H52.

3. Results and discussion

The contents were mixed and transferred to a 1 mL cuvette, and absorbance at 440 nm was measured. One unit (U) of protease activity was defined as the amount of enzyme that caused an increase in 0.1 absorbance unit under the defined assay conditions.

Latex of several plants has been reported to exhibit medicinal properties, including analgesic, anti-inflammatory, procoagulant, and antitumor effects. Some studies have indicated that these properties are mediated by water-soluble proteins present in the latex (Fernandes et al., 2015; Shivaprasad et al., 2010; Kumar et al., 2015; Villanueva et al., 2015). Phytochemical analysis has revealed that the stem bark of T. catharinensis contains primarily monoterpenoid indole alkaloids (Pereira et al., 2004; Nicola et al., 2013), but previous investigations have mostly focused on the specialised metabolites of the whole plant,

2.10. Statistical analysis Statistical analysis was performed by one-way analysis of variance to test differences in absorbance values between latex concentrations, considering a significance of p < 0.05.

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Table 2 MIAs previously isolated or detected from stem barks alkaloidal extract of T. catharinensis. Compound

References

a

Affinisine 16-Epiaffininea Catharinensine Conodurine Coronaridine 3-hydroxycoronaridine Coronaridine-hydroxyindolinine 3-(2-oxopropyl)-Coronaridine Heyneanine Ibogamine Isovoacangine Rupicoline Tabernanthine Voacamidine Voacangine Voacangine hydroxyindolenine Voachalotinea 12-methoxy-4-methyl-Voachalotinea 16-decarbomethoxy-Voacamine Nb-demethyl-Voacamine Voacristine Voacristine-hydroxyindolenine Vobasine

Nicola et al. (2013) Araujo et al. (1984); Gonçalves et al. (2011) and Nicola et al. (2013) Araujo et al. (1984) Araujo et al. (1984) and Gonçalves et al. (2011) Araujo et al. (1984); Braga et al. (1984); Cardoso et al. (1998); Gonçalves et al. (2011) and Pereira et al. (2004) Araujo et al. (1984); Braga et al. (1984); Cardoso et al. (1998); Gonçalves et al. (2011) and Pereira et al. (2004) Nicola et al. (2013) Cardoso et al. (1998) and Gonçalves et al. (2011) Araujo et al. (1984) and Braga et al. (1984) Cardoso et al. (1997) and Cardoso et al. (1998) Araujo et al. (1984) Braga et al. (1984) Cardoso et al. (1998) Braga et al. (1984) Cardoso et al. (1997), 1998 and Pereira et al. (2004) Braga et al. (1984); Cardoso et al. (1997), 1998 and Pereira et al. (2004) Gonçalves et al. (2011) and Nicola et al. (2013) Gonçalves et al. (2011) and Nicola et al. (2013) Araujo et al. (1984) and Gonçalves et al. (2011) Braga et al. (1984) Pereira et al. (2004) Nicola et al. (2013) Nicola et al. (2013)

Bold type: MIAs detected in the alkaloidal extracts from latex of T. catharinensis. a Detected in the others studies using ESI/MS analysis.

Fig. 5. Off-line ESI(+)MS spectrum of MIA-A extract from T. catharinensis latex and structures of identified alkaloids.

respectively. The analysis also revealed that CLTc and CLPTc contained 26.3% and 4.3% carbohydrates, respectively (Table 1). As they were present in small quantities, reducing sugars were not detected in the CLTc and CLPTc samples using colorimetric methodology (Table 1). In this case, reducing sugars represent the amount of reducing terminals present in the sample (Brummer and Cui, 2005), and the inability to detect these indicates they are likely di- or oligosaccharides. CLTc, after determination of monosaccharide composition by GC/MS, displayed a high content of glucose and minor quantities of mannose and galactose.

and several bioactive alkaloids with diverse structures. The primary aim of the present study was to establish the chemical profile of the watersoluble fraction of latex from T. catharinensis, which is used therapeutically in traditional medicine, and evaluate its proteolytic activity. 3.1. Biochemical analysis of T. catharinensis latex The total protein, amino acid, carbohydrate, and reducing sugar concentrations of CLTc and CLPTc were determined. CLTc consisted of approximately 52.5% protein and 21.2% free amino acids, while CLPTc was comprised of 85.6% and 10.1% proteins and free amino acids, 80

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Fig. 6. Off-line ESI(+)MS spectrum of MIA-B extract from T. catharinensis latex and structures of identified alkaloids. Table 3 Chemical composition of MIA-A and MIA-B fractionsof T. catharinensis alkaloidal latex extract by ESI(+)-MS and ESI(+)-MS/MS. Entry

Precursor Ion m/z

Fragmentation MS/MS (%)

Identification

Elementar composition

Mass error (ppm)

References

1 2 3 4 5 6

281.1999 309.1951 325.1895 339.2051 367.2009 381.2166

158.0956(10),144.0801(44), 122.0959(41) 309.1951(100), 291.1857(5), 170.0966(10), 158.0966(15), 138.0968(25) 293.1634(100), 265.1688(24), 152.1062(54), 144.0800(19) 307.1789(13), 279.1841(12), 144.0801(48), 122.0959(14) 337.1898(16), 305.1636(16), 170.0958 (92,7), 158.0958(27) 236.1424(7), 180.1013(31), 170.0958 (100

C19H24N2 C20H24N2O C20H24N2O2 C21H26N2O2 C22H28N2O3 C23H28N2O3

5.95 5.13 6.77 6.05 3.45 3.19

a, a, a, a, a, d

7

395.2311

C43H52N4O5

–

c

8

411.2263

381.2155 (5), 351.2052(5), 321.1582 (10), 220.1108(12), 180.1011(26), 170.0956(100), 152,1063(16) 200,1067(100), 188.1063(10), 180.1013(29)

Ibogamine Affinisine 16-Epi-Affinine Coronaridine Voachalotine 10-hydrdoxy-Na-methylVellosimine* Voacamine* 12-Methoxy-N-methylvoachalotine

C24H31N2O4

5.06

b, c

b, b, b, b, b,

c c c c c

References: a- Nicola et al. (2013); b- Pereira et al. (2008); c -Lépine et al. (2002); d-Marinho et al. (2016). * Suggested structure proposed by the authors.

5.34 ppm, which may be associated with sucrose (Fig. 2A, Table S1 in Supplementary Material 1). In the 13C NMR spectrum of CLTc (Fig. 3A), signals related to anomeric carbon sugars were observed in the δ 105–90 ppm region. Other sugar signals occurred between 65–87 ppm, corresponding to C2C5, and 55–64 ppm, referring to CH2OH (C-6). The region between δ 20–60 ppm in CLTc exhibited some signals related to the aliphatic carbons of amino acids (Fig. 3A); with the aid of the HSQC correlation map (Supplementary Material 2), a signal that may be associated with the methyl groups of proline betaine, betaine, or choline was apparent at 56.6 ppm (Oliveira et al., 2014). The CLPTc13C NMR spectrum showed signals characteristic of a protein molecule (Fig. 3B), where signals of the aromatic amino acids tryptophan and tyrosine were observed between 178.6–115.5 ppm (Oliveira et al., 2014). Fig. 3A shows that the 12 carbon signals in the 13C NMR spectrum characteristic of carbohydrates between 104.0–59.0 ppm were related to fructose and glucose moieties, thus confirming the presence of disaccharide sucrose (Fig. 3A, Table S1 in Supplementary Material 1) as a major carbohydrate component. The assignment of sucrose signals in the CLTc spectrum was also confirmed by comparing its map of correlation with a standard sucrose correlation map (Supplementary Materials 2 and 3). The 1H and 13C NMR assignments of CLPTc also agree with the

3.1.1. NMR analysis NMR spectroscopy (1H and 13C) of CLTc and CLPTc was initially performed to delineate a profile of the major components present in the water-soluble fraction of T. catharinensis latex. Representations of the 1H and 13C NMR spectra of CLTc and CLPTc are displayed in Figs. 2 and 3, respectively. The technique resulted in1H and 13C NMR spectra with good resolution and sensitivity, considering that materials with minimal pretreatment were analysed. The 1H NMR spectrum of CLTc showed signals in the δ 8.5–90 ppm region that are related to amino acid aromatic carbons; upon analysis of the spectrum expansion, it was possible to assign some of the signals to the amino acids tryptophan and tyrosine (Fig. 2A), while the sugar units exhibited signals in the 5.5–3.0 ppm range and signals related to the aliphatic carbons of the amino acids were found in the region between δ 3.5–0.5 ppm (Fig. 2A). The intense singlet may be associated with the methyl group of proline betaine, betaine, or choline (Oliveira et al., 2014). In the CLPTc1H NMR spectrum (Fig. 2B), in addition to the absence of sugars, we observed the characteristic signals of the aromatic amino acids tryptophan and tyrosine (Oliveira et al., 2014). Analysis of the 1H NMR spectra of CLTc (Fig. 2A) showed carbohydrate signals between 3.30 and 4.20 ppm and a doublet that was assigned to the H-1 hydrogen attached to the anomeric carbon at 81

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Fig. 7. Off-line ESI(+)MS spectra of (A) vobasine and (B) voacamine.

339.2051), voachalotine (m/z 367.2009), 12-methoxy-N-methyl-voachalotine (m/z 411.2265), and voacamine (m/z 395.2311). Other compound that had your suggested structure based on their MS fragmentation pathways and MIA structures already isolated from species of the genus Tabernaemontana (Pereira et al., 2008; Zocoler et al., 2005; Marinho et al., 2016) with m/z 381.2159, whose fragmentation profile suggested was 10-hydrdoxy-Na-methyl-vellosimine. Table 3 lists the compounds identified in the alkaloid extract obtained from the latex of T. catharinensis by ESI(+)-MS as well as analysis of the principal precursor ions by ESI(+)-MS/MS. MIAs identified in the alkaloid extracts of T. catharinensis latex were not fully consistent with previous studies of isolation or detection of alkaloids from T. catharinensis stem bark (Table 2). However, this can be explained by the fact that stem bark is composed of several cell types capable of accumulating MIAs, but the latex is stored specifically in ducts known as laticifers, which accumulate normal or specifically toxic MIAs, which are exuded only following insult (Wittstock and Gershenzon, 2002). An interesting observation was the detection of dimeric MIA voacamine, which in previous studies was associated with the anti-plasmodium activity of stem and root bark from T. catharinensis (Federici et al., 2000). The mass spectrum of this compound was obtained in the positive ESI mode. The mass spectrum of voacamine (molecular mass 704 Da) does not present the [M + H+]1+ ion at m/z 705.1, but only the doubly charged ion at m/z 353.3 [M + 2 H]2+. As this dimeric alkaloid contains a moiety derived from vobasine, MS/MS analysis was performed to see if this compound produces ions that could be attributed to the vobasine subunit. The tandem mass spectra of the [M + 2 H]2+ ion contained an abundant ion atm/z 180.1, indicating that structure attribution is compatible with the voacamine structure (Fig. 7), as previously observed by Lépine et al. (2002). However, the latex from T. catharinensis is a non-woody renewable source of biologically active monoterpenoid indole alkaloids.

biochemical analysis because the presence of proteins was evidenced by the characteristic spectral profile and aromatic and aliphatic amino acid signals. 3.2. Analysis of n-alkanes by GC/MS HETCL provided a yield of 0.2% and the preliminary 1H and 13C NMR analysis suggested that the main components were n-alkanes. Then, HETCL was submitted to GC/MS analysis. The individual components of HETCL were identified by comparison of their mass spectra and the GC retention data of compounds previously analysed in the data system. Other identifications were made by comparison of mass spectra with those in data system libraries or cited in the literature (Smith and Strickland, 2007). The most significant points emerging from the data in Fig. 4 is that hydrocarbons were comprised of a series (C14-C33) of normal alkanes with odd carbon numbered chains dominant. The main n-alkanes found in HETCL were C25H52, C26H54, C27H56, C30H62, and C31H64 (Fig. 4). 3.3. Analysis of total monoterpenoid indole alkaloids by off-line ESI/MS Plant latex is a complex mixture of bioactive compounds, including alkaloids, cardiac glycosides, phenolics, terpenes, sugar, and proteins (Hagel et al., 2008; Konno, 2011). Several indole alkaloids have been isolated from the stem bark of T. catharinensis (Table 2), including the leishmanicidal compounds coronaridine and voacangine, and the antimalarial compound voacamine (Soares et al., 2003; Federici et al., 2000), but to date there are no reports in the literature of studies aimed at the extraction and identification of alkaloids obtained exclusively from T. catharinensis latex. In this study, T. catharinensis latex was obtained after cutting the stem and allowing it to flow into tap water, after which it was submitted to an acid-based method of alkaloid extraction. ESI(+)-MS analysis of MIA-A and MIA-B from latex of T. catharinensis revealed the presence of chemicals with m/z ranging from 250 to 747 (Figs. 5 and 6, Supplementary Material 4). The results obtained in this study, taking into account previous reports of T. catharinensis (Pereira et al., 2004; Nicola et al., 2013) indicated the presence of the following alkaloids: ibogamine (m/z 281.1999),affinisine (m/z 309.1951), 16-epi-affinine (m/z 325.1895), coronaridine (m/z

3.4. Proteolytic activity CLTc is a relatively milky fluid with a complex mixture of constituents, although the major components are sucrose and proteins. The presence of proteins such as proteases appears to be significant for plant 82

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defence mechanisms (Vierstra, 1996). Proteases are enzymes that catalyse the hydrolysis of covalent peptide bonds and are essential for many aspects of physiology and development in living organisms. Recently, the use of proteases in industrial processes has significantly increased (Vierstra, 1996; Li et al., 2013). The proteins present in latex (CLTc) are proteolytic and were precipitated with acetone (CLPTc); this extract is richest in proteins that exhibited high specific activity compared with CLTc and trypsin (Table 1). In this regard, the proteolytic activity in T. catharinensis latex is comparable to trypsin, which is widely used in the fields of biotechnology and medicine (Macalood et al., 2013). The aqueous extract of T. catharinensis has been used in traditional medicine as an antidote for snakebites, and some tests have shown it is effective against extracts of the root of T. catharinensiscould inhibit the lethal and myotoxic activity of Crotalus durissus terrificus venom (Batina et al., 2000). The main chemical components present in the venom of snakes of the genus Crotalus are as follows: crotoxin (protein), crotamine (polypeptide), convulxin (glycoprotein), and girotoxin (glycoprotein) (Soares et al., 2010).As these toxins are proteins, the proteolytic activity of its latex could explain the use of the aqueous extract of T. catharinensis in traditional medicine as an antidote for snakebites.

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4. Conclusions The results of the present study showed that CLTc exhibits proteolytic activity. The proteins present in CLTc were important for proteolytic activity. The results support latex as a rich source of molecules, such as sugars, amino acids, n-alkanes, alkaloids, and proteins, with interesting properties. CLTc can be considered a target for fractionation by chromatography to purify and identify the molecules responsible for proteolytic activity. The use and management of T. catharinensis latex as well as the ecological characteristics of the species indicates potential for programs for sustainable production. This is the first study investigating activity in the protein latex content obtained from T. catharinensis. Acknowledgements The authors would like to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brazilian National Council for Scientific and Technological Development) (CNPq, process no. 481915/2013-3) as well as the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Coordination for the Improvement of Higher Education Personnel) and the Complexo de Centrais de Apoio à Pesquisa (Research Support Center Complex) of the State University of Maringá. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2018.11.036. References Agrawal, A.A., Konno, K., 2009. Latex: a model for understanding mechanisms, ecology, and evolution of plant defense against herbivory. Annu. Rev. Ecol. Evol. Syst. 40, 311–331. Almeida, L., Cintra, A.C., Veronese, E.L., Nomizo, A., Franco, J.J., Arantes, E.C., Giglio, J.R., Sampaio, S.V., 2004. Anticrotalic and antitumoral activities of gel filtration fractions of aqueous extract from Tabernaemontana catharinensis (Apocynaceae). Comp. Biochem. Phys. 137C, 19–27. Araujo, A.R., Kascheres, C., Fujiwara, F., Marsaioli, A.J., 1984. Catharinensine, an oxindole alkaloid from Peschiera catharinensis. Phytochemistry 23, 2359–2363. Batina, M.F.C., Cintra, A.C.O., Veronese, E.L.G., Lavrador, M.A.S., Giglio, J.R., Pereira, P.S., 2000. Inhibition of the lethal and myotoxic activities of Crotalus durissus terrificus venom by Tabernaemontana catharinensis A. DC. (Apocynaceae). Identification of one the active components. Planta Med. 66, 424–428. Braga, R.M., Filho, H.F.L., Reis, F.A.M., 1984. 13C NMR analysis of alkaloids from Peschiera fuchsiaefolia. Phytochemistry 23, 175–178.

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