sdv.sp.med sdv.bbm.bc
R G ; Ferraz 
  
C A A ; Oliveira 
  
A P ; Alencar-Filho 
  
J M T ; Araújo 
  
E C C ; Nunes 
  
X P ; Picot 
  
L ; Lopes 
  
N P ; Braz-Filho 
  
R ; Rolim 
  
L A ; Almeida 
  
Raimundo G De Oliveira-Júnior 
  
Christiane Adrielly 
  
A Ferraz 
  
Ana Paula De Oliveira 
  
José Marcos 
  
T De Alencar-Filho 
  
Edigênia C C Araújo 
  
Xirley P Nunes 
  
Laurent Picot 
  
Norberto P Lopes 
  
Raimundo Braz- Filho 
  
Larissa A Rolim 
  
Jackson Roberto 
  
G Da 
  
S Almeida 
  
  
  
  
Chemical constituents of non-polar fractions obtained from Cnidoscolus quercifolius Pohl (Euphorbiaceae), a Brazilian medicinal plant native to Caatinga biome

Keywords: medicinal plants, Caatinga, terpenoids, Cnidoscolus Cnidoscolus quercifolius Pohl plantas medicinais, Caatinga, terpenoides, Cnidoscolus

Cnidoscolus quercifolius Pohl is a Brazilian medicinal plant from Caatinga biome and possesses several pharmacological properties. In this paper, we describe the identification of compounds from non-polar fractions of the leaves (Hex-Le) and stem-barks (Hex-Sb) from C. quercifolius. Chemical analysis were performed by GC-MS approach. Chemical constituents were identified based on spectral data obtained and by comparison with literature data. Fragmentation profile of the main compounds was proposed and presented in this paper. Hydrocarbons (46.84%), triterpenes (32.60%) and carotenoid derivatives (0.83%) were considered the major constituent of Hex-Le. In contrast, hydrocarbons (41.89%), diterpenes (12.83%) and triterpenes (7.0%) were found in Hex-Sb. Four diterpenes (dehydroabietane, sandaracopimaradiene, kaur-16-ene and 13methyl-17-norkaur-15-ene) and one triterpene (diploptene) are reported for the first time in Cnidoscolus.

Introduction

Caatinga is an exclusively Brazilian biome, which occurs predominantly in the Northeastern Region and comprises approximately 10% of the Brazilian territory. This biome usually presents dry vegetation, characterized by semiarid climate, low and irregular rainfall and fertile soil. [START_REF] Santos | Rheological and some physicochemical characteristics of selected floral honeys from plants of Caatinga[END_REF][START_REF] Albuquerque | Caatinga Revisited: Ecology and Conservation of an Important Seasonal Dry Forest[END_REF] Despite the adverse climatic conditions, Caatinga flora is very diverse and includes numerous species used as food source or in folk medicine. In a previous study, the use of 385 Caatinga species for medicinal purposes was verified. These species were distributed in about 265 genera and 91 different families. [START_REF] Albuquerque | Medicinal plants of the Caatinga (semi-arid) vegetation of NE Brazil: a quantitative approach[END_REF] Although the Caatinga biome presents several species with therapeutic potential, chemical and pharmacological investigations involving these plants are still scarce, [START_REF] Menezes | Biogeochemical cycling in terrestrial ecosystems of the Caatinga Biome[END_REF][START_REF] Albuquerque | Re-examining hypotheses concerning the use and knowledge of medicinal plants: a study in the Caatinga vegetation of NE Brazil[END_REF] which makes this biome one of the least studied in the world.

Cnidoscolus quercifolius (syn. C. phyllacanthus (Mull. Arg.) Pax & L. Hoffm.) is a medicinal plant native to Caatinga, popularly known as favela, faveleira or urtiga-branca. In folk medicine, its leaves and stem barks are used to treat hemorrhoids, renal problems, ophthalmic diseases, injury, skin problems, urinary tract infection and inflammatory processes. [START_REF] Albuquerque | Medicinal plants of the Caatinga (semi-arid) vegetation of NE Brazil: a quantitative approach[END_REF] In fact, pharmacological investigations have demonstrated that extracts or isolated compounds from C. quercifolius have some therapeutic properties, such as antinociceptive, [START_REF] Gomes | Antinociceptive activity of the ethanolic extract from barks and leaves of Cnidoscolus quercifolius (Euphorbiaceae) in mice[END_REF] antiinflammatory, [START_REF] Gomes | Phytochemical screening and anti-inflammatory activity of Cnidoscolus quercifolius (Euphorbiaceae) in mice[END_REF] antioxidant, 8-10 cytotoxic [START_REF] Peixoto-Sobrinho | Antiproliferative activity of species of the genus Cnidoscolus against HT-29, Hep-2 and NCI-H292 cells[END_REF] and antimicrobial [START_REF] Paredes | Screening of Bioactivities and Toxicity of Cnidoscolus quercifolius Pohl[END_REF] activities.

The therapeutic properties of C. quercifolius are justified by the presence of bioactive secondary metabolites, mainly terpenoids. Phytochemical studies have resulted in the isolation of new tricyclic benzocyclohepten diterpenes, called favelins, a reference to the popular name of the species. [START_REF] Endo | novel tricyclic benzocycloheptenes with cytotoxic activities from Brazilian plant, Cnidoscolus phyllacanthus[END_REF][START_REF] Lemos | Terpenoids from Cnidoscolus phyllacanthus Pax et Hoff[END_REF][START_REF] Paula | Constituintes Químicos e Atividade Citotóxica de Cnidoscolus phyllacanthus[END_REF] Endo et al. [START_REF] Endo | a novel tetracyclic clyclopropane derivative from the Brazilian plant, Cnidoscolus phyllacanthus[END_REF] also reported the isolation of favelanone and neofavelanone, new cyclopropane and cyclobutene tetracyclic derivatives, respectively. To date, these diterpenes have been reported exclusively in C. quercifolius, indicating that these compounds can be considered its chemotaxonomic markers. However, its chemical composition is still poorly known. In this paper, we describe the identification of compounds from leaves and stem-barks nonpolar fractions of C. quercifolius, including compounds reported for the first time in the genus. 

Experimental

Plant material

Chemicals

Homologous series of n-alkanes (C 9 H 20 -C 40 H 82 ) were purchased from Merck ® (Germany). All solvents (ethanol, hexane, chloroform, ethyl acetate and methanol) were purchased from Synth ® (Brazil).

Extraction and fractionation

The dried and pulverized leaves (482 g) and stem-barks (1,481 g) of C. quercifolius were separately subjected to maceration with 95% ethanol for 72 h. After, the solution was filtered and concentrated under reduced pressure on a rotatory evaporator at 50 °C, producing 63 and 392 g of ethanol extract from leaves (EtOH-Le, 13.07%) and stem-barks (EtOH-Sb, 26.46%). Subsequently, an aliquot of EtOH-Le (15 g) and EtOH-Sb (50 g) was fractionated by liquid chromatography under vacuum (LCV), using silica gel 60 as stationary phase. Hexane (Hex), chloroform (CHCl3), ethyl acetate (AcOEt) and methanol (MeOH) were used as mobile phase, in increasing order of polarity, resulting in the respective fractions, as shown in figure 1.

Hexane fractions (Hex-Le and Hex-Sb) were chosen for GC-MS analysis. 

Gas Chromatography -Mass Spectrometry (GC-MS) analysis

Qualitative and quantitative determination of the chemical constituents present in Hex-Le and Hex-Sb was performed by GC-MS, using a Shimadzu ® gas chromatograph (QP-2010) interfaced with a mass spectrometer, employing the following chromatographic conditions: Phenomenex ® ZB-5MS Zebron column (30.0 m x 0.25 mm x 0.25 mm); helium (99.999%) carrier gas at a constant flow of 1.1 ml.min -1 ; 1 μl injection volume; injector split ratio of 1:40; injector temperature 240 °C; electron impact mode at 70 eV; ion source temperature 280 °C. The oven temperature was programmed at 100 °C (isothermal for 5 min), with an increase 

Identification of compounds

Constituents were identified by comparison of their mass spectra with those of authentic compounds or with reference spectra in the computer library (Wiley7lib and NIST08lib). For some identified secondary metabolites (triterpenes, diterpenes and carotenoid derivatives), the fragmentation profile was proposed based on the peaks observed in the mass spectrum.

Results and Discussion

GC-MS chromatogram of Hex-Le revealed the presence of 49 peaks, of which 30 were identified, corresponding to 80.27% of its total chemical composition (Figure 2A). Among the identified compounds, 0.83% were carotenoid derivatives, 46.84% were long chain hydrocarbons or derivatives, and 32.60% corresponded to squalene, a triterpene considered the major constituent of the sample. Regarding Hex-Sb, the chromatogram showed 74 peaks (Figure 2B), of which 46 were identified, corresponding to 61.72% of its chemical composition. Most of the compounds were long chain hydrocarbons or derivatives (41.89%), followed by diterpenes (12.83%) and triterpenes (7.0%). Carotenoid derivatives were not identified in this sample. The major chemical constituents of Hex-Sb were hexadecane (7.48%) and sandaracopyramadiene (7.53%). All identified compounds are described in Table 1. Two carotenoid derivatives were identified in Hex-Le, β-ionone (0.26%) and dihydroactinidiolide (0.57%) (Figure 3). These constituents are considered products of carotenoid degradation through the action of carotenoid dioxygenase enzymes. [START_REF] Lima | Efeito de baixas doses de irradiação nos carotenoides majoritários em cenouras prontas para o consumo[END_REF][START_REF] Uenojo | Carotenóides: propriedades, aplicações e biotransformação para formação de compostos de aroma[END_REF] In fact, carotenoid degradation products indicate that the plant is in an oxidative stress condition, often associated with the presence of singlet oxygen (O 2 . ). [START_REF] Ramel | Carotenoid oxidation products are stress signals that mediate gene responses to singlet oxygen in plants[END_REF][START_REF] Havaux | Carotenoid oxidation products as stress signals in plants[END_REF] A summary of the biosynthesis as well as the proposed fragmentation profile for β-ionone and dihydroactinidiolide are shown in figure 4. Initially, an α-carotene molecule is used by the carotenoid dioxygenase enzyme as substrate, producing α-ionone and 10'-apo-β-10'-carotenal, which is then converted to β-ionone (m/z 192). In the mass spectra, β-ionone loses a methyl group by a cleavage adjacent to the annular double bond, yielding a new fragment (m/z 177), compatible with the base peak (Figure 3). In an oxidative stress situation, βionone molecule is readily oxidized to 5,6-epoxy-β-ionone and, after a rearrangement step, leads to the formation of dihydroactinidiolide (m/z 180). [START_REF] Lima | Efeito de baixas doses de irradiação nos carotenoides majoritários em cenouras prontas para o consumo[END_REF][START_REF] Uenojo | Carotenóides: propriedades, aplicações e biotransformação para formação de compostos de aroma[END_REF] Rupture of the lactone ring of this molecule releases a stable fragment (m/z 111), compatible with the base peak observed in the mass spectra (Figure 3). The triterpene squalene was found in lower amount in Hex-Sb (4.65%). However, other triterpenes were identified in this sample (diploptene 0.20%, lupeol 2.15%) and its mass spectra are shown in Figure 5.

Squalene is considered the precursor of diplopteno and lupeol, as shown in Figure 6. In its mass spectrum, m/z 69 (base peak) and 81 were the most stable fragments, already described in the literature as characteristic fragments of this molecule. [START_REF] Oyugi | Biological activity and mass spectrometric Analysis of Vernonia amygdalina fractions[END_REF] For lupeol and diploptene, spectral data contributed significantly to the characterization of these molecules, such as molecular ion (m/z 426 and m/z 410, respectively) and base peaks (m/z 191 and m/z 218, respectively). [START_REF] Carvalho | Screening of Filamentous Fungi to Identify Biocatalysts for Lupeol Biotransformation[END_REF][START_REF] Bezerra | Constituintes químicos isolados de três espécies do gênero Sclerolobium[END_REF] Besides these, other fragments are shown in figure 7.

Paula et al. [START_REF] Paula | Constituintes Químicos e Atividade Citotóxica de Cnidoscolus phyllacanthus[END_REF] have reported the presence of lupeol and derivatives in C. quercifolius extracts. However, there are no reports of the presence of diploptene in Cnidoscolus species to date. GC-MS analysis also showed abietane (dehydroabietane 0.24%), pimarane (sandaracopimaradiene 7.53%) and kaurene (kaur-16-ene 4.38%, 13-methyl-17-norkaur-15-ene 0.68%) diterpenes in Hex-Sb, reported for the first time in Cnidoscolus. 

Conclusion

In summary, this study reported a chemical investigation of non-polar fractions obtained from leaves and stem-barks of C. quercifolius. We describe for the first time the identification of diterpenes dehydroabietane, sandaracopimaradiene, kaur-16-ene and 13-methyl- species. Indicators of oxidative stress were also identified (β-ionone and dihydroactinidiolide), in addition to triterpenes (lupeol and diploptene) commonly found in plant species. In this sense, our investigation has contributed to the chemical knowledge of C. quercifolius, a Brazilian medicinal plant from Caatinga biome.

  Leaves and stem-barks of Cnidoscolus quercifolius Pohl were collected in the city of Petrolina, in a Caatinga area (coordinates 09° 03' 55.30'' S and 40° 20' 06.90'' W), State of Pernambuco, Brazil, in February 2013. A voucher specimen (n° 19202) was deposited at the Herbário do Vale do São Francisco (HVASF), of the Universidade Federal do Vale do São Francisco (UNIVASF).

Figure 1 .

 1 Figure 1. Fractionation of EtOH-Le and EtOH-Sb by liquid chromatography under vacuum (LCV). Hexane fractions (Hex-Le and Hex-Sb) were selected for GC-MS analysis.

Figure 2 .

 2 Figure 2. GC-MS chromatograms obtained for Hex-Le (A) and Hex-Sb.

Figure 3 .

 3 Figure 3. Mass spectrum obtained for β-ionone and dihydroactinidiolide identified in Hex-Le.

Figure 4 .

 4 Figure 4. Biosynthesis and proposed fragmentation of carotenoid derivatives β-ionone and dihydroactinidiolide.

Figure 5 .Figure 6 .Figure 7 .

 567 Figure 5. Mass spectra obtained for the triterpenes identified in Hex-Sb.

Figure 8

 8 shows the mass spectra of sandaracopimaradiene as well as the proposed fragmentation profile for the molecule. An abundant fragment (m/z 257) was registered after loss of the methyl group, while the base peak (m/z 137) was originated after a B-ring cleavage, as shown in the figure.

Figure 8 .

 8 Figure 8. Mass spectra and fragmentation proposed for sandaracopimaradiene, identified in Hex-Sb.

Figure 9 Figure 9 .

 99 Figure9shows dehydroabietane mass spectra and its fragmentation profile. Initially, the molecule loses a methyl group, yielding a stable fragment, compatible with the base peak (m/z 255). From that fragment, one isopropyl group is removed, affording another fragment (m/z 213). In addition, A-ring cleavage has been proposed, yielding a fragment (m/z 185) which is then further cleaved, resulting in a new fragment (m/z 159).

Figure 10 .Figure 11 .

 1011 Figure 10. Mass spectra and fragmentation proposed for kaur-16-ene, identified in Hex-Sb.

  -17-norkaur-15-ene m/z 272

  

  CHCl 3 , AcOEt and MeOH of 10 °C.min -1 to 250 °C (isothermal for 5 min) and 10 °C.min -1 to 280 °C (isothermal for 5 min). A mixture of linear hydrocarbons (C 9 H 20 -C 40 H 82 ) was injected under the same experimental conditions.

	EtOH-Le		EtOH-Sb	
	(15 g)		(50 g)	
	LCV	LCV
	Silica gel 60	Silica gel 60
	Hexane, CHCl 3 , AcOEt and MeOH	Hexane,
	Hex-Le	MeOH-Le	Hex-Sb	MeOH-Sb
	(0.17 g)	(10.34 g)	(0.13 g)	(21.44 g)
	CHCl 3 -Le	AcOEt-Le	CHCl 3 -Sb	AcOEt-Sb
	(2.06 g)	(1.98 g)	(16.59 g)	(10.26 g)

Table 1 .

 1 Chemical constituents of non-polar fractions from leaves (Hex-Le) and stem-bark (Hex-Sb) of Cnidoscolus quercifolius. Compounds were identified by CG-MS analysis

	Phytochemical	Compound	Hex-Le		Hex-Sb
			RT (min) Content (%)	RT (min)	Content (%)
	Carotenoid derivatives	β-ionone	22.84	0.26	ND	ND
		Dihydroactinidiolide	24.41	0.57	ND	ND
	Identified (%)		-	0.83	-	ND
	Hydrocarbons and derivatives	1-tridecene	ND	ND	19.45	0.28
		Tetradecane	19.80	0.56	19.82	3.00
		2,3,7-trimethyl-decane	ND	ND	22.27	0.12
		2-methyl-tetradecane	ND	ND	22.37	0.13
		3-methyl-tetradecane	ND	ND	22.65	0.13
		2-methyl-hexadecan-1-ol	ND	ND	23.48	0.21
		Pentadecane	23.81	0.99	23.82	1.99
		5-methyl-pentadecane	ND	ND	25.83	0.29
		2-methyl-pentadecane	ND	ND	26.29	0.24

Acknowledgments

The authors thanks to FACEPE (Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco) for financial support.