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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Histochemical investigation of Cochlospermum regium (Schrank) Pilg. leaves and chemical composition of its essential oil a
b
Marielle Cascaes Inácio , Tiago Antunes Paz , Bianca Waléria c
a
Bertoni , Maria Aparecida Ribeiro Vieira , Márcia Ortiz Mayo d
Marques & Ana Maria Soares Pereira
c
a
Departamento de Produção Vegetal, Faculdade de Ciências Agronômicas, Universidade Estadual Paulista, Botucatu, SP, Brazil b
Departamento de Química Orgânica, Instituto de Química, Universidade Estadual Paulista, Araraquara, SP, Brazil c
Departamento de Biotecnologia Vegetal, Unidade de Biotecnologia, Universidade de Ribeirão Preto, Avenida Costábile Romano, n° 2201 Ribeirânia, 14096-900 Ribeirão Preto, SP, Brazil d
Instituto Agronômico de Campinas, Centro de Pesquisa e Desenvolvimento de Recursos Genéticos Vegetais, Campinas, SP, Brazil Published online: 25 Feb 2014.
To cite this article: Marielle Cascaes Inácio, Tiago Antunes Paz, Bianca Waléria Bertoni, Maria Aparecida Ribeiro Vieira, Márcia Ortiz Mayo Marques & Ana Maria Soares Pereira (2014) Histochemical investigation of Cochlospermum regium (Schrank) Pilg. leaves and chemical composition of its essential oil, Natural Product Research: Formerly Natural Product Letters, 28:10, 727-731, DOI: 10.1080/14786419.2013.879133 To link to this article: http://dx.doi.org/10.1080/14786419.2013.879133
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Natural Product Research, 2014 Vol. 28, No. 10, 727–731, http://dx.doi.org/10.1080/14786419.2013.879133
Histochemical investigation of Cochlospermum regium (Schrank) Pilg. leaves and chemical composition of its essential oil Marielle Cascaes Ina´cioa, Tiago Antunes Pazb, Bianca Wale´ria Bertonic, Maria Aparecida Ribeiro Vieiraa, Ma´rcia Ortiz Mayo Marquesd and Ana Maria Soares Pereirac* Departamento de Produc a˜o Vegetal, Faculdade de Cieˆncias Agronoˆmicas, Universidade Estadual Paulista, Botucatu, SP, Brazil; bDepartamento de Quı´mica Orgaˆnica, Instituto de Quı´mica, Universidade Estadual Paulista, Araraquara, SP, Brazil; cDepartamento de Biotecnologia Vegetal, Unidade de Biotecnologia, Universidade de Ribeira˜o Preto, Avenida Costa´bile Romano, n8 2201 Ribeiraˆnia, 14096-900 Ribeira˜o Preto, SP, Brazil; dInstituto Agronoˆmico de Campinas, Centro de Pesquisa e Desenvolvimento de Recursos Gene´ticos Vegetais, Campinas, SP, Brazil
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a
(Received 30 September 2013; final version received 20 December 2013) Essential oil from Cochlospermum regium (Schrank) Pilg. leaves (CR-EO) has been extracted by hydrodistillation; we analysed the CR-EO by gas chromatography coupled with mass spectrometry. We also conducted histochemical analysis on cross-sections of the central vein of young and adult leaves. A total of 32 compounds were qualitatively and quantitatively analysed, which represented 94.87% of the total CR-EO oil content. The CR-EO basically consisted of sesquiterpenes (96.87%); its main component was b-copaen-4-a-ol (18.73%), followed by viridiflorol (12.67%). The histochemical analyses identified the main classes of compounds present in both young and adult leaves. Keywords: Bixaceae; b-copaen-4-a-ol; sesquiterpenes; GC – MS analysis; Cochlospermum regium
1. Introduction Cochlospermum regium (Schrank) Pilg., Bixaceae, is a medicinal plant species which occurs in the Brazilian savanna, known as Cerrado. According to Zachos and Habel (2011), this biome constitutes a hotspot which occupies 21% of the Brazilian territory and contains 4400 endemic plant species. In traditional folk medicine, C. regium roots are used to treat infections of the female reproductive system (Nunes et al. 2003; Souza & Felfili 2006; Moreira & Guarim-Neto 2009). Therefore, its medicinal potential makes this plant an extensively investigated species. Various chemical compounds have been isolated from C. regium roots, including dihydrokaempferol-3-O-b-glucopyranoside (Lima et al. 1996; Solon et al. 2012). The essential oil of the radicular system, which showed antimicrobial activity against Staphylococcus aureus and Salmonella typhimurium, contains the sesquiterpene b-selinene as a major compound (Honda et al. 1997). The hydroalcoholic extracts of roots, which also showed antimicrobial activity against S. aureus and Pseudomonas aeruginosa, contained ellagic acid; gallic acid, dihydrokaempferol, dihydrokaempferol-3-O-b-(600 -galloyl)-glucopyranoside, pinoresinol, excelsin and the triacylbenzenes, cochlospermine A and B (Solon et al. 2012). Till date, no studies have evaluated the chemical composition of C. regium leaves.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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M.C. Ina´cio et al.
Therefore, we aimed to analyse the chemical composition of the essential oil from C. regium leaves (CR-EO). We also conducted phytochemical screening of CR-EO using histochemical tests.
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2. Results and discussion 2.1. CR-EO composition This is the first investigation on the essential oil obtained from dried C. regium leaves (CR-EO). Table 1 presents the chemical composition of this essential oil. We identified 94.87% of the CR-EO constituents, which corresponded to 32 compounds (Table 1). The major CR-EO components were b-copaen-4-a-ol (18.73%), viridiflorol (12.67%), bicyclogermacrene (8.26%) and longiborneol (7.13%). The CR-EO extraction procedure yield was 0.2%. Honda et al. (1997) analysed the essential oil extracted from the roots of this plant species (radicular system) and identified b-selinene as the main constituent (34.1%). The CR-EO consisted mainly of sesquiterpenes (96.87%); myrcene was the only constituent that did not belong to this class. The few phytochemical studies on leaves of plants of the genus Cochlospermum show that sesquiterpenes are the major constituents of the essential oil. Essential oil from the leaves of Cochlospermum angolense contains 68.8% of sesquiterpenes in which the major constituents are germacrene D (9.4%), a-cadinol (7.4%) and 10-epi-cubenol (6.2%) (Leonardi et al. 2012). The major components of the essential oil from leaves of Cochlospermum vitifolium, which also occurs in Brazil, are sesquiterpenes, including b-caryophyllene (46.5%), a-humulene (26%) and b-pinene (10.6%) (Almeida et al. 2005). Sesquiterpenes are essential for plant survival: they attract natural enemies of herbivores, act in the general plant defence against pathogens and play a role in plant–plant signalling. (Holopainen 2004; Unsicker et al. 2009). Hence, considering that we collected C. regium from its natural habitat, it is not surprising that sesquiterpenes were the main constituents of CR-EO – in its natural environment, this plant is susceptible to adverse biotic and abiotic factors. Table 1. Composition of the essential oil obtained from dried C. regium leaves (CR-EO). Component 1. Copaen-4-a-ol , b . 2. Viridiflorol 3. Bicyclogermacrene 4. Longiborneol 5. trans-Caryophyllene 6. Humulene , a . 7. Cubenol 8. Myrcene 9. Cadinol , epi-a. 10. Germacrene B 11. Cedr-8(15)-EN-9-a-ol 12. Muurola-4,10(14)-dien-1-b-ol 13. Guaiol 14. Humulene epoxide II 15. Spathulenol 16. Cubenol ,1-epi-. 17. Khusimone 18. Longipinanol , epi-. 19. Eudesmol ,10-epi-g.
Percentage
Tabulated Kovats index
Calculated Kovats index
Molecular formula
Molecular mass
18.73 12.67 8.26 7.13 4.49 4.44 4.44 3.82 3.14 2.96 2.87 2.86 2.61 2.16 2.07 1.77 1.51 1.31 1.29
1584 1590 1494 1592 1418 1454 1642 991 1640 1556 1644 1631 1595 1606 1576 1627 1593 1561 1619
1578 1585 1495 1591 1418 1452 1652 988 1639 1554 1636 1631 1600 1605 1580 1622 1593 1562 1619
C15H24O C15H26O C15H24 C15H26O C15H24 C15H24 C15H26O C10H16 C15H26O C15H24 C15H24O C15H24O C15H26O C15H24O C15H24O C15H26O C14H20O C15H26O C15H26O
220 222 204 222 204 204 222 136 222 204 220 220 222 220 220 222 204 222 222
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Table 2. Histochemical tests on the central vein of young and adult C. regium leaves.
Dragendorff
Bouchardt
K2Cr2O7
FeCl31%
Genotype
Leaves
C
T
C
T
C
T
C
T
C
T
C
T
01
Young Adults Young Adults Young Adults Young Adults
2 2 2 2 2 2 2 2
þ þ þ þ þ þ þ þ
2 2 2 2 2 2 2 2
þ þ þ þ þ þ þ þ
2 2 2 2 2 2 2 2
þ þ þ þ þ þ þ þ
2 2 2 2 2 2 2 2
þ þ þ þ þ þ þ þ
2 2 2 2 2 2 2 2
þ þ þ þ þ þ þ þ
2 2 2 2 2 2 2 2
þ þ þ þ þ þ þ þ
02 03 04
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Sudan III
Vanillinhydrochloric acid
Note: C, control; T, treatment; 2, negative; þ, positive.
Figure 1. Histochemical tests on C. regium leaves. (A) Lipids, (B) total phenols, (C) tannins and (D) alkaloids.
2.2. Histochemical analysis Histochemical tests on the cross-sections of the central veins of C. regium leaves using the reactant Sudan III indicated that the epidermis contained lipids: application of iron(III) chloride (FeCl3) and potassium dichromate (K2Cr2O7) in this region rendered a dark (almost black) and brown-reddish colour, respectively, indicating a positive reaction for total phenols. Vanillinhydrochloric acid indicated the presence of tannins in the parenchymal cells of the leaves’ central vein. The Dragendorff and Bouchardt test evidenced nitrogen compounds in the same region (Table 2). The main classes of active compounds were present in both young and adult C. regium leaves (Table 2 and Figure 1). However, histochemical tests only evidence the classes of secondary metabolites, such as total phenols, nitrogen compounds, lipids and lignin (Kraus & Arduin 1997; Ricco et al. 2002). In other words, this technique only serves as a means to screen compounds, making further studies necessary to detail the chemical composition of the plant tissues.
3. Experimental 3.1. General description of the assays The C. regium plant material used for histochemical tests and extraction of the essential oil from leaves was collected from Reserva EcoCerrado Brasil, located in the city of Araxa´, MG, Brazil (Latitude: 198360 48.900 , Longitude: 428080 20.800 , altitude: 929 m), at 9 am. The specimen was identified by Dr Lin Chau Ming (Departamento de Produc a˜o Vegetal, UNESP, Botucatu, SP,
730
M.C. Ina´cio et al.
Brazil). A voucher specimen (No. 1463) has been deposited at the Herbarium of Medicinal Plants of the University of Ribeira˜oPreto (HPM-UNAERP, Ribeira˜o Preto, SP, Brazil). For the histochemical analysis, leaves were classified as young (maximum length ¼ 5 cm) and adults (maximum length ¼ 9 cm).
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3.2. Extraction and analysis of the CR-EO C. regium leaves were dried in an oven at 438C, under circulating air. Then, 26 g of this material was subjected to hydrodistillation in a Clevenger apparatus for 120 min. Compounds were identified and quantified by gas chromatography coupled with mass spectrometry (GC– MS equipment Shimadzu QP-5000), using a silica-fused capillary OV-5 (30 m £ 0.25 mm £ 0.25 mm, Ohio Valley Specialty Chemical, Inc., Marietta, OH, USA), operated by electron spray (70 eV). Analytical conditions were as follows – injector temperature: 2408C; detector temperature: 2308C; carrier gas: He; gas flow: 1.0 mL min21; dilution: 1 mL of the essential oil in 1.0 mL of ethyl acetate (AcoEt); injection volume: 1 mL; split: 1/20; temperature program: from 60 to1658C at 38C min21 and from 165 to 2408C at 108C min21. The oil constituents were identified by matching their mass spectra with NIST 12. Lib., NIST 62. Lib., Wiley 139. Lib. and reported data (Adams 1995). 3.3. Histochemical test Histochemistry was conducted on four C. regium specimens. The cross-sections were manually cut on the central vein region; leaves were separated into young (buds) and adult. The following reagents were employed: Sudan III, to test lipids (reddish colour); FeCl3 1% (dark blue/black colour) and K2Cr2O7 (brown-reddish colour), to detect total phenols; vanillin-hydrochloric acid, to test tannins (reddish colour) and Dragendorff (potassium iodobismuthate, brown-reddish) and Bouchardt (potassium iodate, brown-reddish), to detect alkaloids. All the tests were conducted using a control sample in which the sections were bleached and subjected to the same procedures. 4. Conclusions This is the first report on the chemical composition of the essential oil extracted from C. regium leaves. This oil contains sesquiterpenes as the main constituents, and its major compound is b-copaen-4-a-ol. In addition, histochemical analysis showed that regardless of the maturity stage, C. regium presented the main class of secondary metabolites produced by angiosperms, such as lipids, phenolic compounds, tannins and nitrogen compounds. Acknowledgement We thank the Fundac a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP, grant no. 2008/52719-0) for financial support.
References Adams RP. 1995. Identification of essential oil components by gas chromatography/mass spectroscopy. Wheaton (IL): Allured Publishing Corporation; p. 1 –69. Almeida SCX, Lemos TLG, Silveira ER, Pessoa ODL. 2005. Volatile and non-volatile chemical constituents of Cochlospermum vitifolium (Willdenow) Sprengel. Quı´m Nova. 28:57–60. Holopainen JK. 2004. Multiple function of inducible plant volatiles. Trends Plant Sci. 9:529– 533. Honda NK, Brum RL, Hess SC, Cruz AB, Moretto E. 1997. Antibacterial activity of Cochlospermum regium essential oil. Fitoterapia. 68:79–80.
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Kraus JE, Arduin M. 1997. Manual ba´sico de me´todos em morfologia vegetal [Basic manual of methods in plant morphology]. Rio de Janeiro: Edur. Leonardi M, Giovanelli S, Cioni PL, Flamini G, Pistelli L. 2012. Evaluation of volatile constituents of Cochlospermum angolense. Nat Prod Commun. 7:629–632. Lima DP, Castro MSA, Mello JCP, Siqueira JM, Kassab NM. 1996. A flavanone glycoside from Cochlospermum regium. Fitoterapia. 66:545–546. Moreira DL, Guarim-Neto G. 2009. Usos mu´ltiplos de plantas do Cerrado: um estudo etnobotaˆnico na comunidade sı´tio Pindura, Rosa´rio Oeste, Mato Grosso, Brasil [Multiple uses of Cerrado plants: an ethnobotanical study on the community site Pindura West Rosary, Mato Grosso, Brazil]. Polibota´nica. 27:159–190. Nunes GP, Silva MF, Rezende UM. 2003. Plantas medicinais comercializadas por raizeiros no Centro de Campo Grande, Mato Grosso do Sul [Medicinal plants from herb sellers operating in downtown Campo Grande, Mato Grosso do Sul, Brazil]. Rev bras farmacogn. 13:83– 92. Ricco RA, Sena GA, Vai VM, Wagner ML, Gurni AA. 2002. Taninos condensados de Ephedra chilensis K. Presl. (E. andina Poepp. ex May.) (Ephedraceae). Dominguezia. 18:17–25. Solon S, Carollo CA, Branda˜o LFG, Macedo CS, Klein A, Dias-Junior CA, Siqueira CM. 2012. Phenolic derivatives and other chemical compounds from Cochlospermum regium. Quı´m Nova. 35:1169–1172. Souza CD, Felfili JM. 2006. Uso de plantas medicinais na regia˜o de Alto Paraı´so de Goia´s, GO, Brasil [The utilization of medicinal plants in the region of Alto Paraı´so of Goia´s, GO, Brazil]. Acta Bot Bras. 20:135–142. Unsicker SB, Kunert G, Gershenzon J. 2009. Protective perfumes: the role of vegetative volatiles in plant defense against herbivores. Curr Opin Plant Biol. 12:479– 485. Zachos FE, Habel JC. 2011. Biodiversity hotspots: distribution and protection of conservation priority areas. New York: Springer.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Histochemical investigation of Cochlospermum regium (Schrank) Pilg. leaves and chemical composition of its essential oil a
b
Marielle Cascaes Inácio , Tiago Antunes Paz , Bianca Waléria c
a
Bertoni , Maria Aparecida Ribeiro Vieira , Márcia Ortiz Mayo d
Marques & Ana Maria Soares Pereira
c
a
Departamento de Produção Vegetal, Faculdade de Ciências Agronômicas, Universidade Estadual Paulista, Botucatu, SP, Brazil b
Departamento de Química Orgânica, Instituto de Química, Universidade Estadual Paulista, Araraquara, SP, Brazil c
Departamento de Biotecnologia Vegetal, Unidade de Biotecnologia, Universidade de Ribeirão Preto, Avenida Costábile Romano, n° 2201 Ribeirânia, 14096-900 Ribeirão Preto, SP, Brazil d
Instituto Agronômico de Campinas, Centro de Pesquisa e Desenvolvimento de Recursos Genéticos Vegetais, Campinas, SP, Brazil Published online: 25 Feb 2014.
To cite this article: Marielle Cascaes Inácio, Tiago Antunes Paz, Bianca Waléria Bertoni, Maria Aparecida Ribeiro Vieira, Márcia Ortiz Mayo Marques & Ana Maria Soares Pereira (2014) Histochemical investigation of Cochlospermum regium (Schrank) Pilg. leaves and chemical composition of its essential oil, Natural Product Research: Formerly Natural Product Letters, 28:10, 727-731, DOI: 10.1080/14786419.2013.879133 To link to this article: http://dx.doi.org/10.1080/14786419.2013.879133
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 10, 727–731, http://dx.doi.org/10.1080/14786419.2013.879133
Histochemical investigation of Cochlospermum regium (Schrank) Pilg. leaves and chemical composition of its essential oil Marielle Cascaes Ina´cioa, Tiago Antunes Pazb, Bianca Wale´ria Bertonic, Maria Aparecida Ribeiro Vieiraa, Ma´rcia Ortiz Mayo Marquesd and Ana Maria Soares Pereirac* Departamento de Produc a˜o Vegetal, Faculdade de Cieˆncias Agronoˆmicas, Universidade Estadual Paulista, Botucatu, SP, Brazil; bDepartamento de Quı´mica Orgaˆnica, Instituto de Quı´mica, Universidade Estadual Paulista, Araraquara, SP, Brazil; cDepartamento de Biotecnologia Vegetal, Unidade de Biotecnologia, Universidade de Ribeira˜o Preto, Avenida Costa´bile Romano, n8 2201 Ribeiraˆnia, 14096-900 Ribeira˜o Preto, SP, Brazil; dInstituto Agronoˆmico de Campinas, Centro de Pesquisa e Desenvolvimento de Recursos Gene´ticos Vegetais, Campinas, SP, Brazil
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a
(Received 30 September 2013; final version received 20 December 2013) Essential oil from Cochlospermum regium (Schrank) Pilg. leaves (CR-EO) has been extracted by hydrodistillation; we analysed the CR-EO by gas chromatography coupled with mass spectrometry. We also conducted histochemical analysis on cross-sections of the central vein of young and adult leaves. A total of 32 compounds were qualitatively and quantitatively analysed, which represented 94.87% of the total CR-EO oil content. The CR-EO basically consisted of sesquiterpenes (96.87%); its main component was b-copaen-4-a-ol (18.73%), followed by viridiflorol (12.67%). The histochemical analyses identified the main classes of compounds present in both young and adult leaves. Keywords: Bixaceae; b-copaen-4-a-ol; sesquiterpenes; GC – MS analysis; Cochlospermum regium
1. Introduction Cochlospermum regium (Schrank) Pilg., Bixaceae, is a medicinal plant species which occurs in the Brazilian savanna, known as Cerrado. According to Zachos and Habel (2011), this biome constitutes a hotspot which occupies 21% of the Brazilian territory and contains 4400 endemic plant species. In traditional folk medicine, C. regium roots are used to treat infections of the female reproductive system (Nunes et al. 2003; Souza & Felfili 2006; Moreira & Guarim-Neto 2009). Therefore, its medicinal potential makes this plant an extensively investigated species. Various chemical compounds have been isolated from C. regium roots, including dihydrokaempferol-3-O-b-glucopyranoside (Lima et al. 1996; Solon et al. 2012). The essential oil of the radicular system, which showed antimicrobial activity against Staphylococcus aureus and Salmonella typhimurium, contains the sesquiterpene b-selinene as a major compound (Honda et al. 1997). The hydroalcoholic extracts of roots, which also showed antimicrobial activity against S. aureus and Pseudomonas aeruginosa, contained ellagic acid; gallic acid, dihydrokaempferol, dihydrokaempferol-3-O-b-(600 -galloyl)-glucopyranoside, pinoresinol, excelsin and the triacylbenzenes, cochlospermine A and B (Solon et al. 2012). Till date, no studies have evaluated the chemical composition of C. regium leaves.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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M.C. Ina´cio et al.
Therefore, we aimed to analyse the chemical composition of the essential oil from C. regium leaves (CR-EO). We also conducted phytochemical screening of CR-EO using histochemical tests.
Downloaded by [Tulane University] at 02:07 04 September 2014
2. Results and discussion 2.1. CR-EO composition This is the first investigation on the essential oil obtained from dried C. regium leaves (CR-EO). Table 1 presents the chemical composition of this essential oil. We identified 94.87% of the CR-EO constituents, which corresponded to 32 compounds (Table 1). The major CR-EO components were b-copaen-4-a-ol (18.73%), viridiflorol (12.67%), bicyclogermacrene (8.26%) and longiborneol (7.13%). The CR-EO extraction procedure yield was 0.2%. Honda et al. (1997) analysed the essential oil extracted from the roots of this plant species (radicular system) and identified b-selinene as the main constituent (34.1%). The CR-EO consisted mainly of sesquiterpenes (96.87%); myrcene was the only constituent that did not belong to this class. The few phytochemical studies on leaves of plants of the genus Cochlospermum show that sesquiterpenes are the major constituents of the essential oil. Essential oil from the leaves of Cochlospermum angolense contains 68.8% of sesquiterpenes in which the major constituents are germacrene D (9.4%), a-cadinol (7.4%) and 10-epi-cubenol (6.2%) (Leonardi et al. 2012). The major components of the essential oil from leaves of Cochlospermum vitifolium, which also occurs in Brazil, are sesquiterpenes, including b-caryophyllene (46.5%), a-humulene (26%) and b-pinene (10.6%) (Almeida et al. 2005). Sesquiterpenes are essential for plant survival: they attract natural enemies of herbivores, act in the general plant defence against pathogens and play a role in plant–plant signalling. (Holopainen 2004; Unsicker et al. 2009). Hence, considering that we collected C. regium from its natural habitat, it is not surprising that sesquiterpenes were the main constituents of CR-EO – in its natural environment, this plant is susceptible to adverse biotic and abiotic factors. Table 1. Composition of the essential oil obtained from dried C. regium leaves (CR-EO). Component 1. Copaen-4-a-ol , b . 2. Viridiflorol 3. Bicyclogermacrene 4. Longiborneol 5. trans-Caryophyllene 6. Humulene , a . 7. Cubenol 8. Myrcene 9. Cadinol , epi-a. 10. Germacrene B 11. Cedr-8(15)-EN-9-a-ol 12. Muurola-4,10(14)-dien-1-b-ol 13. Guaiol 14. Humulene epoxide II 15. Spathulenol 16. Cubenol ,1-epi-. 17. Khusimone 18. Longipinanol , epi-. 19. Eudesmol ,10-epi-g.
Percentage
Tabulated Kovats index
Calculated Kovats index
Molecular formula
Molecular mass
18.73 12.67 8.26 7.13 4.49 4.44 4.44 3.82 3.14 2.96 2.87 2.86 2.61 2.16 2.07 1.77 1.51 1.31 1.29
1584 1590 1494 1592 1418 1454 1642 991 1640 1556 1644 1631 1595 1606 1576 1627 1593 1561 1619
1578 1585 1495 1591 1418 1452 1652 988 1639 1554 1636 1631 1600 1605 1580 1622 1593 1562 1619
C15H24O C15H26O C15H24 C15H26O C15H24 C15H24 C15H26O C10H16 C15H26O C15H24 C15H24O C15H24O C15H26O C15H24O C15H24O C15H26O C14H20O C15H26O C15H26O
220 222 204 222 204 204 222 136 222 204 220 220 222 220 220 222 204 222 222
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Table 2. Histochemical tests on the central vein of young and adult C. regium leaves.
Dragendorff
Bouchardt
K2Cr2O7
FeCl31%
Genotype
Leaves
C
T
C
T
C
T
C
T
C
T
C
T
01
Young Adults Young Adults Young Adults Young Adults
2 2 2 2 2 2 2 2
þ þ þ þ þ þ þ þ
2 2 2 2 2 2 2 2
þ þ þ þ þ þ þ þ
2 2 2 2 2 2 2 2
þ þ þ þ þ þ þ þ
2 2 2 2 2 2 2 2
þ þ þ þ þ þ þ þ
2 2 2 2 2 2 2 2
þ þ þ þ þ þ þ þ
2 2 2 2 2 2 2 2
þ þ þ þ þ þ þ þ
02 03 04
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Sudan III
Vanillinhydrochloric acid
Note: C, control; T, treatment; 2, negative; þ, positive.
Figure 1. Histochemical tests on C. regium leaves. (A) Lipids, (B) total phenols, (C) tannins and (D) alkaloids.
2.2. Histochemical analysis Histochemical tests on the cross-sections of the central veins of C. regium leaves using the reactant Sudan III indicated that the epidermis contained lipids: application of iron(III) chloride (FeCl3) and potassium dichromate (K2Cr2O7) in this region rendered a dark (almost black) and brown-reddish colour, respectively, indicating a positive reaction for total phenols. Vanillinhydrochloric acid indicated the presence of tannins in the parenchymal cells of the leaves’ central vein. The Dragendorff and Bouchardt test evidenced nitrogen compounds in the same region (Table 2). The main classes of active compounds were present in both young and adult C. regium leaves (Table 2 and Figure 1). However, histochemical tests only evidence the classes of secondary metabolites, such as total phenols, nitrogen compounds, lipids and lignin (Kraus & Arduin 1997; Ricco et al. 2002). In other words, this technique only serves as a means to screen compounds, making further studies necessary to detail the chemical composition of the plant tissues.
3. Experimental 3.1. General description of the assays The C. regium plant material used for histochemical tests and extraction of the essential oil from leaves was collected from Reserva EcoCerrado Brasil, located in the city of Araxa´, MG, Brazil (Latitude: 198360 48.900 , Longitude: 428080 20.800 , altitude: 929 m), at 9 am. The specimen was identified by Dr Lin Chau Ming (Departamento de Produc a˜o Vegetal, UNESP, Botucatu, SP,
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Brazil). A voucher specimen (No. 1463) has been deposited at the Herbarium of Medicinal Plants of the University of Ribeira˜oPreto (HPM-UNAERP, Ribeira˜o Preto, SP, Brazil). For the histochemical analysis, leaves were classified as young (maximum length ¼ 5 cm) and adults (maximum length ¼ 9 cm).
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3.2. Extraction and analysis of the CR-EO C. regium leaves were dried in an oven at 438C, under circulating air. Then, 26 g of this material was subjected to hydrodistillation in a Clevenger apparatus for 120 min. Compounds were identified and quantified by gas chromatography coupled with mass spectrometry (GC– MS equipment Shimadzu QP-5000), using a silica-fused capillary OV-5 (30 m £ 0.25 mm £ 0.25 mm, Ohio Valley Specialty Chemical, Inc., Marietta, OH, USA), operated by electron spray (70 eV). Analytical conditions were as follows – injector temperature: 2408C; detector temperature: 2308C; carrier gas: He; gas flow: 1.0 mL min21; dilution: 1 mL of the essential oil in 1.0 mL of ethyl acetate (AcoEt); injection volume: 1 mL; split: 1/20; temperature program: from 60 to1658C at 38C min21 and from 165 to 2408C at 108C min21. The oil constituents were identified by matching their mass spectra with NIST 12. Lib., NIST 62. Lib., Wiley 139. Lib. and reported data (Adams 1995). 3.3. Histochemical test Histochemistry was conducted on four C. regium specimens. The cross-sections were manually cut on the central vein region; leaves were separated into young (buds) and adult. The following reagents were employed: Sudan III, to test lipids (reddish colour); FeCl3 1% (dark blue/black colour) and K2Cr2O7 (brown-reddish colour), to detect total phenols; vanillin-hydrochloric acid, to test tannins (reddish colour) and Dragendorff (potassium iodobismuthate, brown-reddish) and Bouchardt (potassium iodate, brown-reddish), to detect alkaloids. All the tests were conducted using a control sample in which the sections were bleached and subjected to the same procedures. 4. Conclusions This is the first report on the chemical composition of the essential oil extracted from C. regium leaves. This oil contains sesquiterpenes as the main constituents, and its major compound is b-copaen-4-a-ol. In addition, histochemical analysis showed that regardless of the maturity stage, C. regium presented the main class of secondary metabolites produced by angiosperms, such as lipids, phenolic compounds, tannins and nitrogen compounds. Acknowledgement We thank the Fundac a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP, grant no. 2008/52719-0) for financial support.
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