Phytochemical screening and isolation of triterpenes and sterols from leaves of Clusia minor L.



Phytochemical screening and isolation of triterpenes and sterols from leaves of Clusia minor L.


Tamizaje fitoquímico y aislamiento de triterpenos y esteroles de las hojas de Clusia minor L.



Raisa Mangas Marín1*
José Carlos Leyva García1
Regla María Becerra García1
Adonis Bello Alarcón2

1 Food and Pharmacy Institute, University of Havana. Havana, Cuba.
2 Faculty of Chemical Sciences, University of Guayaquil. Ciudadela Universitaria Dr. Salvador Allende, Guayaquil, Ecuador.




Introduction: Clusia is one of the most widely studied genera in the Clusiaseae family. Clusia minor L. is a wild shrub with scant phytochemical studies. The latex and leaves from this species are used in traditional medicine to treat warts and sores, respectively.
Objective: Evaluate the chemical composition of extracts obtained from leaves of Clusia minor L.
Methods: The leaves were selected and dried before the extraction process. The extracts were prepared by maceration using solvents of varying polarity (hexane, ethyl acetate and methanol) and by direct extraction with ethanol. All the extracts underwent phytochemical screening. The ethyl acetate extract was fractionated by column chromatography with silica gel. Structural elucidation of the compounds isolated was based on spectroscopic methods and comparison with nuclear magnetic resonance (NMR) data reported for these compounds in the literature.
Results: The following secondary metabolites were identified in the extracts: anthraquinones, triterpenes, steroids, reducing sugars, phenolic compounds and flavonoids. Phytochemical analysis of hexane and ethyl acetate extracts led to isolation of pentacyclic triterpenes, friedelin and betulinic acid, as well as a mixture of the sterols β-sitosterol and stigmasterol.
Conclusions: Phytochemical screening revealed the presence of mainly phenolic compounds in all the extracts. The ethanolic extract was found to contain the greatest variety of secondary metabolites. This was the first time that betulinic acid was isolated from this species.

Key words: Clusia minor L., phenolic compounds, sterols, flavonoids, pentacyclic triterpenes.


Introducción: El género Clusia es uno de los más estudiados de la familia Clusiaseae. Clusia minor L. es un arbusto silvestre con escasos estudios fitoquímicos. El látex y las hojas de esta especie se emplean en la medicina tradicional para el tratamiento de verrugas y llagas, respectivamente.
Objetivo: Evaluar la composición química de los extractos obtenidos de las hojas de Clusia minor L.
Métodos: Se seleccionaron las hojas y se secaron antes del proceso de extracción. Los extractos se prepararon mediante maceración empleando disolventes de diferente polaridad (hexano, acetato de etilo y metanol) y mediante extracción directa con etanol. Se realizó el tamizaje fitoquímico de todos los extractos. Se fraccionó el extracto de acetato de etilo mediante cromatografía en columna con gel de sílice. La elucidación estructural de los compuestos aislados se realizó mediante métodos espectroscópicos y comparación con los datos de resonancia magnética nuclear (RMN) reportados en la literatura para estos compuestos.
Resultados: En los extractos se identificaron los siguientes metabolitos secundarios: antraquinonas, triterpenos, esteroides, azúcares reductores, compuestos fenólicos y flavonoides. El estudio fitoquímico de los extractos de hexano y acetato de etilo permitió aislar triterpenos pentacíclicos, friedelina y ácido betulínico, y una mezcla de esteroles β-sitosterol y estigmasterol.
Conclusiones: El tamizaje fitoquímico permitió identificar principalmente compuestos fenólicos en todos los extractos. El extracto etanólico mostró la mayor variedad de metabolitos secundarios. Por primera vez se aisló el ácido betulínico de esta especie.

Palabras claves: Clusia minor L.; compuestos fenólicos; esteroles; flavonoides; triterpenos pentacíclicos.




The genus Clusia (Clusiaceae) is a rich source of secondary metabolites, including xanthones, flavonoids, biflavonoids, organic acids.1-5 Also the principal metabolites isolated from the species of this genus are polyprenylated benzophenones and triterpenes.6-10 Some of these metabolites are structurally complex and biologically active. The principal biological activities reported are antimicrobial, antioxidant, anti-inflammatory and antitumor properties.3,5,8,11-15

Clusia minor L. (quiripití) is a plant used traditionally to treat sores (latex) and warts (leaves).16

Previous phytochemical studies of the fruits of this species led the isolation of three benzophenones,17 and the analysis by CG/MS of the hexanic extract of the leaves identified terpenes, sterols and vitamin E.18 However, studies about the chemical composition of this species are scarce.

The present work describes the evaluation of chemical qualitative composition and the isolation from the C. minor leaves of friedelin, mixture of β-sitosterol and stigmasterol and betulinic acid. The chemical identification of these compounds was carried out by 1H and 13C NMR data and comparison with those reported in the literature.



Plant material

The leaves of Clusia minor L. (Clusiaceae) were collected at the National Botanical Garden, Havana, Cuba during September 2015 and the identification was realized by Dr. Cristina Panfet. A voucher specimen (Herbarium No. 482) was deposited in the Herbarium of the Tropical Agricultural Fundamental Research Institute. The leaves were dried in a stove at 40 °C during 15 days and subsequently fragmented.

Extraction methodology

The dried and powered leaves (485 g) of Clusia minor L. were successively macerated with n-hexane (extract A), ethyl acetate (extract B) and methanol (extract C) at room temperature three times during seven days each. The ethanolic extract (extract E) was obtained directly following a similar methodology. The extracts were filtered using filter paper and then concentrated to dryness using a rotary vacuum evaporator at 40 ºC. The dried extracts provide 8.84 g, 27.57 g, 23.45 g and 39.65 g, respectively.

Phytochemical screening

The four extracts were evaluated for the existence of different compounds following standard methods19-24. The screening was performed for alkaloids, flavonoids, coumarins, anthraquinones, phenolic compounds, triterpenes/steroids, saponins, cardiac glycosides and reducing sugars.

General experimental procedures

NMR spectra were recorded at room temperature on a Bruker Avance III-300 and 400 MHz spectrometers in CDCl3. The chemical shifts are expressed in parts per million (ppm) and tetramethylsilane (TMS) as internal standard. The coupling constants (J values) have been reported in hertz (Hz).

General isolation procedure

During the process of concentration of the hexane extract, a yellow solid precipitated. This solid was filtered and washed with hexane. The residue was crystallized twice from a mixture of hexane: ethyl acetate (1:1) to afford a colorless crystals identified as friedelin, 1 (33.6 mg).

Part of the ethyl acetate extract (10 g) was fractionated on a column chromatographic and eluted with hexane, ethyl acetate, methanol and mixture of them. Column chromatography (5 x 26 cm) was performed on silica gel 60 F254 (Merck) to afford 298 fractions of 20 ml each. Before the fractions were collected, the solvent was removed on a rotary vacuum evaporator and the residue was recovered with acetone. Thin layer chromatography (TLC) was performed in silica gel 60 F254 plates (aluminum sheets, Macherey-Nagel) on each fraction and the plates were visualized by spraying with ceric sulfate/H2SO4 reagent followed by heating on a hot plate. According to the similar TLC profile, fractions eluted were pooled, affording 31 fractions (1-31). Fractions 4 (115 mg) and 6 (235 mg) were purified on a silica gel 60 F254 column resulting to the isolation of known compounds: a mixture of β-sitosterol and stigmasterol, 2 (6.6 mg) and betulinic acid, 3 (3.3 mg), respectively. All compounds were identified by 1H and 13C NMR data and by comparison with literature data.



Phytochemical screening of four extract obtained from the leaves of C. minor L. showed a variety chemical composition (Table).

From the hexane extract of C. minor leaves was crystallized one triterpene (1). The ethyl acetate extract yielded, after repeated silica gel column chromatography, a mixture of sterols (2) and one triterpene (3) (Figure).



Phytochemical screening

For phenolic compounds, the ferric chloride reagent showed a positive result in the four extracts. This type of metabolites is distributed in the genus Clusia, principally represented by benzophenones and flavonoids.2,4,16,25-28 The occurrence of flavonoids was suggestive in the extracts B, C and E. In the extracts B and E, the predominant flavonoids were the anthocyanidins, however catechins were no detected in the extracts. These results indicated that besides flavonoids, other phenolic compounds are present.

Also all extracts showed purple color for Liebermann-Buchard reaction. These results demonstrated the abundance of triterpenes and/or steroids in this plant. The presence of these compounds in the extract A confirmed the previous study by GC/MS of this specie leaves.18

Many pharmacological activities are attributed to phenolic compounds, triterpenes and sterols, fundamentally. The most representative are antioxidant, anti-inflammatory, antitumoral and antimicrobial properties.29-32 Due to its possible that we are in presence of a plant with potential medicinal activity.

Saponins and reducing sugars were verified only in the extracts C and E. The presence of anthraquinones was revealed by Bornträger reaction in the extracts A, B and E that showed a pink color, meanwhile coumarins was identified in the extracts B and E.

This screening corroborated the absence of alkaloids and cardiac glycosides in this genus. In the literature, no studies exist regarding the occurrence of these secondary metabolites.

The extract E presented a major number of different types of compounds. The assays for determining steroids/sterols and phenols demonstrated that these metabolites are the most abundance in this extract.

Characterization of isolated compounds

The 1H NMR spectrum of compound 1 evidenced the friedelane type carbon skeleton. The spectrum showed signals of aliphatic protons between δ 0.7 and 2.5. The protons at δ 0.7-1.18 as singlets suggestive the presence of eight methyl groups. In addition, the spectrum displayed a broad multiplet between δ 2.20-2.50 for α-proton ketone at position 2 and 4. The 13C NMR and DEPT experiments revealed 30 carbon signals corresponding to eight methyls, eleven methylenes, four methines and seven quaternary carbons (one ketone carbonyl carbon). The 13C NMR spectrum corroborated the analysis of 1H NMR spectrum. The carbon signal at δ 213.2, the most very low field shifts carbon signal, evidenced the presence of the keto group at position 3. The spectrum also displayed two carbon signals at δ 41.4 and 58.1, corresponding to methylene carbons connected to a carbonyl group (C-3). The identity of this compound was confirmed as friedelin (3-oxo-friedelan) by comparison of its spectral data with published values.33

Compound 2 showed a 1H NMR spectrum with aliphatic region very complex and characteristic of steroid skeleton. Six methyl groups were observed in the 1H NMR spectrum as singlet at δ 0.68, 0.80, 0.84, 1.01 and doublet at δ 0.82 (J=6.5 Hz, Me-26) and 0.92 (J=6.5 Hz, Me-21). The proton multiplet at δ 3.52 was attributed to the presence of typical H-3 of sterol nucleus. The olefinic proton region showed three signals at δ 5.35 (t, J=4.8 Hz), 5.15 and 5.05 (dd, J=15.3, 8.1Hz). These signals were assigned for H-6, H-22 and H-23, respectively. The presence of a Δ5-double bond was evident by resonances at δ 140.6 and 121.56 in the 13C NMR spectrum, assigned to C-5 and C-6, respectively. The signal at en δ 71,6 corroborated the presence of the hydroxyl group at C-3 position. The structure of compound 2 was consistent to mixture of β-sitosterol (stigmast-5-en-3β-ol) and stigmasterol (stigmast-5,22-dien-3β-ol) by comparison with the reported literature data34. According to the literature, these compounds were found in mixture commonly. The spectrum analysis and signals intensity suggesting stigmasterol was in minor proportion respect to β-sitosterol. Because in the 13C NMR spectrum, the signals corresponding positions 22 and 23 don't appeared and the protons δ 5.15 and 5.05 are in very low intensity.

The 1H NMR spectrum of compound 3 was consistent with triterpene skeleton. It exhibited signals for six methyl groups: five singlets at δ 0.7, 0.8, 0.93, 0.96 and 1.67 and one doublet at δ 0,91 (J=3.6 Hz). The chemical shifts of methyl proton at δ 1.67 were indicative of the presence of an allylic methyl. The multiplet proton at δ 3.10 was attributed to carbinolic hydrogen of position 3. The singlets at δ 4.56 and 4.69 corresponded with terminal methylene protons typical of lupane type skeleton. Additionally, the spectrum presented two signals at δ 1.97 and 2.25 corresponding to α-carbinolic hydrogens of C-2. The 13C NMR presented signals for 30 carbons. It presented a signal at δ 78.4 corroborating the presence of carbinolic carbon. The spectrum also showed a signal at δ 178.3 characteristic of the carbonyl of carboxylic group. The signals at δ 150.6 and 109.1 were attributed to terminal olefinic carbons of positions 20 and 29, respectively. The compound 3 was identified as betulinic acid (3β-hydroxy-lup-20(29)-en-28-oic acid) by comparison of these data with corresponding literature values.35 This compound was identified for first time from the species C. minor.

These results corroborated the presence of triterpenes and steroids obtained in the previous phytochemical screening. The isolated compounds are widely distributed in the plant kingdom and in this genus. All of them exhibit a variety of biological properties. Betulinic acid has reported to exhibit anti-inflammatory, cytotoxic, antiviral, antitumoral and antioxidant properties37. Besides some of these actions, stigmasterol and β-sitosterol have hypoglicemic and hypercholesterolemic activities.37-38 The existence of these metabolites in the leaves of C. minor suggests that it is a possible source of bioactive compounds with a suitable profile for the development as effective medicinal agents.


The authors are thankful to the staff of the Laboratory 1C from the Faculty of Chemistry of the National Autonomous University of Mexico (UNAM) and PhD. Guillermo Delgado-Lamas.

Conflicto de intereses

Los autores declaran que no tienen conflicto de intereses.



1. Ribeiro PR, Ferraz CG, Guedes MLS, Martins D, Cruz FG. A new biphenyl and antimicrobial activity of extracts and compounds from Clusia burlemarxii. Fitoterapia. 2011;82:1237-40.

2. Ferreira RO, de Carvalho MG, Sarmento TM. Ocorrência de biflavonoides em Clusiaceae. Aspectos químicos y farmacológicos. J Clin Invest 2012;116(8):2262-71.

3. Silva EM, Araújo RM, Freire LG, Silveira ER, Lopes NP, de-Paula JE, et al. Clusiaxanthone and tocotrienol series from Clusia pernambucensis and their antileishmanial activity. J Braz Chem. Soc 2013;24(8):1314-21.

4. Lins ACS, Agra MF, Conceição DCO, Pinto FCT, Camara CA, Silva TMS. Chemical Constituents and Antioxidant Activity from Aerial Parts of Clusia paralicola and Vismia guianensis. Rev Virtual Quim. 2016;8(1):157-68.

5. Duprat RC, Anholeti MC, de Sousa BP, Pacheco JPF, Figueiredo MR, Kaplan MAC, et al. Laboratory evaluation of Clusia fluminensis extracts and their isolated compounds against Dysdercus peruvianus and Oncopeltus fasciatus. Rev Bras Farmacogn.

6. Cuesta-Rubio O, Vélez H, Frontana BA, Cárdenas J. Nemorosone, the major constituent of floral resins of Clusia rosea. Phytochemistry 2001;57(2):279-83.

7. Piccinelli AL, Cuesta O, Barrios M, Mahmood N, Pagano B, Pavone M, et al. Structural elucidation of clusianone and 7-epi-clusianone and anti-HIV activity of polyisoprenylated benzophenones. Tetrahedron. 2005;61(34):8206-11.

8. Anholeti MC, Duprat RC, Figueiredo MR, Kaplan MAC, Santos MG, Gonzalez MS, et al. Biocontrol evaluation of extracts and a major component, clusianone, from Clusia fluminensis Planch & Triana against Aedes aegypti. Mem I Oswaldo Cruz. 2015;110,629-35.

9. Ferreira RO, Sarmento TM, de Carvalho MG. New polyprenylated phloroglucinol and other compounds isolated from the fruits of Clusia nemorosa (Clusiaceae). Molecules. 2015;20:14326-33; doi:10.3390/molecules200814326

10. Bailón-Moscoso N, Romero-Benavides JC, Sordo M, Villacís J, Silva R, Celi L, et al. Phytochemical study and evaluation of cytotoxic and genotoxic properties of extracts from Clusia latipes leaves. Rev Bras Farmacogn. 2016;26:44-9.

11. Rojas NM, Cuesta O, Avilas A, Lugo D, Avellaneda S. Actividad antimicrobiana de nemorosona aislada de Clusia rosea. Rev Cub Farm. 2001;35(Suplemento especial):197-9.

12. Peraza-Sánchez R, Pacheco F, Noh A. Leishmanicidal evaluation of extracts from native plants of the Yucatan peninsula. Fitoterapia. 2007;74(4):315-8.

13. Muñoz U, Jancovski N, Kennelly EJ. Polyisoprenylated benzophenones from Clusiaceae: potential drugs and lead compounds. Curr Top Med Chem. 2009;9:1560-80.

14. Díaz-Carballo D, Gustmanna S, Haydar A, Bardenheuera W, Buehlera H, Jastrowb H, et al. 7-epi-nemorosone from Clusia rosea induces apoptosis, androgen receptor down-regulation and dysregulation of PSA levels in LNCaP prostate carcinoma cells. Phytomedicine. 2012;19:1298- 306.

15. Ferreira RO, de Carvalho AR, Riger CJ, Castro RN, da Silva TMS, de Carvalho MG. Constituintes químicos e atividade antioxidante in vivo de flavonoides isolados de Clusia lanceolata (Clusiaceae). Quim Nova. 2016;39(9):1093-7.

16. Lastres M, Ruiz-Zapata T, Castro M, Torrecilla P, Lapp M, Hernández-Chong L, et al. Knowledge and use of medicinal plants of the Valle de la Cruz community, State Aragua. Pittieria. 2015;39:59-89.

17. Mangas R, Alarcón A, Cuesta-Rubio O, Piccinelli AL, Rastrelli L. Polyprenylated benzophenones derivates from Clusia minor L. fruits. Lat Am J Pharm. 2008;27(5):762-5.

18. Mangas R, Montes-de-Oca R, Bello A, Vázquez AN. Caracterización por Cromatografía de Gases/Espectrometría de Masas del extracto apolar de las hojas de Clusia minor L. Lat Am J Pharm. 2008;27(5): 47-51.

19. Debiyi OO, Sofowora FA. Phytochemical screening of medical plants. Iloyidia. 1978;3:234-46.

20. Sofowora A. Phytochemical Screening of Medicinal Plants and Traditional Medicine in Africa, Nigeria: Spectrum Books Ltd; 1993.

21. Harborne JB. Phytochemical methods: A guide to modern techniques of plant analysis. 3rd ed. London, New York: Chapman and Hall; 1998.

22. Miranda MM, Cuéllar AC. Manual de prácticas de laboratorio de Farmacognosia y productos naturales. Ciudad Habana: Félix Varela; 2000, 25-49.

23. Schenkel EP, Gosmann G, Athayde ML. Saponinas. In: Simoes CM, Schenkel G, Gosmann G, de Mello JC, Mentz LA, Petrovick PR, editors. Farmacognosia: da planta ao medicamento. 6th ed. Porto Alegre: UFRGS, 2007;711-40.

24. Evans WC. Trease and Evans' pharmacognosy. 16th ed. London: Sauders Elsevier; 2009.

25. Bagget S, Mazzola EP, Kennelly EJ. The benzophenones: isolation, structural elucidation and biological activities. Stud Nat Prod Chem. 2005;32:721-71.

26. Cuesta-Rubio O, Piccinelli AL, Rastrelli L. Chemistry and biological activity of poyisoprenylated benzophenone derivatives. Stud Nat Prod Chem. 2005;32:671-720.

27. Anholeti MC, de Paiva SR, Figueiredo MR, Kaplan MAC. Chemosystematic aspects of polyisoprenylated benzophenones from the genus Clusia. An Acad Bras Ciênc. 2015;87(1):289-301.

28. Bailón-Moscoso N, Romero-Benavides JC, Sordo M, Villacís J, Silva R, Celi L, et al. Phytochemical study and evaluation of cytotoxic and genotoxic properties of extracts from Clusia latipes leaves. Rev Bras Farmacogn. 2016;26:44-9.

29. Ivanova D, Gerova D, Chervenkov T, Yankova T. Polyphenols and antioxidant capacity of Bulgarian medicinal plants. J Ethnopharmacol. 2005;96:145-50.

30. Kirca A, Arslan E. Antioxidant capacity and total phenolic content of selected plants from Turkey. Int J Food Sci Technol. 2008;43:2038-46.

31. Biju J, Sulaiman CT, Satheesh G, Reddy VRK. Total phenolics and flavonoids in selected medicinal plants from Kerala. Int J Pharma Pharm Sci. 2014;6:406-8.

32. Wen L, You L, Yang X, Yang J, Chen F, Jiang Y, et al. Identification of phenolics in litchi and evaluation of anticancer cell proliferation activity and intracellular antioxidant activity. Free Radic Biol Med. 2015;84:171-84.

33. Ouédraogo N, Hay AE, Ouédraogo JCW, Sawadogo WR, Tibiri A, Lompo M, et al. Biological and phytochemical investigations of extracts from Pterocarpus erinaceus Poir (Fabaceae) root barks. Afr J Tradit Complement Altern Med. 2017;14(1):187-95.

34. Chundattu SJ, Agrawal VK, Ganesh N. Phytochemical investigation of Calotropis procera. Arabian Journal of Chemistry. 2016;9:230-34.

35. Tala MF, Tchakam PD, Wabo HK, Talontsi FM, Tane P, Kuiate JR, et al. Chemical constituents, antimicrobial and cytotoxic activities of Hypericum riparium (Guttiferae), Rec Nat Prod. 2013;7(1):65-68.

36. Moghaddam MG, Ahmad FBH, Samzadeh-Kermani A. Biological activity of betulinic acid: A review. Pharmacology & Pharmacy. 2012;3:119-23.

37. Moghadasian MH. Pharmacological properties of plant sterols: In vivo and in vitro observations. 2000;67(6):605-615.

38. Zeb MA, Khan SU, Rahman TU, Sajid M, Seloni S. Isolation and biological activity of β-sitosterol and stigmasterol from roots of Indigofera heterantha, Pharm Pharmacol Int J. 2017;5(5):00139.



Recibido: 21/06/2018
Aprobado: 26/06/2018



Raisa Mangas Marín. E-mail: