A pentacyclic triterpene from Lippia origanoides H.B.K and its cytotoxic activity
Arvind Kumar1, Elizabeth Quintero Rueda2, Jairo Rene Martínez3, Raquel Elvira Ocazionez2, Elena E Stashenko3
1 Department of Chemistry, Hemwati Nandan Bahuguna Garhwal University, Srinagar, Uttarakhand, India; Centro de Investigacion en Cromatografia y Espectrometria de Masas, CROM.MASS, Universidad Industrial de Santander, Bucaramanga, Colombia
2 Laboratorio de Arbovirus, Centro de Investigaciones en Enfermedades Tropicales (CINTROP), Universidad Industrial de Santander, Bucaramanga, Colombia
3 Centro de Investigacion en Cromatografia y Espectrometria de Masas, CROM-MASS, Universidad Industrial de Santander, Bucaramanga, Colombia
|Date of Submission||28-May-2020|
|Date of Decision||13-Jul-2020|
|Date of Acceptance||11-Aug-2020|
|Date of Web Publication||30-Nov-2020|
Department of Chemistry, Hemwati Nandan Bahuguna Garhwal University, Garhwal, PO Box 63, Srinagar - 246 174, Uttarakhand
Elena E Stashenko
Centro de Investigacion en Cromatografia y Espectrometria de Masas, CROM-MASS, Universidad Industrial de Santander, Carrera 27, Calle 9, Edificio 45, Bucaramanga-680002
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Lippia origanoides H. B. K. (Verbenaceae) is an aromatic small shrub appreciated in the traditional systems of medicine. L. origanoides essential oil is an ingredient of commercial poultry feed products and its postdistillation residual biomass is an interesting source of bioactive compounds. During our search for the valorization of this residual biomass, supercritical-CO2(SC-CO2)extraction afforded a mixture that was subjected to an investigation of phytochemicals and of cytotoxicity, which was not reported previously. Objectives: The current study was designed to investigate the phytochemicals from the steam-distilled residual biomass of thymol- and carvacrol-rich L. origanoides chemotypes and to evaluate their in vitro cytotoxic activity. Materials and Methods: Steam-distilled aerial parts of L. origanoides chemotypes were extracted with SC-CO2to obtain a greenish-yellow extract with a strong aromatic odor. The SC-CO2extract was subjected to column chromatography, and the isolate obtained was screened in vitro for cytotoxicity against human normal embryonic kidney 293, MRC-5, THP-1, and XP4PA cell lines, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Results: A pentacyclic triterpene, friedelan-3-one (1) was isolated for the first time from L. origanoides chemotypes. The structure of the isolate was elucidated with spectroscopic (ultraviolet, infrared, mass spectra, and nuclear magnetic resonance) techniques. The in vitro cytotoxic activity of the isolated compound was determined. The results showed no significant results against the selected cell lines using the MTT assay. Conclusion: The main significance of the present study was to develop promising routes to utilize the residual biomass for value-addition. This is the first report of friedelan-3-one isolation from the genus Lippia.
Keywords: Cytotoxicity, friedelan-3-one, Lippia origanoides, residual biomass, supercritical-CO2
|How to cite this article:|
Kumar A, Rueda EQ, Martínez JR, Ocazionez RE, Stashenko EE. A pentacyclic triterpene from Lippia origanoides H.B.K and its cytotoxic activity. Phcog Mag 2020;16, Suppl S2:513-6
|How to cite this URL:|
Kumar A, Rueda EQ, Martínez JR, Ocazionez RE, Stashenko EE. A pentacyclic triterpene from Lippia origanoides H.B.K and its cytotoxic activity. Phcog Mag [serial online] 2020 [cited 2022 Jan 21];16, Suppl S2:513-6. Available from: http://www.phcog.com/text.asp?2020/16/5/513/301878
- Valorization of plant residual biomass
- Pentacyclic triterpene, friedelan-3-one was isolated and characterized from the steam distilled supercritical-CO2 extract of Lippia origanoides for the first time
- Friedelan-3-one showed no significant cytotoxicity against the two normal (human normal embryonic kidney 293 and MRC-5) and abnormal (THP-1 and XP4PA) human cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay.
Abbreviations used: MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; DMSO: Dimethyl sulfoxide; SC-CO2: Supercritical-CO2; TLC: Thin-layer chromatography; CC: Column chromatography; NMR: Nuclear magnetic resonance; TMS: Tetramethylsilane; ppm: Parts per million; ESI: Electrospray ionization; m/z: Mass-to-charge ratio; HEK293: Human normal embryonic kidney; MRC-5: Human normal fibroblasts from embryonic lung; THP-1: Human monocytic leukemia; XP4PA: Human dermal fibroblasts from Xeroderma pigmentosum; DMEM: Dulbecco's minimal essential medium; CC50: 50% Cytotoxic concentration.
| Introduction|| |
Lippia origanoides H. B. K. (Verbenaceae family) is an aromatic shrub up to 3 m tall, widely distributed from Central America (Mexico, Guatemala, and Cuba) to northern South America, with prominent occurrence in the Amazonian region of Brazil, the Guianas, Venezuela, and Colombia. In Colombia, L. origanoides is commonly called “Oregano del monte” (mountain oregano) and distributed in the Andean States and in the Northern peninsula of Guajira at altitudes from 400 m to 2500 m., Conventionally, L. origanoides is used in the treatment of stomach ache, indigestion, nausea, flatus, and as a general antiseptic for the mouth, throat, and wounds. Previous phytochemical investigations of this essential oil have characterized its chemical composition and determined its biological activity.,,,,,, Several phenolic compounds were detected in the L. origanoides extracts by gas chromatography-mass spectrometry (GC-MS) and high-pressure liquid chromatography
(HPLC) methods., In the course of our search for the valorization of residual biomass from thymol- and carvacrol-rich L. origanoides chemotypes by means of a green extraction process, supercritical-CO2(SC-CO2) extraction was used to obtain a compound mixture. An isolate was obtained using column chromatography (CC) and was subjected to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay to examine cytotoxic activity against two normal human cell lines (human normal embryonic kidney 293 [HEK293] and MRC-5) and two abnormal human cell lines (THP-1 and XP4PA).
| Materials and Methods|| |
General experimental procedures
Nuclear magnetic resonance (NMR) experiments were recorded on a Bruker Avance III-400 MHz spectrometer (1 H: 400 MHz;13 C: 101 MHz; CDCl3) using TMS as the internal standard. Mass spectra (MS) spectra were obtained on an Ultra HPLC (UHPLC)-electrospray (ESI)-high resolution mass spectrometer Exactive Plus orbitrap system (Thermo Scientific, Bremen, Germany) in positive mode. Melting point (uncorrected) was determined on a capillary point apparatus equipped with a digital thermometer. Ultraviolet (UV) spectrum was recorded on a Flame Ocean Optics spectrophotometer (Ocean Optics, Ostfildern, Germany), and Infrared (IR) spectrum was acquired with a Cary 630 Fourier-transform IR spectrometer (Agilent Technologies, USA). Silica gel (SiO2) (0.063–0.200 mm, Merck, Darmstadt, Germany) was used for CC and precoated silica gel 60 F254 plates (0.2 mm, Merck) were used for thin-layer chromatography (TLC) analysis.
Aerial parts (including flowers, leaves, and stems) of thymol- and carvacrol-rich L. origanoides were collected from the experimental plot of CENIVAM, Universidad Industrial de Santander, Bucaramanga, Colombia in June 2018 and authenticated by Prof. José Luis Fernández Alonso (National University, Bogota, Colombia). A voucher specimen (COL519799) was deposited at the Colombian National Herbarium (Bogota).
Extraction and isolation
Fresh plant material (16 kg) was chopped and steam-distilled to obtain essential oil (yield 1.3%, w/w). Chromatographic (GC-flame-ionization detection, GC-MS) analysis of essential oil exhibited thymol (32.4%) and carvacrol (12.1%) as major constituents [data depicted in [Figure S1] and [Table S1]; see Supplementary Material]. Distilled vegetal material was further air-dried at room temperature and coarsely ground (particle size <1 mm, 460 g), extracted with CO2 at 333 K and 50 MPa in a 2 L extraction chamber (Thar SFE 2000-2-FMC50, Thar Instruments, Pittsburgh, Pennsylvania, USA). The initial static period (20 min) was followed by a continuous flow of CO2(40 g/min, 120 min), which connected to a vortex collection chamber, where pressure was reduced to 0.1 MPa. The extract (yield 1.4%; w/w) obtained after 2 h of CO2 recirculation followed by depressurization was dissolved in ethanol (3–5 mL, Merck), centrifuged to remove the fatty layer, and evaporated with a rotary evaporator. The SC-CO2 extract (3.5 g) was subjected to CC over silica gel (45 g, 50 cm × 1.5 cm) eluted with CHCl3 to yield fractions 1–35. All fractions were monitored by TLC in solvent systems of n-hexane-EtOAc (98:2–85:15) and detected with UV light and visualized by spraying with 5% aq. H2 SO4, followed by heating at 110°C until the characteristic color. Fractions (7–9) were further column chromatographed on silica gel (20 g, 25 cm × 1 cm) eluted with CHCl3 to give 1 (15 mg; 0.43%, w/w).
Thein vitro cytotoxicity of compound 1 against human normal embryonic kidney (HEK293, ATCC® CRL-1573™), human normal fibroblasts from embryonic lung (MRC-5, ATCC® CCL-171™), human monocytic leukemia cells (THP-1, ATCC® TIB-202™), and human dermal fibroblasts from Xeroderma pigmentosum (XP4PA, kindly provided by Dr. Carlos F. Martins, from Institute of Biomedical Sciences, University of São Paulo, Brazil). HEK293 was cultured in Dulbecco's minimal essential medium (DMEM-F12), MRC-5 and XP4PA cells were cultivated in DMEM/high glucose, and THP-1 was cultured in RPMI 1640. The cells were supplemented with 10% fetal bovine serum (Gibco, USA) and 1% antibiotics penicillin (100 U/mL)/streptomycin (100 mg/mL) under a humidified incubator at 37°C in 5% CO2 for 24 h.
Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (tetrazolium) assay
The cell viability of isolated compound 1 was investigated by MTT colorimetric assay. Cells were seeded onto 96-well micro-titer plastic plates (1 × 104 cells/well) treated with varying concentrations of the compound (6.25, 12.5, 25, 50, 75, and 150 μM) maintained in a humidified atmosphere at 37°C in 5% CO2 and incubated for 72 h. The culture medium was removed and 5 mg/mL MTT (Sigma-Aldrich, USA) solution was added to each well, and incubation was continued for 3 h. After incubation, the formed formazan crystals were dissolved in 1 mL of dimethyl sulfoxide and absorbance of wells was measured at a wavelength of 550 nm with an automated spectrophotometric plate reader (Multiskan Go, Thermo Scientific, USA). All experiments were carried out in triplicate. Data analysis was performed using the R Project for statistical computing software (R Development Core Team . R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org).
| Results and Discussion|| |
The SC-CO2 extract of steam-distilled residual biomass of thymol- and carvacrol-rich L. origanoides was selected for phytochemical investigation. The obtained SC-CO2 extract subjected to CC afforded compound 1 [Figure 1] and structure was elucidated by IR, MS and NMR spectroscopic techniques. The NMR key correlations of1 H-1 H COSY and HMBC are shown in [Figure 1]. Thein vitro cytotoxicity of 1 showed no significant results against HEK293, MRC-5, THP-1, and XP4PA cell lines using the MTT assay, and results are depicted in [Figure 2].
|Figure 2: Cytotoxicity of compound 1 against human normal embryonic kidney 293, normal embryonic kidney cells; XP4PA, fibroblast from Xeroderma pigmentosum patients; THP-1, monocyte from a leukemia patient, and MRC-5, fibroblast from normal embryonic lung. Data are shown as means ± standard deviation of three-independent experiments and presented relative to untreated control cells|
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Structural elucidation of compound 1
The UHPLC-ESI(+)-orbitrap-MS data for compound 1 showed the protonated molecule ion (M + H) + m/z 427.38940 (calcd. m/z 427.39344 for C30H51O), indicated six degrees of unsaturation. IR spectrum showed the strong absorption of a ketone group at 1713 cm−1. The compound gave purple color on TLC with 5% aq. H2 SO4, and positive Liebermann-Burchard reaction indicated that the compound possessed a triterpene skeleton. The1 H and13 C-NMR spectra of 1 showed 50 proton signals corresponding to 30 carbons.1 H NMR spectrum showed eleven methylene groups at H-1α,β (2H, m), H-2α,β (2H, m), H-6α,β (2H, m), H-7α,β (2H, m), H-11α,β (2H, m), H-12α,β (2H, m), H-15α,β (2H, m), H-16α,β (2H, m), H-19α,β (2H, m), H-21α,β (2H, m) and H-22α,β (2H, m), eight methyl groups at δH0.89 (3H, d, J = 6.4), 0.75 (3H, s), 0.91 (3H, s), 1.03 (3H, s), 1.07 (3H, s), 1.20 (3H, s), 1.02 (3H, s) and 0.96 (3H, s) and four methine protons at δH2.26 (1H, q), 1.41 (1H, dd), 1.56 (1H, m) and 1.57 (1H, m), indicating that compound was probably a pentacyclic triterpene.13 C NMR spectrum showed a high downfield carbonyl signal at δC213.9 (C-3) correlation with secondary methyl at δH0.89 (J = 6.4 Hz, H-23). This spectrum displayed 30 carbons including eleven sp3 methylene signals (δC22.9, 42.2, 41.9, 18.9, 36.3, 31.2, 33.1, 36.7, 36.0, 33.5, and 39.9) confirmed with DEPT-135° experiment [Figure S2], eight methyls (δC7.5, 15.4, 18.6, 20.9, 19.4, 32.8, 32.5, and 35.7), six sp2 quaternary (δC42.8, 38.1, 40.4, 38.9, 30.4, and 28.9) and four sp2 methine signals (δC58.9, 53.8, 60.2, and 43.5). The COSY (1 H-1 H) correlations showed between H-1 to H-2 and H-10; H-7 to H-6 and H-8; H-11 with H-12; H-15 with H-16; H-18 with H-19, and H-21 with H-22, and HMBC correlations [Figure 1] from H-1α to C-10, H-2 β to C-3; H-4 to C-3 and C-5, H-8 to C-7, C-9, C-14 and C-25, H-10 to C-5 and C-9, H-11 to C-13, H-12 to C-13 and C-14, H-18 to C-13, C-14, C-17, C-20 and C-28, H-19 to C-29, H-21 to C-29 and C-30, H-22 to C-20, H-23 to C-3, C-4 and C-5, H-24 to C-4, C-5, C-6 and C-10, H-25 to C-8, C-9, C-10 and C-11, H-26 to C-8, C-14 and C-15, H-27 to C-12, C-13 and C-14, H-28 to C-16, C-17 and C-22, H-29 to C-20, H-30 to C-19, C-20 and C-29. The HSQC spectrum revealed the direct-CH signals at δH2.26 (H-4) with δC58.9 (C-4), δH1.41 (H-8) with δC53.8 (C-8), δH1.56 (H-10) with δC60.2 (C-10), and δH1.57 (H-18) with δC43.5 (C-18) [Figure S3]. Based on the above discussion and comparison with literature data,,, the structure of compound 1 was assigned as 4, 4a, 6b, 8a, 11, 11, 12b, 14a-octamethylicosahydropicen-3 (2 h)-one, named friedelan-3-one (friedelin).
Friedelan-3-one (1): White needle crystals dissolved in chloroform. Melting point 260°C –262°C. TLC Rf 0.38 (n-hexane: EtOAc, 90:10). UV (CHCl3) λmax(log ε): 245 (0.48), 287 (0.37) nm. IR (neat, νmax, cm−1): 2925, 2867, 1713 (C = O), 1459, 1387. GC-MS: TR87.914 min. MS (EI, 70 eV), m/z (Irel,%): 426 ([M] +, 14), 273 (24), 205 (26), 163 (31), 125 (53), 123 (64), 109 (72), 107 (42), 95 (87), 81 (72), 69 (100), 55 (75). UHPLC-ESI+-orbitrap-MS m/z 427.38940 (M + H) + (calcd. m/z 427.39344).1 H-NMR (400 MHz, CDCl3) δH1.98 (m, H-1α), 1.73 (m, H-1 β), 2.39 (m, H-2α), 2.28 (m, H-2 β), 2.26 (q, H-4), 1.76 (d, H-6α), 1.29 (d, H-6 β), 1.49 (m, H-7α), 1.38 (m, H-7 β), 1.41 (dd, H-8), 1.56 (m, H-10), 1.47 (m, H-11α), 1.24 (m, H-11 β), 1.31 (m, H-12α), 1.30 (m, H-12 β), 1.48 (m, H-15α), 1.28 (m, H-15 β), 1.59 (m, H-16α), 1.36 (m, H-16 β), 1.57 (m, H-18), 1.40 (m, H-19α), 1.25 (m, H-19 β), 1.51 (m, H-21α), 1.32 (m, H-21 β), 1.54 (m, H-22α), 0.98 (m, H-22 β), 0.89 (d, J = 6.4 Hz, H-23), 0.75 (3H, s, H-24), 0.91 (3H, s, H-25), 1.03 (3H, s, H-26), 1.07 (3H, s, H-27), 1.20 (3H, s, H-28), 1.02 (3H, s, H-29), 0.96 (3H, s, H-30).13 C-NMR (101 MHz, CDCl3) δC22.9 (C-1), 42.2 (C-2), 213.9 (C-3), 58.9 (C-4), 42.9 (C-5), 41.9 (C-6), 18.9 (C-7), 53.8 (C-8), 38.1 (C-9), 60.2 (C-10), 36.3 (C-11), 31.2 (C-12), 40.4 (C-13), 38.9 (C-14), 33.1 (C-15), 36.7 (C-16), 30.4 (C-17), 43.5 (C-18), 36.0 (C-19), 28.9 (C-20), 33.5 (C-21), 39.9 (C-22), 7.5 (C-23,-CH3), 15.4 (C-24,-CH3), 18.6 (C-25,-CH3), 20.9 (C-26,-CH3), 19.4 (C-27,-CH3), 32.8 (C-28,-CH3), 32.5 (C-29,-CH3), 35.7 (C-30,-CH3) [Detailed spectral information is provided in Supplementary Material].
Measurement of cytotoxic activity
The cytotoxic activity of compound 1 was studied using the MTT assay against HEK293, MRC-5, XP4PA, and THP-1 cell lines. Significant cytotoxic effect was not observed; the viability of all four cells was reduced between 14% and 37% after treatment with the highest concentration of the compound [Figure 2]. Hence, the 50% cytotoxic concentrations (CC50) were above of or higher than 150 μM. XP4PA cells were deficient against the nucleotide excision repair pathway, which involved the recognition and removal of the DNA lesions. Results suggested that the test compound did not block pathways related to the survival of human cells derived from the embryonic kidney (HEK293), fibroblast from embryonic lung (MRC-5), monocyte from a leukemia patient (THP-1), and fibroblasts from Xeroderma pigmentosum patients (XP4PA). Previous report on compound 1 showed low toxicity against the Vero cells using MTT assay at LC50>200 μg/mL, while not toxic against the formaldehyde-fixed red blood cells using the hemagglutination assay. However, compound 1 possesses several biological activities such as anti-inflammatory, analgesic, antipyretic, antioxidant, and liver protective.
| Conclusion|| |
The present work described the phytochemical investigation on steam-distilled plant residual biomass of L. origanoides used for the isolation of a pentacyclic triterpene ketone, friedelan-3-one (1) for the first time from the genus Lippia. The structure of the isolate was verified based on various spectroscopic techniques and comparison with literature. Results of cytotoxicity of naturally occurring compound 1 showed no significant effects against four (HEK293, MRC-5, XP4PA, and THP-1) cell lines. A valuable and sustainable way for the management of biomass residues has been proposed.
The authors are highly grateful to Prof. Daniel Ricardo Molina at the NMR facilities. The assistance of Mr. Andrés Cáceres for SC extraction of plant samples is also acknowledged. Author (AK) also thankful to the Vicerrectoria de Investigación y Extensión, UIS for awarding the postdoctoral fellowship (Apoyo a estancias postdoctorales).
Financial support and sponsorship
This research was funded by “Patrimonio Autónomo Fondo Nacional de Financiamiento para la Ciencia, la Tecnología y la Innovación, Francisco José de Caldas” Bogotá, Colombia (Grant No. RC-FP44842-212-2018).
Conflicts of interest
There are no conflicts of interest.
| Supplementary Material|| |
Characterization data Ultraviolet/Vis, Infrared, HR.mass spectrometry, and nuclear magnetic resonance (1D and 2D) are available in supplementary material, alongside, Table S1 and Figures S1-S3.
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[Figure 1], [Figure 2]