Toxicological evaluation of the natural products and some semisynthetic derivatives of Heterotheca inuloides Cass (Asteraceae)

Article abstract of DOI:10.1016/j.jep.2015.08.055

Ethnopharmacological relevance Heterotheca ineuloides Cass (Asteraceae), popularly known as árnica mexicana, is widely used in Mexican traditional medicine to treat bruises, dermatological problems, rheumatic pains, and other disorders as cancer. The major constituents in H. inuloides are cadinane type sesquiterpenes, flavonoids and phytosterols. Compounds with a cadinane skeleton have been proved to possess cytotoxic activity against human-tumor cell lines and brine shrimp, and display toxic effects in different animal species. Although this plant has been widely used, there is little available information on the safety and toxicity especially of pure compounds. Aim of this study Evaluate the potential toxicity of the natural products isolated from H. inuloides and some semisynthetic derivatives. Materials and methods The toxic aspects of the following natural products isolated from dried flowers of H. inuloides: 7-hydroxy-3,4-dihydrocadalene (1), 7-hydroxycadalene (2), 3,7-dihydroxy-3(4H)-isocadalen-4-one (3), (1R,4R)-1-hydroxy-4H-1,2,3,4- tetrahydrocadalen-15-oic acid (4), D-chiro-inositol (5), quercetin (6), quercetin-3,7,3′-trimethyl ether (7), quercetin-3,7,3′,4′-tetramethyl ether (8), eriodictyol-7,4′-dimethyl ether (9), α-spinasterol (10), caryolan-1,9β-diol (11) and 7-(3,3-dimethylallyloxy)-coumarin (12) as well as the toxic aspects of the semisynthetic compounds 7-acetoxy-3,4-dihydrocadalene (13), 7-benzoxy-3,4-dihydrocadalene (14), 7-acetoxycadalene (15), 7-benzoxycadalene (16), quercetin pentaacetate (17), 7-hydroxycalamenene (18), 3,8-dimethyl-5-(1-methylethyl)-1,2-naphthoquinone (19), and 4-isopropyl-1,6-dimethylbenzo[c]oxepine-7,9-dione (20). Toxic activities of compounds were determined by sulforhodamine B (SRB) assay, Artemia salina assay, RAW264.7 macrophage cells. Additionally, the acute toxicity in mouse of compound 1, the major natural sesquiterpene isolated from the acetone extract, was evaluated. Results The best cytotoxicity activity was observed for mansonone C (19) on K562 cell line with IC₅₀ 1.45±0.14 μM, for 7-hydroxycadalene (2) on HCT-15 cell line with IC₅₀ 18.89±1.2 μM, and for quercetin pentaacetate (17) on MCF-7 cell line with IC₅₀ 22.57±2.4 μM. Sesquiterpenes mansonone C (19) and 7-hydroxy-3,4-dihydrocadalene (1) caused the strongest deleterious effects against A. salina with IC₅₀ 39.4±1.07, and 45.47±1.74 μM, respectively. The number of viable RAW 264.7 cells was reduced with sesquiterpenes 1 and 2 by more than 90%. In addition, the acute study of 1 revealed no lethal effects at 300 mg/kg body weight, however, a reduction in the body weight of mice, morphological changes in the tissues of the liver and kidney and toxic signs were observed at very high doses (2000 mg/kg). Conclusion The results provided evidence for the cytotoxicity of Mexican arnica (H. inuloides) metabolites and may be correlated with one of the popular uses of this plant, in traditional Mexican medicine, as anticancer remedy. Among the active compounds contained in the acetone extract, the cytotoxic activity is mainly ascribable to cadinene type sesquiterpenes. In addition, evidence of acute toxicity suggests that 7-hydroxy-3,4-dihydrocadalene (1) may lead to toxicity at very high doses.

Full text of DOI:10.1016/j.jep.2015.08.055

Author’s Accepted Manuscript
Toxicological evaluation of the natural products
and some semisynthetic derivatives of Heterotheca
inuloides Cass (Asteraceae)
José Luis Rodríguez-Chávez, Elvia Coballase-
Urrutia, Gloria Sicilia-Argumedo, Teresa Ramírez-
Apan, Guillermo Delgado
www.elsevier.com
PII:
S0378-8741(15)30121-5
http://dx.doi.org/10.1016/j.jep.2015.08.055
JEP9720
DOI:
Reference:
To appear in:
Journal of Ethnopharmacology
Received date: 26 May 2015
Revised date: 6 August 2015
Accepted date: 30 August 2015
Cite this article as: José Luis Rodríguez-Chávez, Elvia Coballase-Urrutia, Gloria
Sicilia-Argumedo, Teresa Ramírez-Apan and Guillermo Delgado, Toxicological
evaluation of the natural products and some semisynthetic derivatives o
Heterotheca inuloides Cass (Asteraceae), Journal of Ethnopharmacology
http://dx.doi.org/10.1016/j.jep.2015.08.055
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Toxicological evaluation of the natural products and some semisynthetic derivatives of
Heterotheca inuloides Cass (Asteraceae)
José Luis Rodríguez-Chávez, 1 Elvia Coballase-Urrutia,2 Gloria Sicilia-Argumedo,3 Teresa Ramírez-
Apan,1 and Guillermo Delgado1*
1Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad
Universitaria, Coyoacán 04510, México, D.F., México.
2Laboratorio de Neuroquímica, Instituto Nacional de Pediatría, Insurgentes Sur 3700C Insurgentes
Cuicuilco, Coyoacán, 04530 México D.F., México.
3Departamento de Patología, Instituto Nacional de Pediatría, Insurgentes Sur 3700C Insurgentes
Cuicuilco, Coyoacán, 04530 México D.F., México.
Running title: Toxicology of products from H. inuloides
abstract
Ethnopharmacological relevance: Heterotheca inuloides Cass (Asteraceae), popularly known as
árnica mexicana, is widely used in Mexican traditional medicine to treat bruises, dermatological
problems, rheumatic pains, and other disorders as cancer. The major constituents in H. inuloides
are cadinane type sesquiterpenes, flavonoids and phytosterols. Compounds with a cadinane
skeleton have been proved to possess cytotoxic activity against human-tumor cell lines and brine
shrimp, and display toxic effects in different animal species. Although this plant has been widely
used, there is little available information on the safety and toxicity especially of pure compounds.
Aim of this study: Evaluate the potential toxicity of the natural products isolated from H.
inuloides and some semisynthetic derivatives
Materials and methods: the toxic aspects of the following natural products isolated from dried
flowers of H. inuloides: 7-hydroxy-3,4-dihydrocadalene (1), 7-hydroxycadalene (2), 3,7-
dihydroxy-3(4H)-isocadalen-4-one (3), (1R,4R)-1-hydroxy-4H-1,2,3,4- tetrahydrocadalen-15-oic
acid (4), D-chiro-inositol (5), quercetin (6), quercetin-3,7,3’-trimethyl ether (7), quercetin-
3,7,3’,4’-tetramethyl ether (8), eriodictyol-7,4’-dimethyl ether (9), -spinasterol (10), caryolan-

1,9-diol (11) and 7-(3,3-dimethylallyloxy)-coumarin (12) were isolated from the acetonic
extract of H. inuloides as well as the toxic aspects of the semisynthetic compounds 7-acetoxy-
3,4-dihydrocadalene (13), 7-benzoxy-3,4-dihydrocadalene (14), 7-acetoxycadalene (15), 7-
benzoxycadalene (16), quercetin pentaacetate (17), 7-hydroxycalamenene (18), 3,8-dimethyl-5-
(1-methylethyl)-1,2-naphthoquinone (19), and 4-isopropyl-1,6-dimethylbenzo[c]oxepine-7,9-
dione (20). Totoxic activities of compounds were determined by sulforhodamine B (SRB) assay,
Artemia salina assay, RAW264.7 macrophage cells. Additionally, the acute toxicity in mouse of
compound 1, the major natural sesquiterpene isolated from the acetone extract, was evaluated.
Results: The best cytotoxicity activity was observed for mansonone C (19) on K562 cell line
with IC50 1.45±0.14 M, for 7-hydroxycadalene (2) on HCT-15 cell line with IC50 18.89±1.2
M, and for quercetin pentaacetate (17) on MCF-7 cell line with IC50 22.57±2.4 M.
Sesquiterpenes mansonone C (19) and 7-hydroxy-3,4-dihydrocadalene (1) caused the strongest
deleterious effects against A. salina with IC50 39.4±1.07, and 45.47±1.74 M, respectively. The
number of viable RAW 264.7 cells was reduced with sesquiterpenes 1 and 2 by more than 90%.
In addition, the acute study of 1 revealed no lethal effects at 300 mg/kg body weight, however, a
reduction in the body weight of mice, morphological changes in the tissues of the liver and
kidney and toxic signs were observed at very high doses (2,000 mg/kg).
Conclusion: The results provided evidence for the cytotoxicity of Mexican arnica (H. inuloides)
metabolites and may be correlated with one of the popular uses of this plant, in traditional
Mexican medicine, as anticancer remedy. Among the active compounds contained in the acetone
extract, the cytotoxic activity is mainly ascribable to cadinene type sesquiterpenes. In addition,
evidence of acute toxicity suggests that 7-hydroxy-3,4-dihydrocadalene (1) may lead to toxicity
at very high doses.

*To whom correspondence should be addressed. Instituto de Química, Universidad Nacional Autónoma
de México. Tel.: +52-55-5622-4446; Fax: +52-55-5616-2217 E-mail address: delgado@unam.mx
Key Words: Heterotheca inuloides; Mexican arnica, cytotoxicity, cadinanes, RAW264.7 macrophages,
acute toxicity.
Chemical compounds studied in this article: 7-hydroxy-3,4-dihydrocadalene (PubChem CID:
163563874), 7-hydroxycadalene (PubChem CID: 608115); quercetin (PubChem CID: 5280343);
quercetin-3,7,3’-trimethyl ether (PubChem CID: 12303); quercetin-3,7,3’,4’-tetramethyl ether (PubChem
CID: 5352005), eriodictyol-7,4’-dimethyl ether (PubChem CID: 320054); -spinasterol (PubChem CID:
5281331), 7-(3,3-dimethylallyloxy)-coumarin (PubChem CID: 320362); quercetin pentaacetate
(PubChem CID: 14005), 3,8-dimethyl-5-(1-methylethyl)-1,2-naphthoquinone (PubChem CID: 21742).
1. Introduction
Plants synthesize an array of metabolites that are released in response to damage or abiotic stress factors,
and in the presence of pathogens or herbivores (Feng et al., 2007; Zhao et al., 2005). Some of these
metabolites are harmful to mammals (Durmic and Blache, 2012; Iason, 2005). Mexican traditional
medicine employs approximately 4,000 plants (both native and introduced), with 85% of traded species
coming from wild collections, some of which have never had their medicinal potential studied in detail
(Juárez-Rosete et al., 2013). Certain plants used in traditional medicine in Mexico contain compounds
that can cause adverse effects or affect the pharmacokinetics of various drugs (Cruz et al., 2012;
Rodríguez-Fragoso et al., 2008). For this reason, the toxicities of several plants have been evaluated to
determine their efficacy and safety (Aoki et al., 2005; Déciga-Campos et al., 2007).
Heterotheca inuloides Cass (Asteraceae), popularly known as árnica mexicana, is a plant native to Mexico
(Nesom, 1990), widely used in Mexican traditional medicine and by and the Mexican-American
community to treat bruises, dermatological problems, rheumatic pains, and other disorders as cancer and
diabetes (Alonso-Castro et al 2011; Comisión Nacional Forestal, 2010; González-Stuart et al, 2006;
Johnson et al, 2006; Zurita-Esquivel et al., 1986). Mainly the dried flower heads (inflorescences) are used
in the form of infusion, although the leaves and stems are sometimes used (Aguilar et al, 1994; Comisión
Nacional Forestal, 2010; Winkelman, 1986). This plant species grows in highlands and in the cooler and

temperate regions of Mexico (Nesom, 1990) and contains an array of bioactive compounds like cadinane
type sesquiterpenes, phytosterols, and flavonoids (Bohlmann and Zdero, 1976; Delgado et al., 2001;
Willuhn and Schneider, 1987). It has been reported that the extracts and some natural products from H.
inuloides display a variety of biological activities such as anti-inflammatory, antibacterial, reactive
oxygen species scavengers, and antiparasitic activity (Coballase-Urrutia et al., 2010; Delgado et al., 2001;
Rodríguez-Chávez et al, 2015). H. inuloides was included in the extra-pharmacopoeia section of the
Mexican Herbal Pharmacopoeia published by the Ministry of Health of Mexico due to a lack of sufficient
scientific research (chemical or clinical) to support its therapeutic use (Secretaría de Salud, 2001). The
herbal concoction containing H. inuloides combined with other plants has been asociated with bleeding
risks to perioperative patients (Wong et al, 2012) and recently, it has been reported that acetone and
methanol extracts of Heterotheca inuloides display mutagenic activity (Ruiz-Pérez et al., 2014), however,
the safety aspects of the pure components isolated and characterized from organic extracts of H.
inuloides, including acute and subacute toxicity, have not been sufficiently evaluated.
The objective of this study was to investigate the toxicological aspects of the metabolites of H. inuloides
using various human tumor cell lines, Artemia salina, and RAW 264.7 murine cell line; and the acute oral
toxicity of the major natural constituent of the extract in mice. Based on the observed bioactivities, the
effect of the modification of some chemical structures on biological activity was also discussed. The
findings from this study contribute to a better understanding of the bioactivity of the natural products of
H. inuloides, which is one of the plant species most widely used for traditional medicinal purposes in
central Mexico.
2. Material and methods
2.1.General
All chemicals were analytical-reagent grade. mCPBA, TCA, DMSO, Tris, SRB, ethanol, K2Cr2O7,
Tween® 80, NaH2PO4, Na2HPO4, H3PO4, MTT, hematoxylin solution, CHCl3, Na2SO3, NaHCO3, NaCl,
RPMI 1640 medium, and formaldehyde were purchased from Sigma-Aldrich (MO, USA). Amphotericin
B was purchased from Gibco, Invitrogen Corporation (California, USA). Sodium pyruvate (ATCC30-
2002) and heat-inactivated fetal bovine serum were purchased from ATTC (VA USA). Melting points
were measured in a Fisher Scientific apparatus with a 300 °C thermometer (Cole-Parmer Instrument

1
13
Company, IL. USA). H NMR (500 MHz) and C NMR (125 MHz) spectra were obtained with Varian
Unity INOVA equipment (Varian Co. CA. USA) using TMS as internal standard. IR spectra were
recorded on a Bruker Tensor 750 FT-IR (Bruker Co., MA. USA) spectrophotometer with KBr pellets. UV
spectra were obtained using a Shimadzu-UV160 spectrophotometer (Shimadzu Corporation, Kioto,
Japan). Resolution mass spectra were obtained on a JEOL AccuTOF JMS-T100LC mass spectrometer
(JEOL Ltd., Tokyo, Japan). Thin layer chromatography was performed using TLC Silica gel 60 F254,
aluminium sheets 20 x 20 cm (Merck KGaA, Darmstadt, Germany). Compounds 5, 7, 8, 9, 11, 12 and 20
were not tested in all assays due to a shortage of material.
2.2.Plant material
Flowers of Heterotheca inuloides (Common name: árnica mexicana, acahual, akawtomitl, acahualli,
xochihuepal, cuauteteco, falsa árnica, hornilla, tabaco de las montañas, re-emn, riemn, and árnica del país;
common English name: false goldenaster, telegraph plant) were provided by MIXIM Laboratories,
Naucalpan, Mexico, and collected in 2010, in the town of Mesas Altas de San Juan Xoconusco,
municipality of Donato Guerra (State of Mexico). The flowers were authenticated by Prof. Abigail
Aguilar-Contreras. A voucher of plant material was deposited under code IMSSM-16064 in the Medicinal
Plant Herbarium of the Instituto Mexicano del Seguro Social (IMSS, Mexico City). The accepted plant
name was verified in www.theplantlist.org (accessed in May 2015).
2.3.Cell lines
Central leukemia (K562), colon (HCT-15) and breast (MCF-7) human tumor cell lines were supplied by
the National Cancer Institute (NCI, Maryland, USA). The RAW264.7 murine macrophages (ATCC, TIB-
71) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).
2.4.Experimental animal 1
Artemia salina (Linnaeus) cysts (Eclosión Azul®, Mexico) were obtained from a local store. The cysts
were hatched in Petri dishes containing artificial seawater made by dissolving the commercial marine salt
Instant Ocean® (Aquarium Systems, Inc. Blacksburg, VA) in deionized water (3.7% w/v with final pH 8)
and incubated at 25 ± 1° C for 48 h. Newly hatched brine shrimps were separated from their shells and the
remaining cysts transferred to freshly-prepared seawater with a Pasteur pipette, and readily used for
bioassays.

2.5.Experimental animal 2
Female CD-1 mice weighing 20 to 25g were obtained from the Production of Laboratory Animals Unit
(UPEAL) from Universidad Autónoma Metropolitana, Mexico City. Prior to their acquisition, mice were
kept in pathogen-free conditions in an HEPA filtered macro environment, at 21 ± 2 °C temperature, with
45-55% relative humidity, 12:12 light/dark cycles, and noise <85 db. Mice were fed ad libitum with
nutritious formula LabDiet-5001® (Purina, St. Louis, MO) and were supplied with ozonated water low in
salts, and housed in acrylic boxes (18.4 cm width × 29.2cm length × 12.7cm height) with the floor
covered by a 5 cm bed of fine sawdust sterilized under UV light irradiation (Crigamex, Mexico).
2.6.Compounds from H. inuloides
The compounds 7-hydroxy-3,4-dihydrocadalene (1), 7-hydroxycadalene (2), 3,7-dihydroxy-3(4H)-
isocadalen-4-one (3), (1R,4R)-1-hydroxy-4H-1,2,3,4-tetrahydrocadalen-15-oic acid (4), D-chiro-inositol
(5), quercetin (6), quercetin-3,7,3’-trimethyl ether (7), quercetin-3,7,3’,4’-tetramethyl ether (8),
eriodictyol-7,4’-dimethyl ether (9), -spinasterol (10), caryolan-1,9-diol (11), and 7-(3,3-dimethyl
allyloxy)-coumarin (12) were isolated from the acetone extract of dried flowers of H. inuloides in a
previous study. The semisynthetic compounds 7-acetoxy-3,4-dihydrocadalene (13), 7-benzoxy-3,4-
dihydrocadalene (14), 7-acetoxycadalene (15), 7-benzoxycadalene (16), quercetin pentaacetate (17) and
7-hydroxycalamenane (18) were obtained by conventional chemical procedures as previously described
(Rodríguez-Chávez et al., 2015).
2.7.Synthesis
3,8-Dimethyl-5-(1-methylethyl)-1,2-naphthoquinone (19), and 4-isopropyl-1,6-dimethylbenzo[c]
oxepine-7,9-dione (20) were obtained by treating the natural product 2 with mCPBA. The reaction was
carried out employing the microwave reactor Monowave 300 (Anton Paar GmbH, Graz, Austria). In a
G10 reaction vial equipped with a stir bar, 2 (83 mg) was mixed with CHCl3 (6 mL) solution of mCPBA
(320mg). The vial was sealed and the reaction mixture was exposed to microwave irradiation. The
reaction mixture was stirred at a fixed rate of 200 rpm at 55 °C for 4 min. The reactor temperature was
simultaneously monitored by an external infrared sensor and by an internal fiber-optic temperature probe
(ruby thermometer) inserted directly in the reaction mixture. After cooling, reaction mixture was washed
with 5% Na2SO3, 5% NaHCO3, and saturated NaCl, and dried with anhydrous sodium sulfate. The solvent
was evaporated and the residue was purified by column chromatography on silica gel using a Hexane-
EtOAc gradient to provide derivatives 19 (Krishnamoorthy and Thomson, 1971) and 20 in 29% and 63%
yields, respectively. Compound 20 is a novel compound that has not reported previously in the literature.
The reaction was monitored by thin-layer chromatography (TLC) and the TLC plates were visualized

with UV light (254 and 360 nm) and ceric ammonium sulfate solution. Column chromatography was
carried out on a column packed with silica-gel 60N spherical neutral size 230-400 mesh.
Mansonone C (19). Red-orange solid, mp 133-135°C [lit: 134-135°C] (Krishnamoorthy and Thomson,
1
1971); H NMR (CDCl3, 500 MHz): δH 7.18 (1H, br, H-2), 7.42 (1H, br, H-3), 7.65 (1H, s, H-5), 2.62
(3H, s, H-9 ) 3.38 (1H, m, H-10), 1.29 (3H, d, J = 6.9 Hz, H-11), 1.28 (3H, d, J = 6.9 Hz, H-12), and 2.07
(3H, s, H-13). 6-isopropyl-4,9-dimethylbenzo[c]oxepine-1,3-dione (20): oily substance; IR (KBr) max
2965, 2929, 2873, 1782,1740, 1568, 1451, 1414, 1383, 1257, 1232, 1212, 1181, 1154, 1090, 1055, 1003,
1
7806, 742 cm-1. UV (MeOH)  max (log ): 242.2 (4.129), 216.4 (4.30). H NMR (500 MHz): δH 7.38
(1H, d, H-7) and δ 7.28 (1H, d, H-8), δ 7.24(1H, s, H-5), δ 3.19 (1H, m, H-13), δ 2.40 (3H, s, H-12), δ
13
2.26 (3H, d, H-16), δ 1.25 (3H, s, H-14), and δ 1.25 (3H, s, H-15). C NMR (CDCl3, 125 MHz): δC
161.72 (C-3), 163.02 (C-1), 144.70 (C-6), 134.62 (C-10), 132.89 (C-5), 132.39 (C-7), 131.07 (C-11),
130.77 (C-4), 128.57 (C-9), 128.57 (C-8), 29.35 (C-13), 23.62 (C-15), 23.62 (C-14), 21.98 (C-12), 19.77
(C-16). EIMS m/z 245 [M]+ (35). HRFABMS m/z 245.1011 [M]+ (calc for C15H16O3 + H, 245.1178).
2.8.Cytotoxicity by sulforhodamine B (SRB) assay
The cytotoxic activities of compounds were determined using the protein-binding dye SRB in a
microculture method (Skehan et al., 1990). This assay is highly sensitive, its staining is not cell-line
dependent, is independent of cell metabolic activity, and the end-point measurement is not time-critical,
in addition, it has been adopted for routine use in the National Cancer Institute for in vitro antitumor
evaluation (Keepers et al., 1991; Vichay and Kirtikara, 2006). Preliminary screening was carried out at 50
M and the IC50 values were determined for those compounds that inhibited cell growth at least 50%.
Cells were cultured in RPMI 1640 medium, supplemented with 10% (v/v) inactivated fetal bovine serum,
100 U/mL penicillin and 100 µg/mL streptomycin sulfate, 2 μM/L glutamine, and 0.25 g/mL
amphotericin B. Cells were maintained in a humidified atmosphere (95% humidity) with 5% CO2 at 37
°C for 24 h prior to the addition of experimental compounds. The viability of the cells used in the
experiments exceeded 95%, as determined with a trypan blue assay. The cells were removed from the
tissue culture flasks by treatment with trypsin and diluted with fresh media. Cell suspensions (100 L),
containing 5000 or 7500 cells per well, were pipetted into 96-well plates plates (Corning, NY), and
incubated at 37◦ C for 24 h in a 5% CO2 atmosphere to reach exponential growth. After 24 h, a 100 L
aliquot of the test compounds or positive control was added (all test substances were dissolved in DMSO
at a maximum concentration of 0.5%, at which DMSO was completely innocuous) to each well. The
cultures were exposed to the compounds for 48 h. After the incubation period, cells were fixed in situ by
adding of 50 mL of cold 50% aqueous TCA and incubated at 4◦ C for 1 h. The supernatant was discarded,

and the plates were washed with tap H2O, and air-dried. The TCA-fixed cells were stained by the addition
of 0.4% SRB. Free SRB solution was then removed by washing with 1% aqueous acetic acid. The plates
were then air-dried, and the bound dye was solubilized by the addition of 10 mM unbuffered Tris base
(100 L). The plates were placed on a shaker for 5 min, and the absorption was determined at 515 nm
using an Ultra microplate reader (ELx808, Bio-Tek Instruments, Inc. Vermont, USA). The concentration-
response curve was plotted for each compound. Results were expressed as the concentration giving 50%
inhibition (IC50), and were estimated from linear regression equations. The IC50 values (mean ± standard
error) for compounds and the positive control are reported in Table 1.
2.9.Viability of RAW264.7 macrophages by MTT assay
Mitochondrial function was used to establish how RAW 264.7 cell macrophages were affected after
exposure to H. inuloides metabolites. This cell line offers advantageous characteristics, such as its ease of
culturing, its adherence capability and its moderate growth which simpliy the experimental procedures
(Wiese and Seydel, 1996), and commonly utilized as an in vitro biological model to determine effects of
toxicants on cells. The cell respiration was determined by the mitochondrial-dependent reduction of MTT
to formazan (Mosmann, 1983). The total amount of formazan is directly proportional to the number of
viable cells (Keepers et al., 1991). The RAW264.7 cells were cultured in Dulbecco´s Modified Essential
Medium (DMEM) with 4 mM L-glutamine, 4.5 g/L glucose, and 1mM sodium pyruvate (ATCC30-2002)
supplemented with 10% heat-inactivated fetal bovine serum (ATTC30-2020) and viability was
determined by trypan blue exclusion. Cells were maintained at 37 °C, 5% CO2 and used for experiments
between passage 5 and 18. Reagents for cell culture were purchased from ATCC. 100 µL/well of 2x106
cells/ml were seed in 96 well microtitre plates and incubated for 2 h. Cells were treated with or without 50
µL of each test compounds dissolved in DMSO and serially diluted at a decreasing concentration of 100-
3.1µM. The final concentration of DMSO in the cell culture supernatant was ≤ 0.1% (v/v). Controls also
received the same amount of DMSO. Cells were then incubated at 37° C, 5% CO2 for 24h. After 24 h of
exposure, the supernatant from each well was removed and briefly, 10 µL of MTT was added to the
remaining cell in 96 well microtitre plates, and incubated for 4 h at 37°C. Incorporated dye was dissolved
in DMSO (100 µL) and their absorbance was measured on a microplates reader at 570 nm using an
ELISA plate reader (Syner/HT: Biotek Instruments). Results were expressed as the percentage of MTT
incorporated into treated cells compared with control cells.
2.10. Artemia salina assay
Brine shrimp lethality assay was carried out to investigate the general toxicity of the metabolites of H.
inuloides. Assays were performed in a 96-well microtitration plate (Corning, NY), as described elsewhere

(Solis et al., 1993) with 10-20 brine shrimp larvae in 100 μL of seawater per well. The samples (in
triplicate) to be assayed were dissolved in ethanol-DMSO (50/50 v/v) and diluted serially in seawater to
give assay concentrations adjusted of 1, 10, or 100 M. Standard potassium dichromate solutions (1, 10,
and 100 M) were used as positive controls. Due to the low solubility of the compounds, the maximum
concentration tested in the brine shrimp toxicity assay was 100 M. Seawater and deionized water blank
controls were also tested. After 24 h, survivors were counted using a Nikon Diaphot 300 inverted
microscope (Nikon Co. Tokyo, Japan) and the mortality rate (%M) of each dose was calculated and
compared with the control. The mortality rate was calculated as the number of dead nauplii divided by the
initial number of nauplii (10) and multiplied by 100.
2.11. Acute oral toxicity in mice
The number of animals used was kept to a minimum. The experimental protocol was approved by the
Institutional Ethical Animal Committee of the Instituto de Química-UNAM (Protocol number
IQUNAM/CICUAL/01/GDL/15). In adittion, the handling and euthanasia of animals were conducted out
in strict accordance with the technical specifications for the production, care and use of laboratory
animals, included in the Mexican Official Standard NOM-062-Z00-1999 (Secretaría de Agricultura,
2001), and Guiding Principles of the Use of Animals in Toxicology, adopted by the Society of
Toxicology (USA) in July 1989 (amended in December 2008). The acute oral toxicity of compound 1 was
evaluated according to the procedures outlined by the OECD (Organization for Economic Cooperation
and Development, 2001). Mice were fasted 3-4 h prior to dosing but were administration water. The mice
were randomized into 3 groups with six mice each (n = 6). Group I (control) animals were administered
only electrolyte solution. Group II animals were administered orally with 300 mg/kg body weight of
compound 1. Group III animals received 2000 mg/kg body weight of the same compound 1. The test
compound was suspended in an electrolyte solution containing glucose, 20g; KCl, 1.5g; NaCl, 3.5g;
Na2H(C3H5O(COO)32H2O, 2.9 g/L (Apotex Mexico), and 2% Tween-80® and was administered in a
single dose by gavage using a suitable intubation cannula. After the fasting period, the animals were
weighed and administered with the substance. After dosing, the fasting was continued for 1 h and mice
were observed continuously for physiological and behavioral changes during the first 4 h and periodically
during the first 24 h. They were then kept under observation for up to 14 days. During this period the
animals were observed daily for mortality, behavioral profile (stereotypes, irritability, sedation),
neurological profile (seizures, motor incoordination), and autonomic profile (salivation, piloerection,
abdominal contortion, diarrhea), and weighed daily. On day 14, the animals were euthanized.
2.12. Justification for dose selection

The acute toxicity study of 7-hydroxy-3,4-dihydrocadalene (1) was evaluated in mice according to the
OECD guideline 423. The guideline states on paragraph 19 that “when there is no information on a
substance to be tested, for animal welfare reasons it is recommended to use the starting dose of 300 mg/kg
body weight”. Since there is insufficient information on the toxicity of 7-hydroxy-3,4-dihydrocadalene
(1), the starting dose of 300mg/kg body weight for a period of 14 days was selected. Due to limited
availability of the substance for further experimentacion, the sub-acute assay could not be assessed in the
present study.
2.13. Histopathological analysis
After sacrificing the mice, parts of the liver and kidney tissues were collected for the histological studies.
The organs were observed macroscopically for the presence or absence of alterations and their appearance
was compared between the treated and control groups. The organs were washed in buffer solution and
fixed immediately with 10% buffer/formaldehyde (v/v) for 24 h, dehydrated with a graded series of ethyl
alcohol, and embedded in paraffin. The paraffin-embedded tissues were cut into 4–5 μm sections with a
microtome (Leica Biosystems, Nussloch Germany) and mounted in glass, and stained with hematoxylin
and eosin (H&E) for examination under a microscope. Histological slides were examined under a light
Olympus BX51 microscope (Olympus Co. Tokyo, Japan) equipped with a digital camera, Micro-
FireCX9000 (mbf Bioscience, Vermont, USA), using the digital image analysis system Picture Frame
(TM) Applications 2.3 070207-05A (IEEE-1394PCI Card).
2.14. Statistical analysis
Experimental values are expressed as the mean ± the standard deviation. Data were analyzed using the
one-way analysis of variance (ANOVA) and Student´s-t test when necessary. Statistical significance was
set at p ≤ 0.05. All calculations were performed using Origin 8 (OriginLab Co. Massachusetts, USA)
software. The 50% lethal concentrations (LC50) were obtained from the data according to the Reed-
Muench method (Reed and Muench, 1938). For IC50 values, data were subjected to ANOVA followed by
Dunnett´s test. Values of p  0.05 (*) and p  0.01 (**) were considered significantly different from the
control (K2Cr2O7). The individual weight of animals was recorded before the administration of the
compound on the 1st day of the study and 14th day of the experiment before sacrificing the animals.
Changes in the weight were compared with that of the control animals. The experimental results were
expressed as the mean ± SEM. Dunnett´s test was performed for the sample treated and control groups.
3. Results
3.1.Identification of compounds.

Two compounds were obtained and isolated from the reaction of compound 2 with mCPBA using
microwaves as the heating source. Compound 19 was purified as an orange solid and its IR spectrum
1
showed the absorption of two carbonyl groups at 1665 and 1670 cm-1. The H and 13C spectral data
showed signals comparable with those for 2, except for the replacement of an aromatic proton at H 7.25
and phenol group at C7 in 2, and displaying carbonyl carbon at C182.0 and 182.8, suggesting a cadinane
1
type o-naphtoquinone. The H and 13C spectroscopic data, melting point and comparisons with data
reported in the literature allowed the identification of the compound as 5-isopropyl-3,8-
dimethylnaphthalene-1,2-dione (mansonone C, 19). Compound 20, obtained as an oily substance, had a
molecular ion peak (M+ + H) at m/z 245, indicating the molecular formula C15H16O3. The structural
elucidation of this compound was achieved on the basis of 1H and 13C NMR spectra, 2D COSY, NOESY,
HSQC and HMBC experiments as well as HR-ESI-MS data (molecular ion peak at m/z 245.1100
[M+H]+). The 1H NMR spectrum displayed signals corresponding to two ortho- coupled aromatic protons
at δH 7.28 (1H, d, J = 8.1, H-8) and δH 7.38 (1H, d, J = 8.1, H-7). There was one singlet signal of aromatic
protons at δH 7.24 (1H, q, J = 8.1, H-5), with the chemical shift indicating that it was next to a carbonyl
group. The presence of two methyl groups at δH 2.40 (3H, s, H-12) and δH 2.26 (3H, d, J = 1.6, H-16) and
one isopropyl moiety at δH 3.18 (1H, m, H-13), δH 1.256 (6H, d, J = 6.8 Hz, H-14 and H-15) supported the
13
structure. The C NMR spectrum displayed signals for 15 carbons, which were assigned by the DEPT
spectrum as four methyls, four methines and two carbonyl carbons. The remaining five signals appearing
in the broad-band spectrum were attributed to the quaternary carbon atoms. IR absorption bands at 1741
and 1782 cm-1 revealed the presence of an anhydride and this functional group was further confirmed by
13
the C NMR spectrum, with signals at δC 161.71 (C-3) and δC 163.02 (C-1). The elucidated structure of
this compound (20) is depicted in Fig. 1.
3.2.Cytotoxicity in cell lines.
To evaluate the cytotoxicity of the compounds from H. inuloides, K652 (leukemia), HCT-15 (colon), and
MFC7 (breast cancer) human cancer cell lines were treated with varying concentrations of these products
compounds and the sulforhodamine B assay was performed. Seven of the samples tested were active in at
least two cell lines with IC50 values ranging from 1.45 ± 0.14 to 79.65 ± 2.1 μM. The acquired
cytotoxicity data revealed that compound 19 (mansonone C) was the most active compound against the
tested K652 cell line, followed by compound 2 and their acetylated derivative 15, which were moderately
active. Compounds 1, 13, and 9 also showed activity, although less. For the HCT-15 cell line, the most
active compound was 2 (7-hydroxycadalene), followed by 19 and 17. Finally, for the MCF cell line the

most active compound was 17 (quercetin pentaacetate), followed by 2. Other compounds showed IC50
values greater than 50 M and some had no activity (see Table 1).
3.3.Artemia salina assay.
The effect of the metabolites of H. inuloides and semisynthetic analogous on the lethality of brine shrimp
was also determined. The LC50 values of the brine shrimp for these compounds and the positive control
are shown in Table 1. The exposure of A. salina to some of the compounds produced toxic effects.
Mansonone C (19) displayed the highest activity, followed by compounds 1, 2, 11, and 18. The LC50
value obtained according to the Reed-Muench method for 19 was 39.4 ± 1.07 M. The remaining
compounds were not toxic to A. salina, and the LC50 values of these compounds were presumably higher
than 100 M. The reference standard, potassium dichromate, had an LC50 value of 385.65 ± 1.07 M.
There was no mortality in the negative control group.
3.4.Viability of RAW264.7 macrophages.
The viability of RAW264.7 macrophages in the presence of metabolites (25 μg/mL) of H. inuloides was
measured by the MTT assay. The results showed compounds 1 and 2 and their acetylated derivatives 13
and 15 produced a significant decrease in the viability of macrophages at 25 μg/mL (Table 2).
Compounds 3,7-dihydroxy-3(4H)-isocadalen-4-one (3), (1R,4R)-1-hydroxy-1,2,3,4-tetrahydrocadalen-
15-oic acid (4), quercetin (6), quercetin-3,7,3’-trimethyl ether (7), quercetin-3,7,3’,4’-tetramethyl ether
(8), eriodictyol-7,4’-dimethyl ether (9), 7-benzoxy-3,4-dihydrocadalene (14), and 7-benzoxycadalene (16)
showed no significant macrophage toxicity (Table 2), and the percentage of living cells examined by
MTT assay was greater than 80% in most cases.
3.5.Acute oral toxicity.
After 14 days of administration of 1 (7-hydroxy-3,4-dihydrocadalene), at a dose up to 300 mg/kg body
weight, there were no toxic symptoms or mortality observed in mice. No seizures or motor incoordination
occurred and other signs of toxicity, such as dyspnea, lethargy, and grunting, were not detected in mice
during the first 4 h or at 24 h after administration. There were no significant differences in behavior, skin,
respiratory or postural abnormalities between the treated and control groups. A significant difference was
observed between treated animals and controls when compound 1 was administered at a dose of 2,000
mg/kg. Mice treated showed piloerection, breathing difficulties and some toxicity signs, such as dyspnea,
lethargy and grunting during the first 4 h. Short term sluggishness was observed in some mice after oral
gavage in the first hour. The animals were observed for 14 days after administration of compound 1, and
no deaths occurred. The corporal weight of the animals was recorded. Weight gain was noticed in the

group treated with 300 mg/kg of compound 1, but the gain was not significantly different compared with
the control group (Fig. 2). Compound 1 caused an abnormal increment in body weight and death in some
mice of the group treated with 2,000 mg/kg, during the 14-day observation period. The macroscopic
examination of the organs of animals treated with the compound 1 showed no change in color compared
with organs from the controls.
3.6.Histopathological analysis.
The liver sections of the control group (Group I) animals showed a typical histo-architecture with a
normal arrangements of cells in the liver lobules with a central vein, radiating hepatic cords, and sinusoids
intact (H&E magnification 40X). Light microscopy demonstrated a well-preserved and homogeneous
cytoplasm of hepatocytes (Fig. 3). In groups II and III (treated with 300 and 2,000 mg/kg body weight of
compound 1, respectively), light microscopy showed distortion in the architecture, the presence of a
chronic inflammatory infiltrate composed of mononuclear cells (Kupffer cells), and vacuolization of
hepatocytes. No morphopathological change was found in the control group, kidneys, which showed
numerous glomeruli, renal tubules and interstitial tissues with normal histological structures. Treatment
with compound 1 (300 mg/kg and 2,000 mg/kg body weight) led to moderate damage. The proximal
convoluted tubules presented sligth alterations in the morphological structure and distension of
Bowman’s capsule, these changes were more evident with the dose of 2,000 mg/kg body weight dose
(Fig. 3).
4. Discussion
Through oxidation of the sesquiterpene 2 with mCPBA using the microwave method, we obtained
compound 19 (mansonone C) and the novel compound 20. Compound 19 is found naturally in the
heartwood of plants of the genera Ulmus and Mansonia, and has been synthesized from sesquiterpene 2
by oxidation with Fremy’s salt (Alexander and Krishna-Rao, 1971; Krishnamoorthy and Thomson, 1971)
and by oxidation in silica (Strunz et al., 1989). It was also obtained by cyclization of the p-benzoquinone
hydroxyperezone (García et al., 1997). Using these conventional methods, the reported yield is low and
the preparation is lenghtly. The synthesis in the microwave required irradiation at 55 °C required only 4
min.
In Mexico, H. inuloides is used in traditional medicine as a remedy for of bruises, inflammatory processes
and other ailments as cancer (Alonso-Castro et al, 2011; Zurita-Esquivel et al., 1986). Previous reports
mentioning that petrol, ethanol, and dichloromethane extracts of H. inuloides displayed cytotoxic action

against murine leukemia (P388 cells) (Villarreal et al., 1992). Additionally, sesquiterpenoids 1 (7-
hydroxy-3,4-dihydrocadalene), 2 (7-hydroxycadalene), -caryophyllene and -caryophyllene epoxide
isolated from dried flowers exhibited cytotoxic activity against breast carcinoma (BT-20 cells), epitheloid
cervix carcinoma (HeLa cells), melanoma skin cells from human (B-16 cells), and melanoma skin cells
from mouse (Kubo et al., 1996). This study compares the cytotoxic activity of components isolated from
acetone extract in order to increase the knowledge on the pharmacological potential of H. inuloides. The
sulforhodamine B assay showed that cadinene type sesquiterpenes are considered to be the main active
constituents. Replacement of hydrogen at position 7 in the sesquiterpenes 1 (7-hydroxy-3,4-
dihydrocadalene) and 2 (7-hydroxycadalene) by an acetyl group reduced the activity (of their acetylated
derivatives 13 and 15, respectively) against the HCT-15 and MCF-7 cell lines. Substitution of the
hydroxyl group at position 7 resulted in a complete loss of activity in the benzoyl-substituted analogs
(compounds 14 and 16). This effect could be attributed to steric hindrance arising from the increase in the
volume of the derivatives. Previous reports indicated that having OH at C-7 in structurally similar
compounds modifies the biological activity (Lee et al., 2007). Conversely, the acetylated derivative (17)
of compound 6 (quercetin) showed greater activity against the cell lines tested. It has been reported that
ester-based derivatives enhanced the bioavailability of quercetin (Biasutto et al., 2007). This effect could
be attributed to the increased solubility of the acetyl derivative compared with the natural compound.
Compounds 1 and 2 were significantly toxic to A. salina larvae. It was observed that replacement of the
phenolic hydrogen in the sesquiterpenes 1 and 2 by acetyl groups in the compounds 13 and 15 did not
modify the toxic effect; however, substitution with benzoyl groups in compounds 14 and 16 reduced the
toxic effect. The other compounds tested did not show toxic effect on the brine shrimp. It is noted that
compounds 1 and 2 were toxic to brine shrimp and were also active against tumor cell lines. These results
are consistent with previously published data indicating that positive results in brine shrimp assays are
associated with pharmacological potential, including anti-tumor activity (Meyer et al., 1982). Also,
compounds with a cadinane skeleton, such as 10-hydroxyamorphane-4-en-3-one, 4-methylcadinane-
,2,10-triol, 10-hydroxyamorphane-4-en-3-one, -cadinol, and parviflorene, have shown
significant cytotoxic activity against human-tumor cell lines and were toxic to brine shrimp (He et al.,
1997; Takahashi et al., 2003).
When tested with the murine peritoneal macrophage cell line RAW264.7, the sesquiterpenes 1 and 2
showed toxicity. Modification of the molecular structures of 1 and 2 changed their toxicity. The viability
values of the acetylated derivatives showed no statistically significant differences from their natural

precursors. However, benzoylated derivatives showed a significant decrease in toxicity in macrophages.
In the present study, the tested flavonoids did not show significant macrophage toxicity.
Acute toxicity tests represent the first stage in the evaluation of the toxic effects of new substances for
therapeutic purposes and provide a preliminary identification of the species, target organ and dose
specific toxic effects. Occasionally, the tests may confirm the short-term and long-term safety of such
products before they are brought to market (Denny and Stewart, 2013; Parasuraman, 2011). In this study
the process of the OECD was selected, because it offers the advantage that adequate information is
obtained using only a few animals. Due to the shortage of compounds, only the acute toxicity of 7-
hydroxy-3,4-dihydrocadalene (1) was evaluated. This cadinane sesquiterpene compound was the major
constituent in the extract from H. inuloides flowers (Delgado et al., 2001). Information in the literature
indicated that compounds with a cadinane skeleton display toxic effects in different animal species; for
example, 9-oxo-10,11-dehydroageraphorone, a cadinene sesquiterpene isolated from Eupatorium
adenophorum (Bhardwaj et al., 2001), and zederone, isolated from Curcuma elata (Pimkaew et al., 2013),
are hepatotoxic. The first compound also causes cholestasis in rodents, and high concentrations of
gossypol produces acute signs of poisoning (Gadelha et al., 2014).
CD-1 albino mice treated with 7-hydroxy-3,4-dihydrocadalene (1) at 300 mg/kg doses did not show lethal
effects, and there were no statistically significant changes in body weight, behavioral profiles, and
appetite suppression. Thus, there were no deleterious effects. This could indicate that at this dose, this
compound (1) did not produce a marked disturbance in metabolism. The highest dose (2,000 mg/kg body
weight) was associated with loss of weight in the first 7 days, with subsequent recovery; as a result, the
weight was similar to the control group in the last 2 days (Fig. 3). Changes in weight have been accepted
as a sensitive indicator of chemically induced changes in the body and may reflect the degree of toxicity
after exposure to a toxic substance (Michael et al., 2007). According to the chemical labeling and
classification of acute systemic toxicity recommended by GHS (UN, 2011), compound 1 (7-hydroxy-3,4-
dihydrocadalene) was assigned class 4 status (LD50 = 500 mg/kg). Histopathological analysis of mice in
the control group revealed there was a characteristic histoarchitecture of a normal liver; while mice
exposed to compound 1, displayed distortion of the hepatic histo-architecture, the presence of a chronic
inflammatory infliltrate, and microvesicular lipidosis in the cytoplasm of hepatocytes. These pathological
signs have been associated with liver toxicity and failure caused by exogenous compounds (Roberts et al.,
2007; Sanyal, 2002), suggesting that compound 1 has a mild to moderate toxic effect, with changes more
visible with 2,000 mg/kg dose. However, the presence of larger nuclei in cells suggests that they may
undergo a regeneration process. Proximal convoluted tubules and the Bowman's capsule processes are

involved in the excretion of endogenous and exogenous compounds, including metabolites and drugs.
However, exposure to certain substances produced alterations in the histostructure (Abdelhalim and
Jarrar, 2011; Hanika et al., 1986). The histopathological study showed after exposure of mice to
compound 1, there were alterations in the morphological structure of the proximal convoluted tubules and
Bowman's capsule, which could be associated with a toxic effect on the kidney tissue that increases with
dose (Fig. 4).
In published studies, the values of the amount of 7-hydroxy-3,4-dihydrocadalene (1) isolated from this
Mexican plant ranged from 0.0009 to 1.67769 mg/g dry weight (Delgado et al., 2001; Kubo et al., 1994;
Maldonado-López et al., 2008; Rodríguez-Chávez et al.; 2015). The extractability of the compounds
depends on the parameters, including the temperature, time and the solvent type and polarity (Tan et al.,
2014). The infusions of H. inuloides used in folk medicine are made with about 3 grams of flowers per
liter of boiled water (Comisión Nacional Forestal, ), while commercial tea infusions (200 mL) prepared
with only one tea bag containing 1 gram of plant (Muñoz-Velázquez et al., 2012) , and the recommended
doses is 3 cups a day during 15 days. Considering that tea infusions are dilute solutions of soluble
constituents, and certain compounds of biological relevance may be not extracted, it's ingestion may not
cause any adverse effects because of the minimal amount ingested. However, further studies are necessary
to evaluate subacute and chronic toxicity to better understand the risks and benefits of using H. inuloides
in medicine.
5. Conclusions
The toxicity of different metabolites isolated from the acetone extract of Heterotheca inuloides and some
semisynthetic derivatives was assessed. It was demonstrated the in vitro activity efficacies of some
flavonoid and cadinene type compounds against diferent cancer cell lines. Among the compounds of H.
inuloides the best cytotoxicity was observed for mansonone C (19) on K562 cell line with IC50 1.45±0.14
M, for 7-hydroxycadalene (2) on HCT-15 cell line with IC50 18.89±1.2 M, and for quercetin
pentaacetate (17) on MCF-7 cell line with IC50 22.57±2.4 M. The strongest deleterious effects against A.
salina were observed for sesquiterpenes mansonone C (19) and 7-hydroxy-3,4-dihydrocadalene (1) with
IC50 39.4±1.07, and 45.47±1.74 M, respectively. Hydroxycadalene (2) also showed a strong toxic effect
on A. salina (IC50=53.92±1.04 M). The number of viable RAW 264.7 cells was reduced with
sesquiterpenes 1 and 2 by more than 90% compared to untreated controls. In the same biological assays
the flavonoid type compounds did not exert toxic effect. In addition, the substituents play an important
role in the activity of 7-modified 1 and 2 derivatives. Single dose acute toxicity of 7-hydroxy-3,4-
dihydrocadalene (1), the main H. inuloides cadinene sesquiterpene component, was examined to identify

potent toxicity. At a dose of 2,000 mg/kg, compound 1 significantly caused changes in body weight of
mice, histomorphological changes in the liver and kidney, and lethal effects in mice. However, the
mechanism of action involved in the toxicity is not yet known, therefore, complementary studies are
needed to clarify the mechanism of action of the compounds. According to the GHS guidelines for the
chemical labeling and classification, compound 1 (7-hydroxy-3,4-dihydrocadalene) was assigned class 4
status (LD50 = 500 mg/kg). This study is the first to show that Heterotheca inuloides, which is claimed to
be a treatment for imflamatory process and other ailments, is a medicinal plant that contains compounds
that may cause side effects, particularly at very high doses, suggesting that should be used with caution.
Abbreviations:
mCPBA,
TCA,
Meta-chloroperoxybenzoic acid
trichloroacetic acid
DMSO,
Tris,
dimethyl sulfoxide
tris(hydroxymethyl) aminomethane
sulforhodamine B
SRB,
K2Cr2O7
Tween® 80
NaH2PO4
Na2HPO4
H3PO4
MTT
potassium dichromate
polyoxyethylenesorbitan monooleate
sodium dihydrogen phosphate
sodium hydrogen phosphate
orthophosphoric acid
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
chloroform
CHCl3
Na2SO3
NaHCO3
NaCl
sodium sulfite
sodium bicarbonate
sodium chloride
NMR,
ATCC,
HEPA,
UV,
nuclear magnetic resonance
American Type Culture Collection
High Efficiency Particle Aarrestance
ultraviolet
IR,
infrared
TLC,
thin-layer chromatography
electron ionization mass spectroscopy
EIMS,
HRFABMS, high resolution fast-atom bombardment mass spectrometry
v/v, volume/volume

MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
Organization for Economic Cooperation and Development
hematoxylin and eosin
OCDE,
H-E,
COSY,
NOESY,
HSQC,
HMBC,
DEPT,
LD50,
correlation spectroscopy
nuclear Overhauser effect spectroscopy
heteronuclear single-quantum correlation spectroscopy
heteronuclear multiple-bond correlation spectroscopy
distortionless enhancement by polarization transfer
median lethal dose
GHS,
Globally Harmonized Classification System
Conflicts of interest
The authors declared no potential conflicts of interest with respect to the research, authorship, and
publication of this article.
Ethical approval
All applicable institutional and/or national guidelines for the care and use of animals were followed.
Funding
Part of this work was supported by Universidad Nacional Autónoma de México-DGAPA-PAPIIT [grant
IG200514].
Acknowledgemements
J.L.R.-Ch. thanks Consejo Nacional de Ciencia y Tecnología (CONACYT, México) and Programa de
Doctorado en Ciencias Biomédicas de la Universidad Nacional Autónoma de México (UNAM) for
financial support for doctoral studies. We thank Chemist Jorge Ebrard (Laboratorios Mixim, Mexico
City) for providing the plant material and Prof. Abigail Aguilar-Contreras (Instituto Mexicano del Seguro
Social) for the identification of the species. We also thank Antonio Nieto, Ángeles Peña, María Isabel
Chávez, Beatriz Quiroz, Héctor Ríos, Rocío Patiño, Javier Pérez, and Luis Velasco from Instituto de
Química, UNAM, for technical assistance.

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Fig. 1. The natural products and derivatives of H. inuloides assayed

Fig. 2. Changes in Body weight of mice exposed to compound 1 (7-dydroxy-3,4-dihydrocadalene) at 300
and 2000 mg/kg body weight. Each point represents the mean of six mice. * p < 0.05 with respect to the
control.

Fig. 3. Representative Hematoxylin & Eosin (H&E) staining liver and kidney sections (5 m) from mice
treated with electrolite solution (control I) or 7-hydroxy-3,4-dihydrocadalene (1) (II and III) obtained on
day 15 of treatment. Figure abbreviations: cv (central vein), Kc (Kupffer cells), Bc (Bowman´s capsule),
Gl (glomerulus), pct (proximal convoluted tubule). (H&E magnification 40X).

Table 1. Activity of H. inuloides metabolites against cancer cell lines and A. salina (IC50 in M).
Cell lines
HCT-15
Artemia
salina
Compound
K562
MCF-7
1
2
3
4
49.35±3.6 59.37± 4.5 61.37± 4.3
45.47±1.74
53.92±1.04
>100
26.41±2.0
18.89±1.2
55.34±2.8
Nd
30.± 0.87
51.76±2.2
Nd*
Nd
Nd
>100
>100
Nd
Nd
5
>100
6
70.3±4.6
>100
>100
>100
>100
7
8
9
--
--
--
--
--
--
51.48±3.5
Nd
57.67±0.6
Nd
62.53±3.6
Nd
10
Nd
Nd
Nd
Nd
Nd
Nd
54.13±1.07
>100
11
12
46.43±3.4
Nd
71.56±2.4
Nd
79.65±2.1
Nd
>100
>100
13
14
27.54±0.9
Nd
55.93±3.5
Nd
63.62±5.8
Nd
>100
--
15
16
>100
17
18
19
35.31±2.9
Nd
28.57±2.1
Nd
22.44±2.6
--
22.57±2.4
Nd
70.16±1.08
39.4±1.07
>100
1.45±0.14
--
--
--
20
Controlsa
0.59 ± 0.02b 0.13 ± 0.05b 0.16 ± 0.01b
385.6±1.07
Results represent the mean ± SD of three independent experiments.
a Camptothecin (CPT) for cytotoxicity in tumor cell lines and potassium dichromate (K2Cr2O7) for A.
salina. b CPT Values are nanomolar range (nM).
*Not determined: at the concentrations tested the activity of compounds was low.
Table 2. Effect of H. inuloides metabolites on the RAW264.7 cells viability.
Compound
% Viability
(25 µg/mL)
1
2
3
4.0±0.5**
3.6±0.1**
92.5±7.5
100
5
6
100
7
8
9
89.7±10.3
79.6±0.1
100
13
14
5.2±0.09**
100

15
16
3.7±0.06**
100
91.92±1.4
Oleic acida
aNegative control. ** p< 0.05 with respect to the control.
grphical abstract