Isolation and In Vitro Biological Evaluation of Triterpenes from Salacia grandifolia Leaves

Salacia grandifolia is naturally found in the Atlantic Forest regions of Brazil. Despite the pharmacological potential of plants from the Salacia genus, phytochemical studies on this species have not been reported in literature. A new triterpene, 28-hydroxyfriedelane-3,15-dione (1), and seven known compounds (friedelan-3-one (2), friedelan-3β-ol (3), friedelane-3,15-dione (4), 15α-hydroxyfriedelan-3-one (5), 28-hydroxyfriedelan-3-one (6), 30-hydroxyfriedelan-3-one (7), and 29-hydroxyfriedelan-3-one (8)) were obtained from the hexane extract of Salacia grandifolia leaves. These isolated compounds and three extracts, hexane (EH), chloroform (EC), and ethyl acetate (EAE), were assessed for their potential biological activities, which consisted in the evaluation of antiviral activity against a murine coronavirus, mouse hepatitis virus 3 (MHV-3), antibacterial activity against the susceptible and methicillin-resistant Staphylococcus aureus (MRSA), and antileukemia activity against the THP-1 and K-562 cell lines. The extracts EH and EAE along with the triterpenes 1 and 6 exhibited moderate to high antiviral activity, with emphasis on 6, which presented an EC50 value of 2.9 ± 0.3 μM. None of the compounds presented antibacterial activity against the tested strains. The evaluated compounds 1, 4, 6 and 7 exhibited low cytotoxic activity against the tested leukemia cell lines. Taken together, this study comprises an overview for the potential of the Salacia grandifolia biological activities, including a new isolated triterpene.


■ EXPERIMENTAL SECTION
Column chromatography (CC) was carried out using silica gel 60 (70−230 mesh or 230−400 mesh, Merck, Darmstadt, Germany) as the stationary phase.The eluents used were hexane, chloroform, ethyl acetate, and methanol, either pure or in increasing polarity mixtures.Thin layer chromatography (TLC) was carried out on plates coated with silica gel 60 using a solution of 1:1 v/v vanillin (vanillin, absolute ethanol, 1% m/ v) and percloric acid (3% v/v), followed by heating to 100 °C.The melting points of the isolated compounds were determined using a Microquı ́mica apparatus, model MQAPF-302 (Microquı ́mica, Palhoca, Brazil), without correction for normal temperature and pressure conditions.Infrared spectra were obtained using a Shimadzu IR-408 spectrometer (Shimadzu, Kyoto, Japan) with KBr pellet samples.1D/2D NMR spectra were acquired using Bruker Avance DRX-400 and DRX-600 spectrometers (Bruker, Billerica, USA), with CDCl 3 as solvent.Chemical shifts (δ) were measured in ppm, using tetramethylsilane (TMS; δ H = δ C = 0) as internal reference standard, and coupling constants (J) were calculated in Hz.The mass spectrum was acquired in a Shimadzu IT-TOF high-resolution mass spectrometer (Shimadzu, Kyoto, Japan) using ESI as an ionization font.
Plant Material.The leaves of Salacia grandifolia were collected in Embu-Guacu, Saõ Paulo, Brazil, in December 2020.A voucher was collected and deposited at the Herbarium of the University of Saõ Paulo under the number "Antar 3242".The plant material was registered at Conselho de Gestaõ do Patrimonio Genetico (CGEN/SisGen), Brazil, under the number AEC7E65.
Extraction and Isolation.The leaves of S. grandifolia were dried at room temperature and subsequently grounded.The powder (384.5 g) was subjected to extraction by maceration with hexane, chloroform, ethyl acetate, and methanol, separately.During the partial removal of the hexane in a  Antiviral Assay.The antiviral activity of isolated compounds and extracts was evaluated against the murine coronavirus MHV-3.Initially, the viability of L929 (mouse adipose fibroblasts; ATCC CCL-1) cells was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Thermo Fisher Scientific, USA) assay. 11Cells were cultured at 37 °C and 5% CO 2 in Dulbecco's modified Eagle medium (DMEM; Cultilab, Brazil) supplemented with 5% fetal bovine serum (FBS; Cultilab, Brazil), 100 IU/mL of penicillin (Cellofarm, Brazil), 100 μg/mL of streptomycin (Merck, Germany) and 0.25 μg/mL of amphotericin B (Cultilab, Brazil).Cells were seeded in 96-well microplates (4 × 10 4 cells per well) and added 200 μL of DMEM with 1% de FBS containing the diluted compounds and extracts to be tested (100 to 1.56 μM or 200 to 3.125 μg/mL).Serial dilutions of DMSO were used as a vehicle control.Additionally, a 10% v/v inhibition control of DMSO was used.After 72 h of incubation under the same conditions, media was removed and 100 μL of MTT diluted in DMEM (0.5 mg/mL) were added to each well and incubated for 3 h.Last, media was removed, and 100 μL of DMSO were added to each well to solubilize the formazan crystals.The absorbance was read at 570 nm using a spectrophotometer (Versamax, Molecular Devices, EUA).The percentages of cell viability inhibition were calculated as the ratio between the absorbance of cells treated with the compounds relative to the vehicle control.Linear regression was used to calculate CC 50 values, considering only those data for which r 2 > 0.9.The 50% cytotoxic concentration (CC 50 ) is defined as the highest concentration of a specific compound that reduces cell viability by 50%.All conditions were tested in two independent assays in triplicate.
Subsequently, MTT assays were also performed to determinate the effective concentration at which compounds were effective in protecting cells from viral infection by 50% (EC 50 ), as described. 11Here, the same conditions as that used for CC 50 were used, with the only difference being the concentrations of the samples (up to 8-fold dilution from the CC 50 values for the compounds) diluted in 100 μL and added to 100 μL of viral suspension at a multiplicity of infection (MOI) of 0.1, that is, one viral particle per 10 cells in culture.Ribavirin was used as the positive control.The EC 50 value was calculated as the percentage ratio between the absorbance of infected cells treated with the compounds and cells treated only with the vehicle.Last, the selectivity index (SI) was calculated using the ratio between CC 50 and EC 50 .All conditions were tested in two independent assays in triplicate.
Antibacterial Activity Evaluation.Compounds were evaluated against S. aureus (ATCC 29213) MRSA (ATCC 43300) strains.The antibacterial activity was evaluated with the broth microdilution method in 96-well microplates as described, 12 in accordance to the Clinical and Laboratory Standards Institute (CLSI) protocol.Briefly, compounds were diluted in Mueller Hinton broth (MHB; Oxoid, Thermo Scientific, UK) to the concentration of 200 μM or 400 μg/mL, and 100 μL of the dilutions were added to each well and the same volume of a bacterial suspension containing 1 × 10 5 CFU/mL, that is, 100,000 colony forming units per milliliter.Penicillin G and vancomycin were used as positive controls for S. aureus and MRSA, respectively.After 24 h incubation at 37 °C, the microplates were inspected visually for inhibition of bacterial growth, and the absorbance at 600 nm of each well was read using a microplate reader spectrophotometer (VersaMax, Molecular Devices, CA, USA).All conditions were tested in two independent assays in triplicate.
Cytotoxic Assay.The cytotoxicity of compounds was evaluated against the K-562 (chronic myeloid leukemia, ATCC CCL-243) and THP-1 (acute monocytic leukemia, ATCC TIB-202) cell lines.Cell viability was determined by MTT assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich, Saint Louis, EUA).For the cytotoxicity assessment, cells were planted in 96-well microplates (1 × 10 4 cells per well) containing RPMI 1640 medium (Roswell Park Memorial Institute, Cultilab, Campinas, SP, Brazil) supplemented with 10% FBS (Cultilab, Campinas, SP, Brazil), 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 mM HEPES, pH 7.4.The cells were incubated in a humidified incubator at 37 °C with 5% CO 2 until the assay was performed.The cytotoxic assays were performed with the samples and positive controls, imatinib and cytarabine, diluted in culture medium containing 1% FBS at concentrations of 100, 10, 1, and 0.1 μg/mL.After 48 h of incubation, 100 μL of MTT salt at a concentration of 0.5 mg/mL was added to each well.The plate was then incubated for 3 h.After the incubation period, the supernatant was removed, and 50 μL of DMSO was added to each well to solubilize the formazan crystals.The absorbance per well was measured at a wavelength of 550 nm using a microplate reader (Versamax, Molecular Devices, USA).The minimum concentration that inhibited 50% of cell viability (IC 50 ) in the presence of the tested compounds was determined by comparing it with cells cultured without the compounds (considered 100% viable).
Both active triterpenes (1 and 6) have a hydroxyl group at C-28, and the monocarbonyl triterpene 6 showed a 23-times increase in activity compared to the new isolated triterpene 1.Interestingly, compound 4, a nonhydroxylated analog of compound 1, showed no antiviral activity up to 100 μM.The other triterpenes that lack the hydroxyl group at C-28 were also inactive.These results suggest the presence of the hydroxyl group at C-28 is essential for the antiviral activity  against MHV-3, and that the presence of a second carbonyl group reduces activity.Therefore, further testing with other hydroxylated triterpenes at C-28 is needed to establish a better structure−activity relationship and to investigate the mechanism behind the observed antiviral activity.Cytotoxic Assay.All tested compounds (1, 4, 6 and 7) showed high IC 50 values, ranging from 350 to 623 μM against THP-1 cells (positive control Cytarabine, IC 50 of 41 ± 8 μM), and from 259 to 593 μM against K-562 cells (positive control Imatinib, IC 50 of 35 ± 4 μM).The compound 4 (friedelane-3,15-dione) exhibited the highest cytotoxic activity against THP-1 cells, with an IC 50 value of 350 ± 43 μM.On the other hand, the most active compound against K-562 cells was the new triterpene 1 (28-hydroxyfriedelane-3,15-dione), with an IC 50 of 259 ± 33 μM (Table 3).

■ CONCLUSIONS
The phytochemical study of Salacia grandifolia leaves led to the isolation of eight pentacyclic triterpenes with a friedelane skeleton.To the best of our knowledge, the NMR data (1D and 2D) of 28-hydroxyfriedelane-3,15-dione (1) is herein reported for the first time.All tested compounds showed low cytotoxic activity against THP-1 and K-562 leukemia cells.Compound 6 (28-hydroxyfriedelan-3-one) exhibited higher antiviral activity compared to 1 against a mouse coronavirus, and both essentially did not reduce the viability of L929 cells.
The IR and NMR spectra of compounds 1-8 and the HR-IT-TOF-MS analysis of compound 1, are available in the Supporting Information (Figures S1−S28), as well as the NMR data comparison of compound 1 (Table S1).(PDF)

Table 2 .
Biological Evaluation of Compounds against MHV-3 in L929 Cells a a NA: not active.