Ursolic Acid Derivatives Down Regulate Inflammatory Mediators

Abstract

Ursolic acid (UA) is being investigated due to its anti-inflammatory potential, and structural modifications can enhance its biological activities. The aim of this study was to evaluate the immunomodulatory effect of the ursolic acid derivatives (UAD) in macrophages and in the carrageenan-induced paw edema model. RAW264.7 cells were cultured in the presence or absence of UA or UAD (1-18). Nitric oxide (NO), nuclear factor kappa B (NF-κB), tumor necrosis factor (TNF), and cellular viability were measured. 30 min before the carrageenan-induced paw edema, the UAD1 and UAD2 (200 mg kg^-1) were administered intraperitoneally. The results showed that UAD2-4, UAD7, UAD9-11 had half maximal inhibitory concentration (IC₅₀) greater than 90 µM and were able to reduce NO, NF-κB and TNF production. Moreover, UAD1 and UAD2 reduced paw edema and IL-6 production. In conclusion, the results obtained demonstrated a variation in the response between the derivatives due to the chemical modifications, showing potential to reduce the inflammatory mediators, deserving further investigations.

Keywords:
ursolic acid; macrophage; carrageenan; inflammation

Introduction

The inflammation processes are associated with the development of several diseases, acute or chronic, many of them of unclear origin, such as rheumatoid arthritis, multiple sclerosis and Crohn‘s disease.¹,² The activation of macrophages is related to the production of inflammatory cytokine and transcription factor expression, such as tumor necrosis factor (TNF) and nuclear factor kappa B (NF-κB), respectively, playing a major role in the establishment of the inflammation.

The cytokines are involved in the activation of endothelial cells and leukocyte infiltration.³ The TNF is a pro-inflammatory cytokine that has several functions, such as inducing the production of other cytokines and lipid mediators of inflammation, proliferation, cell differentiation and apoptosis.⁴ Although the macrophage lineage cells are the main source of TNF, in inflammatory diseases, it can be produced by a wide variety of cells including neutrophils, fibroblasts and endothelial cells.⁵

The transcription factor NF-κB stands out, which plays a crucial role in the initiation and amplification of inflammation. NF-κB consists of a protein assembly located in the cytoplasm of cells and its activity is controlled by a family of inhibitor proteins, denominated IκB, which bind to the NF-κB dimmer, promoting its inhibition. This factor regulates the expression of several genes that code inducible nitric oxide synthase (iNOs), cyclooxygenase-2 (COX-2), TNF-α, interleukin-1-beta (IL-1β) and interleukin-6 (IL 6), which are involved in the pathogenesis of inflammatory diseases.⁶-⁸

Nitric oxide (NO) is a powerful inflammatory mediator, produced by macrophages and is involved in the regulation of several physiological processes. NO can be generated in excess during the host’s response against damage caused by pathogens or other substances, contributing to the pathogenesis of various inflammatory disorders including tissue damage, septic shock, and rheumatoid arthritis.⁹,¹⁰ Therefore, lipopolysaccharide (LPS)-induced NO production may reflect the degree of inflammation. The inhibition of NO production may demonstrate the ability of a substance to act as possible anti-inflammatory agent, although other mediators needed to be evaluated.¹⁰,¹¹

Macrophages, when sensing pathogen-associated molecular patterns (PAMPs) by their pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), induce the synthesis of endogenous pyrogenic cytokines, among them interleukin 6 (IL-6). IL-6 constitutes an important inflammatory marker secreted by macrophages in response to pathogen invasion during acute inflammation.¹²,¹³ Thus, IL-6 has been considered as an important mediator of fever and the acute phase response.¹⁴,¹⁵

The up-regulation of these inflammatory mediators, produced by the defense, is related to signs and symptoms, such as pain, fever, edema and, sometimes, irreversible tissue damage.¹⁶,¹⁷ Thence, the down-modulation of the inflammatory mediators can be valuable to prevent and control the development of inflammatory diseases.¹,²,¹⁸ In this context, a disordered and excessive production of pro-inflammatory mediators may be related to the pathogenesis of several diseases and, therefore, these mediators are possible targets of therapeutic substances.¹⁹

The inhibition of inflammatory mediators and the immune response regulation, through immunosuppression activity, has been shown as a result of the treatment with ursolic acid (UA).²⁰-²⁴ Among the different mechanisms of action of UA, it is possible to highlight its inhibitory action on the expression of NF-κB.²⁵,²⁶

Taking into consideration the promising activity of UA, the aim of this study was to synthesize ursolic acid derivatives (UAD) and to evaluate the improvement in its biological activity. The chemical modifications in the UA structure, create semisynthetic derivatives that demonstrated enhanced cytotoxic activity against tumor cell lines.²⁷ In a continuation of the work of Scherrer et al.,²⁷ this study evaluated the immunomodulatory effect of the UAD in macrophage response and in carrageenan-induced paw edema model.

Experimental

Reagents

Ursolic acid was purchased from Sigma-Aldrich (Saint Louis, MO, USA). The reagents and the solvents were used directly from the manufacturers. Tetramethylsilane (TMS) and CH₃I (Sigma-Aldrich), acetone, EtOAc, t-BuOOH, n-hexane, CH₂Cl₂, anhydrous diethyl ether, acetic anhydride and tween 20 (Merck, Darmstadt, Germany); K₂CO₃, NaHCO₃, NaClO₂, Na₂SO₄, LiAlH₄, BF₃-Et₂O, NaCl and pyridine (Sigma-Aldrich, Saint Louis, MO, USA) were employed to obtain ursolic acid derivatives. Roswell Park Memorial Institute (RPMI)-1640 (Gibco, Grand Island, USA); RPMI-1640 (Lonza, Basel, Switzerland); bovine serum albumin (BSA), L-glutamine, streptomycin-penicillin, sulfanilamide, N-(1-naphthyl)-ethylenediamine hydrochloride, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT), fetal bovine serum, H₃PO₄, carrageenan, phenylmethylsulfonyl fluoride (PMSF), benzethonium chloride, ethylenediaminetetraacetic acid (EDTA) and aprotinin (Sigma-Aldrich, Saint Louis, MO, USA); and IL 6, TNF and NF-κB (PS529)-PE 558423 (BD, Biosciences Pharmingen, San Diego, CA, USA) were used for the biological assays. All the other materials and solvents were of analytical reagent grade and used as received.

Spectral data

Nuclear magnetic resonance (NMR) spectra were recorded in CDCl₃ on an AC200 (Bruker Corporation, Billerica, USA) at 200 MHz for ¹H and 50 MHz for ¹³C, using TMS as internal reference for both nuclei. For each peak, chemical shift values are expressed in parts per million, followed by multiplicity, relative peak integration (when appropriate) and coupling constants (J) in hertz. High resolution mass spectra (HRMS) were obtained using a QSTAR XL spectrometer (Applied Biosystems, Foster City, USA). The spectra in the infrared (IR) were obtained in Spectrum One apparatus (PerkinElmer, Wellesley, USA) coupled to the diffuse reflectance accessory (ATR). The specific rotational power values [α]_D²⁵ were measured on a 241 polarimeter (PerkinElmer, Wellesley, USA) at 20 ºC. Column chromatography was performed on Silica Gel 60 (230-400 mesh, Merck, Darmstadt, Germany), whereas thin-layer chromatography was carried out on Silica Gel 60 F254 plates (0.25 mm thick, Merck, Darmstadt, Germany). Solvents and reagents were used directly from the manufacturer or purified by standard procedures when required.

Urs-12-ene-3β,28-diol (UAD9)

The ursolic acid (60 mg, 0.13 mmol) was reduced with LiAlH₄ (90 mg, 2.37 mmol) in diethyl ether, at room temperature for 20 h. At the end of this period, the excess LiAlH₄ was consumed with a drop of wet ether and then with a drop of water. The reaction mixture was extracted with saturated NaCl solution; the organic phase was dried over anhydrous Na₂SO₄ and the solvent eliminated in a rotary evaporator to afforded the white solid UAD9 (50 mg, 86.5% yield). FTIR (ATR) ν_max / cm^-1 3642 (O-H), 2921 (C-H), 1045 (C-O); [α]_D²⁵ +71.10 (c 1.00, CHCl₃);¹H NMR (200 MHz, CDCl₃) δ 5.13 (t, 1H, J 3.6, H12), 3.54 (d, 1H, J 10.9 Hz, H28a), 3.20 (d, 1H, J 10.9 Hz, H28b), 3.18 (m, 1H, H3), 1.91 (dd, 1H, J 3.8, 8.8 Hz, H18), 1.10 (s, 3H, CH₃), 1.00 (s, 3H, CH₃), 0.99 (s, 3H, CH₃), 0.95 (s, 3H, CH₃), 0.93 (s, 3H, CH₃), 0.82 (s, 3H, CH₃), 0.79 (s, 3H, CH₃); ¹³C NMR (50 MHz, CDCl₃) δ 138.77 (C13), 125.06 (C12), 79.04 (C3), 69.93 (C28), 55.19 (C5), 54.05 (C18), 47.70 (C9), 42.08 (C14), 40.06 (C8), 39.39 (C20), 39.39 (C19), 38.81 (C1), 38.03 (C4), 36.90 (C10), 36.90 (C17), 35.24 (C22), 32.85 (C7), 30.68 (C21), 28.18 (C23), 27.27 (C15), 26.05 (C2), 26.05 (C27), 23.33 (C11), 23.33 (C16), 21.39 (C30), 18.37 (C6), 17.42 (C29), 16.83 (C26), 15.69 (C25), 15.69 (C24); HRMS m/z, calcd. for C₃₀H₅₀O₂Na: 465.7136, found: 465.3730.

Methyl 3-oxours-12-en-28-oate (UAD10)

Oxidation of the ursolic acid methyl ester UAD1 (500 mg, 1.06 mmol) was carried out with pyridinium dichromate (PDC) (716 mg, 3.3 mmol) in anhydrous CH₂Cl₂ at room temperature under constant stirring for 3 h. The crude reaction product was chromatographed over silica gel (eluent n-hexane/EtOAc 8:2) to afford UAD10 (371.9 mg, 74.7% yield). FTIR (ATR) ν_max / cm^-1 2978 (C H), 1726 (C=O), 1706 (C=O), 1241 (C-O); [α]_D²⁵ +89.20 (c 1.00, CHCl₃); ¹H NMR (200 MHz, CDCl₃) δ 5.26 (t, 1H, J 3.4, H12), 3.60 (s, 3H, COOCH₃), 2.23 (d, 1H, J 11.0 Hz, H18), 2.05 (s, 3H, COOCH₃), 1.07 (s, 3H, CH₃), 1.03 (s, 3H, CH₃), 0.94 (s, 3H, CH₃), 0.94 (d, 3H, J 6.1 Hz, CH₃), 0.85 (d, 3H, J 6.2 Hz, CH₃), 0.78 (s, 3H, CH₃); ¹³C NMR (50 MHz, CDCl₃) δ 217.75 (C3), 178.02 (C28), 138.29 (C13), 125.35 (C12), 55.26 (C5), 52.95 (C18), 51.49 (COOCH₃), 48.14 (C17), 47.41 (C4), 46.78 (C9), 42.15 (C14), 39.47 (C8), 38.47 (C10), 39.32 (C1), 39.06 (C19), 38.88 (C20), 36.64 (C22), 34.21 (C2), 32.52 (C7), 30.68 (C21), 28.04 (C15), 26.57 (C23), 24.22 (C16), 23.48 (C27), 23.48 (C11), 21.53 (C24), 21.20 (C30), 19.62 (C6), 17.09 (C29), 16.90 (C26), 15.21 (C25); HRMS m/z, calcd. for C₃₁H₄₈O₃Na: 491.7082, found: 491.3449.

Methyl 3,11-dioxours-12-en-28-oate (UAD11)

The derivative UAD10 (100 mg, 0.21 mmol) was submitted to oxidation with NaClO₂/t-BuOOH for 12 h. After 12 h of reaction, aqueous solution was added of 10% Na₂SO₃ and extracted with EtOAc. The organic phase was washed successively with saturated aqueous NaHCO₃ solution and water, dried on anhydrous Na₂SO₄ and the solvent was removed in a rotary evaporator to give the white solid UAD11 (91 mg, 88.4% yield). FTIR (ATR) ν_max / cm^-1 2928 (C-H), 1726 (C=O), 1658 (C=O), 1199 (C-O); [α]_D²⁵ +57.10 (c 1.00, CHCl₃); ¹H NMR (200 MHz, CDCl₃) δ 5.61 (s, 1H, H12), 3.60 (s, 3H, COOCH₃), 2.37 (s, 1H, H9), 1.27 (s, 3H, CH₃), 1.25 (s, 3H, CH₃), 1.24 (d, 3H, CH₃), 1.22 (d, 3H, J 6.2 Hz, CH₃), 1.05 (d, 3H, J 7.1 Hz, CH₃), 0.85 (d, 1H, J 6.1 Hz, CH₃); ¹³C NMR (50 MHz, CDCl₃) δ 217.35 (C3), 199.23 (C11), 177.25 (C28), 163.43 (C13), 130.61 (C12), 60.78 (C9), 55.41 (C18), 52.81 (C5), 51.96 (COOCH₃), 47.70 (C17), 47.70 (C4), 44.54 (C14), 43.88 (C8), 39.76 (C1), 38.66 (C19), 38.66 (C20), 36.79 (C10), 35.94 (C22), 34.29 (C2), 32.41 (C7), 30.32 (C21), 28.48 (C15), 26.46 (C23), 23.88 (C16), 23.88 (C6), 21.46 (C27), 21.02 (C30), 21.02 (C24), 18.78 (C26), 15.58 (C25); HRMS m/z, calcd. for C₃₁H₄₆O₄Na: 505.6918, found: 507.3442.

28-Methoxy-11,28-dioxo-3,4-secours-12-ene-3,4-lactone (UAD12)

Derivative UAD11 (50 mg, 0.10 mmol) was dissolved in CH₂Cl₂ (5 mL), following the addition of 77% meta-chloroperoxybenzoic acid (MCPBA) (55.3 mg, 0.25 mmol) and NaHCO₃ (147.2 mg, 1.75 mmol). After 24 h under magnetic stirring at room temperature, an additional quantity of 77% MCPBA (47.3 mg, 0.21 mmol) was added. The reaction mixture was maintained under the same conditions for an additional 16 h. EtOAc was added to the mixture, and it was sequentially washed with saturated aqueous NaHCO₃ and NaCl solutions, dried over anhydrous Na₂SO₄, and evaporated to dryness. The crude product (40.4 mg) was filtered over silica gel (eluent n-hexane/EtOAc 1:1) to afford UAD12 (24.9 mg, 48.2% yield). FTIR (ATR) ν_max / cm^-1 2923 (C-H), 1721 (C=O), 1654 (C=O), 1232 (C-O), 1030 (C O); [α]_D²⁵ +135.00 (c 0.5, CHCl₃); ¹H NMR (200 MHz, CDCl₃) δ 5.65 (s, 1H, H12), 3.61 (s, 3H, COOCH₃), 2.61 (m, 2H, H2a and H2b), 2.43 (d, 1H, J 11.1 Hz, H18), 1.47 (s, 3H, CH₃), 1.44 (s, 3H, CH₃), 1.37 (s, 3H, CH₃), 1.30 (s, 1H, CH₃), 0.98 (s, 1H, CH₃), 0.96 (s, 3H, CH₃), 0.88 (s, 3H, CH₃); ¹³C NMR (50 MHz, CDCl₃) δ 198.53 (C11), 177.21 (C3), 175.67 (C28), 163.21 (C13), 130.79 (C12), 85.66 (C4), 61.11 (C9), 54.45 (C5), 52.73 (C18), 51.96 (COOCH₃), 47.74 (C17), 44.61 (C14), 43.99 (C8), 39.69 (C10), 38.88 (C1), 38.70 (C20), 38.70 (C19), 35.98 (C22), 32.34 (C7), 32.34 (C2), 30.35 (C21), 28.37 (C15), 26.05 (C24), 23.92 (C16), 23.92 (C6), 22.12 (C27), 20.98 (C30), 18.41 (25), 17.49 (C29), 17.12 (C26); HRMS m/z, calcd. for C₃₁H₄₆O₅Na: 521.6912, found: 521.3265.

3β-Acetoxyurs-12-en-28-oic acid (UAD13)

Derivative UAD13 was prepared under standard acetylation conditions using acetic anhydride in pyridine. After 24 h of reaction, ice was added ground and stirred for 15 min. The reaction mixture was extracted with EtOAc and washed with 0.5 N HCl. The organic phase was dried over anhydrous Na₂SO₄ and the solvent eliminated in rotary evaporator being obtained as white crystals (194.8 mg, 89.2% yield). FTIR (ATR) ν_max / cm^-1 3661 (O-H), 2924 (C-H), 1733 (C=O), 1694 (C=O), 1242 (C-O), 1455 (C-O-H), 1369 (C-O); [α]_D²⁵ +54.30 (c 1.00, CHCl₃); ¹H NMR (200 MHz, CDCl₃) δ 5.29 (t, 1H, J 3.6 Hz, H12), 4.49 (m, 1H, H3), 2.17 (d, 1H, J 11.5 Hz, H18), 2.04 (s, 1H, OCOCH₃), 1.08 (s, 3H, CH₃), 0.94 (s, 3H, CH₃), 0.86 (d, 3H, J 6.1 Hz, CH₃), 0.86 (s, 3H, CH₃), 0.83 (s, 3H, CH₃), 0.80 (s, 3H, CH₃); ¹³C NMR (50 MHz, CDCl₃) δ 172.96 (C28), 171.12 (OCOCH₃), 137.42 (C13), 126.24 (C12), 80.96 (C3), 55.35 (C5), 52.70 (C18), 49.35 (C17), 47.52 (C9), 42.22 (C14), 39.65 (C8), 39.14 (C19), 38.77 (C20), 38.40 (C1), 37.74 (C4), 36.90 (C10), 35.83 (C22), 33.04 (C7), 30.50 (C21), 28.11 (C23), 28.11 (C15), 24.25 (C16), 23.59 (C11), 23.59 (C2), 23.37 (C27), 21.39 (COCOCH₃), 21.16 (C30), 18.22 (C6), 17.27 (C29), 17.01 (C26), 16.79 (C25), 15.62 (C24); HRMS m/z, calcd. for C₃₀H₅₀O₂Na: 465.7136, found: 465.3730.

(13S)-3β-Acetoxyurs-11-ene-28,13-lactone (UAD14)

The oxidation of derivative UAD13 (54 mg, 0.10 mmol) with NaClO₂/t-BuOOH for 12 h afforded the white solid 3β-acetoxy-11-oxours-12-en-28-oic acid (50 mg, 90.1% yield). ¹H NMR (200 MHz, CDCl₃) δ 5.56 (s, 1H, H12), 4.48 (dd, 1H, J 5.5, 10.9 Hz, H3), 3.57 (s, 3H, COOCH₃), 2.29 (s, 1H, H9), 2.02 (s, 3H, OCOCH₃), 1.22 (s, 3H, CH₃), 1.13 (s, 3H, CH₃), 0.96 (s, 3H, CH₃), 0.88 (s, 3H, CH₃). Then, the reduction of 3β-acetoxy-11-oxours-12 en-28 oic acid with LiAlH₄ in diethyl ether, at 0 ºC for 1 h, was followed by acetylation with acetic anhydride in pyridine and esterification with CH₃I/K₂CO₃. The final reaction product (43.3 mg) was chromatographed over silica gel (eluents n-hexane/EtOAc 9:1, 8:2 and 6:4) to afford UAD14 (10 mg, 23% yield). FTIR (ATR) ν_max / cm^-1 2923 (C-H), 1755 (C=O), 1728 (C=O), 1238 (C-O); [α]_D²⁵ +41.00 (c 0.5, CHCl₃); ¹H NMR (200 MHz, CDCl₃) δ 5.95 (dd, 1H, J 1.5, 10.4 Hz, H12), 5.54 (dd, 1H, J 3.0, 10.4 Hz, H11), 4.50 (m, 1H, H3), 2.05 (s, 3H, COOCH₃), 1.16 (s, 3H, CH₃), 1.15 (s, 3H, CH₃), 1.05 (s, 3H, CH₃), 1.00 (d, 3H, CH₃), 0.94 (s, 3H, CH₃), 0.94 (s, 3H, CH₃), 0.86 (s, 3H, CH₃); ¹³C NMR (50 MHz, CDCl₃) δ 180.00 (C28), 171.11 (OCOCH₃), 133.36 (C12), 128.95 (C11), 89.70 (C13), 80.66 (C3), 60.59 (C18), 54.86 (C5), 52.96 (C9), 45.13 (C17), 41.97 (C14), 41.75 (C8), 38.70 (C19), 38.15 (C20), 38.00 (C1), 37.89 (C4), 36.31 (C10), 31.35 (C7), 31.20 (C22), 30.87 (C21), 27.78 (C23), 25.58 (C15), 23.37 (C2), 22.86 (C16), 21.39 (COOCH₃), 19.22 (C30), 18.96 (C26), 18.04 (C25), 17.93 (C29), 17.60 (C6), 16.09 (27), 16.09 (C24); HRMS m/z, calcd. for C₃₂H₄₈O₄Na: 519.7186, found: 519.3459.

3-Hydroxy-oxime-urs-12-en-28-oato (UAD15)

Derivative UAD1 (500 mg, 1.06 mmol) was dissolved in CH₂Cl₂ (20 mL), following the addition of PDC (716 mg, 3.3 mmol). The reaction mixture was maintained for 3 h under magnetic stirring at room temperature. The crude product was filtered over silica gel (eluent n-hexane/EtOAc 1:1) to afford methyl 3-oxours-12-en-28-oate (371.9 mg). Then, methyl 3-oxours-12-en-28-oate (100 mg, 0.21 mmol) was dissolved in EtOH (10 mL) following the addition of anhydrous pyridine (0.1 mL) and (NH₃OH)Cl (25 mg, 0.36 mmol). The reaction mixture was maintained for 3 h under magnetic stirring at reflux and 95-100 ºC temperature. The EtOH was evaporated and EtOAc was added to the mixture, than it was sequentially washed with saturated aqueous NaCl solution, dried over anhydrous Na₂SO₄, evaporated to dryness to afford UAD15 (98 mg). FTIR (ATR) ν_max / cm^-1 3660 (O-H), 2945 (C-H), 1721 (C=O), 931 (N-O); [α]_D²⁵ +22.00 (c 1.00, CHCl₃); ¹H NMR (200 MHz, CDCl₃) δ 5.24 (sl, 1H, H12), 3.59 (s, 3H, COOCH₃), 3.06 (m, 1H, H2b), 2.22 (m, 1H, H2a), 2.22 (d, 1H, J 11 Hz, H18), 1.14 (s, 3H, C27H₃), 1.04 (s, 3H, C25H₃), 1.02 (d, 3H, C23H₃), 0.93 (s, 3H, C30H₃), 0.84 (d, 3H, J 6.2 Hz, C29H₃), 0.76 (s, 3H, C26H₃), 0.76 (s, 3H, C24H₃); ¹³C NMR (50 MHz, CDCl₃) δ 178.10 (C28), 166.85 (C3), 138.26 (C13), 125.51 (C12), 55.82 (C18), 52.96 (C5), 51.52 (COOCH₃), 44.14 (C17), 47.11 (C9), 42.11 (C14), 40.35 (C4), 39.58 (C8), 39.06 (C19), 38.88 (C20), 38.66 (C1), 37.04 (C10), 36.64 (C22), 32.74 (C7), 30.68 (C2), 30.68 (C21), 28.00 (C15), 27.34 (C23), 24.25 (C16), 24.25 (C11), 23.33 (C27), 21.20 (C24), 21.20 (C30), 19.07 (C6), 17.01 (C26), 17.01 (C29); HRMS m/z, calcd. for C₃₂H₄₈O₄Na: 506.3610, found: 507.3305.

3β-Hydroxy-11-oxours-12-en-28-oate (UAD16)

Derivative methyl 3-oxours-12-en-28-oate (UAD10, 100 mg, 0.21 mmol) was dissolved in EtOAc (5 mL), following the addition of 0.2 mL of t-BuOOH 6 M at n-decane (1.20 mmol) and NaClO₂ (66 mg; 0.73 mmol). The reaction mixture was maintained for 12 h under magnetic stirring at reflux and 100-110 ºC temperature. After this time, Na₂SO₃ aqueous solution 10% was added and extracted with EtOAc. The organic phase was washed successively with saturated aqueous NaHCO₃ solution and water; dried over anhydrous Na₂SO₄ and the solvent was removed on a rotary evaporator to afford 3,11-dioxours-12-en-28-oate (91 mg). Then, the derivative 3,11-dioxours-12-en-28-oate (UAD11, 50 mg, 0.10 mmol) was dissolved in 5 mL of the mixture of MeOH/THF (1:1) following the addition of CeCl₃.7H₂O (164 mg; 0.44 mmol) and 22 mg of NaBH₄ (0.57 mmol). The reaction mixture was maintained for 6 h under magnetic stirring at room temperature. The crude product was filtered over silica gel (eluent n-hexane/EtOAc 1:1) to afford methyl 3-oxours-12-en-28-oate (371.9 mg). The solvent was evaporated, added Et₂O and it was washed with saturated aqueous NaCl solution, dried over anhydrous Na₂SO₄, evaporated to dryness to afford UAD16 (32.25 mg). FTIR (ATR) ν_max / cm^-1 3675 (O-H), 2970 (C-H), 1725 (C=O), 1659 (C=O); [α]_D²⁵ +108.57 (c 0.7, CHCl₃); ¹H NMR (200 MHz, CDCl₃) δ 5.60 (s, 1H, H12), 3.61 (s, 3H, COOCH₃), 3.22 (m, 1H, H3), 2.80 (m, 1H, H2a), 2.75 (m, 1H, H2b), 2.42 (d, 1H, J 11.2 Hz, H18), 2.30 (s, 1H, H9), 1.12 (s, 3H, C27H₃), 0.99 (s, 3H, C25H₃), 0.96 (d, 3H, J 5.0 Hz, C30H₃), 0.90 (s, 3H, C23H₃), 0.86 (d, 1H, J 6.1 Hz, C29H₃), 0.79 (s, 3H, C26H₃), 0.79 (s, 3H, C24H₃); ¹³C NMR (50 MHz, CDCl₃) δ 200.01 (C11), 177.29 (C28), 162.96 (C13), 130.73 (C12), 78.83 (C3), 61.52 (C9), 55.01 (C18), 52.76 (C5), 51.92 (COOCH₃), 47.70 (C17), 44.72 (C14), 43.77 (C8), 39.17 (C1), 38.66 (C19), 38.66 (C20), 37.19 (C10), 37.19 (C4), 36.01 (C22), 33.07 (C7), 30.35 (C21), 28.44 (C15), 28.15 (C23), 27.34 (C2), 23.99 (C16), 21.05 (C30), 21.05 (C27), 18.89 (C26), 17.49 (C6), 17.16 (C24), 16.28 (C29), 15.65 (C25); HRMS m/z, calcd. from C₃₁H₄₄O₄ [M + Na]: 503.3137, found: 503.3442.

3-Oxo,11-hydroxyurs-12-en-28-oate (UAD17)

To a solution of the derivative 3-oxours-12-en-28 oate (50 mg, 0.11 mmol) in 4.0 mL of CH₂Cl₂ was added 5,10,15,20-tetrakis (pentafluorophenyl) porphyrin (3.5 mg, 3 mol%) and MCPBA 55% (44.32 mg, 0.26 mmol). The reaction mixture was maintained under stirring and inert atmosphere at -78 °C for 30 h. At the end of this period, saturated aqueous solutions of NaHCO₃ and Na₂S₂O₃ were added, followed by extraction with EtOAc. The organic phase was washed with saturated aqueous NaCl solution, dried over anhydrous Na₂SO₄ and the solvent removed on a rotary evaporator, yielding 44.9 mg of a residue. This was chromatographed on a silica gel column (packed column dimensions: 10 × 220 mm) using n-hexane:EtOAc mixtures (9:1; 8:2; 1:1) as eluents. The fractions were pooled according to the profiles at three groups: starting material (the derivative 3-oxours-12-en-28-oate, 17.4 mg) was recovered from the first combined fractions group, while the second combined fractions group provided 13.3 mg (30%) of the derivative 3,11-dioxours-12-en-28-oate, and the third combined fractions group provided 25.1 mg of the UAD17 (56%) mixture of isomers. FTIR (ATR) ν_max / cm^-1 3646 (O-H), 2921 (C-H), 1726 (C=O), 1706 (C=O), 1241 (C-O); [α]_D²⁵ +65.10 (c 1.00, CHCl₃); ¹H NMR (200 MHz, CDCl₃) δ 5.30 (d, 1H, J 3.6 Hz, H12), 4.29 (dd, J 3.6 and 8.8 Hz, 1H, H11), 3.61 (s, 3H, COOCH₃), 2.27 (d, 1H, J 10.4 Hz, H18), 1.10 (s, 3H, C27H₃), 1.06 (s, 3H, C25H₃), 0.96 (d, 3H, J 5.3 Hz, C30H₃), 0.94 (s, 3H, C23H₃), 0.91 (s, 3H, C26H₃), 0.88 (d, 3H, J 6.4 Hz, C29H₃), 0.84 (s, 3H, C24H₃); ¹³C NMR (50 MHz, CDCl₃) δ 217.91 (C3), 178.03 (C28), 139.88 (C13), 129.55 (C12), 68.27 (C11), 55.62 (C9), 52.18 (C5), 51.78 (C18), 51.71 (COOCH₃), 47.74 (C17), 47.37 (C4), 42.89 (C8), 42.89 (C14), 40.68 (C1), 38.73 (C19), 38.73 (C20), 38.26 (C10), 36.42 (C22), 34.54 (C7), 34.43 (C2), 30.57 (C21), 28.07 (C15), 26.94 (C23), 24.14 (C16), 23.44 (C27), 21.31 (C30), 19.69 (C6), 18.78 (C29), 18.22 (C26), 17.16 (C24), 17.01 (C25); HRMS m/z, calcd. for C₃₁H₄₄O₄ [M + Na]: 503.3137, found: 503.3453.

3β,28-Dihydroxyurs-9,12-diene (UAD18)

In a suspension of LiAlH₄ (62 mg, 1.63 mmol) and 2.0 mL of dry ethyl ether was added the derivative 3,11-dioxours-12-en-28-oate (62 mg, 0.13 mmol), solubilized in 4.0 mL of dry ethyl ether. The reaction mixture was kept under stirring at room temperature for 4 h. At the end of this period, the excess LiAlH₄ was removed by dripping with wet ether followed by water trickling. 2 mol L^-1 HCl was added, extracted with EtOAc, and the organic phase was washed with saturated aqueous NaCl solution. The organic phase was dried over anhydrous Na₂SO₄, and after removal of the solvent on a rotary evaporator, 44 mg of a residue was obtained. This was subjected to fractionation on a silica gel column, using the n-hexane:EtOAc (8:2 and 1:1) and EtOAc as eluents. The fractions were pooled according to the profiles and the derivative UAD18 (5.2 mg, 9%) was obtained from the first fractions group. FTIR (ATR) ν_max / cm^-1 3662 (O-H), 2941 (C-H), 1690 (C=C), 1240 (C-O); [α]_D²⁵ +7.80 (c 3.5, CHCl₃); ¹H NMR (200 MHz, CDCl₃) δ 5.58 (d, 1H, J 6.0 Hz, H11), 5.51 (d, 1H, J 6.0 Hz, H12), 3.60 (d1, 3H, J 11 Hz, H28a), 3.24 (d, 1H, J 11 Hz, H28b), 3.22 (m, 1H, H3), 1.21 (s, 3H, C27H₃), 1.16 (s, 3H, C25H₃), 1.03 (s, 3H, C23H₃), 0.94 (d, 3H, J 5 Hz, C30H₃), 0.95 (s, 3H, C26H₃), 0.83 (d, 3H, J 5 Hz, C29H₃), 0.82 (s, 3H, C24H₃); ¹³C NMR (50 MHz, CDCl₃) δ 155.09 (C9), 140.19 (C13), 123.45 (C12), 115.29 (C11), 78.76 (C3), 70.21 (C28), 52.37 (C18), 51.12 (C5), 43.11 (C8), 40.72 (C14), 39.36 (C19), 38.73 (C20), 38.07 (C4), 38.07 (C17), 37.30 (C22), 35.17 (C1), 31.93 (C7), 30.61 (C21), 29.77 (C15), 28.26 (C23), 27.93 (C2), 25.47 (C16), 25.47 (C27), 21.53 (C30), 18.33 (C6), 17.66 (C26), 17.31 (C29), 15.69 (C25), 15.69 (C24); HRMS m/z, calcd. for C₃₀H₄₄O₂ [M + Na]: 459.3239, found: 459.3553.

Cell culture

RAW264.7 macrophage cell lines were placed in 96 well plates, containing RPMI-1640 supplemented (2.0 mM L-glutamine, 100.0 µg mL^-1 of streptomycin and penicillin, 5% fetal bovine serum), at 1 × 10⁶ cells mL^-1 or 2 × 10⁵ cells mL^-1 by 3 or 48 h, respectively. Cells were incubated at 37 °C in a 5% CO₂ atmosphere in the presence or absence of the UA or ursolic acid derivatives (UAD1, UAD2, UAD3, UAD4, UAD5, UAD6, UAD7, UAD8, UAD9, UAD10, UAD11, UAD12, UAD13, UAD14, UAD15, UAD16, UAD17 or UAD18) at 15, 30, 60 or 90 µM. The compounds have been solubilized in DMSO, never exceeding 0.1% (v/v), and diluted in RPMI-1640 before the use. The DMSO concentration was determined to allow the solubilization of the UAD and UA but without affecting the RAW264.7 viability.¹⁸

MTT assay

Cellular viability was measured using the MTT assay. After 48 h of culture, the supernatants were removed and the cells were incubated with 100 µL of supplemented RPMI-1640 and 10 µL of MTT (5.0 mg mL^-1), during 4 h at 37 ºC in a 5% CO₂ atmosphere. After purple formazan crystal formation, the supernatants were gently removed and crystal products were solubilized with DMSO. Complete solubilization was obtained by shaking the plates. The optical density (OD) values were determined in the Multiskan microplate reader (Multiskan™ FC Microplate Photometer, Thermo Scientific, Waltham, MA, USA) at 560 nm wavelength. The cellular viability was presented as the half maximal inhibitory concentration (IC₅₀) calculated using Prism software (GraphPad Prism 5.00, San Diego, CA, USA).²⁸

Nitric oxide concentration in RAW264.7

The NO concentration was measured by the Griess method, in the supernatant of the 48 h of culture. To perform the test, 100 µL of the supernatant from each well was transferred to 96-well plates and an equal volume of Griess reagent (1% sulfanilamide, 0.1% N-(1 naphthyl)-ethylenediamine hydrochloride, 5% H₃PO₄) was added. The NO concentration was determined by comparison with a standard sodium nitrite solution and the values were determined by the Multiskan microplate reader (Multiskan™ FC Microplate Photometer, Thermo Scientific, Waltham, MA, USA) at 540 nm wavelength.

NF-κB concentration in RAW264.7

The cells that were cultured for 3 h were detached after this period and stained for the analysis of the p65 expression (indirectly NF-κB),²⁷ following the instructions of the manufacturer. The cells were acquired in the FACSVerse (BD, Biosciences Pharmingen, San Diego, CA, USA) and analysed in the FCS Express software (De Novo software, Pasadena, CA, USA).²⁹

Induction of acute inflammation by carrageenan-induced paw edema

BALB/c (female) mice 6-8 weeks old (n = 6 per group) were obtained from the animal care facilities of the Federal University of Minas Gerais, Belo Horizonte, MG, Brazil, and maintained in micro isolator cages. All procedures were in accordance with the principles of the Brazilian Code for the Use of Laboratory Animals and were approved by the committee on the use of laboratory animals from UFVJM (Protocol No. 03/2018r). The mice were monitored for clinical signs of toxicity after the treatments. Initially, the mice were weighed (26.57 ± 0.31 g) and their right and left paws were measured (0 h) with a pachymeter (Mitutoyo, Kawasaki, Japan). The treatments, phosphate buffered saline (PBS), dexamethasone (0.5 mg kg^-1), UA (200 mg kg^-1), UAD 1 (200 mg kg^-1) and UAD2 (200 mg kg^-1), were administered intraperitoneally (100 µL) 30 min before the induction of the edema. Dexamethasone was used as a positive control treatment due to its anti-inflammatory activity. Carrageenan (2.5%) was dissolved in PBS, and 20 µL injected into the left footpad, and 20 µL of PBS into the right footpad of all groups. The left and the right paws were measured after 1, 2, 3 and 4 h after the injection of carrageenan and the differences were calculated. The magnitude of the carrageenan-induced paw edema was determined as follows: [paw edema / mm] = [footpad thickness of carrageenan / mm] - [footpad thickness of PBS / mm].³⁰

Cytokine production

Cytokine production was assayed by enzyme-linked immunosorbent assay (ELISA) using commercially available antibodies according to the instructions of the manufacturer (BD Biosciences Pharmingen, San Diego, CA, USA). The plates were read at the Multiskan microplate reader (Multiskan™ FC Microplate Photometer, Thermo Scientific, Waltham, MA, USA) at 450 nm wavelength.¹⁸ Supernatants of the 48 h macrophage culture were used to evaluate the TNF. The TNF production (%) was calculated using the formula (X1/X2) × 100, considering X1 the TNF production (pg mL ¹) of stimulated and treated cells and X2 the mean TNF production (pg mL^-1) of stimulated and untreated cells. The IL-6 productions were measured in the mice paw and lymph nodes, after euthanasia. Paw tissue and lymph nodes were removed and homogenized (100 mg mL^-1) in the extraction solution containing 0.4 M NaCl, 0.05% Tween 20, 0.5% BSA, 0.1 M PMSF, 0.1 M benzethonium chloride, 10 mM EDTA and 20 kIU mL^-1 aprotinin. The homogenate was centrifuged at 2000 ×g for 15 min at 4 °C and supernatants were collected to determine the concentration of IL-6.

Statistical analysis

The results represent at least three independent experiments and are presented as the mean ± standard error of the mean (SEM). All data were analyzed using two-way analysis of variance (ANOVA) followed by Bonferroni posttests (GraphPad Prism 5.00, San Diego, CA, USA),²⁸ and the differences were considered significant at p < 0.05.

Results and Discussion

The ten UA semisynthetic derivative compounds were synthesized (Figures 1 and 2) with previously described structural modifications. The synthesis of derivatives UAD1 to UAD8 was previously published by Scherrer et al.²⁷ The structures were elucidated by NMR spectra recorded in CDCl₃ on a Bruker AC200 at 200 MHz for ¹H and 50 MHz for ¹³C. The obtained spectra of the compounds correspond to the data in the literature.³¹,³² Spectral data of the methyl 3β-hydroxyurs-12-en-28-oate (UAD1), methyl 3β-acetoxy-11-oxours-12-en-28-oate (UAD2), methyl 3β-acetoxy-11-oxours-12-en-28-oate (UAD3) and methyl 3β-acetoxyurs-9(11), 12-dien-28 oate (UAD4), urs-12-ene-3β,11,28-triol (UAD5), (17S)-3β hydroxy-22 (17 → 18)-abeours-11-en-28-al (UAD6), (urs-11, 13(18)-diene-3β, 28-diyl diacetate) (UAD7), (13S)-13, 28-epoxyurs-11-en- 3β-ol (UAD8) were previously published.²⁷

Figure 1
Synthesis of the ursolic acid derivatives. Reaction conditions i: pyridinium dichromate (PDC), room temperature; ii: NaClO₂/t-BuOOH; iii: MCPBA/NaHCO₃, room temperature; iv: LiAlH₄, room temperature; v: acetic anhydride/pyridine, room temperature; vi: NaClO₂/t-BuOOH, 100 °C; vii: LiAlH₄, 0 °C; acetic anhydride/pyridine; CH₃I/K₂CO₃, room temperature.

Figure 2
Synthesis of the ursolic acid derivatives. Reaction conditions i: pyridine/(NH₃OH)Cl, 95-100 °C; ii: EtOAc/t-BuOOH, n-decane/NaClO₂/Na₂SO₃, 100-110 °C (reflux); iii: MeOH/THF, CeCl₃.7H₂O/NaBH₄, room temperature; iv: LiAlH₄/diethyl ether, room temperature; v: 5,10,15,20-tetrakis(pentafluorophenyl) porphyrin/MCPBA, -78 °C.

Biological activity

The cytotoxicity index (IC₅₀) and NO production by RAW264.7 macrophages, treated with UA or the UADs, were shown in the Table 1. The IC₅₀ was obtained by the MTT test of the unstimulated cells treated with UA or UADs and the respective controls corresponding to untreated cells. Besides the viability, the Table 1 shows the results of NO production by RAW264.7 macrophages treated with UA or the UADs, stimulated with LPS and interferon (IFN)-γ after 48 h of culture.

Table 1
NO production by RAW264.7 macrophages treated with UA or UAD, after 48 h of culture and cytotoxicity index (IC₅₀)

Compounda	NO production / µM
	15 µMb	30 µMb	60 µMb	90 µMb	IC50 / µM
UA	9.65 ± 0.51c	3.05 ± 1.16c	1.99 ± 0.15c	2.12 ± 0.83c	17.06
UAD1	3.98 ± 0.44c	4.69 ± 0.07c	2.63 ± 0.37c	1.66 ± 0.07c	9.64
UAD2	8.05 ± 0.31c	8.55 ± 0.47c	6.69 ± 1.10c	4.75 ± 0.61c	> 90
UAD3	9.99 ± 1.85c	9.13 ± 1.03c	7.96 ± 1.33c	6.10 ± 1.76c	> 90
UAD4	14.32 ± 0.63	10.47 ± 0.93c	8.05 ± 1.35c	8.94 ± 0.27c	> 90
UAD5	11.41 ± 1.33	8.39 ± 1.10c	2.98 ± 0.38c	4.56 ± 0.27c	39.89
UAD6	10.72 ± 1.63c	10.75 ± 0.19c	4.08 ± 0.72c	0.93 ± 0.21c	74.62
UAD7	11.51 ± 2.40	8.18 ± 2.22c	7.09 ± 0.99c	5.83 ± 1.53c	> 90
UAD8	10.79 ± 1.03c	9.85 ± 0.60c	3.63 ± 0.53c	3.46 ± 0.70c	47.25
UAD9	10.98 ± 1.75c	10.87 ± 0.67c	5.95 ± 0.30c	5.51 ± 0.95c	> 90
UAD10	12.19 ± 1.11	9.86 ± 0.87c	5.96 ± 0.15c	6.05 ± 2.12c	> 90
UAD11	10.11 ± 2.37	10.28 ± 2.82	9.15 ± 0.08c	9.89 ± 1.26c	> 90
UAD12	21.06 ± 1.02	13.23 ± 1.55	12.77 ± 1.21	12.55 ± 0.54	88.76
UAD13	20.44 ± 3.29	15.32 ± 4.58	13.32 ± 1.35	8.57 ± 0.75c	34.60
UAD14	16.33 ± 1.41	13.77 ± 1.89	10.98 ± 1.61	7.25 ± 1.92c	> 90
UAD15	14.76 ± 0.34	17.34 ± 2.24	10.82 ± 1.70	10.59 ± 2.22	> 90
UAD16	13.59 ± 0.57	10.28 ± 4.06	9.02 ± 0.07c	7.62 ± 1.83c	24.60
UAD17	12.46 ± 2.61	10.45 ± 0.63c	8.91 ± 1.26c	6.01 ± 0.42c	80.05
UAD18	12.46 ± 1.38	10.52 ± 1.35	8.58 ± 1.30c	6.98 ± 0.56c	63.40

• ^a
  Compounds: ursolic acid (UA) and ursolic acid derivative (UAD);
• ^b
  treatment concentration;
• ^c
  p < 0.05 in relation to the nitric oxide (NO) production of the untreated stimulated RAW264.7 cells (14.77 ± 2.52). IC₅₀: half maximal inhibitory concentration.

The compounds UA, UAD1, UAD2, UAD3, UAD6, UAD8 and UAD9 were able to reduce the NO production, compared to the untreated stimulated RAW264.7 cells at all tested concentrations. The compounds UAD4, UAD5, UAD7, UAD10, UAD17 and UAD18 reduced the NO production at 30, 60 and 90 µM. Only the UAD12 and UAD15 were not able to reduce NO production (Table 1). The IC₅₀ was greater than 90 µM when the cells were treated with UAD2, UAD3, UAD4, UAD7, UAD9, UAD10, UAD11, UAD14 and UAD15. The UAD1 was the closest to the IC₅₀ of the UA (Table 1).

The percentage of NF-κB expression by RAW264.7 was shown in Table 2. The UA, UAD1, UAD2, UAD3, UAD4, UAD5, UAD6, UAD7, UAD8, UAD11, UAD12, UAD13, UAD14, UAD15 and UAD18 inhibited the NF κB percentage expression at all concentrations tested. The UA, UAD1, UAD2, UAD3 showed a dose-dependent inhibition (Table 2).

Table 2
Percentage of the NF-κB expression by RAW264.7 macrophages treated with UA and UAD, after 3 h of culture

Compounda	NF-κB expression / %
	15 µMb	30 µMb	60 µMb	90 µMb
UA	13.59 ± 0.29c	7.91 ± 0.11c	8.15 ± 0.31c	6.95 ± 0.20c
UAD1	12.77 ± 0.32c	11.98 ± 0.51c	9.65 ± 0.08c	3.03 ± 0.11c
UAD2	15.11 ± 0.28c	14.78 ± 0.19c	11.27 ± 0.32c	8.45 ± 0.08c
UAD3	15.06 ± 0.15c	14.45 ± 0.09c	10.43 ± 0.22c	8.76 ± 0.20c
UAD4	9.77 ± 0.37c	11.11 ± 0.20c	9.17 ± 0.26c	10.47 ± 0.16c
UAD5	15.42 ± 0.20c	12.22 ± 0.23c	12.75 ± 0.18c	13.96 ± 0.24c
UAD6	12.08 ± 0.12c	12.08 ± 0.06c	13.97 ± 0.16c	13.93 ± 0.27c
UAD7	12.09 ± 0.11c	11.65 ± 0.16c	11.10 ± 0.18c	11.84 ± 0.14c
UAD8	13.64 ± 0.19c	15.12 ± 0.16c	14.96 ± 0.12c	14.54 ± 0.15c
UAD9	16.15 ± 0.48	16.27 ± 0.19	11.75 ± 0.12c	15.52 ± 0.24c
UAD10	14.66 ± 0.23c	17.73 ± 0.29	16.67 ± 0.34c	18.40 ± 0.35c
UAD11	12.71 ± 0.27c	14.81 ± 0.29c	15.26 ± 0.31c	14.04 ± 0.17c
UAD12	9.04 ± 0.55c	13.66 ± 0.54c	11.29 ± 0.86c	11.98 ± 0.11c
UAD13	9.64 ± 0.23c	11.62 ± 0.24c	11.21 ± 0.23c	8.96 ± 0.09c
UAD14	11.07 ± 0.19c	9.968 ± 0.06c	11.79 ± 0.26c	9.37 ± 0.28c
UAD15	10.08 ± 0.17c	9.708 ± 0.19c	10.36 ± 0.18c	9.34 ± 0.19c
UAD16	22.69 ± 0.46	16.29 ± 0.21	15.66 ± 0.08	3.38 ± 0.11c
UAD17	17.20 ± 0.50	15.14 ± 0.23c	15.12 ± 0.29c	13.39 ± 0.10c
UAD18	11.27 ± 0.03c	11.72 ± 0.19c	12.12 ± 0.14c	11.49 ± 0.18c

• ^a
  Compounds: ursolic acid (UA) and ursolic acid derivative (UAD);
• ^b
  treatment concentration;
• ^c
  p < 0.05 NF-κB expression by RAW264.7 macrophages treated with UA or UADs versus RAW264.7 not treated (NF-κB expression control: 17.24 ± 0.41). NF-κB: nuclear factor kappa B.

The percentage of TNF produced by RAW264.7 was inhibited by the UAD7, UAD12, UAD13, UAD14, UAD15 and UAD18 in all concentrations tested. The UAD1, UAD10, UAD11 and UAD16 reduced the TNF production in concentrations above 15 µM. All the compounds reduced the production of TNF at 60 and 90 µM (Table 3).

Table 3
Percentage of the TNF production by RAW264.7 macrophages treated with UA and UADs

Compounda	TNF production / %
	15 µMb	30 µMb	60 µMb	90 µMb
UA	> 100	90.01 ± 8.08c	26.13 ± 5.98 c	23.98 ± 2.09c
UAD1	> 100	74.55 ± 9.52c	21.07 ± 0.33 c	17.98 ± 1.79c
UAD2	> 100	89.05 ± 6.69	31.73 ± 2.18 c	30.41 ± 1.36c
UAD3	> 100	> 100	33.48 ± 2.08 c	33.01 ± 2.72c
UAD4	> 100	91.13 ± 15.32	41.80 ± 2.78c	32.64 ± 4.11c
UAD5	> 100	> 100	48.02 ± 1.28c	32.38 ± 1.60c
UAD6	> 100	> 100	36.66 ± 2.82c	28.24 ± 1.03c
UAD7	67.02 ± 1.72c	37.97 ± 3.85c	15.78 ± 0.81c	14.82 ± 2.14c
UAD8	> 100	79.98 ± 12.35	19.55 ± 0.63c	14.43 ± 0.71c
UAD9	> 100	> 100	19.83 ± 0.63c	14.56 ± 1.83c
UAD10	84.01 ± 6.38	42.33 ± 0.96c	17.58 ± 1.13c	16.26 ± 0.69c
UAD11	77.09 ± 8.78	55.80 ± 3.63c	21.72 ± 1.52c	16.87 ± 0.56c
UAD12	49.87 ± 1.07c	19.48 ± 1.19c	6.50 ± 0.25c	4.71 ± 0.43c
UAD13	51.30 ± 2.07c	24.66 ± 1.48c	9.69 ± 0.55c	12.39 ± 1.03c
UAD14	53.41 ± 2.69c	16.34 ± 0.73c	10.92 ± 1.41c	10.81 ± 0.59c
UAD15	61.05 ± 3.85c	27.68 ± 2.01c	11.50 ± 1.14c	10.46 ± 0.60c
UAD16	93.81 ± 4.97	50.35 ± 1.39c	10.81 ± 0.92c	4.67 ± 0.46c
UAD17	> 100	> 100	74.88 ± 0.55c	40.87 ± 3.34c
UAD18	69.51 ± 1.20c	71.82 ± 4.95c	61.87 ± 4.40c	25.20 ± 2.09c

• ^a
  Compounds: ursolic acid (UA) and ursolic acid derivative (UAD);
• ^b
  treatment concentration;
• ^c
  p < 0.05 percentage of the tumor necrosis factor (TNF) production by RAW264.7 macrophages treated with UA and UADs versus RAW 264.7 not treated (100%).

A study³³ has already shown that NF-κB is a well-known transcription factor in the inflammatory process, responsible for inducing the transcription of several proand anti-inflammatory mediator genes, including TNF. In the present work, the reduction in the expression of NF κB was accompanied by the reduction in the production of TNF, probably due to the incorporated transformations to obtain the UADs, which improved the compounds capacities to act on the NF-κB, partly reported by other authors.²⁰,³⁴ Also, these modifications generated UADs with lower cytotoxicity than UA, except for the UAD1.

The structural modification of the UAD2, consists of the esterification of both C-3 and C-28 carbons,³⁵ which may be the factor that improved its capacity to reduce NO, being the compound that best reduced NO at 90 µM, without altering cell viability. Also, it was able to inhibit the expression of NF κB in a dose response manner and to reduce the production of TNF at concentrations of 60 and 90 µM.

The UAD1 and UAD2 were chosen to be used as a treatment on carrageenan-induced paw edema, for its similar results to UA in inhibiting inflammatory mediators, such as NF-κB and NO, at the lowest concentrations tested. However, it is possible to recognize the potential in others UAD that deserves to be tested in the inflammatory models.

Both, UAD1 and UAD2, showed an effective reduction of paw edema of mice, as well as dexamethasone, compared to carrageenan group, who received only PBS treatment. The reduction in paw edema is already observed in the 2^nd h and persists until the 4^th h (Figure 3).

Figure 3
Carrageenan-induced paw edema. Female BALB/c mice (n = 6 per group) were treated intraperitoneally (i.p.) (100 µL) with PBS (carrageenan group), UAD1 (200 mg kg^-1), UAD2 (200 mg kg^-1), ursolic acid (200 mg kg^-1) or dexamethasone (0.5 mg kg^-1), 30 min before the induction of the model. Paw edema [Paw edema / mm] = [footpad thickness of carrageenan / mm] - [footpad thickness of PBS / mm] were monitored until 4 h. *p < 0.05 UAD1, UAD2, UA and dexamethasone versus carrageenan group.

Regarding the analysis of IL-6 in the supernatant of the paw and lymph node macerates, a reduction of IL-6 was observed in the groups treated with UA and the derivatives UAD1 and UAD2 compared to the carrageenan group (Figures 4 and 5). Different studies³⁶-³⁸ have shown that UA and derivatives are able to reduce the inflammatory marker IL-6. Rapid IL-6 production is also known to contribute to host defense during infection and tissue injury, but excessive IL-6 synthesis and dysregulation of IL-6 receptor signaling are involved in inflammatory and autoimmune disorders. Thus, various therapeutic agents have been evaluated to inhibit the cytokine itself or targets associated with its signaling pathway.³⁹,⁴⁰

Figure 4
IL-6 in paw macerates. Female BALB/c mice (n = 6 per group) were treated i.p. (100 µL) with PBS (carrageenan group), UAD1 (200 mg kg^-1), UAD2 (200 mg kg^-1), ursolic acid (UA 200 mg kg^-1) or dexamethasone (DEXA 0.5 mg kg^-1) and not treated group negative control (NC). After euthanasia, the paws were macerated and the supernatant was collected to dose IL-6 by ELISA. *p < 0.05 UAD1, UAD2, UA, dexamethasone and NC versus carrageenan group.

Figure 5
IL-6 in lymph node. Female BALB/c mice (n = 6 per group) were treated i.p. (100 µL) with PBS (carrageenan group), UAD1 (200 mg kg^-1), UAD2 (200 mg kg^-1), ursolic acid (UA 200 mg kg^-1) or dexamethasone (DEXA 0.5 mg kg^-1) and not treated group negative control (NC). After euthanasia, the lymph nodes were macerated and the supernatant was collected to dose IL-6 by ELISA. *p < 0.05 UAD1, UAD2, UA, dexamethasone and NC versus carrageenan group.

Conclusions

The results obtained demonstrated a variation in the response between the derivatives, due to their chemical modifications, but with potential to reduce the inflammatory mediators evaluated. Regarding paw edema, the UAD1 and UAD2 proved to be as efficient as dexamethasone, an anti-inflammatory used in clinical practice. Furthermore, the investigation of the other derivatives, in in vivo models of inflammation, deserves to be expanded.

Supplementary Information

Supplementary information about the compounds spectrum data (Figures S1-S35) are available free of charge at http://jbcs.sbq.org.br as PDF file.

Acknowledgments

This work was supported in part by Grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (431816/2018-2; 460184/2014 w8, CNPQ-NANO 550321/2012-8, 308278/2020-8 and 437418/2018-9), CT-INFRA 2013/FINEP (FINEP 0633/13); Fundação de Amparo à Pesquisa do Estado de Minas Gerais (APQ 02506-22; APQ 02423-18; APQ03152-18; APQ 03536-16 and APQ-01293-14) and Federal University of Juiz de Fora.

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Edited by

Editor handled this article: Brenno A. D. Neto

Publication Dates

• Publication in this collection
  25 Aug 2023
• Date of issue
  2023

History

• Received
  10 Nov 2022
• Published
  14 Mar 2023