Anti-inflammatory and antioxidative effects of six pentacyclic triterpenes isolated from the Mexican copal resin of Bursera copallifera

Background Bursera copallifera (Burseraceae) releases a resin known as “copal ancho” which has been used, since pre-Colombian times, as ceremonially burned incense and to treat tooth ache, tumors, arthritis, cold, cough, and various inflammatory conditions; however, its anti-inflammatory potential is poorly studied. The aim of the present study was to isolate, quantify, and to investigate the anti-inflammatory activity of triterpene compounds isolated from the copal resin of B. copallifera. Methods The constituents present in the total resin of B. copallifera were obtained by successive chromatographic procedures, and quantitative analysis was performed by High Performance Liquid Chromatography (HPLC). Anti-inflammatory effects of the isolated triterpenes were investigated to determine their inhibitory effects on phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced edema in mice, viability and nitric oxide (NO) production inhibition on lipopolysaccharide (LPS)-activated RAW 264.7 macrophages, and inhibition of cyclooxygenase (COX)-1, COX-2 and secretory Phospholipase A2 (sPLA2) activities in vitro. Results Quantitative phytochemical analysis of the copal resin showed the presence of six pentacyclic triterpenes of which, 3-epilupeol (59.75 % yield) and α-amyrin (21.1 % yield) are the most abundant. Among the isolated triterpenes, 3-epilupeol formiate (Inhibitory Concentration 50 % (IC50) = 0.96 μmol), α.amyrin acetate (IC50 = 1.17 μmol), lupenone (IC50 = 1.05 μmol), and 3-epilupeol (IC50 = 0.83 μmol) showed marked inhibition of the edema induced by TPA in mice. α-amyrin acetate and 3-epilupeol acetate, at 70 μM, also inhibited the activity of COX-2 by 62.85 and 73.28 % respectively, while α-amyrin and 3-epilupeol were the best inhibitors of the production of NO in LPS-activated RAW 264.7 cells with IC50 values of 15.5 and 8.98 μM respectively, and did not affected its viability. All compounds moderately inhibited the activity of PLA2. Conclusions This work supports the folk use of B. copallifera and provides the basis for future investigations about the therapeutic use of this resin in treating inflammation.


Background
Bursera species are the dominant woody taxa in dry forests of México, where this genus reach its maximum diversity and abundance with about 84 species being present, 80 of which are endemic to the country [1][2][3]. These plants release a resin known as copal, derived from the Nahuatl language word "copalli" meaning incense [4]. This genus has been taxonomically related to Commiphora and Boswellia, which also produce resins known as myrrha and frankincense, respectively [5].
The resins obtained from Bursera spp. play an important role in the economy of rural families in México, and they are particularly identified with the aromatic resins used by the cultures of pre-Columbian Mesoamerica as ceremonially burned incense and other purposes. Copal is still used by a number of peoples of México and Central America as incense and during sweat lodge ceremonies, and the trees where the resins are obtained are today cultivated in many regions of México [4,6]. Copal, as a traditional natural medicine, has been used to treat various diseases, such as tooth ache, tumors, fever, and inflammatory conditions. Tea made with the resin is a traditional remedy as analgesic and has been used to clean wounds and sores, and to cure bronchitis, cough and rheumatism since pre-Columbian time, and it is still used [7][8][9].
Among various resins collected by local people of Morelos state of México, "copal ancho" (Bursera copallifera, DC, Bullock) is considered as a source of high grade copal resin, and it is commonly used against rheumatoid arthritis, cold, cough, for stroke and dental pain, and for hasten wound healing [10,11].
Previous studies have demonstrated the cytotoxic activity of the chloroform extracts obtained from the stems and fruits of B. copallifera [12]. More recently, our research team showed that the hydroalcoholic extract of the stems as well as the dichloromethane: methanol extract from the leaves inhibited the mouse ear inflammation in response to topical application of TPA by 54.3 and 55.4 % respectively, at the dose of 0.5 mg/ear [13]. Further, in this work, the mechanism for this anti-inflammatory effect was related to the direct inhibition of COX-1 and moderate of COX-2, which are associated with inflammatory diseases. However, the anti-inflammatory potential of the resin and its constituents are still unknown.
The ethnomedicinal importance of B. copallifera and its components, prompted us to undertake detailed investigation on the constituents of the resin and their anti-inflammatory activity in order to evaluate its antiinflammatory potential and compare with those described for the other parts of the plant. Although the TPA-induced mouse ear model of inflammation is nonspecific, it is widely used for acute anti-inflammatory screening because TPA activates PLA 2 , [14] and the resulting edema is primarily mediated by prostaglandin E2 (PGE2) [15]. Thus, both PLA 2 and COX are involved in this model, and it has been demonstrated that the organic extracts of B. copallifera interfere with these enzymes to inhibit TPA-induced inflammation.
In this paper, we report the isolation and identification of six triterpenes (1-6) with anti-inflammatory activity, isolated from the n-hexane soluble fraction of the resin of B. copallifera. In addition, quantification of the active components in the resin was also performed by HPLC analysis. The anti-inflammatory activity of the six isolated compounds was evaluated in the mice model of TPA-induced edema. Furthermore, we examined the inhibitory effects of these triterpenes on cell viability and NO production in LPS-stimulated RAW 264.7 macrophages. Also, their inhibitory effects over COX-1, COX-2, and sPLA 2 activities in vitro were assayed.

Plant material
The resin of B. copallifera (DC.) Bullock was collected in August 2011 at El limón de Cuahuchichinola (N 18°31′ 16.5"), in the Reserva de la Biósfera Sierra de Huautla (REBIOSH) by M. C. Teresita Rodríguez López. Voucher specimen No. 31809 was deposited at the Herbarium of the University of Morelos (HUMO) in the Centro de Investigación en Biodiversidad y Conservación (CIByC) at the Universidad Autónoma del Estado de Morelos (UAEM).

Quantitative analysis by HPLC
Resin dried and pulverized (3 mg) and standards (3 mg) were prepared by sonicating (10 min) in methanol (1 mL) and analyzed by HPLC. All samples were filtered through 0.45 μm syringe filter and injected into HPLC. Chromatography was carried out on a Waters 600E gradient module HPLC system, Waters 717 plus Autosampler, waters 996 photodiode array detector and computer with EmpowerPro of waters. The column used was a reversed phase Chromolith SpeedROD RP-18e (50 mm × 4.6 mm), from Merck. The separation was carried out isocratically using Acetonitrile:Water (95:05) as the mobile phase (40 min). The system was operated at room temperature and monitored at 210 nm. The flow rate was 0.5 mL/min and the injection volume was 20 μL. The standard samples of lup-20(29)-en-3α-ol formiate (1), ursan-3β-ol acetate (2), lup-20(29)-en-3α-ol acetate (3), lup-20(29)-en-3-one (4), lup-20(29)-en-3α-ol (5) and α-amyrin (6) compounds were isolated from the resin of B. copallifera and characterized in our laboratory. The purity (>98) of the isolated compounds was confirmed by HPLC and 1 H NMR analysis. Quantification was performed by comparing their retention times with the standards and calculating the concentration from the respective calibration curves. The assay was performed in triplicate.

In vivo anti-inflammatory activity TPA-induced mouse ear edema
Mouse ear edema was evaluated following the described protocol [16]. All experiments were carried out using six animals per treatment. Adult male CD-1 mice with a body weight ranging from 25 to 30 g were used. Experiments were performed according to the Official Mexican Rule: NOM-062-ZOO-1999 Guidelines (Technical Specifications for the Production, Care, and Use of Laboratory Animals) and international ethical guidelines for the care and use of experimental animals. The experimental protocol followed was approved by Comité de Experimentación del Bioterio of the Universidad Autónoma del Estado de Morelos (BIO-UAEM) (Approval number: BIO-UAEM: 009:2013). Mice were maintained under standard laboratory conditions (Bioterio at the Universidad Autónoma del Estado de Morelos) at 22°C ± 3°C, 70 % ± 5 % of humidity, 12 h light/dark cycle and food/water ad libitum. A negative control group received acetone as vehicle and indomethacin was used as anti-inflammatory drug as positive control group. Finally, compounds were tested by separate treatment groups. Animal ear inflammation was induced with 2.5 μg of TPA dissolved in 20 μL of acetone applied to the internal and external surface of the right ear to cause edema. Sample doses of 1, 0.75, 0.50, 0.25 and 0.125 mg/ear of the compounds, as well as the anti-inflammatory drug of reference (indomethacin) were applied. All the samples of the different treatments were dissolved in (20 μL of acetone or ethanol) depending on the solubility of the specified compound and applied topically on the right ear immediately after TPA application; on the left ear acetone or ethanol was applied as vehicle. Four hours after application of the samples of interest as possible anti-inflammatory agents, the animals of each treatment were sacrificed by cervical dislocation. Circular sections of 6 mm in diameter were taken from both: the treated (t) and the non-treated (nt) ears, which were weighed to determine the inflammation. Percentage of inhibition was determined by the formula expressed below: where Δw = w t − w nt ; being w t the weight of the section of the treated ear and w nt the weight of the section of the non-treated ear. The IC 50 values of the antiinflammatory activity obtained at the doses of 1, 0.75, 0.50, 0.25 and 0.125 mg/ear were calculated using GraphPad Prism® software by lineal regression analysis.

In vitro anti-inflammatory activities Cell culture
Murine macrophage cell line RAW 264.7 (Tib-71tm from ATCC) were grown in DMEM/F12 medium supplemented with 7.5 % heat-inactivated FBS, GlutaMax, without antibiotics. Cells were plated and incubated in a humidified atmosphere containing 5 % CO 2 at 37°C. Cells were sub-cultured by scraping and seeding them in 75 cm 2 flasks or 24-wells plates.

Treatment of macrophages with LPS
RAW 264.7 cells (1.4 × 10 5 cells/well) were plated and incubated into 24-well plates in 0.5 mL of DMEM/F12 medium supplemented with 7.5 % heat-inactivated FBS, for 24 h, at 37°C with 5 % CO 2 . After that, macrophages were incubated for two hours with the test compounds (2-6) at various concentrations (0-70 μM) or vehicle (Dimethyl sulfoxide (DMSO), 0.5 %, v/v) or indomethacin (30 μg/mL). Then, macrophages were incubated with LPS (10 μg/mL) in the presence or absence of test compounds, indomethacin or vehicle and without LPS at 37°C for 20 h to stimulate NO production. Finally, cell-free supernatants were collected and were kept at -20°C until NO quantification. The suppressive effect of compounds 2-6 on NO production was assessed using the Griess reagent

Determination of NO concentration
Nitrite, the stable end product of NO, was used as an indicator of NO production in the culture medium. Nitrite released in the culture medium was measured according to Griess reaction. Briefly, 50 μL of each cell culture supernatants were mixed with 100 μL of Griess reagent (50 μL of 1% sulfanilamide and 50 μL of 0.1% N-(1-naphtyl) ethylenediamine dihydrochloride in 2.5 % phosphoric acid), for 10 min at room temperature. The optical density at 540 nm (OD 540 ) was measured with a microplate reader and nitrite concentration in the samples were calculated by comparison with the OD 540 of a standard curve of NaNO 2 prepared in fresh culture medium [17].

MTS-tetrazolium salt assay
Cell viability was measured based on the formation of blue formazan metabolized from colorless MTS by mitochondrial dehydrogenases, which are active only in live cells. RAW 264.7 macrophages were plated in 96-well plates at a density of 1.2 × 10 4 cells per well for 24 h. The cells were treated and incubated with various concentrations of test compounds (0-70 μM) for 24 h. Cell viability was determined by MTS assay, using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation assay (Promega). Briefly, 20 μl of MTS was added to each well, and the cells were incubated for another 4 h at 37°C with 5 % CO 2 . The optical density was measured at 490 nm on a microplate reader.

Statistical analysis
The results shown were obtained at least by three independent experiments and are presented as means ± SDs. Statistical analyses were performed by one-way analysis of variance (ANOVA) with Tukey's post hoc test. All statistical analyses were performed using the OriginLab (Massachustts USA), version 8.0 software. P values 0.05 were considered to indicate statistical significance.

In vivo anti-inflammatory activity of the resin
The resin of B. copallifera was dissolved with a mixture of dichloromethane:acetone (8:2) at room temperature, this extract showed inhibition on TPA-induced auricular edema in mice (50% inhibitory dose (ID 50 ) = 0.7071 mg/ear).

In vivo anti-inflammatory activity of triterpenes
The isolated triterpenes 1-6 were evaluated at different concentrations in TPA-induced auricular edema in mouse model. Table 1 illustrates the anti-inflammatory activity displayed by these compounds. Except for compounds 3 and 6, all the triterpenes showed marked anti-inflammatory activity with ID 50 values ranging from 0.83 to 1.17 μmol.

In vitro anti-inflammatory activity of triterpenes
The effects of triterpenes 1-6 on the viability of the RAW 264.7 cells was determinated at different concentrations (4.37, 8.75, 17.5, 35.0, and 70.0 μM) (Fig. 2), and all the triterpenes did not exhibit a significant reduction in viability of macrophages compared con the positive control, up to the concentration of 70 μM.
To assess the effect of the triterpenes (1-6) isolated from B. copallifera resin on production of NO in LPSinduced RAW 264.7 cells, cells were treated with/without natural products (4.37, 8.75, 17.5, 35.0, and 70.0 μM) for 2 h and then stimulated with LPS (10 μg/ml) for 24 h. The amount of nitrite, a stable metabolite of NO, was used as the indicator of NO production in the medium. Among the isolated triterpenes, it has been described that lupenone (4) reduced NO production with an IC 50 value of 10.81 μM, in LPS-stimulated RAW 264.7 cells [25], and was included as the positive control. The experimental results showed that NO level was increased in LPS-stimulated RAW cells, and this effect was decreased significantly by treatment with compounds 1-6 (P < 0.001) (Fig. 3). The IC 50 values are gathered in Table 2 and it can be seen that α-amyrin (6) was the most active compound with IC 50 value of 8.98 μM, while 3-epilupeol formiate (1) was the less active one with IC 50 value of 43.31 μM.

Discussion
The resin of B. copallifera, known as "copal ancho", has been used to treat various inflammatory diseases  [10,11,13]. Despite the importance of this plant species, there is little knowledge about the anti-inflammatory activity and the potential anti-inflammatory components.
Except for compounds 3 and 6, all the triterpenes showed marked anti-inflammatory activity when tested in the TPA-induced ear edema in mice. Triterpenes with  the lupane skeleton showed the best activity, being 3-epilupeol (5, ID 50 = 0.83 μmol/ear), together with its 3-formyl ester (1, ID 50 = 0.96 μmol/ear) the most active compounds. 3-epilupeol acetate (3) (ID 50 = > 2.13 μmol), and α-amyrin (6) (ID 50 = > 2.34 μmol) were the less active. In contrast, α-amyrin acetate (2) was active with ID 50 value of 1.17 μmol/ear. A survey of the literature, about the anti-inflammatory properties of the isolated compounds, revealed that compounds 2, 4, 5 and 6 were previously evaluated in the TPA-induced edema in mice [37,[41][42][43][44]. The results obtained in this work matched well with those described, except for α-amyrin (6) probably because its poor solubility. Further, we evaluated the effects of these natural triterpenes on the production of NO in RAW 264.7 macrophages. For comparison, the activity of lupenone (4) was included as positive control. The cytotoxicities of compounds in RAW 264.7 cells were also assessed using MTS assay [45]. In all cases, the natural triterpenes exhibited potent NO production inhibitory activities, and did not affect the cell viabilities in either the presence or absence of LPS, even at a concentration of 70 μM, indicating no significant effect of exposure of the cells to LPS at the concentrations used (Fig. 2). The results indicate that all compounds are effective inhibitors of LPSinduced NO production in these cells. Indeed, as is shown in Fig. 3, the production of NO was markedly elevated in response of LPS. However, the application of the triterpenes 1-6 inhibited the production of NO by LPS in a concentration-dependent manner, and as shown in Table 2 (4) inhibited NO production at 10.81 μM in the previous report [25], the IC 50 value obtained in our assay (20.8 μM) was of equivalent order of magnitude.
Accumulating evidence has indicated that NO is well known for its involvement in the development of inflammation [49,50]. NO is an important intra-and intercellular signaling molecule in cardiovascular, nervous, and immunological systems. NO is involved in various biological reactions including vasorelaxation, inhibition of platelet aggregation, neurotransmission, inflammation, and immunoregulation [51,52]. Therefore, identifying new agents capable of lowering the production of this proinflammatory agent is regarded as an essential requirement for the alleviation of a number of inflammation-related disorders attributed to macrophage activation [53]. Similarly, COX and PLA 2 are key enzymes in the synthesis of inflammatory prostaglandins which contributes to pathogenesis of various inflammatory diseases, edema, angiogenesis, invasion, and growth of tumor. COX-1 is a constitutively expressed enzyme with general housekeeping functions. COX-2 is an inducible enzyme that catalyzes biosynthesis of PGE2 [54,55]. PLA 2 catalyze the hydrolysis of the phospholipid sn-2 ester bond, generating a free fatty acid and a lysophospholipid. The PLA 2 reaction is the primary pathway through which arachidonic acid (AA) is liberated from phospholipids. Free AA is the precursor of the eicosanoids, which include the prostaglandins, generated through the COX reaction, and the leukotrienes, generated through the lipoxygenase reaction [56].

Conclusions
In conclusion the total resin of B. copallifera possess significant and promising anti-inflammatory activity. In this study, we showed that in LPS-stimulated macrophages, the isolated compounds 1-6 dose-dependently inhibited NO and triterpenes α-amyrin acetate (2) and 3-epilupeol acetate (3) inhibited the activity of COX-2, while all of them showed moderate inhibitory activity of PLA 2 enzyme, suggesting that this was the mechanism underlying the observed anti-inflammatory activity observed in vivo.
The study also signifies that isolated constituents could be responsible, at least in part, for its anti-inflammatory activity. The study verifies traditional use of B. copallifera for the treatment of rheumatism, asthma, and other inflammatory disorders.

Funding
The authors declare that they have received funding by Consejo Nacional de Ciencia y Tecnología (CONACyT).

Availability of data and materials
All data and materials are contained and described within the manuscript, except the spectroscopic data of isolated compounds, which are available at request.
Authors' contributions MLG-R conducted the animal experiments and analyzed the data. AR-E and VR-L performed phytochemical and HPLC analyses of the resin, and participated in the correction of the manuscript. JG-C and AM-M conducted the in vitro studies and analyzed data. LA and SM participated in design of the study and preparation of the manuscript. All the authors read and approved the final manuscript.

Competing interests
The authors declare that there is not conflict of interests regarding the publication of this paper.

Consent for publication
Not applicable.