Analytical-grade petroleum ether, diethyl ether, n-hexane, ethyl acetate and dichloromethane were used in this work. Silica gel 0, 06-0,2 mm (70-230 Mesh, Merck) were used for column chromatography and TLC was performed on 0,2 mm-thick Kiesegel 60 F254 layers (Merck). IR spectra were recorded in a WQF-SLO FT-IR (Rayleigh) spectrophotometer in KBr pellets. 1D ¹H, ¹³C, 2D gCOSY, gHSQC, y gHMBC NMR spectroscopy were performed in a Varian Inova spectrometer to 300 y 400 MHz. The spectra were obtained using chloroform (CDCl₃) as solvent.

Nature of carbon atoms was assessed by DEPT spectrum edition technique with proton pulse at 135⁰. Chemical shifts (δ) were expressed in ppm while coupling constants (J) in Hz. δ values were referred to tetramethylsilane as internal reference.

The bark of M. elaeodendroides. was recollected in the zone of “La Coca” dam, Campo Florido, Havana, Cuba, and identified by botanic Dr. Pedro Herrera Oliver from the Ecology and Systematic Institute (Havana, Cuba), where a voucher was deposited at the Herbarium under number HAC41417.

The bark of M. elaeodendroides was dried under air circulation (40 ⁰C). Dried-bark powder (950 g) was macerated with petroleum ether/diethyl ether (1:1) at room temperature for nine days. The product of maceration was filtered and concentrated under reduced pressure, yielding 10 g (1,05 %) of the crude of petroleum ether/diethyl ether (1:1). This crude was chromatographed on a silica gel column, using increasing-polarity mixtures of n-hexane/ethyl acetate as eluent to obtain 5 fractions. Fractions F₁ and F₂ were rich in 3β-friedelinol and betulin, respectively. [15]Fraction F₃ was subjected to preparative HPTLC developed with n-hexane/CH₂Cl₂/Et₂O (2.5:1.5:1.5) resulting in compounds 1 (40 mg) and 2 (32 mg). Compounds 3 (35 mg) and 4 (57 mg) were purified by flash chromatography on silica gel of the fractions F₄ (petroleum ether/CH₂Cl₂/Et₂O, 3.0:3.0:1.0) and F₅ (n-hexane/Et₂O, 6.0:4.0), respectively.

The extract obtained was screened for their topical anti-inflammatory activity at doses of 200 mg/kg; 400 mg/kg and 800 mg/kg. It was used to the compounds 2 and 3 doses of 1, 5 y 10 mg/kg. Croton oil and products under test as well as diclofenac (reference drug) were dissolved in acetone. Diclofenac was used at a dose of 20 mg/kg (≈ 0,5 mg/right ear).

Swiss mice (25-30 g), provided by CENPALAB (National Center for Laboratory Animal Production, Cuba), were kept for a week, before the experiment, at constant conditions of temperature (21±1 ⁰C) and humidity (60-70 %), and in light/dark cycle. Water and food were supplied ad libitum, before the experiment. Each experiment group consisted of five animals.

Edema was only induced on the right ear of each mouse. Croton oil (0.125μg/μL) was applied topically to both inner (10 μL) and outer (10 μL) surface of the right ear (total volume administration of 20 μL) simultaneously with the products under study and diclofenac.

Four hours after administration mice were sacrificed by cervical rupture. One circular portion (6 mm diameter) of each ear was cut and weighted in analytical balance. Since in all cases left ear only received vehicle (acetone, 20 μL), subtracting left ear portion weight from right ear portion weight indicated the extent of edema in each mouse. The extent of inflammation was measured as edema weight through arithmetic mean and standard error calculated for each animal group.

Anti-inflammatory activity was assessed by percentage inhibition of inflammation with regard to negative control group. A 100 value was assigned to the group receiving only Croton oil, and the reduction attributed to each of the treated groups was expressed as %. The data was accompanied by the standard error.

ANOVA method, followed by multiple comparisons (Student-Newman- Keuls), was used to analyze the results. The statistical significance considered was p < 0,05.

Table 1 reports the edema weight average and standard desviation for each experimental group. Inflammation seen in the negative control group was significantly higher than that seen for the different doses of crude (p< 0,05). The highest dose (800 mg/kg) showed higher inflammation values and had statistical difference with regard to doses of 200 and 400 mg/kg.

Table 1

Groups	Doses (mg/kg)	Extent of inflammation [edema weight average (mg) ± SD]
Control (Croton oil)	-	118,4±11,78
Croton oil + diclofenac	20	14,8±2,17a*
Croton oil + petroleum ether/ethyl ether (1:1) crude	200	19,6±1,95a*
Croton oil + petroleum ether/ethyl ether (1:1) crude	400	17,0±7,21a*
Croton oil + petroleum ether/ethyl ether (1:1) crude	800	38,0±5,83b*

Different letters represent significant differences (p < 0,05)

Figure 1 shows the results of inflammation inhibition % by three doses of petroleum ether/diethyl ether (1:1) extract tested.

No significant differences were obtained among the inhibition exhibited by diclofenac (87,5%) and doses of 200 (83,4%) and 400 mg/kg (85,6%). However, there were significant differences (p <0,05) between these groups (diclofenac and doses of 200 and 400 mg/kg) and the one which received a dose of 800 mg/ kg.

It is not clear why the dose of 800 mg/kg of the crude exhibited a lower anti-inflammatory activity compared to doses of 200 and 400 mg/kg despite that a dose of 800 mg/kg still showed an anti-inflammatory response.

Fig. 1

Spengler et al., 2002, reported the isolation of triterpene betulin ¹⁶, which anti-inflammatory activity was reported in the literature by Reyes et al., 2006 using the method of production of nitric oxide and prostaglandins (PGE₂) by stimulation of macrophages.¹⁷

The fractions F1 and F2 rich in 3β-friedelinol, cafeate of betulin and betulin were studied by Spengler et al., 2002.¹⁶ Compounds 1 and 2 were isolated from the fraction F3, whereas compounds 3 and 4 were isolated from the fractions F4 and F5, respectively.

The structures of compound 1-4 were elucidated by IR, mono and bi-dimensional NMR spectroscopy, as in earlier reports. ^(15,16

1:IR (KBr) (ν, cm^-1): 3443; 2939; 2871; 1715; 1631; 1464; 1381; 1044; 844.¹ H NMR (d-CHCl₃), 500 MHz: 4,61(1H,d, J=2,04 Hz, H-29b), 4,51 (1H,m, H-29a), 3,72 (1H,dd, J=10,75 Hz, H-28b),3,27 (1H,d, J=10,75 Hz, H-28a), 2,49 (1H, m, H-2 b), 2,40 (1H, m, H-2 a), 1,90 (1H, m, H-1 b),1,70 (1H, brd, J= 15,0 Hz, H-12 b), 1,64 (1H, m, H-13), 1,61 (3H, s, H-30),1,57 (1H, t, J= 12,5 Hz, H-15 b), 1,48 (2H, m, H-6), 1,43 (2H, m, H-7), 1,42 (1H, m, H-11 b), 1,38 (1H, m, H-1 a), 1,36 (1H, m, H-9), 1,30 (1H, m, H-5), 1,30 (1H, m, H-15 a),1,26 (1H, m, H-11 a), 1,04 (1H, m, H-12 a),1,02 (3H, s, H-23), 0,99 (3H, s, H-26), 0,95 (3H, s, H-24),0,91 (3H, s, H-27), 0,85 (3H, s, H-25).¹³C NMR (d-CHCl₃), 500 MHz: 218,0 (C-3),150,0 (C-20), 109,7 (C-29), 60,5 (C-28), 54,8 (C-5), 49,7 (C-9), 48,6(C-18), 47,7 (C-17), 47,3 (C-4, C-19), 42,8 (C-14), 40,9 (C-8), 39,8 (C-1), 37,4 (C-13), 36,8 (C-10), 34,0 (C-2), 33,9 (C-22), 33,5 (C-7), 29,7 (C-21), 29,1 (C-16), 27,0 (C-15), 26,6 (C-23), 25,2 (C-12), 21,3 (C-11), 21,0 (C-24), 19,6 (), 19,0, 15,9 (C-26), 15,8 (C-25), 14,7 (C-27).

2:IR (KBr) (ν, cm^-1): 3354; 2939; 2871; 1705; 1641; 1458; 1383; 1021; 884.¹ H NMR (d-CHCl₃), 500 MHz: 4,68 (1H, brs, H-29 b), 4,58 (1H, brs, H-29 a), 3,60 (1H, dd, J= 11,2, 4,4 Hz, H-16), 2,50 (1H, td, J=11,4, 5,4 Hz H-19), 2,49 (1H, ddd, J= 16,0, 9,5, 7,5 Hz, H-2 b), 2,40 (1H, ddd, J= 16,0, 8,5, 4,5 Hz, H-2 a), 1,98 (1H, m, H-21 b), 1,90 (1H, ddd, J= 13,0, 7,5, 4,5 Hz, H-1 b), 1,70 (1H, brd, J= 15.0 Hz, H-12 b), 1,68 (3H, brs, H-30), 1,65 (1H, m, H-22 b), 1,64 (1H, m, H-13), 1,57 (1H, t, J= 12,5 Hz, H-15 b), 1,48 (2H, m, H-6), 1,43 (2H, m, H-7), 1,42 (1H, m, H-11 b), 1,40 (1H, m, H-18), 1,40 (1H, m, H-21 a), 1,38 (1H, m, H-1 a), 1,36 (1H, m, H-9), 1,30 (1H, m, H-5), 1,30 (1H, m, H-15 a), 1,28 (1H, m, H-22 a); 1,26 (1H, m, H-11 b), 1,06 (6H, s, H-23 y H-26), 1,04 (1H, m, H-12 a), 1,02 (3H, s, H-24), 1,01 (3H, s, H-27), 0,93 (3H, s, H-25), 0,81 (3H, s, H-28). ¹³C NMR (d-CHCl₃), 500 MHz: 218,0 (C-3), 149,9 (C-20), 109,8 (C-29), 76,9 (C-16), 54,9 (C-5), 49,3 (C-9), 48,6 (C-17), 47,6 (C-18), 47,5 (C-19), 47,3 (C-4), 44,1 (C-14), 40,8 (C-8), 39,6 (C-1), 37,7 (C-22), 37,4 (C-13), 36,8 (C-10), 36,8 (C-15), 34,1 (C-2), 33,5 (C-7), 29,8 (C-21), 26,6 (C-23), 24,8 (C-12), 21,4 (C-11), 21,0 (C-24), 19,6 (C-6), 19,3 (C-30), 16,1 (C-27), 16,0 (C-25), 15,8 (C-26), 11,7 (C-28).

3:IR (KBr) (ν, cm^-1): 3400; 2940; 2870; 1690; 1638; 1458; 1383; 1021; 873.¹ H NMR (d-CHCl₃), 500 MHz: 4.64 (1H, m, H-29b), 4,53 (1H, dd, J= 2,29, 1,44 Hz, H-29a), 3.53 (1H, dd, J= 11,1, 4,9 Hz, H-16), 3,16 (1H, dd, J= 11,5, 4,9 Hz, H-3), 2,49 (1H, m, H-19), 1,67 (1H, m, H-1b), 1,62 (3H, brs, H-30), 1,60 (1H, m, H-2b), 1,56 (1H, m, H-2a), 1,51 (1H, m, H-6b), 1,41 (1H, m, H-11b), 1,39 (1H, m, H-6a), 1,39 (2H, brs, H-7), 1,27 (1H, m, H-9), 1,23 (1H, m, H-11a), 1,19 (2H, m, H-22), 0,98 (3H, s, H-26 ), 0,93 (3H, s, H-27 ),0,91 (3H, s, H-23 ), 0,90 (1H, m, H-1a), 0,76 (3H, s, H-25 ), 0,73 (3H, s, H-24 ), 0,70 (3H, s, H-28), 0,60 (1H, d, J= 10,69 Hz, H-5). ¹³C NMR(d- CHCl₃), 500 MHz: 150 (C-20), 109,6 (C-29), 78,8 (C-3), 76,9 (C-16), 55,6 (C-5), 49,4 (C-9), 48,6 (C-17), 47,6 (C-18), 47,4 (C-19), 44,1 (C-14), 40,8 (C-8), 38,8 (C-4), 38,7 (C-1), 37,7 (C-22), 37,4 (C-13), 36,8 (C-10), 36,8 (C-15), 34,1 (C-7), 29,8 (C-21), 28,0 (C-23), 27,3 (C -2), 24,8 (C-12), 21,4 (C-11), 19,0 (C-30), 18,2 (C-6), 16,0 (C-25, C-26, C-27), 15,0 (C-24), 11,6 (C-28).

4:IR (KBr) (ν, cm^-1): 3400; 2940; 2870; 1686; 1638; 1458; 1383; 1021; 873.¹H NMR (d-CHCl3), 500 MHz: 4,74 (1H, brs, H-29b), 4,62 (1H, brs, H-29a), 2,50 (1H, td, J=11,4, 5,4 Hz H-19), 2,49 (1H, ddd, J= 16,0, 9,5,7,5Hz, H-2 b), 2,40 (1H, ddd, J=16,0, 8,5, 4,5 Hz, H-2a), 1,98 (1H, m, H-21b), 1,90 (1H, ddd, J= 13,0, 7,5, 4,5 Hz, H-1b), 1,70 (1H, brd, J= 15,0 Hz, H-12b), 1.70 (3H, brs,H-30), 1.64 (1H, m, H-13), 1.48 (2H, m, H-6), 1.43 (2H, m, H-7), 1.42(1H,m, H-11b), 1,38 (1H, m, H-1a), 1,36 (1H, m, H-9), 1,30 (1H, m, H-5),1,26 (1H, m, H-11a), 1,07 (3H, s, H-23), 1,04 (1H, m, H-12a), 1,02(3H, s, H-24), 0,99 (3H, s, H-26), 0,98 (3H, s, H-27), 0,93 (3H, s,H-25).¹³C NMR (d-CHCl3), 500 MHz: 218,3 (C-3), 181,2 (C-28), 150,5 (C-20), 109,9(C-29), 56,5(C-17), 55,1 (C-5), 50,0 (C-9), 49,3 (C-18),47,5 (C-19),47,0 (C-4),42,6 (C-14),40,8 (C-8),39,8 (C-1),38,6 (C-13), 37,2 (C-7), 37,1(C-10), 34,3 (C-2), 33,8 (C-22),32,2 (C-16),30,7 (C-21),29,8 (C-15),26,8(C-23),25,7 (C-12),21,5 (C-11),21,2 (C-24),19,8 (C-6),19,5 (C-30),16,1(C-25),16,0 (C-26),14,8 (C-27).

Compounds 1 to 4 respectively correspond to: 1 (28-hydroxy-3-oxo-20(29)-lupene), 2 (16-hydroxy-3-oxo-20(29)-lupene), 3 (3ihydroxy-20(29)-lupene) and 4 (acid 3-oxo-20(29)-lupene-28-oic) (figure 2).

Compound 1 (28-hydroxy-3-oxo-20(29)-lupene) was previously isolated from other species of Maytenus genus ⁽¹⁴, and it is now isolated for the first time from M. elaeodendroides.

Compound 2 (16-hydroxy-3-oxo-20(29)-lupene) was firstly isolated from Florensia resinosa S.F Blake plant and, later on, from Acacia cedilloi (L) Rico by Pech et al., 2002.¹⁸ It is interesting to point out that this is not only the first report of the isolation of this compound from M. elaeodendroides but also from the Celastraceae family.

Compound 3 (3dihydroxy-20(29)-lupene) exhibits interesting medicinal properties such as anti-inflammatory, anti-edematous, antitumoral and anti-HIV activities.^19-22 The topical anti-inflammatory activity against the Croton oil-induced ear edema in mice has been reported for this compound, ID₅₀ = 0,2.²³ This compound was isolated, for the first time, from M. elaeodendroides.

Compound 4 (acid 3-oxo-20(29)-lupene-28-oic) was isolated from Corma and from other plants belonging to different families, including Celastraceae. The compound has shown different kinds of biological activity, including: cytotoxic activity against a wide range of tumor cells, and antibacterial, anti-malarial and anti-inflammatory activities.²⁴ This compound was isolated, for the first time, from M. elaeodendroides.

Fig. 2

As no report on biological activity had been found for compound 2; while it has been reported anti-inflammatory activity for compounds 1, 3, and 4, we decided to perform an assay for anti-inflammatory activity of compounds 2 and 3 because their structures differs only in the substituent on C-3.

Table 2 shows the extent of inflammation in terms of edema weight average and standard deviation for pure compounds 2 and 3, evaluated independently for their topical anti-inflammatory activity in the Croton oil-induced ear edema model in mice.

Table 2

Groups	Doses (mg/kg)	Extent of inflammation [edema weight average (mg) ± SD]
Control (Croton oil)	-	118,4±11,78
Croton oil + diclofenac	20	14,8±2,17a*
Croton oil + compound 2	1	46,8±10,76d*
Croton oil + compound 2	5	29,8±8,26e*
Croton oil + compound 2	10	30,4±1,82e*
Croton oil + compound 3	1	115,2±7,12b
Croton oil + compound 3	5	98,2 ±6,98 c*
Croton oil + compound 3	10	40,6±5,94d*

Different letters represent significant differences (p < 0,05).

Table 2 shows that both compounds 2 and 3 significantly decrease inflammation. All doses of compound 2 showed anti-inflammatory activity compared to control group (Croton oil), whereas only the lowest dose of compound 3 (1 mg/kg) did not show a significant decrease in inflammation.

Diclofenac showed the strongest anti-inflammatory activity compared to the doses evaluated for compounds 2 and 3.

Figure 3 shows the results of percentage of inflammation inhibition at different doses of compounds 2 and 3. It was seen an anti-inflammatory activity for both triterpenes. However, diclofenac showed 87,5 % of inflammation inhibition, differing significantly from doses of 1,0, 5,0, 10,0 mg/kg for both compounds. Between the studied triterpenoid compounds best results were obtained for doses of 5,0 (74,83,%) and 10,0 mg/kg (74,3 %) of compound 2, so this compound has a marked anti-inflammatory effect. Only a dose of 10,0 mg/kg of compound 3 reached more than 50,0 % of inflammation inhibition (65,7 %).

Since the structures of compounds 2 and 3 are almost the same, only differentiated in C-3, we have suggested that the increase in anti-inflammatory activity for triterpene 2 is due to the presence of a carbonyl group in C-3 in its structure instead of the hydroxyl group found in triterpene 3.

Fig. 3

Compounds 1, 3 and 4 as well as betulin ¹⁵ have previously been proven to have anti-inflammatory properties. Herein we have demonstrated that compound 2 has also the same biological activity.

We consider that their presence in the bark of M. elaeodendroides plays an important role in the anti-inflammatory activity found in the extract of petroleum ether /diethyl ether (1:1).