Anti-inflammatory activity of Maytenus senegalensis root extracts and of maytenoic acid.

Abstract

Maytenus senegalensis (Lam.) Excell (Celastraceae) root extracts were investigated for their topical anti-inflammatory properties by measuring the inhibition of the Croton oil-induced ear oedema in mice. The highest anti-inflammatory activity was detected in the chloroform extract, which reduced the oedematous response with a potency similar to that of the NSAID reference drug indomethacin ([ID.sub.50] = 84 and 93 [micro]g/[cm.sup.2], respectively). Fractionation of the chloroform and of the hexane extracts led to the isolation of maytenoic acid (1), which exhibited a dose-dependent antiphlogistic effect ([ID.sub.50] = 0.11 [micro]mol/[cm.sup.2]) twice that of indomethacin ([ID.sub.50] = 0.26 [micro]mol/[cm.sup.2]) and only three times lower than that of hydrocortisone ([ID.sub.50] = 0.04 [micro]mol/[cm.sup.2]).

[c] 2006 Elsevier GmbH. All rights reserved.

Keywords: Maytenus senegalensis; Anti-inflammatory activity; Croton oil ear test; Triterpenes; Maytenoic acid

Introduction

Maytenus senegalensis (Lam.) Excell (Celastraceae) is a shrub or tree, growing in the semidesertic regions of both Asia and Africa (Kokwaro, 1976). Its roots and bark are traditionally used in the folk medicine of some African regions for the treatment of a number of ailments, including chest pains, rheumatism, snakebites, diarrhoea, eye infection, dyspepsia and wounds (Matu and van Staden, 2003; Okine et al., 2005). It was suggested that the addition of M. senegalensis root to milk and meat-based foods by Maasai and Batemi populations of Tanzania is associated with a low incidence of cardiovascular diseases despite their high intake of fats and cholesterol (Johns et al., 1999). In recent years some biological activities of M. senegalensis extracts were confirmed by pharmacological studies. Thus, following the traditional use of the plant in Sudan, Kenya or Tanzania, it was demonstrated that leaf, root and stem bark extracts of M. senegalensis possess in vitro antiplasmodial (Gessler et al., 1994, 1995; El Tahir et al., 1999), antileishmanial (El Tahir et al., 1998), and antibacterial activities (Matu and van Staden, 2003). The antileishmanial and antiplasmodial properties were associated with the terpenoid content of the most active fractions of M. senegalensis (El Tahir et al., 1998, 1999). On the other hand, screening studies on antitumor properties of M. senegalensis root and stem extracts revealed in vitro cytotoxic activity against carcinoma cells (Tin-Wa et al., 1971; Gessler et al., 1995) and in vivo antileukemia effects (Tin-Wa et al., 1971). Furthermore, an antiviral activity, as HIV-1 protease inhibition, was demonstrated for hydrophilic extracts of the stem and for their phenolic constituents (Otake et al., 1995; Hussein et al., 1999a, b).

Interestingly, plants of the genus Maytenus are used in South America to prepare infusa or decoctions as anti-inflammatory and analgesic remedies for oral and/or topical administration (Correa, 1984; Bueno et al., 2005). For some species, these activities have also been experimentally demonstrated. In particular, the triterpenoid constituents of Maytenus undata aerial parts were shown to inhibit thromboxane [B.sub.2] and superoxide anion formation (Muhammad et al., 2000), while antinociceptive, anti-inflammatory as well as antiulcerogenic activities were found in the hexane and ethyl acetate extracts of Maytenus ilicifolia leaves (Jorge et al., 2004). An analgesic property was also documented for the aqueous extract of Maytenus aquifolium leaves (Gonzales et al., 2001). For M. senegalensis, only one activity related to a hypothetical anti-inflammatory effect has been reported: its bark and leaf extracts were shown to inhibit cyclo-oxygenase-1, an enzyme responsible for the synthesis of inflammatory mediators, such as prostaglandins and thromboxanes (Matu and van Staden, 2003). However, no studies demonstrated an actual in vivo anti-inflammatory effect of constituents of this plant.

[FIGURE 1 OMITTED]

We report here evidence for the in vivo anti-inflammatory activity of M. senegalensis root extracts and of maytenoic acid (1) (Fig. 1) isolated from them. The topical anti-inflammatory activity was evaluated as inhibition of the Croton oil-induced ear oedema in mice (Tubaro et al., 1985).

Materials and methods

Plant material

M. senegalensis roots were collected in the Bamako region of Mali in 2000. The plant material was identified by Prof. N'Golo Diarra, Botanist of DMT, Bamako, Mali. A voucher specimen is kept in the Dipartimento di Chimica Organica e Industriale, University of Milan, Italy.

Instruments and chemicals

Thin layer chromatography (TLC) was performed on silica gel [F.sub.254] precoated aluminum sheets (0.2 mm layer, Merck, Darmstadt, Germany); components were detected by spraying a ceric sulphate/ammonium molybdate solution, followed by heating to ca. 150 [degrees]C. Silica gel (Merck, 40-63 [micro]m) and Sephadex LH-20 (Pharmacia) were used for flash chromatography and column chromatography, respectively. Preparative TLC was performed on precoated silica gel 60 [F.sub.254] plates (1 mm layer thickness, Merck). [1.sup.H] and [13.sup.C] NMR spectra were recorded in CD[Cl.sub.3] at 400.132 and 100.613 MHz, respectively, on a Bruker AVANCE 400 spectrometer using an Xwin-nmr software package. EI MS spectra were recorded on a VG 7070 EQ mass spectrometer.

Croton oil, hydrocortisone and indomethacin were obtained from Sigma-Aldrich (Milan, Italy). Ketamine hydrochloride was purchased from Virbac (Milan, Italy). The other reagents and solvents, of analytical grade, were purchased from Carlo Erba (Milan, Italy).

Extraction and isolation procedure

Powdered M. senegalensis roots (100g) were sequentially extracted with n-hexane, chloroform, chloroform-methanol (9:1, v/v), and methanol by means of a Soxhlet apparatus. The extracts were filtered and evaporated to dryness under reduced pressure to give the corresponding residues in amounts of 0.98, 0.63, 0.55 and 11.40 g, respectively.

A portion of the hexane extract (440 mg) was flash chromatographed using hexane-ethyl acetate (7:1) as eluent. Fractions (8 ml) were collected and monitored by TLC (eluent: hexane-acetone-chloroform 8:2:1). Fractions 4-6 furnished a residue (14 mg) which was submitted to flash chromatography (eluent: hexane-ethyl acetate, 15:1) followed by preparative TLC (eluent: hexane-ethyl acetate, 20:1) to give 6mg of lupenone (2) (Prashant and David Krupadanam, 1993). Fractions 25-32 gave pure [beta]-amyrin (3, 81 mg) (Bhattacharyya and Barros, 1986). Fractions 37-50 gave a mixture of compounds (297 mg), which was passed through a Sephadex LH-20 column (eluent: hexane-chloroform-methanol 2:1:1) giving two sets of fractions. Set 28-36 (85 mg) was flash chromatographed (eluent: hexane-ethyl acetate 5:1) furnishing pure [beta]-sitosterol (4, 24 mg) (Furuya et al., 1987); set 44-48 (160 mg) was submitted to a further Sephadex LH-20 separation (eluent: hexane-chloroform-methanol 2:1:1) to give maytenoic acid (1, 13 mg) (Abraham et al., 1971; Itokawa et al., 1991).

A portion (335 mg) of the chloroform extract was fractioned by flash chromatography eluting with hexane containing increasing amount of ethyl acetate (from 20% to 100%). The following compounds were isolated which were further purified by preparative TLC using hexane-acetone-chloroform 8:2:1 as eluent: maytenoic acid (1, [R.sub.f] 0.27, 30 mg); [beta]-sitosterol (4, [R.sub.f] 0.40, 5 mg); [beta]-amyrin (3, [R.sub.f] 0.50, 10 mg) and lupenone (2, [R.sub.f] 0.65, 5 mg).

All compounds were identified by mass spectrometry and mono- and bi-dimensional NMR spectroscopy.

Anti-inflammatory activity assay

Male CD-1 mice (28-32 g; Harlan-Italy, Udine, Italy) were kept for 1 week, before the experiment, at constant conditions of temperature (21 [+ or -] 1 [degrees]C) and humidity (60-70%) and at fixed artificial light cycle (07.00-19.00 h). Inflammation was induced always in the late morning (10.00-12.00 h) in mice anaesthetised with ketamine hydrochloride (145 mg/kg, intraperitoneally). Inflammatory response was induced on the inner surface of the right ear (surface: about 1 [cm.sup.2]) by application of 80 [micro]g of Croton oil dissolved in an appropriate vehicle, as reported below. Control animals received only the irritant solution, whereas the other animals received the irritant together with the tested substances. The vehicles used were acetone or, for the methanol extract and its controls, 42% aqueous ethanol (v/v). At the maximum of the oedematous response, i.e. 6 h later, mice were sacrificed and a plug (6 mm [empty set]) was removed from both the treated (right) and the untreated (left) ears. Oedema was measured as the weight difference between the two plugs. The anti-inflammatory activity was expressed as percentage of the oedema reduction in treated mice compared to the control mice. As a reference, the non-steroidal anti-inflammatory drug (NSAID) indomethacin and the steroid hydrocortisone were used.

All animal experiments complied with the Italian D.L. n. 116 of 27 January 1992 and associated guidelines in the European Communities Council Directive of 24 November 1986 (86/609 ECC).

Statistical analysis

Pharmacological data were analysed by one-way analysis of variance followed by the Dunnett's test for multiple comparisons of unpaired data, and a probability level lower than 0.05 was considered as significant. [ID.sub.50] values (dose giving 50% oedema inhibition) were calculated by graphic interpolation of the dose-effect curves.

Results and discussion

The roots of M. senegalensis (100 g) were successively extracted with n-hexane, chloroform, chloroform-methanol (9:1, v/v) and methanol to obtain the corresponding extracts, which were screened for their topical anti-inflammatory activity at the dose of 300 [micro]g/[cm.sup.2]. As reported in Table 1, each extract induced a significant reduction of the oedematous response, that ranged from 20% (methanol extract) to 92% (chloroform extract). The chloroform extract, being the most active, was further investigated to evaluate its effect at three different doses, in comparison with the reference NSAID indomethacin and to the steroidal anti-inflammatory drug hydrocortisone. The extract dose-dependently reduced oedema formation and showed a potency similar to that of indomethacin and five times lower less active than hydrocortisone (Fig. 2). In fact, its [ID.sub.50] value (dose inducing 50% oedema inhibition) was 84 [micro]g/[cm.sup.2], i.e. slightly lower than that of indomethacin ([ID.sub.50] = 93 [micro]g/[cm.sup.2]), while [ID.sub.50] of hydrocortisone was 16 [micro]g/[cm.sup.2].

[FIGURE 2 OMITTED]

Phytochemical investigation of both the hexane and chloroform extracts led to the isolation of three triterpene derivatives, maytenoic acid (1), lupenone (2) and [beta]-amyrin (3), besides the steroidal compound [beta]-sitosterol (4) (Fig. 1). The triterpenes, isolated in sufficient amounts for the pharmacological assay, were evaluated for their anti-inflammatory activity in comparison to the non-steroidal and steroidal anti-inflammatory drugs indomethacin and hydrocortisone. Maytenoic acid (1) induced a strong dose-dependent oedema inhibition since at the dose of 0.03 [micro]mol/[cm.sup.2] it reduced the oedematous response by 29%, while more than 90% reduction was observed with the highest administered dose (1 [micro]mol/[cm.sup.2]). Lupenone (2) induced 26% oedema reduction at 0.1 [micro]mol/[cm.sup.2] and reached 65% reduction at the highest administered dose (1 [micro]mol/[cm.sup.2]), similarly to [beta]-amyrin (3), which, at same dose levels, provoked oedema inhibitions ranging from 19% to 62% (Table 2). The dose-activity relationships of the tested compounds allowed to calculate their [ID.sub.50] values as index of their anti-inflammatory potency. Maytenoic acid showed an [ID.sub.50] of 0.11 [micro]mol/[cm.sup.2], being twice as active as indomethacin ([ID.sub.50] = 0.26 [micro]mol/[cm.sup.2]) and only three times less potent than hydrocortisone ([ID.sub.50] = 0.04 [micro]mol/[cm.sup.2]). Moreover, it was four and five times more potent than lupenone ([ID.sub.50] = 0.49 [micro]mol/[cm.sup.2]) and [beta]-amyrin ([ID.sub.50] = 0.57 [micro]mol/[cm.sup.2]), respectively. Despite the lowest activity observed for the last two triterpenes, also these compounds are of interest being only about twice less potent than the reference NSAID indomethacin (Table 2).

Considering its high anti-inflammatory activity, maytenoic acid plays a primary role in the antiphlogistic effect of the whole chloroform extract of M. senegalensis roots. However, also lupenone and [beta]-amyrin, even though less active than maytenoic acid, could give a significant contribution to the anti-inflammatory activity of the extract. Although not yet demonstrated, they could probably act in a synergistic way with maytenoic acid.

This study demonstrated the topical anti-inflammatory properties of M. senegalensis root extracts and, in particular, of those obtained with lipophilic solvents (n-hexane and chloroform). Such extracts contain maytenoic acid (1), which was found to be an anti-inflammatory triterpene twice as active as the NSAID indomethacin and only three times less active than hydrocortisone. This is the first report on the anti-inflammatory property of maytenoic acid in vivo and our findings justify the traditional use of M. senegalensis against topical inflammatory-based diseases. Furthermore, it has to be noted that lipophilic preparations could be more useful as practical anti-inflammatory remedy than the traditional infusa and decoctions.

In conclusion, M. senegalensis roots could be employed in therapeutic preparations for the treatment of inflammatory-based diseases, at least topically against skin and mucous membranes affections. The plant material can be also regarded as a source of the potent anti-inflammatory principle maytenoic acid.

Acknowledgement

This work was supported by a grant of the Italian Ministry of Instruction, University and Research (MIUR).

References

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S. Sosa (a,*), C.F. Morelli (b), A. Tubaro (a), P. Cairoli (b), G. Speranza (b), P. Manitto (b)

(a) Dipartimento dei Materiali e delle Risorse Naturali, Universita degli Studi di Trieste, via Valerio 6, 34127 Trieste, Italy

(b) Dipartimento di Chimica Organica e Industriale, Universita degli Studi di Milano, via Venezian 21, 20133 Milano, Italy

*Corresponding author. Tel.: + 39 040 558 3165; fax: + + 39 040 558 3215.

E-mail address: [e-mail] (S. Sosa).

Table 1. Anti-inflammatory activity of Maytenus senegalensis root
extracts

                                                            Oedema
                  Dose ([micro]g/  No.  Oedema (mg)         inhibition
Substance         [cm.sup.2])      an.  mean [+ or -] S.E.  (%)

Controls           --              10   7.8 [+ or -] 0.2    --
Hexane extract    300              10   3.0 [+ or -] 0.1*   62
Chloroform        300              10   0.6 [+ or -] 0.1*   92
  extract
Chloroform-       300              10   5.7 [+ or -] 0.3*   27
  methanol
  extract
Controls           --              10   6.9 [+ or -] 0.1    --
Methanol extract  300              10   5.5 [+ or -] 0.3*   20

*p < 0.05, at the analysis of variance, as compared with controls.

Table 2. Anti-inflammatory activity of maytenoic acid, lupenone and
[beta]-amyrin

                    Dose                   Oedema (mg)        Oedema
                    ([micro]mol/           mean [+ or -]      inhibition
Substance           [cm.sup.2])   No. an.  S.E.               (%)

Controls            --            40       6.9 [+ or -] 0.2   --
Maytenoic acid (1)  0.030         10       4.9 [+ or -] 0.3*  29
                    0.100         10       3.7 [+ or -] 0.4*  46
                    0.300         20       2.6 [+ or -] 0.1*  62
                    1.000         10       0.4 [+ or -] 0.1*  94
Lupenone (2)        0.100         10       5.1 [+ or -] 0.2*  26
                    0.300         10       4.4 [+ or -] 0.3*  36
                    1.000         10       2.4 [+ or -] 0.2*  65
[beta]-Amyrin (3)   0.100         10       5.6 [+ or -] 0.2*  19
                    0.300         10       4.5 [+ or -] 0.2*  35
                    1.000         10       2.6 [+ or -] 0.3*  62
Indomethacin        0.120         10       5.3 [+ or -] 0.3*  23
                    0.250         10       3.2 [+ or -] 0.4*  54
                    0.500         10       2.2 [+ or -] 0.2*  68
Hydrocortisone      0.025         10       4.3 [+ or -] 0.1*  38
                    0.050         10       3.4 [+ or -] 0.4*  51
                    0.100         10       2.0 [+ or -] 0.2*  71

                    [ID.sub.50]
Substance           ([micro]mol/[cm.sup.2])

Controls
Maytenoic acid (1)  0.11
Lupenone (2)        0.49
[beta]-Amyrin (3)   0.57
Indomethacin        0.26
Hydrocortisone      0.04

*p < 0.05 at the analysis of variance, as compared with controls.