Triterpenoids from Vitellaria paradoxa Stem Barks Reduce Nitrite Levels in LPS-Stimulated Macrophages

Vitellaria paradoxa C. F. Gaertn is widely used in African traditional medicine as an anti-inflammatory remedy to treat rheumatism, gastric problems, diarrhea, and dysentery. The phytochemical investigation of the ethyl acetate extract of V. paradoxa stem bark collected in Burkina Faso led to the isolation of eight known and two triterpenes undescribed to date (7 and 10), in the free alcohol form or as acetyl and cinnamyl ester derivatives. The stereostructures of the new compounds were elucidated using HR-ESIMS and 1D and 2D NMR data. The isolated compounds were evaluated in vitro for their inhibitory effect on nitrite levels on murine macrophages J774 stimulated with the lipopolysaccharide (LPS). Among all the compounds tested, lupeol cinnamate (3) and betulinic acid (5) showed a beneficial effect in reducing nitrite levels produced after LPS stimulation.


Introduction
Vitellaria paradoxa (V. paradoxa) C. F. Gaertn (syn. Butyrospermum parkii (G.Don) Kotschy) is a tall (up to 14 m) tree belonging to the Sapotaceae family, occurring over nearly 1 million km 2 within 18 African countries, but predominantly found in in the dry savannah belt of West Africa. V. paradoxa is strictly intertwined with the life of African populations, especially because of the largely used shea butter (Francophone usage: beurre de karité), the reason V. paradoxa is commonly known as the "shea butter tree". This is an edible ivory-colored fat consisting of olein and stearin fractions along with nonsaponifiable compounds, extracted by crushing and boiling the fermented kernel. It is used as an ingredient in the production of cosmetics to protect the skin in sun-protection or post-sun-exposure; to promote wound healing; to soothe skin irritation, chapping, and dermatitis; and other skin ointments [1]. The shea butter methanolic extract has been shown to exert a regulatory effect on LPS induced inflammatory responses through the inhibition of NF-κB activation, suppression of IκBα phosphorylation, and through the downregulation of mRNA and the protein expressions of pro-inflammatory cytokines and interleukins [2]. Besides shea butter, the trunk and stem barks of V. paradoxa macerated in traditional alcoholic beverages are widely used in traditional medicine for the treatment of pain and as an anti-inflammatory remedy to treat rheumatism, gastric problems, diarrhoea, and dysentery [1]. The methanolic stem bark extract showed significant anti-inflammatory and anti-arthritic effects in carrageenan-induced inflammation and CFA-induced arthritic animal models [3], and the stem bark ethyl acetate extract was also demonstrated to exert significant anti-inflammatory effects on adjuvant-induced arthritis with the alleviation 2 of 9 of paw edema and hematological disorder associated with this condition [4]. Different studies have demonstrated that the anti-inflammatory activity of stem bark extracts is mainly ascribed to their rich triterpenoid composition [4], which have been proposed to act by inhibiting pro-inflammatory cytokines and iNOS and COX-2 expression [5]. Recently, a V. paradoxa triterpene-rich extract was shown to suppress pro-inflammatory mediators and attenuate the cartilage degradation and pain in osteoarthritis in an obesity rat model [6].
The present study aimed at obtaining a detailed phytochemical characterization of the triterpenoid composition of the stem barks of V. paradoxa collected in the Baskoure Area of Burkina Faso. This is a rural area located in the East-Centre region of the country, where the plant is widely used by local traditional healers, especially to treat gastrointestinal diseases, which are very common in this area [1]. The isolated compounds were assayed for their activity on reducing the nitrite level in macrophages after a lipopolysaccharide (LPS) insult, an activity related to the anti-inflammatory potential.
Plants 2021, 10, x FOR PEER REVIEW 2 of 10 duced arthritic animal models [3], and the stem bark ethyl acetate extract was also demonstrated to exert significant anti-inflammatory effects on adjuvant-induced arthritis with the alleviation of paw edema and hematological disorder associated with this condition [4]. Different studies have demonstrated that the anti-inflammatory activity of stem bark extracts is mainly ascribed to their rich triterpenoid composition [4], which have been proposed to act by inhibiting pro-inflammatory cytokines and iNOS and COX-2 expression [5]. Recently, a V. paradoxa triterpene-rich extract was shown to suppress pro-inflammatory mediators and attenuate the cartilage degradation and pain in osteoarthritis in an obesity rat model [6]. The present study aimed at obtaining a detailed phytochemical characterization of the triterpenoid composition of the stem barks of V. paradoxa collected in the Baskoure Area of Burkina Faso. This is a rural area located in the East-Centre region of the country, where the plant is widely used by local traditional healers, especially to treat gastrointestinal diseases, which are very common in this area [1]. The isolated compounds were assayed for their activity on reducing the nitrite level in macrophages after a lipopolysaccharide (LPS) insult, an activity related to the anti-inflammatory potential.
Compound 10 was isolated as a white amorphous powder with a molecular formula of C 39 H 56 O 3 , determined by HR-ESIMS. An analysis of the 1D NMR signals was carried out by extensive application of 2D NMR spectroscopy (COSY, HSQC, HMBC), which yielded to the assignment of 10 as a close analogue of β-amyrin cinnamate [5], previously characterized from shea fat. The single difference between the two series of signals were located in the downfield region of the spectrum, where compound 10 showed the presence of one additional oxymethine signal (δ H 4.55, H-11) and the downfield shift of the H-12 (δ H 5.36). The COSY cross-peaks between δ H 4.55 and δ H 5.36 confirmed the location of the additional hydroxy group at C-11, whose α-orientation was assigned on the basis of the H-9/H-11 coupling constant (J = 10 Hz). A further support to the stereostructure assignment of compound 10 came from a comparison of the 1 H/ 13 C NMR data and proton-proton coupling constants with those reported for 11α-hydroxy-β-amyrin isolated from Stautonia hexaphylla [14].
Triterpene alcohols such as α-amyrin, β-amyrin, lupeol, and butyrospermol, most of which occur as acetyl and cinnamyl ester derivatives, have been previously reported to be the main non-glyceride constituents of V. paradoxa kernel [5,6,15,16], leaves [17], and stem bark [4,10,18] extracts. Typically, the kernel fat contains ca. 0.5-6.5% of triterpene esters depending on the geographic site; for instance, shea samples from the western area of the shea belt (Cote d'Ivoire, Ghana, and Nigeria) and from Cameroun generally contain highest percentages of these compounds (2.0-6.5%) [19]. Some of these metabolites exhibited different biological properties such as cytotoxicity [10,18], a significant in vitro and in vivo antitrypanosomal activity [17], inhibitory effects on TPA-induced inflammation in mice and on skin tumor promotion in an in vivo two-stage carcinogenesis test [5], and other bioactivities demonstrating the bio-pharmacological potential of these compounds [16].

Biological Activity of Triterpenoids
Given the remarkable anti-inflammatory action of stem bark extracts [3][4][5][6]19], we initially tested the effect of seven pure compounds among those isolated in this study (compounds 7 and 10 were not tested because of the low amount available, while 2 underwent degradation during tests) on the nitrite levels produced in response to LPS stimulus in murine macrophages J774A.1 Among all of the compounds tested, lupeol cinnamate (3), betulinic acid (5), and α-amyrin cinnamate (6) at 10 µM showed a beneficial effect in reducing the nitrite levels produced after LPS stimulation ( Figure 2). Because of availability reasons, compounds 3 and 5 were selected for further pharmacological studies. Of note, at a 30 µM concentration, none of the tested compounds affected the cell viability after 24 h of treatment, thus excluding the likelihood that their effect in reducing nitrite levels may be because of a non-specific cytotoxic effect in macrophages (data not shown). murine macrophages J774A.1 Among all of the compounds tested, lupeol cinnamate (3), betulinic acid (5), and α-amyrin cinnamate (6) at 10 µM showed a beneficial effect in reducing the nitrite levels produced after LPS stimulation ( Figure 2). Because of availability reasons, compounds 3 and 5 were selected for further pharmacological studies. Of note, at a 30 µM concentration, none of the tested compounds affected the cell viability after 24 h of treatment, thus excluding the likelihood that their effect in reducing nitrite levels may be because of a non-specific cytotoxic effect in macrophages (data not shown). LPS stimulus (1 µg/mL) given for 24 h to murine macrophages determined an increase in nitrite levels, while a pre-treatment (1-30 µM 30 min before LPS) with lupeol cinnamate (3) ( Figure 3A) and/or betulinic acid (5) (Figure 3B), significantly and in a concentration dependent manner, lowered LPS-induced nitrite production. The IC50 (concentration that caused the 50% inhibition of nitrite production) calculated for these compounds were 9.5 ± 0.11 µM for lupeol cinnamate (3) ( Figure 3C) and 12.2 ± 0.11 µM for betulinic acid (5) ( Figure 3D).
As some triterpenoids, including β-amyrin, have been demonstrated to exert analgesic and anti-inflammatory pharmacological effects via indirect cannabimimetic mechanisms by inhibiting the degradation of the endocannabinoid 2-arachidonoylglycerol [20], we tested the active compounds in presence of AM251 (selective CB1 receptor antagonist) or SR144528 (selective CB2 receptor antagonist), but no significant change in the activity was detected when co-administered with lupeol cinnamate (3) ( Figure 4A) and/or betulinic acid (5) ( Figure 4B). AM251 and SR144528, at the concentrations used, did not alter the nitrite levels produced in response to LPS ( Figure 4A,B).
The selective activity shown by compounds 3, 5, and 6 points to the existence of structural requirements that are not easy to unveil from the limited number of compounds  (compounds 1, 3-6, and 8-9) on nitrite levels in the supernatant collected from J774A.1 murine macrophages stimulated with lipopolysaccharide (LPS, 1 µg/mL) for 24 h. The compounds used in this study were supplemented to the cell media 30 min before LPS stimulus. Results are reported as percentage of nitrites and are expressed as mean ± SEM of four independent experiments (in quadruplicate). § p < 0.0001 vs. control (cells without LPS); * p < 0.05, ** p < 0.01, and **** p < 0.0001 vs. LPS as assessed by one-way ANOVA, followed by Dunnett's multiple comparisons test.
As some triterpenoids, including β-amyrin, have been demonstrated to exert analgesic and anti-inflammatory pharmacological effects via indirect cannabimimetic mechanisms by inhibiting the degradation of the endocannabinoid 2-arachidonoylglycerol [20], we tested the active compounds in presence of AM251 (selective CB 1 receptor antagonist) or SR144528 (selective CB 2 receptor antagonist), but no significant change in the activity was detected when co-administered with lupeol cinnamate (3) ( Figure 4A) and/or betulinic acid (5) ( Figure 4B). AM251 and SR144528, at the concentrations used, did not alter the nitrite levels produced in response to LPS ( Figure 4A,B).
The selective activity shown by compounds 3, 5, and 6 points to the existence of structural requirements that are not easy to unveil from the limited number of compounds tested. Compared with 1 and 4, the presence of a cinnamate ester in 3 seems to be beneficial for the bioactivity. This is confirmed by previous studies, which show that esterifications at 3-OH of pentacyclic triterpenes seem to enhance the antinflammatory activity [5]. It is also interesting to note the higher activity of betulinic acid compared with ursolic acid, clearly evidencing that details in the triterpene skeleton can have a major impact on the activity. tested. Compared with 1 and 4, the presence of a cinnamate ester in 3 seems to be beneficial for the bioactivity. This is confirmed by previous studies, which show that esterifications at 3-OH of pentacyclic triterpenes seem to enhance the antinflammatory activity [5]. It is also interesting to note the higher activity of betulinic acid compared with ursolic acid, clearly evidencing that details in the triterpene skeleton can have a major impact on the activity.

General Experimental Procedures
Optical rotations (CHCl3) were measured at 589 nm on a P2000 Jasco polarimeter. The 1 H (500 MHz) and 13 C (125 MHz) NMR spectra were measured at room temperature on a Varian INOVA 500 MHz spectrometer. Chemical shifts were referenced to the resid- Results are reported as a percentage of nitrites and are expressed as means ± SEM of three or four independent experiments. § p < 0.0001 vs. control (cells without LPS); ** p < 0.01 and **** p < 0.0001 vs. LPS as assessed by one-way ANOVA, followed by Tukey's multiple comparisons test.

General Experimental Procedures
Optical rotations (CHCl 3 ) were measured at 589 nm on a P2000 Jasco polarimeter. The 1 H (500 MHz) and 13 C (125 MHz) NMR spectra were measured at room temperature on a Varian INOVA 500 MHz spectrometer. Chemical shifts were referenced to the residual solvent signal (CDCl 3 : δH 7.26, δC 77.0). Homonuclear 1 H connectivities were determined using the COSY experiment, one-bond heteronuclear 1 H-13 C connectivities were determined using the HSQC experiment, and two-and three-bond 1 H-13 C connectivities were determined using gradient-HMBC experiments optimized for a 2,3 J of 8 Hz. Low-and highresolution ESI-MS spectra were performed on a LTQ OrbitrapXL (Thermo Scientific) mass spectrometer with a triple quadrupole analyzer. Medium-pressure liquid chromatography (MPLC) was performed on a Büchi (Switzerland) apparatus using a silica gel (7-230 mesh). Separations were monitored by TLC on Merck 60 F254 silica gel (0.25 mm) plates; HPLC was achieved on a Knauer 1800 instrument equipped with a refractive index detector. LUNA (normal phase, SI60, or reverse-phase RP18, 250 × 4 mm; Phenomenex) columns were used, with 1 mL/min as the flow rate, with isocratic elution at room temperature (see Section 3.3 for solvents).

Plant Material
The stem barks of Vitellaria paradoxa were collected in the summer of 2008 in Baskoure and Songretenga rural areas located in Kourittenga Province, in the East-Centre Region of Burkina Faso, as described in the literature [1]. The plant identification was done by Prof. Joseph Issaka Boussim (University of Ouagadougou), and voucher specimens (V-08-01) were deposited into the herbarium of the Botanical Laboratory of the University of Ouagadougou.

Statistical Analysis
Data are expressed as mean ± SEM of n experiments. To determine statistical significance, one-way ANOVA followed by Tukey's multiple comparisons test and/or Dunnett's multiple comparisons test was used for the comparison of multiple groups. The IC 50 (concentration that caused 50% inhibition of nitrite production) ±95% confidence interval values were calculated by non-linear regression analysis using the sigmoid concentrationresponse curve equation (GraphPad Prism 7).

Conclusions
In conclusion, a detailed phytochemical investigation on the stem barks obtained from the African plant V. paradoxa resulted in the isolation of ten triterpenoids belonging to three different pentacyclic structural families, namely lupane, ursane, and oleanane classes. Among these compounds, we characterized two unprecedented triterpenoids, the ursane ursaldehyde cinnamate (7) and the oleanane 11-hydroxy-β-amyrin cinnamate (10). Three of the isolated compounds induced a reduction in nitrite levels in LPS-stimulated macrophages, an activity related to the anti-inflammatory potential. These results suggest the possible response of the anti-inflammatory properties demonstrated for the stem bark extract [4], although a synergistic action of the complex triterpenoid mixture is likely.