Discovery of Anti-Inflammatory Triterpenoid Glucosides from the Heritiera littoralis Dryand

Two new triterpenoid glucosides, Heritiera A (1) and Heritiera B (2), and six known triterpenoid analogs (3–8) were isolated from Heritiera littoralis Dryand. Their structures were identified by comprehensive spectroscopic analyses and comparisons with the literature. The anti-inflammatory activity of the isolates from H. littoralis was evaluated using a lipopolysaccharide (LPS) stimulated RAW 264.7 cells model. The result showed that four triterpenoids exhibited potent anti-inflammatory activity. Among these compounds, compound 2 substantially inhibits the release of nitric oxide (NO) with an IC50 value of 10.33 μM. The triterpenoids from H. littoralis could be used as potential candidates for the development of new anti-inflammatory agents.


Introduction
Inflammation is a host defense response to danger signals from pathogens, damaged cells, irradiation, etc. [1]. The characteristic signs of acute inflammation include redness, swelling, warmth, and pain, which occur during various acute infections or tissue damage [2]. In the inflammatory response, there is an increase in the permeability of endothelial lining cells and influxes of blood leukocytes into the interstitium, oxidative burst, and release of cytokines (interleukins and tumor necrosis factor-α). At the same time, there is also induction of the activity of several enzymes (oxygenases, NO synthases, and peroxidases) as well as the arachidonic acid metabolism [3]. NO is a gaseous short-lived free radical that is considered to be a mediator of inflammation. Regulating the biosynthesis or activity of NO results in the amelioration of the acute inflammation model [4]. Thus, inhibitors of some pro-inflammatory mediators from folk herbs have potential anti-inflammatory activities, which are of great interest to scientific researchers.
At present, the main drugs used in the treatment of inflammation are non-steroidal antiinflammatory drugs, steroidal anti-inflammatory drugs, and traditional Chinese medicine. As synthetic anti-inflammatory drugs used in clinical currently have obvious adverse reactions, however traditional Chinese medicines with less toxicity and side effects have attracted more and more attention [5,6]. Natural plant components, such as triterpenoids and their glycosides, have various structural types, strong biochemical specificity, good curative effect, and low drug resistance, which are good resources for the development of potentially effective anti-inflammatory drug candidates [7,8]. These compounds have a good inhibitory effect on acute and chronic inflammation, which show an anti-inflammatory activity by inhibiting the production of inflammatory factors, and oleanane and ursane triterpenoids exhibit the most significant hypoglycemic activity, especially among triterpenoids [9][10][11].
show an anti-inflammatory activity by inhibiting the production of inflammatory factors, and oleanane and ursane triterpenoids exhibit the most significant hypoglycemic activity, especially among triterpenoids [9][10][11].
The genus Heritiera belongs to the family Malvaceae growing in tropical and subtropical regions of Asia [12]. It consists of 35 species, of which 3 species can be found in the provinces of Guangdong, Guangxi, Hainan, and Yunnan in China. The genus Heritiera has a long history of medicinal use in China, especially for folk use [13,14]. Heritiera littoralis Dryand., a semi-mangrove plant, has been used as a traditional medicine for the Jing nationality [15]. This plant has been reported for its wide range of activities, including the treatment of hematuria, diarrhea, dysentery, oral infection, and toothache [16][17][18]. So far, Heritiera littoralis has been reported to have significant anti-inflammatory activity in the folk [14]. The isolation and characterization of the chemical constituents from H. littoralis have yielded a range of classes of natural products, such as triterpenoid, flavonoid, phenylpropanoid-glycerol, etc. [19][20][21][22][23]. However, there have been few reports on the biologically active constituents of H. littoralis [24].
To explore the anti-inflammatory lead compounds from the genus Heritiera, the active ingredients were separated from the leaves of H. littoralis. The structures of all isolated constituents were characterized by comprehensive spectroscopic analyses and comparisons with the literature. These findings have led to the isolation of two new triterpenoid glucosides, Heritiera A (1) and Heritiera B (2), and six known triterpenoid analogs (3-8) Figure 1. Furthermore, the isolates were evaluated for their anti-inflammatory activities against the production of the NO in LPS-induced inflammation in RAW264.7 macrophage cells. Herein, the isolation, purification, and determination of these isolates and the assays used to determine the production of NO in RAW264.7 macrophage cells of the constituents are described. The research provided the basis for expanding the utilization and development of this medicinal plant.

Elucidation of the Chemical Structure of Heritiera A (1) and Heritiera B (2)
Heritiera A (1) was obtained as a colorless gum whose molecular formula was determined to be C42H66O15 from the HRESIMS data at m/z 834.4338 [M + Na] + with ten indices of hydrogen deficiency. The 1 H NMR data Table 1 13 C NMR spectra also showed the signals of two anomeric carbons at δH 6.48/δC 96.9 (C-1′) and δH 6.35/δC 95.7 (C-1′′) indicating the pres-

Elucidation of the Chemical Structure of Heritiera A (1) and Heritiera B (2)
Heritiera A (1) was obtained as a colorless gum whose molecular formula was determined to be C 42 13 C NMR spectra also showed the signals of two anomeric carbons at δ H 6.48/δ C 96.9 (C-1 ) and δ H 6.35/δ C 95.7 (C-1") indicating the presence of two sugar moieties in 1. Furthermore, D-glucose was identified by the acid hydrolysis solution of 1 by comparison with authentic sugar samples in an HPLC assay [25]. Moreover, five of the ten indices of hydrogen deficiency were ascribed to two carboxyl groups, one double bond, and two hexacylic rings, with the remaining five indices indicating the presence of a pentacyclic system. With the aid of 2D NMR experiments, the 1 H− 1 H COSY (Figure 2A, bold lines) and HSQC data revealed the partial structures -CH 2 (1)−CH 2 (2)−CH 2 (3)−, -CH 2 (6)−CH 2 (7), −CH (9)−CH 2 (11)−CH 2 (12)−CH (13)−CH (18)−CH (19)−CH 3 (29), -CH 2 (15)−CH 2 (16) and -CH (21)−CH 2 (22)−. Detailed analyses of the abovementioned NMR data showed that the data of 1 were highly similar data to those of 3β-hydroxy-18α,19α-urs-20-en-28-oic acid [26,27], except for the presence of a carboxyl carbon at C-23 (δ C 177.8 ppm) and two glycosyl units in 1. Furthermore, the HMBC correlations from the anomeric protons H-1 (δ H 6.48, dd, J = 8.0, 2.8 Hz) to C-23 (δ C 177.8) and H-1" (δ H 6.35, dd, J = 8.0, 2.8 Hz) to C-28 (δ C 174.8) suggested that the two D-glucoses are situated at C-23 and C-28, respectively. The configuration of the sugar was assigned as β on the basis of coupling constant values (J = 8.0 Hz) of anomeric protons [28]. In addition, the relative stereochemistry was deduced from ROESY spectrum (double arrows in Figure 3A Heritiera B (2) was obtained as a colorless gum whose molecular formula was determined to be C 36  respectively, and one olefinic proton (δ H 5.45, 1H, d, J = 8.0 Hz), one anomeric proton δ H 6.40 (1H, d, J = 8.0 Hz), as well as overlapping hydrogen signals in oxygenated carbons perhaps belonging to sugar moieties. The 13 C NMR and DEPT data (Table 1) revealed the existence of thirty-six carbon resonances, including six methyls, eleven methylenes (one olefinic at δ C 117.6), eleven methines (one olefinic at δ C 158.5), and eight quaternary carbons (one carbonyl at δ C 178.1). The 1 H and 13 C NMR spectra also showed the signals of one anomeric carbon at δ H 6.40 (d, J = 8.0 Hz)/δ C 96.9 (C-1 ), indicating the presence of one sugar moiety in the molecule, after acid hydrolysis of 2, the D-glucose existed in 2 by analysis of HPLC [25]. In addition, the configuration of the sugar was assigned as β on the basis of the coupling constant value (J = 8.0 Hz) of the anomeric proton [28]. Three of the eight indices of hydrogen deficiency were ascribed to one carboxyl group, one double bond, and one hexacylic ring, with the remaining five indices indicating the presence of a pentacyclic system. Above mentioned facts suggested that the aglycone part of 2 was similar to that of erythrodiol [29]; the differences existed mainly in the C (23), C (21), and one more glycosyl unit in 2. The C (23) was a carboxyl carbon with δ C 178.1 ppm, and the C (21) was an oxygenated carbon with δ C 73.1 ppm. Furthermore, in the HMBC spectrum (arrows in Figure 2B), the anomeric proton at δ H 6.40 (H-1 ) showed a correlation with δ C 178.1 (C-23), which indicated that the sugar moiety is situated at C-23. In addition, the relative stereochemistry was deduced from the ROESY spectrum (double arrows in Figure 3B    Heritiera B (2) was obtained as a colorless gum whose molecular formula was determined to be C36H58O10 from the HRESIMS data at m/z 673.3920 [M + Na] + with eight indices of hydrogen deficiency. The 1 H NMR data (Table 1) displayed six methyl singlets (δH 0.92, s, 3H; δH 0.96, s, 3H; δH 1.00, s, 3H; δH 1.20, s, 3H; δH 1.34, s, 3H; δH 1.60, s, 3H) respectively, and one olefinic proton (δH 5.45, 1H, d, J = 8.0 Hz), one anomeric proton δH 6.40 (1H, d, J = 8.0 Hz), as well as overlapping hydrogen signals in oxygenated carbons perhaps belonging to sugar moieties. The 13 C NMR and DEPT data (Table 1) revealed the existence of thirty-six carbon resonances, including six methyls, eleven methylenes (one olefinic at δC 117.6), eleven methines (one olefinic at δC 158.5), and eight quaternary    Heritiera B (2) was obtained as a colorless gum whose molecular formula w determined to be C36H58O10 from the HRESIMS data at m/z 673.3920 [M + Na] + with eig indices of hydrogen deficiency. The 1 H NMR data (Table 1)

Anti-Inflammatory Assay of the Isolates
The RAW264.7 cell viability assays showed that the survival rate was greater than 90% after treatment with all isolates at different concentrations from 0 to 50 µM. The effects of all compounds on the production of NO by LPS-induced RAW 264.7 cells are shown in Table 2. Compound 2 substantially inhibited the release of NO, with an IC 50 value of 10.33 µM. The value is slightly lower than that of the positive control, dexamethasone, with an IC 50 value of 6.39 µM. Compounds 1, 3, and 4 showed moderate effects with IC 50 values of 32.11, 39.32, and 29.98 µM, respectively. Compounds 5, 6, 7, and 8 showed no significant effects against LPS-induced nitric oxide production in RAW264.7 macrophages. However, the anti-inflammatory mechanism of compounds needs to be further discussed. We will conduct research in the next step.

Similarities and Differences of Some Triterpenoids and Their Anti-Inflammatory Activity from Malvaceae
It was reported that some triterpenoids isolated from Malvaceae exhibited good antiinflammatory activity, such as taraxerol (oleanane triterpenoid) and lupeol (lupane triterpenoid) from Grewia flava roots yielded promising IC 50 values of 21.88 ± 0.02 and 14.2 ± 0.01 µg/mL against NO production in RAW264.7 cells respectively [36]. Betulinic acid (lupane triterpenoid) and taraxasterol (ursane triterpenoid) were isolated from Luehea ochrophylla Mart [37]. The literature reported that betulinic acid was justified by inhibiting the release of pro-inflammatory mediators, mainly NO, IL-1β, TNF-α, and reduction of COX-2 levels [38,39]. Taraxasterol had efficacy comparable to prednisolone in a paw edema model induced by formalin [40]. Compound 2, as an oleanane triterpenoid, showed anti-inflammatory activity and inhibited the release of NO, with an IC 50 value of 10.33 µM too. These results support our initial investigation to obtain more anti-inflammatory active triterpenoids from this experiment.

Extraction and Isolation
Yield extract % = weight of dry extract/weight of dried plant material × 100% (1)

Enzymatic Hydrolysis of Compounds 1-2
Acidic hydrolysis of compounds 1-2 was carried out according to the method described previously [25]. The configuration of the sugar moiety was determined by com- 3.6. Cell Viability and Anti-Inflammatory Activity Test [41,42] All terpenoids isolated from the leaves of H. littoralis were subjected to MTT assays to assess the cell viability of LPS-stimulated RAW264.7 cell models.
Anti-inflammatory activity test: The anti-inflammatory activity of the isolates was evaluated by production of NO in RAW 264.7 cells by the Griess assay. Incubated procedure of cells was same as cell viability assay, a series of compounds treated with cell after 24 h, then production of NO in cell supernatant was determined based on the Griess reaction, and the absorbance was measured at 550 nm in a microplate reader. The final IC 50 values were calculated (n = 5). The IC 50 values are presented as the mean ± SD (standard deviation of the average value) from five independent experiments.

Conclusions
To explore the anti-inflammatory lead compounds of H. littoralis (Malvaceae), a phytochemical investigation of n-BuOH extract from the leaves of H. littoralis was carried out. Two new compounds, Heritiera A (1) and Heritiera B (2), and six known triterpenoids (3)(4)(5)(6)(7)(8) were isolated from the leaves of H. littoralis. Four triterpenoids decreased the production of NO on LPS-stimulated RAW 264.7 cells. Among these compounds, compound 2 substantially inhibits the release of NO, with an IC 50 value of 10.33 µM. Compounds 1, 3, and 4 showed moderate effects with IC 50 values of 32.11, 39.32, and 29.98 µM, respectively. Collectively, H. littoralis leaves contain abundant triterpenoids that affect the production of NO in RAW 264.7 cells, which could be meaningful for the development of new anti-inflammatory agents.