Plumeriapropionics A–E, Carboxyl-Substituted Phenylpropionic Acid Derivatives with Anti-Inflammatory Activity from Plumeria rubra L.

Abstract

Five rare carboxyl-substituted phenylpropionic acid derivatives, plumeriapropionics A–E (1–5), together with one known analog, cerberic acid B (6), were isolated from flowers of Plumeria rubra L. Their structures were elucidated using comprehensive spectroscopic methods. To date, only one compound of this structural type has been reported. The inhibitory activities of compounds 1–6 against nitric oxide (NO) production induced by lipopolysaccharide (LPS) were evaluated in vitro using mouse macrophage RAW264.7 cells. Compounds 1–6 showed remarkable inhibitory activities on NO production, with IC₅₀ values in the range of 6.52 ± 0.23 to 35.68 ± 0.17 µM. These results indicate that the discovery of carboxyl-substituted phenylpropionic acid derivatives from the flowers of P. rubra, which show significant anti-inflammatory properties, could be of great importance for the research and development of novel natural anti-inflammatory agents.

1. Introduction

The genus Plumeria, belonging to the Apocynaceae family, consists of approximately ten species native to tropical America, which are mainly distributed in tropical and subtropical regions of Asia [1]. There is one species and one variant in China, mainly growing in Fujian, Guangdong, Guangxi, Yunnan and Hainan provinces [2]. Previous chemical investigations on the plants from the genus Plumeria have led to the isolation and identification of a variety of natural products, including iridoids [3,4,5], triterpenoids [4,5,6,7,8], ferulic acids [9], and flavones [5,10], which display various biological activities, such as anti-diabetic [5], anti-tumor [3], and anti-inflammatory [11,12]. Among the genus Plumeria, P. rubra L. is a deciduous shrub or small tree widely planted in the south of China as an ornamental shrub. The flowers of P. rubra are often used in folk medicine for the treatment of various diseases, such as enteritis, acute bronchitis, bacillary dysentery, and infectious hepatitis [2].

Our preliminary experimental results showed that the 75% ethanol extract of the flowers of P. rubra showed an inhibitory effect against NO production induced by LPS in mouse macrophage RAW 264.7 cells with an IC₅₀ value of 22.89 ± 0.16 µg/mL in vitro. In order to deeply explore the enormous potential of China’s unique tropical medicinal plants in treating and preventing major human diseases, a chemical investigation on the flowers of P. rubra was carried out. Bioassay-guided fractionation of the bioactive extract led to the identification of five new carboxyl-substituted phenylpropionic acid derivatives named plumeriapropionics A–E (1–5), together with one known analogue, cerberic acid B (6) (Figure 1). Structure elucidations of these compounds were clarified by comprehensive spectroscopic analyses, including NMR spectral data, HR-ESI-MS data, IR, optical rotations, and comparisons with the spectral data reported in the literature. Compounds 1–6 are a kind of rare carboxyl-substituted phenylpropionic acid derivatives. Although phenylpropanoid is a common component in plants, carboxyl-substituted phenylpropanoid is very rare in the plant kingdom. So far, only one compound of this structural type has been reported. To explore the potential of these isolated carboxyl-substituted phenylpropionic acid derivatives for the development of anti-inflammatory drugs, compounds 1–6 were evaluated for their anti-inflammatory activities in vitro. Herein, we will report the isolation, identification, and pharmacological activity of these compounds.

2. Results and Discussion

2.1. Phytochemical Investigation

The dried flowers of P. rubra were extracted with 75% ethanol, and the extract was subjected to column chromatography over a silica gel, octadecylsilyl silica gel (ODS), Sephadex LH-20, as well as semi-preparative high-pressure liquid chromatography (HPLC) to yield five new carboxyl-substituted phenylpropionic acid derivatives, plumeriapropionics A–E (1–5), and one known analog, cerberic acid B (6). Compounds 1–6 are rare natural products isolated from the genus Plumeria for the first time.

Compound 1 was obtained as a white amorphous powder. The molecular formula of C₁₈H₁₆O₆ (11 degrees of unsaturation) was deduced by high-resolution electrospray ionization mass spectrometry (HRESIMS) combined with ¹H- and ¹³C-NMR data (

Table 1. ¹H and ¹³C NMR spectral data of compounds 1 and 2.

Position	1 a		2 a
	δH, Mult, (J in Hz)	δC	δH, Mult, (J in Hz)	δC
1	-	137.3	-	136.5
2	8.00 (br, s)	130.4	7.96 (s)	130.6
3	-	129.7	-	128.7
4	7.82 (dd, 7.8, 1.2)	127.8	7.83 (d, 7.8)	128.0
5	7.45 (dd, 7.8, 7.2)	128.9	7.44 (dd, 7.8, 7.8)	128.7
6	7.64 (d, 7.2)	134.5	7.57 (d, 7.8)	134.0
7	3.38 (dd, 14.4, 4.2) 3.30 (dd, 14.4, 7.8)	36.4	3.37 (dd, 14.4, 4.8) 3.31 (dd, 14.4, 7.8)	36.4
8	5.36 (dd, 7.8, 4.2)	73.1	5.46 (dd, 7.8, 4.8)	72.9
9	-	170.4	-	169.5
10	-	165.2	-	165.1
9-OMe	-	-	3.66	52.3
10-OMe	3.83 (s)	52.2	-	-
1′	-	130.9	-	130.9
2′,6′	7.94 (m)	129.4	7.95 (m)	129.4
3′,5′	7.48 (m)	128.9	7.49 (m)	129.0
4′	7.65 (m)	133.8	7.67 (m)	134.0
7′	-	165.2	-	165.1

^a measured in DMSO-d₆ at 600 MHz.

). The ¹H/¹³C-NMR and 135DEPT data revealed one 1,3-disubstituted benzene ring δ_H 8.00 (1H, br s, H-2), 7.82 (1H, dd, J = 7.8, 1.2 Hz, H-4), 7.64 (1H, d, J = 7.2 Hz, H-6), 7.45 (1H, dd, J = 7.8, 7.2 Hz, H-5) and δ_C 137.3 (C-1), 134.5 (C-6), 130.4 (C-2), 129.7 (C-3), 128.9 (C-5), 127.8 (C-4); one benzoyl group δ_H 7.94 (2H, m, H-2′,6′), 7.65 (1H, m, H-4′), 7.48 (2H, m, H-3′,5′) and δ_C 165.2 (C-7′), 133.8 (C-4′), 130.9 (C-1′), 129.4 (C-2′,6′), 128.9 (C-3′,5′); one oxygenated methine proton δ_H 5.36 (1H, dd, J = 7.8, 4.2 Hz, H-8) and δ_C 73.1 (C-8); one methoxy group δ_H 3.83 (3H, s, 10-OMe), δ_C 52.2 (10-OMe); one methylene group δ_H 3.38 (1H, dd, J = 14.4, 4.2 Hz, H-7b), 3.30 (1H, dd, J = 14.4, 7.8 Hz, H-7a) and δ_C 36.4 (C-7); two carboxyl carbons δ_C 170.4 (C-9) and 166.3 (C-10). The ¹H-¹H COSY correlation of H-7/H-8 combined with the HMBC correlations from H-7 and H-8 to C-9 indicated the presence of fragments from C-7 to C-9 (Figure 2). The HMBC correlations of H-2/4 with C-10 and H-7 with C-1/2/6 suggested the linkages of C-3–C-10 and C-7–C-1. The HMBC correlations of H-8 with C-7′ indicated that the benzoyl group was linked at C-8 by an ester bond. The location of the methoxy group at C-10 was confirmed by the HMBC correlation between 10-OMe and C-10. Therefore, the planar structure of 1 was determined. Owing to a putative shared biosynthesis pathway with 1 and 6, the absolute configuration of 1 was tentatively assigned as 8R-form. To substantiate the aforementioned conclusion, the ECD spectra of 1 and 6 were analyzed, revealing comparable characteristics within the 200–400 nm range (see Figure S35). Thus, compound 1 was identified as a rare carboxyl-substituted phenylpropionic acid. We named compound 1 plumeriapropionic A.

Compound 2 was also obtained as white amorphous powder and determined to be C₁₈H₁₆O₆ by HRESIMS spectrum (m/z 327.0878 [M-H]⁻; calcd for C₁₈H₁₅O₆, 327.0874), corresponding to 11 degrees of unsaturation. The first preliminary investigation of its ¹H and ¹³C NMR showed that 2 was closely related to 1. The differences in chemical shift were at the methoxy group (δ_H 3.83 for 1 vs. 3.66 for 2). The location of the methoxy group at C-9 was confirmed by the HMBC correlations from 9-OMe to C-9. This indicates that the methoxy group is substituted at C-9 instead of C-10 in compound 2. Detailed analysis of 2D NMR (HSQC, ¹H-¹H COSY, and HMBC) spectra confirmed that the remaining parts of the molecule were identical to those observed in compound 1. Compound 2 has a similar optical rotation value to compounds 1 and 6. The ECD spectra of 2 and 6 showed similar features in the 200–400 nm range (Figure S36). Therefore, the stereostructure of C-8 was also assigned as the R-form. Thus, compound 2 was also identified as a rare carboxyl-substituted phenylpropionic acid. We named compound 2 plumeriapropionic B.

The molecular formula of 3 was determined as C₁₂H₁₄O₅ based on the HRESIMS (m/z 239.0911 [M + H]⁺; calcd 239.0914). Its ¹H/¹³C-NMR data (

Table 2. ¹H and ¹³C NMR spectral data of compounds 3–5.

Position	3 a		4 a		5 a
	δH, Mult, (J in Hz)	δC	δH, Mult, (J in Hz)	δC	δH, Mult, (J in Hz)	δC
1	-	138.5	-	139.9	-	138.3
2	7.83 (s)	130.2	7.84 (s)	130.7	7.81 (s)	130.5
3	-	129.5	-	129.8	-	130.7
4	7.81 (d, 8.0)	127.2	7.79 (d, 7.6)	127.4	7.80 (d, 8.0)	127.5
5	7.42 (dd, 8.0, 8.0)	128.5	7.41 (dd, 7.6, 7.6)	128.8	7.39 (dd, 8.0, 7.6)	128.4
6	7.49 (d, 8.0)	134.4	7.51 (d, 7.6)	134.9	7.45 (d, 7.6)	134.0
7	3.03 (dd, 14.0, 4.8) 2.90 (dd, 14.0, 8.4)	39.7	3.04 (dd, 13.6, 4.0) 2.81 (dd, 13.6, 8.4)	40.3	3.02 (dd, 13.6, 4.8) 2.90 (dd, 13.6, 8.0)	39.9
8	4.29 (dd, 8.4, 4.8)	71.0	4.06 (m)	71.6	4.28 (dd, 8.0, 4.8)	71.1
9	-	173.8	-	175.6	-	173.9
10	-	166.4	-	166.9	-	167.6
9-OMe	3.62 (s)	51.5	-	-	3.61 (s)	51.5
10-OMe	3.84 (s)	52.1	3.84 (s)	52.5	-	-

^a measured in DMSO-d₆ at 400 MHz.

) closely resembled those of cerberic acid B (6), except for the presence of two methoxy group signals (δ_H/C 3.84/52.1 and 3.62/52.1) in 3. The location of the two methoxy groups at C-9 and C-10 was confirmed by the HMBC correlations from 9-OMe to C-9 and from 10-OMe to C-10. Detailed analysis of 2D NMR (HSQC, ¹H-¹H COSY, and HMBC) spectra confirmed that the other parts of the molecule were the same as those of 6. Compound 3 has the 8R-form based upon the positive optical rotation of 3 and the reported similar compounds in the literature [13,14]. It was also confirmed by the ECD spectra of 1 and 6. The ECD spectra of 3 and 6 showed similar features in the 200–400 nm range (Figure S37). Thus, compound 3 was identified as a rare carboxyl-substituted phenylpropionic acid and named plumeriapropionic C.

Compound 4 was also isolated as a white amorphous powder. Its molecular formula was determined to be C₁₁H₁₂O₅ by HRESIMS data at m/z 223.2028 (calcd 223.2026 for C₁₂H₁₁O₅). The ¹H^/13C NMR data closely resemble that of 3 except for the absence of a methoxy group signal (δ_H/C 3.62/51.5). It also can be confirmed by comparing the chemical shift of 4 with compound 3 at C-9 (δ_C 173.8 for 3 vs. 175.6 for 4). Detailed analysis of 2D NMR (HSQC, ¹H-¹H COSY, and HMBC) spectra confirmed that the other parts of the molecule were the same as those of 3. Owing to a putative shared biosynthesis pathway with 1–3 and 6, the absolute configuration of 4 was tentatively assigned as the 8R-form. The ECD spectra of 4 and 6 showed similar features in the 200–400 nm range (Figure S38). The absolute configuration of 4 was also determined as the 8R-form based on the positive optical rotation. Thus, compound 4 was also identified as a rare carboxyl-substituted phenylpropionic acid and named plumeriapropionic D.

Compound 5 was also obtained as a white amorphous powder. Its molecular formula of C₁₁H₁₂O₅ was determined by HRESIMS data at m/z 223.2021 (calcd 223.2026 for C₁₂H₁₁O₅). Its ¹H and ¹³C-NMR data also closely resembled those of compound 3 except for the absence of a methoxy signal (δ_H/C 3.84/52.1) in 5. This was confirmed by comparing the chemical shift at C-10 of compound 5 with that of compound 3 (δ_C 166.4 for 3 vs. 167.6 for 5). The absolute configuration of 5 was also determined as the 8R-form based on the positive optical rotation and the ECD spectra of 5 and 6 (Figure S39). Thus, compound 5 was also identified as a rare carboxyl-substituted phenylpropionic acid and named plumeriapropionic E.

In addition, the known carboxyl-substituted phenylpropionic acid 6 was isolated and identified as cerberic acid B [13] by comparing the experimental spectral data with the reported spectra data in the literature.

2.2. Anti-Inflammatory Activity

All carboxyl-substituted phenylpropionic acids 1–6 were evaluated for their anti-inflammatory effects by testing their inhibitory activities against NO production by LPS in mouse macrophage RAW 264.7 cells in vitro. The MTT assay was used for measuring the cytotoxic activities of compounds 1–6 against mouse macrophage RAW 264.7 cells. The results are shown in

Table 3. Anti-inflammatory activities of compounds 1–6.

Compound	IC50 (µM)	Compound	IC50 (µM)
1	28.26 ± 0.15	4	33.16 ± 0.18
2	6.52 ± 0.23	5	7.13 ± 0.16
3	35.68 ± 0.17	6	6.68 ± 0.22
Hydrocortisone a	5.61 ± 0.12

^a Positive control.

. Compounds 2, 5, and 6 exhibited especially potent inhibitory activities against NO production, with IC₅₀ values of 6.52 ± 0.23, 7.13 ± 0.16 and 6.68 ± 0.22, respectively. Compounds 1, 3, and 4 showed weaker activities than 2, 5, and 6. These results suggest that the carboxylic group at C-3 can be important for anti-inflammatory activity. In addition, no cytotoxicity was observed in the macrophage RAW 264.7 cells treated with compounds 1–6 (cell viability > 95%)

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotations of 1–6 were measured on a JASCO P-1020 digital polari meter (Jasco Corp., Tokyo, Japan). The NMR spectra of compounds 3–6 were recorded on a Bruker AV spectrometer (400 MHz for ¹H and 100 MHz for ¹³C, Bruker Corp., Karlsruhe, Germany). The NMR spectra of compounds 1 and 2 were recorded on a JEOL JEM-ECP NMR spectrometer (600 MHz for ¹H and 150 MHz for ¹³C, JEOL, Tokyo, Japan). HRESIMS spectra of compounds 1–5 were measured on a Q-TOF Ultima Global GAA076 LC mass spectrometer (Waters Corp., Milford, MA, USA). CD spectra were recorded on a MOS-450 spectrometer. Semi-preparative HPLC was performed on an Agilent 1260 LC (Agilent Corp., Santa Clara, CA, USA) series with a DAD detector using an Agilent Eclipse XDB-C₁₈ (Agilent Corp., Santa Clara, CA, USA) column (9.4 × 250 mm, 5 μm). Silica gel ODS and Sephadex LH-20 (Qing Dao Hai Yang Chemical Group Co., Qingdao, China) were used for open-column chromatography (CC). Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co., Yan Tai, China; G60, F-254) were utilized for thin-layer chromatography (TLC).

3.2. Plant Material

The flowers of P. rubra (Apocynaceae) were collected from Haikou City, Hainan Province, China, in April 2022 and were authenticated by Professor Yu-Kai Chen (School of Hainan Normal University, Hainan, China). The specimens (No JDH20220428) were deposited at the Key Laboratory of Tropical Medicinal Resource Chemistry of the Ministry of Education, Hainan Normal University (Hainan, China).

3.3. Extraction and Isolation

The air-dried flowers of P. rubra (5.3 kg) were extracted with 75% EtOH (4 × 20 L) at room temperature. A dark brown crude extract (0.66 kg) was obtained after concentration in vacuo to remove most of the EtOH. The EtOH extract (87 g) was subjected to silica gel CC (100–200 mesh) eluted by petroleum ether/ethyl acetate (100:0 to 0:100, v/v) to afford nine major fractions (Fr. 1–9). Fr. 3 exhibited an inhibitory effect against NO production induced by LPS in mouse macrophage RAW 264.7 cells. Therefore, systematic separation and purification were carried out against this fraction. The Fr. 3 (32 g) was separated using a silica gel column and eluted with gradient mixtures of petroleum ether/ethyl acetate (10:1 to 1:1, v/v) to obtain five fractions (Fr. 3-1–3-5). Fr. 3-2 (4.8 g) was purified on Sephadex LH-20 (CHCl₃:MeOH, 1:1) and further isolated with semi-preparative HPLC (RP C₁₈ column, 2.5 mL/min, detected at 210, 230, 254, and 280 nm, CH₃CN/H₂O, 30:70 v/v) to obtain 1 (12 mg), 2 (15 mg), and 3 (21 mg). Fraction Fr. 3-3 (5.2 g) was subjected to ODS CC and eluted with MeOH/H₂O (25:75), and further isolated using semi-preparative HPLC (RP C₁₈ column, 2.5 mL/min, detected at 210, 230, 254, and 280 nm, CH₃CN/H₂O, 25:75 v/v) to obtain 4 (5 mg) and 5 (25 mg). Fr. 3-4 (5.6 g) was repeatedly purified with ODS CC and eluted with MeOH/H₂O (15:85) to obtain compound 6 (58 mg).

Plumeriapropionic A (1): white amorphous powder; [α]²⁵_D + 18.4 (c 0.10, MeOH); CD (c 1.0 × 10⁻⁴, MeOH) λ_max (Δε) 288 (3.23), 262 (2.83), 250 (−6.69), 240 (15.99), 223 (26.01); IR (KBr) ν_max 3326, 1741, 1708 and 1681 cm; ¹H-NMR (600 MHz, DMSO-d₆) and ¹³C-NMR (150 MHz, DMSO-d₆), see Table 1; HRESIMS m/z 327.0876 (calcd for C₁₈H₁₅O₆, 327.0874).

Plumeriapropionic B (2): white amorphous powder; [α]²⁵_D + 22.1 (c 0.10, MeOH); CD (c 1.0 × 10⁻⁴, MeOH) λ_max (Δε) 283 (3.36), 260 (2.92), 248 (−4.73), 237 (25.44), 219 (58.31); IR (KBr) ν_max 3328, 1742, 1706 and 1685 cm; ¹H-NMR (600 MHz, DMSO-d₆) and ¹³C-NMR (150 MHz, DMSO-d₆), see Table 1; HRESIMS m/z 327.0878 (calcd for C₁₈H₁₅O₆, 327.0874).

Plumeriapropionic C (3): white amorphous powder; [α]²⁵_D + 13.2 (c 1.0, MeOH); CD (c 1.0 × 10⁻⁴, MeOH) λ_max (Δε) 264 (1.02), 253 (−2.18), 238 (8.38), 218 (6.81); IR (KBr) ν_max 3334, 1744, 1738 and 1680 cm; ¹H-NMR (400 MHz, DMSO-d₆) and ¹³C-NMR (100 MHz, DMSO-d₆), see Table 2; HRESIMS m/z 239.0911 (calcd 239.0914 for C₁₂H₁₅O₅).

Plumeriapropionic D (4): white amorphous powder; [α]²⁵_D + 11.1 (c 1.0, MeOH); CD (c 1.0 × 10⁻⁴, MeOH) λ_max (Δε) 284 (5.51), 260 (3.57), 248 (−9.26), 238 (12.54), 223 (23.43); IR (KBr) ν_max 3336, 1743, 1682 and 1595 cm; ¹H-NMR (400 MHz, DMSO-d₆) and ¹³C-NMR (100 MHz, DMSO-d₆), see Table 2; HRESIMS m/z 223.2028 (calcd 223.2026 for C₁₁H₁₁O₅).

Plumeriapropionic E (5): white amorphous powder; [α]²⁵_D + 12.3 (c 1.0, MeOH); CD (c 1.0 × 10⁻⁴, MeOH) λ_max (Δε) 287 (5.31), 264 (6.90), 253 (−0.30), 242 (4.75), 233 (−8.16), 220 (24.19); IR (KBr) ν_max 3335, 1737, 1681 and 1597 cm; ¹H-NMR (400 MHz, DMSO-d₆) and ¹³C-NMR (100 MHz, DMSO-d₆) see Table 2; HRESIMS m/z 223.2021 (calcd 223.2026 for C₁₁H₁₁O₅).

Cerberic acid B (6): white amorphous powder; [α]²⁵_D + 9.8 (c 1.0, MeOH); CD (c 1.0 × 10⁻⁴, MeOH) λ_max (Δε) 285 (10.54), 261 (12.80), 252 (0.57), 239 (44.49), 220 (50.97), 206 (−38.00).

3.4. Anti-Inflammatory Bioassays

All isolated compounds 1–6 were evaluated for their inhibition of NO production in RAW264.7 cells activated by LPS using the Griess assay with hydrocortisone as a positive control [15,16]. All experiments were carried out in triplicate, and each experiment was repeated three times. Data analysis was carried out using SPSS statistical package version 22.0 (SPSS, Inc., Chicago, IL, USA). The IC₅₀ values (the concentration of drug necessary to induce 50% inhibition) were measured for all of the tested drugs using the Probit test in SPSS software.

4. Conclusions

In this study, the phytochemisty study on the flowers of P. rubra was carried out and led to the isolation of five rare carboxyl-substituted phenylpropionic acids, plumeriapropionics A–E (1–5), together with one known analog, cerberic acid B (6). Carboxyl-substituted phenylpropanoid is very rare in the plant kingdom. So far, only one compound of this structural type has been reported. The discovery of five rare carboxyl-substituted phenylpropionic acids 1–5 can help to extend the phytochemical knowledge of the genus Plumeria. All isolated compounds were investigated for their anti-inflammatory effects and were proven to be useful. Compounds 2, 5, and 6 showed notable inhibitory effects against NO production. These isolated carboxyl-substituted phenylpropionic acids, with potent inhibitory activities on NO production, could be used for the development of new anti-inflammatory agents.