Carabrachiatanins A and B: Two New Phenylpropanoid-Substituted Catechins of Carallia brachiata Merr

Abstract

Objectives: Carallia brachiata Merr. is a Vietnamese medicinal plant, used to treat mouth ulcers, tongue sores, sore throats, and malaria. This paper focuses on the chemical constituents of C. brachiata and their inhibition of NO production. Methods: The dried and minced sample was extracted with methanol. The crude extract was isolated by various chromatographic methods. The structures of the isolates were determined by HR-ESI-MS, NMR, and CD spectra. The NO inhibition activity was evaluated in LPS-activated RAW264.7 cells. Results: Two new catechin derivatives (carabrachiatanins A (1) and B (2)) and seven known proanthocyanidins (3-9) were isolated from C. brachiata by bioguided fractionation. All isolates significantly showed NO inhibition effects with IC₅₀ values ranging from 13.72 ∼ 34.28 µM. Conclusions: Two new and seven known compounds were isolated from C. brachiata. This is the first report of compounds 3-9 from C. brachiata. Compounds 7 and 8 inhibited NO production with their IC₅₀ values of 16.31 ± 0.61 µM and 16.53 ± 0.83 µM, respectively, compared to that of dexamethasone, which showed IC₅₀ value of 14.94 ± 1.11 µM.

Keywords

rhizophoraceae catechin derivative proanthocyanidin nitric oxide inhibition carabrachiatanin

Introduction

Carallia brachiata Merr. belongs to the family Rhizophoraceae (Vietnamese name is “săng mã”).¹ The leaves and wood of this plant have been used for a long time in Vietnamese folk medicine to treat mouth ulcers, tongue sores, sore throats, and malaria. The previous publications have reported megastigmane, 1,2-dithiolane derivative, flavonoids, tannins, glyceroglycolipids, aromatic compounds, alkaloid, and lignans isolated from this plant.^2-5 Some of these compounds showed antiinflammatory and antimicrobial activity.^2-5 The present paper reports two new (1 and 2) and seven known compounds (3-9) isolated from the leaves and branches of C. brachiata. In addition, their effects on NO production in LPS-activated RAW264.7 cells were evaluated and discussed herein.

Results and Discussion

The dried leaves and branches of C. brachiata were extracted with methanol. The methanolic extract was suspended with water and then successively separated with n-hexane and ethyl acetate to give n-hexane soluble fraction, ethyl acetate soluble fraction, and water-soluble fraction. These fractions were then evaluated for their NO inhibitory activity to give an active fraction (ethyl acetate fraction). The ethyl acetate fraction was subjected to isolation and purification by chromatographic methods to obtain nine secondary metabolites (1-9). The known compounds were identified to be ent-epiafzelechin-(2α→O→7,4α→8)-ent-epiafzelechin-(2α→O→7,4α→8)]-ent-afzelechin (3),⁶ carallidin (4),⁷ mahuannin C (5),^8,9 geranin D (6),¹⁰ cinchonain (7),¹¹ epiafzelechin-(4β→8,2β→O→7)-ent-afzelechin (8),¹² ent-epiafzelechin-(2α→O→7,4α→8)-ent-afzelechin (9).¹³ The chemical structures of these compounds were determined by HR-ESI-MS, 1D and 2D NMR, and CD spectra in comparison to those data reported in the literature.

The HR-ESI-MS of 1 exhibited an ion peak at m/z 481.1113 [M-H]⁻ (calcd. for C₂₅H₂₁O₁₀: 481.1135) indicating its molecular formula of C₂₅H₂₂O₁₀ with 15 degrees of saturation. The ¹H NMR spectrum of 1 showed signals due to an ABX coupled aromatic protons [δ_H 6.76 (1H, dd, 8.0, 1.8 Hz, H-6′), 6.80 (1H, d, 8.0 Hz, H-5′), and 6.87 (1H, d, 1.8 Hz, H-2′)], three singlet aromatic protons [δ_H 6.13 (H-6), 6.48 (H-3″), and 6.66 (H-6″)], two methine carbinol protons [δ_H 4.76 (d, 6.6 Hz, H-2) and 4.04 (ddd, 7.8, 6.6, 4.8 Hz, H-3)], two sp³ methylene groups [δ_H 2.85 (dd, 16.2, 4.8 Hz)/2.62 (16.2, 7.8 Hz, H₂-4) and 2.49 (dd, 14.4, 7.2 Hz and 2.65 (dd, 14.4, 4.8 Hz), H₂-10)], a sp³ methine group (δ_H 4.46, dd, 7.2, 4.8 Hz, H-9), and a methoxy group (δ_H 3.40, 3H, s). The ¹³C NMR spectrum of 1 indicated 25 carbons including 15 of the catechin part, 9 of a phenylpropanoid moiety, and a methoxy group (Table 1).¹⁴ A carboxylate group was identified at δ_H 174.3, and C-2, C-3, and C-4 of the catechin part were identified at δ_C 82.7 (CH), 68.5 (CH), and 28.3 (CH₂), respectively. The NMR data of 1 was closely resembling those of catiguanin A suggesting that they have the same planar structure.¹⁴ This was further confirmed by HSQC, COSY, and HMBC correlations as shown in Figure 2. However, the carbon chemical shifts at C-2 (82.7), C-3 (68.5), and C-4 (28.3) of 1 were different from those of catiguanin A [C-2 (80.1), C-3 (66.9), C-4 (29.5)]. In addition, the large J_2,3 value in 1 (6.6 Hz) compared to broad singlet of H-2 in catiguanins A and B suggested trans configuration of H-2 and H-3.¹⁴ Furthermore, the NOESY spectrum of 1 showed NOEs correlation between H-9 (δ_H 4.46) and H-2′ (δ_H 6.87) and between H-3 (4.04 and H-6′ (6.76). This evidence indicated H-9, H-2′, and H-3 were on the same side of the molecule. The absolute configuration of 1 was revealed by comparison of its experimental CD spectrum with the theoretical calculation spectra for two possible enantiomers, (2R,3S,9R)-1a and (2S,3R,9S)-1b. The experimental CD spectrum of 1 observed Cotton effects with a maximum positive sign (+31.2 mdeg) at a wavelength of 202 nm and a minimum negative sign (−10.9 mdeg) at a wavelength of 236 nm, which consisted of the calculated ECD spectrum of (2R,3S,9R)-enantiomer and opposed to (2S,3R,9S)-enantiomer (Figure 4). Thus, the chemical structure of 1 was determined as shown in Figure 1, by a new compound named carabrachiatanin A (Figures S1-S9).

Figure 1.

Structures of 1-9 from Carallia brachiata.

Figure 2.

Key HMBC and COSY interactions of 1 and 2.

Table 1.

¹H and ¹³C NMR Data for 1 and 2.

Pos.	1		2			Glabraoside D¹⁵
Pos.	δ _C ^a	δ_H^a (mult., J in Hz)	δ _C ^a	δ_H^a (mult., J in Hz)	δ _C ^b	δ _C ^b	δ_H^b (mult., J in Hz)
2	82.7	4.76 (d, 6.6)	80.9	4.82 (d, 7.5)	78.3	79.4	4.76 (d, 7.5)
3	68.5	4.04 (ddd, 7.8, 6.6, 4.8)	75.6	4.01 (ddd, 7.8, 7.5, 5.4)	71.8	72.3	3.84 (m)
4	28.3	2.85 (dd, 16.2, 4.8) 2.62 (dd, 16.2, 7.8)	27.6	2.87 (dd, 16.2, 5.4) 2.78 (dd, 16.2, 7.8)	26.0	26.5	2.76 (dd, 16.0, 5.5) 2.56 (dd, 16.0, 8.5)
4a	103.9	-	103.8	-	102.1	102.1	-
5	156.1	-	156.1	-	154.5	154.5	-
6	95.9	6.13 (s)	96.0	6.14 (s)	94.6	94.6
7	153.1	-	153.1	-	150.8	150.9	-
8	104.3	-	104.2	-	102.0	101.9	6.11 (s)
8a	153.1	-	153.1	-	151.3	151.4	-
9	31.4	4.46 (dd, 7.2, 4.8)	31.4	4.46 (dd, 7.2, 4.8)	29.3	29.2	4.20 (dd, 8.5, 3.5)
10	44.9	2.49 (dd, 14.4, 7.2) 2.65 (dd, 14.4, 4.8)	45.1	2.50 (dd, 13.8, 7.2) 2.58 (dd, 13.8, 4.8)	43.4	43.0	2.40 (dd, 14.5, 8.5) 2.71 (dd, 14.5, 3.5)
11	174.3	-	174.2	-	171.2	171.3	-
1′	132.2	-	131.8	-	129.7	129.7	-
2′	114.9	6.87 (d, 1.8)	114.8	6.88 (d, 1.8)	113.6	113.9	6.76 (d, 2.0)
3′	146.3	-	146.4	-	145.0	145.1	-
4′	146.2	-	146.3	-	144.9	145.1	-
5′	116.2	6.80 (d, 8.0)	116.1	6.80 (d, 8.0)	115.3	115.2	6.70 (d, 8.0)
6′	119.8	6.76 (dd, 8.0, 1.8)	119.6	6.76 (dd, 8.0, 1.8)	117.5	117.8	6.63 (dd, 8.0, 2.0)
1″	116.1	-	116.2	-	113.6	113.3	-
2″	146.6	-	146.5	-	143.9	144.0	-
3″	104.2	6.48 (s)	104.3	6.48 (s)	103.2	103.2	6.40 (s)
4″	146.0	-	146.1	-	144.8	144.8	-
5″	142.3	-	142.3	-	141.1	141.1	-
6″	115.3	6.60 (s)	115.2	6.66 (s)	114.1	114.1	6.51 (s)
OCH₃	51.9	3.40 (s)	51.9	3.44 (s)	51.0	51.1	3.46 (s)
3-O-Rha
1″′			102.1	4.39 (br s)	99.7	100.0	4.13 (br s)
2″′			72.1	3.60 (m)	70.3	70.2	3.32 (m)
3″′			72.3	3.62 (dd, 9.0, 3.0)	70.5	70.5	3.32 (m)
4″′			74.0	3.82 (m)	71.8	71.8	3.12 (t, 9.0)
5″′			70.4	3.68 (m)	68.9	68.9	3.46 (m)
6″′			17.9	1.27 (d, 6.6)	17.8	17.8	1.11 (d, 6.0)

Measured in ^aMethanol-d₄ and ^bDMSO-d_6.

The HR-ESI-MS spectrum of 2 exhibited ion peaks at m/z 627.1712 [M-H]⁻ (calcd. for C₃₁H₃₁O₁₄: 627.1714) and m/z 663.1474 [M + Cl]⁻ (calcd. for C₃₁H₃₂O₁₄Cl: 663.1481) confirming its molecular formula of C₃₁H₃₂O₁₄. The NMR spectra of 2 were very similar to those of 1 except for additional signals due to a rhamnopyranose sugar [δ_C 102.1, 74.0, 72.3, 72.1, 70.4 (5xCH) and 17.9 (CH₃); δ_H 4.39 (brs, H-1″′), 3.60-3.80 (4H), and 1.27 (3H, d, 6.6 Hz, H-6″′)] (Table 1). The NMR assignments of 2 were revealed by HSQC, COSY, and HMBC as shown in Figure 2. The sugar moiety was attached to C-3 as evident by HMBC correlations from H-1″′ to C-3 (δ_C 75.6) and from H-3 (δ_H 4.01) to C-1″′ (δ_C 102.1). The methoxy group linked to C-11 (C = O) as determined by HMBC correlation from methoxy protons (δ_H 3.44) to C-11 (δ_C 174.2). The large J_2,3 value (7.5 Hz) in 2 was similar to that of glabraoside D (H-2/H-3 trans)¹⁵ and differed from cis-configuration as in catiguanins A and B (broad singlet, J_2,3∼0 Hz) suggested trans configuration of H-2 and H-3.^14,15 The NOESY cross-peaks from H-9 (δ_H 4.46) to H-2′ (δ_H 6.88) and from H-3 (δ_H 4.01) to H-6′ (δ_H 6.76) were observed indicating that these protons were in the same side of the molecule (Figure 3).¹⁴ The above data suggested the planar structure of 2 was similar to that of glabraoside D.¹⁵ However, the relative and the absolute configuration of glabraoside D were not determined. To know if compound 2 was glabraoside D, the NMR spectra of compound 2 were measured in DMSO-d₆, the same solvent as glabraoside D (Table 1).¹⁵ In this case, the NMR data of 2 did not match the NMR data of glabraoside D (Table 1). Therefore, the absolute configuration of 2 was revealed by the experimental CD spectrum. The CD spectrum of 2 observed Cotton effects with a maximum positive sign (+20.4 mdeg) at a wavelength of 206 nm and a minimum negative sign (−9.3 mdeg) at a wavelength of 242 nm, which were similar to those of 1 suggesting the same 2R, 3S, and 9R configurations. The broad singlet observed for anomeric proton at δ_H 4.39 suggested α-form of the glycosyl linkage. The presence of a L-rhamnose sugar in 2 was determined by acid hydrolysis.¹⁶ Thus, the chemical structure of 2 was determined as shown in Figure 1, by a new compound named carabrachiatanin B. (Figures S10-19).

Figure 3.

Key NOESY correlations of 1 and 2.

Figure 4.

Experimental CD of 1 and 2 and calculated curves for possible enantiomers (2R,3S,9R)-1a and (2S,3R,9S)-1b.

Continuing the bio-guided fractionation, the isolates were further screened for their NO production effects in LPS-activated RAW264.7 cells. At a concentrated solution of 100 µM, except for compounds 3 and 4, other compounds did not significantly show cytotoxicity to the cells (Table S1). Therefore, the NO inhibitory effects of compounds 1, 2, 5-9 were not caused by cytotoxicity. Compounds 1, 2, 5-8 exhibited inhibitory activity over 50% (from 70.62 ± 2.18% to 97.63 ± 2.01%) and were further performed dose-dependent study at diluted concentrations of 0.8-100 µM. As shown in Table 2, compounds 7 and 8 highly inhibited NO production with their IC₅₀ values of 16.31 ± 0.61 µM and 16.53 ± 0.83 µM, respectively, which are similar to the inhibitory level of dexamethasone (IC₅₀ 14.94 ± 1.11 µM). Therefore, further study on antiinflammatory activity of compounds 7 and 8 should be continued. The above results suggested that proanthocyanidins and phenylpropanoid-substituted catechins play important roles in NO inhibition activity of this plant.

Table 2.

NO Production Inhibitory Effects of the Isolates on the RAW264.7 Cell.

Comp.	IC₅₀ (µM)
1	43.18 ± 1.69
2	51.91 ± 2.83
5	26.24 ± 1.55
6	33.58 ± 1.74
7	16.31 ± 0.61
8	16.53 ± 0.83
9	>100
Dex*	14.94 ± 1.11

*Dexamethasone was used as a positive control.

Material and Methods

General

The equipment and experimental procedures are provided in supporting materials.

Plant Materials

Carallia brachiata Merr. (leaves and branches) was collected in Trieu Son, Thanh Hoa in March 2021 and identified by Dr Do Van Hai at the Institute of Ecology and Biological Resources, VAST. The specimen (VAST.04.02/22-23) was kept at the Institute of Marine Biochemistry, VAST.

Extraction and Purification

This section is provided in supporting materials.

Carabrachiatanin A (1)

A yellow solid; $[α]_{D}^{25}$ : −28.5 (c 0.1, MeOH); CD (MeOH) λ (θ): 202 (+31.2), 236 (−10.9) mdeg; HR-ESI-MS m/z 481.1113 [M-H]⁻ (calcd. for C₂₅H₂₁O₁₀: 481.1135), m/z 517.0888 [M + Cl]⁻ (calcd. for C₂₅H₂₂O₁₀Cl: 517.0901), m/z 483.1282 [M + H]⁺ (calcd. for C₂₅H₂₃O₁₀: 483.1291), m/z 505.1095 [M + Na]⁺ (calcd. for C₂₅H₂₂O₁₀Na: 517.1111); ¹³C NMR (150 MHz) and ¹H NMR (600 MHz) data were given in Table 1.

Carabrachiatanin B (2)

A yellow amorphous solid; $[α]_{D}^{25}$ : −31.7 (c 0.1, MeOH); CD (MeOH) λ (θ): 206 (+20.4), 242 (−9.3) mdeg; HR-ESI-MS m/z 627.1712 [M-H]⁻ (calcd. for C₃₁H₃₁O₁₄: 627.1714), m/z 663.1474 [M + Cl]⁻ (calcd. for C₃₁H₃₂O₁₄Cl: 663.1481); ¹³C NMR (150 MHz) and ¹H NMR (600 MHz) data were given in Table 1.

Calculation Theoretical CD spectra

An enantiomer (2R,3S,9R)-1a was generated and conformationally searched by Spartan′18 program. Twenty conformers having the lowest molecular energy and over 1% Boltzmann distribution in Merck molecular force field calculation were extracted and subjected for calculation of their CD curves using Gaussian 16 (Revision C.01 for Windows Operation). At first, each conformer was optimized with DFT/B3LYP density functional and 6-31G(d,p) polarization. Methanol was chosen to calculate the solvent effect in the IEFPCM model. Next, the optimized conformers were subjected for TD-DFT calculation with wb97xd density functional and 6-31G(d,p) polarization. Methanol was set as solvent effect in the IEFPCM model and CD spectrum was calculated at 30 excited states. The CD spectrum of each conformer was then extracted and combined together based on their Boltzmann weighting factor using the SpecDis 1.71 tool.

Conclusions

Two new phenylpropanoid-substituted catechins (1 and 2) and seven proanthocyanidins (3-9) were isolated from the leaves and branches of C. brachiata. The chemical structures of the isolates were elucidated by HR-ESI-MS, 1D and 2D NMR spectra. The absolute configurations of compounds 1 and 2 were determined based on their experimental CD spectra comparison with the theoretical calculation CD spectra. Compounds 1, 2, 5-8 significantly showed NO inhibition in LPS-activated RAW264.7-cells with IC₅₀ values ranging from 16.31 ∼ 51.91 µM. These results suggested that further investigation of antiinflammatory activity should be continued.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231219074 - Supplemental material for Carabrachiatanins A and B: Two New Phenylpropanoid-Substituted Catechins of Carallia brachiata Merr

Supplemental material, sj-docx-1-npx-10.1177_1934578X231219074 for Carabrachiatanins A and B: Two New Phenylpropanoid-Substituted Catechins of Carallia brachiata Merr by Trinh Thi Thanh Van, Nguyen Hoang Nam, Nguyen Thi Hue, Le Nguyen Thanh, Do Thi Trang, Bui Huu Tai, Nguyen Le Tuan, Nguyen Thi Viet Ai, Pham Van Cuong, Nguyen Quoc Vuong and Phan Van Kiem in Natural Product Communications

Footnotes

Acknowledgments

We would like to thank Physician Bui Thi Vui, at Trieu Thanh, Trieu Son, Thanh Hoa for providing the plant sample and Dr Do Van Hai for the plant identification.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: We would like to thank Vietnam Academy of Science and Technology for financial support (VAST 04.02/22-23).

Ethical Approval

Our institution does not require ethical approval for reporting individual cases or case series.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

ORCID iDs

Nguyen Thi Hue

Bui Huu Tai

Pham Van Cuong

Phan Van Kiem

Supplemental Material

Supplemental material for this article is available online.

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