Alkaloids and Iridoids From the Aerial Parts of Rauvolfia cambodiana Pierre ex Pit. and Their Acetylcholinesterase Inhibitory Activity

Abstract

Objective: Rauvolfia cambodiana has been used as a Vietnamese folk medicine to treat high blood pressure and for sedation and memory loss diseases. However, to date, there have been few studies on the chemical constituents and biological activity of this medicinal plant. This study aimed to find the chemical constituents of R cambodiana and evaluate their enhancement memory properties. Methods: The aerial parts of R cambodiana were powdered and ultrasonically extracted with methanol. The methanol extract was suspended in water and successively partitioned with dichloromethane and ethyl acetate to give dichloromethane, ethyl acetate, and water-soluble extracts. Each extract was fractionated and purified by column and HPLC chromatographic methods. The chemical structures of the isolated compounds were elucidated by analysis of HRESIMS, 1D and 2D NMR spectra, and experimental and TD-DFT calculated ECD spectra. The enhancement memory property of the compounds was evaluated by their in vitro acetylcholinesterase inhibitory activity. Results: Five alkaloids (1-5) and 2 iridoids (6 and 7), including one new one named rauvolcambonine (1), and 6 known compounds, perakine (2), ajmaline (3), 10-hydroxy-16-epi-affinine (4), sitsirikine (5), loganic acid (6), and secoxyloganin (7) were isolated and identified from the aerial parts of R cambodiana. Compounds 4 and 5 significantly showed acetylcholinesterase inhibitory effects in vitro with IC₅₀ values of 30.1 ± 1.4 µM and 65.5 ± 3.3 µM, respectively. The other compounds inhibited acetylcholinesterase with IC₅₀ values ranging from 111.0 ± 6.0 µM to 197.6 ± 19.8 µM. Conclusion: One new (1) and 3 other compounds (2, 4-7) were recorded from R cambodiana for the first time. The NMR spectroscopic data of compound 4 are reported herein for the first time. Our study preliminarily revealed that the vobasenal framework type alkaloids could be active ingredients of Rauvolfia species and responsibility for the enhancement memory property of these plants.

Introduction

Rauvolfia cambodiana Pierre ex Pit. (family Apocynaceae) is a shrub, up to 1.5 m tall and distributed in the wild in central Vietnam, such as Quang Tri, Thua Thien Hue, Quang Nam, Quang Ngai, Kon Tum, Gia Lai, and Dak Lak provinces. ¹ In Vietnamese traditional medicine, the stems, roots, and leaves of this plant are used to treat some diseases such as high blood pressure, sedation, and memory loss. ² The stem bark of this plant contains alkaloids (2.95%-6.40% of the dry weight), including isoreserpiline, reserpine, and ajmaline.^3‐5 However, to date, there have been few studies on the chemical constituents and biological activity of R cambodiana. This paper reports the isolation, structure determination, and acetylcholinesterase inhibition activity of one new (1) and 6 known compounds (2-7), including 5 alkaloids (1-5) and 2 iridoids (6 and 7) from the aerial parts of R cambodiana. The acetylcholinesterase inhibitory activity of the isolates is also evaluated and discussed herein.

Results and Discussion

Compound 1 was obtained as a pale-yellow solid. Its molecular formula, C₂₁H₂₄N₂O₃, was determined by HRESIMS (found m/z 353.1878 [M + H]⁺, calcd for [C₂₁H₂₅N₂O₃]⁺: 353.1860, Δ = +5.1 ppm; m/z 375.1667 [M + Na]⁺, calcd for [C₂₁H₂₄N₂O₃Na]⁺: 375.1679, Δ = −3.2 ppm). The ¹H NMR spectrum of 1 showed signals of a 1,2-substituted aromatic ring [δ_H 7.44 (d, J = 8.0 Hz), 7.21 (t, J = 8.0 Hz), 7.38 (t, J = 8.0 Hz), and 7.60 (d, J = 8.0 Hz)], 6 sp³ methine protons [δ_H 2.02 (m, H-20), 2.39 (ddd, J = 6.0, 6.0, 0.6 Hz, H-16), 2.60 (m, H-15), 3.24 (m, H-19), 3.59 (dd, J = 6.0, 5.4 Hz, H-5), and 5.02 (d, J = 0.6 Hz, H-17), 2 sp³ methylene groups [δ_H 2.78 (dd, J = 11.4, 4.8 Hz)/1.59 (d, J = 11.4 Hz) and 1.80 (m)/1.84 (m)], one oxygenated methylene group [δ_H 3.77 (dd, J = 9.6, 3.6 Hz, H_a-21)/3.88 (dd, J = 9.6, 5.4 Hz, H_b-21], an acetyl methyl group [δ_H 2.16 (s)], and a secondary methyl group [δ_H 1.22 (d, J = 7.2 Hz, H-18)]. The ¹³C NMR and HSQC spectra of 1 indicated 21 carbons, including 4 quaternary, 11 CH, 3 CH₂, 2 CH₃, and one carbonyl group. The NMR data of 1 were similar to those of raucaffrinoline isolated from Rauwolfia balansae.^6,7 However, the carbon chemical shifts of C-18, C-19, and C-20 of 1 (δ_C 14.3, 51.8, 41.8) were quite different from those of raucaffrinoline (δ_C 18.3, 53.2, 45.7), but similar to those of vinmajine B (14.0, 51.7, 41.6, respectively) ⁸ measured in the same solvent, CDCl₃ (Table 1). The above observation suggested the cis configuration of H-19 and H-20 in 1 (Figure 1). The COSY and key HMBC correlations of 1 were analyzed and are shown in Figure 2. This evidence verified that the chemical structure of 1 was almost similar to that of raucaffrinoline with the acetyl group linked to C-17 and the hydroxy group at C-21 (Table 1). The relative configurations of 1 were determined from the NOESY spectrum (Figure 3). The observed NOESY cross peaks of H-5 (δ_H 3.59)/H-18 (δ_H 1.22), H-18/H-21 (δ_H 3.77/3.88), and H-16 (δ_H 2.39)/H-21 confirmed that H-16, H-18, and H-21 were on one side of the molecule (β-orientation). In addition, the NOESY cross peak of H-3 (δ_H 4.15) and H-19 (δ_H 3.24) was clearly observed indicating that these protons were in an α-orientation. Finally, the absolute configurations of 1 were determined from its ECD spectrum, which showed identical Cotton effects with the TD-DFT calculated ECD spectrum of the enantiomer (3S,5S,7R,15S,16S,17R,19S,20S) (Figure 4). From the above evidence, the chemical structure of 1 was determined as shown in Figure 1, and named as rauvolcambonine.

Figure 1.

Chemical structures of 1-7 and 1a.

Figure 2.

Key ¹H-¹H COSY and HMBC correlations of 1.

Figure 3.

Key NOESY correlations of 1.

Figure 4.

Comparison between experimental and td-DFT calculated ECD spectra of 1 and its enantiomers.

Table 1.

¹H NMR and ¹³C NMR Spectroscopic Data for 1 and 1a.

Pos.	1		1a
	a,b δ C	a,cδH (mult, J in Hz)	a δ C 6
2	183.7	-	183.6
3	57.0	4.15 (d, 8.0)	57.2
5	51.2	3.59 (dd, 6.0, 5.4)	51.2
6	38.1	2.78 (dd, 11.4, 4.8) 1.59 (d, 11.4)	37.5
7	65.0	-	65.0
8	136.6	-	136.5
9	123.7	7.44 (d, 8.0)	123.8
10	125.4	7.21 (t, 8.0)	125.4
11	128.6	7.38 (t, 8.0)	128.6
12	120.9	7.60 (d, 8.0)	120.9
13	156.6	-	156.5
14	27.9	1.80 (dd, 10.8, 9.0) 1.84 (ddd, 10.8, 5.4, 1.8)	21.7
15	26.3	2.60 (dd, 9.0, 5.4)	26.6
16	43.5	2.39 (ddd, 6.0, 6.0, 0.6)	49.7
17	78.6	5.02 (d, 0.6)	78.2
18	14.3	1.22 (d, 7.2)	18.3
19	51.8	3.24 (dq, 9.6, 7.2)	53.2
20	41.8	2.02 (m)	45.7
21	61.8	3.77 (dd, 9.6, 3.6) 3.88 (dd, 9.6, 5.4)	61.7
C = O	170.0	-	169.9
OCH3	21.2	2.16 (s)	21.1

^a

Measured in CDCl3.

^b

150 MHz.

^c

600 MHz.

All the NMR data assignments were made from ¹H NMR, ¹³C NMR, HSQC, HMBC, COSY, and NOESY spectra.

Compounds 2, 3, and 5-7 were identified as perakine (2), ⁹ ajmaline (3), ¹⁰ sitsirikine (5), ¹¹ loganic acid (6), ¹² and secoxyloganin (7) ¹³ by comparing their NMR data with those reported in the literature and found to match. Furthermore, the chemical structures, as well as their NMR assignments, were confirmed by HSQC and HMBC spectra. This is the first report of compounds 2 and 5-7 from the genus Rauvolfia. Compound 3 was measured in CDCl₃; however, some proton signals were not detected. Therefore, it was remeasured in DMSO-d₆ to determine accurately its chemical structure and assign the NMR data (Table 1). By detailed analysis of the 1D and 2D NMR spectra, compound 4 was indentified as 10-hydroxy-16-epi-affinine, which was reported from Rauvolfia vomitoria. ¹⁴ However, to the best of our knowledge, its ¹³C NMR data have not been reported as yet.

The HR-ESI-MS, IR, CD, and NMR spectra of compounds 1-7 are shown in Supplemental Figures S1-S10, and the NMR data of compounds 2-7 are shown in Tables S1-S3.

The aerial parts of R cambodiana are used in Vietnamese folk medicine to enhance memory. In addition, some monoterpene indole alkaloids from the genus Rauvolfia have been reported to exhibit acetylcholinesterase inhibitory activity. ¹⁴ Therefore, compounds 1-7 were evaluated for their ability to inhibit acetylcholinesterase in vitro. As shown in Table S4, compounds 4 and 5 showed highly inhibitory effects with IC₅₀ values of 30.1 ± 1.4 µM and 65.5 ± 3.3 µM, respectively; the positive control, galantamine, had an IC₅₀ value of 6.17 ± 0.28 µM. The other isolated compounds inhibited acetylcholinesterase with IC₅₀ values ranging from 111.0 ± 6.0 µM to 197.6 ± 19.8 µM. Our results indicated that the high ring junction of monoterpene indole alkaloids (1-3) showed less acetylcholinesterase inhibitory activity in comparison with the vobasenal-type alkaloid (4). Additionally, the remarkable acetylcholinesterase inhibitory activity of alkaloid 4 in our study agreed with a previous report on the significant acetylcholinesterase inhibitory activity of vobasenal type alkaloids. ¹⁴ Therefore, the vobasenal framework type alkaloids could be active ingredients of Rauvolfia species and responsible for the enhancement memory property of these plants.

Materials and Methods

General Experimental Procedures

The used characterization equipment is the same as that described in our previous work.^15,16

Refer to Supplemental Material.

Plant Materials

The aerial parts of R cambodiana were collected from Dakrong Nature Reserve, Quang Tri Province, Vietnam, and identified by Dr Bui Van Thanh, Institute of Ecology and Biological Resources, VAST. The voucher specimen (NCCT-P141) has been deposited at IMBC, VAST.

Extraction and Isolation

Refer to Supplemental Material.

Rauvolcambonine (1)

Pale yellow solid, $[\alpha {]}_D^{25}$ : −21.3 (c 0.03, MeOH); UV (MeOH) λ_max (log ɛ): 220 (2.20), 280 (1.79); IR ν_max (KBr) 3341, 2933, 1744, 1592, 1454, 1235, 1031 cm⁻¹; CD (MeOH): λ nm (rel-Δε) 234 (−6.68), 252 (+18.80), 275 (−3.21); HRESIMS m/z 353.1878 [M + H]⁺, calcd for [C₂₁H₂₅N₂O₃]⁺: 353.1860, Δ = +5.1 ppm; m/z 375.1667 [M + Na]⁺, calcd for [C₂₁H₂₄N₂O₃Na]⁺: 375.1679, Δ = −3.2 ppm; ¹H NMR (CD₃OD, 600 MHz) δ (ppm), ¹³C NMR (CD₃OD, 150 MHz) δ (ppm) data shown in Table 1.

Acetylcholinesterase Inhibitory Assay

Refer to Supplemental Material.

Theoretical Calculation of ECD Spectra for Compound 1¹⁶

Refer to Supplemental Material.

Conclusions

In this study, one new alkaloid named rauvolcambonine (1), and 6 known compounds, perakine (2), ajmaline (3), 10-hydroxy-16-epi-affinine (4), sitsirikine (5), loganic acid (6), and secoxyloganin (7) were isolated from the aerial parts of R cambodiana. Compounds 2 and 4-7 were isolated from R cambodiana for the first time, and the NMR data of compound 4 is reported herein for the first time. All the isolates showed acetylcholinesterase inhibitory effects in vitro with IC₅₀ values ranging from 30.1 ± 1.4 µM to 197.6 ± 19.8 µM.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231153732 - Supplemental material for Alkaloids and Iridoids From the Aerial Parts of Rauvolfia cambodiana Pierre ex Pit. and Their Acetylcholinesterase Inhibitory Activity

Supplemental material, sj-docx-1-npx-10.1177_1934578X231153732 for Alkaloids and Iridoids From the Aerial Parts of Rauvolfia cambodiana Pierre ex Pit. and Their Acetylcholinesterase Inhibitory Activity by Ninh Khac Thanh Tung, Nguyen Chi Mai, Duong Thi Hai Yen, Bui Huu Tai, Ninh Khac Ban, Jong Seong Kang and Phan Van Kiem in Natural Product Communications