Diterpenoids and Flavonoids from Andrographis paniculata

Abstract

Chemical investigation of the aerial parts of Andrographis paniculata resulted in isolation of nine compounds, including a new ent-labdane diterpenoid, andrographic acid methyl ester (1), a new chalcone glucoside, pashanone glucoside (5), and seven known metabolites, andrograpanin (2), andrographolide (3), andropanolide (4), andrographidine A (6), andrographidine F (7), 6-epi-8-O-acetyl-harpagide (8), and curvifloruside F (9). Their chemical structures were elucidated based on comprehensive analyses of the spectroscopic data, including NMR and MS. Among the isolated compounds, andropanolide exerted cytotoxicity toward LNCaP, HepG2, KB, MCF7, and SK-Mel2 carcinoma cells, with IC₅₀ values ranging from 31.8 to 45.9 µM. In addition, andropanolide significantly inhibited the overproduction of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, with an IC₅₀ value of 13.4 µM.

Introduction

The herbaceous plant Andrographis paniculata (Burm. f.) Nees (Acanthaceae) is the well-known medicinal plant that is widely found in Asian countries, such as India, China, Malaysia, Indonesia, Philippines, and Vietnam. It has long been used in the Vietnamese traditional medicine to treat fever, flu, cough, sore throat, pneumonia, tonsillitis, urethritis, vaginitis, cervical ulcers, inflammatory bowel disease, hypertension, etc.¹⁾ As reported by the pharmacological investigations, A. paniculata possesses anti-microbial, anti-inflammatory, hypotensive, antihyperglycemic, antioxidant, anti-atherosclerotic, anti-malarial, anti-human immunodeficiency virus (HIV), antiplatelet aggregation, hepatoprotective, and anti-cancer activities.²⁾ Previous chemical studies have indicated that ent-labdane diterpenoids and flavonoids are the major chemical constituents and these compounds were considered to be responsible for the pharmacological effects of this medicinal plant.^2,3) In the present study, we report isolation and structural elucidation of a new ent-labdane diterpenoid (1), a new chalcone glucoside (5), and five known compounds from the methanol extract of the aerial parts of A. paniculata growing in Vietnam. In addition, cytotoxic and nitric oxide (NO) inhibitory effects of the isolated compounds were also evaluated.

Results and Discussion

The aqueous part obtained from the methanol extract of A. paniculata aerial parts was subjected to combined chromatographic separations to afford nine compounds (1–9) (Fig. 1). Their chemical structures were unambiguously deduced by detailed analyses of the spectroscopic evidence as well as comparison with the data reported in the literature.

Fig. 1. Chemical Structures of Compounds 1–9

Compound 1 was obtained as a colorless gum. Its high resolution-electrospray ionization (HR-ESI)-MS exhibited a quasi-molecular ion [M + Na]⁺ at m/z 401.1931 (calcd for C₂₁H₃₀NaO₆⁺, 401.1935), corresponding with the molecular formula of C₂₁H₃₀O₆ (seven indexes of hydrogen deficiency). The ¹H-NMR spectrum contained olefinic proton signals characteristic of three double bonds, including an exocyclic methylene [δ_H 4.57 and 4.75 (each d, J = 1.5 Hz, H_a-17 and H_b-17)], a trans-disubstituted olefin [δ_H 6.31 (1H, dd, J = 10.0, 16.0 Hz, H-11) and 6.11 (1H, d, J = 16.0 Hz, H-12)], and a trisubstituted double bond [δ_H 5.47 (1H, s, H-14)] (Table 1). The ¹H-NMR spectrum additionally exhibited signals for one oxymethine group at δ_H 3.41 (H-3), one oxymethylene group [δ_H 4.14 and 3.37 (each d, J = 11.0 Hz, H_a-19 and H_b-19), two methyl groups at δ_H 1.24 (3H, s, H₃-18) and 0.83 (3H, s, H₃-20), and one methoxy group at δ_H 3.70 (3H, s). Analysis of ¹³C-NMR and heteronuclear single quantum coherence (HSQC) spectra pointed out the presence of eight sp² carbons [including two carbonyl carbons at δ_C 168.4 (C-15) and 175.2 (C-16) and six olefinic carbons] and 13 sp³ carbons of which one oxymethine at δ_C 81.3 (C-3), one oxymethylene at δ_C 65.0 (C-19), two methines, two methylenes, and three methyls were recognized (Table 1). This spectroscopic evidence suggested that 1 possesses the ent-labdane diterpenoid skeleton which has been demonstrated as one of the major chemical types of A. paniculata.^2,3) Comparative analysis of the ¹H- and ¹³C-NMR spectroscopic data of 1 with those of the reported ent-labdane derivative, andrographic acid revealed that both structures are closely related, except that 1 has an additional methoxy group [δ_H 3.70 (s)/δ_C 51.7] at C-15 position.⁴⁾ This feature was confirmed by an heteronuclear multiple bond connectivity (HMBC) correlation from δ_H 3.70 to δ_C 168.4 (C-15) (Fig. 2) observed in the HMBC spectrum of 1. The relative configuration of 1 was deduced by analysis of the nuclear Overhauser effect spectroscopy (NOESY) spectrum in comparison with that of andrographic acid⁴⁾ (Fig. 2). In the NOESY spectrum, nuclear Overhauser effect (NOE) correlations between H-3 and H-5, between H-3 and H₃-18, between H-5 and H-9, and between H-9 and H-12 were observed, suggesting that H-3, H-5, H-9, and H₃-18 are β-oriented. On the opposite spatial side of the structure, NOE interactions observed between H₂-19 and H₃-20 and between H₃-20 and H-11 enabled to identify α-orientation for H₂-19 and H₃-20. The Z geometry of the 13,14-double bond was assigned by an NOE correlation between H-12 and H-14. Thus, the structure of 1 was established as shown in Fig. 1, named andrographic acid methyl ester. It is noted that the natural occurrence of 1 was confirmed by its presence in both of MeOH and EtOH extracts, as deduced by HPLC analysis (Figs. S15 and S16, Supplementary material).

Table 1. ¹H- and ¹³C-NMR Data for Compounds 1 and 5

Position	1		Position	5
	δCa,b)	δHa,c,d) mult. (J in Hz)		δCa,b)	δHa,c,d) mult. (J in Hz)
1	39.4	1.19 td (5.0, 13.5)	1	136.8	—
		1.60 dt (3.5, 13.5)
2	28.9	1.72	2	129.9	7.42 dd (2.0, 9.0)
3	81.3	3.41	3	130.0	7.76 t (9.0)
4	43.8	—	4	131.3	7.41 t (9.0)
5	55.9	1.25 dd (2.5, 13.0)	5	130.0	7.76 t (9.0)
6	24.4	1.40	6	129.9	7.42 dd (2.0, 9.0)
		1.82
7	37.8	2.08 td (5.0, 13.0)	α	129.1	8.12 d (15.5)
		2.46
8	149.7	—	β	144.3	7.74 d (15.5)
9	62.4	2.43	C=O	195.3	—
10	39.9	—	1′	109.3	—
11	140.6	6.31 dd (10.0, 16.0)	2′	158.0	—
12	132.3	6.11 d (16.0)	3′	132.8	—
13	157.8	—	4′	159.5	—
14	113.0	5.47 s	5′	92.8	6.58 s
15	168.4	—	6′	157.2	—
16	175.2	—	3′-OCH3	61.0	3.79 s
17	109.6	4.57 d (1.5)	4′-OCH3	56.7	3.96 s
		4.75 d (1.5)
18	23.3	1.24 s	1″	102.8	5.19 d (7.0)
19	65.0	4.14 d (11.0)	2″	75.1	3.55 dd (7.0, 9.0)
		3.37 d (11.0)
20	16.3	0.83 s	3″	78.5	3.54 dd (9.0, 9.5)
15-OCH3	51.7	3.70 s	4″	71.6	3.38 dd (9.0, 9.5)
			5″	78.9	3.54
			6″	62.7	3.70 dd (6.5, 12.0)
					3.94 dd (2.0, 12.0)

a) Recorded in CD3OD, b) 125 MHz, c) 500 MHz, d) overlapped signals are shown without multiplicity.

Fig. 2. Key HMBC and COSY Correlations of 1 and 5 and NOESY Interactions of 1

Compound 5 was obtained as a yellow, amorphous powder. Its molecular formula was determined to be C₂₃H₂₆O₁₀ by a sodium adduct ion [M + Na]⁺ at m/z 485.1414 (calcd for C₂₃H₂₆O₁₀Na⁺, 485.1418) found in the HR-ESI-MS, along with analysis of the ¹H- and ¹³C-NMR spectroscopic data. The ¹H-NMR spectrum showed signals for a trans-configured double bond [δ_H 8.12 and 7.74 (each d, J = 15.5 Hz, H-α and H-β], an unsubstituted phenyl ring [δ_H 7.42 (2H, dd, J = 2.0, 9.0 Hz, H-2 and H-6), 7.76 (2H, t, J = 9.0 Hz, H-3 and H-5), and 7.41 (1H, t, J = 9.0 Hz, H-4)], a singlet characteristic of a pentasubstituted aromatic ring at δ_H 6.58 (1H, H-5′), and two methoxy groups at δ_H 3.79 (3H, s, 3′-OCH₃) and 3.96 (3H, s, 4′-OCH₃). A signal of an anomeric proton at δ_H 5.19 (d, J = 7.0 Hz, H-1″) observed in the ¹H-NMR spectrum was indicative of the presence of a sugar unit which was further determined to be glucose by the typical carbon signals at δ_C 102.8 (C-1″), 75.1 (C-2″), 78.5 (C-3″), 71.6 (C-4″), 78.9 (C-5″), and 62.7 (C-6″) observed in the ¹³C-NMR spectrum.⁴⁾ The relative large coupling value (J = 7.0 Hz) of the anomeric proton was characteristic of the β configuration of the glucose. Analysis of the ¹³C-NMR and HSQC spectra showed that, in addition to six typical carbon signals of a the glucose, 17 signals of the aglycone, including a carbonyl carbon at δ_C 195.3 (C=O), one double bond, one phenyl ring, one 1,2,3,4,6-pentasubstituted aromatic ring, and two methoxy groups are recognized, suggesting that 5 possesses the chalcone carbon skeleton.⁵⁾ Comparison of the ¹H- and ¹³C-NMR data of 5 with those of the reported chalcone, pashanone revealed that the structures of both compounds are relatively similar, except for the additional occurrence of the glucose moiety in 5.⁵⁾ This was supported by analysis of the HMBC spectrum (Fig. 2). The anomeric proton of the glucose at δ_H 5.19 (H-1″) was shown to have an HMBC correlation with a non-protonated carbon signal at δ_C 157.2 (C-6′), indicating that the glucose is located at C-6′ position. This was supported by an HMBC correlation from H-5′ to C-6′ as well as an NOE correlation observed between H-1″ and H-5′. Besides, HMBC cross-peaks from δ_H 3.79 to δ_C 132.8 (C-3′) and from δ_H 3.96 to δ_C 159.5 (C-4′) along with an NOE interaction between H-5′ and δ_H 3.96 (4′-OCH₃) enabled to confirm the location of the two methoxy groups at C-3′ and C-4′ positions, respectively. On the basis of the spectroscopic evidence, compound 5 was identified as pashanone glucoside.

By using the same spectroscopic methods and comparing with the reported data, the known metabolites were identified as: andrograpanin (2),⁶⁾ andrographolide (3),⁷⁾ andropanolide (4),⁸⁾ andrographidine A (6),⁴⁾ andrographidine F (7),⁹⁾ 6-epi-8-O-acetyl-harpagide (8),¹⁰⁾ and curvifloruside F (9).¹¹⁾ The previous pharmacological studies have shown that andrograpanin (2) exerts anti-inflammatory activity through down-regulating the p38 mitogen-activated protein kinase (MAPKs) signaling pathways¹²⁾ and selective enhancement of chemokine SDF-1α-induced leukocyte chemotaxis¹³⁾ and an anti-proliferative effect toward HL-60 cells¹⁴⁾; andrographolide (3) possesses various pharmacological effects such as anti-inflammatory and anti-cancer activities^2,3); andropanolide (4) shows a beneficial effect in the treatment of progressive forms of multiple sclerosis¹⁵⁾; while andrographidine A (6) exhibits an anti-proliferative effect against HL-60 cells.¹⁶⁾ Therefore, except 2 and 3, all of the isolates were evaluated for their cytotoxicity toward five human cancer cell lines (including LNCaP, HepG2, KB, MCF7, and SK-Mel2) using sulforhodamine B (SRB) assay^17,18) and their NO inhibitory effects (at the noncytotoxic concentrations) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells using Griess assay.¹⁹⁾ As the result, among the compounds tested, andropanolide (4) was shown to exert cytotoxic effect toward all five human cancer cell lines, including LNCaP, HepG2, KB, MCF7, and SK-Mel2 cells, with IC₅₀ values of 31.8, 43.5, 37.9, 45.9, and 42.1 µM, respectively. In addition, 4 also significantly inhibited the overproduction of NO in LPS-stimulated RAW264.7 macrophages, with an IC₅₀ value of 13.4 µM.

Acknowledgments

This work was financially supported by Vietnam Academy of Science and Technology (VAST) under Grant number KHCBHH.02/18-20.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Material

The online version of this article contains supplementary materials.

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