Triterpenoids from Brazilian Ginseng, Pfaffia paniculata

Abstract

Two new nortriterpenoids, pfaffine A and B (1–2), were isolated from the roots of Pfaffia paniculata Kuntze, along with ten known compounds including four ecdysteroids, ecdysone (3), 20-hydroxyecdysone (4), pterosterone (5), rapisterone (6), five triterpenoids, pfaffic acid (7), pfameric acid (8), mesembryanthemoidigenic acid (9), calenduloside E 6′-methyl ester (10), oleanolic acid 28-O-β-D-glucopyranoside (11), and one monoterpene glycoside (+)-angelicoidenol-2-O-β-D-glucopyranoside (12). The structures of the new compounds were elucidated as 3β,16β-dihydroxy-30-norolean-12,20(29)-dien-28-oic acid (1), and 3β-hydroxy-30-norolean-12,20(29)-dien-28-oic acid-28-O-β-D-glucoside (2) through the extensive analysis of 1D- (¹H, ¹³C, DEPT) and 2D-NMR (COSY, HSQC, HMBC, NOESY) spectra, as well as by a chemical method.

Pfaffia paniculata Kuntze (Amaranthaceae) is the most employed of the Pfaffia species in commercial preparations in Brazil and is popularly known as “Brazilian ginseng” for three centuries [1]. It is also named “paratudo” which means “for all things” since the roots of this plant have been used by native Brazilians as a tonic, an aphrodisiac, and a remedy for many types of illnesses, such as diabetes, ulcers, cancer, and others [2]. Now more than fifty preparations containing P. paniculata are available on the US market, but no analytical method existed to identify P. paniculata raw materials and its products. Thus, the lack of marker compounds and chemical analytical methods, as well as the increasing popularity of the marketed products elicited the need for phytochemical studies of this plant.

Previously two classes of principle ingredients have been reported from this species, including phytosterols (mainly β-ecdysone), as well as pfaffic acid (hexacyclic nortriterpene) and its glycosides, named pfaffosides A–F (saponins) [3–5]. Herein, the detailed phytochemical investigation of P. paniculata was carried out. Two new nortriterpenoids pfaffine A and B (1–2), along with ten known compounds were isolated from its roots. The structures of the new compounds were elucidated on the basis of extensive spectroscopic (1D, 2D-NMR, HR-ESI-MS) and chemical degradation studies. The known compounds were identified as ecdysone (3) [6], 20-hydroxyecdysone (4) [7], pterosterone (5) [8], rapisterone (6) [9], pfaffic acid (7) [4], pfameric acid (8) [10], mesembryanthemoidigenic acid (9) [11], calenduloside E 6′-methyl ester (10) [12], oleanolic acid 28-O-β-D-glucopyranoside (11) [13], and (+)-angelicoidenol-2-O-β-D-glucopyranoside (12) [14] by comparison of spectroscopic data with those reported in the literature. All twelve compounds except ecdysone and 20-hydroxyecdysone are reported for the first time from this plant.

Compound 1 was obtained as a white powder, [α]_D + 11.3 (c 0.1, MeOH). Its molecular formula C₂₉H₄₄O₄ was deduced from the negative-ion high-resolution electrospray ionization mass spectrum (HR-ESI-TOF-MS), showing a pseudomolecular ion [M-H]⁻ at m/z 455.3186 (calcd. for 455.3161), together with ¹³C-NMR spectrum (29 carbon signals). Its IR spectrum exhibited absorption bands due to free hydroxyl (3357 cm⁻¹) and carbonyl groups (1650 cm⁻¹). The ¹H-NMR spectrum of 1 displayed five singlet resonances for tertiary methyl groups at δ_H 1.36, 1.27, 1.06, 1.06, and 0.92, two singlets at δ_H 4.81 and 4.77 assigned to an exomethylene group, two oxymethine protons at δ_H 3.48 and 4.66, and an olefinic proton at δ_H 5.52 (brd). The ¹³C-NMR spectrum showed 29 carbon signals including five methyls, ten methylenes, six (two of which are oxygenated) methines, and eight quaternary carbons. The two signals at δ_H 122.3 and 144.4 ascribable to C-12 and C-13 suggested a Δ¹² oleanene skeleton [15]. On the basis of the ¹H-¹H COSY, HSQC, DEPT spectra, and molecular formula C₂₉H₄₄ O₄, compound 1 was suggested to be noroleanic acid type triterpene. The 20(29)-exomethylene group was ascertained by long-range correlations between H-19α, β (δ_H 2.75, 2.29) and C-20 (δ_C 150.1), H-19α, β and C-29 (δ_C 106.6), H-21α (δ_H 2.60) and C-20,29, H-29a, b (δ_H 4.81, 4.77) and C-19 (δ_C 42.3), H-29a, b and C-21 in the heteronuclear multiple-bond connectivity (HMBC) spectrum of 1 (Fig. 1). The positions of the two hydroxyl groups were established on the basis of the HMBC and NOESY experiments. The HMBC spectrum of 1 showed correlations between the oxymethine proton at δ_H = 3.48 and the carbons C-4 (δ_C 39.5), C-23 (δ_C 28.9), and C-24 (δ_C 16.7), confirming the location of one hydroxyl group at C-3. The 3β-OH equatorial orientation was established using H-3 α coupling constant (Jtrans 10.8 Hz) and the correlation observed in the NOESY spectrum, between the two axial protons H-3α (δ_H 3.48) and H-5 (δ_H 0.89). Furthermore, the HMBC spectrum exhibited correlations between the second oxymethine proton at δ_H 4.66 and the carbons C-17 (δ_C 50.8), C-22 (δ_C 33.9), and C-28 (δ_C 183.0). These data suggested the second hydroxyl group to be located at C-16. The NOESY spectrum displayed important correlations between the proton H-16 (δ_H 4.66) and the methyl protons CH₃-27 (δ_H 1.36), which indicated the β orientation of the 16-hydroxyl group. The β-orientation of H-18 and 28-COOH was evidenced by the NOE correlation between H-16 and H-19 α (δ_H 2.75), and H-21 α (δ_H 2.60). Therefore, the structure of compound 1 was established as 3β, 16β-dihydroxy-30-norolean-12, 20(29)-dien-28-oic acid, for which the name pfaffine A was adopted.

Fig. 1.

Key HMBC and NOE correlation of compound 1. Single arrow indicates key HMBC; double arrow shows NOE correlation.

Compound 2 was obtained as a white powder, [α]_D + 8.7 (c 0.46, MeOH). The positive HR-ESI-MS of compound 2 gave an [M + Na]⁺ ion at m/z 625.3706 consistent with the pseudomolecular formula C₃₅H₅₄O₈Na (calcd. for [M+Na]⁺ 625.3711). Compound 2 displayed 35 carbon resonances in its ¹³C NMR spectrum, of which 29 were assigned to a noroleanic acid type triterpene moiety and 6 to a monosaccharide portion. For the aglycon portion along with five methyl resonances (δ_C 28.9, 26.1, 17.6, 16.6, 15.7), four sp²-hybridized carbon signals (δ_C 123.4, 143.5, 107.4, 148.6) and one signal for hydroxymethine group (δ_C 78.7) could be observed. These data, when correlated with information from ¹H NMR spectrum [five methyl singlets at δ_H 1.26, 1.24, 1.13, 1.05, 0.93, one olefinic proton at δ_H 5.48, and one oxymethine at δ_H 3.46], confirmed that the aglycon of 2 possessed a noroleanic acid type triterpene skeleton. A detailed analysis of the NMR spectroscopic data (¹H, ¹³C, HSQC, and H-H COSY) of the aglycon moiety of 2 in comparison with those of 1 showed the difference of 2 from 1 only for the absence of the secondary alcoholic function at C-16. The ¹H NMR spectrum for the sugar portion of compound 2 showed one anomeric proton signal at δ_H 6.31 (1H, d, J = 8.4 Hz). The chemical shifts of all the individual protons and carbons of the sugar unit were attributed on the basis of COSY and HSQC spectral analysis. The sugar unit, identified as β-glucopyranosyl which was deduced from its ³J_H-1,H-2 coupling constant of 8.4 [16], was placed at C-28 of the aglycon on the basis of the HMBC correlation between the anomeric proton at δ_H 6.31 and the C-28 carbon resonance at δ_C 175.8. The configuration of the glucose unit was established as D after hydrolysis of 2 with 1 N HCl, tri-methylsilation, and determination of retention time by GC [17]. Thus the structure of compound 2 was deduced as 3β-hydroxy-30-norolean-12,20(29)-dien-28-oic acid-28-O-β-D-glucoside based on the above evidence and was given the trivial name pfaffine B.

The two new compounds along with the known compounds reported herein can be used as markers for the qualification and quantification of preparations claimed to contain P. paniculata.

Materials and Methods

Plant material

The root of P. paniculata was purchased commercially. The species was identified morphologically as P. paniculata in comparison with a voucher specimen [No. MRC060100(60)] deposited at the NCNPR (National Center for Natural Products Research) repository. Microscopic analysis was also conducted. The microscopic characteristics, such as cortical cells and vascular bundles in commercial sample, were identical to those in the voucher specimen. In addition, TLC profiles of commercial and voucher samples were similar under normal- and reverse-phase conditions.

General experimental conditions

Optical rotations were measured in MeOH using a Rudolph Research Auto Pol IV polarmeter with a sodium lamp (589 nm) and a 0.5 dm microcell. High-resolution mass spectra (HR-ESI-MS) were performed on Bruker MicroTOF. NMR spectra were recorded in pyridine-d₅ on either a Varian 600 or a Bruker Avance 400 NMR spectrometer. All chemical shifts (δ) are given in ppm with reference to the solvent, and coupling constant (J) is given in Hz. IR spectra were obtained on a Perkin Elmer 100 FT-IR spectrometer. Column chromatography was carried out on silica gel (40 μm for flash chromatography; J.T. Baker) and C18-reversed phase silica (230–400 mesh; Sigma-Aldrich). The fractions were monitored by TLC on silica gel plates (0.25 mm; Merck) as well as on RP-18 F₂₅₄ plates (0.25 mm; Merck). HPLC was performed on an ODS column [Phenomenex prodigy 5u ODS (3) 100A] and elutes were monitored with a UV detector at 240 nm. L-cysteine methyl ester hydrochloride, dioxane, as well as D- and L-glucose were obtained from Sigma-Aldrich.

Extraction and isolation

The dried powder of the roots (2.4 kg) of P. paniculata was extracted by stirring in MeOH (3 × 2 L) at room temperature three hours each time and the MeOH extracts were combined. The MeOH was evaporated under vacuum to get a brown viscous residue (420 g). The concentrate was suspended in water and then partitioned with hexanes, chloroform, and n-BuOH successively. The n-BuOH soluble fraction (72.1 g) was subjected to vacuum liquid chromatography (VLC) on silica gel (600 g) eluting with CHCl₃ (2 L), CHCl₃-MeOH-H₂O [930:80:10 (1 L), 830:180:10 (1 L), 680:300:50 (2.5 L)], and MeOH (1.5 L) successively to give six fractions (A–F). Fraction B [CHCl₃-MeOH-H₂O 830:180:10 (1 L), 2.5438 g] was subjected to reversed-phase C-18 flash chromatography (100 g, column: 4 × 15 cm) eluting with a gradient of MeOH-H₂O (40:60 to 100:0 with elution volume of 1603 mL) to afford twelve fractions. Subfractions 26–33 (between 156 to 198 mL, 25.6 mg) were further purified by reversed-phase C-18 flash chromatography (Biotage Flash 12+M, 9 g, column: 1.2 × 15 cm) eluting with MeOH-H₂O mixtures of increasing polarity (40:60 to 60:40 with elution volume of 2000 mL) to afford pfaffine A (1, between 1638 to 1716 mL, 4.5 mg). Fraction C [CHCl₃-MeOH-H₂O 680:300:50 (0.5 L), 0.4916 g] was subjected to normal-phase silica gel VLC (500 g, column: 4 × 15 cm) eluting with CHCl₃-MeOH-H₂O (7:2:1, 3 L with fraction volume of 15 mL for each) to afford four subfractions. Subfraction II (between 915 to 1110 mL, 103.7 mg) was subjected to RP-C18 VLC [10 g, column: 3 × 13 cm] eluting with a gradient of MeOH-H₂O [74:26 (200 mL), 75:25 (100 mL), 78:22 (200 mL), and MeOH (50 mL)], 86 fractions were collected. Fractions 56–66 (between 285 to 355 mL, 13.7 mg) were purified by preparative HPLC with a gradient of MeOH-H₂O as eluting solvent (60:40 to 90:10) to obtain pfaffine B (2, 4.0 mg).

Pfaffine A (1): white powder; ${[\alpha ]}_{\text{D}}^{25}$+ 11.3 (c 0.1, MeOH); IR ν_max 3357 (OH), 2937, 1650 (C=O), 1443, 1031 cm⁻¹; ¹H- (600 Hz) and ¹³C- NMR (150 Hz) data, see Table 1; HR-ESI-MS: m/z = 455.31863 [M-H]⁻ (calcd. for 455.31614).

Table 1.

NMR data of compound 1 in pyridine-d₅.

No.	1H [δ (ppm), mult., J (Hz)]	13C [δ (ppm)]	COSY	HMBC
1α	1.02 (1H, m)	39.0	H-2
1β	1.57 (1H, m)		H-2
2	1.82 (2H, m)	28.2	H-1α, β, 2
3	3.48 (1H, dd, 10.8, 3.6)	78.1	H-2	C-4, 23, 24
4		39.5
5	0.89 (1H, brd, 10.8)	55.9	H-6α, 6β	C-3, 4, 6, 7, 9, 10, 23, 24, 25
6α	1.60 (1H, m)	18.9	H-5, 6β
6β	1.39 (1H, m)		H-5, 6α	C-5
7α	1.57 (1H)	33.5		C-5, 8
7β	1.40 (1H)			C-5
8		39.8
9	1.65 (1H, dd, 10.2, 7.2)	47.4	H-11	C-5, 8, 10, 11, 14, 25, 26
10		37.4
11	1.90 (1H, m)	23.9	H-9, 12	C-9
12	5.52 (1H, brd)	122.3	H-11	C-9, 11, 14, 18
13		144.4
14		44.6
15α	1.81 (1H, m)	38.9	H-16,15β	C-14, 16, 17, 27
15β	2.44 (1H, t, 12.0)		H-16,15α	C-14, 16, 17, 27
16	4.66 (1H, dd, 11.4, 4.2)	65.5	15α, β	C-17, 22, 28
17		50.8
18	3.53 (1H, dd, 13.8, 4.8)	50.3	H-19α, β	C-12, 13, 14, 16, 17, 19, 28
19α	2.75 (1H, t, 13.8)	42.3	H-18, 19β	C-17, 20, 29
19β	2.29 (1H, dd 13.8, 4.8)		H-18,19α	C-17, 20, 21, 29
20		150.1
21α	2.60 (1H, brt, 13.8)	30.7	H-21β, 22α, β	C-20, 22, 29
21β	2.28 (1H, brd, 16.2)		H-21α,22α, β
22α	3.08 (1H, d 12.0)	33.9	H-21α, β, 22β	C-17, 20, 21
22β	1.81 (1H, m)		H-21α, β, 22α	C-17, 20
23	1.27 (3H, s)	28.9		C-3, 4, 5
24	1.06 (3H, s)	16.7		C-3, 4, 5
25	0.92 (3H, s)	15.7		C-1, 5, 9, 10
26	1.06 (3H, s)	17.8		C-7, 9, 14
27	1.36 (3H, s)	27.0		C-8, 14, 15
28		183.0
29a	4.81 (1H, s)	106.6		C-19, 20, 21
29b	4.77 (1H, s)			C-19, 20, 21

Pfaffine B (2): white powder; ${[\alpha ]}_{\text{D}}^{25}$+ 8.7 (c 0.46, MeOH); IR ν_max 3356 (OH), 2928, 1731 cm⁻¹ (C=O), 1380, 1027; ¹H- (600 Hz) and ¹³C- NMR (150 Hz) data, see Table 2; HR-ESI-MS: m/z = 625.37055 [M + Na]⁺ (calcd. for 625.37109).

Table 2.

NMR data of compound 2 in pyridine-d₅.

No.	1H [δ (ppm), mult., J (Hz)]	13C [δ (ppm)]	HMBC	COSY	NOSEY
1α	1.00 (1H, dt)	39.1		H-1β, 2	H-3
1β	1.56 (1H, m)			H-1α
2α	1.84 (1H, m)	28.3		H-1, 3	H-3
2β	1.84 (1H, m)
3	3.46 (1H, brd, 7.2)	78.2	H-23, 24	H-2	H-1α, 2α, 5, 23
4		39.5	H-23, 24
5	0.87 (1H, d, 11.4)	55.9	H-23,24, 25	H-6β	H-1α, 3, 23, 9, 6α, 7α
6α	1.55 (1H, m)	18.9	H-5	H-6β	H-5, 23
6β	1.37 (1H, m)			H-5, 6α	H-26, 24, 25
7α	1.50 (1H, brd 14.4)	33.2	H-26	H-7β	H-5, 27, 9
7β	1.37 (1H, m)			H-7α
8		40.0	H-6α, 7β, 9, 26, 27
9	1.66 (1H, t, 8.7)	47.8	H-25, 26	H-11	H-1α, 5, 7α, 11, 27
10		37.4	H-5, 9, 25
11	1.92 (2H, m)	23.9	H-9	H-9, 12
12	5.48 (1H, brd)	123.4	H-11, 18	H-11	H-11, 18, 19β
13		143.5	H-11, 18, 27
14		42.2	H-15β, 26, 27
15α	1.22 (1H)	28.2	H-27	H-15β
15β	2.38 (1H, dt)			H-15α, 16α	H-26, 16β
16α	2.20 (1H, dt)	23.7	H-18, 22β	H-15β,16α	H-27
16β	2.09 (1H, m)			H-16α	H-15β
17		47.4	H-18, 22β, 19β
18	3.16 (1H, dd, 13.8, 4.8)	48.2		H-19α, β	H-19β, 22β
19α	2.63 (1H, t, 13.8)	41.8	H-18, 29α, β	H-18, 19β	H-27
19β	2.23 (1H, m)			H-18, 19α	H-12, 29α
20		148.6	H-19α, 19β, 21β
21α	2.23 (1H, m)	30.2	H-19β, 22β, 29α, β	H-21β, 22β
21β	2.07 (1H, m)			H-21α
22α	2.03 (1H, m)	37.7	H-21α, 16β	H-22β
22β	1.71 (1H, dt)			H-21α, 22α	H-18
23	1.26 (3H, s)	28.9	H-3, 5, 24		H-5, 24, 6α
24	1.05 (3H, s)	16.6	H-3, 5, 23		H-25, 23
25	0.93 (3H, s)	15.7	H-5, 9		H-24, 26
26	1.13 (3H, s)	17.6	H-9		H-24, 6β, 15β
27	1.24 (3H, s)	26.1	H-15β		H-9, 7α, 16α, 19α
28		175.8	H-18, 22β, 1′
29a	4.78 (1H, s)	107.4	H-19α, 21α		H-19β
29b	4.71 (1H, s)				H-21β, α
1′	6.31 (1H, d, 8.4)	95.9	H-2′	H-2′	H-5′ ,3′
2′	4.21 (1H, t, 8.4)	74.2		H-1′,3′	H-4′
3′	4.29 (1H, t, 9.0)	78.9	H-2′	H-2′, 4′	H-1, 5′
4′	4.35 (1H, t, 9.0)	71.2		H-3′, 5′	H-2′
5′	4.03 (1H, brd, 9.0)	79.4		H-4′, 6′ α, β	H-1′ ,3′ ,6′ α, β
6′α	4.40 (1H, dd, 11.4, 3.6)	62.4		H-5′ ,6′ β	H-5′
6′β	4.47(1H, brd, 10.2)			H-5′ ,6′ β	H-5′
3-OH	5.75		C-2, 3, 4

A solution (3.0 mg) of compound 2 in 1 N HCl (1.5 mL) was stirred at 80°C 4 h. After cooling, the solution was neutralized with NH₄OH and extracted with EtOAc (2 mL). The aqueous layer after drying was dissolved in pyridine (0.3 mL), and 0.1 M L-cysteine methyl ester hydrochloride in pyridine (1.0 mL) was added. The mixture was heated at 60°C for 1 h and an equal volume of Ac₂O was added and heated one more hour. The acetylated thiazolidine derivatives were analyzed by GC using a capillary column DB-5 ms (30 m × 0.25 mm × 0.25 μm). A temperature gradient system was used starting at 100°C for 1 min and increasing to 250°C at a rate of 10°C/min. D-glucose was identified by the comparison of its retention time with that of the authentic sample after being treated in the same manner.