Anti-inflammatory effect and isolation of phenylethanoid and acylated flavone glycosides from Panzeria alaschanica

1 Introduction

Panzeria alaschanica Kupr. is a member of the Labiate family and is distributed predominantly in Eerduosi of Inner Mongolia, China. It is used as a remedy for postpartum abdominal pain, irregular menstruation and dysmenorrhea due to its effects with menstruation and blood circulation [1, 2]. It is widely used in Mongolia as a substitute for Leonurus artemisia, which is used in the treatment of irregular menstruation, dysmenorrhea, amenorrhea, endless lochia and acute nephritis edema. However, there are few reported phytochemistry studies [3–5] to support these claimed therapeutic and medicinal effects. Recently, we conducted a systematic chemical study on the aerial parts of P. alaschanica, which resulted in the isolation of two new acylated flavone glycosides and a known phenylethanoid [6–9]. Their structures are shown in Fig. 1. This paper describes the isolation, structure elucidation and the anti-inflammatory activity of all compounds.

Fig. 1:

Structures of compounds 1–3.

2 Results and discussion

Compound 1 was obtained as a pale yellow powder. The positive reaction to the Molish and HCl–Mg tests suggested that the compound was a flavonoid glycoside. The molecular formula was determined to be C₃₀H₂₆O₁₂ by HR–ESI–MS at m/z = 577.1338 [M–H]⁻. The analysis of the aromatic region of the ¹H NMR spectrum of 1 (Table 1) confirmed that the compound was an apigenin derivative, with characteristic resonances for H-3 at δ_H = 6.84 ppm (1H, s, δ_C = 103.1 ppm by HSQC), H-6 at δ_H = 6.48 ppm (1H, d, J = 1.5 Hz, δ_C = 99.9 ppm), H-8 at δ_H = 6.82 ppm (1H, d, J = 1.5 Hz, δ_C = 99.9 ppm), H-2′,6′ at δ_H = 7.94 ppm (2H, d, J = 8.5 Hz, δ_C = 129.0 ppm) and H-3′,5′ at δ_H = 6.92 ppm (2H, d, J = 8.5 Hz, δ_C = 116.5 ppm). The ¹³C NMR signals (δ_C = 164.7 (C-2), 130.5 (C-3), 182.4 (C-4), 161.8 (C-5), 99.9 (C-6), 163.2 (C-7), 95.2 (C-8), 157.4 (C-9), 105.8 (C-10), 121.4 (C-1′), 129.0 (C-2′), 116.5 (C-3′), 161.6 (C-4′), 116.5 (C-5′) and 129.0 (C-6′) ppm also proved the presence of an apigenin carbon skeleton. The remaining resonances in the aromatic region were those of an (E)-coumaroyl group, with two spin-spin coupled doublets at δ_H = 7.37 ppm (2H, d, J = 8.0 Hz, δ_C = 130.6 ppm by HSQC) and 6.67 ppm (2H, d, J = 8.0 Hz, δ_C = 116.1 ppm) and characteristic α- and β-H resonances at δ_H = 7.50 ppm (1H, d, J = 16.0 Hz, δ_C = 145.4 ppm) and 6.34 ppm (1H, d, J = 16.0 Hz, δ_C = 114.2 ppm), respectively.

The identity of the acyl group was confirmed by complete assignment of its ¹H and ¹³C resonances from COSY, HSQC and HMBC data. The final group of resonances in the ¹H NMR spectrum of 1 (Fig. 2) were from a sugar with an anomeric proton at δ_H = 5.17 ppm (1H, d, J = 7.0 Hz). The HMBC correlation from δ_H = 5.17 ppm (1H, d, J = 7.0 Hz) to C-7 revealed that the sugar moiety was linked to the C-7 of the aglycone. The assignment of the remaining ¹H and ¹³C resonances of the sugar and the value of the ³J_{H-1″,H-2″} coupling constant indicated that the sugar was β-glucopyranose. Of particular interest were the downfield-shifted resonances of 6″-CH₂ (δ_H = 4.44 and 4.26 ppm) and C-6″ (δ_C = 63.9 ppm), which allowed the site of acylation to be identified as C-6″. The structure of 1 was determined to be 5,7,4′-trihydroxyflavone-7-O-(6″-O-[E]-coumaroyl)-β-glucopyranoside.

Fig. 2:

Some key HMBC correlations of 1 and 2.

Compound 2 was obtained as a pale yellow powder. The positive reactions to the Molish and HCl-Mg tests suggested that the compound was a flavonoid glycoside. The molecular formula was determined to be C₃₉H₃₂O₁₄ by HR-ESI-MS at m/z = 723.1705 [M–H]⁻. The ¹H and ¹³C NMR spectra of compound 2 (Table 1) are similar to those of compound 1, except for the appearance of an additional coumaroyl group with two spin-spin coupled doublets at δ_H = 7.59 ppm (2H, d, J = 9.0 Hz, δ_C = 130.7 ppm by HSQC) and 6.69 ppm (2H, d, J = 9.0 Hz, δ_C = 116.3 ppm) and characteristic α- and β-H resonances at δ_H = 7.62 ppm (1H, d, J = 15.5 Hz, δ_C = 145.1 ppm) and 6.47 ppm (1H, d, J = 15.5 Hz, δ_C = 115.1 ppm). The downfield-shifted resonances of 2″-CH (δ_H = 5.12 ppm) and C-2″ (δ_C = 77.3 ppm) in the sugar moiety of 2 suggested that the coumaroyl group was linked to C-2″. The correlations observed in the HMBC spectrum (Fig. 2), 6″-CH₂ (δ_H = 4.46 and 4.23 ppm) with C-9″′ (δ_C = 166.9 ppm) and 1″-CH (δ_H = 5.39 ppm) with C-7 (δ_C = 162.9 ppm) and 2″-CH (δ_H = 5.12 ppm) with C-9″″ (δ_C = 166.6 ppm), confirmed the structure of 2. The structure of compound 2 was determined to be 5,7,4′-trihydroxyflavone-7-O-(2″,6″-O-[E]-dicoumaroyl)-β-glucopyranoside.

No mortality was observed in the groups of rats treated with compounds 1–3. LD₅₀ values for compounds 1–3 were more than 200 mg kg⁻¹.

The development of edema induced by carrageenan corresponded to the events in the acute phase of inflammation, mediated by histamine, bradykinin and prostaglandin that were produced under an effect of cyclooxygenase. Both compounds possessed anti-inflammatory activity (Table 2). Compound 3 had a higher anti-inflammatory potential than diclofenac (NSAID), while the activity of compounds 1 and 2 were similar to that of diclofenac.

Compounds 1–3 exhibited a significant suppression of inflammation in a dose-dependent manner in rats when compared to the control. As shown in Table 3, compounds 1 and 3 had higher anti-inflammatory potential than acetylsalicylic acid (ASA), while the activity of compound 2 was similar to that of ASA.

3 Experimental section