Efficiently prepared ephedrine alkaloids-free Ephedra Herb extract: a putative marker and antiproliferative effects

Abstract

Ephedrine alkaloids (EAs) have been considered the main pharmacologically active substances in Ephedra Herb (, Mao; EH) since they were first identified by Prof. N. Nagai, and are known to induce palpitation, hypertension, insomnia, and dysuria as side effects. Therefore, the administration of drugs containing EH to patients with cardiovascular-related diseases is severely contraindicated. While our previous studies suggest that some of the effects of EH may not be due to EAs, considering their side effects would be expedient to develop a new EAs-free EH extract (EFE). Here, we established a preparation method for EFE and revealed its chemical composition, including the content of herbacetin, a flavonoid aglycon present in EH and a potential putative marker for EFE quality control. In addition, we showed the antiproliferative effects of EFE against the H1975 non-small cell lung cancer (NSCLC) cell line. EFE was prepared from EH extract using the ion exchange resin SK-1B. LC/Orbitrap MS analysis revealed the removal of EAs, 6-methoxykynurenic acid, and 6-hydroxykynurenic acid from the original extract. Quantitative analysis of herbacetin using LC/MS in acid-hydrolyzed EFE showed that its content was 0.104 %. Although several alkaloidal constituents were removed from EH extract, the antiproliferative effect of EFE against H1975 cells was comparable to that of EH extract. These results indicate that EFE retained the anticancer effect of EH and demonstrated its potential for future development as a new herbal medicine with reduced side effects.

Keywords: Antiproliferative effect; Chemical composition; Ephedra Herb; Ephedrine alkaloids-free Ephedra Herb extract; Herbacetin.

Fig. 1

Residual ratio of ephedrine alkaloids (EAs) exposed to each cation exchange resin (%). Amounts of ephedrine and pseudoephedrine in Ephedra Herb (EH) corresponding to 100 % of the vertical axis were 74.0 and 22.1 mg, respectively

Fig. 2

LC/Orbitrap MS analyses of a Ephedra Herb (EH) extract and b ephedrine alkaloids-free EH extract (EFE): a photodiode array (PDA, 254 nm) and b total ion chromatogram (TIC). Peak 1 6-methoxykynurenic acid; peak 2 6-hydroxykynurenic acid; peak 3 trans-cinnamic acid

Fig. 3

Extracted ion chromatograms of a Ephedra Herb (EH) extract and b ephedrine alkaloids-free EH extract (EFE) samples. m/z 166.12 [M+H]⁺, dl-ephedrine/dl-pseudoephedrine; m/z 180.14 [M+H]⁺, dl-methylephedrine/dl-pseudomethylephedrine; m/z 152.12 [M+H]⁺, dl-norephedrine/dl-pseudonorephedrine

Fig. 4

Structures of compounds 1, 6-methoxykynurenic acid; 2, 6-hydroxykynurenic acid; 3, trans-cinnamic acid; 4, herbacetin

Fig. 5

Ephedra Herb (EH) extract and ephedrine alkaloids-free EH extract (EFE) prevented proliferation of the H1975 non-small cell lung cancer (NSCLC) cell line