Comparison of chemical profiles and effectiveness between Erxian decoction and mixtures of decoctions of its individual herbs: a novel approach for identification of the standard chemicals

The effects of different decoction methods on chemical profiles of anti-menopausal EXD formula have been demonstrated by HPLC, with the six selected chemicals that have been present at HPO-axis in vivo revealed from our previous publication [6]. Results obtained from HPLC profile revealed that all the six chemicals including mangiferin, ferulic acid, icariin, jatrorrhizine, palmatine and berberine were higher in content in EXD-S than that in EXD-C (Figs. 2, 3), the HPLC profile of EXD-S is the same as that in our previous publication [5]. Such changes in chemical profiles may be due to the interaction of different components during the decoction process. For instance, the chemical components may enhanced the solubility of each other when decocted together thus increasing the final content of chemicals in the extract [8]. On the contrary, they may precipitate with each other forming insoluble complex leading to loss of bioactive components [8]. It is known that alkaloids like berberine, palmatine and jatrorrhizine would form precipitate with the flavone baicalin [9]. Alkaloids may also precipitate with organic acids forming insoluble salts [8]. It is possible that the alkaloids species in combined decoction of EXD (EXD-C) may precipitate with organic acid from its different ingredient herbs like ferulic acid, flavonoid compounds such as icariin or other undetected flavone species and are lost from EXD-C. Also, bioactive components can be converted by chemical reactions like hydrolysis of glycosides. In the combined decoction of EXD (EXD-C), hydrolysis may be facilitated to remove the sugar units from the flavonoids glycoside icariin, leading to decrease in its content [2].

Mangiferine, ferulic acid, jatrorrhizine and palmatine were confirmed as the key chemical markers for quality control of anti-menopausal EXD according to our proposed approach. Because (i) they have been present at HPO axis, revealed by our previous study; (ii) their pharmacological effects are related to menopause. As it has been reported that along with aging and menopause, the antioxidant enzymes is down-regulated, and the estrogen secretion through aromatase is hampered [6]. These four chemical markers possess antioxidant activities [10–13]. Besides, ferulic acid were also reported to have estrogenic properties, its treatment increases the bone mineral density in ovariectomized female rats of the Sprague-Dawley strain with slightly increasing the serum levels of estrogen [14]. In addition, it has been shown to be effective in treating hot flashes in menopausal women [15]; (iii) the amounts of these four chemical markers in EXD-S are twofold higher than those in EXD-C, thus different decoction methods could be easily revealed by different amounts of these four markers in HPLC profile (Fig. 3). In particular, palmatine is almost 30.38-fold higher in EXD-S compared with EXD-C (Fig. 3), which will be further biologically characterized in our further experiment.

Besides chemical analysis, the effects of EXD-S and EXD-C on ovarian aromatase mRNA expression and hepatic antioxidant enzymes were evaluated, which has also been proven as the targets of EXD by our group previously [5]. As anticipated, EXD stimulated ovarian aromatase (Cyp19) expression the transcriptional level at high dose (1.76 g/kg). The up-regulation of Cyp19 mRNA level in EXD-S-treated rats was significantly almost twofold higher than that of EXD-C, which may have been due to the overall increase in bioactive components in EXD-S as revealed from the HPLC profiles. It is known that the bioactive components in EXD such as mangiferin, berberine, palmatine and jatrorrhizine possess antioxidant activities [10–13]. Besides, icariin and ferulic acid were also reported to have estrogenic properties [14, 16]. The lower amounts of these bioactive compounds in EXD-C may explain the decreased bioactivity of EXD-C in vivo. The effects of EXD-S on mRNA level of hepatic antioxidants are in line with our previous findings [5]. In our previous study, EXD could significantly up-regulate CAT expression at the transcriptional level [5]. In this study, both EXD-S and EXD-C elicited around 1.5-fold of increase in the mRNA level of CAT, although no statistical significance was detected. Consistent with the results of Cyp19 expression, EXD-S showed a stronger tendency of stimulation of CAT and GPx-1 than EXD-C. The mRNA level of SOD-1 in EXD-S-treated group was significantly higher than that of the group treated with low dosages of EXD-C. These again support the better pharmacological properties of EXD-S than EXD-C.

In a TCM formula, the complexity of chemical components imposes difficulties in the identification of standard chemicals for quality control. The observation of differences in the chemical profiles of EXD-S and EXD-C in relation to their bioactivity has opened up the possibility of a novel and rapid approach to identify the standard chemicals in CM formulas. Since the pharmacological properties of a medicinal formula are conferred by the chemical components, which may change as a result of different decoction conditions. By comparing the HPLC profiles of the decoctions, the differentially extracted components would be those responsible for the observed discrepancy in bioactivity. This would facilitate the identification and selection of bioactive components as standard chemicals out of the complex herbal mixture. In a study on Radix Scutellariae (Huangqin) decoction, an increase in the amount of the bioactive compound baicalin was observed in the combined decoction [17]. In another study on Tangkuei Liu Huang Decoction, the amount of baicalin was higher  in separate decoctions than that of combined decoction [18]. These findings suggest that the bioactive components in a herbal extract can be affected by the decoction method as well as the herbal interaction between different herbs. The decoction methods would also affect the pharmacological properties. In some studies, the combined decoction may have better therapeutic efficacy and vice versa [19, 20]. Whether the component herbs of a Chinese medicinal formula should be decocted separately or in combination together depends on different individual formulas, but the decoction of herbal materials is often an inevitable process for the preparation of most of the CM prescriptions. The evaluation of chemical profiles as well as the pharmacological properties of different processing methods may indicate a novel approach for identifying the standard chemicals of the CM formula. In this study, the feasibility of such approach is evaluated by differential comparison of the HPLC profile of EXD-S and EXD-C in relation to the pharmacological properties. Eventually, this approach can be coupled with analytic techniques to identify the differentially extracted components obtained by different decoction methods.

In future developments, such approach may be polished by further validations with a more comprehensive pharmacological screening platform, and further evaluation of the feasibility of this approach can be conducted with other Chinese medicinal formulas. The four key chemicals, including mangiferine, ferulic acid, jatrorrhizine and palmatine, found in EXD could be further investigated in vitro and in vitro to identify their combined effects as a mixture of four in treating menopausal syndromes.