Ading to loss of bioactive components [8]. It really is known that alkaloids like berberine, palmatine and jatrorrhizine would kind precipitate together with the flavone baicalin [9]. Alkaloids may also precipitate with organic acids forming insoluble salts [8]. It is achievable that the alkaloids species in combined decoction of EXD (EXD-C) may well 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 elements may be converted by chemical reactions like hydrolysis of glycosides. Within the combined decoction of EXD (EXD-C), hydrolysis may possibly be facilitated to get rid of the sugar units in the flavonoids glycoside icariin, major to lower in its content material [2]. Mangiferine, ferulic acid, jatrorrhizine and palmatine have been confirmed because the crucial chemical markers for high-quality handle of anti-menopausal EXD according to our proposed approach. Simply because (i) they have been present at HPO axis, revealed by our previous study; (ii) their pharmacological effects are associated to menopause. Since it hasCheung et al. Chin Med (2017) 12:Web page five ofFig. 1 Overlaid HPLC chromatograms of a EXD-S [4] and b EXD-C from three repeated injections extracted at 345 nm. The peaks of six common chemical compounds have been annotated as mangiferine, ferulic acid, icariin, jatrorrhizine, palmatine and berberine, in a chorological order of retention timebeen reported that as well as aging and menopause, the antioxidant enzymes is down-regulated, plus the estrogen secretion through aromatase is hampered [6]. These fourchemical markers possess antioxidant activities [103]. Apart from, ferulic acid have been also reported to have estrogenic properties, its therapy increases the bone mineralCheung et al. Chin Med (2017) 12:Web page six ofTable 1 The contents of six standard chemical substances of EXD in three injections of EXD-S and EXD-CInjection EXD-S1 EXD-S2 EXD-S3 Mean RSD ( ) EXD-C1 EXD-C2 EXD-C3 Mean RSD ( ) Mean ratio Mangiferin (mg/g) 1.368 1.371 1.382 1.374 0.57 0.6581 0.6610 0.6583 0.6591 0.24 2.085 Ferulic acid (mg/g) 0.4871 0.4896 0.4996 0.4921 1.34 0.1983 0.1980 0.2034 0.1999 1.53 2.462 Icariin (mg/g) 1.731 1.744 1.745 1.740 0.46 1.498 1.493 1.479 1.490 0.65 1.168 Jatrorrhizine (mg/g) 0.1004 0.1010 0.1014 0.1010 0.48 0.02865 0.03009 0.02947 0.02940 2.45 three.435 Palmatine (mg/g) 1.083 1.090 1.092 1.089 0.46 0.03577 0.03661 0.03592 0.03610 1.23 30.17 Berberine (mg/g) 1.615 1.617 1.628 1.620 0.43 1.000 1.001 1.003 1.001 0.17 1.The results are expressed as mg or chemical substances per g of EXD extract.PD-1, Human (CHO, Fc) RSD values had been calculated for each chemical from 3 injections and also the imply ratio represents the ratio of quantity of chemical substances in EXD-S to that of EXD-CFig. 2 The ratio of six common markers in EXD-S and EXD-C Fig.TL1A/TNFSF15, Mouse (Biotinylated, HEK293, His-Avi) 3 The fold differences of standard markers in EXD-S/EXD-Cdensity in ovariectomized female rats in the SpragueDawley strain with slightly rising the serum levels of estrogen [14].PMID:23800738 Furthermore, it has been shown to become productive in treating hot flashes in menopausal ladies [15]; (iii) the amounts of these 4 chemical markers in EXD-S are twofold higher than those in EXD-C, as a result diverse decoction techniques may very well be quickly revealed by different amounts of those four markers in HPLC profile (Fig. 3). In particular, palmatine is pretty much 30.38-fold higher in EXD-S compared with EXD-C (Fig. three), which will be additional biologically characterized in our additional experiment. In addition to chemical analysi.