The risk of cardiovascular disease in older women is one of the major concerns in menopausal health management. It has been reported that about 42% of women older than 65 years of age exhibit a substantial increase in serum level of TC, LDL-C [11] and TG [3]. In this study, we investigated the effects of EXD on serum lipid profiles and its underlying mechanism. HPLC was used to ensure that there were no variations in pharmacological effects of EXD due to sample preparation.
Sample consistency analysis of EXD and its fractions
The comprehensive HPLC chromatograms of EXD and its fractions were generated and compared with the peaks of seven standards, mangiferin, ferulic acid, icariine, curculigoside, jatrorrhizine, palmatine and berberine (Figure 1, 2 and 3). These chromatographic fingerprints with seven marker compounds were used as reference standards, indicating the purity, identity and quality consistency among EXD and its fractions. The relative standard deviations of the amount of the five standards were less than 5% in three batches of EXD and their constituent fractions (Tables 1, 2 and 3), indicating quality consistency among the different batches of EXD and constituent fractions as well as excluding the influence of any unknown variability or instability found in the composition of the active constituents in the pharmacological test of EXD and its fractions.
The BU fraction (Figure 1, 2 and 3, Table 3) contained most of the marker chemicals (including fenolic acids, flavonoids and alkaloids). The relative contents of the seven marker compounds were much higher compared with EXD (original formula) (Table 1), while the EA fraction (Table 2) showed a much higher content of the small phenols, such as mangiferine and ferulic acid. Most of the compounds were detected at 283 nm. The alkaloids (such as berberine, jatrorrhizine and palmatine) displayed a higher absorption at 345 nm, while phenolic acid (such as mangiferin and ferulic acid) showed ideal absorption at 258 nm. The chromatogram of water fraction (WA) at three different UV detection wavelengths indicated that almost all of the compounds could be completely extracted with ethyl acetate and n-butanol, and the residual chemicals in the WA fraction were presumably some small-molecular-weight metabolites, such as sugars, amino acids, organic acids and other trace elements.
Effects of EXD and its fractions on serum lipid profile
The correlation of low estrogen level with serum lipid profile [11] as well as the stimulatory effect of EXD on ovarian estrogen synthesis[6] suggest that EXD may prevent adverse changes in the lipid profile of a menopausal rat model. The serum TC level in the EXD group was significantly reduced after treatment. The constituent fractions of EXD were less effective in lowering the serum TC level. Thus, we suggest that the hypocholesterolemic effect of EXD is exerted by the complete formulation of EXD rather than any constituent compounds. In the premarin-treated group (human equivalent dosage), the TC level was decreased by an insignificant degree.
On the other hand, this study found that the effects of EXD on the serum TG level were insignificant. In all groups treated with EXD and its constituent fractions, TG level was slightly increased. Interestingly, the increase after EXD treatment was less pronounced compared with its constituent fractions. While EXD did not improve the serum TG level, it did not lead to an increase of TG as its bioactive fractions did. In the premarin-treated group, the TG level was comparable to the control group. The effect of premarin on the serum TG level was similar with some other studies of conjugated estrogen on postmenopausal women, in which conjugated estrogen alone may increase the circulating TG level [5, 12, 13].
The effect of EXD on the serum HDL-C level was not obvious. The serum HDL-C in all groups treated with EXD or its fractions did not change significantly, while in the groups treated with the constituent fractions the serum HDL-C level decreased slightly, thereby worsening the lipid profile. While EXD cannot improve the serum HDL-C level, it can prevent the lipid profile from worsening in comparison with its constituent fractions. The serum HDL-C level in the premarin-treated group also decreased slightly.
However, EXD could improve the serum lipid profile in our study model by decreasing the serum LDL-C level. As an increased LDL-C level is a risk factor for CVD, our results suggested that EXD may be able to ameliorate the CVD risk in the menopausal model. Again, effects were observed only in the EXD-treated group, while the individual constituent fractions did not significantly reduce the serum LDL-C level. However, similar to the results on the serum HDL-C, oral administration of premarin did not improve the serum lipid profile. The serum LDL-C level in the premarin-treated group was comparable to the control group and no beneficial effects were noted.
Our results on the serum lipid level showed that EXD may act as a preventive medicine for reducing CVD risk in the rat menopausal model by reducing serum TC level and LDL-C level while preventing the HDL-C and TG levels from worsening. However, treatment with premarin did not manifest significant improvement on the serum lipid profile in the rat model. According to studies about hormone replacement therapy on menopausal women, supplements with estrogen could increase the serum HDL-C level and reduce LDL-C level thus reducing the CVD risk factors [5, 12]. One possible reason is that, the laboratory animals used in this study were kept in a cage and remained physically inactive. Physical inactivity is suspected as one of the risk factors for adverse lipid profile [14]. Therefore, in the case of this study the hypolipidemic effect of premarin was possibly counteracted. Also, due to different metabolism between rats and human, the metabolism of estrone and equilin in premarin may not be the same in rats and human. Further investigation is needed before it can be confirmed.
The improvement of lipid profile in the EXD-treated group but not in the premarin-treated group raises a question of whether the positive action of EXD is due to its estrogenic effect or is estrogen-independent. Our previous study showed that EXD increased the circulating estrogen level by stimulating ovarian biosynthesis [6]; however, this study did not indicate whether this estrogenic effect improved the lipid profile due to the negative response in the premarin-treated group. In future studies, 17-beta-estradiol may be used as a positive control to facilitate comparison. Also, an ovariectomized rat model may be included to for further elucidation of the possible role of stimulated endogenous estrogen production by EXD.
Regulation of HMGCR and LDL-R by EXD at translational levels
In order to elucidate the underlying mechanism of the significant improvement in serum profiles of TC and LDL-C demonstrated by EXD, we examined the protein levels of HMGCR and LDLR using Western blot. The levels of HMGCR and LDLR are crucial in maintaining the TC and LDL-C balance. HMGCR is the rate limiting enzyme that controls de novo synthesis of cholesterol through mevalonate pathway [15]. HMGCR catalyses the conversion of HMG-CoA into mevalonate, ultimately leading to cholesterol synthesis [16]. LDLR maintains cholesterol homeostasis by a receptor-recycling pathway, in which circulating LDL-C is returned into the liver by receptor-mediated endocytosis [16–18].
Only EXD-treated rats displayed a significantly decreased protein level of HMGCR, indicating a decrease in the de novo synthesis of TC. The HMGCR protein levels in the groups treated with other constituent fractions were comparable to each other and showed no significant differences from the control. The protein level of HMGCR in the premarin-treated group was also comparable to those of the control groups. This is consistent with the results of the serum TC level, which did not change significantly in the premarin group. In fact, a previous study suggested that estradiol regulates HMGCR through the activation of AMP-activated protein kinase which phosphorylates HMGCR and inactivates its catalytic property [19] rather than acting at the transcription level.
As the HMGCR level in the EXD-treated group decreased, it is anticipated that the LDLR level would rise. From the results, the increase in LDLR protein level in the EXD-treated group would lead to an elevated clearance of LDL-C, which is in line with the decreased serum LDL-C level. The effect of the constituent fractions of EXD was not as pronounced as that of EXD, suggesting the combined effect of the component fractions in EXD. The increase of protein level of LDL-R in the premarin-treated group was not significant, as expected from the results on serum lipid level and HMGCR level.
Although EXD treatment can significantly suppress the protein level of HMGCR and elevate LDLR level, the effect of EXD on the serum TC and LDL-C levels was not as prominent as that on the relevant regulatory proteins. Such discrepancy may be attributed to the complex regulation of HMGCR and LDLR activity. Moreover, the expression level of HMGCR, the regulation of HMGCR activity involves mechanism such as phosphorylation/dephsphorylation by AMP-dependent kinase (AMPK)/protein phosphatase 2A (PP2A); transcriptional regulation of sterol regulatory element-binding proteins (SREBPs) or protein degradation [15, 20]. Pallottini et al. revealed that while the protein level of HMGCR remained unchanged, its degradation rate reduced and the enzyme was fully active in aged rats [15]. Whether the discrepancy between the protein level and serum lipid profile is related to the above factors was not assessed in this study.
Our results demonstrated that EXD improved the serum lipid profile in a rat menopausal model through the modulation of the protein levels of HMGCR and LDLR, thus lowering serum TC and LDL-C. Further studies on the synergistic effects of EXD on serum lipid profile and more detailed studies on the mechanisms of HMGCR and LDLR regulation by EXD will be implemented by us in the near future.