DESIGNER Extracts as Tools to Balance Estrogenic and Chemopreventive Activities of Botanicals for Women's Health

Abstract

Botanical dietary supplements contain multiple bioactive compounds that target numerous biological pathways. The lack of uniform standardization requirements is one reason that inconsistent clinical effects are reported frequently. The multifaceted biological interactions of active principles can be disentangled by a coupled pharmacological/phytochemical approach using specialized ("knock-out") extracts. This is demonstrated for hops, a botanical for menopausal symptom management. Employing targeted, adsorbent-free countercurrent separation, Humulus lupulus extracts were designed for pre- and postmenopausal women by containing various amounts of the phytoestrogen 8-prenylnaringenin (8-PN) and the chemopreventive constituent xanthohumol (XH). Analysis of their estrogenic (alkaline phosphatase), chemopreventive (NAD(P)H-quinone oxidoreductase 1 [NQO1]), and cytotoxic bioactivities revealed that the estrogenicity of hops is a function of 8-PN, whereas their NQO1 induction and cytotoxic properties depend on XH levels. Antagonization of the estrogenicity of 8-PN by elevated XH concentrations provided evidence for the interdependence of the biological effects. A designed postmenopausal hop extract was prepared to balance 8-PN and XH levels for both estrogenic and chemopreventive properties. An extract designed for premenopausal women contains reduced 8-PN levels and high XH concentrations to minimize estrogenic while retaining chemopreventive properties. This study demonstrates the feasibility of modulating the concentrations of bioactive compounds in botanical extracts for potentially improved efficacy and safety.

Scheme 1. Specialized and DESIGNER Extracts as Tools for Phytomedical and Natural Product Research

Standardized extracts optimized for desired bioactivities can be generated from crude plant material in three main steps: (1) selection of plant material, (2) extraction via a specific procedure, (3) application of the DESIGNER concept. The resulting DESIGNER extracts can be standardized to desired concentrations of different bioactive compounds and represent materials with potentially more targeted biological profiles.

Figure 1

Major bioactive phytoconstituents of spent H. lupulus.

Scheme 2. Concept of Targeted Application

DESIGNER extracts are used to optimize the bioactivity profiles of hops by balancing two dual bioactive constituents: the potent phytoestrogen 8-PN and XH, the major cytoprotective constituent of hops. Targeted and selective depletion/enrichment of 8-PN, IX, and/or XH allows the design of extracts with various biological properties, such as menopausal, Ex3, versus pre-menopausal DESIGNER extract (Ex5+Ex7). Presented are the symbols for XH and 8-PN in the size that depicts their estimated mass %. Other hops constituents are omitted for clarity. The optimal holistic hop extract (wellness) might be standardized to multiple bioactive hop constituents (metabolome), leading to polypharmacological activities.

Figure 2

Estrogenic activity of hop DESIGNER extracts is mainly a function of 8-PN concentration. Induction of alkaline phosphatase in Ishikawa cells by (A) the major bioactive constituents in hops (8-PN, IX, XH, 6-PN) and E₂; (B) 8-PN and the hop DESIGNER extracts. Results were normalized to the control (0.5 nM E₂) and are shown as % of control.

Figure 3

XH decreased and IX increased the estrogenic efficacy of 8-PN. (A) Equivalent concentrations of Ex7 (5 μg/mL) [8-PN (6.9 nM), XH (2.5 μM), and their combination]; (B) equivalent concentrations of 0.09 μg/mL of Ex1 [8-PN (2.5 nM), IX (158 nM), and their combination]. *Indicates significance of at least p < 0.05.

Figure 4

Cytotoxicity of the extracts depends mainly on the XH concentration. Cytotoxicity was performed in parallel to the AP assay with the SRB assay in Ishikawa cells. Cytotoxicity (A) of the pure compounds, 8-PN, IX, and XH; (B) of the DESIGNER hop extracts.

Figure 5

NQO1 induction was a function of XH concentration. (A) Linear regression of NQO1 induction activity in Hepa1c1c7 cells by DESIGNER hop extracts and XH. Results are shown as fold induction and are the means ± SEM of at least three independent determinations in duplicate. Linear regression was performed with Graph-Pad Prism 6. (B) Linear regression of the NQO1 induction potency, presented as 1/CD values of the hop extracts as a function of the corresponding XH% (r² = 0.91). CD values (concentration to double NQO1 activity) were generated from three different independent evaluations in duplicate.

Figure 6

Comparison of estrogenic (AP fold induction, 1 μg/mL) and chemopreventive activity (NQO1 fold induction, 1.25 μg/mL) of (A) Ex3 (postmenopausal extract) and (B) Ex5 (premenopausal extract).