Biological and chemical standardization of a hop (Humulus lupulus) botanical dietary supplement

Abstract

Concerned about the safety of conventional estrogen replacement therapy, women are using botanical dietary supplements as alternatives for the management of menopausal symptoms such as hot flashes. Before botanical dietary supplements can be evaluated clinically for safety and efficacy, botanically authenticated and standardized forms are required. To address the demand for a standardized, estrogenic botanical dietary supplement, an extract of hops (Humulus lupulus L.) was developed. Although valued in the brewing of beer, hop extracts are used as anxiolytics and hypnotics and have well-established estrogenic constituents. Starting with a hop cultivar used in the brewing industry, spent hops (the residue remaining after extraction of bitter acids) were formulated into a botanical dietary supplement that was then chemically and biologically standardized. Biological standardization utilized the estrogen-dependent induction of alkaline phosphatase in the Ishikawa cell line. Chemical standardization was based on the prenylated phenols in hops that included estrogenic 8-prenylnaringenin, its isomer 6-prenylnaringenin, and pro-estrogenic isoxanthohumol and its isomeric chalcone xanthohumol, all of which were measured using high-performance liquid chromatography-tandem mass spectrometry. The product of this process was a reproducible botanical extract suitable for subsequent investigations of safety and efficacy.

Keywords: 8-prenylnaringenin; Ishikawa cells; LC/MS-MS; botanical dietary supplements; estrogen replacement; hops; standardization; xanthohumol.

Figure 1

Chemical structures of hop prenylated phenols and the internal standard, 8-isopentylnaringenin.

Figure 2

Alkaline phosphatase (AP) induction in Ishikawa cells after 96 h incubation with the hop extract. AP induction by 17β-estradiol, 8-PN and XH are shown for comparison.²⁹ The data are averages of at least three independent determinations carried out in triplicate ± SD.

Figure 3

LC/MS-MS chromatograms of an ethanolic extract of spent hops obtained using negative ion electrospray, collision-induced dissociation and selected reaction monitoring (SRM).

Figure 4

Calibration curve for the most estrogenic hop flavonoid, 8-PN, obtained using LC/MS-MS.