Auto-hydrolysis of red clover as “green” approach to (iso)flavonoid enriched products

Abstract

Optimal parameters for the auto-hydrolysis of (iso)flavone glycosides to aglycones in ground Trifolium pratense L. plant material were established as a “green” method for the production of a reproducible red clover extract (RCE). The process utilized 72-h fermentation in DI water at 25 and 37 °C. The aglycones obtained at 25 °C, as determined by UHPLC-UV and quantitative ¹H NMR (qHNMR), increased significantly in the auto-hydrolyzed (ARCE) (6.2–6.7% w/w biochanin A 1, 6.1–9.9% formononetin 2) vs a control ethanol (ERCE) extract (0.24% 1 0.26% 2). After macerating ARCE with 1:1 (v/v) diethyl ether/hexanes (ARCE-d/h), 1 and 2 increased to 13.1–16.7% and 14.9–18.4% w, respectively, through depletion of fatty components. The final extracts showed chemical profiles similar to that of a previous clinical RCE. Biological standardization revealed that the enriched ARCE-d/h extracts produced the strongest estrogenic activity in ERα positive endometrial cells (Ishikawa cells), followed by the precursor ARCE. The glycoside-rich ERCE showed no estrogenic activity. The estrogenicity of ARCE-d/h was similar to that of the clinical RCE. The lower potency of the ARCE compared to the prior clinical RCE indicated that substantial amounts of fatty acids/matter likely reduce the estrogenicity of crude hydrolyzed preparations. The in vitro dynamic residual complexity of the conversion of biochanin A to genistein was evaluated by LC-MS-MS. The outcomes help advance translational research with red clover and other (iso)flavone- rich botanicals by inspiring the preparation of (iso)flavone aglycone-enriched extracts for the exploration of new in vitro and ex vivo bioactivities that are unachievable with genuine, glycoside-containing extracts.

1. Introduction

Trifolium pratense L. from the Fabaceae family, commonly known as red clover, is both a forage plant and a medicinal herb used in dietary products and for the alleviation of menopausal symptoms, premenstrual syndrome, mastalgia, and high cholesterol [1,2]. Like other food plants, T. pratense produces and/or stores phytochemicals in the form of glycosides, and β-glucosidases in the apoplast of the vegetal cells [3,4]. The preparation of crude extracts includes grinding of the dry plant material into small particles to increase the contact area between particles and solvent, providing an efficient diffusion process across cell walls during solid-liquid extraction. Rupturing the apoplast of cells during grinding also liberates enzymes. Isoflavonoid glycosides can be readily converted into their corresponding aglycones (Fig. 1A) via the native β-glucosidase activity present during primarily aqueous extraction, for example with 80% ethanol [5].

Fig. 1.

A: Structures of the discussed isoflavonoid glycosides and aglycones; B: Metabolic conversion of 1 to 3.

Isoflavonoids have structural similarities to human estrogens. Genistein, for example, an isoflavonoid present in soybean and red clover mainly in its glycosidic form, is one of the most estrogenic isoflavonoids. Many other isoflavones are present in leguminous plants, particularly soybean [6,7] and red clover [1,2,8]. Isoflavonoids have recently attracted considerable attention because of their reported potentially beneficial effects in osteoporosis, cardiovascular disease, and alleviation of menopausal symptoms [9–11]. Some studies have already focused on the hydrolysis of (iso)flavonoid glycosides, to generate aglycones [12–14]. It has been reported that isoflavonoid aglycones exhibited higher serum levels after oral consumption compared to isoflavonoid glycosides [13]. In fact, (iso)flavonoid aglycones in leguminous plants occur at low concentrations or even below typical detection limits. However, the yield of isoflavonoid aglycones such as biochanin A (1), formononetin (2), genistein (3), daidzein (4), and prunetin (5) in an extract will dramatically increase when removing the sugar moieties from their corresponding isoflavonoid glycosides: sissotrin, ononin, daidzin, genistin, and prunetrin, respectively. Moreover, the quantitative determination (chemical standardization) of multiple individual (iso)flavonoid glycosides in plant materials is highly demanding, due to their large number [15]. Therefore, the glycosides are normally hydrolysed and the resulting aglycones are subsequently identified and quantified.

Methods for acid hydrolysis of (iso)flavonoid glycosides from vegetables and berries have been published previously [16,17], and glycosidic cleavage usually require relatively high concentrations (1–2 M) of mineral acids under reflux conditions [17,18]. However, the conditions resulting in optimal breakdown of glycosides are too non-selective for cleavage of just the glycosidic bond and/or too harsh for some of the aglycones and other phenolic compounds present in the same plant material, thereby leading to degradation and generation of numerous by-products. Another issue that prevents the preparation of materials for human consumption by mineral acid hydrolysis is the high concentration of the residual mineral acid and/or the base used for neutralization. The more concerning impact, however, is the stability of bioactive compounds/aglycones and potential adverse effects of the degradation products [19–21]. Our Botanical Center has previously been working with a clinical red clover extract (RCE), which was prepared at an industrial scale in a single batch using auto-hydrolysis. Accordingly, it represented an (iso)flavonoid aglycone-rich, chemically and biologically standardized clinical RCE containing 61.7% (iso)flavonoids, exhibited a high concentrations of biochanin A (1) and formononetin (2), and contained minor percentages of genistein (3), daidzein (4), prunetin (5), calycosin (6), and irilone (7) [1,2,8].

In the context of chemical changes over time, dynamic residual complexity (DRC) is a concept of relevance for complex natural product extracts (go.uic.edu/residualcomplexity). DRC can be defined as compound(s) levels changing over time, as a result of any chemical or environmental factor. DRC may be generated by degradation, biocatalytic reactions, rearrangement, or tautomeric equilibria of any species, in a given sample under storage or biological assay conditions [22–24]. The relevance of DRC for red clover was recently demonstrated for 45% EtOH tinctures as traditional preparations of T. pratense, by showing that the percentages of aglycones and glycosides exhibited a substantial change over time, thereby presenting a case of DRC by chemical or enzymatic modifications [25]. On the other hand, DRC could also be occurring during in vitro experiments during evaluation of the bioactive compounds in the bioassay media and incubation time frame [26]. For example, 1 and 2 show low estrogenic activity, but are quickly metabolized in vivo by P450s to the more potent phytoestrogens, 3 and 4, respectively (Fig. 1B) [1,27,28]. Compounds 3 and 4 are known to exhibit considerable estrogenic activities in both in vitro and in vivo studies [29,30]. In particular, 3 demonstrates estrogen receptor (ER) β-preferential activity, suggesting a better safety profile than estradiol [31,32]. In addition, bone protective, together with triglyceride- and cholesterol-lowering properties have been demonstrated for certain isoflavonoids, especially for 3 [13,33,34]. Based on these properties, botanical dietary supplements (BDS) rich in 3 and 4 and their precursors, 1 and 2, respectively, have been proposed to alleviate menopausal symptoms, and osteoporosis [27,35]. Analogous health implications are frequently found for isoflavone-rich “functional food” products.

Analytically, the time-resolved capture of isoflavone (glycoside) structural parameters requires orthogonal analytical methods, such as nuclear magnetic resonance (NMR) and liquid chromatography hyphenated with mass spectrometry (LC-MS). Combined with chemometric analysis and pattern recognition, these analytical methods are capable of generating metabolomic profiles of complex mixtures such as RCEs [36,37]. Being a non-invasive and non-destructive analytical methodology, ¹H NMR offers vital information on metabolite structure [38]. It is also an unbiased method that reveals the overall metabolic profile of biofluids or tissue extracts [39]. NMR-based metabolomic analysis is a powerful technique, which not only enables the parallel assessment of the levels of a broad range of metabolites, but also plays an important role in the investigation of physiologic or pathologic states and their relevant pathways. Multivariate data analysis discriminates samples by comparing metabolomes, in part, by visualization of clustering between different groups. Principal Component Analysis (PCA) is the primary mode of multivariate NMR data analysis [40], used to investigate general interrelation between groups, including clustering and outliers among the samples. For example, PCA was used to investigate NMR spectra of 28 wine samples collected from one tank over 207 days [41–43].

In the present study, ¹H NMR metabolomics was applied to investigate the metabolic profiles and bioactive compounds of RCE hydrolysis [41,44]. Owing to the therapeutically relevant biologic activities of a auto-hydrolyzate, (iso)flavonoid-aglycone rich clinical RCE, the necessity of developing a model-laboratory and “green” auto-hydrolysis method useful for any medicinal plant that does not result in degradation of the aglycone became apparent. The objective of this study was to optimize parameters of auto-hydrolyzing red clover plant material as a proof-of-concept. For this purpose, auto-hydrolysis at 25 °C and 37 °C was investigated, followed by identification and quantification of the (iso)flavonoids and related compounds generated from the auto- hydrolysis process using three orthogonal methods: qHNMR, HPLC and LC-MS. In order to develop a standard sample preparation procedure to generate the most amount of (iso)flavonoid aglycones and avoid degradation of the aglycones themselves, the effects of different auto-hydrolysis conditions (temperature at 25 °C and 37 °C as well as the ratio between weight of plant material/volume of water and time over 3 days; taking multiple measurements during the 3 days) were evaluated as a “green” alternative for the preparation of enriched RCEs suitable for clinical trials. It should be noted that, while the isoflavone aglycones represent the main therapeutic target compounds, the non-isoflavonoids represent near ubiquitous “companion” compounds in higher plants and, therefore, are also affected by (auto-)hydrolysis procedures. This almost certainly alters the bioactivity profiles of the non-isoflavonoids, which span a vast range of biological endpoints.

The second main step of this investigation involved the depletion of fatty acids by macerating each dry auto-hydrolyzed red clover extract (ARCE) with diethyl ether/hexanes 1/1 (ARCE-d/h) to further enrich the (iso)flavonoid content. Furthermore, the composition and biological activities of the auto-hydrolyzed red clover extract (ARCE) and auto- hydrolyzed red clover extract macerated with diethyl ether/hexanes 1/1 (ARCE-d/h) were compared. In the evaluation of estrogenic activity of these extracts, the ARCE exhibited more activity than ERCE, and ARCE-d/h exhibited better activity than ARCE. ARCE-d/h showed a similar activity to the clinical RCE [1,2,8].

2. Experimental section

2.1. Chemicals and reagents

Organic solvents and reagents were purchased from Thermo Fisher Scientific (Hanover Park, IL, USA) and Sigma-Aldrich Inc. (St. Louis, MO, USA), if not otherwise specified. Ethanol 200 proof (100% absolute with CAS–N. 64–17–5) was purchased from Decon Laboratories Inc. (King of Prussia, PA, USA). Isoflavonoid reference standards of 1, 2, 3, 4, and 5 were purchased from Indofine Chemical Co. (Hillsborough, NJ, USA). Standard compound 1 for bioassays was purchased from Sigma- Aldrich Inc. and repurified as outlined below. Isoflavonoid 7 was purified using Centrifugal Partition Chromatography (CPC) as previously reported [25]. The external calibrant, dimethyl sulfone (Code N. 048–33,271; 100%, CRM), was a gift from Wako Pure Chemical Ind., Ltd. (Osaka, Japan). The internal calibrant, 3,5-dinitrobenzoic acid (CAS–N. 99–34–3; 99.3% by qHNMR/100% method) was purchased from Fluka Analytical (Buchs, Switzerland). Deuterated dimethyl sulfoxide (DMSO-d₆, 99.9% D) was obtained from Cambridge Isotope Laboratories Inc. (Andover, MA, USA).

2.2. Plant material

The aerial parts of T. pratense L. were purchased from Mountain Rose Herbs (Eugene, OR, USA) in April 2013. A voucher specimen (code: 719 BCE) is kept in the UIC Botanical Center collection (Chicago, IL, USA). The plant material was powdered using a mill grinder (KitchenAid, St. Joseph, MI, USA). A tea sieve was used to obtain uniform size particles, and the collected powder was stored at room temperature.

2.3. Instrumentation

The qHNMR experiments were performed on a JEOL Resonance Inc. (Akishima, Tokyo, Japan) 400 MHz NMR spectrometer, model JNM- ECZ400S/L1 at 25 °C (298 K) equipped with a liquid N₂ SuperCOOL probe (NM-Z161331TH5SC). The NMR data were analyzed and processed with JEOL Delta v5.3.1 (Delta™ NMR Data Processing Software, Akishima, Tokyo, Japan) and/or MestReNova 11.0.4 software from Mestrelab Research S. L. (Santiago de Compostela, Spain). UHPLC-UV analysis was performed on a Kinetex 1.7 μm XB-C₁₈ 100 Å column (50 mm × 2.1 mm, Phenomenex, Torrance, CA, USA) with a Shimadzu (Kyoto, Japan) Nexera UHPLC DAD system. Shimadzu LabSolutions was used for UHPLC system operation and data analysis. The column oven, detector cell, and autosampler temperature were maintained at 40 °C, 40 °C and 4 °C, respectively, throughout the UHPLC analysis periods.

The SCPE-250 centrifugal partition chromatography (CPC) extractor from Gilson Inc. (Middleton, WI, USA) had a 264 mL total volume, 220 mL of which was attributed to cell volume. The CPC was operated at a flow rate of 50 mL/min and a rotation speed of 2500 rpm, with a pressure maximum of 300 bar. The Spot Prep peripheral operating system was equipped with a 50 mL sample loop, binary pump, a 4 wavelength UV/VIS detector, a sample collector, and data collection software. A miVac centrifugal vacuum concentrator (Speed Vac, Genevac LTD. Ipswich, UK) was used to dry each collected sample. Each sample was further dried for two or three days on a Labconco™ Bench- top FreeZone™ freeze-dry system (Kansas City, MO, USA) before performing gravimetry.

LC-MS-MS analyses were carried out using a Waters 2695 (Milford, MA, USA) solvent delivery system connected to a Waters SYNAPT quadrupole/time-of-flight (Q/TOF) mass spectrometer operated in the positive ion electrospray mode. In qHNMR quantitation methods, DMSO₂ was used as an external and 3,5-dinitrobenzoic acid as an internal calibrant [2,25,45].

2.4. Acquisition of qHNMR spectra

Samples were dissolved in 250 μL DMSO-d₆ of which only 200 μL was delivered into a 3 mm NORELL NMR tube (Landisville, NJ, USA) with a 1000 μL analytical syringe. NMR spectra were acquired at 298 K. The qHNMR spectra of extracts were acquired using standard qHNMR parameters [46–48], which included a 60 s relaxation delay (D1), 46 receiver gain (RG), 32 scans (NS), flip angle (P1) 90° (6.4 μs), and automatic gradient shimming. NMR data was processed with a Lorentzian-Gaussian window function (exponential factor − 0.3, Gaussian factor 0.05), and a baseline correction (fifth order polynomial). The residual DMSO-d₅ signal at 2.500 ppm was used for chemical shift referencing.

2.5. Ethanol red clover extract (ERCE)

10.1 g of dry powdered red clover plant material was macerated with 3 × 100 mL of absolute ethanol for two days each time. The three extracts were combined, and the solvent was removed by rotary evaporation. Subsequently, the material was freeze-dried until the qHNMR experiment was performed.

2.6. Auto-hydrolysis of red clover at 25 °C and 37 °C

The methodology is illustrated in Fig. 2 and described as follows. (i) 10.0–10.2 g of dried powdered plant material was transferred to a glass tube (180 mL) containing 120 mL of milliQ water. Each sample was homogenized and incubated in a Hybridization Incubator (Lab-Line Instruments, Inc. Melrose Park, IL, USA) under 20 rpm at 25 °C for 1, 2 and 3 days. Parallel experiments were also performed at 37 °C. (ii) After incubation, the sample was filtered under vacuum. (iii) Each filter residual plant material (RPM) was collected and dried in an oven at 40 °C for 4 days. (iv) Accordingly, each filtrate (aqueous sugar-enriched solution) was collected and dried separately. (v) Each RPM was divided in two parts: one portion was extracted with 20 mL of EtOH (“e”) producing auto-hydrolyzed red clover extracts (ARCE/e: Et1d, Et2d and Et3d, Fig. S8 and Fig. S9). (vi) Another portion was extracted with 20 mL of a mixture of EtOH and i-PrOH (“ei”; 4:1 v/v), to produce a more enriched extract that corresponded to an auto-hydrolyzed red clover EtOH/i-PrOH extract (ARCE/ei: EtIs1d, EtIs2d and EtIs3d; Fig. S10 and Fig. S11). The organic solvents of filtered extracts were removed on a rotary evaporator at 40 °C under vacuum. These dried samples of ARCE/ e and ARCE/ie were further freeze-dried to remove residual water and then stored at −20 °C until qHNMR analysis. (vii) After taking a 15 mg aliquot for qHNMR analysis, each dried ARCE/e and ARCE/ie was macerated overnight with 20 mL of diethyl ether/hexanes 1:1 to remove fatty acids and related materials. (viii) Subsequent filtration produced three ARCE/e-d/h extracts (Et1d-d/h, Et2d-d/h, and Et3d-d/h; Fig. S12 and Fig. S13), as well as three ARCE/ei-d/h extracts (EtIs1d-d/h, EtIs2d- d/h, and EtIs3d-d/h); while ARCE/e-d/h and ARCE/ei-d/h represented the residual solids remaining on the filter paper (Fig. S14 and Fig. S15). ARCE/e-d/h and ARCE/ei-d/h were dried in a fume hood for three days and stored at −20 °C until UHPLC-UV and qHNMR investigation (Fig. 2).

Fig. 2.

Preparation of ARCE/e (Et1d, Et2d and Et3d) representing auto-hydrolysis products of crude red clover extract subsequently extracted with EtOH (Fig. S8 and Fig. S9), and ARCE/ei (EtIs1d, EtIs2d and EtIs3d), representing auto-hydrolysis products of red clover extract subsequently extracted with EtOH/i-PrOH 4/1 (Fig. S10 and Fig. S11)]. Further maceration of the ARCEs with diethyl ether/hexanes (1:1) yielded the ARCE/e-d/h materials (Et1d-d/ h, Et2d-d/h, and Et3d-d/h; Fig. S12 and Fig. S13) from ARCE/e, and the ARCE/ei-d/h materials (EtIs1d-d/h, EtIs2d-d/h, and EtIs3d-d/h; Fig. S14 and Fig. S15) from ARCE/ei.

2.7. Multivariate analysis

The percent content of target analytes was determined by qHNMR using DMSO₂ as an EC. Each target (iso)flavonoid and related compound with one specific ¹H NMR signal for quantification [2,49] was quantitated (% w/w). The EC calculation sheet available online for this purpose [50] was used. All the spectra were manually corrected for phase and baseline distortions (5th order polynomial), and used the same window function such as Lorentzian (− 0.30 Hz) and Gaussian (0.05 Hz). The content of (iso)flavonoids and related compounds were expressed in % w/w with reference to the EC. The percentages (% w/w) were imported into Origin software (OriginLab, Northampton, MA, USA) for processing Principal Component Analysis (PCA).

2.8. Purification of commercially-sourced biochanin a 1

16.2 mg of 1 (Sigma Aldrich Inc., St. Louis, MO, USA) was purified using Centrifugal Partition Chromatography (CPC) to remove impurities such as 3 (Fig. S1–Fig. S4), using a previously described solvent system and conditions [45].

2.9. Induction of an estrogen-responsive alkaline phosphatase (AP) enzyme in Ishikawa cells

The Ishikawa cell line was provided by Dr. R. B. Hochberg (Yale University, New Haven, CT, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM/F12) containing 1% sodium pyruvate, 1% nonessential amino acids, 1% Glutamax, 0.05% insulin, and 10% heat-inactivated FBS (Gemini Bioproducts) as previously described [51,52]. Two days before treating the cells, the growth medium was replaced with phenol-red-free DMEM/F12 medium containing charcoal/dextran- stripped FBS (Gemini Bioproducts) and supplements as mentioned above. The Ishikawa cell line is a well-established ERα (+) endometrial cancer cell line useful for the evaluation of estrogens [52,53]. This cell line was authenticated via determination of the short tandem repeat profile using the StemElite ID System by Promega (performed at the Research Resources Center, DNA Services, UIC, Chicago, IL), and in accordance with the Ishikawa cell line according to the Health Protection Agency Culture Collection in the UK.

2.10. Alkaline phosphatase (AP) enzyme induction assay

The protocol by Pisha et al. was used as described previously with minor modifications [52,53]. Briefly, Ishikawa cells were pre-incubated in estrogen-free medium for 24 h and plated in 96 well plates (3.9 × 10⁴ cells/well). After another 24 h, test samples were dissolved in DMSO (< 0.1%), and added in parallel to the positive control (estradiol, 0.50 nM), and the negative control (DMSO), to the 96 well plate. After an incubation time of 96 h at 37 °C, cells were washed with PBS and lysed by adding 0.01% Triton X-100 in 0.10 M Tris buffer (pH 9.8), followed by a freeze and thaw cycle at − 80 °C and 37 °C, respectively. Following, p-nitrophenol phosphate (phosphatase substrate; 2.69 mM) was added to each well, and the alkaline phosphatase activity was measured by reading the formation of p-nitrophenol at 405 nm every 15 s for 16 readings using a Power Wave 200 microplate scanning spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA). The maximum slope of the kinetic curves was calculated for each experimental well. The percent induction of alkaline phosphatase for every treatment, compared to that of the estradiol control, was calculated using the following equation:

$$\begin{array}{c}\left[\left({\text{slope}}_{\text{sample}}-{\text{Slope}}_{\text{DMSO}}\right)/\left({\text{slope}}_{\text{estrogen}}-{\text{slope}}_{\text{DMSO}}\right)\right]\times 100\\ =\text{fold estrogenic induction}\end{array}$$

2.11. Metabolism of 1 to 3 in Ishikawa cells

Ishikawa cells were treated with purified 1 (10 and 20 μM) for 96 h in 96 well plates, as described under “alkaline phosphatase assay”. Cell media were collected and analyzed for 1 and its metabolites using LC- MS-MS. Metabolites were separated on an Agilent RRHD 2.1 × 100 mm reversed phase C₁₈ column (1.8 μm particle size) using a mobile phase consisting of 0.01% formic acid (solvent A) and acetonitrile (solvent B) and a linear gradient 12–72% B over 15 min at a flow rate of 0.25 mL/min. The column temperature was 40 °C. 3 was quantified by positive ion electrospray with SRM, monitoring transitions from 271 to 153.

3. Results and discussion

3.1. Native ethanolic red clover extracts (ERCE)

Plant material (10.1 g) was extracted three times with ethanol, to yield 1.95 g (19.2%) of ethanolic crude extract. This extract was highly concentrated in isoflavonoid glycosides (sissotrin, ononin, genistin, daidzin, etc.) and sugars [25,54]. Additionally, the aglycone percentages of 1 (0.24% w/w) and 2 (0.26% w/w) in the extract were determined by EC qHNMR and confirmed by UHPLC-UV (1: 0.25%; 2: 0.29%; Table 1 and Fig. 3).

Table 1.

Results of the EC-qHNMR quantification of the isoflavonoids and related compounds. 10 and 11 have low estrogenic activity, and these two aglycones are also present at low levels in ARCE/e-d/h and ARCE− /ei-d/h.

%w/w*	1	2	3	4	5	6	7	8	9	10	11
ERCE	0.24	0.26	–	–	–	–	–	–	–	–	–
RCE	13.9	14.2	0.24	0.29	0.82	0.34	2.61	0.65	0.34	0.63	0.85
Et1d	6.41	9.67	0.18	0.06	0.17	0.22	0.42	0.23	0.23	0.00	0.00
Et2d	6.65	9.93	0.20	0.10	0.28	0.13	0.62	0.22	0.19	0.00	0.00
Et3d	6.21	9.36	0.25	0.14	0.28	0.19	0.76	0.15	0.28	0.04	0.08
EtIs1d	6.57	8.41	0.22	0.10	0.27	0.21	0.52	0.46	0.38	0.00	0.00
EtIs2d	6.32	8.90	0.18	0.07	0.35	0.15	0.81	0.19	0.16	0.00	0.00
EtIs3d	6.55	6.07	0.19	0.05	0.36	0.06	0.63	0.10	0.22	0.06	0.00
Et1d-d/h	15.7	18.3	0.47	0.26	0.95	0.26	1.56	0.72	2.01	0.09	0.00
Et2d-d/h	14.5	16.9	0.24	0.49	0.79	0.65	2.78	0.69	1.23	0.05	0.12
Et3d-d/h	13.1	16.8	0.66	0.42	0.92	0.59	1.92	0.49	0.62	0.03	0.11
EtIs1d-d/h	16.7	18.2	0.39	0.24	0.82	0.25	1.48	1.30	1.95	0.00	0.00
EtIs2d-d/h	14.8	17.9	0.91	0.51	1.87	1.05	3.11	0.97	1.04	0.02	0.00
EtIs3d-d/h	13.8	14.9	0.60	0.45	0.72	0.29	1.66	0.68	1.18	0.06	0.05

^*Compounds (%w/w): biochanin A 1, formononetin 2, genistein 3, daidzein 4, prunetin 5, calycosin 6, irilone 7, 8, pratensein 9, kaempferol 10 and quercetin 11.

Fig. 3.

A: The ¹HNMR spectrum of a crude red clover EtOH extract (ERCE; 6.92 mg/150 μL of DMSO-d₆). B: UHPLC-UV chromatogram of the ERCE (injection: 1.0 μL of 3.15 mg/mL in MeOH) using a previously documented method [1,2].

3.2. Auto-hydrolysis of red clover extract (ARCE)

An auto-hydrolysis model was employed for investigation regarding hydrolysis of (iso)flavonoid glycosides. In order to optimize the operation conditions, a series of single-factor experiments were carried out for (a) temperature (°C), (b) ratio of material to water (g/mL), (c) and auto- hydrolysis time (h).

1. Auto-hydrolysis at 37 °C hydrolysed the glycosides rapidly, but it also promoted degradation. HPLC profiles showed that (iso)flavonoids and related compounds were extensively decomposed at 37 °C, likely caused by both chemical reactions and enzymatic transformations that decompose these compounds (Supporting information Fig. S5–Fig. S7). The content of the two major isoflavonoids was also determined using HPLC as described in Table S1. By comparison, the same experiments operated at 25 °C provided a milder auto-hydrolysis as evident from stacked chromatograms (Fig. 4).

2. For adequate hydrolysis during fermentation, the raw plant material must be suspended completely in the aqueous medium. Based on a series of observations, an adequate amount of water must be provided to distribute the plant material in the suspension. A total volume of less than 120 mL for a 10 g sample generated precipitation in the glass tube. As a result, the ratio of botanical material to water should be equal or more than 1:12 (g/ mL). To explore this concept, experiments were also performed by changing the ratio of plant material and water to 1:15 (g/mL), giving the same results as for 1:12 (g/mL), at 25 °C, for 72 h. Average yields for ARCE/e and ARCE/ie were 1.12 g (11.0%) and 1.07 g (10.5%), respectively (Table S2).

3. To determine the efficiency of auto-hydrolysis, 1 and 2 were used as target analytes. The quantity of 1 and 2 in ARCE/e and ARCE/ ei was compared to that in ERCE. After one-day (Et1d) of aqueous auto-hydrolysis, a 26.7-fold and 37.2-fold enrichment of 1 and 2 was realized in ARCE/e (ethanol), respectively (Table 1). In a two-day (Et2d) auto-hydrolysis assay, a 27.7-fold enrichment of 1 and 38.2-fold enrichment of 2 was achieved. After the third day (Et3d) of auto-hydrolysis, the concentration of 1 was 25.9-fold and 2 36.0-fold greater than in ERCE. Similar results were found for ARCE/ei (EtOH/i-PrOH 4:1). During the first day (EtIs1d) of aqueous auto-hydrolysis, the concentration of 1 increased by 27.4-fold and 2 by 32.4-fold. Similarly, during the second day (EtIs2d) of auto-hydrolysis, the concentration of 1 increased by 26.3-fold as well as for 2 by 34.2-fold. The third day (EtIs3d), a 27.3-fold enrichment was observed for 1 and a 32.4-fold enrichment for 2. This indicates that after 1 day of auto- hydrolysis, the maximum extent of hydrolysis of (iso)flavonoid glycosides is achieved already, and further fermentation does not increase hydrolysis efficiency. Both methods of extraction post hydrolysis, ARCE/e versus ARCE/ei, resulted in similar extraction efficiency (Table 1). Yields of each extract following auto- hydrolysis (ARCE/e and ARCE/ei) were determined by gravimetric methods (Table S2). On the other hand, aqueous solutions (Aq1d and Aq2d; Fig. 2) were also analyzed by qHNMR in order to evaluate percentages of (iso)flavonoids in the RPM filtrate (Fig. S22). It was observed that Aq1d contained 0.14% w/w of 1 and 0.24% w/w of 2. The other filtrate, Aq2d, contained 0.27% w/w and 0.37% w/w of 1 and 2, respectively. Additionally, anomeric hydrogens corresponding to free sugars (post hydrolysis) were also observed in these filtrates.

Fig. 4.

The qHNMR profiles of RCE, ARCE/e, ARCE/ei, ARCE/e-d/h, ARCE/ei-d/h. Blue boxes indicated the regions used to integrate after normalization. For example, for the reference clinical RCE, integration of the olefinic hydrogens region between 5.00 and 5.50 ppm gave an integral of 138, whereas the sum of the integrals of the aliphatic region at 2.55–3.00 ppm plus 0.00–2.40 ppm gave an integral of 2615.

3.3. Fatty acids in ARCE/e and ARCE/ei vs. ARCE/e-d/h and ARCE/ei-d/h

According to the qHNMR data, both ARCE/e and ARCE/ei contained substantial amounts of fatty (acid) material (Fig. 4). Generally, EtOH extracts contain a wide range of plant metabolites, including fatty acids and their derivatives. The content of (iso)flavonoids and related compounds in crude RCEs is low compared with that of an isoflavone aglycone-rich clinical RCE. The ¹H NMR spectra showed a greater quantity of fatty acids in ARCE/e and ARCE/ei, and less in the ARCE/e- d/h and ARCE/ei-d/h samples. After removal of the fatty components, the concentration of (iso)flavonoids increased considerably. The clinical RCE (15.2 mg/mL in DMSO‑d₆) exhibited an integral value of 138 (5.20–5.56 ppm, Fig. 4) for the α/β hydrogens of unsaturated carbons, as well as an integral of 2615 (sum of 0.00–2.45 and 2.53–3.00 ppm) for hydrogens attached to saturated carbons. On the other hand, the extracts Et1d, Et2d, and Et3d gave integrals in the range 376–462 (green font, Fig. 4) for the α/β hydrogens of unsaturated carbons, and 6629–7554 for the hydrogens attached to saturated carbons. Interestingly, EtIs1d, EtIs2d and EtIs3d almost followed the same tendency in a range between 464 and 806 (red font, Fig. 4) for the α/β hydrogens of unsaturated carbons, and 3101–9068 for the hydrogens attached to saturated carbons. In ARCE/e-d/h and ARCE/ei-d/h, the integral corresponding to fatty acids was reduced significatively through the corresponding resonances: 40–57 (purple font, Fig. 4) for α/β hydrogens of unsaturated carbons, and 1804–3433 for the saturated range. ARCE/ei-d/h showed a very similar trend with integrals of 40–55 (blue font, Fig. 4) for unsaturated vs. 1831–3764 for saturated functions.

3.4. ARCE/e-d/h and ARCE/ei-d/h

The percentage of (iso)flavonoids and related compounds were enriched in ARCE/e-d/h and ARCE/ei-d/h by depleting fatty acids with a maceration procedure. The average yield was determined to be 64.2% (0.72 g) for ARCE/e-d/h vs. 0.68 g (63.5%) for ARCE/ei-d/h (Table S2). In ARCE/e-d/h, the concentration of (iso)flavonoids increased 65.3-fold for 1 and 70.5-fold for 2 after one day (Et1d-d/h) when compared with ERCE (Table 1). Extracts (ARCE/e-d/h) made over two or three days contained slightly more fatty acids compared with the one day extracts. As determined by qHNMR, aglycone enrichment was 60.3-fold for 1 and 65.2-fold for 2 after two days (Et2d-d/h), as well as 54.4-fold for 1 and 64.6-fold for 2 on the third day (Et3d-d/h). The ARCE/ei-d/h series of extracts gave similar results compared to ARCE/e. For instance, 69.7- fold of 1 and 70.2-fold of 2 was determined after one day (EtIs1d-d/h) of fermentation as well as 61.9-fold for 1 and 68.9-fold for 2 during the second day (EtIs2d-d/h). On the last day (EtIs3d-d/h) of maceration provided a 57.7-fold for 1 and 57.5-fold for 2. These data reveal that the concentration of (iso)flavonoids and related (potentially more bioactive) aglycone compounds significantly increased after diethyl ether/ hexanes maceration. Residual isoflavonoids in the enriched fatty extracts were also analyzed by qHNMR to evaluate their loss from ARCE/e or ARCE/ei in the maceration. As shown on Fig. S21, only traces (below the limit of quantitation) can be observed of 2, whereas signals corresponding to fatty acids exhibit relatively high intensity.

3.5. Percentage of (iso)flavonoids and related compounds

A total of eleven compounds at different levels were identified and quantified from each auto-hydrolysis extract (ARCE/e, ARCE/ei, ARCE/ e-d/h and ARCE/ei-d/h). ARCE/e and ARCE/e-d/h contained 1 (6.41 to 15.7% w/w), 2 (9.67 to 18.3% w/w), 3 (0.18 to 0.47% w/w), 4 (0.06 to 0.26% w/w), 5 (0.17 to 0.95% w/w), 6 (0.22 to 0.26% w/w), 7 (0.42 to 1.56% w/w), pseudobaptigenin (8; 0.23 to 0.72% w/w), pratensein (9; 0.23 to 2.01% w/w), kaempferol (10; 0.00 to 0.09% w/w), and quercetin (11; 0.08 to 0.12% w/w). As presented above, a dramatic enrichment of these isoflavonoids after the maceration was observed. However, such an enrichment did not apply to each metabolite in the extract. Compound 11 increased as much as 5-fold from 0.01–0.08% (w/ w) to 0.05–0.12%, while 10 did not follow this trend (Tables 1, 0.04–0.06% in ARCE/e and ARCE/ei to 0.02–0.09% in ARCE/e-d/h and ARCE/ei-d/h).

3.6. UHPLC-UV profiles of ARCE/e-d/h and ARCE/ei-d/h

The profiles of the samples after fatty acid depletion were generated by UHPLC-UV chromatography of ARCE/e-d/h and ARCE/ei-d/h. According to the UHPLC-UV data, all profiles were similar (Fig. S18–Fig. S19). The calibration curves of 1 (5.49 min) and 2 (2.865 min) were established using authentic reference standards. The curves were used to quantify 1 and 2 in each defatted extract (ARCE/e-d/h and ARCE/ei-d/ h). By UHPLC-UV, these aglycones were identified based on their retention times: 4 (1.21 min), 3 (1.98 min), 7 (3.88 min), and 5 (5.27 min), which were almost identical to the UHPLC-UV chromatogram of the clinical RCE (Fig. S18). Moreover, ARCE/e and ARCE/ei experiments were repeated three times to evaluate the reproducibility of the auto-hydrolysis method, and the results were consistent (Fig. S16, Fig. S17 and Table S2, Table S3).

3.7. Principal Component Analysis (PCA) of ARCE/e, ARCE/ie, ARCE/ e-d/h and ARCE/ei-d/h

PCA was used to study relationships in the observed percentage contents (Table 1) of (iso)flavonoids and related compounds in ARCE/e and ARCE/ei, before and after maceration with diethyl ether/hexanes (1:1; ARCE/e-d/h and ARCE/ei-d/h). Principal component score plots (crude extracts in red colour) and loading plot vectors (name of compounds in blue colour) for the extracts (ARCE/e, ARCE/ei, ARCE/e-d/h and ARCE/ei-d/h) were generated according to the contents of (iso) flavonoids and related compounds. Based on the constituents listed in Table 1, thirteen principal components (PC) were extracted, collectively accounting for 83.3% of the variance in the 11 variables (PC1 = 65.7%, and PC2 = 17.7%). PC1 vs. PC2 scores plotted in Fig. 5 reflect the clustering of ARCE/e and ARCE/ei samples, discriminated by auto- hydrolysis and maceration with diethyl ether/hexanes (1:1) to yield ARCE/e-d/h and ARCE/ei-d/h, based on their content in (iso)flavonoids and related compound. As evident in Fig. 5, the ARCE/e and ARCE/ei materials grouped into six distinct clusters of Et1d, Et2d, Et3d, EtIs1d, EtIs2d and EtIs3d, indicating that 11 variables show close similarity across all ARCE/e and ARCE/ei samples. On the other hand, Et3d-d/h, EtIs3d-d/h, and Et2d-d/h were closest to the reference clinical RCE with regard to their content (% w/w) in compounds 1–9. The Et1d-d/h and EtIs1d-d/h samples resembled each other closely, exhibiting high concentrations of 1, 2, 8, and 9. Sample EtIs2d-d/h contained the highest concentration of 3–8 compared with the reference clinical RCE. The highest concentrations of 10 and 11 were found in the reference clinical RCE, therefore the two vectors appeared in the direction of the clinical RCE on Fig. 5.

Fig. 5.

Loading plot vector (name of compounds in blue) and score points (crude extracts in red) of the percent content of the target compounds in each extract as determined by EC-qHNMR analysis. The score of the clinical RCE is separated from the rest as it contains more 10 and 11 than ARCE/e, ARCE/ei, ARCE/e-d/h, and ARCE/ei-d/h. Both the Et1d-d/h and EtIs1d-d/h samples are close to each other with high concentrations of 1, 2, 8, and 9.

3.8. Induction of an estrogen-responsive alkaline phosphatase (AP) enzyme in Ishikawa cells

After the generated extracts were standardized chemically via qNMR and LC-MS, a biological standardization in an estrogenic cell-based bioassay was performed. Upon treatment with estrogens, the enzyme alkaline phosphatase (AP) can be induced in the endometrial cancer cell line, Ishikawa [1,29]. Previous studies have shown that the clinical RCE shows significant levels of estrogenicity in this assay [1,29]. However, the ERCE precursor did not exhibit any estrogenic activity, which is in line with its negligibly low content in isoflavonoid aglycones (Fig. 6). In accordance with the chemical standardization, the biological evaluation revealed that red clover plant material after auto-hydrolysis for 1, 2, and 3 days yields extracts with the expected estrogenic activities (Fig. 6). These results suggest that these ARCEs contain similar amounts of the estrogenic isoflavonoid aglycones, 3 and 4, and their pharmacokinetic aglycone precursors, 1 and 2, respectively (Table 1).

Fig. 6.

Comparing differences in estrogenic activity between various RC extracts (ERCE, clinicalRCE, and ARCEs [EtOH (“Et”) and EtOH/i-PrOH extraction (“EtIs”) after 1, 2, and 3 days of auto-hydrolysis] and ARCE-d/h [EtOH extraction with diethyl ether/hexanes 1:1 treatment (“Et-d/h”) and EtOH/i- PrOH extraction with diethyl ether/hexanes 1:1 treatment (“EtIs-d/h”) after 1, 2, and 3 days of auto-hydrolysis]). Alkaline phosphatase activity was determined as an estrogenic endpoint in the Ishikawa endometrial cancer cell line as described in the Experimental Section. Cells were treated with <0.05% DMSO as vehicle control, estradiol (E₂; 0.5 nM) as positive control, and 5 μg/mL of different RC extracts. The data represent the means ± SD of three independent determinations. * indicates significant estrogenic activity of the extracts compared to DMSO control (p < 0.05[DBM1], one-way ANOVA with Dunnett’s post-test), and ^a indicates significantly different activity compared to the clinical RCE.

While the “green” ARCEs showed significant estrogenic activity, they exhibited lower potency than the prior clinical RCE. However, after maceration of the ARCEs with diethyl ether/hexanes 1/1, extracts [ARCE-d/h (Et-d/h and EtIs-d/h)] with estrogenic activity similar to that of the clinical RCE reference material were obtained (Fig. 6). This is in line with the results from the chemical standardization, showing an increase of isoflavonoid aglycones in the ARCE-d/h samples, particularly of 1 and 2, likely due to removal of fatty acids through maceration with diethyl ether/hexanes 1/1.

While 1 (EC₅₀: 4.6 μM in Ishikawa cells) shows only moderate estrogenic activity [1], isoflavonoid 3 (EC₅₀: 0.3 μM) is a potent phytoestrogen in red clover [29,30]. However, 3 is only contained in low quantities in red clover extracts (Table 1). It is known that 1 is rapidly metabolized in vivo to 3; although this metabolic pathway is not known for Ishikawa cells. Therefore, metabolism is one element of DRC, involving the conversion of 1 to 3 mediated by Ishikawa cells. DRC cannot only occur through P450 oxidation of the 4^′- methoxy group of 1 to generate 3, but the same metabolism reaction is also possible during conversion of 2 to 4. Similarly DRC pathways can be extended to those (iso)flavonoids in extracts that possess a 4^′-methoxy group, as a result of being subject to O-demethylation by a P450 enzyme.

In order to investigate the possible involvement of chemical impurities as an element of static residual complexity on the biological outcomes, the purity of commercial 1 was evaluated by qHNMR. Although the outcome (96.5%, w/w) reached the value claimed in the certificate of analysis, which was based on an HPLC assay, the material did contain 1.88% w/w of the potent phytoestrogen, 3, in addition to residual solvents (1.43% w/w of diethyl ether and 0.16% w/w of MeOH). Thus, this material was further purified by CPC using the hexanes/EtOAc/MeOH/ water (HEMWat) 5.5/4.5/5/5 (v/v) solvent system following a previously documented procedure [45] in order to reach a purity of 99.3% (w/w) and, importantly, obtain 1 (14.3 mg) that was free of 3. While some residual solvent remained as difficult to remove impurities (0.58% w/w of hexanes, and 0.12% w/w of MeOH), they could be considered impartial to the biological outcomes, both qualitatively and quantitatively. All purity determinations were performed using the 100% qHNMR method. The isoflavonoid 3-free sample was fit for studying the metabolism of 1 to 3 in Ishikawa cells (Fig. S2–Fig. S4).

The LC-MS-MS experiments revealed that purified 1 is weakly metabolized to 3 in Ishikawa cells, suggesting that only some of 1’s activity is due to its metabolism to 3 in this cell line (Fig. 1B). Specifically, after incubation of Ishikawa cells with 10 or 20 μM 1 for 96 h, which is the time at which the estrogenic activity is determined, 51 ± 16 nM or 120.4 ± 23.3 nM 3 were detected in the cell media, respectively (Fig. S4). At this concentration, 3 would only show low estrogenic activity in Ishikawa cells. Similar to the metabolism of 1 to 3, it is likely that also 2 will be partially metabolized to 4 in this cell line, as it would undergo a similar biosynthesis pathway in vivo (Fig. 1). According to the percentages of the red clover isoflavones (Table 1) and their individual activity [1], the estrogenicity of several isoflavonoids including 1 and 2, but also 4, 5, and coumestrol, likely contribute [1] (Table 1) to the overall red clover extract activity as shown in Fig. 6. As in vivo higher metabolism rates for 1 to 3 have been described [1,27,28] the in vivo RCE estrogenic activity is likely to be greater as well.

3.9. Summary

An inexpensive, yet efficient, auto-hydrolysis method was developed and validated that is capable of enriching isoflavone aglycones of dietary and therapeutic relevance in red clover and related crude plant extracts. The method avoids the addition of extraneous enzymes or mineral acids and the employment of excess heat [55]. As part of the validation, the ratio of dry plant material to water, temperature, and time of auto- hydrolysis were optimized. An initial experiment performed using a 1:7 ration (g of plant material/mL of water) showed relatively low yield percentages of 1 (3.8, 4.0 and 3.2%) and 2 (5.0, 5.7 and 4.5%), which was likely the result of the sample not dissolving homogeneously, leading to precipitation (Fig. S23). Analyzing the auto-hydrolysis conditions, it can be concluded that a plant material to water ratio of 1:12 or 1:15 (g/mL) gave the best yield of isoflavonoid aglycones. In addition, a temperature of 25 °C resulted in the highest content of aglycones target compounds without degradation. While the present work focused on (iso)flavonoid glycosides, it might inspire future work on other phenylpropanoic phenols and potentially aliphatic compounds such as terpenoids, which might also be amenable to autohydrolysis.

Detailed chemical and biological analyses revealed that increasing the auto-hydrolysis time from 1 to 2 or even 3 days does not improve the (iso)flavonoid aglycone yield in the ARCEs. The first step of the process can be referred to as a “green method” as 10 g of dry and ground plant material can be submerged in 120 mL of water to obtain extracts rich in aglycones (ARCE) and with significant estrogenic activity. The outcomes show that residual complexity in general, and dynamic specifically, needs to be considered closely during bioassays as compounds can be metabolized from weakly to highly active compounds.

The enrichment method is both economical and “green” as developed, does not require sophisticated laboratory equipment, and can likely be scaled up to an industrial level, allowing for its use in future clinical trials. The established auto-hydrolysis method presents a helpful tool for the assessment of the biological profiles of (iso)flavone-rich plant extracts, due to the elevated quantities of the biologically most relevant aglycones. For example, the presented methodology may also be transferable to Epimedium species, as the enzymes mediating the auto- hydrolysis are likely the same plant-intrinsic enzymes that are involved in the biosynthesis of the glycosides.

Abbreviations:

RCE

red clover extract

qHNMR

quantitative ¹H NMR

ARCE

auto-hydrolyzed red clover extract

ERCE

ethanol red clover extract

ARCE/e

ethanol product of auto-hydrolyzed red clover extract

Et1d

ethanol 1 day

Et2d

ethanol 2 days

Et3d

ethanol 3 days

ARCE/ei

ethanol/isopropanol product of auto- hydrolyzed red clover extract

EtIs1d

ethanol/isopropanol 1 day

EtIs2d

ethanol/isopropanol 2 days

EtIs3d

ethanol/isopropanol 3 days

ARCE/e-d/h

ethanol product of auto-hydrolyzed red clover extract macerated in diethyl/hexanes

Et1d-d/h

ethanol 1 day extract macerated in diethyl ether/hexanes

Et2d-d/h

ethanol 2 days extract macerated in diethyl ether/hexanes

Et3d-d/h

ethanol 3 days extract macerated in diethyl ether/hexanes

ARCE/ei-d/h

ethanol/isopropanol product of auto-hydrolyzed red clover extract in diethyl/hexanes

EtIs1d-d/h

ethanol/isopropanol 1 day extract macerated in diethyl ether/hexanes

EtIs2d-d/h

ethanol/ isopropanol 2 days extract macerated in diethyl ether/hexanes

EtIs3d-d/h

ethanol/isopropanol 3 days extract macerated in diethyl ether/hexanes

ER

estrogen receptor

PCA

principal component analysis

RPM

residual plant material

CPC

centrifugal partition chromatography

DMEM/F12

Dulbecco’s modified Eagle’s medium

AP

alkaline phosphatase

DRC

dynamic residual complexity

HEMWat

hexanes/ethyl acetate/methanol/water