Clinical Study Effects of the Chinese Herbal Formulation

Clinical StudyEffects of the Chinese Herbal Formulation(Liu Wei Di Huang Wan) on the Pharmacokinetics ofIsoflavones in Postmenopausal Women

Wirin Limopasmanee,1 Sunee Chansakaow,2 Noppamas Rojanasthien,1 Maleeya Manorot,1

Chaichan Sangdee,1 and Supanimit Teekachunhatean1,3

1Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand2Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand3Center of Thai Traditional and Complementary Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand

Correspondence should be addressed to Supanimit Teekachunhatean; [email protected]

Received 1 February 2015; Accepted 30 April 2015

Academic Editor: Gail B. Mahady

Copyright © 2015 Wirin Limopasmanee et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

A combination of soy isoflavones and Liu Wei Di Huang Wan (LWDHW) is potentially effective for postmenopausal women withintolerable vasomotor episodes who are not suitable candidates for hormonal therapy.The objective of this open-label, three-phase,crossover studywas to determine the influence of both single andmultiple oral doses of LWDHWon isoflavone pharmacokinetics inhealthy postmenopausal women. Eleven subjects were assigned to receive the following regimens in a fixed sequence with washoutperiods of at least one week: Phase A, a single oral dose of soy milk; Phase B, a single oral dose of soy milk coadministered withLWDHW; and Phase C, multiple oral doses of LWDHW for 14 days followed by a single oral dose of soy milk. Blood samples werecollected and mixed with 𝛽-glucuronidase/sulfatase to hydrolyze isoflavone conjugates to their respective aglycones (i.e., daidzeinand genistein) and were determined using high performance liquid chromatography. The pharmacokinetic parameters analyzedwere maximal plasma concentration (𝐶max), time to reach peak concentration (𝑇max), area under the plasma concentration-timecurve (AUC), and half-life (𝑡1/2).The results found no statistically significant differences in pharmacokinetic parameters of daidzeinand genistein among the three regimens.

1. Introduction

Menopause is a period of time in a woman’s life defined asthe cessation of menstruation for more than 12 months andinvolves permanent failure of ovarian function secondaryto depletion of the follicular pool. This commonly occursin Asian women who are in their mid-to-late 40s [1, 2].Vasomotor episodes are themost characteristicmanifestationof themenopause.These episodes are described as hot flushes(the sensations of warmth, usually felt on the chest, neck,and face), hot flashes (episodes with sweating, sometimesfollowed by a chill), andnight sweats.However, the terms “hotflushes” and “hot flashes” are often used interchangeably [1].The proportion of women who report hot flashes may be as

great as 80% in western countries but as low as 10% in someEast Asian countries. Hormone therapy (HT) diminishesvasomotor symptoms in a dose-dependent fashion. However,the use of estrogen alone in HT increases the risk ofendometrial hyperplasia and cancer, while continuous use ofcombinedHT (estrogen plus progestin) may increase the riskof coronary heart disease, breast cancer, stroke, and venousthrombosis [3–5]. Concern over the potential adverse effectsof HT leads many women to look for natural alternatives thatwould provide benefits comparable to those of estrogen butwithout serious complications.

Soy isoflavones, known as phytoestrogens, have beenshown to reduce vasomotor symptoms significantly withoutan adverse effect on the endometrium or vagina [6, 7].

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015, Article ID 902702, 8 pageshttp://dx.doi.org/10.1155/2015/902702

2 BioMed Research International

In addition, they are considered beneficial for reducingthe incidence of cardiovascular disease, hormone-dependentbreast cancer, colon cancer, and osteoporosis [8–11]. Likewise,Traditional ChineseMedicine (TCM) is one attractive optionfor alleviating menopausal symptoms. According to TCM,vasomotor symptoms (especially hot flushes and nightsweats) are caused by Kidney Yin Deficiency. The Chineseherbal formulation of choice for nourishing Kidney YinDeficiency is “Liu Wei Di Huang Wan” (LWDHW) or “Six-Ingredient Pill with Rehmannia.” This formulation consistsof Shu Di Huang (Radix Rehmanniae Preparata), Shan ZhuYu (Fructus Corni), Shan Yao (Rhizoma Dioscoreae), Ze Xie(Rhizoma Alismatis), Mu Dan Pi (Cortex Moutan), and FuLing (Poria). A modified formulation of LWDHW has beenshown to be significantly effective in reducing the numberof hot flushes without serious adverse reactions [12, 13].LWDHW, in addition to being a popular Chinese herbalformula used in the treatment of vasomotor symptoms, is alsoused in the treatment of other medical conditions such asdiabetes [14], hypertension [15], and disorders of the immunesystem [16].

As both soy isoflavones and LWDHW are potentiallyeffective for management of vasomotor symptoms, the com-bination of both modalities to maximize their efficacy isconsidered a plausible attractive option, especially for someperimenopausal or postmenopausal women who experienceintolerable vasomotor episodes but are not suitable candi-dates for HT; however, no study of the pharmacokineticinteraction between the two interventions has been done.The main objective of this study was to determine theinfluence of single and multiple oral doses of LWDHWon isoflavone pharmacokinetics in healthy postmenopausalwomen.

2. Materials and Methods

2.1. Study Design. This study was an open-label, fixed-sequence, three-phase, crossover study, with washout periodsof at least 1 week. The study was carried out at the Clin-ical Pharmacology Unit, Faculty of Medicine, Chiang MaiUniversity. The study protocol was approved by the HumanResearch Ethics Committee of the Faculty of Medicine,Chiang Mai University, and it complied with the HelsinkiDeclaration. The details of the study were clearly describedto all participants and a signed informed consent was volun-tarily obtained from them prior to their participation in thestudy.

2.2. Participants. The sample size calculation was based onthe assumptions that the content of absorbed isoflavones(equivalent to the area under the plasma concentration-timecurve or AUC) would be the main efficacy criterion, and themean difference inAUC following concurrent administrationof LWDHW and soy milk (𝜇2) in comparison to AUCfollowing consumption of soy milk alone (𝜇1) was assumedto be 0 (i.e., 𝜇2 − 𝜇1 = 0). The noninferiority margin (𝛿)was estimated to be 20%, and the standard deviation (𝜎)was expected to be 18.5. By using the following formula for

noninferiority trials [17], the required sample size to achievean 80% power (𝛽 = 0.2) at 𝛼 = 0.05 for detecting suchdifference was 11 participants. Consider

𝑛1 = 𝑛2 =2 (𝑧𝛼 + 𝑧𝛽)

2𝜎2

(𝜇2 − 𝜇1 − 𝛿)2 .

(1)

Eleven postmenopausal Thai women, aged at least45 years with cessation of menstruation for at least 12months and serum follicle-stimulating hormone concentra-tion >30 IU/L, were enrolled in this study. Their body massindex (BMI) was between 18–25 kg/m2. Each participantunderwent a physical and blood examination includingcomplete blood count (CBC), blood urea nitrogen (BUN),creatinine (Cr), and liver function test (LFT) to confirm theirhealth status. Postmenopausal women with a prior historyof malignancy or renal, liver, pulmonary, cardiovascular, orgastrointestinal diseaseswere excluded from the study aswerewomen with a known history of cigarette smoking, alcoholicconsumption, substance abuse, or addiction. Other exclusioncriteria were the use of antibiotics, laxatives, prebiotics,probiotics, synbiotics, other medications, and food supple-ments within the preceding four weeks. All participants wererequested to abstain from soy-food products, other medica-tions, herbal or food supplements, caffeine-containing bev-erages, prebiotics, probiotics, and synbiotics during all studyperiods.

2.3. Dosage and Drug Administration

2.3.1. Soy Preparation. The soy preparation used in this studywas UHT soy milk (V-Soy, expiration date 5 September 2014,manufactured by Green Spot Co., Ltd, Bangkok, Thailand).The mean isoflavone content of daidzin, genistin, daidzein,and genistein was 37.74± 1.56, 36.95± 1.50, 11.21± 0.40, and6.82 ± 0.24mg/375mL, respectively.

2.3.2. LWDHW. The LWDHWused in this study was manu-factured by the Faculty of Pharmacy, Chiang Mai University,Thailand. It consisted of 24% Radix Rehmannia Preparata,12% each of Fructus Corni and RhizomaDioscoreae, 9% eachof Rhizoma Alismatis, Cortex Moutan, and Poria as well as25% honey. All six crude drugs were purchased fromVejpongPharmacy Co., Ltd (Bangkok, Thailand). Raw materials usedin this formula were evaluated following Chinese Pharma-copoeia 2005 [18]. Chemical control of LWDHW was ana-lyzed by high performance liquid chromatography coupledwith photodiode array (HPLC-PDA) using loganin as theactive marker. The standardized crude drugs of LWHDWwere ground into a fine powder andmixed with heated honeyto make boluses approximately 4mm in diameter. Qualitycontrol of the honey pill (i.e., assessment of weight varia-tion, disintegration, moisture content, and microbial con-tamination) was conducted following WHO guidelines ongood manufacturing practices (GMP) for herbal medicines[19].

BioMed Research International 3

Run-inperiod of≥1wk

Studyparticipation

Phase B Phase CPhase A

Blood samplecollection


Washoutperiod of≥1wk

375mLof soymilk

Day0

Day0 Day

0

6g LWDHW

Washoutperiod of≥1wk

6g LWDHW,orally, 3 times daily

for 14days

375mLof soymilk


375mLof soy milk +

–Day−14

Day−1

Figure 1: Administration of soymilk and/or LWDHWinPhasesA, B, andC (treatment in each phasewas given in a fixed-sequence approach).Phase A, a single oral dose of 375mL of soy milk; Phase B, a single oral dose of 375mL of soy milk coadministered with a single oral doseof 6 g LWDHW; and Phase C, an oral administration of 6 g LWDHW three times daily for 14 days followed by a single oral dose of 375mLof soy milk on the next day. Blood sample collection: before administration of soy milk and at 0.5, 1, 2, 4, 6, 8, 10, 12, 24, and 32 hours afteradministration of soy milk.

2.3.3. Drug Administration. This study consisted of threephases in a fixed-sequence approach (Phases A, B, and C)as shown in Figure 1. After a run-in period of at least oneweek, participants were assigned to receive a single oral doseof 375mL of soy milk (Day0 of Phase A). After a washoutperiod of at least one week, participants underwent PhaseB during which a single oral dose of 6 g LWDHW wascoadministered with a single dose of 375mL of soy milk(Day0 of Phase B). Subsequently, participants underwenta washout period of at least one week and continued toreceive treatment in Phase C during which 6 g LWDHWwas administered orally three times daily (approximatelyone hour before meals) for 14 days, followed by a singleoral dose of 375mL of soy milk on the next day (Day0 ofPhase C). In each phase of this study, soy milk was orallyadministered in themorning after an overnight fast of at leasteight hours. Participants were fasted for two hours and wererequested to remain in an upright position for four hoursfollowing soy milk consumption. Water, lunch, and dinnerwere served at 2, 4, and 10 hours, respectively, followingadministration of the soy milk. Identical isoflavone-free dietsand beverages were served to all participants during thethree phases of this study. All participants were requestedto refrain from isoflavone-containing products as well assome specific foods and beverages (e.g., soy milk, tofu,prebiotics, probiotics, synbiotics, alcohol, caffeine beverages,etc.) throughout the study period. During each phase,blood samples were collected at different time points (seebelow).

2.4. Collection of Blood Samples. On Day0 of each phase,venous blood samples (6mL) for quantification of isoflavonelevels were collected from the forearm by venipuncturethrough an indwelling intravenous catheter prior to anadministration of soymilk and then at 0.5, 1, 2, 4, 6, 8, 10, 12,24 and 32 hours after the dose. Blood samples were collectedin heparinized vacutainer tubes and were centrifuged at2500 rpm for 20 minutes. Thereafter, plasma was separatedand immediately maintained at −70∘C in a freezer untilanalyzed.

2.5. Determination of Plasma Isoflavones byHigh Performance Liquid Chromatography withUV-Diode-Array Detection (HPLC-UV)

2.5.1. Sample Preparation. Plasma sample preparation wasperformed as described by Timan et al., 2014 [20]. Specif-ically, 250𝜇L of plasma was mixed with 150 𝜇L of anenzyme mixture of 𝛽-glucuronidase/sulfatase in order tohydrolyze glucuronide and sulfate metabolites to daidzeinand genistein.The enzymemixture consisted of 0.1 g ascorbicacid in 10mL of 0.1M sodium acetate buffer containing0.01 g ethylenediaminetetraacetic acid (EDTA) and 250 𝜇Lof 𝛽-glucuronidase/sulfatase obtained from Helix pomatia(SIGMA G-0876). To complete the hydrolysis of isoflavoneconjugates, plasma samples with the enzyme mixture wereheated in awater bath at a temperature of 37∘C for 15–18 hoursand then allowed to cool to room temperature. Thereafter,10 𝜇L of 50,000 ng/mL fluorescein in 80% methanol wasused as the internal standard (IS) and added to the plasmasample, followed by 1,000 𝜇L of 1% acetic acid in acetonitrileto deproteinize the samples which were then vortex mixedfor 30 seconds. The samples were centrifuged at 14,000 rpmfor 10 minutes to separate the supernatant and were thenevaporated to dryness by a vacuum concentrator at 60∘Cfor three hours. Subsequently, 50 𝜇L of mobile phase B(described below) was added to reconstitute the residue, and5 𝜇L of the reconstituted sample was injected into the HPLCsystem.

2.5.2. HPLC-UVConditions. Thedetermination of isoflavonelevels in plasmawas performed byHPLC-UV as described byTiman et al., 2014 [20].TheHPLC assay was conducted usinga UV detection wavelength of 259 nm. The chromatographysystem consisted of a 5-𝜇m C-18 column equipped witha guard column (Inersil ODS-3, GL Sciences, Japan). Themobile phases used in this HPLC condition were mobilephase A and B consisting of 60mM ammonium acetate indeionized water/methanol/acetonitrile in the proportions of250/45/45 (v/v/v) and 250/220/255 (v/v/v), respectively. Bothmobile phases contained 250𝜇L of 1.44mM sodium dodecyl


sulfate and 30 𝜇L of perchloric acid. A chromatographicseparation was scheduled using a gradient elution of 100% Aat 0–3min, 70 : 30withA : B at 3.1–9min, 20 : 80 at 9.1–18min,and 85 : 15 at 18.1–25min, respectively. The system was oper-ated with flow rate of 1mL/min at 25∘C. The chromatogramrevealed that retention times of IS, daidzein, and genisteinwere 11.234, 14.357, and 16.389 minutes, respectively.

2.5.3. Assay Validations. Calibration curve plots demon-strated that the linear regressions of daidzein and genisteinconcentrations ranged from 37.5, 75, 150, 300, 600, 1200, and2400 ng/mL versus peak height ratios of isoflavones and ISgave coefficients of determination (𝑟2) greater than 0.9997.

For intraday precision, five replicated samples fromeach of three quality control (QC) samples (112.5, 1125,and 2250 ng/mL) were quantified with a single calibrationcurve, while determinations of five replicated samples ofthe respective QC samples using five concurrent standardcalibration curves on five dependent days were conducted toevaluate interday precision.

Precision was shown as the percentage coefficient ofvariation (% CV) calculated as

% CV

=Standard deviation

Mean value of isoflavone concentrations in plasma

× 100.

(2)

The inaccuracy was presented as the percentage of devia-tion (% deviation) calculated as

% Deviation

=(Measured concentration − Spiked concentration)

Spiked concentration

× 100.

(3)

The % CV of intraday precision for the three respectiveQC samples (112.5, 1125, and 2250 ng/mL) were 3.41, 1.04, and1.82% for daidzein and 2.71, 0.78, and 1.15% for genistein. Forinterday precision, % CV for the three QC samples were 2.46,3.53, and 4.08% for daidzein and 5.93, 3.25, and 3.24% forgenistein.

The percentage deviations of intraday assay for the threeQC samples of daidzein were −3.58, −1.10, and −4.34%, andthose of genistein were 9.13, −4.61, and −4.95%, while thepercentage deviations of interday assay of daidzein were−6.73, 1.46, and −1.95%, and those of genistein were 2.16,−4.62, and −5.02%. The mean recovery values (± standarddeviation) of daidzein, genistein, and IS were 93.45 ± 3.55%,90.57 ± 5.94%, and 97.69 ± 2.38%, respectively.

2.6. Pharmacokinetics Parameters and Statistical Analysis

2.6.1. Pharmacokinetics Parameters. The pharmacokineticparameters analyzed in this study were maximal plasmaconcentration (𝐶max), time to reach the peak concentra-tion (𝑇max), the area under the plasma concentration-time

curve from time 0–32 hours and 0–∞ hours (𝐴𝑈𝐶0–32 and𝐴𝑈𝐶0–∞), and the terminal half-life (𝑡1/2). These parameterswere determined by noncompartmental analysis. The 𝐶maxand 𝑇max were acquired by direct visual inspection of eachsubject’s plasma concentration-time curve profile. The 𝑡1/2was calculated as the ratio of 0.693 to the terminal rate con-stant (𝑘𝑒), while 𝑘𝑒 was obtained from the terminal slope onthe semilog concentration versus time plot. The trapezoidalrule was used for the𝐴𝑈𝐶0–32 calculation.The𝐴𝑈𝐶32–∞ wasextrapolated using the ratio of isoflavone concentration atthe 32nd hour to 𝑘𝑒 (𝐶32/𝑘𝑒). Total 𝐴𝑈𝐶0–∞ was the sumof 𝐴𝑈𝐶0–32 and 𝐴𝑈𝐶32–∞. The analysis for pharmacokineticparameters was performed using the TOPFIT version 2.0pharmacokinetic data analysis program for Windows.

2.6.2. Statistical Analysis. The pharmacokinetic parameterswere expressed as mean ± standard deviation (SD). Thenonparametric Friedman’s test was used to analyze the statis-tical differences among the mean values of pharmacokineticparameters obtained following each of the three study phases.Differences were considered to be statistically significant if 𝑃value <0.05.

3. Results

Eleven healthy Thai postmenopausal women completed thestudy protocol.The average age of the participants was 58.09±5.13 years. Their mean weight, height, and body mass indexwere 56.97 ± 3.95 kg, 1.55 ± 0.04m, and 23.67 ± 0.81 kg/m2,respectively.

The mean plasma concentration-time curves of daidzeinand genistein from the 11 participants receiving the three regi-mens during the respective study phases are shown in Figures2 and 3, respectively. The mean plasma concentration-timeprofiles of both daidzein and genistein obtained followingeach phase showed a biphasic pattern. Notably, the earlierpeaks of both isoflavones occurred at approximately 2 hours,whereas the later peaks occurred at approximately 6–8 hours.

The pharmacokinetic parameters of daidzein and genis-tein in all participants obtained following each of the threestudy phases are demonstrated in Tables 1 and 2, respectively.The mean values of the pharmacokinetic parameters (𝐶max,𝑇max, 𝐴𝑈𝐶0–32, 𝐴𝑈𝐶0–∞, and 𝑡1/2) of daidzein and genisteindid not statistically differ among the three study phases.

It is worth noting, however, that the mean valuesof 𝐴𝑈𝐶0–32 and 𝐴𝑈𝐶0–∞ of both daidzein and genisteinobtained from Phase B were somewhat lower (approximately10–15%) than those from Phase A, although the differenceswere not statistically significant. The mean values of AUCsobtained from Phase C, on the other hand, were somewhatgreater than those of Phase A, although, again, not statisti-cally significant. It is noteworthy, however, that this greaterPhase C enhancement of AUCs than in Phase A was rathermore pronounced for genistein than daidzein (approximately15% versus 3%).

After the 14-day administration of LWDHW, the meanvalues of parameters used to assess safety (i.e., systolic anddiastolic blood pressure, FSH level, complete blood count,


Table 1: Pharmacokinetic parameters of daidzein obtained following each of the three study phases in the 11 participants enrolled in the study.

Pharmacokinetic parameters Treatment phase𝑃 value∗

Phase A Phase B Phase C𝐶max (ng/mL) 772.64 ± 247.30 715.45 ± 207.16 804.00 ± 173.03 0.06𝑇max (h) 6.55 ± 2.02 6.64 ± 2.29 6.55 ± 1.81 0.97AUC0–32 (ng⋅h/mL) 7313.77 ± 3184.04 6234.05 ± 2536.89 7543.97 ± 2638.96 0.06AUC0–∞ (ng⋅h/mL) 8169.06 ± 3056.75 7362.15 ± 2344.35 8416.20 ± 2522.09 0.18𝑡1/2 (h) 4.42 ± 0.88 4.58 ± 1.06 4.14 ± 0.80 0.81∗Friedman’s test.Phase A, a single oral dose of soy milk; Phase B, a single oral dose of soy milk coadministered with LWDHW; Phase C, multiple oral doses of LWDHW for 14days followed by a single oral dose of soy milk.

Table 2: Pharmacokinetic parameters of genistein obtained following each of the three study phases in the 11 participants enrolled in thestudy.

Pharmacokinetic parameters Treatment phase𝑃 value∗

Phase A Phase B Phase C𝐶max (ng/mL) 784.36 ± 371.67 670.36 ± 171.53 873.18 ± 336.34 0.09𝑇max (h) 5.36 ± 2.54 5.73 ± 2.28 6.00 ± 2.19 0.64AUC0–32 (ng⋅h/mL) 9062.53 ± 5237.58 7871.89 ± 3054.41 10534.08 ± 4730.12 0.53AUC0–∞ (ng⋅h/mL) 9956.56 ± 5441.06 8544.75 ± 3053.48 11291.46 ± 5030.50 0.53𝑡1/2 (h) 6.80 ± 0.62 6.69 ± 0.80 6.90 ± 0.89 0.91∗Friedman’s test.Phase A, a single oral dose of soy milk; Phase B, a single oral dose of soy milk coadministered with LWDHW; Phase C, multiple oral doses of LWDHW for 14days followed by a single oral dose of soy milk.

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32Mea

n pl

asm

a con

cent

ratio

nof

dai

dzei

n (n

g/m

L)

Time (h)

Phase APhase B

Phase C

Figure 2: Mean plasma daidzein concentration-time curves fromthe 11 participants receiving a single oral dose of soy milk (Phase A),a single oral dose of soy milk coadministered with LWDHW (PhaseB), and multiple oral doses of LWDHW for 14 days followed by asingle oral dose of soy milk (Phase C). Error bars represent standarddeviation (SD).

and kidney and liver function) did not differ statistically orclinically from the baseline values (data not shown).

4. Discussion

The findings from this study revealed no statistically sig-nificant differences in the pharmacokinetic parameters ofdaidzein and genistein among the three regimens. However,the bioavailability of both isoflavone aglycones followingcoadministration of soy milk and LWDHW (Phase B) wassomewhat lower than that of soy milk alone (Phase A),

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32Mea

n pl

asm

a con

cent

ratio

n of

gen

istei

n (n

g/m

L)

Time (h)

Phase A Phase CPhase B

Figure 3: Mean plasma genistein concentration-time curves fromthe 11 participants receiving a single oral dose of soy milk (Phase A),a single oral dose of soy milk coadministered with LWDHW (PhaseB), and multiple oral doses of LWDHW for 14 days followed by asingle oral dose of soy milk (Phase C). Error bars represent standarddeviation (SD).

while the bioavailability of these aglycones after multiple-dose administration of LWDHWfor 14 days (PhaseC) tendedto be greater than that of Phase A.

Soy milk, rather than other soy products such as soyextract capsules or other solid soy foods, was selected forthis study because the isoflavones in soy milk are completelydissolved and ready to be absorbed, avoiding the need forinvestigation of disintegration and dissolution rates, factorswhich can affect isoflavone absorption via the gastrointestinaltract. The isoflavone dose used in this study was selectedaccording to two main reasons. First, the recommended


dose of isoflavones for relieving vasomotor symptoms inpostmenopausal women is demonstrated to be approximately50mg/day or higher [6, 7, 21–23]. Second, it is generallyaccepted that doses of isoflavones ranging from 40 to160mg/day do not adversely affect endometrial thickness,vaginal cytology, or breast density [23–25]. Thus, the 90-mgtotal isoflavones dose used in the present study is well withinthe range of effective doses commonly used for relievingvasomotor symptoms in actual practice.

The generally recommended dose of LWDHW for reliev-ing vasomotor symptoms (commonly equated to Yin Defi-ciency in Chinese medicine) ranges from 9 to 18 g/day [26]and can be consumed into two or three daily doses. Thus,the dose of 18 g/day used in this study is in accordancewith the dose recommended in Chinese medical practice.Furthermore, the 14-day pretreatment with LWDHW inthis study was appropriate for evaluation of its influenceon isoflavone pharmacokinetics since previous studies havedemonstrated that a 14-day pretreatment period is sufficientto affect the enzyme activities involved in Phase I (CYP1A2,CYP2A6, CYP3A4, CYP2C19, andCYP2D6) andPhase II (N-acetyltransferase2, NAT2) hepatic biotransformation. Thesestudies have shown that LWDHWhas no effect on CYP2C19,CYP2D6, and CYP3A4, whereas it can induce CYP1A2 butcan suppress the activities of CYP2A6 and NAT2 [27, 28].

The mean plasma concentration-time curve profiles aftera single oral dose of soy milk in this study exhibited abiphasic pattern, consistent with previously reported findingsin Thai menopausal women [20, 29–31]. The appearance ofthe first peak, observed at approximately two hours aftersoy milk consumption, corresponds to the initial absorptionof aglycones from the duodenum and proximal jejunum.During this step, lactase-phlorizin hydrolase and cytosolic𝛽-glucosidase enzymes in the intestinal mucosa play animportant role in the hydrolysis of isoflavone 𝛽-glycosides(i.e., daidzin and genistin, which are abundant isoflavoneforms in soy milk) to their respective readily absorbableaglycones [32]. The second peak, occurring at approximately6–8 hours, primarily reflects the capacity of gut microbiotato hydrolyze 𝛽-glycosides to their respective aglycones priorto absorption, which mainly occurs in the lower intestinaltract [33–35]. Additionally, the enterohepatic recirculation ofisoflavones might also be partly involved in the occurrence ofthe second peak [34–36].

The findings from Phase B suggest that the bioavailabilityof both daidzein and genistein obtained following the coad-ministration of soy milk and LWDHW was approximately10–15% lower than for soy milk alone (Phase A) despitethe lack of statistical significance. The reduction in 𝐶max ofthe first and second peaks of the plasma concentration-timecurve (without any alternation in elimination 𝑡1/2) indicatesthat coadministration of LWDHWmight negatively interferesomewhat with isoflavone absorption, either in the upperor lower gastrointestinal tract, or both. Since aglycones arereadily absorbed by passive diffusion without any involve-ment with active or facilitated transport [37], LWDHW isunlikely to interfere with a carrier or the saturation process.A plausible explanation for this phenomenon is that somecomponents in LWDHW might partially protect isoflavone

𝛽-glycosides from conversion to their respective readilyabsorbable aglycones. This possibility is in accordance withprevious data showing that the composition of the foodmatrix (such as fiber and carbohydrates) can affect isoflavonebioavailability, especially in females [38]. Nonetheless, theimpact of a single coadministered oral dose of LWDHW onisoflavone bioavailability seems negligible and should lackclinical significance.

The findings from Phase C, on the other hand, demon-strate a tendency for increased isoflavone bioavailability afterpretreatment with multiple-dose LWDHW for 14 days com-pared to that with soy milk alone (Phase A). The increasedisoflavone bioavailability in Phase C primarily correlatedto an enhancement of 𝐶max and AUC of the second peak,suggesting an impact of pretreatment with LWDHW ongut microbiota responsible for conversion of isoflavone 𝛽-glycosides to their respective absorbable aglycones as men-tioned above. The underlying mechanism possibly involvesthe ability of pretreatment with LWDHW to stimulatethe growth of 𝛽-glucosidase-producing microbiota (suchas lactobacilli and bifidobacteria), especially in the lowergastrointestinal tract, yielding an enhanced hydrolysis ofunabsorbable isoflavone 𝛽-glycosides to absorbable aglyconeforms. This hypothesis is in accordance with previouslyreported data demonstrating that Radix Rehmannia (themain component of LWDHW) can exert a prebiotic effectvia stimulation of Lactobacilli growth [39]. Nonetheless, theenhancement ofAUCs caused by pretreatmentwith LWDHWin this study was rather more pronounced for genistein thandaidzein, a finding which is in agreement with previousstudies showing that prebiotic or probiotic supplementationenhances the absorption of genisteinmore thandaidzein bothin rats [40] and in human subjects [30, 41].

Taken together, the present study demonstrates that nei-ther coadministration of single-dose LWDHW nor pretreat-ment with multiple-dose LWDHW significantly alters thepharmacokinetics and bioavailability of isoflavones. There-fore, concurrent use of LWDHW and isoflavones (fromvarious soy products) is likely to be a simple and satisfactorymethod of enhancing drug compliance. Nonetheless, well-controlled clinical trials to investigate and verify the additiveor synergistic efficacy of this combination should be con-ducted.

Some important limitations regarding the present studyshould be addressed. First, in order to fully avoid a carry-overeffect of concurrent administration of LWDHW (conductedin Phases B and C) on isoflavone pharmacokinetics, thisstudy was designed as a three-phase study with a fixedsequence separated by a washout period of at least oneweek rather than randomizing the sequences. Second, thesample size in this preliminary study was small. Furtherstudies with larger samples to increase the statistical powerto differentiate significant differences among study phasesshould be considered. Third, as this study did not includemeasurement of levels of isoflavones and their metabolitesin urine and feces, the effects of LWDHW on isoflavoneexcretion was not investigated. Finally, this study focusedon the influence of LWDHW on the pharmacokinetics ofisoflavones, but it did not address the question of whether


isoflavones affect the pharmacokinetics of LWDHW (or itsactive ingredients). Further investigation of those issues iswarranted.

5. Conclusions

In healthy postmenopausal women, either coadministrationof single-dose LWDHW or pretreatment with multiple-doseLWDHW for 14 days did not statistically alter the pharma-cokinetic parameters of daidzein or genistein compared tothose with the administration of a single oral dose of soymilkalone.

Conflict of Interests

There is no conflict of interests. None of the authors have adirect financial relation with any of the commercial identitiesmentioned in the paper.

Acknowledgments

Financial support from Faculty of Medicine, Chiang MaiUniversity, is gratefully acknowledged. The authors are alsograteful for the editorial assistance of Dr. G. Lamar Robert,English language consultant, Department of Pharmacology,Faculty of Medicine, Chiang Mai University. Thanks are alsoextended toMs. Sujitra Techatoei for her secretarial support.

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