Estradiol

Loughborough University

Chemistry Department

Analytical Chemistry

Estradiol Genistein

Measurement of Phytoestrogens in Saliva

Taghreed Muhammad Fagieh

Supervisor/ Dr Helen Reid

September- 2008

IsoflavonesMammalian endogenous estrogens

Phytoestrogens are natural compounds found in manyfoods. They are structurally and functionally similar to 17β-estradiol as phytoestrogens can bind to two kinds of estrogen receptors. They show estrogenic and antiestrogenic activity (1). They are classified into many classes(2)

• isoflavones • lignans• coumestans

Isoflavones are the most known class of phytoestrogens thatare naturally occurring and showing estrogenic activity (2).


• Daidzein and genistein are the main isoflavones which are derived from formononetin and biochanin A, respectively (1).

FormononetinGenistein Daidzein


• Isoflavones are metabolized after consuming by mammals in the gastrointestinal tract and converted to heterocyclic phenols which

are structurally similar to estrogens (1).

• Soy products have relatively high concentration of isoflavones in form of β-glycoside conjugates.

• After consumed these conjugates are hydrolyzed resulting in

bioactive aglycones which are considered as weak agonists and antagonists (3).


• Therefore, there has been a dramatic interest in phytoestrogens and their essential role in human health as they are considered as alternative to hormone replacement therapy and estrogen replacement therapy (2).

• It has been suggested that a diet rich in isoflavones is associated with potential beneficial effect on human (3).

• Isoflavones have been analyzed in human urine, plasma and recently in saliva (4). HPLC has been widely used to analyze samples with simple preparation of samples (2).


1. The aim of the project To develop the method used to analyze saliva samples by HPLC

To measure the concentration of isoflavones in saliva using their standard curves

To calculate the recovery percentage of each isoflavone in saliva processed

With alkaline hydrolysis

Without alkaline hydrolysis


2. Material and methods

2.1 Instrumentation• HPLC Agilent Chemstation 1200 series auto sampler, with the column of

150 x 4.60 mm Gemini-NX 5μm C18 110A. • Centrifugation system, Hermle Z 200 A.• PH meter, 3310 JENWAY

2.2 Mobile phase We used gradient mobile phase of 60:40 MeOH:H2O with 0.1% formic acid

to 30:70 MeOH:H2O with 0.1% formic acid. Mobile phase A was used as the solvent

2.3 Stock and standard solutions• 20 ppm of each daidzein and genistein stock solutions was used to made

up the series of known concentration standard solutions of 0.1, 0.2, 0.4, 0.5, 0.7 and 1.0 ppm. Formononetin was used as an internal standard.

• 20.1 ppm formononetin stock solution was used to prepare 1, 2, 4, 5, 7 and 10 ppm standard solutions without internal standard.


Time(min)Solvent BFlow rate

075%1 ml/min

100%1 ml/min

150%1 ml/min

1675%1 ml/min

1975%1 ml/min

Injection volume10 μl

Column temperature37◦ C

Flow rate1 ml/min

wavelength259 nm

Stop time20 min

2.4 Experimental procedures

2.4.1 Separation of the four isoflavones using the gradient mobile phases as shown in table (1).

Under particular conditions as shown in table (2).

Table (1)

Table (2)


2.4.2 Determination of natural isoflavones in saliva

Four participants took a part in this study and produced saliva samples. Naturalisoflavones were measured in each sample which was processed with alkalinehydrolysis.

• Alkaline hydrolysis• 2 ml saliva + 5 ml solvent + 2.5 ml NaOH + 0.5 ml acetic acid• Centrifugation at 3000 rpm for 10 min• Adjustment of pH at 5• Ether extraction• Leave the samples overnight and then reconstitute the dry solid in 2 ml solvent


2.4.3 Measurement of isoflavones in saliva

2.4.3.1 After consuming soya milk Saliva samples were produced every 2 hours and at 24 hr and 48 hr. 2.4.3.2 After ingesting isoflavones tablet Saliva samples were produced every hour and at 24 hr.

All saliva samples were spiked with a known concentration of formononetin as a recover marker and then processed using alkaline hydrolysis.


2.4.4 Recovery experiments

2.4.4.1 Saliva was processed with alkaline hydrolysis All samples were spiked with known concentration of daidzein, genistein

and formononetin. Then they were processed using alkaline hydrolysis.

2.4.4.2 Saliva was processed without alkaline hydrolysis All samples were spiked with known concentration of daidzein, genistein

and formononetin. Then they were processed without alkaline hydrolysis.

Then, the recovery percentage was calculated from the following equation

Final concentration of isoflavones obtained from AUC using calibration curve X final volume of solvent

X100 Initial concentration of isoflavones in 2 ml saliva initial volume of saliva


2.4.5 Improvement of processing of saliva samples 2.4.5.1 Preparation of saliva samples prior to HPLC • saliva was frozen

• then thawed at room temperature• centrifugation at 4000 rpm for 30 min• adjustment of pH at 5• extraction• leave the sample overnight • finally, reconstitute the dry solid in 1 ml solvent

2.4.5.2 Calculation of recovery percentage of isoflavones Solvent composition was changed to • 600 μl methanol with 0.1 % formic acid • 400 μl water with 0.1 % formic acid


3. Results and discussion

3.1 Separation of isoflavones using gradient mobile phase under the previous condition and obtain the following retention times of each isoflavone

IsoflavonesRetention times

Daidzein 8.249min

Glycitein8.656min

Genistein10.196 min

Formononetin12.764 min


3.2 Calibration curves and LOD3.2.1 Daidzein calibration curve and detection limitLOD = 0.012962 LOD = 0.099405

Mean calibration curve of daidzein using gradient

y = 136.19x - 1.5043

R2 = 0.9999

0

20

40

60

80

100

120

140

0 0.2 0.4 0.6 0.8 1 1.2

Con of Daid. in ppm

Mea

n of

AU

C o

f dai

dzei

n

Mean calibration curve of daidzein with formononetin as internal standard

y = 5.8946x + 0.3782

R2 = 0.9919

0

1

2

3

4

5

6

7

0 0.2 0.4 0.6 0.8 1 1.2

Con of daid. in ppm

Mea

n of

ratio

of A

UC

of D

/F


3.2.2 Genistein calibration curve and detection limit

LOD = 0.10709 LOD = 0.00592

Mean calibration curve of Genistein using gradient

y = 107.27x + 3.5612

R2 = 0.991

0

20

40

60

80

100

120

0 0.2 0.4 0.6 0.8 1 1.2

Con. of Gen in ppm

Mea

n A

UC

of G

en

calibration curve of genistein with formo as an internal st

y = 5.3035x - 0.0003

R2 = 1

0

1

2

3

4

5

6

0 0.2 0.4 0.6 0.8 1 1.2

Con of gen in ppm

ratio

of A

UC

of g

en.fo

rmo


3.2.3 Formononetin calibration curve and detection limit

LOD = 0.095893

Mean calibration curve of formo.

y = 13.37x + 0.1947

R2 = 0.9999

0

20

40

60

80

100

120

140

0.00 2.00 4.00 6.00 8.00 10.00 12.00

Con of formo. in ppm

Mean

AU

C o

f fo

rmo

no

neti

n


3.3 Determination of natural isoflavones in saliva

• There were no peaks of isoflavones appeared in two saliva samples of two participants.

• Tiny peaks of genistein, daidzein and glycitein appeared in saliva of a participant who drinks soy milk everyday.

• Peak of daidzein and a tiny peak of formononetin appeared in saliva of the last participant.


3.4 Measurement of isoflavones in saliva

3.4.1 After consuming soya milk There were no peaks of isoflavones came out in the participant’s saliva up to 48hr and formononetin showed recovery percentage of >100% A peak of genistein appeared in her saliva after a week. 3.4.2 After ingesting isoflavones tablet There were no peaks appeared up to 24 hr and formononetin showed

recovery percentage of >100% except in 24 hr saliva.


3.5 Recovery experiments 3.5.1 With alkaline hydrolysis

IsoflavonesStandard sample

Control sample 1

Control sample 2

Saliva 1Saliva2Saliva3

Daidzein39%30%112%63%296%65%

Genistein135.21%101.96%140.59%72.63%106.16%113.40%

Formono122.30%293.58%290.95%132.50%119.85%225.86%

Daidzein showed 64% ± 0.014 Genistein showed 97 ± 21.8 Formononetin gave 159.4% ± 0.58


3.5.2 Without hydrolysis

IsoflavonesStandard sample

Control sampleSaliva 1Saliva 2Saliva3

Daidzein63%29%24%21%22%

Genistein219.44%100.82%85.98%18.10%53.14%

Formono337.62%77.14%77.99%47.10%58.70%

Daidzein showed 23% ± 0.0166 Genistein showed 35.6% ± 24.8

Formononetin gave 61.3% ± 15.6


3.6 Improvement of processing of saliva samples and recovery percentage

IsoflavonesControl

sample 1Control sample

2Saliva 1Saliva2Saliva3

Daidzein24%25%23%20%24%

Genistein79.53%50.49102377.85293362.17616558.32%

Daidzein showed 23% ± 0.0198 Genistein showed 69.47% ± 0.107


4 Conclusion and future perspectives

• Saliva must be clear prior to run by HPLC to avoid problems that may occur in the column

• Further improvement in solvent and its way of adding to the dry solid is needed to obtain more soluble isoflavones and clear saliva

• Daidzein showed 23% ± 0.0166 without hydrolysis and 23% ± 0.0198 using new improved method

• Genistein gave 69.47% ± 0.107 applying new process and 35.6% ± 24.8 without hydrolysis

• Further study is needed to improve the method used to measure isoflavones in saliva and achieve more satisfied results.


References

1. A. L. Murkies, G. Wilcox and S. R. Davis, "Clinical Review 92 Phytoestrogens", J Clin Endocrinol Metab, 1998, 83(2), pp. 297-303.

2. A. L. Ososki and E. J. Kennelly, "Phytoestrogens: a Review of the Present State of Research", Phytother. Res, 2003, 17, pp. 845-869.

3. K. D. R. Setchell, et al, "Bioavailability, Disposition, and Dose-Response Effects of Soy Isoflavones When Consumed by Healthy Women at PhysiologicallyTypical Dietary Intakes", J Nutr, 2003, 133, pp. 1027-1035.

4. D.C. Knight and J.A. Eden, "Phytoestrogens - a short review", Maturitas, 1995, 22,pp. 167-75.

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