1. Loughborough University Chemistry Department Analytical Chemistry EstradiolGenistein Measurement of Phytoestrogens in Saliva Taghreed Muhammad Fagieh Supervisor/ Dr Helen Reid September- 2008

2. Isoflavones Mammalian endogenous estrogens Phytoestrogens are natural compounds found in many foods. 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 that are naturally occurring and showing estrogenic activity (2).

3. Isoflavones Mammalian endogenous estrogens Daidzein and genistein are the main isoflavones which are derived from formononetin and biochanin A, respectively (1). Formononetin Genistein Daidzein

4. Isoflavones Mammalian endogenous estrogens 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).

5. Isoflavones Mammalian endogenous estrogens 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).

6. Isoflavones Mammalian endogenous estrogens 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

7. IsoflavonesMammalian endogenous estrogens 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.

8. Isoflavones Mammalian endogenous estrogens Flow rateSolvent BTime(min) 1 ml/min75%0 1 ml/min0%10 1 ml/min0%15 1 ml/min75%16 1 ml/min75%19 10 μlInjection volume 37 ◦CColumn temperature 1 ml/minFlow rate 259 nmwavelength 20 minStop time 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)

9. Isoflavones Mammalian endogenous estrogens 2.4.2 Determination of natural isoflavones in saliva Four participants took a part in this study and produced saliva samples. Natural isoflavones were measured in each sample which was processed with alkaline hydrolysis. 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

10. Isoflavones Mammalian endogenous estrogens 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.

11. Isoflavones Mammalian endogenous estrogens 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

12. Isoflavones Mammalian endogenous estrogens 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

13. Isoflavones Mammalian endogenous estrogens 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 Retention timesIsoflavones 8.249minDaidzein 8.656minGlycitein 10.196 minGenistein 12.764 minFormononetin

14. Isoflavones Mammalian endogenous estrogens 3.2 Calibration curves and LOD 3.2.1 Daidzein calibration curve and detection limit LOD = 0.012962 LOD = 0.099405

15. Isoflavones Mammalian endogenous estrogens 3.2.2 Genistein calibration curve and detection limit LOD = 0.10709 LOD = 0.00592

16. Isoflavones Mammalian endogenous estrogens 3.2.3 Formononetin calibration curve and detection limit LOD = 0.095893

17. Isoflavones Mammalian endogenous estrogens 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.

18. Isoflavones Mammalian endogenous estrogens 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.

19. Isoflavones Mammalian endogenous estrogens 3.5 Recovery experiments 3.5.1 With alkaline hydrolysis Saliva3Saliva2Saliva 1 Control sample 2 Control sample 1 Standard sample Isoflavones 65%296%63%112%30%39%Daidzein 113.40%106.16%72.63%140.59%101.96%135.21%Genistein 225.86%119.85%132.50%290.95%293.58%122.30%Formono Daidzein showed 64% ± 0.014 Genistein showed 97 ± 21.8 Formononetin gave 159.4% ± 0.58

20. Isoflavones Mammalian endogenous estrogens 3.5.2 Without hydrolysis Saliva3Saliva 2Saliva 1Control sample Standard sample Isoflavones 22%21%24%29%63%Daidzein 53.14%18.10%85.98%100.82%219.44%Genistein 58.70%47.10%77.99%77.14%337.62%Formono Daidzein showed 23% ± 0.0166 Genistein showed 35.6% ± 24.8 Formononetin gave 61.3% ± 15.6

21. Isoflavones Mammalian endogenous estrogens 3.6 Improvement of processing of saliva samples and recovery percentage Saliva3Saliva2Saliva 1 Control sample 2 Control sample 1 Isoflavones 24%20%23%25%24%Daidzein 58.32%62.17616577.85293350.49102379.53%Genistein Daidzein showed 23% ± 0.0198 Genistein showed 69.47% ± 0.107

22. Isoflavones Mammalian endogenous estrogens 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.

23. Isoflavones Mammalian endogenous estrogens 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.