1. Clinical Action and Safety of
Red Clover Isoflavones
Martin Imhof
General Public Teaching Hospital
Korneuburg/Vienna
Medical University - Vienna

2. • Million Women Study Collaborators.
Breast cancer and hormone-
replacement therapy in the Million
Women Study. Lancet 2003; 362:419-
27
• Women´s Health Initiative (WHI) Study
– Interrupted due to severe side
effects

3. Herbal Alternatives
to
Hormone Replacement Therapy

4. How Safe and Effective are
Phytohormones?

5. Are Red Clover Isoflavones
Effective ?
A Double Blind, Randomized,
Placebo-controlled
Cross-Over Study

6. Week 12Week 4 Week 8
-25
-20
-15
-10
-5
0
PercentageReductioninHot
Flushes
Placebo
Active
Baber RJ, et al. Climacteric 1999
Effect of 40 mg red clover extracts over No.
Daily hot flashes and Greene Scores (n=51
postmenopausal)*
*RDBPC study

7. Tice JA, et al. JAMA 2003
Effect of 80 mg vs. 57 mg red clover extracts vs
placebo over No. hot flashes freq and Greene
Scores (n=252 postmenopausal)*
*ICE study (RDBPC)
 After 12 weeks:After 12 weeks:
 80 mg RCE (n=84): 41% reduction80 mg RCE (n=84): 41% reduction
 57 mg RCE (n=83): 34%.57 mg RCE (n=83): 34%.
 Placebo (n=85): 36% (p > 0.05).Placebo (n=85): 36% (p > 0.05).

8. Van Weijer PH, et al. Maturitas 2002
Effect of 80 mg red clover extracts vs
placebo over hot flashes frequenz and
Greene Scores (n=30 postmenopausal)*
 4 weeks placebo run in phase4 weeks placebo run in phase
(16% reduction).(16% reduction).
 After 12 weeks:After 12 weeks:
 Red clover reduced hot flashesRed clover reduced hot flashes
freq 44%.freq 44%.
 Placebo no further reduction.Placebo no further reduction.
*RDBPC study

9. • Placebo-controlled, cross-over, double-blind, randomized
• 113 menopausal women, 109 finished this study
• 25 weeks
• 80 mg isoflavones daily (2 capsules of menoflavon)
• Within the 25 weeks 4 examinations
• Start: November 2002 - End: February 2005
Menoflavon Study 80mg isoflavones /d
of red clover – clinical trial Austria

10. Examinations and questionnaires
 Kupperman scoreKupperman score (index before start >15)(index before start >15)
 Changes inChanges in blood serum levelsblood serum levels (T, E2, FSH, LH,(T, E2, FSH, LH,
SHBG, HDL, LDL, Triglycerides, Lpa, Cholesterol)SHBG, HDL, LDL, Triglycerides, Lpa, Cholesterol)
 Effects on theEffects on the endometriumendometrium Vaginal smearVaginal smear
(cytodiagnosis)(cytodiagnosis)
 Urine-analysisUrine-analysis (urinary excretion of isoflavones)(urinary excretion of isoflavones)
 Depressions scaleDepressions scale after Zerssenafter Zerssen
 Hospital Anxiety and Depression Scale (Hospital Anxiety and Depression Scale (HADS-DHADS-D))
 Observation studyObservation study: effects of menoflavon on skin, hair,: effects of menoflavon on skin, hair,
nails and libido, vaginal atrophy….nails and libido, vaginal atrophy….

11. Demographic and anamnestic data
Group A
(n=50)
Group B
(n=59)
Overall
(n=109)
Mean age (yrs.) 54.5 ± 6.2 53.7 ± 7.8 53.5 ± 7.1
Mean BMI 24.5 ± 3.9 24.9 ± 3.9 24.7 ± 3.9
Hysterectomy (%) 18.0 13.6 15.6

12. Kupperman Index
0
5
10
15
20
25
30
35
40
45
MF11RCE Placebo
gestiegen
gefallen
unverändert
Wilcoxon Test, PVerum < 0,001, PPlacebo < 0,001

13. Reduction of hot flushes
0
5
10
15
20
25
30
35
40
45
MF11RCE Placebo
zugenommen
abgenommen
unverändert
Wilcoxon Test, PVerum < 0,001, PPlacebo = 0,001

14. Hot flushes, Boxplots-comparison
Hot flushes were reduced significantly
with MF11RCE (p<0,001)
No effect under Placebo
before after
MF11RCE
before after
Placebo

15. Reduction sleep disorders
0
10
20
30
40
50
MF11RCE Placebo
Zunahme
Abnahme
keine Veränderung
Wilcoxon Test, PVerum < 0,001, PPlacebo = 0,018

16. Sleeping behavior - Boxplots-comparison
before after
MF11RCE Placebo
afterbefore
Sleeping behavior improved high
significantly with MF11RCE (p<0,001)
No effect under Placebo

17. Mood improvement
0
5
10
15
20
25
30
35
MF11RCE Placebo
verschlechtert
verbessert
unverändert
Wilcoxon Test, PVerum < 0,001, PPlacebo = 0,001

18. Baseline measures
Group A
(n=50)
Group B
(n=59)
Overall
(n=109)
E2
(pg/ml) 36.30 38.31 37.39
T (ng/ml) 0.43 0.63 0.54
LH (mIU/ml) 30.64 29.92 30.25
FSH (mlU/ml) 60.31 60.45 60.38
SHBG (nmol/l) 61.44 57.69 59.41
Group A
(n=41)
Group B
(n=51)
Overall
(n=92)
Endometrium
(mm)
4.3 3.4 3.8
Data are presented as mean values.

19. Wilcoxon-test MF11RCE vs. placebo
N Negative
Ranks
Positive
Ranks
p
E2 109 40 43 0,8610,861
T 109 61 36 0.003
LH 109 64 44 0.861
FSH 109 62 47 0.069
SHBG 109 58 51 0.824
ET 92 15 45 0.001

20. Changes with MF11RCE
Before
MF11RCE
After
MF11RCE
Change
(%)
T (ng/ml) 0.54 ± 0.26 0.66 ± 0.29 22.12
ET (mm) 3.8 ± 1.9 3.2 ± 1.5 -14.69
Data are presented as mean ± SD.

21. Changes in Endometrium after 90 days
of treatment with MF11RCE

22. Changes in Testosteron serum levels
after 90 days of treatment

23. Maturitas. 2006 Aug 20;55(1):76-81.
Effects of a red clover extractEffects of a red clover extract (MF11RCE(MF11RCE))
onon endometriumendometrium and sex hormones inand sex hormones in
postmenopausal women.postmenopausal women.
Martin Imhof, Anca Gocan, Franz Reithmayr, Markus Lipovac,Martin Imhof, Anca Gocan, Franz Reithmayr, Markus Lipovac,
Claudia Schimitzek4, Johannes HuberClaudia Schimitzek4, Johannes Huber

24. Results clinical study - 80 mg
isoflavones – 113 women Austria
 Testosterone levels increasedTestosterone levels increased significantly (p=0,021) Wilcoxon testsignificantly (p=0,021) Wilcoxon test
with MF11RCE, no significant change with placebowith MF11RCE, no significant change with placebo
 Endometrium thicknessEndometrium thickness waswas reducedreduced withwith MF11RCE (p<0.001)MF11RCE (p<0.001)
 Climacteric symptomsClimacteric symptoms as evaluated withas evaluated with Kupperman index wasKupperman index was
reducedreduced in general (p<0.001) with MF11RCE treatment.in general (p<0.001) with MF11RCE treatment.
o Hot flushes and sweating were reducedHot flushes and sweating were reduced statistically significantlystatistically significantly
(p<0.001)(p<0.001)
o Sleeping behavior improvedSleeping behavior improved highly significanthighly significant (p<0.001)(p<0.001)
o DepressionDepression andand nervousnessnervousness were significantly reduced (p<0.001)were significantly reduced (p<0.001)
o Lack of concentrationLack of concentration was significantly reduced (p<0.001)was significantly reduced (p<0.001)
 EstradiolEstradiol,, FSHFSH andand LHLH plasmaplasma levels did not change significantlylevels did not change significantly
comparing both groupscomparing both groups

25. Design clinical study - 80 mg
isoflavones – 60 women Ecuador
 Double-blind, placebo controlled, randomized, cross-overDouble-blind, placebo controlled, randomized, cross-over
 90 days 2 capsules menoflavon/day - wash out - 90 days90 days 2 capsules menoflavon/day - wash out - 90 days
Placebo resp. vice versaPlacebo resp. vice versa
 60 women, average age: 51,3 years60 women, average age: 51,3 years
 Inclusion criteria: Kupperman-Index ≥15, no HRT,..Inclusion criteria: Kupperman-Index ≥15, no HRT,..
 3 examinations (at the beginning, after 90 days, end)3 examinations (at the beginning, after 90 days, end)
 Kupperman-Index, lipids, hormone levels (E2, FSH, LH,Kupperman-Index, lipids, hormone levels (E2, FSH, LH,
Testosterone, SHBG, Lipoprotein a), vaginal cytologyTestosterone, SHBG, Lipoprotein a), vaginal cytology

26. Effect of 80 mg red clover extracts over
Kupperman scoring after 3 months (n=53
postmenopausal)
Hidalgo L, Chedraui P, et al. Gynecol Endocrinol, 2005, In press
0
5
10
15
20
25
30
baseline red clover placebo
baseline red clover placebo

27. The effect of red clover supplementation over lipid profile
a
vs. baseline; b
vs. red clover phase; * p < 0.05;
Parameter
Baseline
n=53
After isoflavone
supplementation n=53
After placebo
n=53
Total cholesterol
(mg/dL)
223.9 ± 37.6
214 ± 32.2 a
* 220.4 ± 34.1 b
HDL-C (mg/dL)
39.7 ± 11.5
40 ± 9.6 a
41.1 ±10 b
LDL-C (mg/dL)
146.8 ± 29.9
129.7 ± 39.4 a
* 140 ± 35.2 b
Triglycerides
(mg/dL)
199.6 ± 77.8
181.1 ± 72.3 a
* 242.7 ± 166.9 b
*
Lipoprotein A
(mg/dL)
41.2 ± 36.9
22.8 ± 26.9 a
* 20.5 ± 25.8 b
Hidalgo L, Chedraui P, et al. Gynecol Endocrinol, 2005, In press

28. The effect of red clover supplementation over hormonal profile
Parameter
Baseline
n=53
After isoflavone
supplementation n=53
After placebo
n=53
SHBG (nmol/L) 50.1 ± 23.7 46.9 ± 21.9 a
49 ± 22 b
FSH (mIU/mL)
63.7 ± 22.6 63.3 ± 26.1 a
62.6 ± 22.1 b
LH (mIU/mL) 27.7 ± 13.8 30.8 ± 13.4 a
28.7 ± 14 b
17 β estradiol
(pg/mL)
23.8 ± 8.8 22.4 ± 7.2 a
20.9 ± 4.6 b
Testosterone
(ng/dL)
24.8 ± 11.2
21.4 ± 5 a
23.2 ± 11.3 b
Hidalgo L, Chedraui P, et al. Gynecol Endocrinol, 2005, In press

29. Results clinical study - 80 mg
isoflavones – 60 women Ecuador
 No significant effect on BMI, weight and blood pressure inNo significant effect on BMI, weight and blood pressure in
both groupsboth groups
 significant effect on Kupperman-Index, lipidssignificant effect on Kupperman-Index, lipids
 Compared to placebo menoflavon significantly decreased:Compared to placebo menoflavon significantly decreased:
• menopausal symptoms, like:menopausal symptoms, like:
• hot flushes, night sweats, sleeping disorders,hot flushes, night sweats, sleeping disorders,
nervousness, depression, dizziness, ....nervousness, depression, dizziness, ....
• Triglyceride levelTriglyceride level
 menoflavon had a positive effect over the vaginamenoflavon had a positive effect over the vagina

30. CONCLUSIONS
 80 mg (MF11RCE) Red Clover does80 mg (MF11RCE) Red Clover does
significantly decrease menopausal complaintssignificantly decrease menopausal complaints
 80 mg (MF11RCE) Red Clover significantly80 mg (MF11RCE) Red Clover significantly
influences Testosterone level andinfluences Testosterone level and
Endometrium thicknessEndometrium thickness
 The action of MF11RCE on MCF 7 and HumanThe action of MF11RCE on MCF 7 and Human
Endothelial Breast Cells does not show a riskEndothelial Breast Cells does not show a risk
of tumour induction in analysis of geneof tumour induction in analysis of gene
activationactivation

31. Phytohormones can stimulate MCF 7
cell proliferation

32. 0,00
10,00
20,00
30,00
40,00
50,00
60,00
70,00
80,00
ASR (W)
Finland Gr eece China Nor ther nEur ope Souther nEur ope East Asia
Pr ostate
Br east
Colon/ RectumMan
Colon/ RectumWoman
Cancer Mondial/ Globocan 2000
Epidemiological Data CancerEpidemiological Data Cancer

33. Cell proliferation is not
sufficient to describe
the risk of tumour development

34. Experiment 1
Cells:
- MCF-7 mamma carcinoma cell line
Phytohormones:
- Red clover - Trifolium pratense, (MF11RCE)
- “synthetic” Genistein and Daidzein (Indofine).
Technology:
- 20K Chip arrays (Affymetrix)

35. Experimental design:
MCF-7 were examined after
12 and 24 hours.
Experimental groups:
- untreated cells
- combination of genistein plus daidzein
(2,5 µg/ml each)
- red clover extract (5 µg/ml)

37. Flowchart of experimental analysis
c-DNA
Scan Hybridize
(16 hours)
mRNA
IVT
(Biotin-UTP
Biotin-CTP)
Labeled fragments
L L
L
L
Cells

38. Comparison of the mRNA expression pattern in MC-F7
cells stimulated with red-clover extract with that of
daidzin and genestin stimulated cells.
22.000 genes were screened by DNA array and for each gene 11 oligos for
perfect match and 11 oligos for mismatch are analyzed. Therefore a total of
500.000 spots were quantitatively analyzed.

39. Results
A significant increase (>2times) in mRNA expression
induced by phytohormones was seen for 117 genes.
The functional repertoire of these genes cover
-Growth arrest genes
-DNA repair genes
(GADD34)
-Growth factors
(insulin-like growth factor binding protein 4)

40. 203720_s _at (ERCC1
207348_s _at (LIG3)
218685_s _at (SMUG1
201236_s _at (BTG2)
204461_x_at (RAD1)
219510_at (POLQ)
201746_at (TP53)
203565_s _at (MNAT1
202239_at (ADPRTL1
204766_s _at (NUDT1
31861_at (SMBP2)
204093_at (CCNH)
207405_s _at (RAD17
205811_at (POLG2)
207598_x_at (XRCC2
219317_at (POLI)
202330_s _at (UNG)
205091_x_at (RECQL
34063_at (RecQ5)
203719_at (ERCC1)
204828_at (RAD9)
202332_at (CSNK1E)
219502_at (FLJ10858
202726_at (LIG1)
205733_at (BLM)
203725_at (GADD45A
218428_s _at (REV1L
218961_s _at (PNKP)
201459_at (RUVBL2)
203210_s _at (RFC5)
201529_s _at (RPA1)
205024_s _at (RAD51
206066_s _at (RAD51
208393_s _at (RAD50
205189_s _at (FANCC
203564_at (FANCG)
203806_s _at (FANCA
205909_at (POLE2)
204603_at (EXO1)
203577_at (GTF2H4)
207891_s _at (TREX2
202453_s _at (GTF2H
204408_at (APEX2)
204884_s _at (HUS1)
203422_at (POLD1)
203616_at (POLB)
207347_at (ERCC6)
208386_x_at (DMC1)
202466_at (POLS)
203409_at (DDB2)
207945_s _at (CSNK1
CLUSTERED DNA REPAIR GENES
Red CloverSynthetic
Phytohormones
unstimulated

41. Y-axis: Affymetrix Experiment, Default Interpretation
Colored by: Control_12
Gene List: Tumor Suppressor (376), 204159_at selected
Control_12 Control_24 G+D_12h G+D_24h redc_12h redc_24h
Sample
0.01
0.1
1
10
100
Control_12 Control_24 G+D_12h G+D_24h redc_12h redc_24h
Sample
0.01
0.1
1
10
100
CDKN2C
Cyclin dependent kinase inhibitor
CLUSTERED TUMOR SUPRESSOR GENES

42. Results
THE GENES DIFFERENTIALLLY UP-REGULATED BY
SYNTHETIC PHYTOESTROGENS ARE
“METALLOTHIONEINS” (MT)
AND ARE BELIVED TO PLAY AN ROLE IN
TUMORGENESIS.

43. Y-axis: Affymetrix Experiment, Default Interpretation
Colored by: G+D_12h
Gene List: like 217165_x_at (MT1F) (0.95) (70)
Control_12 Control_24 G+D_12h G+D_24h redc_12h redc_24h
Sample
0.01
0.1
1
10
100
Control_12 Control_24 G+D_12h G+D_24h redc_12h redc_24h
Sample
0.01
0.1
1
10
100
12h 24h
Red Clover
12h 24h
Synthetic
Phytohormones
12h 24h
unstimulated
DecreaseIIncreaseEXTRACT OF GENES THAT ARE DIFFERENTLY REGULATED

44. Conclusion
Application of red clover and synthetic
phytohormones causes a transcriptional
regulation of 117 genes in MCF-7.
The effect of synthetic Daidzein/Genistein differs
significantly from that of red clover by
upregulation of transcription of 10 genes
(Metallothionins).
This first results do not implicate a major
influence of red clover on the proliferation or
differentiation of MCF-7 cells.

45. Experiment 2
Cells:
- Cell culture from primary mammae epithelial
cells (healthy cells)
Technology:
- 20K Chip arrays (Affymetrix)

46. Experiment 2
Pooled sera of 5 menopausal women . Before and
after intake of MF11RCE (menoflavon) for 3 month
• Control group = before intake of MF11RCE
• Treatment group = after intake of MF11RCE

47. Regulation of Transcription
0
100
200
300
400
500
600
700
800
900
1000
TCF4
TCF7L2
MAFB
MXI1
ID4
HOP
BTF3
BAZ1A
Gene
Estimat
control
treatment

48. Regulation of Transcription
• MF11RCE inhibits the expression of
transcriptional genes
(TCF4,TCF7L2,MAFB,MXI1,ID4,HOP,BTF3,BAZ1A)
and genes in relation to ribosomal subunit
joining and the initiation of translation
(EIF3S5, EIF4EBP, RPL29,RPS16)
= MF11RCE can influence cell proliferation and
differentiation by down regulation of cellular
transcription and translation

49. Genes involved in regulation
of DNA
0
100
200
300
400
500
600
G
M
N
N
H
2AFV
H
IST1H
2
H
M
G
B2
M
C
M
5
PC
N
A
PR
K
D
C
PR
M
1
Gene
Estimat
control
treatment

50. Genes involved in regulation of
DNA
• MF11RCE down regulates genes
involved with DNA replication.
(GMNN,H2AFV,HIST1H2,HMGB2,MC5,PCNA,PRKDC
,RM1)
= MF11RCE has an anti-proliferative
effect through inhibition of DNA
replication

51. Apoptosis
0
200
400
600
800
1000
1200
1400
1600
1800
2000
AFURS1 ENC1 PORIMIN
genes
signalestimates
control
treatment

52. Apoptosis
• MF11RCE induces Expression of
(AFURS1, ENC1/PIG10 p53 induced gene 10)
and inhibits Porimin
= MF11RCE can rise the p53 level
(ENCI/PIG10) and activate
apoptosis.
= MF11RCE can induce senescence
(AFURS1)

53. Regulation of cell proliferation
0
200
400
600
800
1000
1200
JAG
1
RARR
ES
VEG
F
M
ET
genes
signalestimates
control
treatment

54. Regulation of cell
proliferation
• MF11RCE inhibits JAG1,RARRES,
VEGF,MET. (cell proliferation)
and cell differentation (Keratin 10,
Keratin 19).
= MF11RCE inhibits genes of
cell-proliferation and cell-
differentation

55. In a culture of primary mammae epithelial
cells MF11RCE down regulates genes for
–Transcription and translation
–Protein synthesis
–DNA replication
–Cell proliferation
and up regulates genes for
–Apoptosis

56. Conclusion
MF11RCE seems not to be
inductive for tumor and/or tumor
growth in a healthy mamma
epithelial cells culture.
Further study are necessary to
improve if Isoflavones can
protect against tumor
development an/or tumor growth

57. • Georg Steiner
• Michael Loeffler
• Anka Gocan
• Marianne Imhof
• Kristian Hrachowitz
• Sylvia Molzer
• Markus Lipovac
• Johannes Huber