Dietary daidzein, but not genistein, has a hypocholesterolemic effect in non-ovariectomized and ovariectomized female Sprague-Dawley rats on a cholesterol-free diet

Figures & data

Table 1. The primers used for reverse transcription-polymerase chain reaction (RT-PCR) analysis.

Genes		Primer sequence	PCR product (bp)
CYP7A1	Sense	CTG CAA ACT GAT GGG GAA AT	145
	Antisense	TGG GTC AAT GCT TCT GTG TC
CYP8B1	Sense	ATG AAG GCT GTG CGA GAG	127
	Antisense	TCT CTT CCA TCA CGC TGT C
CYP27	Sense	CTC AAG AGA CTG TCG GCA C	114
	Antisense	CTT CCT GGA TCT CTG GGT T
LDLR	Sense	CTT GCC CTG ATG GTA TGC TA	137
	Antisense	CTT GCG CTT CAG TGA CAC A
HMG-CoA-R	Sense	GCT GGT GAG TTG TCC TTG AT	117
	Antisense	CTT CTT GGT GCA TGT TCC CT
Apo-B	Sense	TTG ACA CAC TGA AGT TCC TA	124
	Antisense	ACA TCA AAC CCT GGT ATT AG
LXR	Sense	TGC TAA TGA AGC TGG TGA	144
	Antisense	AGA GGA CAC GGA GAA ACA GT
FXR	Sense	CGT TCA GCG GAG ATT TTC AA	140
	Antisense	ATT TTC AGC TCC CCG ACA CT
ACAT2	Sense	CGGTCATGCTGATCCTCTTT	204
	Antisense	GTGTCACCAGCTCCCAAAAT
IBAT	Sense	TGC CTG GAT CAT TGA ACC	149
	Antisense	GAG TGT TCT GCA TTC CAG TT
MTP	Sense	AGCGACATCACAGTGGACTC	223
	Antisense	GGCTCTCTTTTCTTCTCCGA
GAPDH	Sense	GCCATCAACGACCCCTTCATT	240
	Antisense	CGCCTGCTTCACCACCTTCTT
Cyclophilin	Sense	ATGGTCAACCCCACCGTGTT	206
	Antisense	CGTGTGAAGTCACCACCCT

Notes: CYP7A1, cholesterol 7α-hydroxylase; CYP8B1, sterol 12-alpha-hydroxylase; CYP27, sterol 27-hydroxylase; LDLR, low density lipoprotein receptor; HMG-CoA-R, hydroxymethylglutaryl-coenzyme reductase; Apo-B, apolipoprotein-B; LXR, liver X receptor; FXR, farnesoid X receptor; ACAT, acyl-CoA:cholesterol acyl transferese; IBAT, ileal sodium-dependent bile acid transporter; MTP, Microsomal triglyceride transfer protein; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

Table 2. Effect of daidzein and genistein on body weight, body weight gain, and food intake and serum lipid concentration.^a

		Final body weight^f	Body weight gain^f	Food intake^f	Serum lipid concentration
					Total cholesterol	Triglycerides
		(g)	(g/28)	(g/28)	(mM)
Non-Ovx^b	Control	238 ± 7	82 ± 6	421 ± 10	2.58 ± 0.08	0.94 ± 0.17
	Daidzein	242 ± 6	86 ± 6	431 ± 10	2.31 ± 0.08	0.86 ± 0.15
	Genistein	247 ± 5	91 ± 4	446 ± 10	2.56 ± 0.10	0.95 ± 0.15
	Daidzein and Genistein	239 ± 7	82 ± 6	415 ± 16	2.20 ± 0.09	0.87 ± 0.14
Ovx^c	Control	316 ± 10	178 ± 9	627 ± 25	2.72 ± 0.09	1.32 ± 0.12
	Daidzein	272 ± 7*	133 ± 6*	534 ± 19*	2.24 ± 0.15	1.20 ± 0.07
	Genistein	306 ± 8	168 ± 8	604 ± 20	2.87 ± 0.16	1.42 ± 0.13
	Daidzein and Genistein	289 ± 8	151 ± 9	583 ± 14†	2.39 ± 0.14	0.99 ± 0.10
Three-way ANOVA^d^,^e	p-value
Main effects	OP	<0.01	<0.01	<0.01	NS	<0.01
	D	<0.01	<0.01	<0.01	<0.01	<0.05
	G	NS	NS	NS	NS	NS
Interaction	OP*D	<0.01	<0.01	<0.05	NS	NS
	OP*G	NS	NS	NS	NS	NS
	D*G	NS	NS	NS	NS	NS
	OP*D*G	<0.05	<0.05	<0.05	NS	NS

^a Data are expressed as mean ± SEM (n = 8).

^b Non-Ovx, non-ovariectomized.

^c Ovx, ovariectomized.

^d D, effect of dietary daidzein; G, effect of dietary genistein; OP, effect of operation (Non-Ovx and Ovx).

^e NS, Not significant.

^f Asterisks show a significant difference compared with the control group, and daggers show a significant difference when compared with daidzein group, determined by three-way ANOVA with Bonferroni’s test (p < 0.05).

Table 3. Effect of daidzein and genistein on total white fat (ovary, mesentery, perirenal fat).^a

		Ovary fat	Mesentery fat^f	Perirenal fat	Total white fat
		(g)
Non-Ovx^b	Control	5.7 ± 0.4	4.7 ± 0.3	5.8 ± 0.4	16.3 ± 1.0
	Daidzein	5.6 ± 0.6	4.7 ± 0.2	5.2 ± 0.4	15.5 ± 1.0
	Genistein	6.7 ± 0.4	5.4 ± 0.3	6.2 ± 0.4	18.3 ± 0.8
	Daidzein and Genistein	5.8 ± 0.8	4.7 ± 0.4	5.2 ± 0.8	15.8 ± 1.9
Ovx^c	Control	4.9 ± 0.3	7.9 ± 0.5	8.7 ± 0.6	21.5 ± 0.9
	Daidzein	3.5 ± 0.6	5.4 ± 0.4*	6.1 ± 0.7	15.0 ± 1.5
	Genistein	4.3 ± 0.5	7.7 ± 0.6	9.4 ± 1.3	21.4 ± 2.2
	Daidzein and Genistein	4.6 ± 0.4	6.7 ± 0.6†	7.7 ± 0.7	19.0 ± 1.4
Three-way ANOVA^d^,^e	p-value
Main effects	OP	<0.01	<0.01	<0.01	<0.01
	D	NS	<0.01	<0.01	<0.01
	G	NS	NS	NS	NS
Interaction	OP*D	NS	<0.05	NS	NS
	OP*G	NS	NS	NS	NS
	D*G	NS	NS	NS	NS
	OP*D*G	NS	NS	NS	NS

^a Data are expressed as mean ± SEM (n = 8).

^b Non-Ovx, non-ovariectomized.

^c Ovx, ovariectomized.

^d D, effect of dietary daidzein; G, effect of dietary genistein; OP, effect of operation (Non-Ovx and Ovx).

^e NS, Not significant.

^f Asterisks show a significant difference compared with the control group, and daggers show a significant difference when compared with daidzein group, determined by three-way ANOVA with Bonferroni’s test (p < 0.05).

Table 4. Effect of daidzein and genistein on liver wet weight, total lipids, cholesterol and triglyceride concentration; fecal bile and lipid excretion.^a

						Cholesterol				Fecal excretion^f
		Total liver wet weight^f	Total lipid	Phospholipid^f	Triglycerides	Total	Free	Esterified		Bile acid		Lipid
		(g)	(mg/liver)	(μmol/liver)					(μmol/day)		(mg/day)
Total liver wet weight^f	Total lipid	Phospholipid^f	Triglycerides	Total	Free	Esterified		Bile acid		Lipid
(g)	(mg/liver)	(μmol/liver)					(μmol/day)		(mg/day)
Non Ovx^b	Control	7.1 ± 0.3	390 ± 33	236 ± 11	110 ± 27	42 ± 2	38 ± 1	3.2 ± 0.8		10.5 ± 1.5		18.1 ± 1.3
	Daidzein	7.4 ± 0.3	317 ± 16	231 ± 9	56 ± 9	37 ± 2	32 ± 2	4.4 ± 0.6		21.1 ± 2.3*		25.2 ± 1.7*
	Genistein	7.5 ± 0.3	354 ± 16	227 ± 6	89 ± 9	43 ± 1	36 ± 2	7.0 ± 2.0		12.8 ± 2.4		21.9 ± 1.5
	Daidzein and genistein	7.1 ± 0.3	316 ± 19	210 ± 12	81 ± 13	38 ± 2	30 ± 1	7.3 ± 1.6		26.9 ± 4.7#		24.5 ± 2.2
Ovx^c	Control	10.3 ± 0.6	547 ± 66	314 ± 18	197 ± 25	51 ± 3	43 ± 3	8.7 ± 1.0		2.9 ± 0.6		31.9 ± 2.0
	Daidzein	8.4 ± 0.4*	431 ± 51	248 ± 11*	149 ± 27	43 ± 2	34 ± 2	8.3 ± 1.6		16.3 ± 0.8*		24.8 ± 1.4*
	Genistein	9.8 ± 0.5	518 ± 59	291 ± 15	208 ± 54	45 ± 4	37 ± 2	8.6 ± 2.0		11.3 ± 1.0 *		30.8 ± 2.3
	Daidzein and genistein	8.9 ± 0.4	469 ± 40	255 ± 13 #	165 ± 23	44 ± 3	33 ± 1	11.2 ± 2.3		30.4 ± 2.2#,†		34.2 ± 2.3†
Three way ANOVA^d^,^e					p-value
Main effects	OP	<0.01	<0.01	<0.01	<0.01	<0.01	0.05	<0.01		NS		<0.01
	D	<0.01	NS	<0.01	<0.05	<0.01	<0.01	NS		<0.01		NS
	G	NS	NS	NS	NS	NS	<0.05	<0.05		<0.01		<0.05
Interaction	OP*D	<0.05	NS	<0.05	NS	NS	NS	NS		NS		<0.01
	OP*G	NS	NS	NS	NS	NS	NS	NS		<0.05		NS
	D*G	NS	NS	NS	NS	NS	NS	NS		NS		NS
	OP*D*G	NS	NS	NS	NS	NS	NS	NS		NS		<0.01

^a Data are expressed as mean ± SEM (n = 8).

^b Non-Ovx, non-ovariectomized.

^c Ovx, ovariectomized.

^d D, effect of dietary daidzein; G, effect of dietary genistein; OP, effect of operation (Non-Ovx and Ovx).

^e NS, Not significant.

^f Asterisks show a significant difference compared with the control group, daggers show a significant difference when compared with daidzein group, and number signs show a significant difference when compared with genistein group, determined by three-way ANOVA with Bonferroni’s test (p < 0.05).

Table 5. Effect of daidzein and genistein on the mRNA expression for lipid metabolism genes in the liver.^a,f

		Apo-B	ACAT2	LDLR	HMG-CoA-R	CYP7A1	CYP8B1	CYP27	LXR	FXR
		(×10^–2)	(×10^–2)	(×10^–3)	(×10^−3)	(×10^−2)	(×10^−1)	(×10^−1)	(×10^−1)	(×10^−2)
Non OVX^b	Control	0.9 ± 0.2	2.5 ± 0.4	1.5 ± 0.2	8.5 ± 0.9	1.1 ± 0.2	1.2 ± 0.2	1.2 ± 0.2	1.4 ± 0.2	3.1 ± 0.4
	Daidzein	0.7 ± 0.1	2.5 ± 0.2	1.7 ± 0.2	7.7 ± 0.8	0.9 ± 0.1	0.9 ± 0.1	1.2 ± 0.2	1.2 ± 0.1	3.2 ± 0.2
	Genistein	0.8 ± 0.1	3.1 ± 0.3	1.3 ± 0.3	7.5 ± 0.6	1.3 ± 0.2	1.1 ± 0.1	1.4 ± 0.1	1.4 ± 0.1	3.7 ± 0.3
	Daidzein and genistein	0.8 ± 0.1	3.5 ± 0.2	1.7 ± 0.2	7.7 ± 0.5	1.4 ± 0.2	0.8 ± 0.1	1.3 ± 0.2	1.3 ± 0.1	3.8 ± 0.2
OVX^c	Control	2.2 ± 0.3	4.5 ± 0.5	3.1 ± 0.6	9.7 ± 1.4	1.9 ± 0.4	3.0 ± 0.7	2.3 ± 0.1	2.2 ± 0.2	5.5 ± 0.6
	Daidzein	1.8 ± 0.2	4.6 ± 0.9	4.0 ± 0.8	11.6 ± 1.3	2.0 ± 0.3	2.4 ± 0.2	2.1 ± 0.1	2.1 ± 0.1	5.1 ± 0.5
	Genistein	1.9 ± 0.1	5.0 ± 0.7	3.0 ± 0.4	10.2 ± 2.3	2.2 ± 0.5	2.0 ± 0.3	2.4 ± 0.1	2.4 ± 0.1	5.8 ± 0.6
	Daidzein and genistein	1.5 ± 0.1	5.4 ± 0.6	3.1 ± 0.5	9.6 ± 1.5	2.7 ± 0.5	2.0 ± 0.3	2.2 ± 0.2	2.2 ± 0.1	6.7 ± 0.8
Three-way ANOVA^d^,^e	p-value
Main effects	OP	<0.01	< 0.01	<0.01	<0.05	<0.01	<0.01	<0.01	<0.01	<0.01
	D	<0.05	NS	NS	NS	NS	NS	NS	NS	NS
	G	NS	0.05	NS	NS	0.07	NS	NS	NS	<0.05
Interaction	OP*D	NS	NS	NS	NS	NS	NS	NS	NS	NS
	OP*G	NS	NS	NS	NS	NS	NS	NS	NS	NS
	D*G	NS	NS	NS	NS	NS	NS	NS	NS	NS
	OP*D*G	NS	NS	NS	NS	NS	NS	NS	NS	NS

^a Data are expressed as mean ± SEM (n = 8).

^b Non-Ovx, non-ovariectomized.

^c Ovx, ovariectomized.

^d D, effect of dietary daidzein; G, effect of dietary genistein; OP, effect of operation (Non-Ovx and Ovx).

^e NS, Not significant.

^f Reference gene was cyclophilin. CYP7A1, cholesterol 7α-hydroxylase; CYP8B1, sterol 12-alpha-hydroxylase; CYP27, sterol 27-hydroxylase; LDLR, low density lipoprotein receptor; HMG-CoA-R, hydroxymethylglutaryl-coenzyme reductase; Apo-B, apolipoprotein-B; LXR, liver X receptor; FXR, farnesoid X receptor; ACAT, acyl-CoA:cholesterol acyl transferese.

Table 6. Effect of daidzein and genistein on the mRNA expression for lipid metabolism genes in the small intestinal mucosa.^a,g

		IBAT	HMG-CoA-R	MTP^f	FXR	LXR
Upper intestinal mucosa		(×10^−3)	(×10^−3)	(×10^−1)	(×10^−2)	(×10^−2)
Non Ovx^b	Control	2.7 ± 0.5	5.0 ± 0.3	4.9 ± 0.7	0.9 ± 0.1	5.9 ± 0.3
	Daidzein	2.0 ± 0.5	5.7 ± 0.7	6.7 ± ± 2.5	1.0 ± 0.2	8.1 ± 2.1
	Genistein	3.1 ± 0.5	5.4 ± 0.6	4.8 ± 0.4	0.9 ± 0.1	5.6 ± 0.2
	Daidzein and Genistein	3.1 ± 0.6	5.1 ± 0.4	5.4 ± 0.8	1.1 ± 0.2	6.4 ± 0.2
Ovx^c	Control	0.9 ± 0.1	4.3 ± 1.4	2.1 ± 0.5	1.0 ± 0.6	3.4 ± 0.7
	Daidzein	0.8 ± 0.1	5.0 ± 2.0	2.2 ± 0.6	0.9 ± 0.6	2.8 ± 0.5
	Genistein	0.6 ± 0.1	2.5 ± 0.4	1.2 ± 0.2*	0.3 ± 0.1	4.2 ± 2.2
	Daidzein and Genistein	1.0 ± 0.2	3.3 ± 0.4	1.2 ± 0.2^†	0.4 ± 0.1	2.5 ± 0.2
Three-way ANOVA^d^,^e	p-value
Main effects	OP	<0.01	<0.05	<0.01	NS	<0.01
	D	NS	NS	NS	NS	NS
	G	NS	NS	<0.05	NS	NS
Interaction	OP*D	NS	NS	NS	NS	NS
	OP*G	NS	NS	<0.05	NS	NS
	D*G	NS	NS	NS	NS	NS
	OP*D*G	NS	NS	NS	NS	NS
		IBAT	HMG-CoA-R	MTP	FXR^f	LXR
Lower intestinal mucosa		(×10^−3)	(×10^−3)	(×10^−1)	(×10^−2)	(×10^−2)
Non Ovx^b	Control	2.3 ± 0.4	14.7 ± 3.1	26.4 ± 5.7	4.0 ± 0.4	30.7  ± 4.1
	Daidzein	2.0 ± 0.3	13.0 ± 2.1	26.7 ± 3.6	4.1 ± 0.5	29.6  ± 2.6
	Genistein	2.4 ± 0.3	14.5 ± 2.5	34.8 ± 3.2	5.2 ± 0.4	39.4  ± 4.2
	Daidzein and Genistein	3.4 ± 0.6	18.4 ± 3.1	33.4 ± 5.1	6.0 ± 0.9^†	34.2  ± 4.5
Ovx^c	Control	0.3 ± 0.1	3.4 ± 0.5	10.3 ± 1.8	1.6 ± 0.3	18.2 ± 3.9
	Daidzein	0.4 ± 0.1	4.8 ± 0.9	8.6 ± 1.2	1.4 ± 0.3	16.5 ± 4.3
	Genistein	0.4 ± 0.1	4.2 ± 0.5	7.0 ± 1.5	1.4 ± 0.3	15.3 ± 3.0
	Daidzein and Genistein	0.4 ± 0.1	4.0 ± 0.7	7.0 ± 1.6	1.5 ± 0.4	15.3 ± 2.5
Three-way ANOVA^d^,^e	p-value
Main effects	OP	<0.01	<0.01	<0.01	<0.01	<0.01
	D	NS	NS	NS	NS	NS
	G	NS	NS	NS	<0.05	NS
Interaction	OP*D	NS	NS	NS	NS	NS
	OP*G	NS	NS	<0.05	<0.05	NS
	D*G	NS	NS	NS	NS	NS
	OP*D*G	NS	NS	NS	NS	NS

^a Data are expressed as mean ± SEM (n = 8).

^b Non-Ovx, non-ovariectomized.

^c Ovx, ovariectomized.

^dD, effect of dietary daidzein; G, effect of dietary genistein; OP, effect of operation (Non-Ovx and Ovx).

^e NS, Not significant.

^fAsterisks show a significant difference compared with the control group, daggers show a significant difference when compared with daidzein group, and number signs show a significant difference when compared with genistein group, determined by three-way ANOVA with Bonferroni’s test (p < 0.05).

^g Reference gene was GAPDH. IBAT, ileal sodium-dependent bile acid transporter; HMG-CoA-R, hydroxymethylglutaryl-coenzyme reductase; MTP, Microsomal triglyceride transfer protein; FXR, farnesoid X receptor; LXR, liver X receptor; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

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