Effects of Sanyaku and Its Constituent Diosgenin on the Fasted and

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Bioactive Constituents, Metabolites, and Functions

Effects of sanyaku and its constituent diosgenin on the fasted and postprandial hypertriacylglycerolemia in high fat diet-fed KK-A mice y

Tsutomu Hashidume, Kaori Sasaki, Jun Hirata, Mai Kato, Yuko Yoshikawa, Yusaku Iwasaki, Hidekazu Arai, Shinji Miura, and Noriyuki Miyoshi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03040 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 5, 2018

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Journal of Agricultural and Food Chemistry

Effects of sanyaku and its constituent diosgenin on the fasted and postprandial hypertriacylglycerolemia in high fat diet-fed KK-Ay mice

Tsutomu Hashidumea,b, Kaori Sasakia, Jun Hirataa, Mai Katob, Yuko Yoshikawaa,c, Yusaku Iwasakid, Hidekazu Araia,b, Shinji Miuraa,b, Noriyuki Miyoshia,b, *

a

Graduate School of Integrated Pharmaceutical and Nutritional Sciences, bSchool of Food Nutritional Sciences, University of Shizuoka, Shizuoka, Japan

c

School of Veterinary Medicine, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, Tokyo, Japan

d

Laboratory of Animal Science, Graduate School of Life and Environmental Sciences,

Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan

*

Corresponding author: Noriyuki Miyoshi

Tel: +81-54-264-5531 Fax: +81-54-264-5530 Email: [email protected]

1

ACS Paragon Plus Environment


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Abstract

2

In this study, we examined the fasted and postprandial triacylglycerol (TG) levels in

3

KK-Ay mice fed a high fat diet (HFD) or a HFD containing either 500 ppm (0.05%)

4

diosgenin or 500 ppm (0.05%) diosgenin-containing Chinese yam sanyaku. Oral fat

5

tolerance tests revealed that not only in fasting state, but also after loading of lipid

6

emulsion, plasma levels of TG were significantly reduced in sanyaku- and diosgenin-

7

fed mice. Levels of fat oxidation especially in the dark phase (7 pm to 7 am) were

8

increased in the sanyaku and diosgenin groups. Moreover mRNA levels of lipoprotein

9

lipase and peroxisome proliferative activated receptor, gamma, coactivator 1α were

10

moderately up-regulated in the liver of sanyaku- and diosgenin-ingested mice. These

11

results suggest that consecutive ingestion of diosgenin or diosgenin-containing

12

sanyaku at the dose achievable in human diet potentially ameliorates fasted and

13

postprandial

14

improvement of TG metabolism.

hypertriacylglycerolemia,

which

could be

associated

with

the

15

16

Keywords: Diosgenin, hypertriacylglycerolemia, oral fat tolerance test, lipoprotein

17

lipase

18

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Introduction

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Diosgenin is an aglycone of dioscin, a major bioactive steroidal saponin found

21

in natural plants including tuber of wild yam (Dioscorea villosa Linn) and seed of

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fenugreek (Trigonella Foenum graecum Linn). Diosgenin is neither synthesized nor

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metabolically converted into steroid byproducts in the mammalian body, and hence is

24

considered safe. Several bioactivities of diosgenin have been shown to be beneficial to

25

health in animal model experiments including its efficacy against hyperglycemia 1,

26

hypercholesterolemia

27

modulatory roles of diosgenin on sugar and lipid metabolism, and intestinal absorption,

28

diosgenin has properties that regulate the cell signaling involved with inflammation,

29

oxidative stress, and apoptosis

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associated with beneficial effects induced by diosgenin. In our previous report

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diosgenin and sanyaku, another source of diosgenin (contained at 63.8±1.2 mg/kg dry

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weight) given at 20, 100, and 500 ppm in the diet for 17 weeks significantly inhibited

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azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colon carcinogenesis in

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ICR mice 5. Sanyaku, a freeze-dried Chinese yam (Dioscorea batatas), has been widely

35

used as a traditional Chinese medicine whose benefits include nutritional fortification,

36

tonic, and antitussive effects. It is also used for its antidiarrheal, expectorant, and

2,3

, obesity 4, cancer

5

and dementia

6,7

. In addition to the

8,9

. The activation of these multiple pathways is likely

3



37

hypoglycemic effects 10. The study revealed that the ingestion of diosgenin or sanyaku

38

ameliorated not only AOM-DSS-induced inflammation but also the elevated levels of

39

serum triacylglycerol (TG) involved with lipoprotein lipase (LPL) induction 5.

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Blood TG circulates as lipoprotein particles mainly in the form of chylomicron

41

(CM), and very low density lipoprotein (VLDL). These lipoprotein particles consist of a

42

neuronal lipid core (TG and cholesterol esters) surrounded by a monolayer of

43

phospholipids, free cholesterol and proteins, such as apolipoproteins. TG derived from

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dietary fat is hydrolyzed by lipase to yield fatty acids to be absorbed in the small

45

intestine. TG is reconstructed in enterocytes for the incorporation of CM, then released

46

to the bloodstream via lymphatic vessels. Lipolysis of CMs is mediated by LPL,

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resulting in the formation of smaller remnant CM particles, which are then catabolized

48

in the liver. Another TG-rich lipoprotein is VLDL, which is synthesized in the liver.

49

Secreted VLDLs are also hydrolyzed by LPL to form smaller remnant particles that are

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TG-depleted and enriched with cholesterol esters. In clinical practice, blood levels of

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TG are traditionally measured after overnight fasting, in which values over 150–200

52

mg/dL are diagnosed as hypertriacylglycerolemia although cut-off values vary

53

depending on the guidelines. Higher levels of serum TG are strongly associated with

54

vascular damage, and accordingly with the risk of atherosclerosis and cardiovascular

4


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disease. Additionally, recent prospective epidemiologic studies have shown that

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postprandial rather than fasted hypertriacylglycerolemia is significantly associated with

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hazard ratio for cardiovascular events11,12.

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We have previously shown that daily consumption of diosgenin and sanyaku at

59

doses that would be feasible in a human diet (20, 100, and 500 ppm) ameliorated the

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serum TG level mediated by hepatic gene expression associated with lipid metabolism 5.

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Another study has shown that diosgenin and its glycosylated form dioscin prevented

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intestinal absorption of TG via pancreatic lipase inhibition, although the effective doses

63

(2% and 5% dioscorea powder in diet) were quite high, making its efficacy as a dietary

64

constituent rather limited

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mechanisms of diosgenin- and sanyaku-induced TG reduction, oral fat tolerance tests

66

(OFTT),

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hypertriacylglyceridemic KK-Ay mice were examined. The doses of diosgenin and

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sanyaku used are achievable in a human diet.

respiratory

13

. Therefore, in the present study, to examine the molecular

gas

analysis,

and

hepatic

69

70

71

Materials and Methods

72

Chemicals

5


gene

expression

in


73

Diosgenin was purchased from Sigma Chemical (St. Louis, MO). Sanyaku

74

(Chinese yam), a freeze-dried powder used in traditional Chinese medicine, was

75

obtained from a local pharmacy (Niiya, Shizuoka, Japan). Diosgenin content in sanyaku

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used in this study was 0.0064% w/w. Porcine pancreas lipase was obtained from Sigma.

77

All other chemicals were purchased from Wako Pure Chemical Industries (Osaka,

78

Japan).

79

80

Animal experiments

81

All animal experiments were approved by the animal ethics committee of

82

University of Shizuoka (approved number 135005), and performed according to

83

guidelines for the care and use of laboratory animals at the University of Shizuoka.

84

Hypertriacylglyceridemic and type II diabetic model KK-Ay mice (5-week-old male)

85

were purchased from CLEA Japan Inc. (Tokyo, Japan). All animals were housed

86

individually in plastic cages and had free access to drinking water under controlled

87

conditions of humidity (50±10%), light (12/12-h light/dark cycle) and temperature

88

(23±2 °C). After a 1-week adaptation period with a basal diet (MF, Oriental Yeast, Co.,

89

Ltd., Tokyo, Japan) given ad libitum, mice were randomized by body weight into

90

control, sanyaku, and diosgenin groups (n=7). To evaluate the inhibitory effects of

6


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sanyaku and diosgenin on lipid absorption, the first OFTT was performed on day 0.

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Mice starved of food for 18 hours were orally administered 10 mL/kg body weight of

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lipid emulsion, which was prepared by sonication of 200 mg/mL soybean oil, 12 mg/mL

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egg lecithin, and 22.5 mg/mL glycerol with or without 500 ppm diosgenin or sanyaku

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suspended in MilliQ water according to previous reports with slight modification

96

Blood samples were collected sequentially from the tail vein using heparinized capillary

97

tubes, and determined levels of triglyceride using the Triglyceride E test Wako (Wako

98

Pure Chemical, Japan). The area under the TG concentration-time curve from 0 to 8 h

99

(AUC0–8h) was calculated using the trapezoidal formula 17. Levels of blood glucose were

100

monitored using ACCU-Chek Aviva (Roche). After the first OFTT, mice were given a

101

high fat diet (HFD; QuickFat, CLEA Japan, Inc.) containing 500 ppm sanyaku or 500

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ppm diosgenin until the end of the experiment (Fig. 1). After mice had been fed HFD,

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HFD with sanyaku, or HFD with diosgenin for 8 weeks, the second OFTT was

104

performed in the same manner as first OFTT, but with a lipid emulsion with neither

105

sanyaku nor diosgenin. One week after the second OFTT, levels of oxygen consumption

106

and carbon dioxide productions were measured by mass spectrometer (model

107

ARCO-2000, Arco System). Metabolic parameters were calculated as follows:

108

RQ=VCO2/VO2,

FAT=1.695×VO2-1.701×VCO2

7


14–16

.

(mg/min),


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CHO=4.585×VCO2-3.226×VO2 (mg/min), energy=3.815×VO2+1.232×VCO2 (cal/min).

110

Locomotor activity was measured by counting the frequency of passes into the different

111

regions in the cage every 5 min during the observation period. Mice feces were

112

collected after respiratory gas analysis to determine fecal diosgenin and TG. All mice

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were sacrificed at the age of 17 weeks. Prior to being euthanized, animals were starved

114

overnight. Blood and other organs such as skeleton muscle, liver, and white adipose

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tissue (WAT) were collected. Levels of tissue and fecal triglyceride were also assayed

116

using the Triglyceride E test (Wako). Free fatty acid (FFA) was assayed by NEFA

117

C-test (Wako).

118

119

Lipase assay The activity of pancreas lipase was measured according to the method of Ikeda

120

14,15

121

et al.

122

sonication of 80 mg triolein, 10 mg egg lecithin, 5 mg sodium taurocholate suspended

123

in

124

methyl-2-aminoethanesulfonic acid and 0.1 M NaCl. Then, 200 µL of reaction mixture

125

consisting of 100 µL of lipid emulsion, 10 U of porcine pancreatic lipase dissolved in

126

TES buffer, and diosgenin or MeOH (vehicle final conc. 0.5%) were incubated at 37 °C

TES

with slight modifications. Briefly, a lipid emulsion (9 mL) was prepared by

buffer

(pH

7.0)

consisting

of

0.1

M

8


N-tris

(hydroxymethyl)

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for 30 min. After incubation, 1 nmol heptadecanoic acid (internal standard for LC-MS

128

analysis) was added and then extracted twice by chloroform: MeOH (1:1). Dried

129

samples were dissolved in 100 µL MeOH: isopropanol (6:4), and an aliquot (10 µL)

130

was injected to LC-MS to quantify released fatty acids according to the method of Hao

131

et al.

132

performance liquid chromatography (HPLC) system coupled with G6410B triple

133

quadrupole tandem mass spectrometer. HPLC separation was performed with an X

134

Select CSG C18 column (3.5 µm, 150 mm × 2.1 mm, Waters) at 40 °C. An isocratic

135

elution with MeOH: isopropanol (40:6) at 0.2 mL/min was used. MS was operated in

136

negative ion mode using an electrospray ionization source. Oleic acid and

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heptadecanoic acid were detected in single ion monitoring mode at m/z 281.2 and 269.3,

138

respectively. In this analytical condition, recovery rate and relative standard deviation

139

(RSD) were 89.5% and 3.2%, respectively (n=5).

18

with slight modification. LC-MS consisted of an Agilent 1200 series high

140

141

LC-MS/MS analysis of diosgenin in plasma and feces

142

MicroSmash (Tomy Seiko, Japan) was used to homogenate 10 µL plasma or 10

143

mg feces suspended in 200 µL ethanol with zirconia beads. After centrifugation at

144

21,500×g for 5 min, the supernatant was collected into a new tube. The precipitate was

9



145

further extracted twice with 200 µL ethanol, and these supernatants were combined,

146

dried, and dissolved in 200 µL ethanol. 10 µL samples were injected into LC-MS/MS

147

consisting of an Agilent 1200 series HPLC coupled with a G6410B triple quadrupole

148

tandem mass spectrometer. HPLC separation was performed with a Super-ODS column

149

(2.3 µm, 100 mm × 2.0 mm, TOSOH) at 40 °C. An isocratic elution with MeOH:

150

MilliQ (9: 1) containing 0.1% formic acid at 0.2 mL/min was used. The MRM transition

151

for diosgenin was monitored m/z 415.6>253.2. In this analytical condition, the detection

152

limit was 5 pg (12 fmol)/injection and the recovery rate and RSD were 78.5% and 8.3%

153

in plasma and 98.4% and 3.3% in feces, respectively (n=3).

154

155

Real-time RT-PCR

156

Total RNA was extracted using TRIzol reagent (Invitrogen) from mice skeleton

157

muscle, liver, and WAT stored in RNaseLater solution (Ambion). Total RNA samples

158

(200 ng) were converted into cDNA using PrimeScript RT Master Mix (TaKaRa). To

159

quantitatively estimate the level of each gene, quantitative PCR was performed using

160

gene-specific primers, cDNA and SYBR Premix (TaKaRa). The amplification

161

conditions were as follows: denaturation at 95 °C for 10 s, and annealing and extension

162

at 60 °C for 30 s. The cycle threshold (CT) values of each gene and 18S rRNA detected

10


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by real-time RT-PCR were converted to signal intensities by the delta-delta method,

164

which calculates the difference in one CT-value as a two-fold difference between the

165

signal for each gene and the signal for a normalization gene (18S rRNA). The formula

166

used was: 2(CT each gene-CT 18S rRNA). The sequences of the PCR primer pairs are as follows:

167

cytochrome c oxidase subunit 2 (COX2); 5′-CCG ACT AAA TCA AGC AAC AGT AAC

168

A-3′ and 5′-AAA TTT CAG AGC ATT GGC CAT AG-3′, cytochrome c oxidase subunit

169

4 (COX4); 5′ CTA TGT GTA TGG CCC CAT CC-3′ and 5′-AGC GGG CTC TCA CTT

170

CTT C-3′, mitochondrial transcription factor A (mtTFA); 5′-GGA ATG TGG AGC GTG

171

CTA AAA-3′ and 5′-TGC TGG AAA AAC ACT TCG GAA TA-3′, lipoprotein lipase

172

(LPL); 5′-CCA GGA TGC AAC ATT GGA GA-3′ and 5′-CAA CTC AGG CAG AGC

173

CCT TT-3′, peroxisome proliferative activated receptor, gamma, coactivator 1α

174

(PGC1α); 5′-GAA GTG GTG TAG CGA CCA ATC-3′ and 5′-AAT GAG GGC AAT

175

CCG TCT TCA-3′, 18S rRNA; 5′-CTT AGA GGG ACA AGT GGC G-3′ and 5′-ACG

176

CTG AGC CAG TCA GTG TA-3′.

177

178

Statistical analysis

179

All data are presented as means±SEM. All statistical analyses were performed

180

with EZR (Saitama Medical Center, Jichi Medical University), which is a graphical user

11



181

interface for R (The R Foundation for Statistical Computing). More precisely, it is a

182

modified version of R commander designed to add statistical functions frequently used

183

in biostatistics. Statistical analyses of data were performed by two-way repeated

184

measures ANOVA. One-way ANOVA followed by Bonferroni’s multiple range tests

185

was used to compare multiple test groups. Differences were considered significant at p

186

< 0.05.

187

188

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Results

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Effects of diosgenin and sanyaku on plasma levels of TG

191

Body weight and amount of food intake were not significantly different

192

among groups during the experimental period with the exception of the food intake of

193

sanyaku group at week 0 (Fig. 2), although all KK-Ay mice were fed HFD for 8 weeks

194

to accelerate hyperglycemia (Fig. 3A) and hypertriacylglycerolemia (Fig. 4A). There

195

were no significant differences among the experimental groups in some biological

196

parameters including plasma FFA, WAT weights, TG accumulation in peripheral tissue,

197

and plasma ketone bodies (Fig. 3B-D). Although fasting plasma TG increased

198

age-dependently in the control group, the elevated levels of fasting plasma TG were

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ameliorated by sanyaku or diosgenin supplementation (Fig. 4A). TG in liver and white

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adipose tissue (WAT) and WAT weight tended to be decreased in the sanyaku and

201

diosgenin groups, although that of muscle TG was slightly increased (Fig. 3E-H). The

202

first OFTT was performed at week 0, prior to initiation of HFD or HFD with sanyaku

203

or diosgenin. At the first OFTT, to determine the inhibitory effect of sanyaku or

204

diosgenin on fat absorption, those either sanyaku or diosgenin was mixed with lipid

205

emulsion and then orally administered to mice. As shown in Fig. 4B, levels of plasma

206

TG in control mice rapidly became elevated to 473±55.7 mg/dL at 1 hour after lipid

207

loading, and then gradually declined to basal level within 8 hours. The levels of TG in

208

the sanyaku and diosgenin groups at each time point were slightly higher and lower,

209

respectively (Fig. 4B), however AUC0-8h was not significantly different, indicating that

210

500 ppm sanyaku or diosgenin mixed in lipid emulsion (50 mg/100 mL) did not inhibit

211

lipid absorption (Fig. 4D). The second OFTT was performed at week 8 to determine

212

the effect of daily administration of sanyaku and diosgenin on lipid metabolism.

213

Therefore, either sanyaku or diosgenin-free lipid emulsion was orally administered to

214

all mice at the second OFTT. As shown in Fig. 4C, not only fasting TG levels (time =

215

0) but also after lipid loading plasma TG in sanyaku and diosgenin groups was reduced

216

compared with that of the control, in which Cmax values were 602, 313, and 469 mg/dL

13



217

in the control, sanyaku, and diosgenin groups, respectively. Moreover, as shown in Fig.

218

4E, AUC and also ∆AUC values (data not shown) were decreased in sanyaku and

219

diosgenin groups. Pancreatic lipase activities were significantly inhibited by high doses

220

(more than 1 mM) of diosgenin in in vitro experiments (Fig. 5A). However, the amount

221

of diosgenin in the feces collected from the diosgenin-administered group at week 10

222

was determined as 7.6 pmol/mg feces (Fig. 5B). In addition, levels of TG in feces

223

collected at week 10 were not significantly reduced in sanyaku and diosgenin groups

224

(Fig. 5C). These results suggest that the dose of diosgenin in the intestinal tract was

225

probably far lower than 1 mM. Moreover, lipid absorption would be less affected by

226

diosgenin and sanyaku under the current experimental conditions. Therefore, taken

227

together, these results suggest that reduced levels of TG in the sanyaku and diosgenin

228

groups observed in not only the fasted state but also in the second OFTT could be

229

associated with the improvement of lipid metabolism rather than the inhibition of lipid

230

absorption as a result of daily oral ingestion of sanyaku and diosgenin.

231

232

Diosgenin and sanyaku up-regulated fat oxidation

233

We measured the respiratory gas of KK-Ay mice fed HFD, HFD supplemented

234

with sanyaku or HFD supplemented with diosgenin at week 9. As shown in Fig.6A,

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oxygen consumption (VO2) showed a circadian pattern. The integrated values of VO2

236

for 12 hours in the dark and light phases were not significantly different among groups

237

(data not shown). The respiratory exchange ratio was slightly lower in the diosgenin

238

group than in others (Fig. 6B). Levels of fat oxidation in the dark phase were slightly

239

but not significantly increased in the sanyaku and diosgenin groups, although they

240

were not notable in the light phase (Fig. 6C). Locomotor activity in dark phase was

241

higher in the sanyaku and diosgenin groups compared with the control (p

Effects of Sanyaku and Its Constituent Diosgenin on the Fasted and

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