Effects of Long-Term High-Ergosterol Intake on the Cholesterol and Vitamin D Biosynthetic Pathways of Rats Fed a High-Fat and High-Sucrose Diet

Abstract

Dyslipidemia is a lifestyle-related (physical inactivity or obesity) disease; therefore, dietary foods that can easily be consumed in daily life is important to prevent dyslipidemia. Ergosterol, a precursor of vitamin D₂, is a fungal sterol present in the membranes of edible mushrooms and other fungi. Ergosterol is converted to brassicasterol by 7-dehydrocholesterol reductase (DHCR7), a cholesterol biosynthesis enzyme that converts 7-dehydrocholesterol (a precursor of vitamin D₃) into cholesterol. Previously, we reported that ergosterol increases 7-dehydrocholesterol, decreases cholesterol levels by competitive effect of DHCR7, and reduces DHCR7 mRNA and protein levels in human HepG2 hepatoma cells. Here, we investigated the effects of long-term high ergosterol intake on the cholesterol, vitamin D₂, and D₃ biosynthetic pathways of rats fed a high-fat and high-sucrose (HFHS) diet using GC–MS and LC with tandem mass spectrometry. In HFHS rats, oral ergosterol administration for 14 weeks significantly decreased plasma low-density lipoprotein cholesterol, total bile acid, and cholesterol precursor (squalene and desmosterol) levels and increased 7-dehydrocholesterol levels compared to HFHS rats without ergosterol. Ergosterol, brassicasterol, and vitamin D₂ were detected, cholesterol levels were slightly decreased, and levels of vitamin D₃ and its metabolites were slightly increased in rats fed HFHS with ergosterol. These results showed that ergosterol increased vitamin D₂ levels, inhibited the cholesterol biosynthetic pathway, and possibly promoted vitamin D₃ biosynthesis in vivo. Therefore, daily ergosterol intake may aid in the prevention of dyslipidemia.

INTRODUCTION

Dyslipidemia is a lifestyle-related (physical inactivity or obesity) disease; therefore, not only medicine but also dietary foods and exercise are essential for the prevention and treatment of dyslipidemia.^1,2) Dyslipidemia increases the risk of atherosclerosis and stroke, the leading cause of morbidity and mortality globally.¹⁾ Dyslipidemia occurs due to various lipid abnormalities in the blood, including elevated low-density lipoprotein-cholesterol (LDL-C), elevated triglyceride (TG), or low levels of high-density lipoprotein-cholesterol (HDL-C).^2,3) Dyslipidemia of hypercholesterolemia, or hyper-LDL-cholesterolemia, is widely treated with statins, which inhibit 3-hydroxy-3-methylglutaryl-CoA reductase, an enzyme involved in the presqualene pathway of cholesterol biosynthesis.^2,4) However, treatment with statins often results in intolerance, which is usually diagnosed when a patient is unable to continue statin therapy due to perceived, or objectively documented, adverse effects.⁵⁾ Therefore, investigation of new dyslipidemia agents or dietary foods that can be easily consumed in daily life and can act via mechanisms distinct from those of statins is pertinent.⁶⁾

Ergosterol, a precursor of vitamin D₂, is a common fungal sterol present in the membranes of edible mushrooms and other fungi.^7,8) Ergosterol is converted to brassicasterol by 7-dehydrocholesterol reductase (DHCR7) at various sites, such as skin fibroblasts,⁹⁾ liver,^10,11) and HepG2 hepatoma cells.¹²⁾ DHCR7 is widespread in mammals,¹³⁾ and also catalyzes the reduction of the Δ7 bond of 7-dehydrocholesterol to cholesterol and 7-dehydrodesmosterol to desmosterol in the human liver^9,11,14) (Fig. 1). Both ergosterol and 7-dehydrocholesterol are metabolized by DHCR7 and tend to compete with each other.⁹⁾ Previously, we reported that ergosterol increased 7-dehydrocholesterol and decreased cholesterol levels, and reduced the mRNA and protein levels of DHCR7 and 24-dehydrocholesterol reductase in human HepG2 hepatoma cells.¹²⁾ Additionally, UV light induces the conversion of 7-dehydrocholesterol to vitamin D₃ in the skin¹⁵⁾ (Fig. 1). Thus, ergosterol may have an inhibitory effect on the cholesterol biosynthetic pathway as well as an activating effect on the vitamin D biosynthetic pathway in vivo. However, it has also been reported that most ergosterol administered orally once a day for 1 week in rats is excreted in feces, while the remaining sterol is absorbed through the intestine.¹⁰⁾ Absorbed ergosterol is rapidly metabolized to brassicasterol within 25 h and has no vitamin D biological activity.¹⁰⁾ In another study, wherein C57BL/6NCrl mice (normal mice) received a high dose of ergosterol (7 mg/kg) for 6 weeks, ergosterol was reportedly absorbed at 5.7 ± 2.2 nmol/L, and serum vitamin D₃ level increased.¹⁶⁾ Decreases in the serum level of total cholesterol (T-CHO) due to long-term ergosterol intake has only been reported in pathological rat models in Sprague–Dawley rats fed a high-cholesterol diet.¹⁷⁾

Fig. 1. Cholesterol, Vitamin D₂, and Vitamin D₃ Biosynthesis Pathways

HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; HMGCS1, 3-hydroxy-3-methylglutaryl-CoA synthase 1; HMGCR, HMG-CoA reductase; SQLE, squalene epoxidase; LSS, lanosterol synthase; CYP51, lanosterol 14α-demethylase; DHCR14, 14-dehydrocholesterol reductase; SC4MOL, sterol-C4-methyl oxidase; HSD17B7, hydroxysteroid 17β dehydrogenase 7; NSDHL, NAD(P)-dependent steroid dehydrogenase-like; EBP, sterol-8,7-isomerase; SC5DL, sterol-C5-desaturase; DHCR7, 7-dehydrocholesterol reductase; DHCR24, 24-dehydrocholesterol reductase; CYP2R1, 25-hydroxylase; CYP27B1, 1α-hydroxylase.

Owing to their bioactive ingredients, edible mushrooms have recently emerged as a promising dietary component for preventing hypercholesterolemia.¹⁷⁾ Ergosterol is the main sterol present in most edible mushrooms⁸⁾; however, only one systematic trial on the effects of ergosterol on blood cholesterol have been performed.¹⁷⁾ Additionally, the mechanism whereby ergosterol inhibits cholesterol biosynthesis remains unclear, and vitamin D₃ biosynthesis by ergosterol has not been investigated in pathological models.

Here, we studied the effects of long-term high ergosterol intake on cholesterol and vitamin D₃ biosynthetic pathways in rats fed a high-fat and high-sucrose (HFHS) diet.

MATERIALS AND METHODS

Reagents

Methanol, acetonitrile, formic acid, ethyl acetate, n-hexane, squalene, vitamin D₃, and 5α-cholestane (internal standard, I.S., for ergosterol, brassicasterol, and cholesterol and its precursors) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Lanosterol was purchased from Nagara Science Corporation (Gifu, Japan), and 7-dehydrocholesterol, desmosterol, lathosterol, cholesterol, and vitamin D₂ were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Additionally, 25-hydroxyvitamin D₂ and 1α, 25-dihydroxyvitamin D₃ were purchased from Cayman Chemical (Ann Arbor, MI, U.S.A.), and 1α, 25-dihydroxyvitamin D₂ was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, U.S.A.). 25-Hydroxyvitamin D₃ was purchased from LKT Laboratories, Inc. (St. Paul, MN, U.S.A.), 25-hydroxyvitamin D₂-d₆ (I.S. for vitamin D₂, vitamin D₃, and its metabolites) was purchased from Toronto Research Chemicals (Toronto, ON, Canada), ergosterol (for feeding rats) was purchased from Tokyo Chemical Industry Corporation, Ltd. (Tokyo, Japan), ergosterol (for quantitative analysis) and brassicasterol were purchased from Tama Biochemical Corporation Ltd. (Tokyo, Japan), and a trimethylsilyl derivatizing agent, Tri-Sil HTP reagent (hexamethyldisilazane (HMDS) : trimethylchlorosilane (TMCS) : pyridine = 2 : 1 : 10) was purchased from Thermo Fisher Scientific Corporation (Waltham, MA, U.S.A.). Amplifex^® Diene reagent was purchased from SCIEX (Chemistry and Consumables R&D, Framingham, MA, U.S.A.).

Animal Experiments

Twenty-four 4-week-old Wistar rats were purchased from Japan SLC Inc. (Hamamatsu, Japan) and maintained under controlled temperature and light conditions (23 ± 1 °C and 12 h light: 12 h dark, respectively). All the experimental procedures followed the guidelines for animal experimentation of the Animal Care and Use Committee of Niigata University of Pharmacy and Applied Life Sciences, Japan (2020-7 approved in May 2020). The rats were divided into the following three groups: (i) control animals (n = 8) were fed an AIN-93M standard diet (Oriental Yeast Corporation, Ltd., Tokyo, Japan), (ii) HFHS animals (n = 8) were fed an HFHS diet, and (iii) HFHS + ergosterol animals (n = 8) were fed an HFHS diet supplemented with 1.0% ergosterol dry powder. The composition of each diet is presented in Table 1. As previous reports had experimented with a dosage of 0.5–1.5% ergosterol,¹⁷⁾ rats in this study were fed a median ergosterol dosage of 1.0%. The individual components of the HFHS diet were purchased from Oriental Yeast Corporation, Ltd., except for cholesterol, L-cysteine, and tert-butylhydroquinone, which were purchased from FUJIFILM Wako Pure Chemical Corporation. Ergosterol was added to the HFHS diet components and lipid levels were adjusted to the same values as those in the HFHS diet (Table 1).

Table 1. Dietary Composition of Ergosterol-Supplemented and Control Diets Used in the Feeding Experiment

Component (g/kg)	Control	HFHS	HFHS + ES
Casein lactic	140	235	235
Corn sugar	465.692	—	—
α-Corn sugar	155.00	148.69	148.69
Sucrose	100	200	200
Cellulose	50	50	50
Beef tallow	—	140	140
Lard	—	140	140
Soy oil	40	20	10
Cholesterol	—	15	15
AIN-93 mineral mix	35	35	35
AIN-93 vitamin mix	12.5	12.5	12.5
L-Cysteine	1.80	3.75	3.75
tert-Butylhydroquinone	0.008	0.06	0.06
Ergosterol	—	—	10

Control, AIN-93M standard diet; HFHS, high-fat and high-sucrose; HFHS + ES, high-fat and high-sucrose feed supplemented with 1.0% ergosterol dry powder; —, not applicable.

Following a 1-week habituation period, each group was fed its respective diet for 14 weeks with ad libitum access to tap water and chow. Food intake was measured daily and weight gain was measured once per week. Fourteen weeks later, after fasting overnight, the rats were anesthetized with isoflurane (FUJIFILM Wako Pure Chemical Corporation), blood samples were collected from the jugular vein, and the rats were sacrificed by bleeding from the descending aorta.

Plasma Biomarker Analysis

Plasma levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), TG, T-CHO, free cholesterol (F-CHO), LDL-C, HDL-C, total bilirubin (T-BIL), direct bilirubin (D-BIL), indirect bilirubin (I-BIL), total bile acids (TBA), blood urea nitrogen (BUN), and creatinine (CRE) were determined. AST, ALT, ALP, LDH, and CRE were analyzed using JSCC transferable method; TG, T-CHO, F-CHO, T-BIL, D-BIL, and I-BIL were analyzed using enzyme method; LDL-C and HDL-C were analyzed using direct assay; TBA was analyzed using enzyme cycling method; and BUN was analyzed using urease glutamate dehydrogenase (GLDH) method by Oriental Yeast Corporation Ltd. (Tokyo, Japan).

Measurement of Plasma Ergosterol, Brassicasterol, Cholesterol Precursors, and Cholesterol Levels by GC–MS

Levels of ergosterol, brassicasterol, cholesterol precursors, and cholesterol were measured as previously described.^12,18,19) Briefly, 50 μL of plasma in a glass tube containing 250 ng of 5α-cholestane (I.S.) was saponified with 2 mL of 10 mol/L potassium hydroxide, followed by the addition of 10 mL of methanol. After adjusting the pH to 7.0 with 50% phosphoric acid, 5 mL of H₂O was added to the mixture, and extracted with 10 mL of n-hexane. The n-hexane layer was collected, dried under a stream of nitrogen, and subsequently derivatized for 30 min at 60 °C with a trimethylsilyl ester.

The GC–MS conditions were as follows: capillary column, DB-5MS (30 m × 0.25 mm; particle size, 0.25 µm; Agilent Technologies, Santa Clara, CA, U.S.A.); carrier gas, helium (1 mL/min); column temperature, 180 °C for 1 min → 20 °C/min → 250 °C → 5 °C/min → 280 °C → 3 °C/min → 300 °C for 12 min; inlet temperature, 230 °C; ion source temperature, 250 °C; interface temperature, 250 °C; injection volume, 1 µL; detection mode, selected ion monitoring; GC–MS instrument, GCMS-QP2010Plus (Shimadzu Corporation, Kyoto, Japan); monitoring ions, m/z 363, 468 for ergosterol, m/z 380, 470 for brassicasterol, m/z 69, 81 for squalene, m/z 393, 498, 483 for lanosterol, m/z 458, 459, 255 for lathosterol, m/z 325, 351 for 7-dehydrocholesterol, m/z 343, 456, 253 for desmosterol, m/z 368, 458, 329 for cholesterol, and m/z 217, 357 for 5α-cholestane (bold and underline indicates the quantitative ion).

Vitamins D₂, D₃, and Their Metabolites in Plasma Measured by Liquid Chromatography with Tandem Mass Spectrometry (LC–MS/MS)

Vitamins D₂, D₃, and their metabolites were measured as previously described.²⁰⁾ Briefly, 120 µL of plasma was added to 600 µL of acetonitrile and 25 μL of 25-hydroxyvitamin D₂-d₆ (I.S.), and centrifuged at 2000 × g for 10 min at 4 °C. The supernatant was extracted with 600 μL of ethyl acetate. The upper organic layer was collected, dried under a stream of nitrogen, and subsequently derivatized with Amplifex^® Diene reagent.

LC–MS/MS analysis was performed on a Prominence UFLC (Shimadzu Corporation) equipped with a system controller (CBM-20A), degasser (DGU-20A5), quaternary pump (LC-20AD), autoinjector (SIL-20A), and column oven (CTO-20A), coupled with an API3200 (SCIEX, Framingham, MA, U.S.A.) equipped with an electrospray ionization (ESI) source. Chromatographic separation was performed using an Inert Sustain™ C₁₈ (2.1 × 150 mm; particle size, 5 µm; GL Sciences Inc., Tokyo, Japan). A gradient elution program was conducted for chromatographic separation using mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) as follows: 0–6 min (30–65% B), 6–8 min (65–65% B), 8–12 min (65–100% B), 12–15 min (100–100% B), 15–17 min (100–30% B), 17–19.1 min (30–30% B). The column was maintained at 40 °C and the flow rate was 0.4 mL/min. The injection volume was 20 µL.

The mass spectrometer was operated with an ESI source in positive ion detection mode. The source temperature was set to 700 °C and curtain gas to 25 psi. Quantification was performed with multiple reaction monitoring of the transitions with m/z 728.3→669.3 for vitamin D₂-Amplifex, m/z 744.4→685.4 for 25-hydroxyvitamin D₂-Amplifex, m/z 760.2→701.4 for 1α, 25-dihydroxyvitamin D₂-Amplifex, m/z 716.3→657.3 for vitamin D₃-Amplifex, m/z 732.3→673.3 for 25-hydroxyvitamin D₃-Amplifex, m/z 748.2→689.3 for 1α, 25-dihydroxyvitamin D₃-Amplifex, and m/z 750.3→691.4 for 25-hydroxyvitamin D₂-d₆-Amplifex.

Statistical Analysis

Statistical analyses were performed using the EZR statistical software package (version 1.40).²¹⁾ Values are presented as mean ± standard error. Data were analyzed using the Tukey–Kramer test, and statistical significance was set at p < 0.05.

RESULTS

Effect of Ergosterol-Supplemented Diet on Body Weight, Food Consumption, and Organ and Body Fat Weights in Rats

The body weight gain and food consumption profiles of rats fed the AIN-93M standard diet, HFHS diet, and ergosterol-supplemented HFHS diet (HFHS + ergosterol) are shown in Fig. 2. The body weights of rats in the HFHS and HFHS + ergosterol groups were significantly higher than those of rats in the control group (p < 0.05 at 5 weeks and p < 0.01 at 6–14 weeks for the control group; p < 0.05 at 4 weeks and p < 0.05 at 5–14 weeks for the HFHS group, Tukey–Kramer test, n = 8). The body weights of rats in the HFHS + ergosterol group were slightly lower than those of rats in the HFHS group; however, the difference was not significant (Fig. 2A). A comparison of daily food intake revealed that rats in the control group ate more chow than rats in the HFHS and HFHS + ergosterol groups throughout the experimental period (p < 0.01, Tukey–Kramer test, n = 8) (Fig. 2B).

Fig. 2. Effect of Ergosterol-Supplemented Diet on Body Weight and Food Consumption of Rats

Body weight (A) and total weekly food consumption (B) of rats fed different diets. Values are mean ± standard error (n = 8, each group). * p < 0.05, ** p < 0.01 compared with control, Tukey–Kramer test. Control, AIN-93M standard diet; HFHS, high-fat and high-sucrose; HFHS + ES, high-fat and high-sucrose feed supplemented with 1.0% ergosterol dry powder.

The weights of several organs and types of body fat in each experimental group were compared at the end of the study (Table 2). The liver, heart, and mesenteric, epididymal, perirenal, and visceral fat were heavier in the HFHS group (p < 0.01 for liver, and epididymal, perirenal, and visceral fat; p < 0.05 for heart and mesenteric fat, Tukey–Kramer test, n = 8) than in the control group, indicating that these rats developed obesity.²²⁾ In the HFHS + ergosterol group, liver weight was slightly lower than that in the HFHS group (p = 0.083, Tukey–Kramer test, n = 8) (Table 2). Furthermore, no significant differences were observed between the organ weights of rats in the HFHS + ergosterol group and those of rats in the HFHS group (Table 2).

Table 2. Weights of Organs of Rats Fed High-Fat and High-Sucrose (HFHS) Diet or HFHS Diet Supplemented with Ergosterol

Organ or body fat (g)	Control	HFHS	HFHS + ES
Liver	13.18 ± 0.76	27.81 ± 1.57**	23.48 ± 1.57**
Spleen	0.68 ± 0.04	0.78 ± 0.02	0.76 ± 0.03
Kidney	3.10 ± 0.07	3.41 ± 0.18	3.47 ± 0.12
Heart	1.18 ± 0.05	1.47 ± 0.08*	1.35 ± 0.09
Mesenteric fat	8.47 ± 0.59	12.48 ± 1.14*	12.69 ± 0.93*
Epididymal fat	7.50 ± 0.48	11.90 ± 1.17**	13.47 ± 0.88**
Perirenal fat	9.23 ± 0.89	17.21 ± 1.42**	16.45 ± 1.13**
Visceral fat	25.19 ± 1.69	41.60 ± 3.55**	42.60 ± 2.65**

Values are mean ± standard error. (n = 8, each group); * p < 0.05, ** p < 0.01 compared with control, Tukey–Kramer test. Control, AIN-93M standard diet; HFHS, high-fat and high-sucrose; HFHS + ES, high-fat and high-sucrose feed supplemented with 1.0% ergosterol dry powder.

No deaths were reported in any group during the experimental period, and no significant differences were observed between the body weight and daily food intake of rats in the HFHS + ergosterol group and those of rats in the HFHS group (Fig. 2). Additionally, no weight change or loss of organs due to metabolism and excretion (liver and kidneys), including the spleen, was observed (Table 2), suggesting that 1.0% ergosterol has no lethality and detrimental effect on the liver and kidney functions.^23–25) Furthermore, no behavioral signs of toxicity, such as tremors, convulsions, piloerection, or changes in locomotor activity, were observed in all rats.²³⁾ Our study results imply that 1.0% ergosterol does not have toxicity in rats.

Effect of Ergosterol-Supplemented Diet on Plasma Biomarker Levels in Rats

Plasma levels of AST, ALT, ALP, LDH, T-CHO, F-CHO, LDL-C, HDL-C, T-BIL, D-BIL, I-BIL, and TBA were significantly higher in the HFHS group than in the control group (p < 0.01, Tukey–Kramer test, n = 8). LDL-C and TBA levels were significantly lower (p < 0.05, Tukey–Kramer test, n = 8) in the HFHS + ergosterol group than in the HFHS group (Table 3), and T-BIL and D-BIL levels were slightly lower (p = 0.069 for T-BIL, p = 0.059 for D-BIL, Tukey–Kramer test, n = 8) than in the HFHS group (Table 3). The T-CHO/HDL-C and LDL-C/HDL-C ratios, which are used as clinical predictors of cardiovascular disease risk,^2,32,33) were significantly higher in the HFHS group than in the control group (p < 0.01, Tukey–Kramer test, n = 8). T-CHO/HDL-C and LDL-C/HDL-C ratios were significantly lower in the HFHS + ergosterol group than in the HFHS group (p < 0.01 for LDL-C/HDL-C ratio and p < 0.05 for T-CHO/HDL-C ratio, Tukey–Kramer test, n = 8). Additionally, the plasma level of BUN was significantly lower in the HFHS group than in the control group (p < 0.01, Tukey–Kramer test, n = 8) (Table 3). However, no significant difference in plasma AST, ALT, BUN, and CRE levels were observed in the HFHS and HFHS + ergosterol groups. These results suggest that feeding with 1.0% ergosterol does not have detrimental effects on the liver and kidneys.²⁵⁾

Table 3. Plasma Levels of Specific Biomarkers in Rats Fed High-Fat and High-Sucrose (HFHS) Diet or HFHS Diet Supplemented with Ergosterol

Plasma biomarker (Units)	Associated disease in human	Control	HFHS	HFHS + ES
AST (IU/L)	Hepatic damage17,26,27)	81.75 ± 2.47	1081.13 ± 204.48**	998.50 ± 190.55**
ALT (IU/L)	Hepatic damage17,26,27)	52.38 ± 2.80	1205.38 ± 256.32**	1188.75 ± 278.25**
ALP (IU/L)	Hepatobiliary duct dysfunction,26) cholestasis27–30)	348.75 ± 16.39	922.13 ± 132.62**	664.63 ± 114.93
LDH (IU/L)	Hepatic damage (a non-specific tissue damage biomarker),26,27) myocardial infarction,31) etc.	123.88 ± 12.39	452.00 ± 65.10**	423.50 ± 90.24**
T-CHO (mg/dL)	Hypercholesterolemia3)	76.38 ± 6.15	191.75 ± 19.20**	156.13 ± 11.70**
F-CHO (mg/dL)	—	17.50 ± 1.98	52.63 ± 6.73**	43.00 ± 4.39**
TG (mg/dL)	Hypertriglyceridaemia3)	39.63 ± 4.66	34.63 ± 3.18	39.75 ± 3.53
LDL-C (mg/dL)	Hyper-LDL-cholesterolemia,3) coronary artery disease2)	7.50 ± 0.85	48.38 ± 5.94**	31.38 ± 2.80**, #
HDL-C (mg/dL)	Low-LDL-cholesterolemia3)	23.25 ± 0.80	32.25 ± 1.75**	31.88 ± 1.81**
T-CHO/HDL-C (ratio)	Cardiovascular mortality2,32,33)	3.25 ± 0.17	5.87 ± 0.38**	4.87 ± 0.12**, #
LDL-C/HDL-C (ratio)	Cardiovascular mortality32)	0.32 ± 0.03	1.49 ± 0.17**	0.98 ± 0.05**, ##
T-BIL (mg/dL)	Cholestasis27–30)	0.06 ± 0.01	0.50 ± 0.14**	0.22 ± 0.05
D-BIL (mg/dL)	Cholestasis27–30)	0.02 ± 0.00	0.39 ± 0.11**	0.15 ± 0.04
I-BIL (mg/dL)	Cholestasis27,29)	0.05 ± 0.01	0.11 ± 0.03**	0.07 ± 0.01
TBA (μmol/L)	Hepatic damage,17) cholestasis27,28,30)	12.50 ± 3.10	132.88 ± 32.07**	75.38 ± 20.36**, #
BUN (mg/dL)	Renal damage,17)	14.96 ± 0.73	9.50 ± 0.43**	8.51 ± 0.56**
CRE (mg/dL)	Renal damage17)	0.38 ± 0.02	0.37 ± 0.01	0.37 ± 0.01

Values are mean ± standard error (n = 8, each group); ** p < 0.01 compared with control; ^##p < 0.01, ^#p < 0.05 compared with HFHS, Tukey–Kramer test. Control, AIN-93M standard diet; HFHS, high-fat and high-sucrose; HFHS + ES, high-fat and high-sucrose feed supplemented with 1.0% ergosterol dry powder; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; LDH, lactate dehydrogenase; T-CHO, total cholesterol; F-CHO, free cholesterol; TG, triglyceride; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; T-BIL, total bilirubin; D-BIL, direct bilirubin; I-BIL, indirect bilirubin; TBA, total bile acid; BUN, blood urea nitrogen; CRE, creatinine.

Effect of Ergosterol-Supplemented Diet on the Plasma Levels of Ergosterol, Brassicasterol, Cholesterol Precursors, and Cholesterol in Rats

Our quantitation method using GC–MS revealed that the correlation coefficients of the calibration curves were greater than 0.999 for all compounds. Recovery rates ranged from 95.2 ± 3.7 to 100.9 ± 2.5% (n = 3). The intra-day precision (relative standard deviation, R.S.D.) ranged from 1.5 to 7.1%, whereas the inter-day precision (R.S.D.) ranged from 2.4 to 5.6% (n = 3, respectively) (Table 4). Additionally, the high resolution and sensitivity of GC–MS³⁴⁾ make it an attractive tool to separate commonly similar structures, such as cholesterol, its precursor, plant sterols, or oxysterols.^18,19,35) Our quantitation method was highly specific, sensitive, and reproducible.

Table 4. Validation for Ergosterol, Brassicasterol, Cholesterol Precursors, Cholesterol, Vitamin D₂, D₃, and Their Metabolites in Spiked Rat Plasma

Compounds	Correlation coefficient (R2)	Recovery (%)	Intra-day precision (RSD, %)	Inter-day precision (RSD, %)
Ergosterol	0.999	97.2 ± 6.5	2.3	4.4
Brassicasterol	0.999	100.9 ± 2.5	2.7	4.1
Squalene	0.999	99.0 ± 3.6	1.5	2.4
Lanosterol	0.999	100.6 ± 2.2	7.1	5.6
Lathosterol	0.999	98.5 ± 5.3	4.9	5.3
7-Dehydrocholesterol	0.999	99.3 ± 4.3	7.0	4.4
Desmosterol	0.999	95.2 ± 3.7	7.1	5.5
Cholesterol	0.999	100.3 ± 5.8	3.5	5.2
Vitamin D2	0.999	94.9 ± 4.0	6.6	5.2
25-Hydroxyvitamin D2	0.999	101.1 ± 4.5	3.1	5.7
1α, 25-Dihydroxyvitamin D2	0.998	101.2 ± 5.4	2.6	9.5
Vitamin D3	0.999	97.6 ± 3.2	6.2	5.8
25-Hydroxyvitamin D3	0.999	99.5 ± 4.3	6.9	6.6
1α, 25-Dihydroxyvitamin D3	0.998	93.9 ± 4.4	4.8	9.8

Recovery is represented as the mean ± standard deviation (n = 3). RSD, relative standard deviation.

Ergosterol levels in the control and HFHS groups were below the limit of quantification (LOQ) (0.01 μg/mL) in plasma. However, the ergosterol level in the HFHS + ergosterol group was found to be 0.981 ± 0.08 μg/mL (Fig. 3A). The brassicasterol level increased significantly to 13.44 ± 0.96 μg/mL in the HFHS + ergosterol group compared to the control and HFHS groups (p < 0.01, Tukey–Kramer test, n = 8) (Fig. 3B). Levels of cholesterol precursors (squalene, lanosterol, lathosterol, 7-dehydrocholesterol, and desmosterol) and cholesterol increased significantly in the HFHS group compared to the control group (p < 0.01 for squalene, desmosterol, and cholesterol, and p < 0.05 for lanosterol, lathosterol, and 7-dehydrocholesterol, Tukey–Kramer test, n = 8) (Figs. 3C–H). The squalene level decreased significantly to 65% in the HFHS + ergosterol group compared to the HFHS group (p < 0.05, Tukey–Kramer test, n = 8) (Fig. 3C). No significant difference was observed in lanosterol and lathosterol levels in the HFHS + ergosterol group compared to the HFHS group (Figs. 3D, E). The 7-dehydrocholesterol level increased significantly to 175% in the HFHS + ergosterol group compared to the HFHS group (p < 0.01, Tukey–Kramer test, n = 8) (Fig. 3F). The desmosterol level decreased significantly to 74% in the HFHS + ergosterol group compared to the HFHS group (p < 0.01, Tukey–Kramer test, n = 8) (Fig. 3G). The cholesterol level slightly decreased; however, it did not change significantly in the HFHS + ergosterol group compared with that in the HFHS group (Fig. 3H).

Fig. 3. Effect of Ergosterol-Supplemented Diet on Plasma of Ergosterol, Brassicasterol, Cholesterol Precursor, and Cholesterol Levels in Rats

Ergosterol (A), brassicasterol (B), squalene (C), lanosterol (D), lathosterol (E), 7-dehydrocholesterol (F), desmosterol (G), and cholesterol (H) in rats fed different diets. Values are mean ± standard error (n = 8, each group). * p < 0.05, ** p < 0.01, Tukey–Kramer test. Control, AIN-93M standard diet; HFHS, high-fat and high-sucrose; HFHS + ES, high-fat and high-sucrose feed supplemented with 1.0% ergosterol dry powder; LOQ, limit of quantification.

Effect of Ergosterol-Supplemented Diet on the Plasma Levels of Vitamins D₂, D₃, and Their Metabolites in Rats

Our quantitation method using LC-MS/MS revealed that the correlation coefficients of the calibration curves were greater than 0.998 for all compounds. Recovery rates ranged from 93.9 ± 4.4 to 101.2 ± 5.4% (n = 3). The intra-day precision (R.S.D.) ranged from 2.6 to 6.9%, whereas the inter-day precision (R.S.D.) ranged from 5.2 to 9.8% (n = 3, respectively) (Table 4).

No significant difference in the vitamin D₂ level was observed in the HFHS group compared with the control group; however, the vitamin D₂ level was significantly higher in the HFHS + ergosterol group than in the control and HFHS groups (p < 0.01, Tukey–Kramer test, n = 8) (Fig. 4A). In contrast, vitamin D₃ significantly decreased in the HFHS and HFHS + ergosterol groups compared to the control group (p < 0.01, Tukey–Kramer test, n = 8) (Fig. 4B). The 25-hydroxyvitamin D₃ level significantly decreased in the HFHS group (p < 0.05, Tukey–Kramer test, n = 8). Although vitamin D₃ and 25-hydroxyvitamin D₃ levels did not change significantly, they slightly increased in the HFHS + ergosterol group compared to the HFHS group (Figs. 4B, C). The LOQ of vitamin D_2, vitamin D₃, and 25-hydroxyvitamin D₃ was at 0.02, 1, and 0.5 ng/mL, respectively. The 25-hydroxyvitamin D₂, 1α, 25-dihydroxyvitamin D₂, and 1α, 25-dihydroxyvitamin D₃ concentrations were below the limit of detection (LOD) (0.3, 1, and 3 ng/mL, respectively) in the plasma of all groups. Our results showed that ergosterol increased vitamin D₂ and vitamin D₃ levels; however, 25-hydroxyvitamin D₂, 1α, 25-dihydroxyvitamin D₂, and 1α, 25-dihydroxyvitamin D₃ were under the LOD. This observation of vitamin D₃ may be the result of ergosterol not yet being metabolized into its active form under these conditions, as the plasma levels of 1α, 25-dihydroxyvitamin D₃ were reported as 0.24 ± 0.01 ng/mL, which remained unchanged in the mice fed a high-fat diet for 11 weeks.²⁰⁾ Several metabolites of vitamin D₂ have been reported in rats.³⁶⁾ In a previous study, following supplementation with vitamin D₂, 25-hydroxyvitamin D₂ concentrations gradually increased to plateaus by 6 week in cats.³⁷⁾ Furthermore, differences are observed in the bioavailability of vitamin D₂ by species, such as cats, pigs, or rats³⁷⁾; therefore, it is suggested that the differences in vitamin D₂ metabolism are a result of species variations.

Fig. 4. Effect of Ergosterol-Supplemented Diet on Plasma Vitamins D₂, D₃, and 25-Hydroxyvitamin D₃ Levels in Rats

Vitamin D₂ (A), vitamin D₃ (B), and 25-hydroxyvitamin D₃ (C) levels in rats fed different diets. values are mean ± standard error (n = 8, each group). * p < 0.05, ** p < 0.01, Tukey–Kramer test. Control, AIN-93M standard diet; HFHS, high-fat and high-sucrose; HFHS + ES, high-fat and high-sucrose feed supplemented with 1.0% ergosterol dry powder.

DISCUSSION

HFHS in male rats exhibited obesity characterized by significantly enhanced body weight gain, increased liver weight, and increased mesenteric, epididymal, perirenal, and visceral fat compared to the control groups, which was consistent with the results of a previous study.^22,38) HFHS in male rats have been reported to be the best and most common model for simulating metabolic syndrome, which is characterized by obesity and dyslipidemia in humans.^33,39–41) Furthermore, we used male rats to eliminate the activational effects of estradiol acutely and chronically which influence body weight homeostasis.⁴²⁾ Hence, in this study, we used the HFHS rat model to evaluate ergosterol-mediated inhibition of dyslipidemia. Additionally, HFHS diets induce hepatic damage (indicated by increasing AST, ALT, ALP, and LDH levels at 2.6- to 23.0-fold),^26,27) cholestasis (indicated by increasing ALP, T-BIL, D-BIL, I-BIL, and TBA levels at 2.5- to 23.7-fold),^27–30) hypercholesterolemia (indicated by increasing T-CHO, F-CHO, and LDL-C levels at 2.5- to 6.5-fold),¹⁷⁾ and an increased risk of cardiovascular disease (indicated by increasing T-CHO/HDL-C and LDL-C/HDL-C ratios at 1.8- and 4.7-fold, respectively)^2,32,33) in rats. However, no significant increase in plasma TG levels was observed in this study, which may be associated with TG deposition in the hepatocytes of rats fed the HFHS diet. Lipid metabolism gradually declined after liver damage, and liver TG was not discharged into the blood for decomposition; therefore, the plasma levels declined rather than increased, consistent with the findings of previous studies.^38,43) Furthermore, the plasma levels of cholesterol and its precursors were significantly increased in the HFHS group compared to the control group, suggesting that cholesterol biosynthesis was enhanced after long-term feeding with an HFHS diet. Plasma levels of vitamin D₃ and its metabolite (25-hydroxyvitamin D₃) were significantly lower in the HFHS group than in the control group.^20,44)

Obesity is associated with vitamin D deficiency.⁴⁴⁾ People with obesity have higher adipose stores of vitamin D₃, and the increased adipose mass in these individuals serves as a reservoir of vitamin D₃.^20,44) Moreover, the increased amount of vitamin D₃ required to saturate this reservoir may predispose obese individuals to inadequate plasma vitamin D₃ and 25-hydroxyvitamin D₃ levels.^20,44) Therefore, HFHS rats are suitable for evaluating cholesterol and vitamin D₃ biosynthetic pathways.

The plasma LDL-C, T-CHO/HDL-C, and LDL-C/HDL-C ratios in the HFHS + ergosterol group significantly decreased by 35, 17, and 34%, respectively, compared to those in the HFHS group. The T-CHO/HDL-C ratio is a strong predictor for cardiovascular disease risk and its reduction (34, 16, and 18%, respectively, in the case of statins, simvastatin³²⁾) is associated with a reduction in cardiovascular mortality³²⁾; therefore, our data suggest that ergosterol may contribute a reduction in cardiovascular mortality. A recent study reported that the T-CHO/HDL-C ratio decreased with increasing intake of the Mediterranean diet, which is promoted as the preferred dietary model for cardiovascular disease prevention.⁴⁵⁾ Furthermore, plasma ALP, T-BIL, D-BIL, and TBA levels were lower by 30, 56, 62, and 43%, respectively, in the HFHS + ergosterol group than those in the HFHS group, suggesting that ergosterol might improve severe cholestasis.^28–30) This is because studies have a reduction in ALP, T-BIL, D-BIL, and TBA levels (31, 51, 34, and 39%, respectively), which play a therapeutic role in resisting cholestasis.^26,27,42) Ergosterol, brassicasterol, and vitamin D₂ were detected in the HFHS + ES group, indicating that the HFHS diet with long-term ergosterol intake facilitated the absorption ergosterol, its conversion to brassicasterol by DHCR7,^9–12) and ultimately its conversion to vitamin D₂ by UV light.⁸⁾ Notably, ergosterol intake significantly decreased plasma squalene and desmosterol levels and significantly increased plasma 7-dehydrocholesterol levels compared with the HFHS group. We previously reported that ergosterol increases 7-dehydrocholesterol levels in HepG2 hepatoma cells,¹²⁾ and that it may compete with 7-dehydrocholesterol.¹²⁾ Furthermore, ergosterol slightly decreased cholesterol levels in rats and has the same competitive effect of DHCR7. These effects of ergosterol on 7-dehydrocholesterol led to an increase in vitamin D₃ and 25-hydroxyvitamin D₃ levels. These results indicate that ergosterol has an inhibitory effect on cholesterol biosynthesis and an additional possibility of improving osteoporosis resulting from vitamin D (vitamin D₂, vitamin D₃, or both)^15,46) deficiency by increasing vitamin D₂, D₃, or its metabolite.

In conclusion, we have demonstrated for the first time that long-term ergosterol intake can reduce cardiovascular mortality by reducing plasma LDL-C, T-CHO/HDL-C, and LDL-C/HDL-C ratios and improve serious cholestasis by reducing plasma ALP, T-BIL, D-BIL, and TBA in HFHS-fed rats. Additionally, ergosterol increases plasma 7-dehydrocholesterol levels and decreases plasma desmosterol levels, suggesting that ergosterol alleviates dyslipidemia and inhibits the cholesterol biosynthetic pathway. Furthermore, ergosterol also increases vitamin D₂ levels and perhaps promotes vitamin D₃ biosynthesis in vivo. It is suggested that ergosterol is possibly useful for the prevention of dyslipidemia and osteoporosis from vitamin D deficiency.

Acknowledgments

We would like to thank Fumiko Fuwa for providing experimental assistance.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Agarwala A, Petersen KS, Jafari F, Kris-Etherton PM. Dietary management of dyslipidemia and the impact of dietary patterns on lipid disorders. Prog. Cardiovasc. Dis., 75, 49–58 (2022).
• 2) Blaton V. How is the metabolic syndrome related to the dyslipidemia? EJIFCC, 18, 15–22 (2007).
• 3) Japan Atherosclerosis Society. The dyslipidemia treatment guide for arteriosclerosis prevention 2023. Nishimura-syoten, Tokyo (2023).
• 4) Chrysant SG, Ibrahim M. Niacin-ER/statin combination for the treatment of dyslipidemia: focus on low high-density lipoprotein cholesterol. J. Clin. Hypertens (Greenwich), 8, 493–501 (2006).
• 5) Brown AS, Watson KE. Statin Intolerance. Rev. Cardiovasc. Med., 19 (Suppl. 1), S9–S19 (2018).
• 6) Agabiti Rosei E, Salvetti M. Management of hypercholesterolemia, appropriateness of therapeutic approaches and new drugs in patients with high cardiovascular risk. High Blood Press. Cardiovasc. Prev., 23, 217–230 (2016).
• 7) Hammann S, Lehnert K, Vetter W. Esterified sterols and their contribution to the total sterols in edible mushrooms. J. Food Compost. Anal., 54, 48–54 (2016).
• 8) Koyyalamudi SR, Jeong SC, Song CH, Cho KY, Pang G. Vitamin D2 formation and bioavailability from Agaricus bisporus button mushrooms treated with ultraviolet irradiation. J. Agric. Food Chem., 57, 3351–3355 (2009).
• 9) Honda M, Tint GS, Honda A, Batta AK, Chen TS, Shefer S, Salen G. Measurement of 3β-hydroxysteroid Δ⁷-reductase activity in cultured skin fibroblasts utilizing ergosterol as a substrate: a new method for the diagnosis of the Smith–Lemli–Opitz syndrome. J. Lipid Res., 37, 2433–2438 (1996).
• 10) Tsugawa N, Okano T, Takeuchi A, Kayama M, Kobayashi T. Metabolism of orally administered ergosterol and 7-dehydrocholesterol in rats and lack of evidence for their vitamin D biological activity. J. Nutr. Sci. Vitaminol., 38, 15–25 (1992).
• 11) Tuckey RC, Tang EKY, Chen YA, Slominski AT. Selective ability of rat 7-dehydrocholesterol reductase (DHCR7) to act on some 7-dehydrocholesterol metabolites but not on lumisterol metabolites. J. Steroid Biochem. Mol. Biol., 212, 105929 (2021).
• 12) Kuwabara N, Ohta-Shimizu M, Fuwa F, Tomitsuka E, Sato S, Nakagawa S. Ergosterol increases 7-dehydrocholesterol, a cholesterol precursor, and decreases cholesterol in human HepG2 cells. Lipids, 57, 303–311 (2022).
• 13) Lecain E, Chenivesse X, Spagnoli R, Pompon D. Cloning by metabolic interference in yeast and enzymatic characterization of Arabidopsis thaliana sterol Δ7-reductase. J. Biol. Chem., 271, 10866–10873 (1996).
• 14) Lamberson CR, Muchalski H, McDuffee KB, Tallman KA, Xu L, Porter NA. Propagation rate constants for the peroxidation of sterols on the biosynthetic pathway to cholesterol. Chem. Phys. Lipids, 207 (Pt. B), 51–58 (2017).
• 15) Holick MF, Chen TC. Vitamin D deficiency: a worldwide problem with health consequences. Am. J. Clin. Nutr., 87, 1080S–1086S (2008).
• 16) Baur AC, Kühn J, Brandsch C, Hirche F, Stangl GI. Intake of ergosterol increases the vitamin D concentrations in serum and liver of mice. J. Steroid Biochem. Mol. Biol., 194, 105435 (2019).
• 17) He WS, Cui D, Li L, Tong LT, Rui J, Li H, Zhang H, Liu X. Cholesterol-reducing effect of ergosterol is modulated via inhibition of cholesterol absorption and promotion of cholesterol excretion. J. Funct. Foods, 57, 488–496 (2019).
• 18) Hirayama S, Nakagawa S, Soda S, Kamimura Y, Nishioka E, Ueno T, Fukushima Y, Higuchi K, Inoue M, Seino U, Ohmura H, Yamato S, Miida T. Ezetimibe decreases serum oxidized cholesterol without impairing bile acid synthesis in Japanese hypercholesterolemic patients. Atherosclerosis, 230, 48–51 (2013).
• 19) Ohta-Shimizu M, Fuwa F, Tomitsuka E, Nishiwaki T, Aihara K, Sato S, Nakagawa S. New inhibitory effect of Latilactobacillus sakei UONUMA on the cholesterol biosynthesis pathway in human HepG2 cells. Biol. Pharm. Bull., 44, 485–493 (2021).
• 20) Bonnet L, Margier M, Svilar L, Couturier C, Reboul E, Martin J-C, Landrier J-F, Defoort C. Simple fast quantification of cholecalciferol, 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D in adipose tissue using LC-HRMS/MS. Nutrients, 11, 1977 (2019).
• 21) Kanda Y. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplant., 48, 452–458 (2013).
• 22) Jahn D, Dorbath D, Kircher S, Nier A, Bergheim I, Lenaerts K, Hermanns HM, Geier A. Beneficial effects of vitamin D treatment in an obese mouse model of non-alcoholic steatohepatitis. Nutrients, 11, 77 (2019).
• 23) Mpofana N, Chipangura JK, Paulse M, Yalo M, Gqaleni N, Nxumalo CT, Dlova NC, Hussein AA, Crouch NR. An investigation into the acute and subacute toxicity of extracts of Cassipourea flanaganii Stem Bark In Vivo. Plants (Basel), 12, 2281 (2023).
• 24) Srivastava S, Virmani T, Haque MR, Alhalmi A, Al Kamaly O, Alshawwa SZ, Nasr FA. Extraction, HPTLC analysis and antiobesity activity of Jatropha tanjorensis and Fraxinus micrantha on high-fat diet model in rats. Life (Basel), 13, 1248 (2023).
• 25) Alam N, Amin R, Khan A, Ara I, Shim MJ, Lee MW, Lee UY, Lee TS. Comparative effects of oyster mushrooms on lipid profile, liver and kidney function in hypercholesterolemic rats. Mycobiology, 37, 37–42 (2009).
• 26) Alshehri FS, Alorfi NM. Protective role of resveratrol against VCM-induced hepatotoxicity in male Wistar rats. Front. Pharmacol., 14, 1130670 (2023).
• 27) Lala V, Zubair M, Minter DA. Liver function tests. StatPearls. StatPearls Publishing, Treasure Island, FL: ‹https://www.ncbi.nlm.nih.gov/books/NBK482489/›, accessed 1 September, 2023.
• 28) Li Y, Tian C, Song X, Du M, Zhang G, Liu H. New IMB16-4 hot-melt extrusion preparation improved oral bioavailability and enhanced anti-cholestatic effect on rats. Drug Des. Devel. Ther., 17, 649–657 (2023).
• 29) Fahmy SR, Sayed DA, Soliman AM, Almortada NY, Aal WEA. Protective effect of echinochrome against intrahepatic cholestasis induced by alpha-naphthylisothiocyanate in rats. Braz. J. Biol., 80, 102–111 (2020).
• 30) Liu J, Hou LL, Zhao CY. Effect of YHHJ on the expression of the hepatocellular bile acid transporters multidrug resistance-associated protein 2 and bile salt export pump in ethinylestradiol-induced cholestasis. Exp. Ther. Med., 15, 3699–3704 (2018).
• 31) Farhana A, Lappin SL. Biochemistry, Lactate Dehydrogenase. StatPearls. StatPearls Publishing, Treasure Island, FL: ‹https://www.ncbi.nlm.nih.gov/books/NBK557536/›, accessed 1 September, 2023.
• 32) Gil-Ramírez A, Caz V, Martin-Hernandez R, Marín FR, Largo C, Rodríguez-Casado A, Tabernero M, Ruiz-Rodríguez A, Reglero G, Soler-Rivas C. Modulation of cholesterol-related gene expression by ergosterol and ergosterol-enriched extracts obtained from Agaricus bisporus. Eur. J. Nutr., 55, 1041–1057 (2016).
• 33) Gancheva S, Zhelyazkova-Savova M, Galunska B, Chervenkov T. Experimental models of metabolic syndrome in rats. Scripta Scientifica Medica, 47, 14–21 (2015).
• 34) Lee MS, Jung BH, Chung BC, Cho SH, Kim KY, Kwon OS, Nugraha B, Lee YJ. Metabolomics study with gas chromatography-mass spectrometry for predicting valproic acid-induced hepatotoxicity and discovery of novel biomarkers in rat urine. Int. J. Toxicol., 28, 392–404 (2009).
• 35) Kuwabara N, Fuwa F, Sato S, Hirayama S, Miida T, Nakagawa S. Simultaneous determination of cholesterol precursors, plant sterols, and oxysterols in plasma using one-round pretreatment. Medical Mass Spectrometry, 7, 89–97 (2023).
• 36) Reddy GS, Tserng KY. Isolation and identification of 1,24,25-trihydroxyvitamin D₂, 1,24,25,28-tetrahydroxyvitamin D₂, and 1,24,25,26-tetrahydroxyvitamin D₂: new metabolites of 1,25-dihydroxyvitamin D₂ produced in rat kidney. Biochemistry, 25, 5328–5336 (1986).
• 37) Ruggiero CE, Backus RC. Effects of vitamin D₂ and 25-hydroxyvitamin D₂ supplementation on plasma vitamin D epimeric metabolites in adult cats. Front. Vet. Sci., 8, 654629 (2021).
• 38) Kiyose C, Takeuchi H, Yabe Y, Koike T, Sakiya K, Nagase M, Tanaka-Yachi R, Takahashi-Muto C. Improvement effect of sweet basil (Ocimum basilicum L.) powder intake on obese mice fed a high-fat and high-sucrose diet. J. Oleo Sci., 70, 1317–1323 (2021).
• 39) Wong SK, Chin KY, Suhaimi FH, Fairus A, Ima-Nirwana S. Animal models of metabolic syndrome: a review. Nutr. Metab. (Lond), 13, 65 (2016).
• 40) Suliman HM, Osman B, Abdoon IH, Saad AM, Khalid H. Ameliorative activity of adansonia digitata fruit on high sugar/high fat diet-simulated Metabolic Syndrome model in male Wistar rats. Biomed. Pharmacother., 125, 109968 (2020).
• 41) Zhou X, Han D, Xu R, Li S, Wu H, Qu C, Wang F, Wang X, Zhao Y. A model of metabolic syndrome and related diseases with intestinal endotoxemia in rats fed a high fat and high sucrose diet. PLOS ONE, 9, e115148 (2014).
• 42) Brown LM, Clegg DJ. Central effects of estradiol in the regulation of food intake, body weight, and adiposity. J. Steroid Biochem. Mol. Biol., 122, 65–73 (2010).
• 43) Wang L, Jia Z, Wang B, Zhang B. Berberine inhibits liver damage in rats with non-alcoholic fatty liver disease by regulating TLR4/MyD88/NF-κB pathway. Turk. J. Gastroenterol., 31, 902–909 (2020).
• 44) Carrelli A, Bucovsky M, Horst R, Cremers S, Zhang C, Bessler M, Schrope B, Evanko J, Blanco J, Silverberg SJ, Stein EM. Vitamin D. Storage in adipose tissue of obese and normal weight women. J. Bone Miner. Res., 32, 237–242 (2017).
• 45) Carter SJ, Roberts MB, Salter J, Eaton CB. Relationship between mediterranean diet score and atherothrombotic risk: findings from the third national health and nutrition examination survey (NHANES III), 1988–1994. Atherosclerosis, 210, 630–636 (2010).
• 46) Bandela V, Munagapati B, Karnati RK, Venkata GRS, Nidudhur SR. Osteoporosis: its prosthodontic considerations—a review. J. Clin. Diagn. Res., 9, ZE01–ZE04 (2015).