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Article

Anti-Obesity and Anti-Diabetic Activities of Fermented Schizandrae Fructus Pomace Extract in Mice Fed with High-Fat Diet

by
Md. Mohibbullah
1,
So-Jung Park
2,
Jae-Suk Choi
3,* and
Sae-Kwang Ku
4,*
1
Department of Fishing and Post Harvest Technology, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka 1207, Bangladesh
2
Suheung Research & Development, Daewangpangyoro 700, Korea Bio Park A, 8th fl. Bundangu, Gyeonggido, Seongnam 13488, Republic of Korea
3
Department of Seafood Science and Technology, The Institute of Marine Industry, Gyeongsang National University, 2-9, Tongyeonghaean-ro, Tonyeong-si 53064, Republic of Korea
4
Department of Anatomy and Histology, College of Korean Medicine, Daegu Haany University, Gyeongsan 38610, Republic of Korea
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2023, 13(24), 13307; https://doi.org/10.3390/app132413307
Submission received: 11 November 2023 / Revised: 11 December 2023 / Accepted: 14 December 2023 / Published: 16 December 2023
(This article belongs to the Section Food Science and Technology)
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Versions Notes

Abstract

:
Obesity is implicated as a factor in several serious metabolic conditions, including hypertension, cardiovascular disease, and type II diabetes. This study aimed at the development of more potent and safer alternative medications to address these metabolic diseases with minimal side effects. Following oral administration of 400, 200, and 100 of mg/kg fermented Schizandrae fructus pomace extracts (fSFP) once daily for 84 days to high-fat diet (HFD)-supplied ICR mice, respectively, we measured liver enzyme activity related to glucose metabolism, gene expression related to fat metabolism, anti-obesity effect, liver and kidney protection effect, and anti-diabetic effect to confirm the effect of fSFP on improving related complications including obesity and diabetes. In the HFD control group, significant obesity and type II diabetes symptoms were developed. However, oral administration of 200 mg/kg of fSFP showed a protective effect on kidney damage and diabetes complications related to insulin-resistant type II diabetes mellitus, as well as oxidative stress-induced abnormalities in glucose and fat metabolism, comparable to that of metformin 250 mg/kg, a positive control. The major bioactive substance in fSFP was identified as shizandrin, which was quantitated as 1.25 mg/g (w/w). Therefore, fSFP extracts can be taken as a medicinal food in combating obesity and diabetes, two current major health concerns.

1. Introduction

Obesity serves as a root cause for various metabolic disorders, including hypertension, heart disease, and type II diabetes [1]. In the presence of obesity, adipocytes release diverse adipokines influencing both adipocyte and non-adipocyte metabolism, thereby triggering chronic inflammation and associated metabolic ailments such as insulin resistance [2]. Typically, adipocytes function to store lipids for long-term energy use in individuals with obesity. Consumption of a high-fat diet induces triglyceride (TG) accumulation in multiple tissues, heightens lipolysis, elevates circulating fatty acids, and induces insulin resistance in adipocytes. Consequently, excess fat is deposited in non-adipose tissues like the liver and muscles. The augmented binding and transport of fatty acids increase their uptake in non-adipocytes, particularly muscle cells, negatively impacting insulin-mediated glucose metabolism in cases of adipocyte insulin resistance. This lipotoxic process, due to prolonged exposure to fatty acids, diminishes insulin secretion in the pancreas [3]. TG formation in hepatocytes can lead to non-alcoholic fatty liver disease (NAFLD), characterized by hepatocyte fat accumulation, steatohepatitis, and eventual hepatocyte necrosis and fibrosis, particularly affecting carbohydrate metabolism [4]. Research involving a high-fat diet (HFD) suggests that obese and hyperglycemic animal models can assist in evaluating the efficacy of functional foods to prevent diseases like NAFLD and metabolic syndrome [5]. Hence, the present study adopted a mouse model fed a 45% kcal high-fat diet to investigate the pharmacological action of fSFP in addressing metabolic syndrome, encompassing obesity.
Peroxisome proliferator-activated receptor (PPAR) and 5′ adenosine monophosphate-activated protein kinase (AMPK) are two representative cell-regulating factors that are crucial for biological fat synthesis [6,7,8]. The onset of inflammation and obesity-induced NAFLD is implied by a decline in AMPK activity [9,10]. According to research by Veiga et al. [8] and Xu et al. [11], PPAR is known to play a role in both lipid oxidation and lipid synthesis. PPARγ and PPARα are both known to have a role in lipid oxidation and lipid synthesis, respectively. Efforts are underway to formulate glucosidase inhibitors and potent antioxidants with minimal adverse effects. These developments are crucial for effectively managing oxidative stress, recently identified as a key factor in the pathogenesis of diabetes and its complications. Concurrently, maintaining optimal glycemic control is increasingly recognized as a pivotal strategy in diabetes treatment [5,12,13]. As an example of an oral biguanide-based anti-diabetic medication, metformin is well-known for activating AMPK [14]. Metformin was used as the positive control group in this investigation.
The fruit of Schisandra chinensis (Turcz.) Baill., Schizandrae fructus, has five distinct flavors: sweet, sour, astringent, spicy, and salty. It also contains a significant amount of phenolic compounds, which have been linked to a number of pharmacological effects, including anti-inflammatory and antioxidant effects [15,16]. Asian traditional medicine has employed Schisandra chinensis [17]. It is now known from a recent study that Schisandra chinensis, either alone or in combination with grape seed extract, has excellent anti-obesity and anti-diabetic benefits. It also boosts energy metabolism by increasing PGC-1 expression, which leads to an improvement in muscle strength [18]. In addition, it has been reported that Schisandra chinensis pomace displays pharmacological action that is comparable to that of Schisandra chinensis [19,20]. Consequently, efforts are being made to take steps to develop alternative medicines made from natural ingredients that have few adverse effects and are more potent. In this study, mice fed a 45% Kcal HFD were used to assess the effects of fermented Schisandra chinensis pomace (fSFP) on conditions including adiposity, diabetes mellitus, and related pathophysiological sequelae.

2. Materials and Methods

2.1. Preparation of Test Materials

In order to prepare the test materials, fresh Schizandrae fructus was gathered in Geochang, Jeollabuk-do, Korea, and washed twice with tap water. In order to achieve natural fermentation, white sugar (CJ CheilJedang, Seoul, Republic of Korea) and cleaned Schizandrae fructus were combined in a stainless steel fermentation tank at a ratio of 1:0.7 (kg/kg) and allowed to proceed naturally for 5 months at a temperature of 15 to 25 °C. The fermentation broth was recovered by straining the fermented Schizandrae fructus through a sieve and twice washing it with tap water (10 times volume). Additionally, an 8-h primary extraction process utilizing 8-fold purified water as a solvent was performed on the remaining fruits at temperatures between 95 and 100 °C and pressures between 0.9 and 1.0 kg/m2. After recovering the first extract, the fermented Schizandrae fructus was additionally mixed with 5-fold purified water, and the secondary extraction procedure was carried out for 4 h at 95–100 °C and a pressure of 0.9–1.0 kg/m2. A 1mm membrane filter was used to filter the combined first and second extracts, which were then concentrated to at least 25 brix at 60–65 °C. Maltodextrin (Daesang Co., Seoul, Republic of Korea) was added to this concentrate in a proportion of 60% to the solid content, and light brown powders were made using a spray dryer with an inlet temperature of 170 °C and an exit temperature of 80 °C. This experiment utilized the use of this powdered, fermented Schizandrae fructus pomace extract. Unless otherwise stated, all chemical reagents are purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. HPLC Analysis

An ultrasonic generator (UCS-2, Jeiotech, Daejeon, Republic of Korea) was used to ultrasonically extract 2 g of fermented or fresh Schizandrae fructus after it had been ground for 5 min. A filter (PTFE-Hydrophilic, 0.45 m, Hyundai Micro, Seoul, Republic of Korea) was then used as a sample. Shizandrin standard solution was prepared by properly weighing approximately 2 mg of shizandrin standard and adding methanol to precisely 20 mL. To measure the peak areas of the sample solution and the standard solution, 10 L of each sample solution and 10 L of the standard solution were evaluated using the liquid chromatography. A C18 column (CAPCELL PAK C18 (MGII) for liquid chromatography with an inner diameter of 4.6 mm, a length of 250 mm, and a length of 5 m was utilized. The HPLC apparatus used was an Agilent 1200-DAD device. The mobile phase contained A: 0.05% TFA and B: ACN; the flow rate was 0.7 mL/min; at 0 min, A: 550; at 40 min, A: 55; and at 41 to 55 min, A: 5. With the use of an Agilent 1200 Infinity diode array detector, absorbance was determined at 254 nm.

2.3. Experimental Animal

A total of 80 female ICR mice, aged 6 weeks, were procured from OrientBio in Seungnam, Republic of Korea, and subjected to a 10-day acclimatization period. Following this period, only the experimental animals demonstrating consistent weight gain were selected and distributed into six groups after an additional week of acclimation to the high-fat diet (HFD). The Laboratory Animal Ethics Committee of Daegu Haany University granted prior approval for all experimental procedures, and all animal handling adhered to ethical guidelines for animal testing [Approval No. DHU2021-063, dated 10 August 2021].

2.4. Experimental Group

The following six groups, each with ten mice, were allocated based on their treatments by oral administration as follows:
NFD control = Vehicle (distilled water) 10 mL/kg, accompanied by a supply of NFD
HFD control = Vehicle (distilled water) 10 mL/kg, accompanied by a supply of HFD
Metformin = 250 mg/kg of metformin, accompanied by a supply of HFD
fSFP400 = fSFP 400 mg/kg, accompanied by a supply of HFD
fSFP200 = fSFP 200 mg/kg, accompanied by a supply of HFD
fSFP100 = fSFP 100 mg/kg, accompanied by a supply of HFD

2.5. Measurement of Body and Organ Weights, Daily Food Consumption and Fat Density

To minimize feeding-related variations, all experimental animals underwent a 12-h fast, with restricted access to water, at both the initiation and conclusion of the experiment. Body Weight (BW) increments were quantified throughout the acclimatization phases (BW at the start of administration—BW at the beginning of high-fat diet [HFD] supply) and administration periods (BW at termination—BW at the start of administration). Measurements of liver, pancreas, left kidney, left periovarian fat pads, and abdominal wall deposited fat pads were taken at the time of sacrifice. The BW at sacrifice and absolute weights were used to calculate the relative weights (%) of body parts. The average daily food intake per mouse (expressed in grams/day/mouse) was determined by subtracting the amount of diet left over 24 h after supply from the amount initially provided per head of mice. Using in-live DEXA (InAlyzer, Medikors, Seungnam, Republic of Korea), the average adipose tissue densities within both the whole-body and intra-abdominal regions were determined for each murine subject.

2.6. Blood Biochemical Analysis

Following the 84-day intervention period, mice were anesthetized using 2–3% isoflurane, after which blood samples were collected from the vena cava. Blood glucose levels were assessed utilizing an automatic hematology machine (NX500i, Fuji Medical Systems Co., Ltd., Tokyo, Japan). A portion of the blood was placed in a coagulation-activated serum tube. Subsequently, the blood was separated, and AST, ALT, ALP, LDH, GGT, BUN, creatinine, total cholesterol, TG, LDL, and HDL levels were measured using the automated hematology machine. Serum HbA1c and serum insulin levels were determined using an automated HbA1c assay (Easya1c, Infoopia, Anyang, Republic of Korea) and a mouse insulin ELISA kit (Alpco Diagnostics, Windham, NH, USA), respectively. To analyze the lipid profile in feces, lipids were extracted from the fecal samples collected eight hours subsequent to the final administration. Fecal triglyceride and total cholesterol were quantified through colorimetric analysis using a commercial kit (Ann Arbor, Cayman Islands, MI, USA), following established procedures [5,12,13,21,22].

2.7. Hepatic Lipid Peroxidation and Antioxidant Defense Mechanisms

After measuring the BW of rat liver tissue, MDA and GSH concentrations and CAT and SOD enzyme activities, were evaluated. The amount of hepatic lipid peroxidation was determined by calculating MDA using thiobarbituric acid and a UV/visible spectrophotometer (Optizen POP, Mecasys, Daejeon, Republic of Korea) set to 525 nm absorption [23]. GSH levels were determined spectrophotometrically using 2-nitrobenzoic acid at 412 nm absorbance [24]. The decomposition of H2O2 was monitored using a spectrophotometer in the presence of CAT at 240 nm [25]. Measurement of SOD activity was performed following the method of Sun et al. [26]. SOD activity at 560 nm was detected using spectrophotometry.

2.8. Analysis of Hepatic Glucose-Regulating Enzymes

Hepatic enzyme regions were prepared using the methodology outlined by Hulcher and Oleson [27]. A 0.3 g sample of liver tissue was homogenized in buffer solution and then centrifuged at 1000× g for 15 min at 4 °C. The assay for glucokinase activity was conducted in accordance with the methodology outlined by Davidson and Arion [28], with minor modifications. The reaction commenced with the addition of 10 μL of 5 mM ATP, and the mixture underwent incubation at 37 °C for 10 min, with absorbance recorded at 340 nm. G6pase activity was measured according to the method outlined by Alegre et al. [29], involving pre-incubation of the reaction mixture at 37 °C for 3 min. Subsequently, 5 μL of liver tissue homogenate was introduced, and the mixture underwent incubation at 37 °C for 4 min, with absorbance measured at 340 nm. The enzymatic activity of Phosphoenolpyruvate Carboxykinase (PEPCK) was quantified utilizing the methodology developed by Bentle and Lardy [30]. In this case, 10 μL of liver tissue homogenate was added to the reaction mixture, and the reduction in absorbance at 340 nm was observed with a spectrophotometer for enzyme activity calculation.

2.9. Real-Time RT-PCR Analysis

The assessment of periovarian adipose tissue PPARα, PPARγ, leptin, UCP2, adiponectin, C/EBPα, C/EBPβ, FAS, and SREBP1c mRNA expression followed established protocols as reported previously [8]. RT-PCR was employed to determine the mRNA expression of ACC1, AMPKα1, and AMPKα2 in tissue preparations. In brief, RNA extraction was carried out and the reverse transcription of RNA was performed, following the instructions provided by manufacturer. The quantification of molecular expression was normalized to the vector control. A comprehensive list of PCR oligonucleotide primer sequences is provided in Supplementary Table S1.

2.10. Histopathology

Analysis of body mass involved the examination of specific anatomical structures, including a portion of the left kidney, the left periovarian fat pad, the left lobe of the liver, the left splenic lobe of the pancreas, and a segment of the fat pad adhering to the abdominal wall connected to the quadratus. The lumborum muscle underwent preservation in 10% neutral buffered formalin. Following this, automated tissue processing using Shandon Citadel 2000 (Thermo Scientific, Waltham, MA, USA) was employed for paraffin embedding with 3–4 μm serial sections. Representative areas underwent hematoxylin and eosin staining [2] for light microscopy, utilizing a computer-aided image analyzer. An alternative method involved drying the tissue in a 30% sucrose solution, staining with Oil Red, and subsequent cryosectioning [5,12,13].

2.11. Immunohistochemistry

Immunostaining of pancreatic tissue was conducted using avidin-biotin-peroxidase (ABC) technology [5,12,13]. Rabbit polyclonal glucagon (Abcam, UK, dilution: 1:100) or guinea pig polyclonal insulin antiserum (5:1 dilution) served as primary antisera, fixed at 4 °C overnight. Subsequently, a one-hour incubation with a biotinylated universal secondary antibody (Vector Lab., Burlingame, CA, USA) and ABC reagent (Vectastain Elite ABC Kit, Vector) at a 1:50 dilution ensued. The reaction was developed for 3 min using a peroxidase substrate kit (Vector Lab., Burlingame, CA, USA, dilution 1:50). Each section underwent rinsing three times in 0.01 M PBS between each procedural step. It is noteworthy that the histopathologist was completely blinded to the population distribution during the analysis.

2.12. Statistical Analyses

All data are expressed as the mean ± standard deviation (SD), based on a sample cohort of 10 mice. Multiple comparative experiments were conducted across different dosage groups. Levene’s test was employed to assess the homogeneity of variables. Subsequent data analysis utilized the one-way analysis of variance (ANOVA) test, and the Tukey’s honestly significant difference (THSD) test was applied to determine the significance of group comparisons, if the Levene’s test did not show substantial differences in variable homogeneity. In cases where Levene’s test revealed significant differences between two groups, Dunnett’s T3 (DT3) test was used for pairwise comparisons. The statistical analysis was carried out using SPSS version 18.0 for Windows from IBM-SPSS Inc., Armonk, NY, USA.

3. Results

3.1. Confirmation of Shizandrin Content in fSFP

The shizandrin concentration in the powdered fermented Schisandra fructus pomace extract was determined to be 1.25 mg/g. On the other hand, fresh Schisandra fructus was found to have 0.18 mg/g of shizandrin. Shizandrin, one of the primary bioactive components, was shown to significantly increase while Schisandra fructus through the fermentation process (Figure 1).

3.2. Effects of fSFP on Obesity

3.2.1. Weight Change

Following a high-fat diet for seven days, HFD control group exhibited a significant (p < 0.01) increase in weight in comparison to NFD control group. Weight gain (p < 0.01 or p < 0.05) in fSFP groups was evident at 14 days, 35 days and 35 days after the initiation of fSFP administration, as well as 28 days after the commencement of metformin administration. Throughout the 84-day administration period, all treatment groups, including the metformin-treated group, demonstrated a notable reduction in BW (p < 0.01) compared to the HFD control group (Table S2, Figure 2).

3.2.2. Changes in the Average Intake of Feed

There was a notable decrease in the mean feed consumption in the HFD control group when contrasted with the NFD control group (p < 0.01). Nonetheless, the average feed intake did not show significant variations between the HFD control group and any groups receiving the fSFP treatments (Table S2).

3.2.3. Changes in Fat Accumulation

Comparing the HFD control group to the NFD control, a significant rise (p < 0.01) in body fat and an increase in abdominal fat accumulation were also observed. In contrast, as compared to the HFD control group, all three doses of fSFP orally delivered group, including metformin-treated group, demonstrated a significant decrease (p < 0.01) in body fat and abdominal fat formation, respectively (Figure 3).

3.2.4. Changes in Fat Weight

In comparison to the NFD control group, a significant rise (p < 0.01) in the relative and absolute weight of deposited fat in the periovarian and abdominal wall was observed in the HFD control group. However, significant decreases (p < 0.01 or p < 0.05) were seen in comparison to the HFD control group in the groups provided with all three dosages of fSFP, including the metformin treatment group (Tables S3 and S4; Figure 3).

3.2.5. Changes in the Histology of Fat Accumulation in the Abdominal Wall and Periovarian Region

The accumulation of adipose tissue around the ovaries and abdominal wall, as well as an enlargement in adipocyte size, were significantly greater (p < 0.01) in the HFD control group compared to the NFD control group. Conversely, a significant reduction (p < 0.01) in both periovarian and abdominal adipose tissue thickness and adipocyte diameter was observed in each of the groups treated with varying dosages of fSFP, as well as in the metformin treatment group, relative to the HFD control group (Table S5; Figure S1).

3.2.6. Changes in the Histology of Zymogen Granules in the Exocrine Pancreas

The fraction of zymogen granules in pancreatic exocrine tissue was confirmed to be significantly lower (p < 0.01) in the HFD control group than the NFD control group. However, a significant increase (p < 0.01 or p < 0.05) in the proportion of zymogen granules was demonstrated in all three dosages of fSFP-administered groups, including the metformin treatment group, in compared to the HFD control group (Table 1; Figure S2).

3.3. Effects of fSFP on Anti-Diabetic Activity

3.3.1. Changes in Pancreatic Weight

In the HFD control group, there was a significant reduction (p < 0.01) in the relative weight of the pancreas when compared to the NFD control group. In contrast, all three dosages of the fSFP-administered groups, including the metformin treatment group, exhibited a significant increase (p < 0.01) in pancreatic relative weight compared to the HFD control group. Notably, none of the groups demonstrated significant changes in the absolute weight of the pancreas (Tables S3 and S4).

3.3.2. Changes in Blood Sugar, Insulin, and HbA1c

There were significant elevations (p < 0.01) in the levels of blood glucose, insulin, and Hemoglobin A1c (HbA1c) observed in the HFD control group in comparison to the NFD control group. However, in comparison to the HFD control group, significant (p < 0.01 or p < 0.05) decreases in blood glucose, insulin, and HbA1c were demonstrated in each of the three dosages of fSFP-administered groups, including the metformin treatment group (Table S6, Figure 4).

3.3.3. Changes in the Histology of Pancreatic Islets

Relative to the NFD control group, the HFD control group exhibited a significant increase (p < 0.01) in both the quantity and mean diameter of pancreatic islets, along with a notable proliferation of these islets. Conversely, in each of the three dosage groups receiving fSFP, as well as in the metformin treatment group, when compared to the HFD control group, there were significant reductions (p < 0.01) in both the quantity and diameter of pancreatic islets, observed in a dose-dependent manner (Table 1; Figure S3).

3.3.4. Immunohistochemical Changes in Pancreatic Islets

Relative to the NFD control group, the HFD control group exhibited a significant increase (p < 0.01) in the count of insulin and glucagon-immunoreactive cells, as well as in the insulin-to-glucagon cell ratio. However, in all three doses of the fSFP groups, including the metformin treatment group, there was a notable decrease in both the number of insulin and glucagon immunoreactive cells and the insulin/glucagon cell ratio (p < 0.01) when compared with the HFD control group (Table 1; Figure S3).

3.4. Effects of fSFP on Hyperlipidemia

3.4.1. Changes in Total Cholesterol, TG, LDL and HDL Contents in Blood

In the HFD control group, blood total cholesterol, TG and LDL increased significantly (p < 0.01), and the decrease in HDL content was significant, compared with NFD control. However, an increase in HDL content and a decrease in serum total cholesterol, TG and LDL were observed in the fSFP groups, including metformin treatment group, compared to HFD control (p < 0.01) (Table S6).

3.4.2. Changes in Lipid Content in Feces

Compared with the NFD control group, the total cholesterol and TG content in the feces of the HFD control group were found to be slightly higher. However, there was a significant (p < 0.01) dose increase in fecal total cholesterol and TG content at all three administered doses of fSFP, including metformin, respectively (Figure 5).

3.5. Effects of fSFP on Liver Damage

3.5.1. Changes in Liver Weight

There was a significant elevation in liver weight in the HFD control group as compared to the NFD control group (p < 0.01). However, a notable dose-dependent decrease in liver weight was observed in the fSFP group at all three dosages, including the metformin-treated group (p < 0.01). In contrast, no significant changes in relative liver weight were detected in either treatment group when compared to the HFD control group (Tables S2 and S3).

3.5.2. Changes in AST, ALT, ALP, LDH and GGT Levels in Blood

In comparison to the NFD control group, there was a significant elevation (p < 0.01) in the levels of Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Alkaline Phosphatase (ALP), Lactate Dehydrogenase (LDH), and Gamma-Glutamyl Transferase (GGT) in the blood of the HFD control group. However, in the groups receiving all three doses of fSFP, including the metformin treatment group, a significant reduction (p < 0.01) in serum levels of AST, ALT, ALP, LDH, and GGT was observed when compared to the HFD control group (Table 2).

3.5.3. Histopathological Alterations in the Liver

Hepatocyte hypertrophy caused by fat accumulation in hepatocytes was confirmed in the HFD control group. Additionally, steatohepatitis (percentage of fatty area in the liver parenchyma) and mean hepatocyte diameter were significantly larger than the NFD control (p < 0.01). However, in the group where all three doses were administered, including the metformin treatment group, steatohepatitis and mean hepatocyte diameter were significantly reduced dose-dependently compared to the HFD control group (p < 0.01) (Figure 6 and Figure S4).

3.6. Effects of fSFP on Kidney Damage

3.6.1. Changes in Kidney Weight

The kidney weight in the HFD control group exhibited a significant increase compared to the NFD control group (p < 0.01). However, a notable dose-dependent decrease in kidney weight was observed in the groups receiving each of the three doses of fSFP, including the metformin treatment group, when compared to the HFD control group (p < 0.01). In contrast, there were no significant alterations in relative kidney function between the HFD-fed group and the NFD control group, nor were there any significant changes observed in either of the treatment groups (Tables S2 and S3).

3.6.2. BUN and Creatinine Contents in Blood

Compared with the NFD control group, the blood BUN and creatinine content of the HFD control group increased significantly (p < 0.01). However, there was a significant (p < 0.01 or p < 0.05) decrease in blood BUN and creatinine levels in all three doses of the fSFP group, including the metformin group, compared to the HFD control group (Table 2).

3.6.3. Changes in Renal Histopathology

The number of degenerated renal tubules was significantly increased in the HFD control group compared to the NFD control group (p < 0.01). This type of kidney degeneration is characterized by tubular vacuolization caused by the accumulation of fat droplets. However, compared with the HFD control group, a reduction in the number of degenerative tubules was observed in all three doses of fSFP, including the metformin treatment group (Figure 7 and Figure S5). This decrease was significant (p < 0.01 or p < 0.05).

3.7. Effects of fSFP on the Liver Antioxidant Defense System

The results showed that liver lipid oxidation controlled by HFD was higher than that in the NFD control group (p < 0.01), while GSH content, and CAT and SOD tissue activities were decreased significantly (p < 0.01), respectively. However, all three doses of the fSFP group, including metformin group, were significantly (p < 0.01) recovered, compared with HFD control (Figure 8).

3.8. Effects of fSFP on Liver Enzymes Related to Glucose Metabolism

Compared with the NFD control group, the activity of the glucose-degrading enzyme GK and PEPCK were decreased (p < 0.01) and the activity of the glucose-synthesizing enzyme G6pase was increased in the liver tissue of the HFD control group, respectively. Compared with HFD control group, fSFP treatment groups significantly (p < 0.01 or p < 0.05) reversed all these effects, including metformin group (Table S7).

3.9. Effects of fSFP on Expression of Genes Associated with Lipid Metabolism

3.9.1. Changes in the Expression of mRNA in Liver Tissue

Compared with the NFD control group, the expression of ACC1 mRNA was significantly increased in the HFD control group (p < 0.01), while the expression of AMPK1 and AMPK2 mRNA was decreased. However, all three doses of fSFP administration, including the metformin group, restored all these effects compared with the HFD control group (Table 3).

3.9.2. Changes in the Expression of mRNA in Adipose Tissue

In the HFD control group, there was a significant upregulation (p < 0.01) in the expression of leptin, CCAAT Enhancer-Binding Protein (C/EBPα and C/EBPβ), Fatty Acid Synthase (FAS), Sterol Regulatory Element-Binding Protein 1c (SREBP1c), and peroxisome proliferator-activated receptor (PPAR) compared to the NFD control group. Moreover, a significant (p < 0.01) decreases of UCP2, adiponectin and PPAR mRNA expressions in adipose tissue was observed in HFD control as compared to NFD control. However, administration of all three doses of fSFP, including in the metformin treatment group, reversed these effects, as observed when compared with the HFD control group (Figure 9).

4. Discussion

HFD causes obesity in animals such as mice and is directly associated with type II diabetes, which is characterized by symptoms such as hyperglycemia, insulin resistance, obesity, renal degeneration, and hyperlipidemia [5,12]. In all fSFP dose groups, including 250 mg/kg metformin, there was a decrease in BW over the entire 84-day drug test compared to the HFD control group. Obesity is the most common consequence, and histological studies have shown that it causes adipocyte hypertrophy [5,12,13,22]. In addition to directly affecting adipose tissue, energy storage and endocrine systems, it can also release adipokines. According to Mitchell et al. [31] and Choi et al. [12,13], changes in adipokine secretion can lead to complex diseases, including obesity and insulin resistance. The results of this study were that the fSFP group showed significant adipogenesis and adipocyte hypertrophy inhibition effects at all three doses compared with the HFD control group. It is known that when obesity occurs, the pancreas reduces the number of zymogen granules [32]. Histopathologically, all three doses of fSFP groups increased zymogen granules in the exocrine pancreas; it indicates that this anti-obese effect may be through the production of fat digestive enzymes.
As type II diabetes progresses, insulin resistance often leads to increased serum insulin and HbA1c levels [21]. Furthermore, to sustain glycemic equilibrium, chronic exposure to HFD leads to an increase in the quantity and proliferation of pancreatic cells, including both insulin- and glucagon-secreting cells, as well as a rise in the ratio of insulin/glucagon-producing cells [33]. Blood glucose, blood insulin, and HbA1c levels, as well as histological and immunohistological changes in the pancreatic endocrine region, decreased in all three doses of the fSFP groups; this reveals the effects of pancreatic endocrine function. The hypolipidic effect of the candidate increases the level of low lipoprotein, lower cholesterol and fat (triglycerides). As diabetes worsens, obesity and hepatocyte degradation lead to increased levels of AST, ALT, ALP, LDH, and GGT in the blood, known as the induction of NAFLD [34]. Moreover, fibrosis and hepatocellular damage from abnormal glycolysis can further deteriorate the liver [34]. The results of this study are that the fSFP-administered group showed anti-hyperlipidemic activity at all three doses, which reduced NAFLD in HFD-fed mice.
Diabetic nephropathy is characterized by high blood BUN and creatinine levels and occurs when the diabetic condition progresses to glomerular atrophy and fibrosis, inflammatory cell infiltration, and tubular necrosis [35]. The effects of diabetic nephropathy were dose-dependently reduced in all three doses of the fSFP group, indicating an effect on diabetic nephropathy. It is also known that the development of various types of diabetes is directly related to cellular damage caused by oxidative stress [36]. Many free radicals are produced in diabetes due to strong oxidative stress and inhibition of endogenous antioxidants [37]. In this study, lipid peroxidation, which is associated with interference with antioxidant defense systems, was reduced in the three-dose fSFP group. In rats fed with HFD, it has been shown to exhibit decreased glucose-degrading enzyme and increased glucose-synthesizing enzyme, which often leads to a hyperglycemic state [21]. In addition, fSFP significantly and dose-dependently inhibited glucose metabolism enzymes in the liver.
AMPK is now recognized as one of the most important cellular factors regulating blood sugar and fat metabolism by promoting fat oxidation and inhibiting fat synthesis and glucose production in the liver and adipose tissue [7]. Therefore, it is important to monitor the expression of this AMPK gene and AMPK signaling pathway-related proteins in the liver and adipose tissue. Additionally, PPARs are known to play a role in lipid oxidation and lipid synthesis [8,11]. It is now known that adiponectin produced by adipocytes improves insulin sensitivity and fat oxidation in an AMPK-dependent manner and that UCP2 is a thermogenesis-associated protein [38]. The development of AMPK expression is associated with the development of the antioxidant activity [12,13,14] and positive regulation of glucose metabolism activities [7]. HFD-induced changes in AMPK and lipid metabolism-related mRNA expression in liver and adipose tissue were significantly recovered in all three dose fSFP groups. Additionally, among treatment groups in our study, it was found that the metformin control group was compared with the fSFP 200 mg/kg control group.
There are currently no effective medications available to treat metabolic syndrome [39]. To put it another way, although some medications are used to treat the metabolic syndrome [40]. Additionally, a number of side effects are also problematic, and its usage is restricted. It is vital to produce medications that have minimal side effects even when taken for a long period and can control the entire metabolic syndrome. According to studies by Jeoung et al. [19] and Kim et al. [20], Schizandrae fructus pomace also exhibits pharmacological action similar to that of Schizandrae fructus. One of the main active ingredients in the widely used traditional medicinal plant Schisandra chinensis is shizandrin. Shizandrin was identified and quantified as considerable amount in fSFP in the present study, which indicates that anti-obesity and anti-diabetic activities of fSFP might be, at least in part, potentiated by shizandrin. The usefulness of this substance in treating a variety of liver illnesses is demonstrated by its sedative, hypnotic, anti-aging, antioxidant, and immunomodulatory characteristics, all while maintaining a good safety profile [41]. However, hepatic and intestinal first-pass metabolism have a significant impact on the bioavailability of shizandrin. Therefore, additional research is required to determine the bioavailability of shizandrin in fSFP in HFD mice in order to guarantee its therapeutic effectiveness in the future. Although, the effect of fermented Schizandrae fructus pomace extract on reducing obesity based on by a diet high in carbohydrates was not confirmed by the present investigation. Moreover, additional investigation is necessary to confirm the bioavailability of different ingredients of fSFP in HFD mice in order to guarantee its therapeutic effectiveness in the future.

5. Conclusions

In conclusion, the prolonged oral administration of all three doses of fSFP over 84 days demonstrated significant effectiveness in reducing hyperlipidemia associated with obesity and diabetes, as well as alleviating NAFLD and diabetic nephropathy in a dose-dependent manner. The presence of the bioactive compound shizandrin was quantified in this study, and contributed, at least in part, to the anti-obesity and anti-diabetic properties of fSFP. Consequently, there is potential for the development of fSFP as a potent therapeutic agent or functional food component targeting a range of metabolic diseases, including obesity, diabetes, and NAFLD.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app132413307/s1, Figure S1: Histological images of adipocytes; Figure S2: Histological images of pancreas. Figure S3: Histological images of insulin- and glucagon-immunoreactive cells; Figure S4: Histological images of liver; Figure S5: Histological images of kidney; Table S1: The sequence of oligonucleotides for RT-PCR analysis. Table S2: Mice BW and average daily food consumption; Table S3: Absolute weight of organs; Table S4: Relative weight of organs; Table S5: Histopathology and histomorphometry analyses of periovarian and abdominal wall-deposited fat pads; Table S6: Glucose and serum lipid levels; Table S7: Activity of hepatic glucose-regulating enzymes.

Author Contributions

Conceptualization, J.-S.C. and S.-K.K.; methodology, M.M., S.-J.P. and S.-K.K.; software, S.-K.K.; validation, J.-S.C. and S.-K.K.; formal analysis, S.-J.P. and S.-K.K.; investigation, J.-S.C. and S.-K.K.; resources, S.-K.K.; data curation, J.-S.C. and S.-K.K.; writing—original draft preparation, M.M., J.-S.C. and S.-K.K.; writing—review and editing, M.M., S.-J.P., J.-S.C. and S.-K.K.; visualization, J.-S.C. and S.-K.K.; supervision, J.-S.C. and S.-K.K.; project administration, J.-S.C. and S.-K.K.; funding acquisition, S.-J.P. and S.-K.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

All laboratory animals were treated according to the national regulations of the usage and welfare of laboratory animals, and approved by the Institutional Animal Care and Use Committee in Daegu Haany University (Gyeongsan, Gyeongbuk, Korea) prior to animal experiment (Approval No. DHU2021-063, 10 August 2021).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to privacy.

Acknowledgments

The preparation and processing of the raw materials for this research work were performed by Hye-Rim Park and Beom-Rak Choi of Nutracore Co., Ltd., Gyeong-gi-Do 16514, Republic of Korea, for which the authors are grateful. Those individuals have consented to this acknowledgement.

Conflicts of Interest

Author S.-J.P was employed by the company Suheung Research & Development. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Wendel, A.A.; Purushotham, A.; Liu, L.-F.; Belury, M.A. Conjugated linoleic acid fails to worsen insulin resistance but induces hepatic steatosis in the presence of leptin in ob/ob mice. J. Lipid Res. 2008, 49, 98–106. [Google Scholar] [CrossRef] [PubMed]
  2. Tilg, H.; Moschen, A.R. Adipocytokines: Mediators linking adipose tissue, inflammation and immunity. Nat. Rev. Immunol. 2006, 6, 772–783. [Google Scholar] [CrossRef] [PubMed]
  3. Lebovitz, H.E. Insulin resistance: Definition and consequences. Exp. Clin. Endocrinol. Diabetes 2001, 109, S135–S148. [Google Scholar] [CrossRef] [PubMed]
  4. Angulo, P. Nonalcoholic fatty liver disease. N. Engl. J. Med. 2002, 346, 1221–1231. [Google Scholar] [CrossRef]
  5. Choi, J.-S.; Kim, J.-W.; Park, J.B.; Pyo, S.E.; Hong, Y.-K.; Ku, S.K.; Kim, M.-R. Blood glycemia-modulating effects of melanian snail protein hydrolysates in mice with type II diabetes. Int. J. Mol. Med. 2017, 39, 1437–1451. [Google Scholar] [CrossRef]
  6. Steinberg, G.R.; Kemp, B.E. AMPK in health and disease. Physiol. Rev. 2009, 89, 1025–1078. [Google Scholar] [CrossRef]
  7. Lin, C.-H.; Kuo, Y.-H.; Shih, C.-C. Effects of Bofu-Tsusho-San on diabetes and hyperlipidemia associated with AMP-activated protein kinase and glucose transporter 4 in high-fat-fed mice. Int. J. Mol. Sci. 2014, 15, 20022–20044. [Google Scholar] [CrossRef]
  8. Veiga, F.M.S.; Graus-Nunes, F.; Rachid, T.L.; Barreto, A.B.; Mandarim-de-Lacerda, C.A.; Souza-Mello, V. Anti-obesogenic effects of WY14643 (PPAR-alpha agonist): Hepatic mitochondrial enhancement and suppressed lipogenic pathway in diet-induced obese mice. Biochimie 2017, 140, 106–116. [Google Scholar] [CrossRef]
  9. Hasanvand, A.; Amini-Khoei, H.; Hadian, M.-R.; Abdollahi, A.; Tavangar, S.M.; Dehpour, A.R.; Semiei, E.; Mehr, S.E. Anti-inflammatory effect of AMPK signaling pathway in rat model of diabetic neuropathy. Inflammopharmacology 2016, 24, 207–219. [Google Scholar] [CrossRef]
  10. Xu, X.J.; Gauthier, M.-S.; Hess, D.T.; Apovian, C.M.; Cacicedo, J.M.; Gokce, N.; Farb, M.; Valentine, R.J.; Ruderman, N.B. Insulin sensitive and resistant obesity in humans: AMPK activity, oxidative stress, and depot-specific changes in gene expression in adipose tissue. J. Lipid Res. 2012, 53, 792–801. [Google Scholar] [CrossRef]
  11. Xu, H.; Wu, B.; Wang, X.; Ma, F.; Li, Y.; An, Y.; Wang, C.; Wang, X.; Luan, W.; Li, S. Cordycepin regulates body weight by inhibiting lipid droplet formation, promoting lipolysis and recruiting beige adipocytes. J. Pharm. Pharmacol. 2019, 71, 1429–1439. [Google Scholar] [CrossRef] [PubMed]
  12. Choi, B.-R.; Kim, H.-J.; Lee, Y.-J.; Ku, S.-K. Anti-diabetic obesity effects of Wasabia japonica Matsum leaf extract on 45% Kcal high-fat diet-fed mice. Nutrients 2020, 12, 2837. [Google Scholar] [CrossRef] [PubMed]
  13. Choi, E.-H.; Chun, Y.-S.; Kim, J.; Ku, S.-K.; Jeon, S.; Park, T.-S.; Shim, S.-M. Modulating lipid and glucose metabolism by glycosylated kaempferol rich roasted leaves of Lycium chinense via upregulating adiponectin and AMPK activation in obese mice-induced type 2 diabetes. J. Funct. Foods 2020, 72, 104072. [Google Scholar] [CrossRef]
  14. Ma, A.; Wang, J.; Yang, L.; An, Y.; Zhu, H. AMPK activation enhances the anti-atherogenic effects of high density lipoproteins in apoE−/− mice. J. Lipid Res. 2017, 58, 1536–1547. [Google Scholar] [CrossRef] [PubMed]
  15. Guo, L.Y.; Hung, T.M.; Bae, K.H.; Shin, E.M.; Zhou, H.Y.; Hong, Y.N.; Kang, S.S.; Kim, H.P.; Kim, Y.S. Anti-inflammatory effects of schisandrin isolated from the fruit of Schisandra chinensis Baill. Eur. J. Pharmacol. 2008, 591, 293–299. [Google Scholar] [CrossRef] [PubMed]
  16. Kim, S.H.; Joo, M.H.; Yoo, S.H. Structural identification and antioxidant properties of major anthocyanin extracted from Omija (Schizandra chinensis) fruit. J. Food Sci. 2009, 74, C134–C140. [Google Scholar] [CrossRef]
  17. Panossian, A.; Wikman, G. Pharmacology of Schisandra chinensis Bail.: An overview of Russian research and uses in medicine. J. Ethnopharmacol. 2008, 118, 183–212. [Google Scholar] [CrossRef]
  18. Park, H.J.; Kim, H.-J.; Kim, S.R.; Choi, M.-S.; Jung, U.J. Omija fruit ethanol extract improves adiposity and related metabolic disturbances in mice fed a high-fat diet. J. Nutr. Biochem. 2017, 41, 137–141. [Google Scholar] [CrossRef]
  19. Jeoung, Y.-K.; Kim, J.-H.; Baek, I.-S.; Kang, Y.J.; Chi, J.-H. Effect of schizandra berry dregs and rice bran treatment on γ-aminobutyric acid (GABA) content enhancement in Pleurotus ostreatus. J. Mushroom 2017, 15, 88–93. [Google Scholar]
  20. Kim, M.-S.; Sung, H.-J.; Park, J.-Y.; Sohn, H.-Y. Evaluation of anti-oxidant, anti-microbial and anti-thrombosis activities of fruit, seed and pomace of Schizandra chinensis Baillon. J. Life Sci. 2017, 27, 131–138. [Google Scholar] [CrossRef]
  21. Chung, S.I.; Rico, C.W.; Kang, M.Y. Comparative study on the hypoglycemic and antioxidative effects of fermented paste (doenjang) prepared from soybean and brown rice mixed with rice bran or red ginseng marc in mice fed with high fat diet. Nutrients 2014, 6, 4610–4624. [Google Scholar] [CrossRef] [PubMed]
  22. Kim, J.W.; Lee, Y.S.; Seol, D.J.; Cho, I.J.; Ku, S.K.; Choi, J.S.; Lee, H.J. Anti-obesity and fatty liver-preventing activities of Lonicera caerulea in high-fat diet-fed mice. Int. J. Mol. Med. 2018, 42, 3047–3064. [Google Scholar] [CrossRef] [PubMed]
  23. Jamall, I.S.; Smith, J.C. Effects of cadmium on glutathione peroxidase, superoxide dismutase, and lipid peroxidation in the rat heart: A possible mechanism of cadmium cardiotoxicity. Toxicol. Appl. Pharmacol. 1985, 80, 33–42. [Google Scholar] [CrossRef] [PubMed]
  24. Sedlak, J.; Lindsay, R.H. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal. Biochem. 1968, 25, 192–205. [Google Scholar] [CrossRef] [PubMed]
  25. Aebi, H. Catalase. In Methods of Enzymatic Analysis; Elsevier: Amsterdam, The Netherlands, 1974; pp. 673–684. [Google Scholar]
  26. Sun, Y.I.; Oberley, L.W.; Li, Y. A simple method for clinical assay of superoxide dismutase. Clin. Chem. 1988, 34, 497–500. [Google Scholar] [CrossRef]
  27. Hulcher, F.H.; Oleson, W.H. Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A. J. Lipid Res. 1973, 14, 625–631. [Google Scholar] [CrossRef]
  28. Davidson, A.L.; Arion, W.J. Factors underlying significant underestimations of glucokinase activity in crude liver extracts: Physiological implications of higher cellular activity. Arch. Biochem. Biophys. 1987, 253, 156–167. [Google Scholar] [CrossRef]
  29. Alegre, M.; Ciudad, C.J.; Fillat, C.; Guinovart, J.J. Determination of glucose-6-phosphatase activity using the glucose dehydrogenase-coupled reaction. Anal. Biochem. 1988, 173, 185–189. [Google Scholar] [CrossRef]
  30. Bentle, L.A.; Lardy, H.A. Interaction of anions and divalent metal ions with phosphoenolpyruvate carboxykinase. J. Biol. Chem. 1976, 251, 2916–2921. [Google Scholar] [CrossRef]
  31. Mitchell, M.; Armstrong, D.T.; Robker, R.L.; Norman, R.J. Adipokines: Implications for female fertility and obesity. Reproduction 2005, 130, 583–597. [Google Scholar] [CrossRef]
  32. Wilson, J.S.; Korsten, M.A.; Leo, M.A.; Lieber, C.S. Combined effects of protein deficiency and chronic ethanol consumption on rat pancreas. Dig. Dis. Sci. 1988, 33, 1250–1259. [Google Scholar] [CrossRef] [PubMed]
  33. Noriega-Lopez, L.; Tovar, A.R.; Gonzalez-Granillo, M.; Hernandez-Pando, R.; Escalante, B.; Santillan-Doherty, P.; Torres, N. Pancreatic insulin secretion in rats fed a soy protein high fat diet depends on the interaction between the amino acid pattern and isoflavones. J. Biol. Chem. 2007, 282, 20657–20666. [Google Scholar] [CrossRef] [PubMed]
  34. Wang, L.; Peng, W.; Zhao, Z.; Zhang, M.; Shi, Z.; Song, Z.; Zhang, X.; Li, C.; Huang, Z.; Sun, X. Prevalence and treatment of diabetes in China, 2013–2018. JAMA 2021, 326, 2498–2506. [Google Scholar] [CrossRef] [PubMed]
  35. Niewczas, M.A.; Pavkov, M.E.; Skupien, J.; Smiles, A.; Md Dom, Z.I.; Wilson, J.M.; Park, J.; Nair, V.; Schlafly, A.; Saulnier, P.-J. A signature of circulating inflammatory proteins and development of end-stage renal disease in diabetes. Nat. Med. 2019, 25, 805–813. [Google Scholar] [CrossRef] [PubMed]
  36. Ceriello, A.; Quatraro, A.; Giugliano, D. New insights on non-enzymatic glycosylation may lead to therapeutic approaches for the prevention of diabetic complications. Diabet. Med. 1992, 9, 297–299. [Google Scholar] [CrossRef] [PubMed]
  37. Collier, A.; Small, M.; Wilson, R.; Bradley, H.; Thomson, J.A. Free radical activity in type 2 diabetes. Diabet. Med. 1990, 7, 27–30. [Google Scholar] [CrossRef] [PubMed]
  38. Dulloo, A.G.; Seydoux, J.; Jacquet, J. Adaptive thermogenesis and uncoupling proteins: A reappraisal of their roles in fat metabolism and energy balance. Physiol. Behav. 2004, 83, 587–602. [Google Scholar] [CrossRef]
  39. Tan, Y.; Kim, J.; Cheng, J.; Ong, M.; Lao, W.-G.; Jin, X.-L.; Lin, Y.-G.; Xiao, L.; Zhu, X.-Q.; Qu, X.-Q. Green tea polyphenols ameliorate non-alcoholic fatty liver disease through upregulating AMPK activation in high fat fed Zucker fatty rats. World J. Gastroenterol. 2017, 23, 3805. [Google Scholar] [CrossRef]
  40. Rotman, Y.; Sanyal, A.J. Current and upcoming pharmacotherapy for non-alcoholic fatty liver disease. Gut 2017, 66, 180–190. [Google Scholar] [CrossRef]
  41. Wang, X.; Wang, X.; Yao, H.; Shen, C.; Geng, K.; Xie, H. A comprehensive review on Schisandrin and its pharmacological features. In Naunyn-Schmiedeberg’s Archives of Pharmacology; Springer: Berlin/Heidelberg, Germany, 2023; pp. 1–12. [Google Scholar]
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Figure 1. Standard shizandrin compound (A), fresh Schisandra fructus (B) and fermented Schisandra fructus (C).
Figure 1. Standard shizandrin compound (A), fresh Schisandra fructus (B) and fermented Schisandra fructus (C).
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Figure 2. BW change (A) and BW gain (B) in mice. THSD test results indicated significant differences (p < 0.01) compared to NFD control (a). Analysis underwent DT3 testing, revealing noteworthy distinctions (b) and (c) with p < 0.01 and p < 0.05, respectively, compared to NFD control and (d) with p < 0.01 compared to HFD control. The values are depicted as the mean ± SD for a sample size of 10 mice.
Figure 2. BW change (A) and BW gain (B) in mice. THSD test results indicated significant differences (p < 0.01) compared to NFD control (a). Analysis underwent DT3 testing, revealing noteworthy distinctions (b) and (c) with p < 0.01 and p < 0.05, respectively, compared to NFD control and (d) with p < 0.01 compared to HFD control. The values are depicted as the mean ± SD for a sample size of 10 mice.
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Figure 3. Representative whole-body DEXA images of gross body mass and abdominal fat pads (A), and total body and abdominal fat densities (B) in mice. Analysis underwent DT3 testing, revealing noteworthy distinctions (a) with p < 0.01 compared to NFD control and (b) with p < 0.01 compared to HFD control. The values are depicted as the mean ± SD for a sample size of 10 mice.
Figure 3. Representative whole-body DEXA images of gross body mass and abdominal fat pads (A), and total body and abdominal fat densities (B) in mice. Analysis underwent DT3 testing, revealing noteworthy distinctions (a) with p < 0.01 compared to NFD control and (b) with p < 0.01 compared to HFD control. The values are depicted as the mean ± SD for a sample size of 10 mice.
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Figure 4. Effects on serum insulin and blood HbA1c levels in mice. THSD test results indicated significant differences (p < 0.01) compared to both NFD control (a) and HFD control (b). The values are depicted as the mean ± SD for a sample size of 10 mice.
Figure 4. Effects on serum insulin and blood HbA1c levels in mice. THSD test results indicated significant differences (p < 0.01) compared to both NFD control (a) and HFD control (b). The values are depicted as the mean ± SD for a sample size of 10 mice.
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Figure 5. Effects on total cholesterol and triglycerides in the feces of mice due to fSFP administration. Analysis underwent DT3 testing, revealing noteworthy distinctions (a) with p < 0.01 compared to NFD control and (b) with p < 0.01 compared to HFD control. The values are depicted as the mean ± SD for a sample size of 10 mice.
Figure 5. Effects on total cholesterol and triglycerides in the feces of mice due to fSFP administration. Analysis underwent DT3 testing, revealing noteworthy distinctions (a) with p < 0.01 compared to NFD control and (b) with p < 0.01 compared to HFD control. The values are depicted as the mean ± SD for a sample size of 10 mice.
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Figure 6. Effects on histopathological alterations in the liver steatosis (A) and mean hepatocyte diameters (B) due to fSFP administration. THSD test results indicated significant differences (p < 0.01) compared to both NFD control (a) and HFD control (b). Analysis underwent DT3 testing, revealing noteworthy distinctions (c) with p < 0.01 compared to NFD control and (d) with p < 0.01 compared to HFD control. The values are depicted as the mean ± SD for a sample size of 10 mice.
Figure 6. Effects on histopathological alterations in the liver steatosis (A) and mean hepatocyte diameters (B) due to fSFP administration. THSD test results indicated significant differences (p < 0.01) compared to both NFD control (a) and HFD control (b). Analysis underwent DT3 testing, revealing noteworthy distinctions (c) with p < 0.01 compared to NFD control and (d) with p < 0.01 compared to HFD control. The values are depicted as the mean ± SD for a sample size of 10 mice.
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Figure 7. Effects on histopathological alterations in the kidney due to fSFP administration. DT3 test results indicated significant differences (a) with p < 0.01 compared to NFD control and (b) and (c) with p < 0.01 and p < 0.05, respectively, compared to HFD control. The values are depicted as the mean ± SD for a sample size of 10 mice.
Figure 7. Effects on histopathological alterations in the kidney due to fSFP administration. DT3 test results indicated significant differences (a) with p < 0.01 compared to NFD control and (b) and (c) with p < 0.01 and p < 0.05, respectively, compared to HFD control. The values are depicted as the mean ± SD for a sample size of 10 mice.
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Figure 8. Effects on antioxidant defense systems on malondialdehyde content (A), and glutathione (B), catalase (C) and superoxide dismutase (D) activities in the liver due to fSFP administration. DT3 test results indicated significant differences (a) and (b) with p < 0.01 and p < 0.05 compared to NFD control, and (c) and (d) with p < 0.01 and p < 0.05 compared to HFD control, respectively. The values are depicted as the mean ± SD for a sample size of 10 mice.
Figure 8. Effects on antioxidant defense systems on malondialdehyde content (A), and glutathione (B), catalase (C) and superoxide dismutase (D) activities in the liver due to fSFP administration. DT3 test results indicated significant differences (a) and (b) with p < 0.01 and p < 0.05 compared to NFD control, and (c) and (d) with p < 0.01 and p < 0.05 compared to HFD control, respectively. The values are depicted as the mean ± SD for a sample size of 10 mice.
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Figure 9. Effects on mRNA expressions associated with lipid metabolism in the adipose tissue due to fSFP administration. DT3 test results indicated significant differences (a) with p < 0.01 compared to NFD control, and (b) and (c) with p < 0.01 and p < 0.05 compared to HFD control, respectively. The values are depicted as the mean ± SD for a sample size of 10 mice.
Figure 9. Effects on mRNA expressions associated with lipid metabolism in the adipose tissue due to fSFP administration. DT3 test results indicated significant differences (a) with p < 0.01 compared to NFD control, and (b) and (c) with p < 0.01 and p < 0.05 compared to HFD control, respectively. The values are depicted as the mean ± SD for a sample size of 10 mice.
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Table 1. Changes on histopathology–histomorphometry of the pancreas in mice.
Table 1. Changes on histopathology–histomorphometry of the pancreas in mice.
ItemsZymogen Granules (%/mm2 of Exocrine)Mean Islet Numbers (Numbers/10 mm2)Mean Islet Diameter (μm/Islet)Insulin-IR Cells (Cells/ mm2) [A]Glucagon-IR Cells (Cells/ mm2) [B]Insulin/Glucagon Ratio [A/B]
Groups
Controls
   NFD59.13 ± 11.476.20 ± 1.48109.41 ± 15.21148.40 ± 20.0434.50 ± 5.524.32 ± 0.27
   HFD11.63 ± 2.61 c18.60 ± 1.90 a201.13 ± 14.38 a2531.80 ± 385.04 c368.10 ± 37.72 c6.86 ± 0.53 c
Reference
   Metformin37.97 ± 13.85 ce10.40 ± 2.27 ab163.85 ± 14.40 ab1190.20 ± 347.37 ce257.30 ± 34.93 ce4.56 ± 0.77 e
Test materials—SFP
   400 mg/kg44.96 ± 10.27 e8.60 ± 1.17 b137.21 ± 20.11 ab 375.10 ± 160.13 de 95.50 ± 46.61 ce3.88 ± 0.34 e
   200 mg/kg38.66 ± 12.17 de10.10 ± 1.66 ab161.69 ± 16.97 ab1071.90 ± 253.91 ce249.00 ± 40.49 ce4.27 ± 0.40 e
   100 mg/kg28.64 ± 11.34 cf13.60 ± 2.22 ab175.60 ± 12.99 ab1444.20 ± 300.69 ce284.80 ± 43.86 ce5.05 ± 0.49 de
The values are depicted as the mean ± SD for a sample size of 10 mice. THSD test results indicated significant differences (p < 0.01) compared to both NFD control (a) and HFD control (b). Analysis underwent DT3 testing, revealing noteworthy distinctions (c) and (d) with p < 0.01 and p < 0.05, respectively, compared to NFD control and (e) and (f) with p < 0.01 and p < 0.05, respectively, compared to HFD control.
Table 2. Changes on serum AST, ALT, ALP, LDH, GGT, BUN and creatine levels in NFD- or HFD-supplied mice.
Table 2. Changes on serum AST, ALT, ALP, LDH, GGT, BUN and creatine levels in NFD- or HFD-supplied mice.
ItemsAST
(IU/L)		ALT
(IU/L)		ALP
(IU/L)		LDH
(×10 IU/L)		GGT
(IU/L)		BUN
(mg/dL)		Creatinine
(mg/dL)
Groups
Controls
   NFD78.20 ± 15.6541.80 ± 11.3875.10 ± 15.2471.26 ± 15.635.40 ± 1.5129.60 ± 10.490.64 ± 0.17
   HFD195.60 ± 26.68 a152.20 ± 16.82 a219.60 ± 21.80 a407.86 ± 57.83 c20.40 ± 1.71 a128.80 ± 20.46 a2.24 ± 0.41 c
Reference
   Metformin129.00 ± 15.01 ab97.70 ± 13.84 ab143.40 ± 21.05 ab242.18 ± 72.72 cd11.90 ± 1.29 ab76.10 ± 18.62 ab1.36 ± 0.16 cd
Test materials—fSFP
   400 mg/kg108.20 ± 17.87 ab83.80 ± 16.61 ab121.30 ± 27.04 ab167.05 ± 27.53 cd9.30 ± 1.42 ab62.20 ± 15.35 ab1.15 ± 0.16 cd
   200 mg/kg127.10 ± 16.33 ab95.40 ± 20.28 ab141.80 ± 21.28 ab221.47 ± 58.13 cd11.80 ± 1.99 ab76.70 ± 16.11 ab1.33 ± 0.25 cd
   100 mg/kg144.70 ± 12.24 ab111.60 ± 15.25 ab157.60 ± 17.52 ab287.73 ± 55.39 cd15.20 ± 1.23 ab96.90 ± 8.70 ab1.62 ± 0.09 ce
The values are depicted as the mean ± SD for a sample size of 10 mice. THSD test results indicated significant differences (p < 0.01) compared to both NFD control (a) and HFD control (b). Analysis underwent DT3 testing, revealing noteworthy distinctions (c) with p < 0.01 compared to NFD control, and (d) and (e) with p < 0.01 and p < 0.05, respectively, compared to HFD control.
Table 3. Changes on lipid metabolism-related gene mRNA expressions in liver of mice.
Table 3. Changes on lipid metabolism-related gene mRNA expressions in liver of mice.
ItemsHepatic Tissue (Relative to Control/GAPDH)
GroupsACC1AMPKα1AMPKα2
Controls
   NFD1.00 ± 0.051.00 ± 0.051.00 ± 0.05
   HFD5.25 ± 0.89 a0.26 ± 0.05 a0.24 ± 0.05 a
Reference
   Metformin2.84 ± 0.59 ac0.50 ± 0.11 ac0.43 ± 0.08 ac
Test materials—fSFP
   400 mg/kg1.90 ± 0.28 bc0.68 ± 0.17 ac0.60 ± 0.13 ac
   200 mg/kg2.75 ± 0.77 ac0.50 ± 0.11 ac0.44 ± 0.08 ac
   100 mg/kg3.56 ± 0.61 ac0.44 ± 0.10 ac0.40 ± 0.06 ac
The values are depicted as the mean ± SD for a sample size of 10 mice. Analysis underwent DT3 testing, revealing noteworthy distinctions (a) and (b) with p < 0.01 and p < 0.05, respectively, compared to NFD control, and (c) with p < 0.01, respectively, compared to HFD control.
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Mohibbullah, M.; Park, S.-J.; Choi, J.-S.; Ku, S.-K. Anti-Obesity and Anti-Diabetic Activities of Fermented Schizandrae Fructus Pomace Extract in Mice Fed with High-Fat Diet. Appl. Sci. 2023, 13, 13307. https://doi.org/10.3390/app132413307

AMA Style

Mohibbullah M, Park S-J, Choi J-S, Ku S-K. Anti-Obesity and Anti-Diabetic Activities of Fermented Schizandrae Fructus Pomace Extract in Mice Fed with High-Fat Diet. Applied Sciences. 2023; 13(24):13307. https://doi.org/10.3390/app132413307

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Mohibbullah, Md., So-Jung Park, Jae-Suk Choi, and Sae-Kwang Ku. 2023. "Anti-Obesity and Anti-Diabetic Activities of Fermented Schizandrae Fructus Pomace Extract in Mice Fed with High-Fat Diet" Applied Sciences 13, no. 24: 13307. https://doi.org/10.3390/app132413307

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