To Li, Lihn Xu, Qiojun Yn, Jun Liu,*, Zhengqing Jing,*

a Key Laboratory of Food Bioengineering (China National Light Industry), College of Food Science and Nutritional Engineering,China Agricultural University, Beijing 100083, China

b Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Engineering, China Agricultural University, Beijing 100083, China

Keywords:

Hawthorn leathers

Functional oligosaccharides

In flammatory responses

Glucose and lipid metabolism

Anti-obesity

A B S T R A C T

High sucrose content in traditional hawthorn leathers limits the potential consumption, particularly for elders and diabetics. In this study, sucrose-free hawthorn leathers were formulated with 75% fructooligosaccharides(FOS) and 25% xylooligosaccharides (XOS) (HLF75), which exhibited comparable morphology and sensory properties to the traditional ones. Then, the anti-obesity activity of HLF75 was investigated using high-fat diet (HFD) fed C57BL/6J mice. Comparing with traditional hawthorn leathers, HLF75 supplementation in HFD significantly decreased the levels of blood glucose and serum lipid. The histomorphologies of liver and subcutaneous fat tissues were ameliorated by HLF75, as well as the down-regulated mRNA expression levels of IL-1β, Nos2 and Cox-2 in the liver. M oreover, the protein levels of M yD88 and NF-κB in the liver were suppressed by HLF75 treatment with decreased F4/80-positive macrophage number. Ho wever, the expression levels of PI3K, phosphorylated-AKT (Thr308), and phosphorylated-mTOR (Ser2448) proteins related to glucose metabolism were increased in the liver. Moreover, fat synthesis-related gene expression in HLF75-fed mice was suppressed while expressions of lipolysis genes were improved. Thus, HLF75 supplementation alleviated HFD-induced obesity through the alleviation of inflammation and restoration of the disturbed glucose and lipid metabolism. Functional oligosaccharides could be effective sucrose substitutes in hawthorn leathers and enable their potential utilization as functional foods.

1. Introduction

Hawthorn (Crataegus pinnatifidaBunge.Var. major) belongs to the Rosaceae family and is widely distributed in East Asia, North America, and Europe. The fruit of hawthorn has been approved by the Ministry of Health of China as raw material for functional foods[1,2]. It has attracted much attention due to the documented functional properties (e.g. antioxidant, aid digestion, and anti-diabetic) and proved benefits against cardiovascular diseases [3]. As a good source of antioxidants, the antioxidant activities of hawthorn fruit are thought to be related with the total polyphenols, flavonoids, and triterpenoid acids [4]. Traditionally, dried hawthorn fruit has been used for medicinal purposes to improve digestion and increase appetite [5]. In addition, pectin pentaglaracturonide, a product obtained by enzymatic hydrolyzation of hawthorn pectin, has been reported to inhibit fatty acid synthesis and improve insulin sensitivity in obese mice [6].

Hawthorn fruit has been widely used in the food industry after being processed into foodstuff in the form of jam, jelly, tea, wine,canned fruit, and preserved fruit which is represented by hawthorn leathers [7]. Due to the high contents of organic acids (e.g. citric acid, succinic acid, and malic acid etc.), sucrose, one of the main sweeteners used in foods and beverages, is added into hawthorn leathers to improve the quality attributes and extend shelf life [8].Nevertheless, the added granulated sugar (sucrose, ＞ 50%) in hawthorn leathers could be easily absorbed by the intestine, leading to a series of health risks such as obesity, hypertension, and diabetes [9].Moreover, the high content of sucrose has severely discouraged the potential consumption of hawthorn leathers, which is not conducive for the sustainable development of the food industry [10].Consequently, sucrose replacement in hawthorn leathers is desirable.Sweeteners and/or food raw materials with high-intensity sweetness are commonly used as sucrose substitutes for the preparation of sucrose-free foods. Stevioside, a natural sweetener, has been used as a sucrose alternative in the production of reduced sugar chocolates (50% of cocoa parts) with an enhanced bioactive profile [11]. A report has indicated that sugar alcohols are gaining more popularity as sucrose alternatives amongst individuals [12]. Even though the sweeteners mentioned above are mostly calorie free, while some of them might impart undesirable flavour and aftertaste, especially bitterness. As an alternative, prebiotics, particularly functional oligosaccharides,have shown great potential to deliver texture and structure reinforcing benefits to sucrose-free foods [13]. It has been demonstrated that the implementation of functional oligosaccharides and inulin are sufficient to replace sucrose in various food products such as chocolates and fruits jam [14]. Mango nectar with the highest desirability of 0.85 was produced after the addition of 6% (m/m) inulin without undesirable changes of physicochemical and organoleptic properties [15].

As effective prebiotics, functional oligosaccharides can help to attenuate metabolic disorders through the regulation of gut microbiota [16,17].Both fructooligosaccharides (FOS) and xylooligosaccharides (XOS)have been reported to prevent high-fat diet (HFD)-induced obesity.The intestinal absorption of dietary fat was suppressed by FOS administration, thus inhibited body fat accumulation [18,19]. The supplementation of XOS modulated the cecum microbiome in mice and subsequently decreased visceral fat accumulation [20,21]. When incorporated into food production as sucrose substituents, functional oligosaccharides such as FOS and XOS can impart acceptable sensory properties. Functional sugar-free guava preserves composed of FOS as the sucrose substituent have been designed and the textural properties have not been significantly changed by the addition of FOS [22]. Moreover, the organoleptic properties and stability against food processing conditions enable FOS and XOS as functional food ingredients, as well as their multi-dimensional effects on human health [23].

In the present work, it is hypothesized that FOS and/or XOS, the sucrose substitutes, can impart health beneficial effects to the sucrosefree hawthorn leathers. For this purpose, the sensory characteristics of the formulated sucrose-free hawthorn leathers were evaluated.The preventive effects of sucrose-free hawthorn leathers on HFD-induced obesity were examined as well as the potential mechanisms.Sucrose-free hawthorn leathers with functional oligosaccharides will potentially be suitable for obese individuals as functional foods and thus benefit the hawthorn industry.

2. Material and methods

2.1 Preparation of hawthorn leathers

The preparation of hawthorn leathers has been referred to the method that was previously described by Benjamín García-García et al. [24]. Fresh hawthorn fruits were provided by Yida Food Co., Ltd(Hebei, China). After cleaning and coring, hawthorn fruits (100 g)were cooked (Pressure cooker, 12PLS507A, Midea Group Co., Ltd,China) with 70 g sucrose or sucrose substitutes (Supplementary Table S1) in 55 mL water for 20 min. The mixture was homogenized(1 200 r/min) using a blender (JJ-2, Wuxi Woxin Instrument Manufacturing Co., Ltd, China) and poured onto the metal plate after cooling down to room temperature (23?25 °C). The thickness of puree layer was monitored by the digital Vernier and kept at 3?5 mm.The metal plates were then transferred onto tray dryer and the puree layer was dried for 8 h at 60 °C at an air velocity of 2 m/s. The dried hawthorn leathers were collected and used as samples for subsequent analyses. The main nutritional components (per 100 g) of the prepared hawthorn leathers were measured as carbohydrates (76.06?79.16 g),pectin (15.95?17.88 mg), polyphenols (2.06?2.43 mg), flavonoids(0.41?0.60 mg), citric acid (3.74?3.98 mg), vitamin C (0.37?0.42 mg),and minerals (Ca, Fe, P; 0.35?0.43 mg) (Supplementary Table S2).

2.2 Morphology and sensory evaluation of hawthorn leathers

Micrographs of the prepared hawthorn leathers were acquired by scanning electron microscope (JSM-6380LV, JEOL, Tokyo, Japan).The microscope was operated under low vacuum and the acceleration voltages were set at 10 and 15 kV.

The hedonic evaluation was conducted to evaluate the sensory properties of the prepared hawthorn leathers [25]. Fifteen untrained panelists scored the color, taste, sweetness, acidity, and textural properties of hawthorn leathers with a ten-point hedonic scale, from 0 (very disliked) to 10 (very liked). All prepared hawthorn leather samples were evaluated at 48 h after drying.

2.3 Anti-obesity effects of hawthorn leathers

2.3.1 Animals, diets, and experimental design

C57BL/6J male mice at age of 6 weeks old were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing,China). The mice were housed in cages under 40%?60% humidity and light/dark (12 h/12 h) cycle. The temperature was 23?25 °C and all mice had free access to drinking water. Since arrival, the mice were acclimatized for one week (basal diet), then randomly divided into 5 groups (n= 8/group). Normal control (NC) group: mice fed with standard diet containing 10% of energy from fat, 3.6 total kcal/g;high-fat diet (HFD) group: mice fed with HFD consisted of 60% of energy from fat, 5.0 total kcal/g; metformin (Met) group: mice fed with HFD plus intragastrically administrated Met (400 mg/kg·bw·day);hawthorn leathers formulated with sucrose (HLS) group: mice fed with HFD plus intragastrically administrated homogenization buffer of HLS (6 g/kg·bw·day); hawthorn leathers formulated with 75% FOS and 25% XOS (HLF75) group: mice fed with HFD plus intragastrically administrated homogenization buffer of HLF75(6 g/kg·bw·day). The protocol for maintenance and treatment of all animals was approved by the Animal Ethics Committee of China Agricultural University (approval number: 20185001-3).

The body weights of mice were recorded once a week for 11 weeks. Blood samples were collected and centrifuged (3 000 r/min,4 °C for 10 min) for the separation of serum. Liver and adipose tissues were quickly taken and stored at ?80 °C or by immersion in formaldehyde solution after sacrifice. All mice survived after 11 weeks treatment without casual or obvious signs of toxicity.

2.3.2 Oral glucose tolerance test (OGTT)

The oral glucose tolerance test (OGTT) was performed as previously described [26]. The blood glucose level of mice after overnight fasting was measured (0 min). Then, mice were orally administrated with glucose solution (2 g/kg body weight, 20% glucose) and the blood glucose level was monitored at 30, 60, 90,120, and 150 min, respectively.

2.3.3 Blood lipids and oxidative stress level

Lipids of high-density lipoprotein cholesterol (HDL-C), lowdensity lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triacylglycerols (TG) in the serum were measured using commercially available assay kits. The antioxidant enzyme activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum, superoxide dismutase (SOD) and glutathion peroxidase(GSH-Px) in the liver, and malonaldehyde (MDA) contents were determined by specific diagnostic kits. All measurements were followed the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, China).

2.3.4 Histological analysis

Fresh liver and subcutaneous fat tissues were fixed, embedded,sliced, and then subjected for H&E staining. The size of subcutaneous fat and hepatic steatosis degree of stained tissue samples were recorded using a BA210T microscope (Motic Medical Diagnosis System Co., Ltd, China) equipped with a digital camera (Digital Sight DS-Fi1c, Nikon Corporation, Japan) [27].

2.3.5 Immuno fluorescence analysis

After deparaffination and citrate antigen retrieval, liver tissues were permeabilized with 0.5% Triton in phosphate buffered saline(PBS) and then blocked with 1% bovine serum albumin (BSA) and 2% fetal bovine serum (FBS) in PBS for 60 min. After blocking,the primary antibody of anti-F4/80 (1:200 dilution; Abcam, MA,USA) was applied overnight. All sections were then incubated for 1 h with Alexa Fluor 488 goat anti-rabbit IgG (1:200 dilution;Abcam, MA, USA) followed by incubation with DAPI (Sigma, MO,USA). Immunofluorescence images were obtained using a confocal ultraspectral microscope (FV3000, Olympus, Japan).

2.3.6 Gene expression quantification

Total RNA extraction from liver tissues was conducted using 9 767 mRNA extraction kit (Takara, Japan). The extracted RNA was then reverse-transcribed into cDNA using PrimeScript? RT reagent kit. The expression of interleukin 1 beta (IL-1β), nitric oxide synthase 2 (Nos2), cyclooxygenase 2 (Cox2), toll-like receptor 4 (TLR4),myeloid differentiation factor 88 (MyD88), nuclear factor kappa-B subunit 1 (NF-κB1), phosphoinositide-3-kinase regulatory subunit 1(PI3Kr1), thymoma viral proto-oncogene 1 (Akt1), mechanistic target of rapamycin kinase (mTOR), peroxisome proliferator activated receptor gamma (PPARγ), fatty acid synthase (FASN), peroxisome proliferator activated receptor alpha (PPARα), and hormone sensitive lipase (Lipe) genes were quantified by RT-qPCR with the primers listed in supplementary Table S3. The thermal cycle conditions were as follows: 95 °C for 30 s; 40 cycles of amplification (95 °C for 5 s,60 °C for 30 s); 72 °C for 30 s. Then, melting curves were acquired stepwise from 55 °C to 95 °C. GAPDH was used as a reference for normalization. The expression level of the target gene was measured through comparative CT method.

2.3.7 Protein extraction and western blotting

Frozen colon tissues (100 mg) were homogenized on ice in 700 μL RIPA lysis buffer with protease inhibitor cocktail and phosphatase inhibitor. The lysates were centrifuged at 12 000 ×g(4 °C for 15 min)for collection of supernatants. The total protein content was measured using BCA protein assay kit (Biomiga, USA). An equal amount of protein sample (20 μg) was separated with 8% SDS-PAGE and transferred to nitrocellulose membrane. Then, the membrane was incubated with specific primary antibody (1 : 1 000) against MyD88 (CST, cat. number 4283S), NF-κB (p65) (CST, cat. number 8242S), PI3K (p110β) (Proteintech, cat. number 20584-1-AP), AKT(Proteintech, cat. number 10176-2-AP), Thr308 phosphorylated AKT (p-AKT) (CST, cat. number 13038T), mTOR (Proteintech, cat.number 20657-1-AP), Ser2448 phosphorylated mTOR (p-mTOR)(CST, cat. number 5536T), PPAR-γ (CST, cat. number 2435T),FASN (CST, cat. number 3180T), and HSL (CST, cat. number 18381T) overnight at 4 °C, respectively. The grayscale of protein bands was quantified by the automatic chemiluminescence imaging system (Tanon 4800, Tanon Science and Technology Co., Ltd,China). GAPDH (CST, cat. number 5174T) was used as the internal reference.

2.4 Statistical analysis

All data were expressed as mean ± standard deviation (SD).Statistical difference was determined by one-way analysis variance(ANOVA), along with the Tukey’s test atP＜ 0.05 (SPSS 23.0 package, SPSS Inc, Chicago, IL, USA).

3. Results

3.1 Morphology and sensory evaluation of hawthorn leathers

As shown in Fig. 1A, traditional hawthorn leathers containing sucrose (HLS) showed a bright red color and high transparency.Substitution of sucrose with FOS/XOS (75:25) (HLF75) resulted in slightly lighter color of the hawthorn leathers, while the transparency was not significantly different from that of the HLS group. The substitution of sucrose with XOS significantly reduced the color and transparency of hawthorn leathers. Unfortunately, the puree of hawthorn leathers without sucrose (HL) and hawthorn leathers formulated with 50% FOS and 50% XOS (HLF50) displayed a very high fluidity during the laying process so that it could not be laid(images not shown). HLS presented a porous appearance and a loss in cell wall morphology (Fig. 1B), which was a characteristic of the dehydrated gels of pectin and sucrose. In hawthorn leathers formulated with XOS (HLX), a more compact matrix was formed with few pores.Micrographs of HLF75 and hawthorn leathers containing FOS (HLF)were similar in appearance to those of the HLS group.

Fig. 1. Morphology and sensory evaluation of hawthorn leathers. (A) Gross appearances of hawthorn leathers; (B) Scanning electron micrographs of hawthorn leathers; (C) Radar chart of sensory evaluation; (D) Total scores of sensory evaluation.

In terms of color, HLS exhibited the highest organoleptic quality(9 score), followed by HLF, HLF25, and HLF75 (7 score), while HLX displayed the lowest value (5 score) (Fig. 1C). HLF75 exhibited the best sensory properties in relation to the texture of cohesiveness,springiness, and chewiness, which were similar to HLS. Taking into account the scores of acidity and sweetness, HLS showed the best mouthfeel of all formulations while hawthorn leathers with sucrose substitutes had the discomfort in sour and sweet. From the sensory analysis, it was concluded that HLF75 exhibited similar overall acceptability to that of the HLS (Fig. 1D).

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3.2 HLF75 treatment decreased the fat and body weight of obese mice

As compared with the NC group, HFD significantly increased the body weight of mice (P＜ 0.05) while the body weight of the HLS group was even higher than that of the HFD group (Table 1).HLF75 treatment significantly decreased the body weight when compared with both HFD and HLS groups (P＜ 0.05), which was comparable with that of the Met group. Similarly, the weights of perinephric fat, epididymal fat, and subcutaneous fat were decreased after HLF75 treatment as compared with both HFD and HLS groups(P＜ 0.05), resulting in decreased body fat percentage (P＜ 0.05 versus HLS group).

Table 1Effect of hawthorn leathers treatment on fat and body weight of obese mice.

3.3 HLF75 treatment regulated blood parameters of obese mice

As shown in Table 2, mice fed with HFD plus intragastrically administrated homogenization buffer of HLS (HLS group)significantly increased the levels of ALT and AST as compared with the NC group (P＜ 0.05), which were even higher than those of the HFD group. The contents of TC, TG, and LDL-C in the HLS group were significantly increased (P＜ 0.05), while the HDL-C content was significantly decreased after HLS treatment (2.25 mmol/L)(P＜ 0.05). HLF75 treatment significantly attenuated the levels of ALT and AST in the blood of mice, as well as the decreased contents of TC,TG, and LDL-C. The HDL-C content of HLF75 was also recovered(2.76 mmol/L) to a comparable level of the NC group (2.95 mmol/L).

Table 2Effect of hawthorn leathers treatment on blood parameters of obese mice.

3.4 HLF75 treatment ameliorated glucose tolerance and oxidative stress of obese mice

Comparing with mice in both HFD and HLS groups, HLF75 treatment improved glucose tolerance as evidenced by the plasma glucose levels after glucose challenge (Fig. 2A). The blood glucose level of mice in the HLF75 group at 150 min was markedly reduced by 15.7% as compared with the impaired blood glucose in the HFD group. Consistently, the total areas under the blood glucose curve (AUC) were significantly increased in HFD and HLS groups as compared with that of the NC group. The administration of sucrose-free hawthorn leathers formulated with 75% FOS and 25% XOS (HLF75 group) significantly decreased the AUC which was comparable to that of the NC group.

Fig. 2 Effect of hawthorn leather treatment on oral glucose tolerance and oxidative stress in mice. (A) Blood glucose level of mice; (B) AUC of mice; (C) SOD activity in the liver; (D) The activity of GSH-Px in the liver; (E) The content of MDA in the liver. Data are expressed as mean ± SD (8 mice/group). Values of eachgroup with different letters over the bars are significantly different (P < 0.05) by analysis of one-way ANOVA followed by Tukey’s multiple range test.

Fig. 2 (Continued)

The oxidative stress in the liver of mice was investigated by measuring the activities of SOD and GSH-Px, as well as the content of MDA. As compared with the HFD group, the SOD activity of mice fed with HFD plus HLF75 was significantly increased by 38.9%(P＜ 0.05) (Fig. 2C). Consistently, GSH-Px activity in the HLF75 group was significantly increased by 34.5% (P＜ 0.05) when compared with that of the HLS group (Fig. 2D). Mice fed with HFD plus HLS (HLS group) showed a significantly elevated level of MDA as compared with that of the NC group (P＜ 0.05). However, the HLF75 group significantly suppressed the MDA production induced by HFD, which was significantly reduced by 55.7% against that of the HLS group (Fig. 2E).

3.5 HLF75 treatment improved the histomorphologies of liver and subcutaneous fat tissues

For the NC group, the structure of liver tissue was clear and integrated with the hepatic cells of normal morphology regularly arranged (Fig. 3A). HFD induced disordered cell arrangement, illde fined hepatocytes boundary, fat vacuoles, edema of liver lobules,and hepatic steatosis in the liver tissues. Supplementation of HLS in the HFD (HLS group) severely damaged the liver structure of mice with a large number of vacuoles in the liver cells. However, the intake of HLF75 significantly lowered the degree of hepatic steatosis with a reduced number of fat vacuoles in the hepatic lobule when compared with HLS (P＜ 0.05) (Fig. 3C). Particularly, the degree of disordered hepatic lobule was apparently reduced in the HLF75 group with regularly arranged liver cells.

Fig. 3 Effect of hawthorn leather treatment on histomorphological changes of the liver and subcutaneous fat tissues in mice. (A) Histopathological appearance of liver tissue (H&E staining 200 ×). Vein (black arrow), fat droplets (blue arrow), and the cell nucleus (red arrow); (B) Histopathological appearance of subcutaneous fat (H&E staining 200 ×). The adipocytes (black arrow), and the boundaries between adipocytes (red arrow); (C) Hepatic steatosis grade (1-5 scale); (D) Adipocyte size of subcutaneous fat.

Fig. 3 (Continued)

H&E staining of the subcutaneous fat tissue showed that the adipose size of HFD-fed mice was much bigger than that of the NC group mice (Figs. 3B and 3D). Dietary supplementation with Met or HLF75 in the HFD dramatically reduced the adipose size(P＜ 0.05). Interestingly, when HLS were incorporated into the HFD, the increased adipose size of subcutaneous fat as well as the blurred boundary was displayed when compared with other groups.Thus, the HLF75 treatment attenuated the damage on adipose tissue induced by HFD.

3.6 HLF75 treatment inhibited the inflammatory response of obese mice

Fig. 4 Effect of hawthorn leather treatment on expression levels of inflammatory cytokines and NF-κB signaling pathway in the liver tissue of mice. mRNA expression level of (A) IL-1β, (B) Nos2, (C) Cox2, (D) TLR4, (E) MyD88, and (F) NF-κB; (G) Images of western blotting; Protein expression ratio of (H) MyD88 to GAPDH and (I) NF-κB to GAPDH. Data are expressed as mean ± SD (8 mice/group). Values of each group with different letters over the bars are significantly different (P < 0.05) by analysis of one-way ANOVA followed by Tukey’s multiple range test.

3.7 HLF75 treatment inhibited the number of macrophages of obese mice

The effect of hawthorn leather treatment on the number of macrophages in the liver tissues was then examined to further re flect the inflammatory state. As shown in Fig. 5, the numbers of F4/80-positive macrophages in HFD and HLS groups were significantly increased as compared with the NC group. Interestingly, a very pronounced decrease in the number of F4/80-positive macrophages in Met and HLF75 groups was observed.

Fig. 5 Effect of hawthorn leathers on the number of F4/80-positive macrophages in the liver tissue of mice as measured by immuno fluorescence microscopy.

3.8 HLF75 treatment ameliorated the glucose metabolism in obese mice

Due to the important role in glucose metabolism, the activation of PI3K/AKT/mTOR signaling pathway as affected by hawthorn leathers formulated with sucrose or 75% FOS/25% XOS was investigated. As compared with the NC group, the mRNA expression levels ofPI3Kr1,Akt1, andmTORgenes in liver tissue of HFD group mice were significantly reduced (P＜ 0.05, Figs. 6A-6C).In contrast, after treatment with HLF75, the expression level ofPI3Kr1,Akt1, andmTORgenes was increased by 110.2% (P＜ 0.05),151.2%, and 667.1% (P＜ 0.05), respectively, as compared with the HFD group. Similarly, the protein expression levels of PI3K(p110β), p-AKT (Thr308), and p-mTOR (Ser2448) were significantly suppressed by HFD and/or HFD plus HLS. Nevertheless, HLF75 treatment significantly up-regulated the protein expression levels of PI3K (p110β), p-AKT (Thr308), and p-mTOR (Ser2448) (P＜ 0.05,Figs. 6E-6G).

Fig. 6 Effect of hawthorn leather treatment on glucose metabolism in the liver tissue of mice. mRNA expression level of (A) PI3Kr1, (B) Akt1, and (C) mTOR; (D)Images of western blotting; Protein expression ratio of (E) PI3K to GAPDH, (F) p-AKT to AKT, and (G) p-mTOR and mTOR. Data are expressed as mean ± SD(8 mice/group). Values of each group with different letters over the bars are significantly different (P < 0.05) by analysis of one-way ANOVA followed by Tukey’s multiple range test.

3.9 HLF75 treatment improved the lipid metabolism of obese mice

As presented in Fig. 7A and 7B, HFD promotedPPARγandFASNgenes expression in the liver. The intake of HLS further increased the mRNA expression ofPPARγandFASNgenes when incorporated into the HFD. As compared with the HFD group, the expression level ofPPARγandFASNgenes was decreased by 48.1% and 44.2%,respectively (P＜ 0.05), due to the incorporation of HLF75 in the HFD group. Nevertheless, the expression level ofLipeandPPARαgenes in the HLF75 group was increased by 72.9% and 249.4%,respectively (P＜ 0.05).

Fig. 7 Effect of hawthorn leather treatment on lipid metabolism in the liver tissue of mice. mRNA expression level of (A) PPARγ, FASN, (C) Lipe, and (D)PPARα; (E) Images of western blotting; Protein expression ratio of (F) PPAR-γ to GAPDH, (G) FASN to GAPDH, and (H) HSL to GAPDH. Data are expressed as mean ± SD (8 mice/group). Values of each group with different letters over the bars are significantly different (P < 0.05) by analysis of one-way ANOVA followed by Tukey’s multiple range test.

Fig. 7 (Continued)

The protein expression levels of PPAR-γ and FASN in the HLS group were increased by 7.1% and 2.3%, respectively, when compared to that of the HFD group. However, pronounced downregulation of PPAR-γ and FASN expression in the HLF75 group was observed (P＜ 0.05) (Figs. 7F, 7G). Strikingly, the expression level of HSL in the HLF75 group was increased by 68.4% as compared with the HFD group (P＜ 0.05) (Fig. 7H).

4. Discussion

The reduction of sucrose consumption is one of the major challenges in food industry [15]. Therefore, it is reasonable to replace sucrose with other sweeteners. However, sucrose alternatives should not cause significant changes in the sensory characteristics of food products [11]. In this study, sucrose-free hawthorn leathers formulated with functional oligosaccharides were produced and the anti-obesity properties were evaluated.

Among the prepared sucrose-free hawthorn leathers, HLF75 showed comparable colour and morphology with that of HLS,followed by HLF (Figs. 1A and 1B). Moreover, HLF75 was scored as the best in terms of mouthfeel, flavour, and textural properties among all sucrose-free hawthorn leathers (Figs. 1C and 1D). The substitution of sucrose with FOS or XOS alone affected the hardness, gumminess,and cohesiveness of hawthorn leathers. However, Pereira et al. [22]reported that the addition of FOS did not influence the textural properties of functional sugar-free guava preserves. The decreased sensory properties of formulated sucrose-free hawthorn leathers might be due to the weak ability of single FOS or XOS to bind with pectin, while the greater interaction between pectin and FOS/XOS (75:25) conferred a softer texture to the formulated sucrosefree hawthorn leathers.

Both FOS and XOS have been reported to possess a variety of physiological activities, including anti-obesity activity [21,28].Pectin, flavonoids, and polyphenols in hawthorn were considered to contribute to the health beneficial effects against body weight gain and lipid metabolism dysfunction [29-31]. Based on the nutritional components we measured in hawthorn leathers (Supplementary Table S2), the effective dosage of pectin (150?300 mg/kg·bw),polyphenols (300?400 mg/kg·bw), and flavonoids (5?20 mg/kg·bw)has not been reached for mice gavaged with HLS and HLF [32-34].Thus, FOS and XOS are possibly the dominant ingredients for the observed anti-obesity activity of HFL75. Adipose tissues of epididymal fat, perinephric fat, and subcutaneous fat are considered as predictors of the development of metabolic disorders [35]. In this study, supplementation of HLF75 decreased the weight of adipose tissues in HFD-fed mice (Table 1). This may be due to the supplementation of XOS and FOS in HFD decreased fat accumulation by suppressing the expression of adipogenesis genes and altering intestinal microbial composition [12,36]. It has been proven that obesity is associated with disturbances in glucose and blood lipids such as hyperglycemia and hyperlipidemia [37]. The intake of HLF75 markedly lowered serum lipid contents and ameliorated glucose tolerance in HFD-fed mice (Table 2; Figs. 2A and 2B). Sucrose can be easily absorbed in the intestinal tract [8], resulting in increased the blood glucose level in the HLS group. Furthermore, HLF75 treatment improved SOD and GSH-Px activities, and reduced the MDA level in the liver of mice (Figs. 2C-2E). This indicated that the provision of functional oligosaccharides might be an attractive strategy for ameliorating oxidative damages and further retarding obesityassociated pathological conditions [38].

Liver injury is the main relative factor of obesity [39]. Hong et al. [40]reported that the liver of obese rodents exhibited the accumulation of numerous fat droplets as compared to that of the normal group. Consistently, HFD caused hepatic steatosis, while supplementation of HLF75 exhibited a significant hepatoprotective effect (P＜ 0.05) (Figs. 3A and 3C). H&E staining depicted that the subcutaneous adipose size of HLS-fed mice was even bigger than that of the HFD group (Figs. 3B and 3D). Nevertheless, supplementation of HLF75 in HFD effectively ameliorated liver damage and reduced adipose size, suggesting the protective effects of prebiotic hawthorn leathers in ameliorating metabolism dysfunction induced by HFD.Inflammation has been reported to be associated in liver injury.TLR4, the LPS receptor, triggers the activation of NF-κB via MyD88-dependent pathway, resulting in up-regulation of pro-inflammatory mediators [41]. Previous study demonstrated that a high-glucose medium enhanced the expression of pro-inflammatory cytokines [42].In this study, HLF75 treatment prevented the HFD-mediated increase in the transcription levels ofIL-1β,Nos2, andCox2genes(Figs. 4A-4C). Activation of NF-κB induces the expression of proinflammatory factors encoded by genes [43]. As a negative regulator,HLF75 treatment suppressed the protein expression of MyD88 and NF-κB (p65) (Figs. 4G-4I). For the development of many inflammation related chronic diseases, macrophages are activated,releasing pro-inflammatory factors [44]. Similarly, we found that HLF75 treatment significantly decreased the number of F4/80-positive macrophages in liver tissue (Fig. 5). These results suggested that the anti-inflammatory effect of HLF75 might be associated with the suppression of TLR4/MyD88/NF-κB pathway and inhibition of macrophage activation in liver tissue of mice.

PI3K/AKT/mTOR pathway has been proven to be a classical pathway for activation of insulin receptor to regulate glucose metabolism [45]. With regard to obesity, a down-regulation of PI3K/AKT/mTOR pathway resulting in adverse effects on glucose metabolism has been shown in adipose tissue [46]. Similarly, our results demonstrated that treatment with HLF75 effectively reinstated the expression levels of PI3K/AKT/mTOR pathway associated genes and proteins (Fig. 6), which might be owing to the synergistic effects of XOS and FOS. Notably, a recent study found that the oligosaccharides from seaweedSargassum confusumameliorated the abnormality of blood lipid and impaired glucose tolerance in diabetic animals by up-regulating the PI3K signaling pathways [47].

Activation of PI3K/AKT/mTOR pathway can also improve the disturbance in lipid metabolism [48]. It has been previously demonstrated that PPAR-γ suppression can inhibit glycolysis and accordingly the expression of PI3K/AKT/mTOR signaling pathway [49].PPARγis a key regulation gene in adipogenesis in coordination withFASN, which can stimulate preadipocyte differentiation and the generation of mature adipocytes, thus enhance lipid accumulation [50,51].Circulation of free fatty acid levels is predominantly decided by the metabolism of the stored triglycerides through enzymatic lipolysis [52], whileLipeandPPARαare the primary genes involved in the lipolysis process. In the present study, supplementation of HLF75 significantly inhibited the adipogenic gene expression ofPPARγandFASN(Figs. 7A and 7B), and promoted the expression of lipolysis genes, namelyLipeandPPARα(Figs. 7C and 7D). Dietary supplementation of XOS and FOS has been reported to reduce the expression of genes involved in adipogenesis and fatty acid synthesis [21,53]. Therefore, these changes in gene expression might be responsible for the observed reduction in weights of adipose tissues and body, and recovery of liver injury. The protein expression of HSL was significantly up-regulated, while PPAR-γ and FASN expression levels were down-regulated in the HLF75 group as compared to the HFD group (Figs. 7F-7H). The changes in protein expression might improve fat utilization and inhibit fat synthesis, leading to anti-obesity effects of hawthorn leathers formulated with functional oligosaccharides. Therefore, our findings indicated that hawthorn leathers formulated with 75% FOS/25% XOS can contribute to improved glucose and lipid metabolism mediated by PI3K/AKT/mTOR signaling pathway.

5. Conclusion

Sucrose-free hawthorn leathers containing 75% FOS/25% XOS(HLF75) exhibited comparable sensory properties with traditional hawthorn leathers. HLF75 showed anti-obesity activity with suppressed body weight gain, blood glucose and oxidative stress, and improved the histology of liver and subcutaneous fat tissues. HLF75 supplementation ameliorated inflammatory responses in obese mice through down-regulating expression levels of TLR4/MyD88/NF-κB pathway associated genes and proteins and inhibition of macrophage activation. The restoration of PI3K/AKT/mTOR pathway by HLF75 might improve glucose and lipid metabolism in obese mice. Our findings shed light on the application of functional oligosaccharides as sucrose substitutes for functional food production.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgments

This project was supported by the National Natural Science Foundation of China (31630096).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.fshw.2022.03.033.