Structure characterization of red raspberry ketones
The FT-IR spectra of RRK obtained by ultrasonic-assisted ethanol are shown in Fig. 1A We found that the spectrum of RRK was in line with the standard sample. We observed a strong and wide absorption peak at 3371.21 cm–1, which was consistent with the characteristic absorption peak range of hydroxyl (3000–3750 cm–1). We also observed an absorption peak around 1550 cm–1, which was associated with a benzene ring. We inferred that a phenolic hydroxyl structure was present in RRK [24, 25].
The spectra of raspberry ketone standard obtained from GC–MS are shown in Fig. 1C The peak time of the standard was 4.685 min. The standard curve was Y = 415.14X − 53.714, R2 = 0.9990 (Fig. 1B). As shown in Fig. 1C–D, the shape of the RRK chromatographic peak was similar to the raspberry ketone standard, and the peak time was 4.699 min. The purity of RRK was 80.06 ± 1.19%.
Effect of RRK on the biochemical indicators in serum
As shown in Fig. 2A, the body weight of rats in the high-fat diet group was significantly increased than those in the normal-diet group after they were fed for 4 weeks (260.12 ± 6.41 g vs 288.31 ± 16.60 g, P < 0.01). The HDL-C levels decreased remarkably, and the LDL-C, TC, and TG levels increased significantly (P < 0.05), which indicated that the NAFLD modeling was successful (Fig. 2B) [26].
During the administration period, the rats administered with RRK by oral gavage were more stable, moved freely, and had a better mental state and a shiny coat. Their diet and water intake were normal, and they did not show any abnormalities. However, the rats in the MC group which didn’t administered with RRK had a darker and rougher coat because of the metabolic disorders in liver caused by NAFLD.
After 28 days of treatment, the body weight of rats in the MC group increased significantly (P < 0.05) than those in the NC group. The weight of rats in the PC, HRK, MRK, and LRK groups decreased significantly (P < 0.05) than those in the MC group. This indicated that RRK can decrease the body weight of NAFLD model rats (Fig. 3A). We also observed that the HDL-C levels of the NAFLD model rats increased, and the LDL-C, TC, and TG levels remarkably decreased after the treatment with RRK. (Fig. 3B–E, P < 0.05). This indicated that RRK can promote the breakdown and metabolism of lipids in the serum, and the ability to transport cholesterol and triglycerides was improved in the NAFLD model rats. Thus, RRK has a positive effect on the removal of the deposited lipids from the arterial wall of blood vessels.
Some studies have also found similar results. Reem et al. found Garcinia cambogia (Garcinia gummi-gutta) and raspberry ketone could alleviate hepatocyte inflammation, fatty changes, and oxidative stress caused by a high-fat fructose diet [27]. Moreover, Wang et al. also found that raspberry ketone could protect the liver and reduce fat in the body against nonalcoholic steatohepatitis [28].
AST and ALT were released from the liver after its injury, which reflected the level of liver injury. As shown in Fig. 4A–B, the rats in the MC group showed a large increase in AST levels than those in the NC group (P < 0.01), whereas the AST levels decreased in the PC and RRK groups than those in the MC group (P < 0.01). Moreover, the ALT levels in the MC group also exhibited a remarkable improvement compared with those in the normal group (P < 0.01), whereas the ALT levels in the PC and RRK treatment groups, showed a highly significant decrease compared with those in the MC group (P < 0.01). Thus, RRK can decrease the levels of ALT and AST in serum. We inferred that RRK may repair necrotic hepatocytes to a certain extent and might be able to decrease the permeability of the cell membrane. Overall, RRK can inhibit the further development of NAFLD, which has certain significance in its clinical prevention. These results are consistent with some previous studies. Faheem et al. reported that cranberry extract can decrease ALT and AST levels in serum and can retard the progression of NAFLD [14]. The extract from pomegranate peel can decrease the levels of ALT and AST and can be used in the treatment of NAFLD [29]. AKP is overproduced when the liver cells are damaged. Compared with the NC group, the AKP level in the MC group significantly increased (P < 0.01), and the AKP level in the PC group and the groups with different doses of RRK significantly decreased (Fig. 4C, P < 0.01) compared with the MC group. This indicated that RRK can effectively reduce AKP levels in serum and control the abnormal metabolism and impaired excretion of AKP from the body. These results were consistent with those of other studies [30, 31]. Hence, the results showed that RRK can prevent the inflammatory response in the liver to alleviate NAFLD.
Some studies have reported that abnormal lipid metabolism can trigger an increase in the blood glucose levels of the body and insulin resistance can be present, which can contribute to the risk of type 2 diabetes such as Shah et al.’s and Koh et al.’s studies [32, 33]. Based on this, we speculated that RRK can decrease glucose levels. We found that the glucose levels in the MC group increased significantly compared with those in the NC group (P < 0.01), and the model rats treated with different doses of RRK had decreased glucose levels in the serum, which was consistent with the results of the PC group. The results indicated that RRK can inhibit the secretion of blood glucose in vivo (Fig. 5A). Insulin (INS) is a major hormone that lowers blood glucose levels and promotes glycogen synthesis. An abnormal increase in blood glucose levels leads to an increase in the compensatory secretion of INS. The levels of INS increased significantly in the MC group, and this phenomenon was relieved remarkably in the RRK group in a dose-dependent manner (Fig. 5B, P < 0.05). This finding is consistent with those of other studies. A plant extract, procyanidin B2, can improve insulin resistance and glucose–lipid metabolism during NAFLD and reduce blood glucose levels [34]. You et al. also reported that oat β-glucan can relieve abnormal lipid metabolism, which was associated with a decrease in blood glucose levels [35]. In this study, we found that RRK can decrease the glucose and insulin levels in rats, which indicated that RRK can also increase glucose metabolism in vivo. Therefore, we hypothesized that RRK also has a positive effect on diabetes.
FFA concentration in the blood serum is associated with the glycolipid metabolism and endocrine function in vivo and indicates the degree of liver injury. Figure 5C showed that FFA levels were significantly higher in the MC group than those in the NC group (P < 0.01) and were significantly lower in the rats treated with RRK, which was similar to the results of the PC group. High FFA can cause an inflammatory response, which in turn leads to metabolic disturbances in the body. TNF-α is an important indicator of the inflammatory response, which was significantly higher in the MC group than in the normal rats (P < 0.01). After RRK treatment, TNF-α levels in serum indicated a significant decrease (Fig. 5D, P < 0.05). These results suggested that RRK can decrease the production of TNF-α in the adipose tissue. Ngamlerst et al. reported that Maoberry extracts decreased TNF-α levels in the NAFLD model rats and decreased lipid production [36]. A previous study reported that excessive FFA leads to the overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) [37]. Some studies reported that the increased indicators of the inflammatory response can induce oxidative stress in vivo [38, 39]. We speculated that RRK has a positive effect on lipid metabolism, oxidative stress, and inflammatory response in the liver tissues.
Effect of RRK on the biochemical indicators in the liver
Figure 6A shows some representative indicators of lipid metabolism. Compared with the NC group, the TG levels in the liver were significantly higher in the MC group (P < 0.01), whereas it was markedly decreased (P < 0.01) in the PC, HRK, MRK, and LRK groups. This also showed a certain dose–effect relationship. It indicated that RRK can effectively inhibit the increase of TG and maintain lipid metabolism in the liver of NAFLD rats. This result was consistent with the results of some previous studies [40, 41].
To determine whether RRK can alleviate oxidative stress to relieve NAFLD, we evaluated the levels of MDA, SOD, T-GSH, and GSH-Px to determine the antioxidant capacity of the liver (Fig. 6B–E). MDA is a product of lipid peroxidation in the body, which can reflect the degree of lipid peroxidation. This is an indicator of cell damage and biofilm destruction. We found a remarkable increase in the MDA level of the MC group (P < 0.01), which decreased significantly after RRK treatment compared with that of the model group (P < 0.01). Meanwhile, the level of SOD in the serum of the MC group decreased significantly (P < 0.01), which indicated that the ability of NAFLD rats to remove harmful substances decreased during the metabolic process. The level of SOD in the liver increased distinctly after RRK treatment than those in the model group (P < 0.01). Glutathione (T-GSH) is an important antioxidant and free radical scavenger in living organisms. It can bind to harmful substances, such as free radicals and heavy metals and help in their excretion. Glutathione peroxidase (GSH-Px) can decompose lipid hydroperoxides in organisms and inhibit the damage of fatty acids on biofilms. RRK can significantly increase the GSH and GSH-Px levels in NAFLD rats. This suggested that RRK can repair cell damage and biofilm disruption triggered by abnormal lipid metabolism caused by NAFLD. All these findings showed that RRK can improve the antioxidant capacity of NAFLD rats in the liver. Denise et al. reported that the extract of Citrus maxima leaves showed antioxidant activity in the NAFLD model rats, which was consistent with the results of our study [42]. Furthermore, we also speculated that RRK can also have a positive effect on liver morphology.
Morphological observations of the liver
The results of liver pathology staining are shown in Fig. 7. The hepatic tissues of the NC group have a clear and intact structure, along with a certain regularity of cell arrangement and distribution, normal size of hepatocytes, medium-sized nuclei, and a more homogeneous cell body. In the MC group, we observed that the lobules of the liver were heavily damaged, and most cells varied markedly in size and contained a certain amount of lipid droplet vacuolation within the hepatocytes, along with steatosis and fibrosis. Moreover, we observed varying degrees of cellular inflammation with focal necrosis of the glandular follicles, which is consistent with the liver inflammation indicators. After treatment with RRK, the liver lobules were clearer, the hepatic blood sinusoids were more intact, and the hepatic cords were more regularly arranged. We observed no distinct steatosis or inflammatory infiltration. The hepatocytes were more regularly arranged, and fewer intrahepatic lipid droplets were observed in the HRK group. Also, the hepatic cords were more distinctly defined, and the structural integrity of the hepatic lobules was improved more remarkably. These findings were consistent with those of some other reports [43, 44]. These results indicated that RRK has positive therapeutic effects on NAFLD in a dose-dependent manner.