The herbal formulas were composed of extracts from AJ, AG, AM, LJ, TP and PV, at w/w ratios of 1:1:1:1:1:1, 1:1:1:2:2:2 or 2:2:2:1:1:1. The herbal formulas were termed LCBP-Anocure-16001, 16002 and 16003 (LA16001, LA16002, LA16003), respectively. Herbal medicines were obtained from Kyung Hee Herb Pharm (Wonju,) and Omniherb (Seoul, Korea). LA16001 was composed of 40 g each of AJ, AG, AM, LJ, TP and PV; LA16002 was composed of 25 g each of AJ, AG and AM and 50 g each of LJ, TP and PV; LA16003 was composed of 50 g each of AJ, AG and AM and 25 g each of LJ, TP and PV. The ingredients were placed in 15-fold volume of distilled water and boiled at ~100°C for 3 h. The extracts were filtered using 75 µm microfilters. The filtered residues were placed in a 15-fold volume of distilled water, boiled at ~100°C for 3 h and refiltered through 75-µm microfilters. The extracted liquid was concentrated under low pressure at 60°C. Finally, 81.8 g (LA16001), 84 g (LA16002) and 93.3 g (LA16003) dried extract was obtained; the yields were 34.08, 37.33 and 41.47%, respectively. The extracts were prepared according to the Good Manufacturing Practice guidelines (31,32). SJDBT was obtained from Hanpoong Pharm & Foods Co., Ltd. (Jeonju, Korea) and was prepared as previously described (16). Cisplatin (8 mg/kg) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was dissolved in distilled water.

A total of 98 male BALB/c mice (age, 6 weeks) were purchased from Orient Bio Inc. (Seongnam, Korea). Mice (n=4/group) were randomly assigned to the following groups: Normal, control, cisplatin, LA16001, LA16002, LA16003, SJDBT, MGA (Sigma-Aldrich; Merck KGaA), AJ, AG, AP, LJ, TP and PV, and were maintained at 23±3°C (humidity, 55±15%), under a 12-h light/dark cycle. Access to food and water was ad libitum, apart from during the experimental period. All groups (except the normal group) were fasted for 24 h prior to the start of the experiment (the normal group was used as a control for starvation). The control group did not receive any drug treatment, only starvation; this group was used as a control for the cisplatin group. All groups (except the normal and control groups) were induced to become anorexic through administration of cisplatin. Therefore, the cisplatin group was used as a control for the drug treatments. The drug was administered at the concentration of 1,000 mg/kg on days 1–3. After 24 h, 8 mg/kg cisplatin was intraperitoneally injected into the mice. During fasting, water was continuously provided and food was provided following the oral administration of drugs. Food intake and body weight were measured daily throughout the duration of the study (Fig. 1A). An equal amount of food was given at the same time each day and mice were weighed the following day. All mice were sacrificed at the time labeled ‘Sac’ in the animal study design presented in Fig. 1A. Blood and adipose tissue, stomach, and hypothalamus were obtained from the sacrificed mice.

The present study was approved by the Animal Ethics Committee of Kyung Hee University [approval no. KHUASP (SE)-016-100].

Samples from stomach, fat and hypothalamic tissue were isolated from mice sacrificed 4 h subsequent to the administration of cisplatin (n=4 mice/group), and lysed with radioimmunoprecipitation assay lysis buffer (Biosesang, Inc., Seongnam, Korea). Lysates were centrifuged at 196 × g for 20 min at room temperature, and supernatants were collected and used for the assessment of target proteins using ELISA. The levels of ghrelin (cat. no. EZRGRA-90K; EMD Millipore, Billerica, MA, USA), leptin (cat. no. DY498; R&D Systems, Inc., Minneapolis, MN, USA) and IL-6 (cat. no. 555240; BD Biosciences, San Jose, CA, USA) were measured using ELISA kits according to the manufacturer's protocol. The blood of the mice sacrificed 3 days subsequent to the administration of cisplatin was used. Control group and cisplatin group, LA16001 group mice were used, 4 mice/group. Exsanguination was performed, and whole blood was analyzed using the HEMAVET^® 950 hematology system (Drew Scientific Inc., Miami Lakes, FL, USA). SigmaPlot, version 2001 (Systat Software, Inc., San Jose, CA, USA) was used for analysis of the blood data. To anesthetize, 400 µl/mouse 1.2% avertin solution was intraperitoneally injected [1.2% avertin solution, 0.5 g 2,2,2-tribromoethanol powder (Sigma-Aldrich; Merck KGaA) dissolved in 1 ml 2-methyl-2-butanol (Sigma-Aldrich; Merck KGaA) at 55°C, and 39 ml PBS]. The avertin solution was filtered through a Nalgene 0.22-µm filter (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Adipose tissue was isolated from mice. To obtain protein, the adipose tissue was lysed using radioimmunoprecipitation assay buffer (Biosesang, Inc.) and was centrifuged at 24,562 × g at 4°C for 20 min. Protein concentration was measured using a Bradford assay. Equal amounts of extracted protein samples (20 µg) were separated by 8% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (GE Healthcare Life Sciences, Chalfont, UK). Membranes were blocked in 1% bovine serum albumin (Sigma-Aldrich; Merck KGaA) and 2% skimmed milk for 1 h at room temperature. Membranes were then incubated at 4°C overnight with the following primary antibodies: Anti-phosphorylated (p)-JAK1 (1:1,000; cat no. 3331; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-p-STAT3 (1:1,000; cat no. 9131; Cell Signaling Technology, Inc.) and anti-α-tubulin (1:10,000; cat no. T-5168; Sigma-Aldrich; Merck KGaA). Subsequently, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:2,000 in 1× PBS with 0.05% Tween-20; cat. no. 5450-0010; KPL, Inc., Gaithersburg, MD, USA) for 1 h at room temperature. Protein bands were visualized using Enhanced Chemiluminescence kit solution (DoGen, Seoul, Korea) and quantified using ImageJ software version 1.49v (National Institutes of Health, Bethesda, MD, USA) and normalized to α-tubulin.

The data are expressed as the mean ± standard deviation and experiments were performed on 4 mice/group. The statistical significance of the differences between groups was assessed using Student's t-test. Statistical analysis was performed using Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, USA). P<0.05 was considered to indicate a statistically significant difference. One-way analysis of variance was additionally performed with post hoc test (Tukey), using SPSS software (version 23; IBM Corp., Armonk, NY, USA).

Mice were fasted for 24 h and intraperitoneally injected with 8 mg/kg cisplatin. Experimental drugs were orally administrated 24 h before cisplatin injection. To investigate the effects of the experimental drugs on food intake and body weight, mice were maintained for 3 days following the injection of cisplatin (Fig. 1A). The results of the present study demonstrated that following cisplatin administration, food intake and body weight decreased (Figs. 1B and C, respectively). Notably, LA16001, LA16002, LA16003, SJDBT and MGA alleviated cisplatin-induced loss of appetite (Fig. 2A), and LA16001 and MGA significantly reversed decreases in body weight (Fig. 2B). LA16001 appeared to have more potent orexigenic effects on food intake, compared with all other treatment groups (Fig. 2A). In addition, all treatment groups demonstrated significantly reversed cisplatin-induced loss of appetite, with LA16001 exerting the most pronounced effect (Fig. 3A). Furthermore, LA16001, AP, AG and TP treatment significantly reversed loss of body weight in experimental mice, again with LA16001 being the most effective (Fig. 3B). These results suggested that LA16001 may be able to counteract cisplatin-induced anorexia in vivo.

A total of 4 h post-cisplatin injection, the hormone levels of ghrelin, leptin and IL-6 were assessed in mouse tissue and serum samples. Mice in the cisplatin and LA16001 groups exhibited increased ghrelin levels in stomach tissue samples compared with mice in the fasting control group (Fig. 4A). Cisplatin was demonstrated to reduce leptin levels in fat and hypothalamic tissue, whereas LA16001 appeared to counteract this effect (Figs. 4B and C). IL-6 levels were assessed in serum and fat tissue samples. IL-6 levels in fat tissue were not altered following cisplatin administration; however, they were significantly decreased in serum samples. Notably, LA16001 administration was demonstrated to significantly increase fat tissue and serum IL-6 levels (Figs. 4D and E). These results indicated that LA16001 administration increased the levels of ghrelin, leptin and IL-6 in cisplatin-treated mice.

A total of 4 h post-cisplatin injection, the phosphorylation status of JAK1 and STAT3 was investigated in fat tissue samples. LA16001 administration resulted in significantly increased protein expression levels of p-JAK1 and p-STAT3 compared with the cisplatin group (Figs. 5A and B). These results suggested that the molecular mechanisms underlying the orexigenic effects of LA16001 in cisplatin-induced anorexic mice may involve the activation of the JAK1/STAT3 intracellular signaling pathway.

White blood cell and neutrophil counts were performed 3 days after cisplatin administration to evaluate the effects of LA16001 on chemotherapy-induced immunosuppression. Treatment with cisplatin was demonstrated to reduce the numbers of white blood cells (Fig. 6A) and neutrophils (Fig. 6B) in mouse whole blood samples. Notably, LA16001 administration appeared to counteract the effects of cisplatin on immune cell numbers (Fig. 6). These results suggested that LA16001 may mitigate the immunosuppressive effects associated with cisplatin treatment.