In Vivo Effects of Coffee Containing Javamide-I/-II on Body Weight, LDL, HDL, Total Cholesterol, Triglycerides, Leptin, Adiponectin, C-Reactive Protein, sE-Selectin, TNF-α, and MCP-1

Abstract

Background

Diet plays an unequivocal role in the development of obesity. Interestingly, recent studies have demonstrated that coffee products containing javamide-I/-II may be commonly found in the market. However, there is no information about in vivo effects of coffee containing javamide-I/-II (CCJ12) on obesity-related metabolic factors (body weight, LDL, HDL, total cholesterols, triglycerides, adiponectin, and leptin) in nonobese people.

Objectives

The objective of this study was to investigate in vivo effects of CCJ12 on these metabolic factors as well as inflammatory/cardiovascular disease risk factors [C-reactive protein (CRP), soluble E-selectin (sE-selectin), TNF-α, monocyte chemoattractant protein-1 (MCP-1)] in a nonobese model.

Methods

Sprague-Dawley male rats were fed a complete diet for 20 wk with either drinking water containing CCJ12 [coffee containing javamide-I/-II group (CG), n = 10] or unsupplemented drinking water [water control group (NCG), n = 10]. The amounts of javamide-I/-II in CCJ12 were quantified by HPLC. Water/food consumption and body weight were monitored weekly, and the concentrations of metabolic/inflammatory/cardiovascular disease risk factors were measured by ELISA.

Results

There was no significant difference in water/food consumption between the NCG and CG during the study. Also, no significant difference was found in average body weights between the groups either. In addition, after 20 wk, both groups did not show any significant difference in plasma LDL, HDL, and total cholesterol concentrations. Likewise, adiponectin and leptin concentrations were not significantly different between the groups. As expected, the 2 groups did not show any significant difference in plasma concentrations of CRP and sE-selectin. Furthermore, there was no significant difference in plasma concentrations of TNF-α and MCP-1 between the groups.

Conclusions

The data suggest that CCJ12 may not have significant effects on the metabolic/inflammatory/cardiovascular disease risk factors in the CG, compared with the NCG.

Coffee containing javamide-I/-II is available in the market, but there is limited information related to its health effects. Therefore, its overall effects on metabolic/cardiovascular disease/inflammatory factors were investigated in vivo.

Introduction

Obesity is a serious global health issue (1–3). Worldwide, the prevalence of obesity has more than tripled since 1975, and >340 million children and adolescents (aged 5–19 y) are reported to be overweight or obese (1, 2). Similarly, in the United States, the prevalence of obesity is getting worse, and severe obesity increases currently at an annual rate of >9.0% (3). In fact, increased obesity is believed to be a main contributor to the development/progression of several human chronic diseases such as diabetes, cardiovascular diseases, asthma, hypertension, kidney diseases, osteoarthritis, infection, and cancers (4–12). Particularly, the transition of individuals from the nonobese category [BMI (in kg/m²) < 30] to the obese category (BMI > 30) is occurring at an alarming rate, worsening obesity globally (13, 14). Therefore, it is important to seek approaches to prevent rising obesity in the nonobese population. Although modern lifestyles, high-calorie diets, and genetic factors are considered as major risk factors for obesity, some diet components (e.g., unhealthy meals, drinks) are also blamed for increasing obesity (15, 16). Coffee is one of the popular drinks consumed worldwide including in Europe, Asia, and the United States (17). Particularly, in the United States, >60% of US adults drink coffee daily (17). Related to coffee consumption, there are many positive reports related to several human diseases such as diabetes, cancer, cognitive function, and even all-cause mortality (18–20). However, there are also a limited number of reports suggesting adverse effects of high coffee consumption on high blood pressure, cholesterol, and other disorders (20, 21). For instance, cafestol was reported to increase serum homocysteine and cholesterol concentrations, thereby exerting adverse effects on the cardiovascular system (20). Also, caffeine has been alleged to produce undesirable effects on endothelial function and blood pressure, despite caffeine tolerance in some regular coffee drinkers (21). In addition, some studies have cautiously suggested that high coffee consumption may be associated with some negative metabolic effects (21, 22), indicating that more studies should be conducted to determine potential effects of coffee consumption on metabolic and other factors, particularly related to bioactive compounds in coffee.

Coffee contains numerous bioactive compounds such as caffeine, chlorogenic acids, niacin, and javamide-I/-II. Of particular interest, javamide-I (N-coumaroyltryptophan) and javamide-II (N-caffeoyltryptophan) are tryptophan-conjugated phenolic amide compounds with some health effects (23, 24). Studies have suggested that javamide-I/-II and their derivatives may drive several biological activities including anti-inflammatory activity (24–28). Therefore, there has been increasing interest in their amounts in coffee beans and products found in the market. In fact, my previous studies showed that coffee beans (Arabica and Robusta) could contain javamide-I/-II in the range of 0.03–5.0 mg/g, indicating that many coffee products available in the market may contain javamide-I/-II (23, 24, 29). However, there is currently no information about overall effects of coffee products containing javamide-I/-II on body weight and key metabolic factors [e.g., LDL, HDL, total cholesterol, triglycerides (TGs)], which may be of great interest to coffee drinkers. Therefore, in this study, overall effects of coffee containing javamide-I/-II (CCJ12) on these factors were investigated in vivo using rats fed a normal diet, because coffee health benefits and antiobesity effects would be of interest to the general public including nonobese individuals. In addition, in this study, the effects of CCJ12 on obesity-related adipokines (adiponectin and leptin) and cardiovascular disease/inflammatory risk factors [C-reactive protein (CRP), soluble E-selectin (sE-selectin), tumor necrosis factor-alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1)] were also investigated in vivo, because these risk factors are significantly influenced by obesity (30–49). In particular, the amounts of javamide-I/-II were quantified along with other bioactive coffee compounds (caffeine and chlorogenic acids) in the coffee sample used for this study. Furthermore, the amounts of javamide-I/-II were methodically calculated using the FDA guideline “Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers (50),” providing information about the daily dosages of javamide-I/-II exposed to rats under the study. To the best of my knowledge, this may be the first study about in vivo effects of CCJ12 on body weights, metabolic markers (LDL, HDL, total cholesterol, TGs), adipokines (adiponectin and leptin), and inflammatory/cardiovascular disease risk factors (CRP, sE-selectin, TNF-α, and MCP-1).

Methods

Materials

Ten coffee products (e.g., Folgers, Maxwell House, Nestlé, Starbucks) were purchased from local grocery stores and Internet vendors. Among them, 6 ground coffee samples (6 g) were prepared in 250 mL using a regular coffee maker (Brew N Go coffee maker, Black & Decker). Also, 4 instant coffee samples (2 g) were prepared in 250 mL drinking water. Coumaric acid, caffeic acid, tryptophan, and other chemicals were purchased from Sigma Chemical Co.

HPLC quantification of coffee compounds

For HPLC analysis, caffeine and chlorogenic acids [3-O-caffeoylquinic acid (3-CQA), 4-O-caffeoylquinic acid (4-CQA), 5-O-caffeoylquinic acid (5-CQA)] were purchased from Sigma Chemical Co. and javamide-I/-II standards were prepared as reported previously (23, 24). Briefly, coumaric or caffeic acid in DMSO was converted to the symmetrical anhydride with DIC (N,N′-Diisopropylcarbodiimide) and then tryptophan was added to the reaction mixture. After the reaction proceeded at room temperature for 12 h, the synthesized products were purified by HPLC. Javamide-I/-II, chlorogenic acids (3-CQA, 4-CQA, 5-CQA), and caffeine were analyzed using an HPLC method as previously reported (23, 29). Briefly, Nova-Pak C18 (Waters; 150 mm × 2.1 mm i.d., 4 µm) and a 2-phase linear gradient condition were used to analyze standard and the coffee compounds: buffer A (20 mM NaH₂PO₄, pH 4.3) for 0–2 min, a first linear change from buffer A to buffer B (40% acetonitrile) for 2–18 min, a second linear change from buffer B (40% acetonitrile) to buffer B (60% acetonitrile) for 18–25 min, and buffer B (60% acetonitrile) for 5 min at the flow rate of 1 mL/min. All standards (javamide-I/-II, 3-CQA, 4-CQA, 5-CQA, and caffeine) were prepared in buffer A (20 mM NaH₂PO₄, pH 4.3) and the samples (10 µL) were injected by an autosampler into the Agilent 1260 Infinity Quaternary LC system (Agilent Technologies) and were monitored by a photo diode array detector (1260 DAD) (Agilent Technologies).

Animal study

Twenty-two 8-wk-old Sprague-Dawley male rats (155–175 g; 20 rats for the study and 2 rats as a reserve) were purchased from Charles River and housed in ventilated microisolator racks with an automatic watering system in a room with a 12:12-h light/dark cycle and ambient temperature of 18–23°C with relative humidity of 55.5%. The animals were acclimated to the experimental condition for 2 wk. Then, the animal study was conducted according to an animal protocol approved by the Beltsville Area Animal Care and Use Committee (AUP Approval no. 16-020). Rats were fed an AIN-76A purified diet to provide the recommended allowance of all nutrients required for optimal health. For the study, rats were divided into 2 groups: the water control group (NCG; n = 10) and coffee containing javamide-I/-II group (CG; n = 10), because the preliminary data indicated that 10 rats in each group would provide a power of >0.99 to detect a 20% difference in means of outcome variables with an SD of 15%. For the CG, CCJ12 (javamide-I/-II contents: ∼0.096/0.72 mg, respectively) was prepared in 30 mL water, because the average water intake of Sprague-Dawley rats (average body weight: 330 g) was found to be ∼30 mL/d (for detail, see “Preparation and consumption of CCJ12” in the Results section). CCJ12 was provided to the CG for 20 wk, whereas water was provided to the NCG for the same period. During the 20 wk, food/water consumption and body weight of rats in both groups were monitored weekly. At the end of the experiment, after 12-h deprivation of food, rats (total: 20) were individually killed by carbon dioxide asphyxiation, which is commonly used for killing small rodents. After checking for the absence of vital signs (i.e., heartbeat, respiration, and response to painful stimuli), each animal was removed from the cage and blood was immediately collected into EDTA-coated vials and centrifuged (1300 g at 4°C for 10 min)    for the plasma. All plasma samples were stored at −80°C until analyzed.

Measurements of LDL, HDL, total cholesterol, and TGs

Plasma TGs, HDL cholesterol, and total cholesterol were determined using a cholesterol assay kit (Cayman Chemical Inc.). LDL cholesterol was calculated from TG, total cholesterol, and HDL cholesterol concentrations using the modified Friedewald formula: LDL = total cholesterol − HDL − (TG)/5.

Measurements of adiponectin, leptin, CRP, and sE-selectin

Adiponectin and leptin concentrations were measured using rat adiponectin and leptin ELISA kits (Millipore Corp.), respectively. The concentrations of CRP and sE-selectin were determined using the CRP ELISA kit (Abcam) and sE-Selectin/CD62E ELISA kit (R&D Systems), respectively, according to the manufacturers’ protocols.

Cell culture

Human peripheral blood mononuclear cells (PBMCs) were purchased from the American Type Culture Collection. PBMCs were cultivated in Roswell Park Memorial Institute 1640 medium with 10% FBS, 100 units penicillin/mL, and 100 units streptomycin/mL. The cells were maintained at 37°C in a humidified atmosphere of 5% CO₂. For the experiment, PBMCs (2 × 10) were treated with several concentrations of javamide-I/-II for 18 h. After the treatment, cell culture samples were centrifuged (2800 g at 4°C for 10 min) , and the supernatants were saved/stored at –80°C for assays.

Measurements of TNF-α and MCP-1

The concentrations of TNF-α and MCP-1 were measured in cell culture and blood samples. TNF-α and MCP-1 concentrations were determined using TNF-α and MCP-1 Quantikine ELISA kits (R&D Systems), respectively, according to the manufacturers’ protocols.

Statistical analysis

All statistical analyses were performed with Sigma Plot 11.0 (Systat Software Inc.). P values were calculated using 1-factor ANOVA with the Holm-Sidak method, and P < 0.05 was considered as statistically significant. Data points in all figures represented means ± SDs (n ≥ 5).

Results

HPLC quantification of javamide-I/-II, caffeine, and chlorogenic acids

The amounts of javamide-I/-II, chlorogenic acids, and caffeine were determined in 10 coffee products, as described in the Methods. As shown in Table 1, the amounts of javamide-I/-II were found to be <0.25 and <1.89 mg/cup (250 mL), respectively. Also, chlorogenic acids (3-CQA, 4-CQA, and 5-CQA) and caffeine were found in variable amounts, as follows: chlorogenic acids (22.8–71.5 mg/cup) and caffeine (49.7–113.8 mg/cup) (Table 1). These data suggest that there may be significant variability of coffee compounds in coffee products available in the market, especially for javamide-I/-II.

TABLE 1.

Amounts of coffee compounds (3-CQA, 4-CQA, 5-CQA, caffeine, and javamide-I/-II) in coffee samples¹

Amounts in coffee samples, mg/cup (n = 10)	Amounts in the study sample, mg/cup (n = 10)
6.5 ± 0.6 ≤ 3-CQA ≤ 22.1 ± 2.01	16.7 ± 1.81
6.1 ± 0.6 ≤ 4-CQA ≤ 18.2 ± 1.51	13.9 ± 1.43
10.2 ± 1.1 ≤ 5-CQA ≤ 31.2 ± 3.11	26.2 ± 1.21
49.7 ± 4.2 ≤ Caffeine ≤ 113.8 ± 11.0	89.6 ± 9.10
Javamide-I ≤ 0.25 ± 0.06	0.20 ± 0.05
Javamide-II ≤ 1.89 ± 0.39	1.49 ± 0.21

¹Values are means ± SDs. Coffee samples were prepared as described in the Methods. 1 cup = 250 mL. 3-CQA, 3-O-caffeoylquinic acid; 4-CQA, 4-O-caffeoylquinic acid; 5-CQA, 5-O-caffeoylquinic acid.

Preparation and consumption of CCJ12

Based on Table 1, the coffee sample for the study was prepared with javamide-I (0.20 mg/cup), javamide-II (1.49 mg/cup), chlorogenic acids (56.8 mg/cup), and caffeine (89.6 mg/cup), and the compositional analysis of the coffee sample was performed using an HPLC method (Figure 1). Because 3 cups/d are the daily average for coffee drinkers of many countries including the United States, total amounts of the coffee compounds (javamide-I/-II, chlorogenic acids, and caffeine) in the 3 cups were as follows: javamide-I (0.6 mg), javamide-II (4.47 mg), chlorogenic acids (170.4 mg), and caffeine (268.8 mg). Based on the values for weekly water consumption, the intakes of javamide-I/-II were calculated (Figure 2). The weekly amounts of javamide-I/-II were found to be 0.62–0.98 and 4.5–7.0 mg, respectively, suggesting that the weekly average intakes of javamide-I/-II were ∼0.812 and ∼6.1 mg and the daily intakes ∼0.116 and ∼0.87 mg, respectively. These numbers were a bit higher than the daily amounts of javamide-I/-II (0.096/0.72 mg, respectively) calculated using the FDA guideline. Nonetheless, these data indicated that rats were exposed daily to average amounts of javamide-I/-II (0.116 and 0.87 mg, respectively) during the study.

FIGURE 1.

Compositional analysis of CCJ12. The compositional analysis of CCJ12 was performed using HPLC as described in the Methods. (A) 3-CQA, (B) 5-CQA, (C) 4-CQA, (D) caffeine, (E) javamide-II, and (F) javamide-I.

FIGURE 2.

Effects of coffee containing javamide-I/-II on water intake and calculated amounts of javamide-I/-II. Both the CG and NCG were fed a normal control diet. The amounts of javamide-I/-II were calculated based on water consumption. Data were analyzed using 1-factor ANOVA and water intake was not significantly different between the groups. Data are presented as mean ± SD (n = 10). CG, coffee containing javamide-I/-II group; NCG, water control group.

Effects of CCJ12 on food consumption and body weight

Like water consumption, food consumption was also monitored for 20 wk. As shown in Figure 3, there was a very similar pattern of food consumption in both groups and no significant difference was found in food consumption between the 2 groups. These data suggest that CCJ12 may not induce or suppress the appetite for food in rats of the CG, compared with the NCG. As expected, no difference was found in average body weights between the groups.

FIGURE 3.

Effects of coffee containing javamide-I/-II on food intake and body weight. Data were analyzed using 1-factor ANOVA. Body weight change and food intake were not significantly different between the groups. Data are presented as mean ± SD (n = 10). CG, coffee containing javamide-I/-II group; NCG, water control group.

Effects of CCJ12 on LDL, HDL, total cholesterol, and TGs

Because there was no body weight difference between the groups, the concentrations of cholesterol (total, LDL, and HDL) and TGs were investigated in the plasma samples from both groups to support the finding. As shown in Table 2, there was no significant difference in LDL, HDL, and total cholesterol concentrations between the groups. Although there was a small decrease in the concentration of TGs in the CG, it was found to be nonsignificant. These data show that CCJ12 may not have significant effects on total cholesterol, LDL, HDL, and TG concentrations in the CG, compared with the NCG.

TABLE 2.

Effects of the coffee with javamide-I/-II on plasma lipid profiles¹

Lipids	NCG	CG
LDL-C, mg/dL	21 ± 1.9	21 ± 1.8
HDL-C, mg/dL	41 ± 4.1	40 ± 4.7
Total-C, mg/dL	79 ± 6.4	77 ± 6.6
TG, mg/dL	102 ± 12.3	96 ± 11.5

¹ n = 10. Values are means ± SDs. Lipid profile data were analyzed using 1-factor ANOVA and differences between the groups were found to be statistically nonsignificant. Both the CG and NCG were fed a normal control diet. CG, coffee containing javamide-I/-II group; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; NCG, water control group; TG, triglyceride; Total-C, total cholesterol.

Effects of CCJ12 on plasma leptin and adiponectin

The concentrations of leptin and adiponectin were also measured in the plasma samples from both groups. As shown in Figure 4A, there was no difference in the concentrations of plasma leptin between the 2 groups. Likewise, the concentrations of adiponectin were also not significantly different between the groups (Figure 4B). These data suggest that CCJ12 may not influence body weight and 2 adipokines (adiponectin and leptin) in the CG, compared with the NCG.

FIGURE 4.

Effects of coffee containing javamide-I/-II on plasma leptin (A) and adiponectin (B) concentrations. The data were analyzed using 1-factor ANOVA and plasma leptin and adiponectin concentrations were not significantly different between the NCG and CG. Data are presented as mean ± SD (n = 10). CG, coffee containing javamide-I/-II group; NCG, water control group.

Effects of CCJ12 on plasma CRP

The foregoing data suggest that CCJ12 may have no significant effects on body weight, leptin, and adiponectin. Therefore, the effect of CCJ12 on another obesity-related risk factor (CRP) was investigated to complement the finding (42). As shown in Figure 5A, the plasma concentrations of CRP were not significantly different between the groups. These data suggest that CCJ12 may not have a significant effect on plasma CRP concentration in the CG, compared with the NCG.

FIGURE 5.

Effect of coffee containing javamide-I/-II on plasma CRP (A) and sE-selectin (B) concentrations. The data were analyzed using 1-factor ANOVA and the difference of plasma CRP and sE-selectin concentrations between the groups was found to be nonsignificant. Data are presented as mean ± SD (n = 10). CG, coffee containing javamide-I/-II group; NCG, water control group; CRP, C-reactive protein; NCG, sE-selectin, soluble E-selectin.

Effects of CCJ12 on plasma sE-selectin

The effect of CCJ12 on sE-selectin expression was also investigated in the plasma samples. The data showed that there was no significant difference in plasma sE-selectin concentrations between the CG and NCG (Figure 5B). These data suggest that CCJ12 may not have significant effects on 2 obesity-related atherosclerotic risk factors (CRP and sE-selectin) in the CG, compared with the NCG.

Effects of javamide-I/-II on TNF-α and MCP-1 in PBMCs

Interestingly, several recent studies have suggested that javamide-I/-II may drive selective inhibitory effects on inflammatory cytokines (24–28). Therefore, potential effects of javamide-I/-II on TNF-α and MCP-1 were investigated in PBMCs without stimulation, because rats consuming a normal diet are presumed to be in a basal status of inflammation. Also, their effects on TNF-α and MCP-1 were investigated at concentrations <40 µM, because the maximum concentration of javamide-I/-II in CCJ12 preparation provided to the rats was <70 µM and potential dilutions are expected after intake and distribution into body water. As shown in Figure 6, javamide-I/-II had no significant effects on TNF-α and MCP-1 production in PBMCs. These data indicate that javamide-I/-II at the tested concentrations may not have significant effects on TNF-α and MCP-1 in PBMCs.

FIGURE 6.

Effects of javamide-I/-II on TNF-α (A) and MCP-1 (B) in PBMCs. Data were analyzed using 1-factor ANOVA and the effects of javamide-I/-II on TNF-α and MCP-1 were found to be nonsignificant in PBMCs. Data are presented as means ± SDs (n = 5). MCP-1, monocyte chemoattractant protein-1; PBMC, peripheral blood mononuclear cell.

Effects of CCJ12 on TNF-α and MCP-1 in rats

The foregoing data above suggest that javamide-I/-II in coffee may have no significant effects on TNF-α and MCP-1 ex vivo. Therefore, potential effects of CCJ12 on TNF-α and MCP-1 were investigated in rats. As shown in Figure 7A, there was no significant difference in plasma concentrations of TNF-α between the groups. Likewise, significant difference was not found in plasma MCP-1 concentrations between the groups (Figure 7B). Based on the data, CCJ12 is not likely to have significant effects on both inflammatory cytokines (TNF-α and MCP-1) in the CG, compared with the NCG.

FIGURE 7.

Effects of coffee containing javamide-I/-II on plasma TNF-α (A) and MCP-1 (B) measured using rat plasma samples. Data were analyzed using 1-factor ANOVA and there was no significant difference in TNF-α and MCP-1 concentrations between the NCG and CG. Data are presented as mean ± SD (n = 10). CG, coffee containing javamide-I/-II group; NCG, water control group; TNF-α, tumor necrosis factor-alpha; MCP-1, monocyte chemoattractant protein-1.

Discussion

Metabolic syndrome is a cluster of health/disease-related conditions such as excess abdominal body fat, high blood glucose, abnormal cholesterol/TG concentrations, and high blood pressure. In fact, metabolic syndrome is considered as a major risk factor for diabetes, heart attack, stroke, and others (1–14). Unfortunately, metabolic syndrome is becoming prevalent worldwide owing to increasing obesity (3–10). Especially the transition of nonobese individuals (BMI < 25) to overweight and eventually obese individuals (BMI > 30) is a significant contributor to worsening obesity and metabolic syndromes (13–15). Therefore, there is a consensus that a precision antiobesity strategy should be used to prevent the development of obesity in the nonobese population. For years, modern lifestyles, high-calorie diets, and genetic factors have been considered as major risk factors for increasing obesity. However, several studies have suggested that some diet components (e.g., unhealthy meals and drinks) and their compounds may also play significant roles in the development of obesity-related metabolic conditions (11, 16, 17). Therefore, there is an urgent need to evaluate food/drink components, related to obesity and related disease conditions.

Coffee contains many bioactive compounds such as caffeine, chlorogenic acid, javamide-I/-II, and others (51, 52). Notably, javamide-I/-II are compounds relatively newly found in coffee beans and products (23, 24). Javamide-I/-II and their derivatives have been reported to drive several health-related biological activities (24–28). Interestingly, the previous studies showed that coffee products could contain javamide-I/-II at the range of 0–5.0 mg/g (23, 24, 29). Also, these studies suggested that coffee drinkers may have been exposed to these compounds for years (24, 29). However, there is no information about the potential effects of coffee products containing javamide-I/-II (CCJ12) on key metabolic factors (body weight, LDL, HDL, total cholesterol, TGs). Therefore, in this study, potential effects of CCJ12 on these factors were investigated along with other obesity-related biomarkers (leptin, adiponectin, CRP, sE-selectin, TNF-α, MCP-1) in vivo. In this study, water consumption was carefully monitored to determine the effect of CCJ12 on body weight and other metabolic factors (Table 1), because CCJ12 was supplied in drinking water. Furthermore, the amounts of javamide-I/-II in the drinking water were calculated using the FDA guideline “Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” for this study (50). In fact, this procedure provided essential information about the daily exposure amounts of javamide-I/-II, chlorogenic acids, and caffeine during the study, which have not been well documented in other coffee studies (19–22). Also, in this study, the effects of CCJ12 on water/food consumption were closely monitored, because there was a concern about a potential impact of CCJ12 on water/food consumption at the beginning of this study. However, as shown in Figure 2, average water consumption of the CG was not significantly different from that of the NCG, suggesting that CCJ12 may have little effect on water consumption in the CG, compared with the NCG. Similarly, food consumption was not significantly different between the 2 groups (Figure 3), indicating that CCJ12 may not increase or decrease the appetite for food in the CG, compared with the NCG. Altogether, the data suggest that CCJ12 may have no significant effect on water/food consumption in the CG, compared with the NCG.

Based on these data, significant body weight change was not expected between the 2 groups. As expected, the data did not show a significant difference in average body weights between the groups (Figure 3). Because there was no body weight change, significant differences were not expected in the concentrations of key metabolic factors (e.g., LDL, HDL, total cholesterol, and TG concentrations). As expected, rats from both groups showed no significant difference in plasma LDL, HDL, and total cholesterol concentrations (Table 2). Although there was a small decrease in the concentration of TGs in the CG, it turned out to be statistically nonsignificant. These data clearly suggest that CCJ12 may have no significant effects on body weight and cholesterol concentrations in the CG, compared with the NCG. Like the metabolic factors, the concentrations of adipokines (e.g., leptin and adiponectin) are also influenced by obesity (38–40). For instance, plasma leptin production is markedly increased in large adipocytes (39), whereas plasma adiponectin decreases with obesity (40). Therefore, the effects of CCJ12 on these adipokines were investigated in this study. As shown in Figure 4, no significant difference was found in plasma concentrations of leptin and adiponectin between both groups. However, it should be noticed that coffee is commonly served with a lot of additives such as flavor agents, sugar, cream, and fat. Therefore, there is a possibility that such coffees may show different effects on body weight and metabolic markers.

Because CCJ12 did not have significant effects on the metabolic factors and adipokines, the effect of CCJ12 on another obesity-related atherosclerotic risk factor (CRP) was further investigated, because CRP is commonly used as a reliable atherosclerotic risk factor in patients with progressed cardiovascular disease (42, 50, 53). As shown in Figure 5A, plasma CRP concentrations were not significantly different between both groups. These data suggest that CCJ12 may not have a significant effect on the production of CRP in the CG, compared with the NCG. In fact, these data are in line with the reported systematic meta-analysis data of CRP in coffee drinkers (53), although there was no specific information about the component composition of the coffee used in the study. Like CRP, E-selectin is a good biomarker for assessing the progression of atherosclerosis. E-selectin regulates adhesive interactions between certain blood cells and endothelium. Because E-selectin is only expressed on activated endothelium and its soluble form (sE-selectin) can be found in the plasma (43), the effect of CCJ12 on plasma sE-selectin was investigated. As shown in Figure 5B, the plasma sE-selectin concentration of the CG was found to be slightly lower than that of the NCG, but this was determined to be statistically nonsignificant. These data indicate that CCJ12 may not have a significant effect on plasma concentration of sE-selectin in the CG, compared with the NCG. However, like CRP, there is very limited information about the effect of coffee on E-selectin, particularly related to coffee compounds (43).

Like CRP and E-selectin, production of TNFs and MCP-1 is significantly modulated by obesity (44–47). Because TNF-α is a key inflammatory cytokine involved in several stages of immune processes, its deregulation is critically involved in the initiation and/or progression of several human chronic diseases such as chronic/acute inflammation, diabetes, cardiovascular disease, liver disease, rheumatoid arthritis, and cancer (44, 45). Like TNF-α, the expression of MCP-1 is also modulated by obesity (46, 47). MCP-1 can recruit monocytes and other blood cells into inflamed blood vessel walls, ultimately advancing the progression of plaque instability and vascular injury (46). Interestingly, previous studies have indicated that javamide-I/-II may have selective inhibitory effects on inflammatory cytokines (24–28). Therefore, the effects of javamide-I/-II on TNF-α and MCP-1 were investigated in PBMCs, because the cells are primarily involved in Tthe productions of NF-α and MCP-11 n humans. As shown in   Figure 6, javamide-I/-II did not increase or decrease the concentrations of TNF-α and MCP-1 significantly in PBMCs, suggesting that CCJ12 may have little effect on the production of TNF-α and MCP-1 in the CG. As anticipated, there was no significant difference in plasma TNF-α concentrations between both groups (Figure 7A). Although there was a small decrease of TNF-α production in the CG, the decrease was determined to be nonsignificant. Likewise, there was no significant difference in plasma MCP-1 concentrations between both groups (Figure 7B). The in vivo data were in fact compatible with the PBMC data. Furthermore, our preliminary data suggest that javamide-I/-II themselves may not have significant effects on the cytokine concentrations in rats fed a normal diet. However, there is still very limited information available about potential health effects of coffee components on metabolic/inflammatory biomarkers. Therefore, it may be worthwhile to investigate potential effects of other bioactive coffee compounds on metabolic/inflammatory biomarkers in the future. Altogether, the data in this study suggest that CCJ12 may not have significant effects on body weight, LDL, HDL, total cholesterol, TGs, adiponectin, leptin, CRP, sE-selectin, TNF-α, and MCP-1 in rats fed a normal diet.

In conclusion, the CCJ12 did not have significant effects on the water/food consumption in the CG, compared with the NCG. Accordingly, a significant difference was not found in average body weights between the groups. Also, both groups did not show any significant difference in plasma LDL, HDL, total cholesterol, adiponectin, and leptin concentrations. Furthermore, both groups did not show any significant difference in plasma concentrations of CRP and sE-selectin either. As expected, any significant difference was not found in TNF-α and MCP-1 concentrations between the groups. All these data suggest that CCJ12 may not have significant effects on body weight, LDL, HDL, total cholesterol, TGs, adiponectin, leptin, CRP, sE-selectin, TNF-α, or MCP-1 in the CG, compared with the NCG.

Notes

Supported by USDA Agricultural Research Service appropriated fund 52530-050-00D (to JBP).

Author disclosures: The author reports no conflicts of interest.

Mention of trade names or commercial products in this publication is solely for providing specific information and does not imply recommendation or endorsement by the USDA.

Abbreviations used: CCJ12, coffee containing javamide-I/-II; CG, coffee containing javamide-I/-II group; CRP, C-reactive protein; MCP-1, monocyte chemoattractant protein-1; NCG, water control group; PBMC, peripheral blood mononuclear cell; sE-selectin, soluble E-selectin; 3-CQA, 3-O-caffeoylquinic acid; 4-CQA, 4-O-caffeoylquinic acid; 5-CQA, 5-O-caffeoylquinic acid.

Data Availability

Data described in the article, code book, and analytic code will be available upon request pending application, approval, and generation of appropriate legal agreements. Mention of trade names or commercial products in this publication is only for research purposes. The author has no conflict of interest.