Piper nigrum Extract: Dietary Supplement for Reducing Mammary Tumor Incidence and Chemotherapy-Induced Toxicity

A low piperine fractional Piper nigrum extract (PFPE) was prepared by mixing cold-pressed coconut oil and honey in distilled water, namely, PFPE-CH. In this study, PFPE-CH was orally administered as a dietary supplement to decrease the risk of tumor formation and reduce the side effects of chemotherapeutic drugs during breast cancer treatment. The toxicity study demonstrated no mortality or adverse effects after administrating PFPE-CH at 5000 mg/kg during a 14-day observation period. Additionally, PFPE-CH at 86 mg/kg BW/day did not cause any harm to the kidney or liver function of the rats for six months. In a cancer prevention study, treatment with PFPE-CH at 100 mg/kg BW for 101 days induced oxidative stress and increased the immune response by altering the levels of cancer-associated cytokines (IL-4, IL-6, and IFN-g), leading to a reduction in the tumor incidence of up to 71.4% without any adverse effects. In combination with doxorubicin, PFPE-CH did not disrupt the anticancer effects of the drug in rats with mammary tumors. Surprisingly, PFPE-CH reduced chemotherapy-induced toxicity by improving some hematological and biochemical parameters. Therefore, our results suggest that PFPE-CH is safe and effective in reducing breast tumor incidence and toxicity of chemotherapeutic drugs during cancer treatment in mammary tumor rats.


Introduction
Breast cancer is one of the most common cancers affecting women worldwide. Among females, breast cancer is the most diagnosed cancer, with an estimated 2.3 million new cases (11.7%), and it was the leading cause of death with an estimated 0.7 million deaths (6.9%) in 2020 [1]. The most frequent chemotherapy regimens for breast cancer are doxorubicin, paclitaxel, cyclophosphamide, methotrexate, and 5-fluorouracil [2]. Adjuvant chemotherapy and radiation therapy are the principal treatment options for breast cancer patients. However, the adverse effects, including immunosuppression, myelosuppression, gastrointestinal toxicity, post-treatment toxicity, tumor relapse, and cancer drug resistance, limit their use in a clinical setting [3,4]. The poor side effect profile of cytotoxic pharmaceuticals has considerably diminished the value of therapeutic drugs.
Chemoprevention is a growing area of anticancer research with an increasing number of preclinical and clinical studies of several cancer types [5]. Chemoprevention, a means of cancer control that entirely prevents the occurrence of the disease, slows down or is reversed with the use of nontoxic natural, dietary, or synthetic products, and has emerged as a promising and pragmatic medical approach to reducing the risk of cancer [6]. There

Chemotherapeutic Study
Rats were randomly separated into 8 groups of 10 animals each. Group 1 (control) received no therapy. Group 2 (vehicle) received 5% cold-pressed coconut oil and 20% honey in distilled water. Group 3 (positive control) received doxorubicin (Dox) (Adriamycin ® , Pfizer Thailand LTD, Bangkok, Thailand) at a dose of 2 mg/kg BW. Groups 4 and 5 (single treatment) were given PFPE-CH orally at doses of 100 and 200 mg/kg BW, respectively. Groups 6 and 7 (double combination treatment) were given DOX at a dose of 2 mg/kg BW, followed by orally administered PFPE-CH at doses of 100 or 200 mg/kg BW, respectively. Group 8 (triple combination treatment) was given 100 mg/kg BW of PFPE-CH plus 25 mg/kg BW of Tu, followed by 2 mg/kg BW of DOX. Animals in each group were given 150 mg/kg BW of NMU to induce breast cancer at 50 and 80 days of age. Upon discovery of an initial tumor with a diameter of 2-3 mm, the indicated treatment conditions were administered 3 times a week for 30 days. Tumor volumes were measured as previously described [18]. At the end of the experiment, all rats were sacrificed, and wet tumors and organs were weighed. After blood was collected from the rats via heart puncture, hematological and biochemical analyses, oxidative stress markers, and cytokine studies were performed.

Chronic Toxicity Study
Rats were randomly divided into 6 groups of 10 animals each. Group 1 (Control) received no treatment. Group 2 (vehicle) received 5% cold-pressed coconut oil and 20% honey in distilled water. Groups 3 to 5 (single treatment) were given PFPE-CH orally at doses of 43, 86, and 108 mg/kg BW. Group 6 (combination treatment) was administered 43 mg/kg BW of PFPE-CH plus 11 mg/kg BW of Tu. The treated group was orally given the PFPE-CH extract daily for 180 days (6 months). All rats were observed for apparent signs of toxicity during the experimental period. At the end of the experiment, blood was collected from the rats for hematologic and clinical chemistry study. The organs (heart, kidney, liver, lung, spleen, and stomach) were weighed; the organ weight/body weight ratio was calculated.

Histopathological Study
Mammary tissues were collected from each animal group and fixed in 10% buffered formalin. The tissues were then processed sequentially and washed with phosphatebuffered saline, dehydrated in an ascending series of ethanol, and cleared with acetone and xylene. Furthermore, tissues were embedded in a paraffin block and each block was cut into 5 µm. The specimens were cleaned and deparaffinized with xylene. The slides were sequentially rehydrated in ethanol and rinsed in running water before and after being dipped in hematoxylin dye. The sections were then stained with eosin solution. After being air-dried, they were mounted with DPX (mounting media) and covered with a glass slip; the slides underwent a histopathologic investigation.

Thiobarbituric Acid Reactive Substances (TBARS)
TBARS assay (R&D Systems, Minneapolis, MN, USA) was used to evaluate the lipid peroxidation in biological fluids, which considered the levels of oxidative stress within a biological sample. Briefly, 50 µL of MDA standard (0-64 mM) or sample was mixed with 50 mL of SDS lysis solution and incubated at room temperature for 5 min. After adding the thiobarbituric acid (TBA) reagent, the samples were incubated for 60 min at 95 • C. Next, the samples were centrifuged at 3000× g for 15 min and transferred supernatant to a new tube. Then, an equal volume of n-butanol was added, and the mixture was processed for another 1-2 min. The sample was centrifuged at 10,000× g for 5 min, and the butanol fraction was then measured at a wavelength of 532 nm using a spectrophotometer (Thermo Fisher Scientific, Inc., Watham, MA, USA). The MDA concentration of the sample was calculated as previously described [19].

Cytokine Detection Assay
At the end of the experiment, the blood samples were collected from each animal group. Serum concentrations of interleukin 4 (IL-4), IL-6, IL-10, and interferon γ (IFN-γ) were determined using Cytokine & Chemokine 22-Plex Rat ProcartaPlex TM Panel (Thermo Fisher Scientific, Watham, MA, USA). The protocol was conducted according to the manufacturer's instructions. Samples were analyzed using the Luminex 200 detection system (Thermo Fisher Scientific, Watham, MA, USA), and the data were analyzed using xPONENT MAGPIX software (Thermo Fisher Scientific, Watham, MA, USA).

Statistical Analysis
The results of all experiments were expressed as mean ± S.E.M., except for cytokine results, which were expressed as mean ± S.D. The statistical significance of the difference was estimated by one-way ANOVA. Differences were considered significant at p-values less than 0.05.

GC-MS Analysis Revealed That 41 Compounds Contain PFPE
To quantify the phytochemical contents and chemical compound constituents, the PFPE was analyzed using the GC-MS technique. The composition of PFPE based on mass spectra and retention time indicated the presence of more than 40 phytochemical constituents in 4 groups, including alkaloids (52.822%), terpenes (27.125%), amides (19.650 %), and lignans (0.492%). Among 41 phytochemicals, more than 30 constituents contributed to antioxidant, antimicrobial, antifungal, and anticancer activities (Table S1 ). Due to the crystallization step in the PFPE extraction protocol, the percentage of piperine in the dichloromethane extract of P. nigrum crude extract remained at 19.59%, whereas the extracted solvents, diethyl ether, and dichloromethane were less than 4 femtograms.

PFPE-CH Does Not Induce Acute Toxicity in ICR Mice
The OECD Test Guideline's Up and Down Procedure was used to assess the acute toxicity test at an oral limit dose of 5000 mg/kg BW of the PFPE-CH formulation. After the first 4 h period, the ICR mice did not demonstrate acute toxic signs. Thus, daily monitoring of the long-term results was performed. Throughout the 14-day observation period, the PFPE-CH-treated ICR mice displayed normal behavior with no signs of toxicity. Furthermore, none of the groups had a lower body weight or food intake than the control group ( Figure 1A,B). However, the vehicle and PFPE-CH groups had less effect on water consumption than the control group ( Figure 1C). These findings suggest that PFPE-CH is safe for acute toxicity and that the oral LD 50 in mice is greater than 5000 mg/kg BW in ICR mice. Furthermore, none of the groups had a lower body weight or food intake than the control group ( Figure 1A,B). However, the vehicle and PFPE-CH groups had less effect on water consumption than the control group ( Figure 1C). These findings suggest that PFPE-CH is safe for acute toxicity and that the oral LD50 in mice is greater than 5000 mg/Kg BW in ICR mice. (A) Body weight, (B) food intake, and (C) water consumption of mice that received PFPE-CH at an oral limit dose of 5000 mg/kg BW were recorded during the observation period. Values represent the mean ± SEM; a p < 0.05 was significantly different compared to the control group; b p < 0.05 was significantly different compared to the vehicle group using one-way ANOVA analysis.

PFPE-CH Reduces Mammary Tumor Rat Incidence
To determine the cancer-preventive effect of PFPE-CH, rats were intraperitoneally injected with 2 doses of NMU and then orally treated with PFPE-CH for 101 days. In this study, at least 10% of the rats in both the control and vehicle groups developed their first detectable breast tumor within 37 days of the study period. Rats treated with PFPE-CH at a dose of 100 mg/Kg BW developed the first tumor of 10% on day 44 and accumulated to 20% on the sacrificed date, whereas only a single rat treated with PFPE at doses of 200 and 250 mg/Kg BW was found to have a tumor on day 79 and 44, respectively. Furthermore, the combination of 100 mg/kg BW PFPE-CH with 25 mg/kg BW turmeric resulted in a tumor incidence of 40% in treated animals, with the first tumor appearing on day 58. Moreover, the single PFPE-CH treatment or the combination with turmeric showed no significant change in body weight, food, and water intake from day 0 to day 79 compared to the control group. However, PFPE-CH at doses of 250 mg/Kg BW showed a significant difference in reduced food intake compared to the control group on days 80, 87, and 94 of the treatment period ( Figure 2). Moreover, the organ weight ratio in PFPE-CH treated groups did not significantly change compared to the control and vehicle groups (Table 1). (A) Body weight, (B) food intake, and (C) water consumption of mice that received PFPE-CH at an oral limit dose of 5000 mg/kg BW were recorded during the observation period. Values represent the mean ± SEM; a p < 0.05 was significantly different compared to the control group; b p < 0.05 was significantly different compared to the vehicle group using one-way ANOVA analysis.

PFPE-CH Reduces Mammary Tumor Rat Incidence
To determine the cancer-preventive effect of PFPE-CH, rats were intraperitoneally injected with 2 doses of NMU and then orally treated with PFPE-CH for 101 days. In this study, at least 10% of the rats in both the control and vehicle groups developed their first detectable breast tumor within 37 days of the study period. Rats treated with PFPE-CH at a dose of 100 mg/kg BW developed the first tumor of 10% on day 44 and accumulated to 20% on the sacrificed date, whereas only a single rat treated with PFPE at doses of 200 and 250 mg/kg BW was found to have a tumor on day 79 and 44, respectively. Furthermore, the combination of 100 mg/kg BW PFPE-CH with 25 mg/kg BW turmeric resulted in a tumor incidence of 40% in treated animals, with the first tumor appearing on day 58. Moreover, the single PFPE-CH treatment or the combination with turmeric showed no significant change in body weight, food, and water intake from day 0 to day 79 compared to the control group. However, PFPE-CH at doses of 250 mg/kg BW showed a significant difference in reduced food intake compared to the control group on days 80, 87, and 94 of the treatment period ( Figure 2). Moreover, the organ weight ratio in PFPE-CH treated groups did not significantly change compared to the control and vehicle groups (Table 1).
x FOR PEER REVIEW 7 of 22 Figure 2. Effects of oral administration of PFPE-CH on a chemopreventive study in NMU-induced mammary tumor rats for 101 days. (A) Tumor incidence of rats treated with PFPE-CH, (B) body weight, (C) food intake, and (D) water consumption was recorded during a cancer prevention study in rats. a p < 0.05 was significantly different compared to the control group using one-way ANOVA analysis.   In the leukocyte differentiation, PFPE at a dose of 250 mg/kg BW significantly decreased lymphocyte proportion while the neutrophil proportion significantly increased (Table 1). Additionally, the clinical blood chemistry showed that PFPE at a dose of 250 mg/kg BW significantly increased the level of blood urea nitrogen (BUN) and creatinine while decreasing the level of alkaline phosphatase. Moreover, PFPE-CH at a dose of 200 mg/kg BW affected the reduction in SGOT activity. Therefore, PFPE-CH at a dose of 100 mg/kg BW is the highest dose with no observed adverse effects on behavioral health.

PFPE-CH Did Not Disrupt the Anticancer Effects of Doxorubicin on Mammary Tumor Rats
In a cancer therapeutic experiment, the anticancer effect of PFPE-CH alone and in combination with chemotherapeutic medication (doxorubicin) was assessed by evaluating tumor growth. The tumor volume of the control and vehicle groups increased in a timedependent manner from day 0 to day 24 ( Figure 3A). However, the tumor volume in the vehicle group significantly decreased compared to the control group. Only doxorubicin effectively decreased tumor development at all time points compared to the control group. In the only PFPE-CH therapy, both the doses of 100 and 200 mg/kg BW suppressed tumor development from day 0 to day 18, and significant enlargement was observed on days 20 and 22, respectively. However, the tumor volume in the PFPE-CH-treated group was significantly lower than the vehicle group, when compared at the same time points from day 16 to day 24. On the other hand, the same phenomenon was not observed in the combination treatment. At the last 5 time points (from day 16 to day 24), the tumor volume of each treatment group was normalized with the mean tumor volume of the control group and expressed as the percentage of tumor suppression ( Figure 3B). PFPE-CH at a dose of 100 mg/kg BW showed tumor suppression rates comparable to those of doxorubicin from day 0 to day 18, and thereafter gradually declined from day 20 to day 24. Doxorubicin therapy and the combination of doxorubicin and PFPE-CH did not significantly differ in terms of tumor suppression rates. However, the percentage of tumor reduction rate was decreased in the triple combination treatment. Interestingly, the PFPE-CH at doses 100 and 200 mg/kg BW did not significantly change both the body weight and organ weight/body weight ratio compared to the control and vehicle groups. Meanwhile, doxorubicin significantly increased the liver weight ratio and reduced the spleen weight ratio, as shown in Table 2. However, the size of the spleen returned to normal when treated with PFPE-CH. In contrast, the combination of doxorubicin with 100 mg/kg BW PFPE and 25 mg/kg BW turmeric significantly decreased the spleen weight ratio.   H&E staining of tumor histology, as shown in Figure 3C-K, revealed that the terminal duct lobule (TDL) and fatty tissue were present in the typical histopathological characteristics of normal breast tissue ( Figure 3C). A strong basement membrane encircled the ductal epithelial and myoepithelial cells that made up the terminal duct. In contrast, the tumor histological characteristics of the control ( Figure 3D) and vehicle ( Figure 3E) groups showed tightly packed cells with high-grade ductal carcinoma and invasive ductal carcinoma, with 67-80% of grade III and 20-33% of grade I-II (Table S2 [ 74]). Rats that were given doxorubicin treatment demonstrated a reduction in ductal carcinoma to 17% in grade III and 17% in grade I ( Figure 3F). In the PFPE-CH group that received a dose of 100 mg/kg BW, the ductal carcinoma initially showed 100% grade III ( Figure 3G) manifestation, which decreased to 50% when the dose was 200 mg/kg BW ( Figure 3H). The combination of doxorubicin with PFPE-CH at doses of 100 ( Figure 3I) and 200 mg/kg BW ( Figure 3J) reduced grade III histology to 33% and 17%, respectively. However, the triple combination revealed 100% of grade III carcinoma ( Figure 3K). These findings suggest that PFPE-CH at 200 mg/kg BW is the lowest dose that can reduce breast tumor histology and is more effective when combined with drug therapy than turmeric.
The results from blood parameter analysis demonstrated that treatment with doxorubicin alone and the triple combination treatment affected white blood cells, hemoglobin, hematocrit, total protein, and albumin in rats. Notably, no significant differences in the hematologic and clinical blood chemistry values were observed in the rats treated with PFPE-CH at doses of 100 mg/kg BW compared to the control group. Interestingly, rats that received a combination treatment of doxorubicin and PFPE-CH showed normal levels of white blood cells. However, hemoglobin and hematocrit levels in the combination treatment of doxorubicin with PFPE-CH groups increased compared to those treated with doxorubicin alone (Table 2). These results suggest that PFPE-CH at a dose of 100 mg/kg BW is the highest dose that can inhibit cancer cell growth without any adverse effects on hematological and blood biochemical values. In addition, the combination of PFPE-CH with doxorubicin increased white blood cells, red blood cells, hemoglobin, hematocrit, platelets, total protein, and alkaline phosphatase compared to doxorubicin treatment alone.

PFPE-CH Increased the Stress Condition, Indicated by the Elevation of TBARS Level
At the end of the prevention study period, blood was drawn from the heart on the sacrifice day. Serum samples from each group were pooled and oxidative stress conditions were determined. The results show that in the presence of PFPE-CH at doses of 200 and 250 mg/kg BW and PFPE-CH in combination with turmeric, the TBARS level significantly increased compared to the control and vehicle groups. The increase in TBARS was positively correlated with the tumor observation time ( Figure 4A). The results indicate that PFPE-CH at doses of 200 and 250 mg/kg BW, as well as PFPE-CH combined with turmeric, showed cancer prevention effects through the generation of oxidative stress to induce cancer stress, which can inhibit tumor growth.
were determined. The results show that in the presence of PFPE-CH at doses of 200 250 mg/kg BW and PFPE-CH in combination with turmeric, the TBARS level significa increased compared to the control and vehicle groups. The increase in TBARS was p tively correlated with the tumor observation time ( Figure 4A). The results indicate PFPE-CH at doses of 200 and 250 mg/kg BW, as well as PFPE-CH combined with turm showed cancer prevention effects through the generation of oxidative stress to induce cer stress, which can inhibit tumor growth.
On the 30th day after treatment, the serum was collected in order to determine TBARS level, as shown in Figure 4B. The TBARS level of doxorubicin increased compa to that of the control and vehicle groups. In contrast, the TBARS level decreased at d of 100 and 200 mg/kg BW of PFPE-CH treatment. However, the TBARS level of the PF CH combination with doxorubicin was higher compared to that of the vehicle and con groups. On the contrary, TBARS levels decreased in the triple combination group c pared to that of the control and double combination groups. These findings suggest PFPE-CH at a dose of 100 mg/kg BW is the lowest dose that enhances the effect of d rubicin via increased oxidative stress in cancer cells, resulting in DNA damage and death without any adverse effects. However, the triple combination seems to decrease effectiveness of doxorubicin in eliminating cancer cells.  P100 + Dox2 + Tu25: PFPE-CH 100 + Doxorubicin 2 + Turmeric 25 mg/kg BW. Data are shown as mean ± SEM. The a, b, c, d, and e were represented as p < 0.05, which were significantly different compared to the control, vehicle, P100, P200, and P100 + Dox2 groups, respectively, using one-way ANOVA.
On the 30th day after treatment, the serum was collected in order to determine the TBARS level, as shown in Figure 4B. The TBARS level of doxorubicin increased compared to that of the control and vehicle groups. In contrast, the TBARS level decreased at doses of 100 and 200 mg/kg BW of PFPE-CH treatment. However, the TBARS level of the PFPE-CH combination with doxorubicin was higher compared to that of the vehicle and control groups. On the contrary, TBARS levels decreased in the triple combination group compared to that of the control and double combination groups. These findings suggest that PFPE-CH at a dose of 100 mg/kg BW is the lowest dose that enhances the effect of doxorubicin via increased oxidative stress in cancer cells, resulting in DNA damage and cell death without any adverse effects. However, the triple combination seems to decrease the effectiveness of doxorubicin in eliminating cancer cells.

PFPE-CH Altered the Levels of IL-4, IL-6, IL-10, and IFN-γ
In the prevention study, rat serum determined the levels of IL-4, IL-6, IL-10, and IFN-γ compared to those in the control and vehicle groups. As shown in Figure 5, the levels of IL-4, IL-6, and IFN-γ decreased with PFPE-CH therapy at all treatment dosages.
Curiously, the combination of PFPE-CH and turmeric significantly boosted the levels of IL-10 but only marginally increased the levels of IL-4 and IFN-γ, whereas the IL-6 level remained unchanged. These results indicate that treatment with PFPE-CH alone had a cancer prevention effect by reducing the levels of IL-4, IL-6, and IFN-γ, as well as IL-6-associated TBARS levels.

PFPE-CH Altered the Levels of IL-4, IL-6, IL-10, and IFN-γ.
In the prevention study, rat serum determined the levels of IL-4, IL-6, IL-10, and IFNγ compared to those in the control and vehicle groups. As shown in Figure 5, the levels of IL-4, IL-6, and IFN-γ decreased with PFPE-CH therapy at all treatment dosages. Curiously, the combination of PFPE-CH and turmeric significantly boosted the levels of IL-10 but only marginally increased the levels of IL-4 and IFN-γ, whereas the IL-6 level remained unchanged. These results indicate that treatment with PFPE-CH alone had a cancer prevention effect by reducing the levels of IL-4, IL-6, and IFN-γ, as well as IL-6-associated TBARS levels. Data are shown as mean ± SD. The a, b, c, d, and e were represented as p < 0.05, which were significantly different compared to the control, vehicle, P100, P200, and P250 groups, respectively, using one-way ANOVA.
For the cancer treatment study, IL-4 levels in the double and triple combination treatments significantly decreased compared to those in the control and vehicle groups ( Figure  6A). The level of IL-6 significantly increased in PFPE-CH at a dose of 100 mg/kg BW. However, IL-6 levels decreased when combining PFPE-CH and doxorubicin. Meanwhile, IL-6 levels increased in the triple combination group compared to the control and vehicle groups ( Figure 6B). Treatment with PFPE-CH at a dose of 100 mg/kg BW significantly increased the levels of IL-10 compared to the control and vehicle groups. However, the level of IL-10 decreased in the double combination treatment but not in the triple combination treatment ( Figure 6C). The levels of IFN-γ in the vehicle and doxorubicin treatment were higher compared to those in the vehicle group. Meanwhile, the IFN-γ levels in the presence of PFPE-CH were significantly lower compared to those in the control and vehicle groups. However, IFN-γ level increased in the double and triple combinations compared to a single treatment with PFPE-CH ( Figure 6D). These results indicate that a single treatment with PFPE-CH at a dose of 200 mg/kg BW can reduce breast tumor volume and tumor histology by decreasing IL-4 and IL-6 levels and is more effective when combined with doxorubicin. For the cancer treatment study, IL-4 levels in the double and triple combination treatments significantly decreased compared to those in the control and vehicle groups ( Figure 6A). The level of IL-6 significantly increased in PFPE-CH at a dose of 100 mg/kg BW. However, IL-6 levels decreased when combining PFPE-CH and doxorubicin. Meanwhile, IL-6 levels increased in the triple combination group compared to the control and vehicle groups ( Figure 6B). Treatment with PFPE-CH at a dose of 100 mg/kg BW significantly increased the levels of IL-10 compared to the control and vehicle groups. However, the level of IL-10 decreased in the double combination treatment but not in the triple combination treatment ( Figure 6C). The levels of IFN-γ in the vehicle and doxorubicin treatment were higher compared to those in the vehicle group. Meanwhile, the IFN-γ levels in the presence of PFPE-CH were significantly lower compared to those in the control and vehicle groups. However, IFN-γ level increased in the double and triple combinations compared to a single treatment with PFPE-CH ( Figure 6D). These results indicate that a single treatment with PFPE-CH at a dose of 200 mg/kg BW can reduce breast tumor volume and tumor histology by decreasing IL-4 and IL-6 levels and is more effective when combined with doxorubicin.

PFPE-CH Does Not Cause Chronic Toxicity in Sprague Dawley Rat
A cancer-preventive study of PFPE-CH showed that giving PFPE-CH at doses of 100, 200, and 250 mg/kg BW three times a week to rats could prevent cancer development. Therefore, these doses were fed to rats daily in order to test the chronic toxicity at doses of 43, 86, and 108 mg/kg BW/day, respectively. The experiment lasted for more than 6 months, and throughout the entire trial period, no death occurred among the tested rats. The body weight and organ weight of the PFPE-CH group did not demonstrate any significant differences compared to the control group. However, rats treated with 108 mg/kg BW/day of PFPE-CH showed a reduced level of serum SGOT compared to the control and vehicle groups. Moreover, rats treated with the combination treatment exhibited reductions in serum creatinine levels compared to the control and drug groups (Table 3). Therefore, the suitable doses of PFPE-CH for long-term oral application were daily doses of 43 and 86 mg/kg BW/day or 100 and 200 mg/kg BW three times a week. shown as mean ± SEM. The a, b, c, d, e, f, and g were represented as p < 0.05, which were significantly different compared to the control, vehicle, Dox2, P100, P200, P100 + Dox2, P200 + Dox2, and P100 + Dox2 + Tu25 groups, respectively, using one-way ANOVA.

PFPE-CH Does Not Cause Chronic Toxicity in Sprague Dawley Rat
A cancer-preventive study of PFPE-CH showed that giving PFPE-CH at doses of 100, 200, and 250 mg/kg BW three times a week to rats could prevent cancer development. Therefore, these doses were fed to rats daily in order to test the chronic toxicity at doses of 43, 86, and 108 mg/kg BW/day, respectively. The experiment lasted for more than 6 months, and throughout the entire trial period, no death occurred among the tested rats. The body weight and organ weight of the PFPE-CH group did not demonstrate any significant differences compared to the control group. However, rats treated with 108 mg/kg BW/day of PFPE-CH showed a reduced level of serum SGOT compared to the control and vehicle groups. Moreover, rats treated with the combination treatment exhibited reductions in serum creatinine levels compared to the control and drug groups (Table 3). Therefore, the suitable doses of PFPE-CH for long-term oral application were daily doses of 43 and 86 mg/kg BW/day or 100 and 200 mg/kg BW three times a week. L. and "generally recognized as safe" as categorized by the FDA, and it is widely used in complementary therapy for cancer patients in combination with chemotherapeutic drugs [83]. Therefore, the combination of 100 mg/kg BW of PFPE-CH and 25 mg/kg BW of turmeric was used. The protective effect of PFPE-CH was approximately 100, 200, and 250 mg/kg BW and was able to inhibit breast cancer occurrence at 71%, 80%, and 80%, respectively. Meanwhile, the combination of PFPE with turmeric showed an inhibition rate of 43%. We further evaluated the effect of PFPE-CH on the relationship between tumor formation, reactive oxygen species (ROS), and cancer-related cytokines. Cytokines regulate key immune players in cancer development and progression and are potential targets for treatment [84]. In this study, PFPE-CH effectively induced oxidative stress and decreased the levels of IL-4 and IL-6. These results have positive correlations with the observed tumor time. Moreover, the PFPE-CH group had only a 20% tumor incidence and could slow the onset of cancer compared to the control and vehicle groups. The first detectable tumors in the combination therapy were found later than with PFPE-CH at 100 mg/kg BW alone. However, the number of mammary tumor rats gradually increased to 40% after the first tumor was detected. These results might be consistent with the highest levels of IL-4 and IL-10 that were found under this condition. Breast cancer progression is facilitated by IL4, a pleiotropic cytokine secreted by fibroblasts, Th2, adipose-derived stem cells (ADSCs), as well as breast cancer cells. IL-4 promotes metastatic spreading by activating the MAPK pathway [85]. Moreover, increased IL-10 secretion is associated with aggressive breast cancer via the transformation of M1 macrophage to an M2 angiogenic phenotype [86]. For this reason, cancer cells could evade immunosurveillance and form tumors that were detected during the experiment. However, curcumin is a potent immunomodulatory agent. In breast cancer, curcumin can increase M1 and decrease M2 macrophages, resulting in a decrease in STAT3, IL-10, and arginase I gene expression and secretion in mice with metastatic breast cancer [87]. In our study, turmeric combination with PFPE-CH contradicts the results of a previous study that reported that piperine enhances the absorption of curcumin into the bloodstream and can improve the activity of curcumin [88]. We found that the combination of curcumin and PFPE was less effective in preventing cancer than PFPE-CH alone. This phenomenon could be due to PFPE having a low level of piperine, which might cause low absorption of curcumin, resulting in less effectiveness of this combination in this study. Herein, we concluded that PFPE-CH alone had a secondary chemopreventive effect by decreasing tumor formation, leading to a reduction in tumor incidence.
In addition, PFPE-CH at a dose of 250 mg/kg BW significantly decreased the lymphocyte proportion, whereas the neutrophil significantly increased in the prevention experiment. Lymphocytes can destroy tumor cells without prior sensitization. However, many tumors downregulate the expression of class 1 major histocompatibility complex (MHC) molecules, causing reduced recognition of T lymphocytes and leading to a possible decrease in the lymphocyte count [89]. In the liver function test, PFPE-CH at a dose of 200 mg/kg BW reduced SGOT activity. On the other hand, PFPE-CH at a dose of 250 mg/kg BW significantly decreased the alkaline phosphatase compared to control rats. In fact, the low activity of SGOT and alkaline phosphatase was not a sign of progressive liver damage [90,91].
In part of the chemotherapeutic activity, doxorubicin treatment exhibited increased liver weight due to its side effects such as hematopoietic suppression and hepatotoxicity, and the most serious side effect is life-threatening cardiomyopathy [92]. In addition, doxorubicin also decreased the white blood cells, hemoglobin, hematocrit, total protein, and albumin. This drug causes bone marrow suppression, resulting in anemia, neutropenia, and thrombocytopenia [4]. Our results showed that doxorubicin treatment increased the INF-γ levels. In general, doxorubicin increases INF-γ in tumors by accumulating IL-12 and is a signal transducer and activator of transcription 1 (STAT1)-dependent antitumor efficacy [93]. Moreover, oxidative stress was increased after doxorubicin treatment, resulting in DNA damage and cancer cell death [94,95]. These results are consistent with our results that showed a decrease in tumor volume and the highest tumor suppression rate by reducing IL-4 and IL-6 levels. However, the vehicle treatment significantly decreased the tumor volume compared to the control group. This phenomenon could be due to the effects of honey and cold-pressed coconut oil, as mentioned above. Meanwhile, PFPE-CH alone significantly reduced tumor size compared to the vehicle group. Therefore, the reduction in tumor size was due to the effect of PFPE-CH, which contained various phytochemical compounds, as mentioned in Table S1. Moreover, PFPE-CH has been reported to have anticancer activities in various pathways, both in vitro and in vivo [18][19][20][21] The current study showed that PFPE-CH at a dose of 100 mg/kg BW had an inhibitory effect on tumor growth. However, the tumor volume increased after 18 days of treatment. At this point, we propose that it might be due to the components in PFPE that contain various types of compounds. Caryophyllene and piperine are the bioactive compounds found in PFPE with approximately 13% and 20%, respectively. Caryophyllene has several biological activities, including anti-carcinogenic, anti-inflammatory, and immune-modulatory. The remaining piperine in PFPE-CH also has therapeutic potential against many cancer types. Nevertheless, the pharmacological activities of caryophyllene and piperine are limited due to their low water solubility, high sensitivity to oxidation when exposed to light or oxygen, fast metabolism, and systemic elimination [96][97][98]. These properties could allow tumors to evade attacks from the immune system.
The inhibition of immune cells and soluble cytokines promotes tumor progression. For example, the upregulation of IL-6 increases tumor invasion and epithelial-mesenchymal transition in human breast cancer cells [99,100]. Moreover, cancer-associated fibroblasts (CAFs) from breast tissue can produce IL-10 that disrupts the cytokine balance to stimulate tumor growth by initiating angiogenesis [101]. In our current study, IL-6 and IL-10 levels were increased when treated with PFPE-CH at a dose of 100 mg/kg BW. This result was consistent with the tumor suppression rate at the last three time points (on days 20, 22, and 24). Doxorubicin and PFPE-CH have different tumor-suppressive mechanisms. The side effect of doxorubicin was reduced by PFPE-CH in the combination treatment. IL-4 and IL-10 levels decreased more in the combination treatment than doxorubicin or PFPE alone, which correlated with reduced tumor progression (Table S2). Interestingly, the combination of doxorubicin with PFPE-CH slightly improved some blood parameters (white blood cells, red blood cells, hemoglobin, hematocrits, platelets, total protein, SGOT, and alkaline phosphatase).
Prior to exploring the possible uses of PFPE-CH as preventive and co-treatment agents, acute toxicity was determined according to the standard guidelines. A single oral dose of 5000 mg/kg PFPE-CH caused no death, no significant abnormal change in behavior, and no signs of toxicity signs in mice throughout the 14-day observation period. Therefore, PFPE-CH could be considered safe for acute toxicity, and its oral LD 50 was greater than 5000 mg/kg in mice. Moreover, a chronic study reported that daily doses of 43, 86, and 108 mg/kg BW/day or 100, 200, and 250 mg/kg BW three times a week caused no deaths during the 6 months of the experimental period. PFPE-CH at a dose of 108 mg/kg BW/day was found to affect SGOT and alkaline phosphatase. Meanwhile, the combination of PFPE-CH and turmeric significantly decreased blood creatinine. A low level of creatinine is associated with liver disease and impaired renal function [102]. Therefore, the use of PFPE-CH in combination with turmeric is a concern. These findings suggest that PFPE at a dose of 200 mg/kg BW or 86 mg/kg BW/day was the highest dose that had no adverse effects on behavioral health for 6 months.

Conclusions
The PFPE-CH formulation exhibited secondary chemoprevention type and anticancer effects on NMU-induced rats. It could be considered safe for acute toxicity at doses of up to 5000 mg/kg and there was no evidence of any harm to rats at a dose of 86 mg/kg/day for 6 months. Its effects indicate the potential use of PFPE-CH as an orally administered dietary supplement to decrease the risk of breast tumor formation and support the body during cancer treatment processes. The recommended dose of PFPE administered alone was not more than 43 mg/kg/day or 100 mg/kg BW (3 times/week), which was the no-observed-adverse-effect level (NOAEL) for cancer prevention and anticancer treatment. In combination with PFPE and a chemotherapeutic drug (doxorubicin), the dose recommendation was not more than 86 mg/kg/day or 200 mg/kg BW (3 times/week). However, the effect of PFPE 200 mg/kg BW with doxorubicin on liver function is a concern. In addition, the triple combination of PFPE, curcumin, and doxorubicin is not recommended for use in cancer treatment.