Effects of Highly Bioavailable Curcumin Supplementation on Common Cold Symptoms and Immune and Inflammatory Functions in Healthy Japanese Subjects: A Randomized Controlled Study

Abstract This study aimed to investigate the effects of 12-week consumption of highly bioavailable curcumin (150 mg/day of Theracurmin Super [TS-P1] or Theracurmin [CR-033P]) on common cold symptoms, immune function, and inflammatory markers. A randomized, double-blind, placebo-controlled study was conducted from November 2021 to May 2022 on 99 healthy Japanese adults. Using a computerized random number generator, each subject was randomly assigned to one of the following three groups: TS-P1, CR-033P, or placebo (n = 33 per group). For 12 weeks, each group consumed the four capsules that were given to them daily. The cumulative number of days for which common cold symptoms persisted was set as the primary outcome. Immunity parameters, inflammatory parameters, liver function parameters, and physical examination results were additional outcomes. A safety assessment was also performed. Ninety-four subjects completed the study, and the per protocol set included 30 subjects in the placebo group, 32 subjects in the TS-P1 group, and 33 subjects in the CR-033P group. The cumulative number of days for which common cold symptoms persisted was significantly lower in the TS-P1 and CR-033P groups than that in the placebo group. No adverse effects were observed. Consumption of highly bioavailable curcumin, TS-P1 or CR-033P (150 mg/day), for 12 weeks reduced the number of days for which common cold symptoms persisted in healthy Japanese adults.


Introduction
The common cold is a viral upper respiratory tract infection caused by some viruses, such as rhinovirus, adenovirus, coronavirus, enterovirus, influenza virus, parainfluenza virus, and respiratory syncytial virus (Wine and Alper 2012).Local symptoms in the nose and throat are noted in mild cases, while systemic symptoms with fever and muscle pain are observed in severe cases (Eccles 2005(Eccles , 2009)).The term 'common cold' is ill-defined both subjectively and objectively, and it is frequently used to refer to the manifestations of the cold syndrome, such as sinusitis, pharyngitis, laryngitis, tonsillitis, and otitis media (Eccles 2009).According to research, cold syndrome is characterized by the onset of symptoms such as headache, sneezing, chills, and sore throat, followed by nasal discharge, nasal congestion, coughing, and fatigue (Jackson et al. 1958).There are two pathways that cause cold symptoms after viral infection.Local symptoms, such as sneezing, runny nose, nasal congestion, and coughing, are caused by the production of bradykinin and prostaglandins in the nasal epithelium.In contrast, systemic symptoms, such as fever, myalgia, and headache, are caused by the release of cytokines from dendritic cells, neutrophils, and macrophages in response to the activation of Toll-like receptors (Eccles 2009).Modulating these pathways is therefore anticipated to reduce the symptoms of viral upper respiratory tract infections (also known as cold symptoms).Among food components, polyphenols have been reported to exhibit several immunomodulatory and anti-inflammatory properties (Yahfoufi et al. 2018); therefore, we believe that foods containing polyphenols are effective against the common cold.
Curcumin, a type of polyphenol, has long been used in traditional medicine to treat skin conditions due to its anti-inflammatory properties (Hatcher et al. 2008).Some previous studies showed that curcumin inhibited the production of bradykinin and prostaglandins through its anti-inflammatory effects (Koeberle et al. 2009;Moriyuki et al. 2010;Babaei et al. 2020).Furthermore, other studies reported that curcumin suppressed the release of inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, IL-6, and IL-8 (Babaei et al. 2020;Zahedipour et al. 2020;Thimmulappa et al. 2021), and exhibited antiviral properties (Zorofchian Moghadamtousi et al. 2014;Zahedipour et al. 2020).Therefore, curcumin may be effective in preventing local and systemic symptoms caused by viral upper respiratory tract infections; however, very few studies have examined its effect on common cold symptoms experienced by healthy individuals.
Although curcumin is known to have low availability, the various effects exhibited by curcumin are reportedly beneficial by increasing doses in the nontoxic range or by using processed curcumin to increase availability (Kunnumakkara et al. 2019).There are many ways to increase bioavailability (Anand et al. 2007;Chung et al. 2021;Sharma et al. 2007;Tabanelli et al. 2021), and Theracurmin (CR-033P) is one of the forms of curcumin with enhanced bioavailability (Sunagawa et al. 2015).CR-033P has a high dispersibility, and is prepared by firstly micronizing ordinary crystalline curcumin (mean particle size 22.75 μm) into submicron particles (mean particle size 0.19 μm) using gum ghatti.These submicron particles are then mixed with several excipients to produce CR-033P, which contains approximately 30% curcumin.This modified form has enhanced curcumin absorbability in humans (Sasaki et al. 2011).A randomized, open-label, three-period, six-sequence, crossover study revealed that the maximum blood concentration and area under the concentration-time curve of Theracurmin were 20-and 42-fold higher than those of conventional curcumin, respectively (Chung et al. 2021).Additionally, Theracurmin Super (TS-P1), a highly absorbable form of curcumin, was developed.TS-P1 was shown to increase the blood concentration of curcumin to twice that obtained from CR-033P administration.(Hirose et al. 2022).TS-P1 is prepared firstly by melting ordinary crystalline curcumin into amorphous curcumin (non-crystalline material), which is a form that has a higher solubility and/ or a higher dissolution rate (Schittny et al. 2020).This amorphous curcumin is then mixed with a dispersing agent and an excipient to produce TS-P1, which contains approximately 30% curcumin.These highly absorbable forms of curcumin before mixing with other ingredients are extracted from the rhizomes of Curcuma longa using permitted organic solvents, and they contain more than 85% of curcumin, approximately a total of 10% are dimethoxy curcumin and bisdemethoxycurcumin in their usual forms.Another form of curcumin with increased bioavailability achieved by reducing the particle size significantly shortened symptom recovery time in patients with COVID-19 (Ahmadi et al. 2021;Saber-Moghaddam et al. 2021).Based on the abovementioned previous studies, TS-P1 and CR-033P are expected to alleviate common cold symptoms and modulate immune and inflammatory functions in healthy individuals.
In this study, we investigated the effects of the 12-week daily intake of TS-P1 and CR-033P (each containing 150 mg/day as curcumin, respectively), two forms of curcumin formulations with enhanced bioavailability, on common cold symptoms, immune function, and inflammatory markers in healthy Japanese adults.A safety assessment of TS-P1 and CR-033P was also performed.Regarding the evaluation of cold symptoms, the cold symptoms that were subject to self-judgment were evaluated rather than the cold symptoms based on the doctor's diagnosis.Since this study's aim was to verify whether daily supplementation with curcumin alleviated cold-like symptoms, we used the subject's evaluation of cold symptoms as an outcome assuming a daily environment.

Design
The study was designed as a randomized, placebo-controlled, double-blind, and parallel-group comparison, and the allocation of subjects into three groups (TS-P1, CR-033P, and placebo) was based on a 1:1:1 ratio.The study was approved by the ethics committee of Takara Clinic (Medical Corporation Seishinkai, Tokyo, Japan; date of approval: November 22, 2021, approval ID: 2111-02164-0043-1 C-TC) and was registered at the University Hospital Medical Information Network Clinical Trial Registry (UMIN000046160).The study protocol followed the principles of the Declaration of Helsinki (2013) as well as the Ethical Guidelines for Medical and Biological Research Involving Human Subjects of the Ministry of Education, Culture, Sports, Science, and Technology; the Ministry of Health, Labor, and Welfare; and the Ministry of Economy, Trade, and Industry of Japan.

Subjects
The inclusion criteria were as follows: (1) Japanese, (2) men or women, (3) aged ≥20 years, (4) healthy individuals, (5) subjects who were judged as eligible to participate in this study by the physician, and (6) subjects who underwent examination at screening (Scr) to participate in this study and whose immunological vigor score (Hirokawa, Utsuyama, Kitagawa, et al. 2009, 2014) were within normal range and relatively low at Scr.The exclusion criteria were as follows: (1) subjects who were undergoing medical treatment or had a history of a heart condition, malignant tumor, or myocardial infarction; (2) subjects with a pacemaker or an implantable cardioverter defibrillator (ICD); (3) subjects currently undergoing treatment for cardiac arrhythmia, liver disorder, kidney disorder, cerebrovascular disorder, rheumatism, diabetes mellitus, dyslipidemia, hypertension, gastroesophageal reflux disease, hormone-sensitive diseases, iron deficiency, or any other chronic diseases; (4) subjects who consume 'Foods for Specified Health Uses' , 'Foods with Functional Claims' , or other functional food/beverage daily; (5) subjects currently taking medicines (including herbal medicines) and supplements; (6) subjects allergic to medications and/or the test-food-related products; (7) subjects with COVID-19; (8) subjects who were pregnant, breastfeeding, and planning to become pregnant; (9) subjects who had been enrolled in other clinical trials within the last 28 days before agreeing to participate in this trial or planned to participate in another trial during this trial; (10) subjects who smoked; (11) subjects with autoimmune disease; (12) subjects who used immunosuppressants, such as steroids; (13) subjects who had received vaccination for influenza within the last 3 months; (14) subjects who had undergone surgery within the last 2 weeks; and (15) subjects who were judged as ineligible to participate in this study by the physician.
The subjects were recruited via a website (https://www.go106.jp/)operated by ORTHOMEDICO Inc. (Tokyo, Japan).The study protocol was comprehensively explained to all subjects at the office of ORHOMEDICO Inc.Furthermore, all subjects provided informed consent before participation in the study.No subjects were a part of the sponsor or funding companies.Medical Corporation Seishinkai, Takara Clinic (Tokyo, Japan) evaluated the data obtained and managed the health of the study subjects, and the examinations were conducted at Nerima Medical Association, Minami-machi Clinic (Tokyo, Japan).

Intervention
Subjects in the TS-P1 group or CR-033 group were provided capsules containing excipients (diluents and lubricants) plus 131.7 mg of TS-P1 or CR-033P, respectively, while those in the placebo group were provided capsules in which the functional ingredients in the active food were replaced by excipients.Subjects were instructed to consume two capsules of the allocated test products with water 10-15 min before breakfast.They were instructed to repeat the same before dinner.In total, four capsules were consumed by each subject daily.This intervention was designed to administer 150 mg of curcumin per day in both TS-P1 and CR-033P groups.The consumption of the above test foods began at the start of the survey period and the intervention period lasted for 12 weeks.The ethics committee declared that each capsule was identical in color, odor, and flavor.

Sample size
No study till date has evaluated the effects of the 12-week consumption of either TS-P1 or CR-033P on the cumulative number of days for which common cold symptoms persisted in healthy Japanese adults.Hence, we assumed a large difference in the cumulative number of days for which common cold symptoms persisted between the TS-P1 or CR-033P group and the placebo group and used an effect size (d) of 0.80 as suggested by Cohen (Cohen 1992).The sample size was calculated with an assumed effect size (d) of 0.80, significance level (α) of 0.05, and statistical power (1−β) of 0.80.Consequently, a sample size of 78 was determined (26 subjects per group).To maximize the statistical power (1−β) as much as possible within the budget, the sample size was set at 90 subjects (30 subjects per group), and statistical power (1−β) was recalculated to be 86.1.Eventually, assuming that dropouts or subjects who deviated from protocol were 10% during this trial, the number of subjects included was 99 (33 subjects per group).

Enrollment, randomization, and blinding
Of the 143 subjects who provided informed consent, 99 eligible subjects who were deemed eligible for the study were selected by the physician.The test foods were provided to the contract research organization by the sponsor.An individual in charge of shipping who belongs to the contract research organization gave the code of the test foods to an allocation controller who was not directly involved in the studies after confirming that the test foods (each capsule) were undistinguishable and after entering and verifying the data at Scr. Allocation was performed according to a computer-generated randomization list by this allocation controller.The allocation adjustment factors were sex, age, and immunological vigor score.Subjects were equally but randomly assigned to either the TS-P1, CR-033P, or placebo group (n = 33 per group).Only the person in charge of shipping had access to the allocation table with the coded test foods, and the test foods were sent to each subject according to the table.After the test foods were shipped, the allocation table was kept at a secure location until the subjects for analysis and statistical analysis methods were fixed.The sponsor, principal physician, subphysician, entire contract research organization staff (i.e.director of the trial, director of trial conduction, individual in charge of monitoring, director and staff of statistical analysis, and individual in charge of shipping), medical institution staff, ethics committee members, contract laboratory, and others who were related to this study were not aware of the group assignments.The allocation controller locked the allocation table until the key opening day.

Examination items
The schedule of this study is shown in Table 1.A survey on common cold symptoms, immunity test, measurement of inflammatory markers, physical examination, and peripheral blood examination were conducted to determine the efficacy.In the survey on common cold symptoms, the number of expression days in which subject-selfjudged cold symptoms were expressed was evaluated up to 12 weeks after starting test food consumption.Since the survey on common cold symptoms is based on subject-selfjudged, although it does not imply cold symptoms in medical terms, it includes cold-like symptoms that the subject accidentally experiences.
All subjects were asked to complete a medical questionnaire and Calorie and Nutrition Diary (Suzuki et al. 2019) to enable understanding of their health conditions.In addition, subjects were asked to keep a daily record of their lifestyle, health conditions, and food consumption.Side effects and adverse events were recorded when experienced by the subjects.If symptoms were recognized as persisted adverse events in subject, the physician immediately administered necessary and appropriate treatment and made the decision regarding whether the subject could continue this study and the emergency key should be opened.Furthermore, the physician evaluated and documented the relationship between the adverse event and the test food.Subjects who consumed or used medicines or over-the-counter drugs were required to report information regarding their usage (such as reason of usage, type of medication, date of usage, or amount of usage) to the contract research organization.Subjects who withdrew their consent or deviated from the protocol were excluded from this study.

Primary outcome
Subjects were instructed to record the presence or absence of common cold symptoms in the daily record during the intervention period.'Whole body malaise' , 'chilliness' , 'feverishness' , 'fatigue' , 'sneezing' , 'nasal discharge' , 'blocked nose' , 'throat pain' , 'cough' , 'joint pain' , and 'muscle pain' were among the symptoms of the common cold.Subjects were required to respond to each question on an eight-point scale in which '0' indicated no symptoms and values closer to '7' indicated more severe symptoms.In this study, common cold symptoms were defined as the onset of more than oneof the Note: X indicates timing of implementation.♦ indicates consecutive days of measurement or consumption.Scr, screening (examination before consumption); 0 wks, start of consumption of test food; 8 wks, examination 8 weeks after consumption; 12 wks, examination 12 weeks after consumption.
abovementioned symptoms.The cumulative number of days for which common cold symptoms persisted during the intervention period per subject, was set as the primary outcome.

Secondary outcomes
In a survey on common cold symptoms, the following items were assessed as secondary outcomes: (1) the maximum number of days for which common cold symptoms persisted in the subjects during the intervention period, (2) the cumulative number and maximum number of days for which each common cold symptom persisted in the subjects during the intervention period, and (3) the frequency with which subjects experienced them.
Immunity test was conducted at the Institute for Health and Life Science (Tokyo, Japan).In the immunity test, seven immune subscores, including (1) the number of T cells, (2) ratio of CD4 + /CD8 + T cells, (3) number of naive T cells, (4) ratio of naive/memory T cells, (5) number of B cells, (6) number of NK cells, and (7) number of CD8 + CD28 + T cells, were measured.These measured values were scored from one to three points according to the reference values.The sum of these scores was used as the immunological vigor score (the upper limit is 21 points), which was graded into immunological grades on a five-point scale (Hirokawa, Utsuyama, Kitagawa, et al. 2009, Hirokawa, Utsuyama, Kikuchi, et al. 2009;Utsuyama et al. 2009;Hirokawa et al. 2014).In addition, T lymphocyte age calculated based on the negative correlation found between the number of CD8 + CD28 + T cells and age (Hirokawa, Utsuyama, Kitagawa, et al. 2009, Hirokawa, Utsuyama, Kikuchi, et al. 2009) was assessed.Scr and 8 weeks after consumption (8 wks) served as the assessment timepoints.
In measurement of inflammatory markers, the following items were assessed: levels of interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, and high sensitive C-reactive protein; number of white blood cells; white blood cell differential count (i.e. percentage and numbers of neutrophils, lymphocytes, monocytes, eosinophils, and basophils); and ratio of neutrophils/lymphocytes in the blood.The level of each items was measured by LSI Medience Corporation (Tokyo, Japan), and each item was evaluated in accordance with the global standard.Assessment points for the number of white blood cells, white blood cell differential count, and the ratio of neutrophils/lymphocytes were Scr, 8 wks, and 12 weeks after consumption (12 wks), while assessment points for other items were Scr and 8 wks.
As part of the physical examination, the subjects' weight, body mass index (BMI), body fat percentage, muscle mass, systolic and diastolic blood pressures, and pulse rate were measured.Height was only measured at briefing to calculate BMI.Assessment points of these physical examination parameters were Scr, 8 wks, and 12 wks.The measured values of height, systolic and diastolic blood pressures, and pulse rate were used as the subjects' baseline factors.

Safety evaluation
The incidence of side effects and adverse events were recorded.In addition, the physician evaluated and documented the relationship between the adverse event and the test food.

Statistical analysis
All statistical analyses in this study were two-sided, and the significance level was set at 5%.Data analyses were performed using Windows SPSS, version 23.0 (IBM Japan, Ltd., Tokyo, Japan).Subjects who markedly deviated from the protocol were excluded from the analysis population.A primary outcome-oriented analysis was conducted, and multiplicity in multiple items and multiple time points occurring in secondary outcomes were not considered in this study.The subjects' baseline data were demographically aggregated by the enrolled subjects and the analyzed subjects.The subjects' gender for each group were compared using chi-square test, and the subjects' age, height, weight, systolic and diastolic blood pressures, and pulse rate for each group were compared using a general linear model with group as a factor.
The measured values of the primary and secondary outcomes are shown as mean, standard deviation (SD), group differences, and standard error (SE).Group differences were presented as 95% confidence interval.A group comparison between the TS-P1 or CR-033P group and the placebo group in a survey on common cold symptoms was performed using a general linear model with the group as a factor.A general linear model with group as a factor was used for assessing baseline values at Scr, while a general linear model with baseline values as covariates and group as a factor was used for assessing the measured values at 8 wks and change from baseline to 8 wks in group comparisons between the TS-P1 or CR-033P group and the placebo group for immunity test and measurement of inflammatory markers except for the number of white blood cells, white blood cell differential count, and the ratio of neutrophils/ lymphocytes.On the other hand, in evaluation items except for the immunity test and measurement of inflammatory markers, the number of white blood cells, white blood cell differential count, and the ratio of neutrophils/lymphocytes, a general linear model with group as a factor was used for group comparisons of baseline at Scr, whereas a linear mixed model with baseline values as covariates and time points, groups, interaction between time points and groups, interaction between baseline values and time points, and subjects as factors was used for group comparisons of the measured values at 8 wks and 12 wks and changes from baseline to 8 wks and to 12 wks respectively.These analysis methods were used in both per protocol set (PPS) and subgroup constructed in this study.Only for this subgroup, we additionally analyzed a comparison between the TS-P1 and CR-033P groups in a survey on common cold symptoms, and this analysis method was used in other datasets.

Analysis set
Figure 1 shows the flowchart of this study.Subjects were recruited from November 25, 2021 to January 14, 2022.The test period was from November 25, 2021 to May 31, 2022.Despite the fact that every subject received the treatment prescribed for them, four subjects-one in the TS-P1 group and three in the placebo group-failed to show up for the examination at 8 wks; hence, they were excluded from the analysis dataset.The 95 subjects in the final efficacy and safety analysis datasets were PPS (32 subjects in the TS-P1 group, 33 subjects in the CR-033P group, and 30 subjects in the placebo group).Table 2.1 provides a summary of the subjects' baseline factors.
There was no significant difference in the PPS baseline factors.
Because some subjects used medications to alleviate common cold symptoms, we created a subgroup of subjects who were included with PPS and did not use medications to alleviate common cold symptoms and conducted exploratory verification.The subjects' baseline factors in a subgroup are summarized in Table 2.2.In subgroup baseline factors, significant differences in age and systolic blood pressure (p = 0.048 and p = 0.027, respectively) were observed between the TS-P1 and placebo groups.

PPS analysis
The results of a survey on common cold symptoms, including primary outcomes, are shown in Tables 3 and 4.
In a group comparison between the TS-P1 and placebo groups, the cumulative number of days for which common cold symptoms persisted, defined as the onset of more than one of the symptoms ('whole body malaise' , 'chilliness' , 'feverishness' , 'fatigue' , 'sneezing' , 'nasal discharge' , 'blocked nose' , 'throat pain' , 'cough' , 'joint pain' , and 'muscle pain') during the intervention period per subject and the frequency at which subjects experienced them, in the TS-P1 group were significantly lower than those in the placebo group (p = 0.036, p = 0.038, respectively).The mean ± SD of the cumulative number of days for which common cold symptoms persisted and the frequency at subjects experienced them were 38.6 ± 33.8 days and 45.7 ± 40.1% in the TS-P1 group, and 56.8 ± 32.0 days and 67.0 ± 37.7% in the placebo group each respectively (Table 3).Values are shown as mean ± standard deviation (SD), group differences, and standard error (Se) and 95% confidence interval (95%Ci) for group differences.the cumulative number of days with the common cold symptoms which was defined as the onset of more than one of the each symptoms ('whole body malaise' , 'chilliness' , 'feverishness' , 'fatigue' , 'sneezing' , 'nasal discharge' , 'blocked nose' , 'throat pain' , 'cough' , 'joint pain' , and 'muscle pain'), while the maximum duration of expression days with the common cold symptoms (defined as the total of the maximum duration of the days with each symptom).GD means group difference.†, primary outcome, *p < 0.05.Note: Values are shown as mean ± standard deviation (SD), group differences, and standard error (Se) and 95% confidence interval (95%Ci) for group differences.GD means group difference.
The maximum number of days for which common cold symptoms persisted (defined as the total of the maximum number of days with each symptom) in the TS-P1 group was also significantly lower than that in the placebo group (p = 0.007), and its mean ± SD was 22.3 ± 30.2 days in the TS-P1 group and 45.2 ± 34.7 days in the placebo group (Table 3).
In addition, we found significant differences in terms of the number of days of 'sneezing' , 'nasal discharge' , 'blocked nose' , and 'cough' between the TS-P1 and placebo groups (Table 4).The cumulative number of days for which 'sneezing' persisted (mean ± SD) and the frequency at which subjects experienced them [mean ± SD] in the TS-P1 group (7.9 ± 14.9 days [9.4 ± 17.7%]) were significantly lower than those in the placebo group (18.4 ± 18.6 days [21.7 ± 22.1%]) (p = 0.026 and p = 0.027, respectively).The cumulative number of days for which 'nasal discharge' persisted (mean ± SD) and the frequency at which subjects experienced them [mean ± SD] in the TS-P1 group (9.8 ± 14.8 days [11.7 ± 17.6%]) were significantly lower than those in the placebo group (24.4 ± 30.4 days [28.4 ± 35.2%]; p = 0.029 and p = 0.033, respectively), and the maximum number of days (mean ± SD) in the TS-P1 group (4.2 ± 8.3 days) was also significantly lower than that in the placebo group (15.6 ± 22.3 days) (p = 0.013).The cumulative number of days for which 'blocked nose' persisted (mean ± SD) and the frequency at which subjects experienced them [mean ± SD] in the TS-P1 group (3.0 ± 5.1 days [3.6 ± 6.0%]) were significantly lower than that in the placebo group (14.2 ± 25.6 days [16.3 ± 29.0%]; p = 0.026 and p = 0.029, respectively), and the maximum number of days (mean ± SD) in the TS-P1 group (1.8 ± 3.9 days) was also significantly lower than that in the placebo group (10.1 ± 19.9 days) (p = 0.036).The cumulative number of days for which 'cough' persisted (mean ± SD) and the frequency at which subjects experienced them [mean ± SD] in the TS-P1 group (2.6 ± 8.6 days [3.1 ± 10.3%]) were significantly lower than those in the placebo group (9.0 ± 17.1 days [10.8 ± 20.4%]) (p = 0.047 and p = 0.048, respectively).
In a group comparison between the CR-033P and placebo groups, the cumulative number of days for which common cold symptoms persisted during the intervention period per subject in the CR-033P group was significantly lower than that in the placebo group (p = 0.042), and its mean ± SD was 39.4 ± 34.7 days in the CR-033P group and 56.8 ± 32.0 days in the placebo group (Table 3).The maximum number of days for which common cold symptoms persisted in the CR-033P group was also significantly lower than that in the placebo group (p = 0.037), and its mean ± SD was 27.8 ± 33.1 days in the CR-033P group and 45.2 ± 34.7 days in the placebo group.The frequency at which subjects experienced common cold symptoms in the CR-033P group was also significantly lower than that in the placebo group (p = 0.045), and its mean ± SD was 46.7 ± 41.2% in the CR-033P group and 67.0 ± 37.7% in the placebo group (Table 3).However, no significant difference in any of the symptoms of the common cold was observed among the groups (Table 4).

Immunity test
No clinically significant effects were detected in either TS-P1 or CR-033P group in each item of the immunity test (data not shown).

Measurement of inflammatory markers
No clinically significant effects were detected in either TS-P1 or CR-033P group in each item in the measurement of inflammatory markers (data not shown).

Physical examination and peripheral blood examination
No changes in any of the parameters were found during physical examination and peripheral blood examination (data not shown).

Subgroup analysis
The results of a survey on common cold symptoms in a subgroup of subjects who were included with PPS and did not use medications to alleviate common cold symptoms are shown in Tables 5 and 6.
In a group comparison between the TS-P1 and placebo groups, the cumulative number of days for which common cold symptoms persisted during the intervention period per subject in the TS-P1 group was significantly lower than that in the placebo group (p = 0.041), and its mean ± SD was 26.2 ± 23.0 days in the TS-P1 group and 38.7 ± 21.7 days in the placebo group (Table 5).The maximum number of days for which common cold symptoms persisted in the TS-P1 group was also significantly lower than that in the placebo group (p = 0.009), and its mean ± SD was 16.9 ± 21.4 days in the TS-P1 group and 33.3 ± 24.0 days in the placebo group (Table 5).
In addition, we noted significant differences in 'sneezing' , 'nasal discharge' and 'blocked nose' between the TS-P1 and placebo groups (Table 6).The frequency at which subjects experienced sneezing was significantly lower that in the placebo group (p = 0.008), and its mean ± SD was 7.5 ± 13.4% in the TS-P1 group and 22.8 ± 23.1% in the placebo group.The cumulative number of days for which 'nasal discharge' persisted (mean ± SD) and the frequency at which subjects experienced them [mean ± SD] in the TS-P1 group (6.9 ± 11.5 days [9.8 ± 16.0%]) were significantly lower than those in the placebo group (16.8 ± 20.8 days [29.0 ± 36.8%]; p = 0.049 and p = 0.025, respectively), and the maximum number of days (mean ± SD) in the TS-P1 group (3.3 ± 7.9 days) was also significantly lower than that in the placebo group (12.0 ± 17.5 days) (p = 0.049 and p = 0.038, respectively).The cumulative number of days for which 'blocked nose' persisted (mean ± SD) and the frequency at which subjects experienced them [mean ± SD] in the TS-P1 group (1.6 ± 3.4 days [2.9 ± 5.6%]) were significantly lower than those in the placebo group (11.0 ± 18.3 days [17.9 ± 30.1%]; p = 0.020 and p = 0.020, respectively), and the maximum number of days (mean ± SD) in the TS-P1 group (0.8 ± 1.9 days) was also significantly lower than that in the placebo group (8.0 ± 14.6 days) (p = 0.033).
No significant differences in the overall common cold symptoms and individual common cold symptoms were observed between the CR-033P and placebo groups (Table 5 and Table 6, respectively).

Side effects and adverse events
There were no side effects or adverse events attributable to the test food in this study.In addition, the physician confirmed that continuous consumption of the test food was safe.
Table 5. Cumulative and maximum persisted days for which, and frequency with which common cold symptoms were experienced during the intervention period per subject in the subgroup analysis.Note: Values are shown as mean ± standard deviation (SD), group differences, and standard error (Se) and 95% confidence interval (95%Ci) for group differences.the cumulative number of expression days with the common cold symptoms which was defined as the onset of more than one of the each symptoms ('whole body malaise' , 'chilliness' , 'feverishness' , 'fatigue' , 'sneezing,' 'nasal discharge' , 'blocked nose,' 'throat pain,' 'cough,' 'joint pain' , and 'muscle pain'), while the maximum duration of expression days with the common cold symptoms (defined as the total of the maximum duration of the days with each common cold symptom).GD means group difference.*p < 0.05.The cumulative and maximum number of days for which common cold symptoms persisted and the frequency at which subjects experienced them were significantly lower in the TS-P1 and CR-033P groups than those in the placebo group.Furthermore, the cumulative number of days for which sneezing (group difference from the placebo group −10.5 days, 95%CI [−19.6, −1.3]), nasal discharge (group difference from the placebo group −14.5 days, 95%CI [−27.5, −1.5]), blocked nose (group difference from the placebo group −11.2 days, 95%CI [−21.0,−1.4]), and cough (group difference from the placebo group −6.4,95%CI [−12.7,−0.1]) persisted was significantly shorter in the TS-P1 group than that in the placebo group.The frequency at which subjects experienced sneezing (group difference from the placebo group −12.4%, 95%CI [−23.2, −1.5]), nasal discharge (group difference from the placebo group −16.7%, 95%CI [−32.0,−1.4]), blocked nose (group difference from the placebo group −12.7%, 95%CI [−24.1, −1.3]), and cough (group difference from the placebo group −7.6%, 95%CI [−15.2, −0.1]) persisted were significantly shorter in the TS-P1 group than those in the placebo group.On the other hand, the maximum duration of nasal discharge (group difference from the placebo group −11.4 days, 95%CI [−20.4,−2.5]) and nasal obstruction (group difference from the placebo group −8.4 days, 95%CI [−16.1, −0.6]) were significantly shorter in the TS-P1 group than those in the placebo group.Collectively, our data indicate that consumption of TS-P1 and CR-033P (150 mg/day as curcumin contents, respectively) reduced common cold symptoms.In particular, TS-P1 ameliorated local symptoms, such as sneezing, nasal discharge, nasal obstruction, and cough.Moreover, we analyzed and compared the TS-P1 and CR-033P subgroups on common cold symptoms and found that the maximum number of days on which nasal discharge and blocked nose were expressed was significantly lower in the TS-P1 group than in the placebo group (p = 0.027 and p = 0.049, respectively; Supplementary Table 1).This local symptom-improving effect in said areas may be related to the fact that TS-P1 has twice as higher total curcumin concentration in blood than CR-033P (Hirose et al. 2022); high bioavailability of TS-P1 may have improved local symptoms through efficient and strong effects on immune and inflammatory markers.
Coughing, one of the symptoms of the common cold, is largely caused by the production of bradykinin and prostaglandins (Eccles 2009), which act on airway sensory nerve endings (Eccles and Lee 2004;Jacoby 2004).Bradykinin activates the bradykinin receptor to secrete phospholipase A2, which induces arachidonic acid.Arachidonic acid is then oxidized by cyclooxygenase to form prostaglandins (Babaei et al. 2020).Curcumin is known to exhibit high affinity for bradykinin receptors (Yimam et al. 2016) and inhibitory effects on cyclooxygenase (Rao 2007;Moriyuki et al. 2010) in this pathway.Curcumin has been shown to inhibit inflammatory symptoms by acting on bradykinin receptors and cyclooxygenase in vivo airway investigations (Kumari et al. 2019;Yuan et al. 2018).Meanwhile, common cold symptoms such as sneezing, nasal discharge, and blocked nose involve histamine (Eccles 2009), which is released by immunoglobulin E (IgE)-mediated antigen stimulation that activates mast cells (Tanaka and Furuta 2021).Previous studies reported that curcumin reduced serum histamine levels in a mouse model of IgE/Ag-induced passive systemic anaphylaxis (Li et al. 2014), and administration of curcumin alleviated symptoms in a mouse model of allergic rhinitis (Zhang et al. 2015;Acar et al. 2016).Furthermore, a study on patients with allergic rhinitis revealed that 8 weeks of curcumin administration reduced sneezing, nasal discharge, and nasal obstruction (Wu and Xiao 2016).Therefore, we believed that coughing was suppressed by TS-P1 acting on bradykinin receptors and cyclooxygenase, and sneezing, nasal discharge, and blocked nose were relieved via suppression of histamine production by TS-P1.Moreover, in this study, there was no statistically significant difference in any of the common cold symptoms between the CR-033P and placebo groups; however, the number of days for which common cold symptoms, except chills, persisted in the CR-033P group was marginally shorter than that in the placebo group.This finding indicates that CR-033P could shorten the recovery time of common cold symptoms through the same mechanism of action as TS-P1.
TS-P1 and CR-033P have been shown to affect cytokines in vitro and in vivo (Ohno et al. 2017;Yamaguchi et al. 2017;Mizumoto et al. 2019;Adachi et al. 2020;Park et al. 2020;Kuwabara et al. 2021).In addition, since prostaglandins have been reported to induce IL-6 and IL-8 production in nasal polyp-derived fibroblasts via the prostaglandin receptor/protein kinase B (Akt)/nuclear factor (NF)-κB pathway (Cho et al. 2014), we predicted that curcumin affects cytokines via an antagonist action on prostaglandins.However, the present study could not clarify the effects of TS-P1 and CR-033P on cytokines such as TNF-α, IL-1β, IL-6, and IL-8.A previous study investigating the effect of curcumin on serum cytokines in obese individuals found significant reductions in serum levels of IL-1β and IL-4 but no changes in those of IL-6 and TNF-α (Ganjali et al. 2014).Moreover, a Korean study (Kim et al. 2011) that observed cytokine levels in 110 healthy subjects showed that the IL-1β level was 0.17-39.0pg/mL, IL-6 level was 0.16-37.7 pg/mL, and TNF-α level was 0.86-26.8pg/ mL, indicating that these indices have a wide range of result values.Furthermore, a laboratory (LSI Medience) that measured IL-1β, IL-6, and TNF-α levels set reference values for healthy subjects for each index as <0.928 pg/mL for IL-1β, <7.0 pg/mL for IL-6, and 2.27-11.2pg/mL for TNF-α.In light of these studies, the data in our study were consistent with both reference values, and the data were shown to be within the range for healthy subjects.Therefore, we considered that the test foods had no influence on cytokines because 1) the values were within the range for healthy subjects, 2) these indicators were highly variable, and 3) these cytokines were not adjusted in allocation.However, CR-033P has been reported to modulate cytokines (Ohno et al. 2017;Yamaguchi et al. 2017;Kuwabara et al. 2021;Mizumoto et al. 2019;Adachi et al. 2020;Park et al. 2020).In addition, since TS-P1 was a curcumin with improved bioavailability over CR-033P (Hirose et al. 2022), we believe that TS-P1 will have similar results to those previously reported for CR-033P.Future studies investigating the effects of TS-P1 and CR-033P on serum cytokine levels in humans will need to adjust baseline factors as much as possible.
No statistically significant differences in systemic symptoms, such as fever, myalgia, and headache, were observed partially because at least half of the subjects did not experience any of the symptoms in the relevant categories.Perhaps as a result of the recent increase in hygiene awareness for the prevention of COVID-19 infections, Japan has experienced a sharp decline in the number of influenza outbreaks; 1,876,083 infections were observed in 2019, of which approximately 30% were noted in 2020, and approximately 0.2% of the infections noted in 2020 were observed in 2021 (World Health Organization 2022).In addition, the year-long decline in influenza cases coincided with the intervention period (World Health Organization 2022).Therefore, it is possible that the timing of the study and recent advancements in public health reduced the possibility of viral infection.These may also be the reasons why no significant differences between groups were observed in the present study.However, the findings of the present study have indicated that TS-P1 and CR-033P exert antiviral and anti-inflammatory effects.Future studies should be conducted during the year-end and new year period, when the number of influenza cases is high, to clarify the effects of TS-P1 and CR-033P on systemic symptoms of the common cold and the associated cytokine levels.
Curcumin has long been thought to be effective against the common cold, but this has only been inferred from its in vitro and in vivo functions.This study is the first to obtain evidence using a randomized controlled trial to demonstrate curcumin's efficacy in treating common cold symptoms in healthy humans.However, as the results of this study were analyzed using PPS, the level of evidence is weak.Additionally, because the study was based on the reporting of subjective cold-like symptoms, it is was unclear whether the symptoms were actual subjective symptoms caused by an upper respiratory tract infection.Therefore, it is necessary to verify curcumin's effect on common cold symptoms based on a physician's diagnosis in the future.

Conclusions
We examined the effects of consumption of 150 mg/day curcumin equivalent of TS-P1 or CR-033P for 12 weeks on common cold symptoms, immune function, inflammatory markers, and safety in healthy Japanese adults.The results of the study demonstrated that the cumulative number of days for which common cold symptoms persisted was significantly reduced after consumption of 150 mg/day curcumin equivalent of TS-P1 and CR-033P for 12 weeks, and the maximum number of days for which common cold symptoms persisted was also significantly shortened.Moreover, for each of the common cold symptoms, TS-P1, which showed twice as high total curcumin concentration in blood than CR-033P, significantly decreased 'sneezing' , 'nasal discharge' , 'nasal obstruction' , and 'coughing' in cumulative number of days and significantly shortened 'nasal discharge' and 'nasal obstruction' in maximum number of days.Therefore, our data revealed that consumption of 150 mg/day curcumin equivalent of TS-P1 or CR-033P reduces common cold symptoms.In particular, consumption of 150 mg/day curcumin equivalent of TS-P1 shortens sneezing for approximately 10 days, nasal discharge for approximately 14 days, blocked nose for approximately 11 days, and cough for approximately 6 days.
All study experiments were sponsored by Theravalues Corporation.

About the authors
Yoshitaka Kuwabara is the Executive Director at Theravalues Corporation.He is a leading expert in the pharmaceutical industry and an experienced product strategist.Toshihiro Kakinuma is the manager of the Statistical Analysis Division at ORTHOMEDICO Inc.He has a particular interest in studying dietary supplements and functional foods.

Akiko
Asami Baba is the manager of the Clinical Science Division at ORTHOMEDICO Inc.She has much experience in the research of functionality of antioxidants and a particular interest in studying dietary supplements and functional foods.
Tsuyoshi Takara is the director of Medical Corporation Seishinkai, Takara Clinic.He is a specialist in cancer treatment.He is also actively involved in the research of functionality of food, and has been conducting about 100 clinical trials on food annually as a principal investigator.

Figure 1 .
Figure 1.flowchart of subjects in this study.
Hirose is a member of the Research and Development Department at Theravalues Corporation.She has been conducting researches on curcumin and analyses related to product quality assurance.Hyunjin Lee is an Assistant Researcher of the Research and Development Department at Theravalues Corporation.She has much experience in applied Health Science and a particular interest in studying dietary supplements and functional foods.

Table 1 .
Schedule of enrollment, intervention, and assessment.

Table 2 . 1 .
Baseline characteristics of subjects in ppS analysis.Values of sex are shown as number of cases and percentage of cases in each group.in other items, values are shown as mean and standard deviation (SD).

Table 2 .
2. Baseline characteristics of subjects in subgroup analysis.items Subgroup where subjects who were included with ppS and did not use medications to alleviate common cold symptoms Values of sex are shown as number of cases and percentage of cases in each group.in other items, values are shown as mean and standard deviation (SD).

Table 3 .
Cumulative and maximum persisted days for which, and frequency with which common cold symptoms were experienced during the intervention period per subject in ppS analysis.

Table 4 .
Cumulative and maximum persisted days for which, and frequency with which each symptom was experienced during the intervention period per subject in ppS analysis.

Table 6 .
Cumulative and maximum persisted days for which, and frequency with which each symptom was experienced during the intervention period per subject in the subgroup analysis.

Table 6 .
Continued.DiscussionThe mean cumulative number of days for which common cold symptoms persisted (sum of the number of days for which the following symptoms persisted: whole body malaise, chilliness, feverishness, fatigue, sneezing, nasal discharge, blocked nose, throat pain, cough, joint pain, and/or muscle pain) during the intervention period was