The study subjects were recruited and selected at the Clinical Trials Center for Functional Foods (CTCF2) at Chonbuk National University Hospital from September to November 2015. A total of 80 healthy male and female subjects agreed to participate in this study. A total of 80 participants were randomly assigned into one of the study groups (40 subjects each) using a computer-generated random number table by the Randomization program of the version 9.2 SAS® system (SAS Institute, Cary, NC, USA).
The criteria for the selection and exclusion of participants in this study are described below.
The selection guidelines were as follows:
1) Male and female adult participants aged 20~75 at the time of the screening test.
2) Participants who showed a slight decrease in immunity and had a peripheral blood white blood cell (WBC) level of 3*103 ~ 7*103 cells/μl measured during the screening test.
3) Participants who fully understood the test and decided to participate of their own free will and agreed to the written consent document.
The exclusion criteria were as follows:
1) Male and female adult participants who had a BMI less than 18.5 kg/m2 at the time of the screening test.
2) Participants who had a family history of medicinally or clinically significant hypersensitivity reactions.
3) Participants with thyroid or hypophyseal disorder.
4) Participants with acute severe cardiovascular diseases such as cardiac insufficiency, myocardial infarction, or stroke.
5) Participants with immunological disease, liver or renal failure.
6) Participants who had specific diseases such as a malignant tumor, lung disease, leukemia, collagenosis, multiple sclerosis, allergic skin disease, and other autoimmune disorders or a history thereof.
7) Participants diagnosed with diabetes.
8) Participants who had a “gastroesophageal reflux disease”, such as Crohn’s disease.
9) Participants who, within 2 weeks from the first intake day, had ingested medicine, Chinese medicine, or a health functional food that can affect the immunomodulatory effect of the test product. In the case of health functional foods, it was possible to participate in this program after a one-week wash out period.
10) Participants who had a history of treatment with anti-psychotics within 2 months of the screening test.
11) Participants who had a history of treatment for alcoholism or drug abuse.
12) Those who participated in other clinical studies within 2 months prior to the date of the first clinical test product intake.
13) Participants who had an alanine transaminase (ALT) or aspartate transaminase (AST) plasma level more than three times the guideline of the organization.
14) Female participants who were pregnant or breastfeeding or planned to become pregnant during the test period.
15) Participants who had disqualifying results in the diagnostic test and were inappropriate for other reasons.
All subjects gave written informed consent before entering the study. The study was conducted according to the Declaration of Helsinki. The study protocol was approved by the Functional Food Institutional Review Board (FFIRB) of Chonbuk National University Hospital (FFIRB number 2015–02-010) on August 25, 2015. This study was registered on June 28, 2016 after the termination of the study sub heading: The efficacy and safety of CS mycelium culture extract (P. hepiali, CBG-CS-2) on the promotion of immunity.
This study was an 8 week, randomized, double blind, and placebo-controlled clinical trial that was performed according to a computer-generated randomization schedule designed to assign subjects to the CBG-CS-2 or placebo group. Neither the investigators nor the subjects knew the randomization code or the results of the NK cell activity levels until after statistical analysis was complete. Subjects attended a screening visit (within 4 weeks), at which inclusion and exclusion criteria were assessed. The enrolled subjects were scheduled for their first visit, and subjects were randomly assigned to one of two groups, either the CBG-CS-2 (n = 40) or placebo group (n = 40). Subjects received either the CBG-CS-2 or placebo capsules every week, and all of the subjects were instructed to take either two CBG-CS-2 capsules (twice per day) or two placebo capsules (twice per day) per day (1.68 g/day) after breakfast and dinner for 8 weeks.
During the intervention period of 8 weeks, subjects were asked to continue their usual diets and activity and to not ingest any other functional foods or dietary supplements. Anthropometric and biochemical parameters, vital signs, and nutrient intake were measured before and after the intervention period. Every week, the subjects were asked to report any adverse events or changes in training, lifestyle, or eating patterns and to assess their capsule-dosing compliance.
A CONSORT checklist for the reporting of this study can be found in Additional file 1.
Preparation of test materials
Cordyceps mycelium extract (Paecilomyces hepiali, CBG-CS-2) was provided by Chebeigen, Inc. (Jeonju, Republic of Korea), and CS mycelium culture extracts were prepared as described previously . The main components of the mycelium culture extract were as follows: 32% cordyceps polysaccharide, 7.3% cordycepic acid, 0.13% adenosine, and 0.001% cordycepin . Main component profiles in the CBG-CS-2 were analyzed by high-performance liquid chromatography (HPLC) according to the Korean Food and Drug Administration (KFDA) guidelines, and these cordyceps polysaccharide profiles are shown in (Table 1). The extract was administered as a dark brown powder composed of 85.36% CS mycelium culture extract powder peel, 12.64% microcrystalline cellulose, and 2.0% hydroxylpropyl methylcellulose. The placebo was composed of 84.0% microcrystalline cellulose, 14.2% hydroxylpropyl methylcellulose, and 0.6% orange yellow.
The efficacy evaluation and safety evaluation parameters before the baseline (Week 0) of the human study and after eight weeks of participation were analyzed prior to participation in this human study for all of the subjects. The fasting blood samples of all subjects were collected and centrifuged (Hanil Science Industrial Co., Ltd. Seoul, Korea) at 3000 rpm for 20 min while keeping an empty stomach for more than 12 h from the day before the blood collection, and they were kept frozen at − 80 °C until the analysis. The blood samples were analyzed using a Hitachi 7600–110 analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan). The primary outcomes of this clinical trial measured the changes in natural killer cell activity [28, 34], and the secondary outcomes measured cytokines (IFN-γ and TNF-α, IL-1β, IL-2, IL-4, IL-10, and IL-12). The assessment of the safety parameters was performed through measuring an electrocardiogram (ECG) as well as laboratory tests (WBCs, RBCs, hemoglobin, hematocrit, platelets, total bilirubin, γ-glutamyl transferase (GGT), ALT, AST, alkaline phosphatase (ALP), TC, TG, LDL-C, HDL-C, fasting blood glucose, total protein, albumin, blood urea nitrogen (BUN), creatinine, creatine kinase, lactate dehydrogenase, Na, K, Cl, Ca, and P). Vital signs (systolic blood pressure, diastolic blood pressure, and pulse) of the study subjects were measured at every visit.
All statistical processing was analyzed using SAS version 9.2 (SAS Institute, Cary, NC, USA). All data were expressed as the mean ± standard deviation (SD) for continuous variables and as a frequency (%) for categorical variables. In this study, the intention-to-treat (ITT) population visited at least once and included performing an analysis of the efficacy and safety of those who had undergone measurements on the main evaluation variables after taking the CBG-CS-2 and placebo products. The data on the efficacy evaluation were based on the per-protocol (PP) group as the main analysis target, but a further analysis of the ITT group was implemented for its efficacy. To detect a 7.4% (SD 11.4%) difference in NK-cell activity change between groups with a power of 80% with a two-tailed alpha level of 0.05, a sample size of 40 per group (N = 80) was needed . In the case of the categorical variables, the significance test was applied with the Chi-square test (Fisher’s exact test). Additionally, the baseline and endpoint comparison of the investigation results of the evaluation variables in each group were tested by applying the paired t-test. The mean comparison between the two groups was tested with the independent sample t-test for independent samples, and the outcome variables for repeated measurement of the intake groups were applied with a linear mixed model within the intake groups and between the intake groups. The significance was statistically significant at the level of p < 0.05.