Improvement in Human Immune Function with Changes in Intestinal Microbiota by Salacia reticulata Extract Ingestion: A Randomized Placebo-Controlled Trial

Plants belonging to the genus Salacia in the Hippocrateaceae family are known to inhibit sugar absorption. In a previous study, administration of Salacia reticulata extract in rats altered the intestinal microbiota and increased expression of immune-relevant genes in small intestinal epithelial cells. This study aimed to investigate the effect of S. reticulata extract in human subjects by examining the gene expression profiles of blood cells, immunological indices, and intestinal microbiota. The results revealed an improvement in T-cell proliferation activity and some other immunological indices. In addition, the intestinal microbiota changed, with an increase in Bifidobacterium and a decrease in Clostridium bacteria. The expression levels of many immune-relevant genes were altered in blood cells. We concluded that S. reticulata extract ingestion in humans improved immune functions and changed the intestinal microbiota. Trial Registration: UMIN Clinical Trials Registry UMIN000011732

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Study Outline
Title Evaluation of immunity enhancement by ingestion of a Salacia plant extract-containing supplement Name of products tested Salacia-containing supplement and placebo

Objective
The objective is to determine effects of a "Salacia plant extract-containing supplement" on immunological functions in adult males who were conscious of being prone to tiredness and ingested the supplement for 4 weeks. A placebo-controlled, double-blind study is conducted to evaluate the immunity-enhancing effect of the Salacia plant extract-containing supplement ingested, and blood pressure/pulse measurement, blood testing, immunity measurement, cytokine production measurement, blood gene expression analysis, blood bile acid measurement, intestinal flora analysis, and a questionnaire survey are conducted.

Subjects
Number of subjects: 30 (Group 1: 15, Group 2: 15) Entry criteria (based on subjects' self-reporting) 1) Japanese males who are at least 50 years and less than 60 years of age at the time of consenting ※ "Japanese" herein refers to individuals with Japanese genetic background, regardless of Japanese citizenship. 2) Individuals who have a feeling of tiredness on a daily basis and a feeling of not recovering from tiredness readily.

3) Day-shift, sedentary workers 4) Individuals with a regular life rhythm
Screening criteria 1) Selection is made by the sponsor (immunological scores, etc.) Screening items 1) Body height measurement 2) Body structure measurement (body measurement) 3) Blood pressure/pulse measurement 4) Blood test 5) Blood sample submission 6) Fecal sample submission Exclusion criteria 1) Individuals who have ever medically treated for a heart disease, a renal disorder, a hepatic disorder, a gastrointestinal disease, or some other conditions. 2) Exclusion for diseases, etc. [individuals being treated for atrial fibrillation, arrhythmia, hepatic disorder (especially history or current illness of hepatitis), renal disorder, cerebrovascular disorder, rheumatism, diabetes, dyslipidemia, hypertension, and other chronic diseases 3) Individuals with a gastrointestinal disease (such as extreme constipation and frequent diarrhea) 4) Individuals with severe anemia 5) Individuals taking an medicine (including kampo formulations) or a supplement on a regular basis 6) Individuals who are allergic (to test food-related food/drug) 7) Individuals taking a food/drug with a digestive conditioning effect on a daily basis over the last one month 8) Individuals consuming alcohol excessively 9) Smokers 10) Individuals with diabetes 11) Individuals who have participated within the last 3 months or is currently participating in any other clinical study 12) Individuals with any abnormality found in the screening study 13) Individuals determined to be ineligible by the Principal Investigator Subject allocation method Subjects shall be grouped in a manner such that the resulting groups are as close as possible to each other in the mean immunological score.

Study Title and Study Number
Study title: Evaluation of immunity enhancement by ingestion of a Salacia plant extract-containing supplement Study number: FJFR-13916 3. Study Organization 1) ) ) ) Sponsor

Background
Plants of the genus Salacia have been used for generations as Ayurvedic medicinal plants for prevention of diabetes and obesity. Moreover, recent studies have shown that Salacia plants extracts improved effects on intestinal environment/flora, in addition to the already known improving effects on the blood sugar level and obesity.
In addition, various immunological effects have been demonstrated in animals that received Salacia plant extracts, including enhanced expression of Th1 cell-associated genes, increased growth of intestinal bacteria considered to be involved in immunity, and symptomatic improvement of influenza infection, and immunostimulatory effects of Salacia plant extracts are also expected for humans.
However, effects of Salacia plant extracts on human immune functions have not been determined in any previous studies, and remain to be studied.

Objective
The objective is to determine effects of a "Salacia plant extract-containing supplement" on immunological functions in adult males who were conscious of being prone to tiredness and ingested the supplement for 4 weeks. A placebo-controlled, double-blind study is conducted to evaluate the immunity-enhancing effect of the Salacia plant extract-containing supplement ingested, and blood pressure/pulse measurement, blood testing, immunity measurement, cytokine production measurement, blood gene expression analysis, blood bile acid measurement, intestinal flora analysis, and a questionnaire survey are conducted.

Subjects
※ "Japanese" herein refers to individuals with Japanese genetic background, regardless of Japanese citizenship.
Of SOUKEN registrants who have undergone a questionnaire survey at the time of recruitment, those who self-report to meet the following "Entry criteria (based on subjects' self-reporting)" and not to meet the "Exclusion criteria" are subjected to a screening survey, and 30 of those who meet the "Screening criteria" and were considered appropriate to participate in this study are selected as subjects in this study.
Even if test results, etc. of any selected subject are found contradictory to those self-reported earlier ("Entry criteria (based on subjects' self-reporting)" "Exclusion criteria"), the subject shall be deemed to be approved to participate in the study as long as the subject has not completed the screening survey, and shall not be excluded from the analysis set unless there is a special reason to do so.

1) ) ) ) Entry criteria (based on subjects' self-reporting)
1) Japanese males who are at least 50 years and less than 60 years of age at the time of consenting ※ "Japanese" herein refers to individuals with Japanese genetic background, regardless of Japanese citizenship. 2) Individuals who have a feeling of tiredness on a daily basis and a feeling of not recovering from tiredness readily. 3) Day-shift, sedentary workers 4) Individuals with a regular life rhythm 2) ) ) ) Screening criteria 1) Selection is made by the sponsor (immunological scores, etc.) 3) ) ) ) Screening items 1) Body height measurement 2) Body structure measurement (body measurement) 3) Blood pressure/pulse measurement 4) Blood test 5) Blood sample submission 6) Fecal sample submission 4) ) ) ) Exclusion criteria 1) Individuals who have ever medically treated for a heart disease, a renal disorder, a hepatic disorder, a gastrointestinal disease, or some other conditions. 2) Exclusion for diseases, etc. [individuals being treated for atrial fibrillation, arrhythmia, hepatic disorder (especially history or current illness of hepatitis), renal disorder, cerebrovascular disorder, rheumatism, diabetes, dyslipidemia, hypertension, and other chronic diseases 3) Individuals with a gastrointestinal disease (such as extreme constipation and frequent diarrhea) 4) Individuals with severe anemia 5) Individuals taking an medicine (including kampo formulations) or a supplement on a regular basis 6) Individuals who are allergic (to test food-related food/drug) 7) Individuals taking a food/drug with a digestive conditioning effect on a daily basis over the last one month 8) Individuals consuming alcohol excessively 9) Smokers 10) Individuals with diabetes 11) Individuals who have participated within the last 3 months or is currently participating in any other clinical study 12) Individuals with any abnormality found in the screening study 13) Individuals determined to be ineligible by the Principal Investigator

Consent from Subjects
① Objective and methods of this study ② Explanation about the test products, their effects, and adverse effects predicted to occur ③ Subjects are kept under adequate control by the Principal Investigator during the study. ④ Subjects are free from any penalty even if they do not consent to participate in the study. ⑤ Even after formally consenting to participate in the study, subjects can withdraw it any time. ⑥ Appropriate procedures and therapies that subjects are entitled to receive in case of a health damage related to this study ⑦ Subjects shall receive information that potentially effects subjects' intention for continued participation in this study as soon as such information is obtained. ⑧ Other necessary matters concerning protection of subjects' human rights and disclosure of subjects' information ⑨ What subjects should do and do not ⑩ Establishment of the consultation service at the institution that subjects should contact for more information about this study and subjects' rights or in case of health damages related to this study ⑪ About cooperation expenditures to be paid to subjects

Outline of the Test Products
Test products are those supplied by Fujifilm Corporation (a Salacia-containing supplement and a placebo).

1) ) ) ) Origin and the course of development
The functional food "Metabarrier", which has been released in 2008 from Fujifilm Corporation, contains useful components such as green tea extracts and red wine polyphenols as well as the Salacia plant extract. A test food comprising the Salacia plant extract as the only useful component was prepared to clarify functions of the Salacia plant extract alone.
2) ) ) ) Safety/efficacy data The test product used in this study is prepared from commercially available Metabarrier by removing some components therein, and safety/efficacy information available for Metabarrier may be used as a guide.
No serious adverse event was reported in the results of the human use study conducted by SOUKEN before Metabarrier was launched. Furthermore, no serious health damage has been reported to be caused by Metabarrier components since its release in 2008 through to now. Therefore, the safety of the test product appears to be sufficient.
In addition, the test product is considered to have a mild effect of reducing intestinal sugar absorption via inhibition of the α-glucosidase activity, since it contains the Salacia plant extract as does Metabarrier.
3) ) ) ) Amount to be ingested and underlying rationale The amount of the Salacia plant extract in commercially available Metabarrier is 240 mg as a daily intake. In this study, the test product is formulated in tablets containing the Salacia plant extract in the amount corresponding to that ingested daily as Metabarrier. It should be noted that up to 1000 mg/day can be ingested safely according to the guidelines issued by the Ministry of Health, Labour and Welfare.

Study Methods 1) ) ) ) Study design
Placebo-controlled, double-blind 2) ) ) ) Study schedule The study shall be conducted in the following schedule. ・Number of observation: 2 ・Observation time points: Before ingestion, after 4 weeks of ingestion ・Ingestion period: 28 days ① Acquisition of written consent ② Main study [Number of subjects in the main study] Number of subject: 30 (Group 1: 15, Group 2: 15) ※ For items to be carried out in the main study, see Attachment 1.

3) ) ) ) Subject allocation
Subjects shall be grouped in a manner such that the resulting groups are as close as possible to each other in the mean immunological score.

Endpoints (various tests, measurement items, and survey methods)
Items set forth in Attachment 1 are performed.

Test Product Management 1) ) ) ) Persons in charge of the test products
Fujifilm Corporation Yuriko Oda SOUKEN Co., Ltd.

3) ) ) ) Test product management and storage
Test products are appropriately stored by the Institution (SOUKEN Co., Ltd.).

Subject Management Matters
① Subjects undergo fasting for at least 6 hours before the time of visit. (Example: Subjects scheduled to visit the clinic at 7 am may not eat after 4 am on the day) Water (mineral water) should be taken sufficiently. Subjects are not allowed to consume alcohol and should avoid excessive exercise on the day before a test. ② During the study, subjects are not allowed to take any supplement, except the test product and control product, stated to have immunological/digestive conditioning effects, or food containing an ingredient with the digestive conditioning effect (e.g., Easyfiber/Weider jelly fiber-in/Allbran/Oligo-no-okage/Calpis and the like/Kirin Supli/Kore-cut/Fibe-mini/Any green juice with FSHU stamp/Nisshin oishisa plus psyllium series/apple-flavored black vinegar/Lactic acid bacteria beverage (Yakult; Pirukuru; Mirumiru; Joa; Laurie Ace; etc.)/Yogurt; Yogurt drink (R-1; LG21; Bifidus; Meiji Bulgaria yogurt series; Soful; etc.)). ③ During the study (from 1 week before the screening day to the test day after 4-week ingestion), subjects should avoid irregular ways of living (such as lack of sleep, over-eating/drinking). For diet and exercise, subjects should maintain the same quantity and quality as in daily lives before the start of the study. ④ During the study, drugs considered to affect test results (such as laxatives, intestinal remedies) are prohibited in principle. Record it in web diary if any of these must be taken. ⑤ During the ingestion period, subjects are required to record whether the test food is ingested in the web diary specified by the Institution every day. ⑥ In case of any change in physical conditions during the study, contact the entity managing the study immediately, and follow its instructions on subsequent necessary actions.
※ Although the items listed above on subject management must be complied in principle, they do not apply in cases medically required to do otherwise or other cases in which risks to the body or life are foreseen. If any of the items listed above is violated, the subject should contact the Institution promptly.

Statistical Analysis
① The significance level shall be 5% by a two-tailed test. ② For items/methods for statistical analysis, see Attachment 1.

Final Report 1) ) ) ) A report shall be prepared based on the data tabulated and statistically analyzed at the Institution (Data attachment to the study report).Schedule until the report is prepared (plan)
Work is done according to the following schedule. ※ However, if it takes more than 7 days to obtain a test result, the subsequent schedule is postponed for a considerable period of time (on the following day if it is a holiday or in weekend).
Expected submission date (only business days are counted) Data to be submitted Within 10 days after the end of the study Case conference 10 days after the end of the study Submission of a draft data attachment to the study report Within 14 days after submission of a draft data attachment to the study report The data attachment to the study report to be finalized 25 days after the end of the study Key open Within 14 days after the data attachment to the study report has been finalized Submission of a draft study report Within 14 days after submission of a draft study report The study report to be finalized

Criteria for discontinuation of the study and drop-out cases
In case of any of the following, the study is terminated on the basis of Principal Investigator's medical and ethical judgment. Except for some special circumstances, appropriate medical care is provided to subjects to ensure the safety of the subjects.
① When a serious adverse reaction, subjective/objective symptom, etc. has emerged ② When the subject is difficult to continue the study because of complication with any other disease or progression of a complication ③ When tests are extremely difficult to perform ④ When the whole study is discontinued ⑤ When the Principal Investigator finds the study necessary to be discontinued

Compensation to Subjects
If a damage to a subject has occurred due to the study during the study, or if a subject has filed a law suit seeking compensation for a damage caused by this study, the Principal Investigator shall promptly inform the Sponsor of it.
The Institution shall be responsible for compensations for health damages caused intentionally or negligently by the Institution, while the sponsor shall be entirely responsible for compensations in cases where health damages have occurred due to the test products. However, this does not apply if the health damage was falsely reported or intentionally caused by the subject.

Criteria for Exclusion from Analysis (Reporting) Set
Subjects who meet any of the following criteria are discussed in the case conference and shall be excluded from the analysis (reporting) set unless there is a special reason to do otherwise.
① Subjects found to have substantially deviated from instructions pertinent to subject management matters during the study. ② Subjects with data of which reliability is compromised in a major manner due to, for example, troubles in testing ③ Subjects who have missed ingestion (the amount ingested does not meet the daily dose) in more than 15% of the days scheduled for ingestion ④ Subjects found not to meet entry criteria and to meet exclusion criteria ⑤ Subjects found appropriate to treat as drop-out cases for evident reasons

Note on Completing the Survey Form
A black-ink ballpoint pen shall be used to complete the survey form, and corrections shall be made with double lines.

Modification/Absence of Test Data
If a delay or cancellation of measurement is unavoidable for subject's health conditions or request, subject's health conditions and request shall be given priority in accordance with the spirit of the Declaration of Helsinki. If some data are impossible to collect for these reasons or other unavoidable reasons, they shall be treated as missing data.

Protection of Subjects' Privacy
All personnel involved in this study shall give full considerations to handling of information that can be used to identify individuals. In other records obtained in the course of this study, numbers shall replace names to prevent individuals from being identified. The test subcontractor is not allowed to obtain subjects' personal information under any circumstance.

Ethics 1) ) ) ) Compliance matters
This study shall be conducted in accordance with the Declaration of Helsinki (revised at the Seoul general assembly in 2008), with due ethical considerations.

2) ) ) ) Shiba Palace Clinic Ethical Review Board
Members of the Shiba Palace Clinic Ethical Review Board meet and discuss ethics and validity of the protocol for this study. The study must be conducted based on the protocol approved by the Shiba Palace Clinic Ethical Review Board. Any substantive deviation from the protocol must be approved by the Board. ○ -(S value adopted)