Effect of Sea Grapes-antioxidants Extract in Obese Men
Phase: EARLY_PHASE1
Conditions: Obesity, Aging, Diabetes
Sponsor: State Islamic University of Sunan Kalijaga Yogyakarta

Official Title of the study: 
Effect of Sea grapes-antioxidants Extract in Obese Men : 4 
Weeks Randomized-Double Blind Controlled Trial 
 
Document Type: Study Protocol with SAP and/or ICF 
Date Approval by Ethical Committee: August 23, 2021 
Principle Investigator: 
Fahrul Nurkolis 
Biological Sciences, 
Faculty of Sciences and Technology, 
State Islamic University of Sunan Kalijaga (UIN Sunan Kalijaga Yogyakarta) 
 
 
INTRODUCTION 
 
Peroxisome proliferator-activated receptor (PPAR)-γ coactivator 1 alpha (PGC-1α) plays 
a role in many substantial metabolic processes, energy homeostasis and promotes muscle tissue 
remodeling, which is oxidative and less glycolytic (Liang & Bangsal, 2006). In conjunction with 
PGC-1α on mitochondrial respiration (regulating respiration) in muscle cells, this co-activator also 
induces gene expression for the insulin sensitive glucose transporter (Glut-4) and increases glucose 
uptake (Puigserver & Spiegelman, 2003; S. Yang et al., 2020). PGC-1α also induces brown 
adipose tissue (BAT) which usually declines in aging (Puigserver & Spiegelman, 2003). BAT 
which is a brown adipocyte contains many small droplets and a much higher number of 
mitochondria (containing iron), which gives the tissue its color (Cedikova et al., 2016). Brown fat 
also contains more capillaries than white fat and this supplies tissues with oxygen and nutrients 
and distributes the heat generated throughout the body (Cedikova et al., 2016). Noticing the 
function of PGC-1α in the thermogenic brown adipose tissue (BAT) program, the regulation of 
Glut-4 and mitochondrial oxidation in the muscle, and the dominant role of PGC-1α in hepatic 
gluconeogenesis along promoting muscle tissue remodeling activity, all of which suggest that this 

co-activator can be a target for antiobesity, antidiabetic and antiaging drugs (Chronic Diseases and 
Aging) (Liang & Ward, 2006; Puigserver & Spiegelman, 2003; S. Yang et al., 2020). 
Sea grape (Caulerpa racemosa) or lawi-lawi (local Indonesian term) is a species of green 
algae classified as Caulerpaceae family that had be found in the waters around Sulawesi (Pakki et 
al., 2020). Sea grapes are harvested intensively because they are an important source of 
macronutrients and micronutrients, especially in East and Southeast Asia (commercially grown in 
ponds and consumed in parts of the Philippines, Indonesia and Vietnam) as the main source of 
traditional diets (Chen et al., 2019), although it is still used for health products. Several studies 
have shown that sea grapes contain several bioactive components, such as protein, 
polysaccharides, polyphenols, flavonoids, and antioxidants (P. Yang et al., 2015; Yep et al., 2019; 
Taslim & Fahrul, 2021). In addition, sea grapes contain high antioxidant levels, and may be 
potential as functional foods or nutraceuticals (Tanna et al., 2020; Yep et al., 2019; Nurkolis et al., 
2021). Doses of 150 mg/kg BW (30 mg/200g BW) sea grape extract in a previous pre-clinical trial, 
showed that it could improve blood glucose, total cholesterol and serum PGC-1 levels in rats fed 
a diet high in fat and cholesterol (Kuswari et al., 2021). In addition, you also have hepatoprotective 
activity (aka non-toxic) in diabetic mice (Qudus B Aroyehun et al., 2020). Previous research 
(Kuswari et al., 2021), is an in vivo (Pre-clinical) study that has not represented the benefits or 
efficacy of sea grape extract on variables tested in humans. Therefore, this clinical trial was 
conducted to support the effect of sea grape extract-antioxidant on blood glucose, total cholesterol, 
and PGC-1 levels in obese men for 4 weeks using a Randomized-Double Blind Controlled Trial. 
Methods 
This clinical trial study was conducted at Manado City, Indonesia. 
Extract Creation 

Fresh sea grape (Caulerpa racemosa) is collected from the shallows (5-10 meters above 
sea level) of Mantehage seawater, North Sulawesi, Indonesia. Botanical identification and 
authentication are confirmed in the department of pharmacology, faculty of mathematics and 
natural sciences, Sam Ratulangi University, Indonesia. Specimens are collected for future 
reference. Sea grapes (Whole body) is thoroughly rinsed with water, dried with air at room 
temperature, and in a 40o C oven, then smoothed with an electric grinding. Furthermore, the extract 
preparation, coarse powder (1 kg) is macerated in ethanol 96% for 72 hours with each extraction 
carried out in triple, which results in a yield of 34%. The extract is roughly filtered with Whatman 
41 filter paper. The total filtrate is glued and evaporated at 40o C with the RV 8 IKA rotary 
evaporator under reduced pressure (100 millibars) for 90 minutes and evaporated in a 40oC oven 
to produce the viscous extract. The extract is stored in the refrigerator at a temperature of 10°C 
until used in research. The extract powder is encapsulated in capsules no. 000. 
Dose Conversion 
The best and significant dose in a preliminary study (Pre-clinical) in Ratus novergicus rats 
is 150 mg/kg BW (Kuswari et al., 2021). Afterward, the doses in this clinical trial study applied 
the table (Table 1) "Evaluation of drug activities: pharmacometrics, ed. by D.R. Laurence and A.L. 
Bacharach" (Laurence & Bacharach, 2013). The conversion factor from mouse to human was 56.0. 
However, the dose is still within (mg/kg BW) while the conversion factor for rats must be per 
200gram BW, so 150 divided (1000:200=5) then you get 30 mg/200gram BW. Therefore, it means: 
Human Dose = Rat Dose x Conversion Factor = 30 x 56,0 = 1680 mg/70kg BW  
= 1,68 g/70kg BW 
Antioxidant Test on Sea Grape Extract 

Antioxidant activity is determined by using DPPH (Batubara et al., 2015). Each sample 
consisting of 100 L was placed in microplate 96 and added 100 L DPPH 0.3 mM, then incubated 
for 30 minutes in a dark room. Sample absorption was measured using an ELISA reader at 
wavelength 517 nm. Antioxidant activity is calculated by equation 1 as follows: 
 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 (%) =
("#$"%)
"#
𝑥 100%  
Explanation: 
A0 = Blank Absorption, A1 = Standard or Sample Absorption 
The results of the antioxidant activity test against DPPH resulted in a value of 45.66 ± 0.55% 
(Triplicates/Triplo). 
Caulerpin (PubChem ID: 5326018) with molecular formula: C24H18N2O4 is a typical secondary 
metabolite compound belonging to the algae genus Caulerpa Sp and is a bioactive compound from 
a group of alkaloid compounds that also functions as antioxidants (de Souza et al., 2009; Aguilar-
Santos., 1970; Gorbi et al., 2014). Therefore, a further test was carried out, namely the antioxidant-
caulerpin levels in the extract (sample) using Liquid Chromatography - Mass Spectrometry (LC-
MS) and obtained 398.13278 Mass (397.12257 m/z). This shows that it is true that there is an 
antioxidant compound-caulerpin in the sample extract. 
 
 
 
 
 
 
 

 
 
Study Protocol with SAP and/or ICF 
 
The study was a 4-week, randomized, double-blind, placebo-controlled clinical trial 
followed by a 1-week screening period. Participants who respond to the invitation and meet the 
entry criteria during the telephone screening interview are scheduled for the initial visit. Evaluation 
during the initial visit includes physical examination in the form of BMI (Body Mass Index) based 
on Asia-Pacific guidelines and blood parameter screening tests in the form of blood glucose, 
triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and total cholesterol, 
and PGC-1α were performed on all participants within 1 week of the initial screening. A random 
number between 1 and 70 was generated for each subject and registered participants were 
scheduled for their first visit and randomly assigned to the sea grape extract group (n=35) or 
placebo (n=35). Sea grape extract/placebo tablets/capsules were given to participants every 1 week 

(1 day of consumption per oral) 15 Minutes before eating (According to the diabetes drug guidline 
consumtion).  
During the 4-week intervention period, participants were asked to continue their usual diet 
and not to consume functional foods or other dietary supplements. Anthropometry (BMI), Waist 
Hip Ratio (WHR) , blood parameters, urine profile (Marker of toxicity), and nutrient intake of both 
groups were measured before and after the intervention period. During the trial phase, all 
participants were instructed to maintain their normal diet and physical activity. Each week 
participants were asked to report assessments of side effects or changes in training, lifestyle, or 
diet; and to evaluate tablet compliance. 
Subjects/Participants 
The study participants were recruited during 2021 at RSUP Prof. Dr. RD. Kandou 
(Manado, Republic of Indonesia). A total of 150 participants agreed to participate in the study. 
Only individuals men who are obese (BMI ≥ 25 kg m−2 and Waist Hip Ratio (WHR) ≥ 0.90) 
according to Asia-Pacific guidelines and have not been diagnosed with other diseases were 
included in the study. To meet guidelines for evaluating the efficacy of functional food referring 
to the Korea Food and Drug Administration (because Indonesia does not yet exist and Koreas 
belong to one Asian region so used) (KFDA Guidline, 2021), very obese participants (BMI ≥ 30 
kg m−2) were not included in the study. 
In total, 70 participants met the research criteria (age 29.98 ± 3.26 year; weight, ..... ± .... 
kg; BMI, .... ±.... kg m−2) and were randomly divided into two groups (n = 35 each) given sea grape 
extract 1.68 g/70kg BB day−1 or placebo (1.68 g/70kg BB day−1). The exclusion criteria for this 
study are as follows: (a) significant weight variation (over 10%) in the last 3 months; (b) a history 
of cardiovascular disease including arrhythmia, heart failure, or myocardial infarction, diabetes 

mellitus (DM) and the use of pacemakers; (c) a history of conditions that may interfere with test 
products or inhibit their absorption such as gastrointestinal diseases (Crohn's disease) or surgeries 
that have been experienced (caesarean section or enterocele); (d) participation in other clinical 
trials in the last 2 months; (e) abnormal liver function; (f) a history of kidney disease (eg, acute or 
chronic renal failure and nephrtic syndrome); (g) undergo antipsychotic drug therapy within the 
last 2 months; (h) laboratory test results as well as medical or psychological conditions that may 
interfere with successful participation in research assessed by researchers; (i) a history of alcohol 
or substance abuse; and (j) allergy or hypersensitivity to any of the ingredients in the test product; 
(k) is neither a passive nor an active smoker. All participants give written consent before the 
investigation begins. The research protocol was submitted to the Health Research Ethics 
Commission (KEPK) of The General Hospital of Education Prof. Dr. RD. Kandou (Manado, 
Republic of Indonesia) online at http://sim-epk.keppkn.kemkes.go.id. All protocols referred to The 
Declaration of Helsinki and The Council for International Organizations of Medical Sciences 
(CIOMS). 
Measurement of Efficacy Results 
A total of 70 participants who met the research criteria were asked to visit the clinic once 
every 1 week (0th, 1st, 2nd, 3rd, and 4th/last week of the study period) with a total of 5 clinical 
visits including initial examination. During each visit, the use of the supplement is currently 
reviewed and symptoms or side effects are noted. During screening visits, demographic and 
lifestyle information is collected (age, alcohol consumption, and smoking habits). Medical history 
is taken and urine turbidity tests are carried out. 
The following parameters are assessed; Weight, Height, WHR and BMI during each visit. 
Blood samples were collected after a minimum of 12 hours of fasting during initial screening as 

well as in the 0, 2nd and 4th weeks of the intervention period to obtain blood glucose profiles, total 
cholesterol, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and 
PGC-1α. Blood samples were taken from the arm vein. The arm vein to be punctured is cleaned 
with 70% alcohol and allowed to dry. A tourniquet is placed on the upper arm to expose and 
slightly accentuate the veins. The skin is stretched over the vein with the fingers of the left hand 
so that the vein cannot move. The skin is pierced with a needle and syringe with the right hand 
until the tip of the needle enters the lumen of the vein. The tourniquet is removed and slowly 
withdraw the syringe suction until the desired amount of blood is obtained. A 70% alcohol swab 
is placed over the needle and the syringe and needle are removed. The needle is removed from the 
syringe and the blood sample is transferred into a vacutainer tube. The sample is then centrifuged 
for 20 minutes at a speed of 3000 rpm. Finally, the serum is collected for analysis of blood glucose, 
total cholesterol, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and 
PGC-1α. Blood glucose, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein 
(LDL) and cholesterol levels were tested using COBAS Integra® 400 plus analyzer (Roche). The 
sample was washed with Phosphate Buffered Saline (PBS, pH 7.4) 1% until the liquid was clear. 
The sample was centrifuged at 3000 rpm for 20 minutes to obtain pellets and supernatants. The 
supernatant is taken for PGC-1α examination. The concentration of PGC-1α is measured using the 
Human PGC-1α (PGC1 alpha (PPARGC1A) (NM_013261) Human Untagged Clone kit. 
Evaluation of Safety and Diet 
The safety of the extract is assessed by the following procedure. Urine Test Strips 10 Verify 
Parameters, Parameters examined are: 
- Glucose (50 -100 mg / dl) 
- Protein (7.5 - 15 mg / dl) 

- pH (5-9) 
- Leukocytes (9-15 leu / ul) 
- Nitrite (0.05-0.1 mg/ dl) 
- Urobilinogen (0.2-1.0 mg / dl) 
- Blood (5-10 Ery/ul) 
- Ketones (2.5-5 mg/dl) 
- Bilirubin (0.4-1.0 mg/ dl) 
- Specific Gravity (SG) (1,000-1,030) 
Test the urine parameters above using Urinalysis 10U Reagent Strips Verify (REF U031-
102 exp. 2022-03-05). Pulse rate and blood pressure are measured on each visit after a 5-minute 
break using intelliVue MP70 (Philips, Netherlands). Personal reports are also recorded at these 
times. We kept the subjects maintaining their usual diet and activity, and all participants completed 
diet records on each visit to the clinic during the intervention period to evaluate their energy intake 
and diet quality. Food intake data were analyzed by a nutritionist (Melvin Junior Tanner, S.Gz). 
Statistical Analysis 
Statistical analysis is performed using SPSS software, version 26 (IBM Corporation). Fixed effects 
include treatment groups, treatment visits, and interactions between treatment and visit groups. 
The value p<0.05 is considered statistically significant. 
 
 
 
 
 

 
 
Table 1. Dose Conversion of Experimental Animals with Humans 
 
Mice 
20 g 
Rat 
200 g 
Guinea 
Pig 400 g 
Rabbit 
1,5 kg 
Cat 
2 kg 
Ape 
4 kg 
Dog 
12 kg 
Human 
70 kg 
Mice 
20 g 
1,0 
7,0 
12,25 
27,8 
29,7 
64,1 
124,2 
387,9 
Rat 
200 g 
0,14 
1,0 
1,74 
3,9 
4,2 
9,2 
17,8 
56,0 
Guinea 
Pig 
400 g 
0,08 
0,57 
1,0 
2,25 
2,4 
5,2 
10,2 
31,5 
Rabbit 
1,5 kg 
0,04 
0,25 
0,44 
1,0 
1,08 
2,4 
4,5 
14,2 
Cat 
2 kg 
0,03 
0,23 
0,41 
0,92 
1,0 
2,2 
4,1 
13,0 
Ape 
4 kg 
0,016 
0,11 
0,19 
0,42 
0,45 
1,0 
1.9 
6,1 
Dog 
12 kg 
0,008 
0,06 
0,10 
0,22 
0,24 
0,52 
1,0 
3,1 
Human 
70 kg 
0,0026 0,018 
0,031 
0,07 
0,076 
0,16 
0,32 
1,0 
 

SPECIAL MEDICAL APPROVAL LETTER (INFORM CONSENT) 
 
I Signed Below: 
Name: 
Gender (M/F):  
Age/Date of Birth: 
Address: 
Phone: 
Stating in fact from *Myself/*As a Parent/*Husband/*Wife/*Child/*Guardian of: 
Name: 
Gender (M/F):  
Age/Date of Birth: 
Address: 
Phone: 
 
Hereby agree/refuse to be involved as a volunteer or participant in the study "Supplementation of 
sea grape extract on blood glucose levels, total cholesterol, and PGC-1". 
From the explanation given, I have understood everything related to the disease, as well as the 
medical measures that will be taken and the possibility of post-action that can occur according to 
the explanation given. 
 Manado, …………………..…2021 
 
Doctor, 
 
 
 
 
Subjects, 
 
Sign 
 
 
 
 
 
 
    Sign 
 
 
(……………………)  
 
 
 
(…………………………..) 
* Strikethrough The Unnecessary 
 
 
 

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