Effects of Tetragonula Aff. Biroi Propolis Extract on Lesion Growth, Apoptotic and Inflammatory Activity of the Rat Endometriosis Tissue
Phase: EARLY_PHASE1
Conditions: Endometriosis; Peritoneum, Endometriosis
Sponsor: Indonesia University

 
 
INDONESIA UNIVERSITY 
 
 
 
THE EFFECT OF TETRAGONULA AFF TOLERANT PROPOLIS 
ETHANOL EXTRACT. BUREAU OF ENDOMETRIOSIS LESI 
GROWTH, APOPTOSIS ACTIVITY CHANGE AND NETWORK 
ENDOMETRIOSIS ACTIVITIES 
 
 
STUDY PROTOCOL 
 
 
dr. Herbert Situmorang, SpOG(K) 
 
 
 
FACULTY OF MEDICINE 
MEDICAL SCIENCE DOCTORAL PROGRAM 
 
 
 
 
JAKARTA 
AGUSTUS 2019 
 
 

STUDY PLANNING 
 
The study involved 80 endometriosis mice which were randomly divided into 16 distinct groups: 
the sham (SH) -0, SH-2, SH-4, SH-6, negative control (KN) -2, KN-4, KN groups -6, positive control 
(KP) -2, KP-4, KP-6, Dosage of Propolis 1 (P50) -2, P50-4, P50-6, Dosage Propolis 2 (P100) -2, P100-
4, P100 -6. All mice that will be made endometriosis models are certain to have a normal estrous 
cycle. The first laparotomy was performed to make an endometriosis model according to the 
Vivian technique, which was confirmed to be successful 30 days later with a second laparotomy. 
Subsequently given different treatment according to the study group and carried out a third 
laparotomy at the appropriate time (2, 4, and 6 weeks after the second laparotomy). 
 
TREATMENT OF RAT 
All rats were treated in the ARF IMERI FKUI laboratory. Standard care in research rate follows 
standard maintenance protocols according to Bauman135 to provide optimal well-being for 
these rats, including: the need for social contact, a place to rest, a place to hide, exploration 
needs, the need for nutrition and free from environmental disturbances (e.g temperature, sound, 
etc.) 
 
Sh-2 
Sh-4 
Sh-6 
Sh-0 
WEEKS 
Normal 
Estrus Cycle 
Negative control 
Positive control 
50 mg/kg Propolis
100 mg/kg Propolis
 

ESTRUS CYCLE EXAMINATION 
The estrous cycle of rats was examined by taking vaginal lavage. Each rat in one cage was marked 
to distinguish the first to fifth mice. Vaginal discharge is taken by inserting the tip of a plastic 
pipette containing 10 μL NaCl 0.9% into the rat's vagina, but not too deep. If the rat is considered 
aggressive, sampling can be carried out. The vaginal discharge is then placed on a glass slide. 
Glass slide that is used is different for each cage. 
 
 
PROPOLIS ETANOL EXCRETION 
Ethanol Propolis extract (EEP) is made from fine Propolis extracted with 96% ethanol using the 
method described in Pratami. EEP powder according to the treatment dose was dissolved in 10 
cc of aqua before it was given to rat endometriosis models 
 
1st LAPAROTOMY 
1. Anaesthesia of rats (pentobarbital sodium, 50 mg / kg BW i.m.) 
2. Perform Asepsis and antisepsis procedures in the abdomen of the rat 
3. Incision mediana below the center along the 4 cm 
4. Treat bleeding 
5. Identification of bikornu uterus 
6. Resection of rat uterus by 0.5 x 0.5 cm with regard to the endometrium and serous sides 
7. Suturing the rat's uterus in the right anterior peritoneum 3 cm from the line 
8. incision with the endometrial side facing the peritoneum 
9. After there was no bleeding, the abdominal wall was covered in one layer with PGA 
thread number 2.0 
10. Rat returned to the cage, then complete the research form 

 
Bikornu Uterus Identification 
 
 
Incision in the uterus 
 
 
 
Stitching the uterus into peritoneum 
 

2nd LAPAROTOMY 
1. Anesthesia of rats 
2. Perform Asepsis and antisepsis procedures in the abdomen of the rat 
3. The median incision is below the center 4 cm long 
4. Treat bleeding 
5. After the peritoneum is opened, display the endometriosis lesions that have formed 
6. Do lesion documentation 
7. Excision of the formed lesion, then insert the tissue into the anatomic pathology tube 
8. Cover the entire abdomen using a thread and complete the research form 
 
THE TREATMENT 
The treatment is given by sonde orally 
 
PROPOLIS 
Each mililiters contains 20mg of propolis. The propolis dosage that we use is 50mg/kg BW. So, 
we give 0,2ml for propolis 50 treatment, and 0,4ml for propolis 100 treatment 
 
DIENOGEST 
We use 0,1mg/Kg BW/day dienogest 
 
WATER 
We give 0,2 ml every day for each rat 
 
SPECIMEN COLLECTION 
After the treatment is done (for 2, 4, and 6 weeks), we do the necropsy. We take a picture of the 
lesion (to compare with the beginning lesion), then we take the lesion and peritoneal rinses and 
blood for the protein level examinations. We also take the ovarium, uterus and peritoneum for 
pathologic anatomy examination for each treatment. 
 
SPECIMEN EXAMINATION 
 
Protein Levels of Bax, Bcl, Cleaved Caspase 3, IL-1B, and PGE 2 
Protein samples were obtained from endometriosis tissue taken by the treatment group. The 
lysis network uses a lysis buffer solution at 4 ° C for 30 minutes. The total protein is then broken 
down based on its molecular weight by the SDS-PAGE (Sodium Docecyl Sulphate-Polycramide Gel 
Electrophoresis) method and transferred to the PVDF (Polyvinylidene Difluoride) membrane. The 
membrane is then incubated with primary antibodies (Anti-Bax antibody [Abcam], Anti-Bcl-2 
antibody [Abcam], Anti-Cleaved Caspase-3 antibody [Abcam], Anti-IL-1B antibody [Abcam]), 
followed by incubation with secondary antibodies (Horse-Radish Peroxidase [Santa Cruz 
Biotechnology]). Proteins that have been bound to antibodies are then viewed using enhanced 
chemiluminescent reagent kits [Boster Bio] 
 
 
 

Mollecular docking 
Twenty-three compounds contained in Propolis were tested by screening using rule 5 (Lipinski's 
rule) consisting of: molecular weight <500 Dalton, <10 hydrogen donor bonds, <10 hydrogen 
acceptor bonds, partition coefficient values between -5 to 5. The screening results determine 
which compounds can enter the body and can work in endometriosis tissue. Furthermore, the 
three-dimensional shape is made of compounds which have passed the first stage. This process 
was created using the Marvin Sketch program. Furthermore, the compound that has been 
formed is stored as a ligand with extension pdb (protein data bank). Look for NFĸB receptor 
proteins, estrogen A, estrogen B, progesterone A and progesterone B in the protein data bank, 
which will be stored as protein. Ligands and proteins are tested using standard Lamarckian 
algorithms in the Autodock program. The results obtained are in the form of bond energy (ΔG), 
where smaller results indicate a higher likelihood of interaction. 
 
Examination of PGE2 receptor mRNA expression 
 
Isolation of RNA in Endometriosis Tissue Samples 
The RNA isolation process is carried out by first preparing a mixture consisting of 400 μL RB Buffer 
and 4 μL Beta-Mercaptoethanol. Then, the tissue is removed and placed in a later RNA, then the 
later RNA is removed and mixed with RB buffer and beta mercaptooethanol 
 
RNA spectrophotometry 
The instrument used to measure the concentration and purity of RNA is the Nano Drop 
Spectrophotometer. The device is turned on then the sample place is cleaned with tissue and 
distilled water. After that, blank measurements were taken by dripping RNAse-free water as 
much as 2 μL in the sample place. Next, concentration and purity (A260 / A280) measurements 
were taken in each sample. After the measurement is complete, the sample is again stored at -
20 ° C. 
 
Synthesis of cDNA 
CDNA synthesis was carried out using the ReverTra Ace® qPCR RT Master Mix kit with gDNA 
Remover [Toyobo] based on the ReverTra Ace® qPCR RT Master Mix Instruction Manual protocol 
with gDNA Remover (Toyobo 2017: 2). The first thing to do is calculate the ratio of the volume of 
RNA template and free water in order to obtain a mixture with a final concentration of RNA 25 
ng / μL with the formula: 
 
 
 
Initial RNA concentration 
RNA Volume Used 

RNA templates and reagents to be used are placed on ice and then thawed. After that, Genomic 
DNAse I reaction solution is prepared according to the table 
Composition of Genomic DNAse I solution 
 
 
Genomic DNAse I reaction solution was then incubated at 37 ° C for 5 minutes then immediately 
placed on ice. Then,  the Reverse transcription solution is made according to the following table 
with the sample remaining on the ice: 
 
 
Realtime PCR 
Determination of Melting Temperature (Tm) 
The primary melting temperature (Tm) is determined by the formula: 
 
A = Adenin, T = Thymine, G = Guanin, C = Sitosi 
 
Quantitative Real-time PCR (Q-PCR) samples 
Real-time PCR is done using the QuantBR SYBR Green PCR kit 
Master Mix [Qiagen] is based on the QuantiTech® SYBR® Green PCR Handbook protocol (Qiagen 
2011: 11). Samples and reagents to be used are prepared and then homogenized by spin down. 
After that, the correction mix is made according to the following table: 
Mix-real time PCR reaction composition 
Materials
Materials

 
The reaction mix is then put into a 0.2 ml tube and homogenized by spin down for a few seconds 
then put into the Techne Prime Pro 48 [Techne] PCR machine and set according to the following 
table: 
Real-time PCR cycle 
 
The results are then compared with standard curves and analyzed 
 
DATA ANALYSIS 
Data on lesion area of endometriosis tissue models, Bax / Bcl-2, Caspase 3, IL-1B, and PGE2 ratios 
obtained from In Vivo research are numerical data that will be processed using SPSS 20. The data 
will be presented in the average form if normal distribution or median if not normally distributed. 
The normality of distribution will be known through the Shapiro Wilk Test. Differences in 
endometriosis responses between dose and duration groups were performed with the Anova 
test (normal distribution) or Kruskal Wallis (abnormal distribution). Changes in estrous cycles 
between groups are known through the chi square test. 
To find out the binding of EEP active compounds, it was carried out using the in silico test. 
Compounds are said to bind when the value of ΔG is less than zero or negative. From the binding 
compounds, path analysis will be carried out to determine the relationship of the bonds with 
receptors related to the pathophysiology of endometriosis. 
 
 
Components 
Steps 
Time 
Temperature