Production and characterization of vaginal suppositories with propolis wax as active agent to prevent and treat Fluor albus
Siti Farida, Nurul Azizah, Heri Hermansyah, and Muhamad Sahlan
Citation: AIP Conference Proceedings 1817, 030013 (2017); doi: 10.1063/1.4976782 View online: http://dx.doi.org/10.1063/1.4976782
View Table of Contents: http://aip.scitation.org/toc/apc/1817/1 Published by the American Institute of Physics
Production and Characterization of Vaginal Suppositories with Propolis Wax as Active Agent to Prevent and Treat
Fluor Albus
Siti Farida
1,a), Nurul Azizah
2, Heri Hermansyah
2, Muhamad Sahlan
2, a)1Department of Medical Pharmacy, Faculty of Medicine - Universitas Indonesia, Kampus UI, Salemba 10430, Indonesia
2Bioprocess Engineering, Department of Chemical Engineering, Faculty of Engineering - Universitas Indonesia, Kampus UI Baru, Depok 16424, Indonesia
a)Corresponding author: [email protected]
Abstract. Based on the content contained in propolis wax especially antimicrobial function, it can be analyzed that propolis wax had superiority for Fluor albus. This research was conducted on two formulation of vaginal suppositories with base, supplementary and active agent as a fixed variable: 2% propolis wax (% w/w).
Evaluation of this research were weight variation, melting time, consistency, irritation effect test and physical and chemical stability test (organoleptic, pH and polyphenol content).
Keywords: Propolis wax, vaginal suppositories, Fluor albus
INTRODUCTION
One of reproductive health problems in women is a disease of the reproductive tract infections, Fluor albus.
Based on the data, WHO noted that 5% of adolescent women in the world are infected by sexually transmitted diseases (STDs) with symptoms of vaginal discharge (Fluor albus) annually, while in the United States there are 1 of 8 teenagers experiencing PMS due to vaginal discharge (Fluor albus) [1]. About 90% of Indonesia women might potentially experience Fluor albus. Since Indonesia is a tropical country so fungi are easy to grow in large number [2]. Fluor albus is a symptom of genital disorders experienced by women, in the form of discharge of liquid white yellowish white or gray of the vaginal canal. It has been reported that the Candida sp. is a fungus that is most widely found in the vaginal secretions of women who experience vaginal discharge. Of the 100 species of Candida isolated, 50-60% is Candida albicans [3].
Treating candidiasis infection is usually done with dispensing azol which can inhibit the biosynthesis of ergosterol, which was needed in the preparation of membranes of Candida albicans. However, it was lately known that Candida albicans is resistant to azol [4].
Propolis is honey bee hive product formed from the substance of a resin plant and bee secretions containing polyphenols and flavonoids which have a bitter taste as anti-inflammatory, anti-virus, antioxidant, and antimicrobial [5].
For further utilization, obtained propolis must be purified before it is processed further. Propolis wax is a by- product of the process of purifying propolis which is currently untapped further because the content of polyphenols in propolis wax is less compared to pure propolis (approx. 2.4 – 5%). However, based on very small amounts, the polyphenols in propolis wax was proved to be still effective inhibiting the growth of Candida albicans and three strains of pathogenic bacteria, i.e. Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus mutans [6].
Based on the content contained in the propolis wax especially antimicrobial function, then it can be analyzed that propolis wax had benefits for Fluor albus. Vaginal suppository was selected in this study. Vaginal suppository is a ball-shaped solid tapering-like cones which were used through the vagina. This type was chosen because it is based on several considerations, such as it can be entered directly in the blood so that the drug can effect conferring more quickly than drug use peroral. In addition, the drug can directly pass through the vagina so the treatment process will take place faster. In this research, propolis wax will be the active material while the oleum cacao was base in making of vaginal suppository.
MATERIALS AND METHODS
Materials
Materials in making of vaginal suppositories were propolis wax, oleum cacao/ cocoa butter, cetaceum, cera alba, castor oil, and ethanol 96%. In melting time and irritation effect test, we used 6months-female rabbit (New Zealand) from Bogor Institute of Agriculture.
Preparation of Propolis Wax Suppositories
At the beginning of making vaginal suppositories was mixing all of the materials and division into 2 formulas.
Table 1 shows formulations of vaginal suppository. In this research, materials variated were oleum cacao, cetaceum and cera alba in (% w/w) to get the best result.
TABLE 1. Formulation of Vaginal Suppositories
Material Formula 1
(% w/w) Formula 2
(% w/w)
Propolis wax 2 2
Oleum cacao 58 59
Cera alba 30 27
Cetaceum 10 12
All materials were melted based on their boiling point. After all of the materials were melted, the materials were mixed until homogenous on hot plate at 50°C. The materials of suppositories were filled in mold. The mold was freezed in refrigerator for 15 minutes. All suppositories were packed in aluminum foil and kept in refrigerator for use in evaluation.
Evaluation of Suppositories
The prepared vaginal suppositories were evaluated for weight variation, melting time test, consistency test, irritation effect test, and physical and chemical stability test (organoleptic, pH and polyphenol content).
Weight Variation
Ten vaginal suppositories for each formula were calculated. No suppositories should deviate from average weight by more than 5%.
Melting Time Test
Melting time of vaginal suppositories were evaluated using 6 months-female rabbits (New Zealand) from Bogor Institute of Agriculture (IPB). Vaginal suppositories were entered into rabbit’s vagina. In each formula, there were tests by 3 rabbits for control suppository (commercial product). Melting time can be known by observing the rabbit’s vagina. To detect the melting time of vaginal suppositories, sterilized cotton bud was entered in rabbit’s vagina and checked in every one minute.
Consistency Test
Consistency of vaginal suppositories were tested using penetrometer. 3 samples of each formula of vaginal suppository were set in penetrometer and observed for 5 seconds. If the resulting value was higher, then the resulting consistency was getting smaller and more easily spread in the body.
Irritation Effect Test
In this test rabbit’s eyes were used to determine if the active agent in vaginal suppositories was irritant or not.
Three rabbit’s eyes were prepared for each formula. Left side of rabbit’s eye was tested by expelling a liquid of propolis wax solution (2% propolis wax which was dissolved in castor oil) drop by drop and control (castor oil) was given in right side of rabbit’s eye. Effect of propolis wax solution was observed for 3 minutes, after that observation was conducted to detect the effect shown in rabbit’s eyes. The good effect if when rabbit’s eyes were back to normal condition after 3 minutes given the solution.
Physical and Chemical Stability Test Organoleptic Test
Organoleptic tests were done by the five senses to determine the change of shape, color, smell and fungus presence. Organoleptic of vaginal suppositories were tested in every week for 4 weeks at room temperature.
pH
The pH of vaginal suppositories was tested by pH-indicator strips and tested in every week for 4 weeks. Vaginal suppositories should have melted in advance. At the liquid condition, pH can be checked using pH-indicator strips.
Polyphenol Content
For determination of polyphenol content, 1 gram vaginal suppositories were dissolved in 10 ml ethanol and centrifuged for 15 minutes with 4600 rpm. After centrifugation, the liquid of vaginal suppositories was taken, moved and calculated. The liquid of vaginal suppositories was attached by follin and Na2Co3 reagent. Absorbance of sample of suppositories were measured against blank at 765 nm. The absorbance should be converted into concentration, then it should be calculated based on average weight of each formula.
RESULTS AND DISCUSSION
The weight variation studies for all vaginal suppositories in 2 formulas were found to be within the acceptable range of <5%. The average weight variation shown on Table 2. Melting time test were performed in vagina’s rabbit at 37°C (body temperature of rabbit). Formula 1 average spent time to melt was 17 minutes whereas formula 2 was 5.3 minutes. Control vaginal suppositories (commercial product) were also evaluated and had melting time 6 minutes. Vaginal suppositories were good if they had shortest melting time. The addition of cera alba and cetaceum were different on the two formulas which can affect difference on their melting point. Composition of cera alba in formula 2 was more than formula 1, so formula 2 had shorter time to melt than formula 1. This was due to addition of cera alba experienced increased melting point because of it has melting point (62-64°C) above melting point of oleum cacao (30 - 36°C). The addition of cetaceum also had an effect on melting point of the suppository.
Cetaceum has a melting point 42-50°C. A third of this material changed the melting point of oleum cacao so with that comparison can melt at body temperature (37°C). Thus the concentration of cera alba and cetaceum gave effect on the increase in melting point of the suppository with base-oleum cacao. Because of oleum cacao was not soluble in water, hence the increase in melting point was very influential with its melting time. If the suppository was melted with short time then drugs can more easily soluble in liquid medium [7]. Inconsistency evaluation of vaginal suppositories, it was shown that formula 2 had higher values/lower consistency level than formula 1. On the formula 1, generated average yield value consistency was 20 (1/10 mm), formula 2 was 23 (1/10 mm), and the
control was 16.7 (1/10 mm). Yield value represents the value of the depth of the penetrometer on semi solid material to penetrate within 5 seconds. The higher yield value means the lower level in consistency. Formula 1 had a lower consistency level than control. It is known, that the higher the level of consistency means an increasing strength of material. In a test of consistency, it is known from the lowest to the highest level of consistency in this 3 variants:
formula 2 > formula 1 > control vaginal suppositories. This indicated that the formula 2 was an easy material to disperse in the body compared with the formula 1 and control.
Eye irritation test is a test on the animal (rabbit) to detect toxic effects that appear after exposure on the eyes. The purpose of the test is the eye irritation to obtain information of the possibility of imminent danger arising at the time of the test material exposed on the eyes [8]. Active agent was tested to detect irritation on rabbit’s eyes and it showed that active agent (2% propolis wax in castor oil) did not cause irritation in rabbit’s eyes. It can be seen that rabbit’s eyesreturned to normal condition after 3 minutes administering treatment. It proved that propolis wax is safe to use as active agent in vaginal suppositories.
TABLE 2. Evaluation weight variation, melting time and consistency of vaginal suppositories Formulation code Weight Variation
(average) = (gram)
Melting Time (average) =
(minutes)
Consistency Test (average) =
(10-1mm/ 5 s)
F1 2.3335 ± 0,022 17 20
F2 2.3234 ± 0,018 5.3 23
Control 6 16.7
TABLE 3. Evaluation in irritation effect of propolis wax as active agent
Sample Irritation Effect
Active Agent (2% propolis wax in castor oil)
No irritation after 3 minutes
administering treatment
Before treatment After treatment Castor oil (control) No irritation after 3
minutes administering treatment
Before treatment After treatment
The performance of vaginal suppositories in 2 formulations and control were found that there was no drastic change in the shape, color, smell, and fungus presence. The shape of vaginal suppositories indeed changed in every week because they degraded and not intact as the beginning. Color and odor of vaginal suppositories were also unchanged. However, there were no fungus attached to vaginal suppositories for 4 weeks. Propolis wax vaginal suppositories remained yellow brownish for 4 weeks and have an odor that was similar to mixture of oleum cacao and cera alba. This was due to the most composition of suppositories were oleum cacao and cera alba. Propolis wax was proven capable to inhibit the Candida albicans reached 76% in the concentration 79.43 mg/mL, and other fungus and bacterias [6]. With these studies, propolis wax was then used as antimicrobials to avoid the smell of
rancid due to microbial activity so that the material becomes stable. It also can function as a preservative so that it can be saved for a longer period[9].
TABLE 4. Evaluation of organoleptic stability Stability Test
Week Organoleptic
F1 F2 Control
Week-0
Week-3
Two formulas of vaginal suppositories pH were stable. Vagina has a healthy condition if pH is acid. From evaluation for 4 weeks, vaginal suppositories pH was 5. Control vaginal suppository (vagistin) was also evaluated and its pH was 5. pH condition of all suppositories were evaluated and should be acceptable because the pH was acid. pH stability of all vaginal suppositories can be seen from the graph in Fig. 1.
The last evaluation, two formulas were tested and resulting to almost stable polyphenol content for 4 weeks.
Generally, polyphenol in formula 1 fluctuated but still represented the stability of polyphenol content. Formula 2 was decreased in week-0 until week-1 but stable in week-1 until week-3. Although both formulas contained active agent (2% propolis wax), but the composition of cera alba were different. In formula 1, composition of cera alba was 30% w/w, whereas formula 2 was 27% w/w. This was causing the polyphenol content in formula 1 had higher concentration compared with formula 2, because cera alba also had polyphenol content. With the presence of polyphenol content, it was proven that active agent in vaginal suppositories can prevent and treat Fluor albus. Since polyphenol serves as an anti-microbial agent by inhibiting the growth of the Candida albicans [10], a fungus which is one of the microbial cause of occurrence of Fluor albus in women's vagina. Graph of polyphenol content stability can be seen from the graph in Fig. 2.
FIGURE 1. Graph of pH stability 0
1 2 3 4 5
0 1 2 3 4
Week-
pH
pH Stability
F 1 F 2 Control
FIGURE 2. Graph of polyphenol content stability
CONCLUSION
It can be concluded that formula 2 of propolis wax vaginal suppositories had best result in melting time and consistency test. Active agent (2% propolis wax) was proven to not irritate rabbit’s eye and was safe to use as active agent in vaginal suppositories. Both formulas were stable in pH evaluation and almost stable in polyphenol content for 4 weeks.
ACKNOWLEDGMENTS
This research is funded by Universitas Indonesia for Multidiscipline research grant 2015.
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0.00 0.25 0.50 0.75 1.00 1.25 1.50
0 1 2 3
Concentration of Polyphenol (% w/w)
Week-
Polyphenol Content Stability
F 1 F 2