DESCRIPTION OF PREMARRIAGE PREGNANCY PREPARATION IN DISTRICT SEDAYU
Rohani 1 Irsan pious 2, Theodorus3Salni 4
1
STIKES Mitra Adiguna Palembang, Indonesia
2,3,4
Sriwijaya University in Palembang, Indonesia
ABSTRACT
Candidiasis is a fungal infection in humans the caused by Candida albicans. Incorrect use of antifungal can lead to toxicity and resistance. One of the plants that can Become an antifungal is coriander(Coriandrumsativum Linn). This study aims at finding out the effectiveness of Coriandrum sativum Linn fraction as antifungal of Candida albicans. Laboratory experimental study in vitro has been Carried out in PPS Unsri Laboratory from February to May 2014, with the following phases of study:
extraction by maceration method, fractionation with liquid-liquid fractionation method (FCC), sensitivity test of Candida albicans using nystatin,antifungal activity of fraction test, determination of minimum inhibitory concentration (MIC) of the active fraction, Followed by bioautografi test and class determination of compounds.The Data were Analyzed using unpaired t-test, ANOVA, Posthoc,Linear Regression. The results Showed that the active fraction of coriander seeds is ethyl acetate and the class of compounds found in coriander seeds is phenol. MIC value of ethyl acetate fraction is0.625%.. It can be concluded coriander(Coriandrumsativum Linn) have antifungal activity of the ethyl acetatefraction. It is suggested that a study about pure compounds in the antifungal ethyl acetate fraction of coriander seeds need to be Carried out.
Key Words: antifungal, coriander(Coriandrumsativum Linn), Candida albicans
INTRODUCTION
Whitish pathological can be caused by vaginal candidiasis, vaginal trichomonas, bacterial vaginosis, gonorrhea or foreign bodies. Several investigators have reported that the cause of vaginal discharge that most of the vaginal candidiasis (Darmani, 2003).
From the results of epidemiological studies on the incidence of candidiasis vaginalis in women of reproductive age in India between 2005 and 2006, from 885 women with active sexual showed 20%
were diagnosed with candidiasis vaginalis, 80% of asymptomatic infection (Rathod etal.,2012). In women are expected to suffer from vaginal candidiasis at least once in his life that is about 75%, of which 40-45% of them will experience recurrent infections twice or more (Ratna in Setiawati, 2006).
Based on the results of research conducted by Kumalasari mention that in 2002, 50%
of women have experienced vaginal discharge Indonesia later in 2003, 60%
had experienced vaginal discharge, whereas in 2004 nearly 70% of Indonesian women have experienced vaginal discharge at least once in his life (Munijaya in Kumalasari T, 2005).
Estimated annual incidence of mycosis invasive Candida is 72 to 228 infections per million population (Chandra et al., 2011).
One of the plant as a potential antifungal is coriander(Coriandrumsativum Linn).
Based on the results of the study states that the component linalol and geraniol in coriander is an antifungal (Tabassum &
Vidyasagar, 2013). Essential oil of coriander seeds can be used as an antimicrobial potential to inhibit or preventinfections Candida (Begnani et al., 2010). The methanol extract of Coriandrum sativum showed activity in inhibiting the growth of Candida albicans (Bai & Kanimozhi, 2012). In addition, the research states that extracts of coriander seeds has an activity of inhibiting the
INTERNATIONAL SEMINAR | MIDWIFERY EDUCATION REFORM 147 growth of the Mycobacterium smegmatis,
Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Micrococcus luteus and Candida albicans (Simonati & Maria,2009).
From the above needs to be done research test the effectiveness of antifungal Candida albicans from a fraction of coriander(Coriandrumsativum Linn) in various concentrations to the inhibition of the growth of fungus theCandida albicans and equality with fungal drug nystatin.Expected results of this test can be used as a new drug and can be used as an alternative medicine to treat various diseases caused by the fungus Candida albicans.
RESEARCH METHOD
This study is alaboratory experiments.
invitro This study was conducted at the Laboratory Joint Graduate Program (PPs) Sriwijaya University. The study lasted from February 2014 to May 2014. This research using Candida albicans which is still classified as sensitive obtained from Palembang Health Laboratory.
In this study, the treatment group is the concentration of the solvent in which the concentration gradient 6: 20%, 10% , 5%, 2.5%, 1.25%, and 0.625. For the positive control group used in this study is Nystatin and a solution of DMSO as a negative control.
The equipment used in this study include extractor, autoclave, blender glass, hot plate, a bottle of wattle, Bunsen, petri dish, a funnel, a pumpkin erlenmeyer, electric heaters, test tube rack, glass beaker, measuring cups, incubators, vernier caliper, needle ose, megnetik stirrer, water bath, tweezers, pipette, test tubes, calliper, vacuum liquid chromatography.
The materials used in this study include:
coriander(Coriandrumsativum L.), the test medium,culture Candida albicans as the fungustest, distilled water, alcohol,
aluminum foil, H2SO4 10%, cotton, filter paper, label paper , medium Sabouraud Dextrose Agar (SDA), medium Sabauroud Dextrose Broth (SDB), the solvent DMSO(dimethylsulfoksida),methanol, solvent ethyl acetate, n-hexane, simplisia
(dry powder)
coriander(Coriandrumsativum Linn) and a plate of silica gel F254, nystatin,gloves, masks,
Work procedures antifungal activity test of fraction
Test the antifungal activity of fractions results fractionation was conducted to determine the fraction where the active compound is located. Concentration of fractions tested to Candida albicans was 20% (200 mg / ml) with solvent DMSO(dimethylsulfoxide).Suspension mushrooms put in a petri dish 0.1 ml, then
added SDA medium
(SabouroudDextroseAgar) 10 ml which has not been frozen, with temperatures around 40°C. Furthermore homogenized to freezing. Paper discs measuring 6 mm dripped 20 mL with a micropipette using current from each of the fractions and made as many as five repetitions, then placed on an agar medium and labeled in a petri dish. After it was incubated for 2x24 hours at 37oC. Tests confirmed active antifungal activity when the surrounding discs are clear zone-free growth of Candidaalbicans.
Test bioautografi
Bioautografi test is performed to determine the antifungal activity of a compound using thin layer chromatography. The test procedure is as beriku bioautografit: the active fraction at a concentration of 1% spotted on a plate of silica gel GF254, was then developed with a mobile phase for the separation of the compounds contained in fractions, penotolan active fraction was made double on the chromatogram. The chromatogram was laid in a culture dish containing C.albicansandida,active fraction on the
INTERNATIONAL SEMINAR | MIDWIFERY EDUCATION REFORM 148 chromatogram is left attached to the agar
medium, then removed carefully. Petri dishes containingcultures Candida were incubated for 2x24 hours. Once the medium is incubated observed clear zone that shows growth inhibition Candida albicans and is an area of active compounds are located and calculated the value of Rf his (Betina, 1973).
The determination of the minimum inhibitory concentration active fraction Determination of minimum inhibitory concentration conducted using agar diffusion method using paper discs(paperdisc)diameter of 6 mm. The smallest concentration that inhibits the growth of mold is the value of the minimum inhibitory concentration (MIC).
KHM working procedures, namely: active fractions were made with a concentration of 20%, 10%, 5%, 2.5%, 1.25% and 0.625%. Solvents used were DMSO.
Suspense mushrooms put into a petri dish
0.1 ml, then added
medium(SabouraudDextroseAgar)10 ml which has not been frozen, shake it a petri dish so perfectly mixed and then allowed to stand until frost. Paper disc diameter of 6 mm that has been poured with a solution of fractionation as much as 10 mL / disc inserted into the culture medium and then incubated for 2x24 hours in an incubator at 37°C and measured the diameter of inhibition zone is formed.
RESULTS AND DISCUSSION
Extraction of crude drugs coriander seeds(Coriandrumsativum Linn)
Extraction was done by maceration using methanol. Coriander seeds (Coriandrumsativum Linn) has been cleared weighing 250 grams of dried, obtained a dry weight of 250 grams and then blend until smooth in order to get as much as 250 grams of powder bulbs.
Simplisia inserted into the Erlenmeyer flask and then added a solution of methanol 1000 ml and allowed to stand for
2x24 hours, then filtered to obtain a liquid extract. Based on the results of extraction with methanol using water to simplisia coriander seeds (Coriandrumsativum Linn) showed the extraction of as much as 41.6 grams (16.6%).
Fractionation extracts of coriander(Coriandrumsativum Linn) Results of the extraction of crude drugs coriander seeds (Coriandrumsativum Linn ) gained as much as 41.6 grams of methanol extract, the extract is carried fractionation with fractionation methods Liquid-Liquid (FCC) with the solvent n- hexane, ethyl acetate and water methanol each as much as 1 L gradually, then each of the liquid fraction obtained evaporated in a fume hood to obtain each of the fractions in paste form. The results obtained from the fractionation process as in Table 1.
Table 1.
Results of fractionated extracts of coriander(Coriandrumsativum Linn)
Solvent Weight fraction (g)
Percent (%)
n-hexane, 12.4 29.8
ethyl acetate 3 6 8.6
methanol
water 25.6 61.6
Total 41.6 100
Table 1. it can be seen that the results of fractionation of extracts of coriander(Coriandrumsativum Linn) with methanol has a greater weight than the n- hexane and ethyl acetate. The solvent has the ability to separate compounds in the extract by polarity. Fractionation Liquid- Liquid (FCC) is a simple and common fractionation performed. The basic principle of liquid-liquid fractionation is the process of contact between the solvent and the other one that is not intermingled and have different densities so that the two phases will be formed shortly after the addition of a solvent in a flask and shaking
INTERNATIONAL SEMINAR | MIDWIFERY EDUCATION REFORM 149 separating funnel. This led to mass
displacement from the origin to the solvent extracting solvent (Mirwan and Ariono, 2009).
Test the sensitivity of the fungus Candida albicans
Fungus Candida albicans obtained from the Central Health Laboratory Palembang.sensitivity test done Nystatin through methods Diffusion Agar. The concentration used is 1000 ug / ml with solvent dimethylsulfoxide (DMSO). The result of sensitivity to each bacterium can be seen in Table 2:
Table 2.
The sensitivity of the test resultsfungus Candida albicans with the Nystatin at a concentration of 1000 ug / ml (0.1%)
Antifungal Diameter resistor (mm)
Nystatin 0.1% 30
In Table 2 it can be seen that the nystatin diameterproduce inhibitory effect on fungus theCandida albicans by 30 mm.
This is indicated by the formation of a clear zone on a paper disc that can be seen in Figure 1:
Figure 1. Results of Sensitivity Test with Nystatin at a concentration of 1000 ug / ml againstfungal Candida albicans
Sensitivity test is performed to determine whether the fungus Candida albicans used in this research is still sensitive or resistant, sensitivity of the test results obtained inhibition diameter 30 mm, meaning Candida albicans used is still sensitive. Thus, the fungus Candida albicans can be used for further research in accordance with the opinion of Volk &
Brown (1997) that the outcomes sensitivity tests Nystatin is said to be resistant if the inhibition zone formed <12 mm and senstitif if> 18 mm.
Test the antifungal activity of Candida albicans from fractions of extracts of coriander(Coriandrumsativum Linn) Test the antifungal activity of the fraction of n-hexane, ethyl acetate and water methanol carried by the diffusion method in order to determine which fraction of the active compound is located.
Figure 2.
Extract and coriander seeds in a fraction of DMSO
Methanol extract was created with a concentration of 20% by dissolving in the solvent DMSO. The test results of the antifungal activity of each fraction is shown in Table 3:
INTERNATIONAL SEMINAR | MIDWIFERY EDUCATION REFORM 150 Table 3.
Test results antifungal activity fractions
coriander seed
extract(Coriandrumsativum Linn) at a concentration of 20% of the fungus Candida albicans
Typefraction Diameter inhibitory (mm) methanol extract
N-hexane,
0.00 ± 0.00
± 0.000.00 ethyl acetate 19.4 ± 0.54 methanol water 0.00 ± 0.00
From Table 3 it can be seen that the fraction of ethyl acetate has a diameter of inhibition onfungus Candida albicans sedangan of 19.4 mm in the methanol extract, fraction of n-hexane and water methanol fraction has no inhibitory diameter of the fungus. This is evidenced by the formation of a clear zone on a paper disc that can be seen in Figure 4.3:
Figure 3. Test results antifungal activity of the extract, fraction of n-hexane, ethyl acetate and methanol water at a concentration of 20% of the fungus Candida albicans
Remarks: 1. The methanol extract 2.
fraction n-hexane, ethyl acetate fraction 3., 4. faction methanol, K: control
Measurement results obtained inhibition diameter of 19.4 mm diameter resistor, meaning 20% ethyl acetate fraction has strong inhibitory to the fungus Candida albicans. it showed that of the three
factions found that the active fraction is the fraction of ethyl acetate. This contrasts with the results of research Bai &
Kanimozhi (2012) which states that the methanol extract of Coriandrum sativum showed activity in inhibiting the growth of Candidaalbicans.The antifungal activity of the fractions indicated with resultant inhibition area on the medium that has been dikulturisasi. The more widespread inhibitory regions are generated, the greater the strength of the antifungal. In addition, concentrations fraction also influences the formation of inhibition zone.
This is consistent with the statement of Davis and Stout (1971) who argued that the provision of the strength of an antifungal as follows: the area 20 mm or more barrier means it is very strong, the area 10-20 mm barrier means strong , 5-10 mm and regional barriers means being 5 mm or less being weak. According to Herman et al., (2007) that the interpretation area of antimicrobial growth inhibition refers to the common standards of the Ministry of Health (1988) that is said to be sensitive to the antimicrobial antimicrobial plant origin when a diameter of 12-24 mm pconstraints.
The determination of minimum inhibitory concentration (MIC) of ethyl acetate fraction of coriander(Coriandrumsativum Linn) In this study the determination of minimum inhibitory concentration by decreasing the concentration that starts from 20%, 10%, 5%, 2.5%, 1.250%, and 0.625% with 5 repetitions , The results of the MIC determination ethyl acetate fraction of coriander(Coriandrumsativum Linn) can be seen in Table 4.
Table 4. Mean Inhibitory Diameter (mm) fraction of ethyl acetate coriander seeds(Coriandrumsativum Linn) against
INTERNATIONAL SEMINAR | MIDWIFERY EDUCATION REFORM 151 the fungus Candida albicans at various
concentrations Concentration
Fraction (% )
N
Mean (mm) + SD Diameter resistor 20 5 ± 1.3718.8
10 5 17.4± 0.97
5 5 15.2to 1.65 ±
2.5 5 11.4 ± 0.83
1, 25 5 10.2± 0.65
0.625 5 7.6± 0.54
Antifungal activity of each fraction with various concentrations can be seen in Figure 4:
Figure 4. Determination of ethyl acetate fraction MIC against fungi Candida albicans
Description:
1: Concentration of 20%, 2: Concentrations of 10%, 3: Concentration 5%, 4: Concentration of 2.5%, 5: Concentration of 1.25%, 6: Concentration of 0.625%, K: Controlon
Based Table 4 and Figure 4 shows the diameter of inhibition zone formed around the paper disc which is an indication of the strength ofactivity. Candida albicans Based on these values in mind that the higher the percentage, the greater the concentration of inhibition zone diameter are formed. The diameter of inhibition
zone at a concentration of 20%, ie 18.8 mm, then the diameter of inhibition decreases with decreasing concentration of the fraction. The smallest diameter of inhibition zone at a concentration of 0.625%, ie 7.6 mm. This is in accordance with the opinion of Greenwood (1995) in Syarifah (2006) states that the activity of the fraction decreases with decreasing the concentration, so that the diameter of inhibition zone formed also getting smaller.
The concentration of minimum inhibitory (MIC) of ethyl acetate fraction of coriander(Coriandrumsativum Linn) to the growth of the fungus Candida albicans can be determined by looking at the concentration limit that still contained the growth of fungi that are not contained fungal growth. From Table 4.4 it can be seen that the limit is at a concentration of 0.625% with a 7.6 mm diameter resistor.
At a concentration of 0.625% to barriers fungal growth marked by a clear zone around the disc. According to Bailey and Scott's (1994), the MIC is the minimum concentration that can inhibit the growth of bacteria or prevent multiplication of germs.
With this explanation can be stated that the MIC is located on the last concentration which still contained the growth of germs before the concentration which had not contained the growth of germs. So MIC in ethyl acetate fraction of coriander(Coriandrumsativum Linn) is a concentration of 0.625%. These results are higher than the MIC ethyl acetate fraction of red betel leaf(Piperbetle L) inhibited the growth of Candida albicans which KHM its ethyl acetate fraction was 2.5%
with a 11.4 mm diameter resistor (Reveny, 2011). The smaller the minimum inhibitory concentration of the extract indicates the potential of the extract as an antifungal, because with a small concentration of the extract was able to inhibit fungal pertmbuhan.
Based statistical test oneway ANOVA obtained wasp value = 0,000 with a value
INTERNATIONAL SEMINAR | MIDWIFERY EDUCATION REFORM 152 of α = 0.05(p<α), which means there
differences are striving towards the mean diameter of each inhibitory concentration.
Test bioautografi and grouping the active compound
Ethyl acetate fraction bioautografi test and determination of compounds active plate of silica gel GF254 using the appropriate eluent as mobile phase. Bioautografi test results and determination of classes of active compounds coriander seeds can be seen in Table 6:
Table 6. Test results and grouping bioautografi active compound coriander seeds(Coriandrumsativum Linn)
Type Fraction
eluent
(solvent) Rf Color
active compou
nd Ethyl
acetate
Ethyl acetate : methanol (9.5: 0.5)
0.81 Yellow Phenol
test results obtained bioautografi active compound with Rf value of 0.81 with the eluent ethyl acetate: methanol (9.5: 0.5).
This can be seen by the formation of clear zone (fungal growth inhibition) at Rf 0.81 as shown in Figure 5
Figure 4.5 (a). Bioautografi test results, (b). TLC ethyl acetate fraction coriander
seeds
Sprayed patches with Rf 0.81 H2SO4 was then heated raised yellow patches indicating a phenol compound. This means
the phenolic compounds contained in ethyl acetate fraction of coriander seed is an active compound antifungal Candida albicans.
The chemicals contained in the seeds of Coriandrum sativum Linn are saponins, flavonoids, and tannins (Suharmiati &
Lester, 2005). Coriander seeds also contain essential oils, flavonoids, polyphenols and β-carotenoids (Patel etal.,2013). The content of essential oil of coriander is sabinene, myrcene, alpha- terpinene, ocimene, linalool, geraniol, decanal, desilaldehida, trantridecen, acid petroselinat, acid oktadasenat, D-mannite, scopoletin, p-simena, kamfena, and felandren (Astawan 2009). In this study turns compounds antifungal obtained is phenol.
Research conducted Rajeshwari, et al., (2013) showed that the test results phytochemical the ethyl acetate fraction of coriander seeds is known that the content of secondary metabolites highest phenols, followed flavonoids, and tannins that are the main components of coriander seeds.
This is in line with what researchers found when research using thin layer chromatography (TLC) showed that the compound contains ethyl acetate fraction coriander seeds obtained is phenol.
According Pelczar and Reid (1979) in Poeloengan et al., (2006) , a phenol compound antimicrobial compounds, with the mechanism of inhibition of microbial phenol as follows: (1) damage the cell wall causing lysis or inhibit the process of formation of a cell wall in the growing; (2) changing the permeability of the cytoplasmic membrane that causes leakage of nutrients from the cell; (3) denature cell proteins; (4) the metabolic system damage in cells by inhibiting the action of the enzyme intraseluluer.
Rahmah and Aditya (2010) adds, compounds that are fungistatik eg phenolic compounds can mendenatuasi proteins, which damage the tertiary structure of proteins so that the protein loses its
INTERNATIONAL SEMINAR | MIDWIFERY EDUCATION REFORM 153 properties. Terdenaturasinyacell wall
proteins Candida albicans will lead to fragility in the cell walls so easily penetrated other active substances that are fungistatik. If a protein is denatured protein enzyme, the enzyme cannot work that causes the metabolism and impaired nutrient absorption process.
It is known that the cell wall of Candida albicans is the part that directly interacts with the host cell.cell walls Candida contain substances that are essential for virulence, among other mannoprotein derivative which hasproperties immunosuppressive that enhance the immune defense against the host fungus.
Candida is not only stick, but also penetrate into the mucosa. Aspartyl proteinase enzymes help Candida at an early stage to penetrate the layer of tissue invasion mucocutaneous berkreatin (Bachtiar, 1997).cell wall Candida albicans serves as a protector and also the target of several antimycotics. Cell wall plays a role also in the process of attachment and colonization as well be antigenic. The main function of the cell wall is to give shape to the cell and protect yeast cells from the environment.
The mechanism of action of phenols can form complexes with ergosterol contained in fungal cell membranes, the complex causes of enlarged pores in the yeast cells.
Through the pores is a small component of the fungal cell contents come out as nucleic acids and other proteins. It if continues will lead to the death of the fungus. Phenol complex is in a weakened state, dissociation indirect cause phenol penetrate cells. At high concentration of the lipid, the biggest effect is the ability bengabung phenol with lipid components of the cell. Fungal cell membranes are composed of phospholipids which would cause a disrupted cell membrane permeability so the fungus is inhibited (the Goddess, 2009). In accordance with the opinion of Regezi and Sciubba (1999) which states that Candida albicans is a species that is very sensitive to phenolic compounds.
It is in accordance with the opinion of Jawets, et al., (1996) that the workings of antimicrobial depends on the concentration of antiseptic, time and temperature , At very low concentrations can stimulate the growth of microorganisms, whereas at higher concentrations can inhibit the growth of microorganisms.
The study only looked at the effectiveness of concentration fraction of coriander seeds in inhibiting the growth of the fungus Candida albicans as one of the working mechanism of antifungal, while other factors such as time and the temperature was not observed.
CONCLUSION
Based on the results of research and discussion that has been done, it could be concluded bahwafraksi ethyl acetate coriander seeds(Coriandrumsativum Linn) is active against the fungus Candida albicans with the Minimum Inhibitory concentration (MIC) of 0.625% (6250 ug / ml) of the fungus Candida albicans and ethyl acetate fraction of coriander seeds(Coriandrumsativum Linn) containing the active antifungal compound of phenols.
ACKNOWLEDGEMENTS
Acknowledgements to the chairman STIKES Mitra adiguna Palembang mother Diana H. Soebyakto, Kes and all those who have helped in the implementation of this study the author can not mention one by one.
REFERENCES
Astawan, M. 2009. Ketumbar. Portal CBN, http://cybermed.cbn.net.id diaskses 23 Oktober 2013.
Bahtiar Boy M. 1997. Beberapa faktor yang mempengaruhi Candida albicans pada pathogenesis kandidiasis mulut.