OPTIMIZATION ANTIBACTERIAL OINTMENT FORMULA PEG400 AND PEG4000 BASE OF MANGOSTEEN PEEL (Garciniamangostana Linn) ETHANOLIC EXTRACT WITH DESIGN FACTORIAL METHOD

OPTIMIZATION ANTIBACTERIAL OINTMENT FORMULA PEG400 AND PEG4000 BASE OF MANGOSTEEN PEEL (Garciniamangostana Linn) ETHANOLIC EXTRACT WITH DESIGN FACTORIAL METHOD

Beny Dwi Hatmoko, Suprapto, and Rima Munawaroh

Fakultas Farmasi Universitas Muhammadiyah Surakarta

Jl. A. Yani Tromol Pos 1, Pabelan Kartasura Surakarta 57102

.ABSTRACT

Mangosteenpeel has benefit as an antioxidant, as well as an antibacterial. Compounds that have antibacterial activity is α-mangostin. In order to make it easy the used and effectiveness of antibacterial, mangosteenpeel extract needs to made as ointments. This study purpose to determine the effect of the combination of PEG 400 and PEG 4000 base on the physical characteristic and antibacterial against  Staphylococcus aureus as well as get the optimum formula. Ointments were made into four formulas that have low level 26 g and high level 32 g of PEG 400, low level 30 g and high level 37 g of PEG 4000 with factorial design method. Formula optimization using Design Expert software with the parameters of  the physical characteristic of ointment and antibacterial activity. Factorial design predicted results verifie and analyzed byt-test LSD with 95% confidence level. The optimum combination of PEG 400 and PEG 4000 incomparison of 30,7g and 33g in 100g of ointment. Comparison of prediction and verification showed significant differences in testing parameters pHand not significantly different in dispersive power, adhesion, viscosity and antibacterial. Optimal ointment with a semi-solidform, pH 4,17, viscosity 98,33d.Pas, adhesion 17,33seconds, dispersive power10,36 cm² and inhibition zone diameter of 11 mm.

Keywords: ointments, mangosteenpeel, PEG 400, PEG 4000, antibacterial, factorial design

INTRODUCTION

            The mangosteen  (Garciniamangostana Linn.) contains xanthones compounds such as α-, β- and ɤ- mangostinn, garsinon E, deoxigartinin and gartanin (Perez et al., 2008). The other compounds quinine, carboxilyc acid, and halogenated hydrogen (Putra, 2010). Besides has antioxiandt benefit, mangosteen also has antibacterial benefit. The compound that has antibacterial activity is α-mangostin (Linuma et al., 1996). Based on previous study (Geetha et al., 2011), ethanolic extract of mangosteen peel with the concentration 200 µg could inhibit Staphylococcus aureus.

             In order to make it ease for use, it needs to prepare into ointment. The ointment base that used was a combination of PEG 400 and PEG 4000. PEG is used because it has the strong adhesive power and can be easily spreaded on the skin, not irritating the skin then easily to wash and also did not destruct the gas exchange and perspiration production (Voigt, 1984). The increment of PEG 400 will increase the viscosity, smaller spreadability and greater adhesive power (Charunia, 2009).

To gain the precise and good formula, the optimization of formula is needed by using factorial design method (Proust, 2005 to gain the optimum combination of PEG 400 and PEG 4000 that evaluated by the ointment characteristic and its antibacterial activity.

METHODS

Equipment: Viscotester RION (VT-04E RION), pH meter (Hanna pH 211 instruments), adhesive time tester, spreadability tester, autoclave (MA 672®),oven (Memmert®), Laminar Air Flow (AstariNiagara®), incubator (Memmert®), Analytical scales (Ohaus®), glassware (Pyrex).

Materials: ethanolic extract of mangosteen peel obtained from PT. Lansida Yogyakarta, ethanol 70%, PEG 400, PEG 4000, nipagin, propylene glycol, Staphylococcusaureus ATCC 29213 obtained from Microbiology Laboratory of Pharmacy Faculty of Universitas Muhammadiyah Surakarta, DMSO, distilled water, sterilized NaCl, MuellerHinton medium, standard Mc. Farland solution, dye Gram (A, B, C, D), silica GF 254, blue tips, yellow tips, white tips, chloroform, ethyl acetate, methanol, BHI (Brain Heart Infusion) and formaline 1%.

PROCEDURES

The preparation of Material

The ethanolic extract of mangosteen peel obtained from PT. Lansida Yogyakarta with phenol concentration 0,5%; water concentration 8,15% and ashes concentration 9,2%.

Thin Layer Chromatography test

The identification of α-mangostin in mangosteen peel extract used thin layer chromatography method (TLC) using silica GF₂₅₄ and mobile phase chloroform: ethyl acetate (9:1) v/v (Depkes RI, 2010). The extract was made by diluting 250 mg of extract in 5 mL methanol and diluted to a series of concentration, then it was spotted onto silica and eluted. The spot was observed under UV 254 nm and its Rf was calculated.

Identification of bacteria

The bacteria identification was conducted by Gram method. The smear was shed by formaline 1% for 5 minutes, then dried. The smear was shed with A-Gram dye for 10 minute and the bacteria was identified using Gram test.

The biochemistry test used Manitol salt Agar method that known as a method to determine the type of bacteria by its fermentation to manitol for 18-24 hours. The discoloration of medium indicated the type of bacteria.

Ointment Test

The evaluation of mangosteen peel extract ointment included organoleptic test (form, color, and odor), physical evaluation (pH, viscosity, spreadability, and adhesive time test), and antibacterial activity against Staphylococcusaureusby using in vitro diffusion method.

RESULT AND DISCUSSION

A.      Thin Layer Chromatography (TLC)

The TLC profile of α-mangostin adapted from SuplemenFarmakope Herbal showed Rf 0,53 (DepKes RI, 2010) meanwhile from the sample showed that the spot had Rf 0,58. so it could be concluded that sample contained α-mangostin according Supplemen Farmakope Herbal (Departemen Kesehatan RI, 2010).

B.       Antibacteria Activity Test Result

The antibacterial activity test was to determine the antibacterial activity against Staphylococcus aureus. The obtained result from mangosteen peel extract in concentration 12% w/v with solvent DMSO had antibacterial activity against Staphylococcus aureus which showed by the diameter of inhibition zone was 10,5 mm.

The antibacterial activity test was conducted by agar-well diffusion method and divided to 10 wells in 2 petri dishes.

            The antibacterial activity test was conducted by agar-well diffusion method and divided to 10 wells in 2 petri dishes. First petri dish was added by formula 1, I, KI, FII, KIII, and amoxicillin. The second petri dish was added by FIII, KIII, FIV, KIVand amoxicillin. The ointment weight that added into each well was ± 65 mg. The diameter of inhibition zone for FI, FII, FIII and FIV were 10,67 ± 0,58 mm, 13,33 ± 1,15 mm, 12,67 ± 2,31 mm and 11,83 ± 0,76 mm. Amoxicillin as a positive control had a diameter of inhibition zone as much as  36,67 ± 2,88 mm. The ointment base that used had a few inhibition zone. This happened because PEG that used had a bactericid characteristic although a little bit (Voigt, 1984) and also nipagin(Rowe et al., 2009).In first week, the optimum formula of ointment had the diameter of inhibition zone as much as 10,67±0,58 mm and in fourth week it was 11 ± 1 mm because the viscosity of ointment had been decresing and affected to its diffusion (Puspitasari, 2007).

C.      Ointment Physical Evaluation Result

1.      Organoleptic Test

Organoleptic test results include odor, homogeneity, concistency and color of the ointment preparations with visual observation in the ointment. From the observations, all with the addition of extract ointment and control base had the same shape that was semi-solid. That was conducted to smell the ointment with the addition of the extract in the form of a distinctive odor mangosteen peel. The addition of extracts made of dark brown ointment. Results showed homogeneity ointment, uniform and no coarse particles in the ointment. Homogeneity ointment also could affect the dose of ointment when it used, so that homogeneous ointment would provide a uniform dose.

2.        pH

The pH test is one of criteria should not be underestimated because including physical and chemistry characteristic of a dosage form and predicts the stability of a dosage form. According to Sudjono et al. (2012), the skin pH is ranged from 4 until 6,5.

The ointment pH test results for FI, FII, FIII, FIV and optimum formula were 4,46; 4,47; 4,46; 4,47 and 4,17 and qualified to pH skin (4-6,5). So, the ointment would not irritate the skin.

The equation of factorial design approach for pH :

Y = +4,46  - 1,47 (A) - 5,0 (B) + 1,48 (A)(B) (1)

Notes: Y= obtained respond, (A) = PEG 400, (B) = PEG 4000, (A)(B) = interaction between two factors

From the equation 1, it can be concluded that the interaction coefficient between PEG 400 and PEG 4000 valued +1,48;it meant that the combination of PEG 400 and PEG 4000 could increase pH of ointment. The coefficient of PEG 400 as much as-1,47 meaning that the increment of PEG 400 could reduce pH meanwhile the coefficient of PEG 4000 was-5,0 it meant that the increment of PEG 4000 could reduce the pH of ointment.

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Figure1. Interaction between PEG 400 and 4000 to pH showed that PEG 4000 in lower or higher levelwith the addition of PEG 400 could reduce pH ointment.

           

The interaction of PEG 400 and PEG 4000 was synergic and it was proven with the red and the blue line was in the one line (Figure1). The red one (High level PEG 4000 ) and the blue one which covered by the red line (Low level PEG 4000) showed that by increasing PEG 400 could reduce the pH of ointment. The effect of reducing pH from PEG 400 and PEG 4000 (high level) with PEG 4000 (low level) because the both lines were coincided.

Figure2. Contour plot pH of ointment in red area showed the combination of PEG 400 and PEG 4000 had high pH.

From Figure 2, blue area was for the combination of PEG 400 (high level) and PEG 4000 (high level) could reduce the pH of ointment because the pH of PEG 400 was 4-7 (Rowe et al., 2009). The red area is shown by the combination of PEG 400 (low level) and PEG 4000 (high level) which could increase the pH of ointment because the pH of PEG 4000 is 5 – 7,4 (higher than PEG 400).

3.  Viscosity

To know the viscosity of ointment should be conducted the viscosity test. The viscosity was influenced by temperature storage, and materials (Padmadisastra et al., 2007). The viscosity of ointment also affected to convenience when the ointment used. The lower viscosity of ointment, the easier of its use and will increase the spreadability (Peranda, 2012).

            The result of viscosity test for FI- FIV were 88,67 ± 3,21; 66,67 ± 2,88; 143,57 ± 3,21 and 72,50 ± 2,50 dPas. The optimum formula with viscosity as much as 98,33±2,88 dPas. According to Isnaini (2012), the higher viscosity in ointment was, the lower its spreadability and vice versa. The high viscosity would increase the adhesive time (Rahmawati, 2012).

The equation of viscosity according factorial design approach:

Y = +92,81 - 15,31 (A) + 23,44 (B) - 12,19 (A)(B) (2)

Notes: Y= obtained respond, (A) = PEG 400, (B) = PEG 4000, (A)(B) = interaction between two factors

From the equation above, it could be concluded that the interaction of PEG 400 and PEG 4000 was -12,19, it meant that the combination of both PEG could reduce the viscosity of ointment. The coefficient of PEG 400 was -15,31 that meant could reduce the viscosity value of ointment. The coefficient of PEG 4000 was +23,44 so it could reduce the viscosity value of ointment.

The interaction between PEG 400 and PEG 4000 was antagonist interaction because the lines were not parallel (Figure 3). The red line (High level PEG 4000 ) showed that PEG 400 could reduce the viscosity of ointment, while the black line (Low level PEG 4000) showed that by increasing PEG 400 would reduce the viscosity. The effect of increased viscosity from PEG 400 in high level PEG 4000 was greater than low level PEG 4000 which shown by the slope differences in both of lines. This was due to PEG 4000 in the dosage form would increase the viscosity, adhesive time, and decrease the spreadability of ointment (Charunia, 2009).

Figure3. Interaction between PEG 400 and 4000 to the viscosity of ointment showed that high level PEG 4000 with addition of PEG 400 could reduce the viscosity of ointment.

From contour plot curve (Figure 4), the blue area shown by the combination between high level PEG 400 and low level PEG 4000 would reduce the viscosity, mean while the red area shown by the combination of low level PEG 400 and high level PEG 4000 would increase the viscosity

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Figure4. Contour plot viscosity of ointment in red area showed that the combination of PEG 400 and PEG 4000 could increase the viscosity of ointment.

4. Spreadability

This test aimed to determine the spreadability of ointment in the skin surface when applied. This test was conducted by putting ointment in the petri dish surface and adding 50 – 300 g load. The ointment had better good spreadability with no pressure (Isnaini, 2012).

The spreadability test result for FI until FIV were 8,29 ± 0,25; 13,39 ± 0,17; 6,15 ± 0,55 and 10,40 ± 1,34 cm². The optimum formula had a spreadability15,59 cm². The spreadability was not comparable with viscosity. The greater of viscosity would make the fewer spreadability and vice versa (Isnaini, 2012).

The equation of spreadability according to factorial design approach:

Y = 9,56 + 1,28 (A) -2,34 (B) - 0,21 (A)(B) (3)

Notes: Y= obtained respond, (A) = PEG 400, (B) = PEG 4000, (A)(B) = interaction between two factors

From the equation 3, it could be concluded that the interaction coefficient of PEG 400 and PEG 4000 was -0,21, which meant that the combination of both PEG could reduce the spreadability of ointment. The coefficient of PEG 400 was +1,28; it meant that PEG 400 could increase the spreadability and the coefficient of PEG 4000 was -2,34 had meaning that PEG 4000 could reduce the spreadability of ointment.

The interaction between PEG 400 and PEG 4000 was synergic shown by the parallel lines (Figure5). The red line (high level PEG 4000) showed that by increasing the PEG 400 could increase the spreadability of ointment, meanwhile the black line (low level PEG 4000) showed that by increasing PEG 400 would also increase the spreadability of ointment.

Figure5. The interaction between PEG 400 and PEG 4000 showed that either PEG 400 and 4000 could increase the spreadability.

Figure6. Contour plot spreadability of ointment in red area showed the combination of PEG 400 and PEG 4000 could increase the spreadability of ointment.

From the contour plot curve above (Figure 6), the blue area shown by the combination of low level PEG 400 and high level PEG 4000 had meaning that could reduce the spreadability of ointment whereas the red area could increase the spreadability of ointment shown by the combination of high level PEG 400 and low level PEG 4000.

5. Adhesive time

The adhesive time test is to determine how long the ointment can stick on the skin surface when it applied. The adhesive time is also affected to how many active substance that absorbed in the skin (Fitriyana, 2012).

The adhesive time test results for FI until FIV were 7,88± 0,20; 6,13 ± 0,23; 13,43 ± 1,46 and 17,33 ± 2,51 seconds. The adhesive time of optimum formula was 17,33± 2,51 seconds. The long adhesive time will increase the viscosity of ointment (Rahmawati, 2012).

The equation of adhesive time according to factorial design approach:

Y =+8,59- 1,59 (A) + 2,06 (B) - 1,19 (A)(B) (4)

Notes: Y= obtained respond, (A) = PEG 400, (B) = PEG 4000, (A)(B) = interaction between two factors.

From the equation4it can be concluded that the interaction coefficient between PEG 400 and PEG 4000 was-1,19, and the meaning was the combination of both type of PEG could reduce the adhesive time of ointment. The coefficient of PEG 400 was-1,59 and the meaning of it that PEG could reduce the adhesive time. But, the coefficient of PEG 4000 was +2,06 so, PEG 4000 could increase the adhesive time of ointment. PEG 4000 had more effect in increasing the adhesive time than PEG 400.

Figure7. Interaction between PEG 400 and 4000 to the adhesive time of ointment showed that PEG 4000 (low or high level) with PEG 400 addition could reduce the adhesive time of ointment.

The interaction between PEG 400 and 4000 was antagonist shown by not parallel lines (Figure7). The red line (high level PEG 4000) showed that by increasing PEG 400 could decrease the adhesive time of ointment, meanwhile the black line (low level PEG 4000) showed that by increasing PEG 400 would reduce the adhesive time. The adhesive time decreasing effect of PEG 400 in high level PEG 4000 is greater than in low level PEG 4000, which shown by the differences in slope of both lines. This happened because when the viscosity of ointment is increasing, the adhesive time will also increase.

From the Contour plot curve (Figure8) the red area shown by the combination between low level PEG 400 and high level PEG 4000 which could increase the adhesive time. And the blue area could decrease the adhesive time shown by the combination of high level PEG 400 and low level PEG 4000.

Figure8. Contour plot of adhesive time of ointment in red area showed that the combination of PEG 400 and PEG 4000 could increase the adhesive time of ointment.

6.  Antibacteria Activity Test Result

This test also conducted to determine the antibacterial activity of ointment to inhibit the growth of Staphylococcusaureus.

The test results for FI until FIV were 11,67± 0,58; 13,33 ± 1,15; 12,67 ± 2,31 and 11,83 ± 0,76 mm of diameter of inhibition zone. The optimum formula had a diameter of inhibition zone as much as 10,67± 0,58 mm.

The equation of antibacterial activity according to factorial design approach:

Y = +12,38 + 0,13 (A) - 0,21 (B) - 0,63 (A)(B) (5)

Notes: Y= obtained respond, (A) = PEG 400, (B) = PEG 4000, (A)(B) = interaction between two factors.

From the equation 5can be known that coefficient PEG 400 was+0,13and meant that PEG 400 could increase antibacterial activity. The coefficient of PEG 4000 was -0,21 and meant that PEG 4000 could reduce antibacterial activity of ointment, but with more PEG 400 caused the increasing of antibacterial activity (Figure9). The interaction coefficient between PEG 400 and PEG 4000 was-0,63; it meant that the combination between PEG 400 and PEG 4000 could reduce the antibacterial activity of ointment. PEG 400 had more effect in increasing antibacterial activity than PEG 4000.

Figure 9. Interaction between PEG 400 and 4000 to antibacterial activity of ointment tshowed that high level or low level PEG 4000 with addition of PEG 400 could increase the antibacterial activity of ointment.

The interaction between PEG 400 and PEG 4000 was antagonist which shown by unparallel line (Figure9). The red line (high level PEG 4000) showed that PEG 400 could increase the antibacterial activity, and the blue line (low level PEG 4000) showed that by increasing PEG 400 would increase the antibacterial activity against Staphylococcus aureus.

Figure10. Contour plot of antibacterial activity of ointment in red area showed the combination of PEG 400 and PEG 4000 could increase the antibacterial activity of ointment.

From the contour plot (Figure 10) the red area would increase the antibacterial activity which shown by the combination of high level PEG 400 and low level PEG 4000. The blue area could decrease the antibacterial activity which shown by the combination of low level PEG 400 and high level PEG 4000.

D.    The determination of Optimum Point Based on Factorial Design

This point determination was to know the optimum area in contour plot super imposed. To gain the optimum formula needed to combine the contour plot of parameter of physical evaluation in ointment and its antibacterial activity until obtained the yellow super imposed. This yellow area showed the optimum formula. The criteria of parameters of physical evaluation and antibacterial activity was shown in Table 2.

Table2. Criteria of parameter of physical evaluation and antibacterial activity

Parameter	Criteria	Notes
pH	4 - 6,5	Range
Viscosity (dPa.S)	90	Target
Adhesive time (seconds)	13,43	Maximize
Spreadability (cm2)	13,39	Maximize
Antibakteri (mm)	13,33	Maximize

The pH parameter considered a range 4 – 6,5;  because if the pH of ointment less or more than that range, it can irritate the skin (Sudjono dkk., 2012). The target of viscosity was 90 dPas because from the orientation result showe good consistency around 90 dPas. The maximize criteria of adhesive time was 13,43 seconds because if the adhesive time are more than that time, will cause the hard in releasing of active substance and affected the effect of treatment will be greater. The maximize criteria for spreadability was 13,39 cm²because the active substance will be absorbed faster when the spreadability is good and wide (Ulaen dkk., 2012). The maximize criteria for antibacterial activity was 13,33 mm. Because the greater of the diameter of inhibition zone was, the greater of antibacterial activity of the ointment was in inhibiting the bacteria growth.

Stability test of Ointment

The stability test of ointment was to know the stability of ointment from outer substance affects. This stability test was conducted for 4 weeks. The analyzed result used independent The optimum formula with the prediction of optimum formula in PEG 400 and PEG 4000 (30,7 g and 33 g) composition. The prediction value and desirability obtained was 0,641. When the value approach number 1, showed the best optimization to combine all the function. The optimum formula test result compared to prediction of contour plot super imposed.

Verification of Optimum Point UsingFactorial Design Method

The verification was conducted to know the significance of prediction result in factorial design using SPSS program with 95% confidence interval. From the results, can be concluded that the parameter pH showed the significant value p<0,05 it meant that between the prediction value and verification value had the difference significantly. Meanwhile, for viscosity, adhesive time, spreadability and antibacterial activity test showed p>0,05 (no significantly difference).  T-test was conducted to sample by comparing the result of week 0 until week 4. Adhesive time altered significantly (p<0,05) but in pH, spreadability, viscosity and antibacterial activity did not alter significantly (p>0,05).

A.      CONCLUSION

1.        The combination of PEG 400 and PEG 4000 affected the physical properties and antibacterial activity of ointment. PEG 400 woul reduce the viscosity and increase the spreadability and antibacterial activity. But, PEG 4000 could increase pH and adhesive time of ointment.

2.        The obtained optimum formula from contour plot superimposed with ratio PEG 400 30,7 g and PEG 4000 33 g in 100 g ointment.

B.    SUGGESTION

It should conduct the skin convenience test to many respondents for determining whether the ointment irritate the skin or not.