INTRODUCTION:

Acne vulgaris is a common chronic inflammatory skin disease in pilosebaceous units. The three main factors that play a role in the development of acne include sebaceous glands are hyper seborrhoea, keratinocytes with hyperkeratinisation of pilosebaceous units, and bacterial infection such as from Propionibacterium acnes and Staphylococcus epidermidis. In particular, P. acnes represents less than 2% of skin surface bacteria and S. epidermidis can present in a larger percentage.

Infection of both species maylead to the formation of comedos, papules, and pustules that trigger acne development¹. P. acnes and S. epidermidis are Gram-positive bacteria. Both species of bacteria are commensal bacteria on human skin that can turn out to be opportunistic. P. acnes and S. epidermidis are frequently isolated from lesions caused by acne, and both are thought to contribute to acne inflammation. According to research by Nakase et al. there has been an increasing number of P. acnes and S. epidermidis bacteria strains resistant to clindamycin. It is known that more than 80% of patients who carry clindamycin-resistant P. acnes also carry clindamycin-resistant S. epidermidis. An alternative antibacterial agent, such as that from natural ingredients, is needed in treating acne vulgaris and preventing an increased number of antibiotic-resistant strains².

Cassia alata L., commonly referred to as candle bush, is a type of large shrub growing wildly in tropical and humid climates. In Indonesia, Thailand, and the Philippines, C. alata leaves have empirically been used as a traditional medicine to treat various diseases, including skin diseases³. C. alata leaves extract has been reported to have an inhibiting activity against clinical isolates of Gram-positive and Gram-negative bacteria⁴. According to research by C. alata leaves extract has antibacterial activity against P. acnes at a minimum inhibitory concentration (MIC) of 0.625 mg/mL and minimum bactericidal concentration (MBC) of 1.25 mg/mL, and against S. epidermidis at MIC of 2.5 mg/mL and MBC>5mg/mL⁵. In another study, it was also known C. alata leaves has antibacterial activity at MIC of 31.3mg/mL and MBC of 62.5mg/mL against P. acnes, and at MIC of 15.6mg/mL and MBC of 15.6 mg/mL against S. epidermidis⁶.

Based on previous research, C. alata leaves have the potential to be developed as a treatment for acne vulgaris. Therefore, in the present study we try to develop a topical dosage forms of C. alata leaves extract, investigate its stability, and investigate its antibacterial activity. One example of a semi-solid preparation commonly used in topical medications for acne is cream. Vanishing cream base is suitable for acne-prone skin because it gives a cooling effect, is easy to spread, and doesn't feel or look greasy when applied to the skin⁷.

MATERIALS AND METHODS:

Materials:

Materials for cream preparation and evaluation were Carbopol 940(Lubrizol, South America); isopropyl myristate (MakingCosmetics Inc., United States); stearic acid; glyceryl monostearate (Alfa Aesar, United States); polysorbate 60 (Industria Chimica Panzeri, Italy); cetostearylalcohol (Ecogreen Oleochemical, Singapore); phenoxyethanol; triethanolamine (Merck KGaA, Germany); lanolin (Lux Chemicals, South Africa); propylene glycol, titanium dioxide (Arrow Fine Chemicals, India); and aquadest; and methylene blue dye; buffer solution pH 4.01 (Horiba, Japan); buffer solution pH 7.00 (Horiba, Japan); and KCl solution (Horiba, Japan).

Materials for antibacterial activity study were Mueller Hinton Broth (MHB) (Himedia, India); bacteriological grade agar powder (Himedia, India); and Mc Farland standard solution 0.5(Himedia, India); bacterial isolate Propionibacterium acnes ATCC 6919; isolates of Staphylococcus epidermidis ATCC 12228; ethanol 96%; 0.9% sterile NaCl (Otsuka Pharmaceutical, Japan); and clindamycin (Zhejiang Hisoar Pharmaceutical, China)

Study Design:

This was an experimental study includes the formulation of C. alata extract cream preparation with a varied concentration. The antibacterial activity of C. alata extract and its prepared cream formulation was expressed asminimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) against P. acnes and S. epidermidis.

Preparation of Extract:

The C. alataleaves extract obtained from National Research and Innovation Agency (BRIN) which was made in November 2021 with the same manufacturing method as Angelina et al., (2020), and then stored at -17°C⁸.

Antibacterial Activity of C. alata Leaves Extract:

Antibacterial activity of C. alata leaves extract was assessed against P. acnes ATCC 6919 and S. epidermidis ATCC 12228. The MIC and MBC were determined by the microdilution assay^9,10. First, 10% C. alata leaves extract stock solution was prepared using a mixture of DMSO and sterile 0.9% NaCl (1:10) as a solvent. Bacterial suspensions were prepared by inoculating the bacterial isolates into the sterile 0.9% NaCl. The turbidity of the suspension was adjusted to the 0.5 McFarland standard solution, which was equivalent to 1.5×10⁸ CFU/mL. The MIC was performed using a 96-well microplate, consisted of 100 µL Mueller Hinton Broth (MHB) for each well. Then each well in rows A, B, and C was added with 100µL of suspension of P. acnes or S. epidermidis bacteria according to the bacteria tested so that the finalconcentration, respectively, was 2.5%; 1.25%; 0.625%; 0.312%; 0.156%; 0.078%; 0.039%; 0.020%; 0.010%; and 0.005%. In row H added 100µL of sterile 0.9% NaCl which was homogenized as a color comparison. The microplate was closed and incubated under conditions suitable for the bacteria. P. acnes was incubated at 37°C for 48hours under anaerobic conditions, while S. epidermidis was incubated for at 37°C for 24hours.

Formulation of C. alata Leaves Extract Cream:

C. alata leaf extract was obtained from MBC and varied to 1.25%; 2.5%; and 5% (Table 1). Cream formula was selected by adding and modifying the excipient concentration. The oil phase (isopropyl myristate, stearic acid, glyceryl monostearate, and cetostearyl alcohol) is mixed and melted to a temperature of about 70°C using a water bath. Carbopol 940 was dispersed in 75% of the volume of aquadest to be used in the formula, and heated using a hotplate to 70°C while stirring using a stirrer. The aqueous phase (polysorbate 60, propylene glycol, phenoxyethanol, triethanolamine, and aquadest) was mixed and heated using a water bath to 70°C. At the same temperature, the three phases were mixed and stirred using an overhead stirrer until a homogeneous cream mass was formed (cream A). As much as 10% of the mass of cream A was taken in another container and waited for it to cool down to a temperature of ±45°C. Then, some of the cream mass was mixed with the C. alata leaves extract using an immersion blender (cream B). Cream B is added to cream A while stirring until homogeneous. Titanium dioxide is added little by little using a sieve to not form lumps while stirring until homogeneous¹¹.

Table 1: C. alata Leaves Extract Cream Formulas

Ingredients	Concentration (%)
Ingredients	F0	F1	F2	F3
C. alata leaves extract	0	1.25	2.5	5
Isopropyl myristate	1	1	1	1
Stearic acid	4	4	4	4
Glyceryl monostearate	0.5	0.5	0.5	0.5
Cetostearyl alcohol	1	1	1	1
Carbopol 940	0.3	0.3	0.3	0.3
Polysorbate 60	3	3	3	3
Propylene glykcol	5	5	5	5
Phenoxyethanol	0.4	0.4	0.4	0.4
Triethanolamine	0.2	0.3	0.35	0.45
Titanium dioxide	0.5	0.5	0.5	0.5
Aquadest	ad 100	ad 100	ad 100	ad 100

Evaluation of C. alataLeaves Extract Cream:

1. Organoleptic Test:

Organoleptic examination was carried out by observing cream preparations including color, odor, and softness using the five senses or visually 12.

2. Homogeneity Test:

1g cream preparation placed between two object glasses, then observed under light. In homogeneous preparations, coarse particles or water droplets will not be visible (Dewi et al., 2014).

3. pH Test:

The pH meter was calibrated with a buffer solution of pH 4.0 and 7.0. 1g of cream diluted with 5 mL of distilled water. The pH meter electrode is immersed in the preparation. Evaluation of the cream is eligible if the pH of the preparation meets the skin pH criteria in the interval 4,5 – 6,5 ^12,13.

4. Emulsion Type Test:

Determination of the type of emulsion is carried out by two methods, namely the staining test and the miscibility test^14,15. 0.5g cream preparation sample was weighed and placed on a drip plate. In each row on the drip plate, 3 wells were filled with cream preparation to replicate the test three times. In the staining test method, the dye used is 0,1% methylene blue solution which is soluble in water. To each of the cream preparations on the drip plate, 3 drops of methylene blue were added and stirred. The mixture of cream and dye was taken and observed. If methylene blue is dispersed, then the preparation is an O/W emulsion type. However, if the color is not completely dispersed or there are blue globules, then the preparation is a W/O emulsion type. Water and oil are used in the immiscibility test. In another row on the platters, each cream preparation added with 3 drops of water or oil and stirred. The addition of oil if mixed homogeneously, then the preparation is a W/O emulsion type. However, if the water mixed homogeneously, the preparation belongs to the type of O/W emulsion.

5. Viscosity and Rheology Test:

Cream viscosity was measured using a viscometer and each formula was replicated three times. Preparations as much as 100grams put into a beaker. In this study, spindle 11 was used, the viscosity value and % torque were recorded at the 15th rotation, and the spindle speed variations were 5rpm, 10rpm, 15rpm, 20rpm, 25rpm, 30rpm, 40rpm, 50 rpm, 60rpm, 80rpm, 100rpm, 120rpm, 140rpm, 160 rpm, 180rpm, and 200rpm. After reaching the highest speed the spindle speed is lowered back down to the spindle speed at the initial speed.

6. Centrifugation Test:

Test tubes were filled with 5g cream samples in each and centrifuged at 5000rpm for 30min. If the samples remained homogeneous after 30min, it was considered to have proper stability^16,17.

7. Accelerated Stability Test:

Cream sample was stored in a climatic chamber at a temperature of 40°C and a relative humidity (RH) of 75%(Carstensen and Rhodes, 2000). The container used were aluminium-lined airtight container. Storage in the climatic chamber was carried out for 3 months with tests at week 0^th, 2^nd, 4^th, and 12^th. Physical stability observation data such as pH and viscosity of each cream preparation were analyzed using the IBM SPSS Statistics 25 application with Repeated Measure ANOVA for normally distributed data or Friedman Test for data not normally distributed (α = 0.05). If the results of statistical analysis obtained the value of Sig.<0.05 then there is a significant difference during storage time and the preparation is said to be unstable during storage time. Descriptive data in the form of organoleptic, homogeneity, and emulsion type were observed visually.

Antibacterial Activity Test of C. alata Leaves Extract Cream:

The antibacterial activity test of C. alata cream formulation against P. acnes and S. epidermidis were determined by well-diffusion method. The method same with the extract antibacterial activity test above.

RESULTS AND DISCUSSION:

Antibacterial Activity of C. alata Leaves Extract:

According to Bury-Moné, an agent can be considered a bactericide if the MBC value is not more than four times the MIC value. Based on the MIC and MBC values obtained in this study (Table 2), it can be said that C. alata leaves extract is bactericidal against S. epidermidis and bacteriostatic against P. acnes. Although considered bacteriostatic against P. acnes, the extract still exhibited bacterial killing activity in vitro¹⁸.

Table 2: Antibacterial Activity of C. alata Leaves Extract

Bacteria	MIC	MBC
Propionibacterium acnes	0,156%	1,25%
Staphylococcus epidermidis	0,078%	0,312%

According to Angelina et al., C. alata leaves extract contained emodin, kaempferol, kaempferol-3,7-diglucoside, and kaempferol-3-O-β-D-glucopyranoside¹⁰. Kaempferol is a chemical compound that has the potential to treat acne because it has the advantage of being fat-soluble¹⁹. Kaempferol has a MIC value of 64µg/mL and a MIC value of 256µg/mL against clindamycin-resistant P. acnes strains. Aloe-emodin is also one of the chemical compounds obtained from the extract of C. alata²⁰. Aloe-emodin has a MIC value of 32g/mL and a MIC value of 128g/mL against S. epidermidis, the antibacterial activity was observed using a scanning electron microscope (SEM), known that aloe-emodin has a mechanism by damaging the outer membrane of S. epidermidis cells²¹.

Formulation and Evaluation of C. alata Leaves Extract Cream:

In preparing the cream formulations, the addition of C. alata leaves extract with concentration of 1.25%; 2.5%; and 5% causes the cream to become darker as the higher concentration of extract is added. The dark brown color of the cream is considered aesthetically lacking, so an attempt was made to lighten the color with the addition of titanium dioxide. Titanium dioxide is a compound that has a high refractive index, so its light-scattering properties can be utilized in its use as a white pigment²². Titanium dioxide is practically insoluble in water. The addition of titanium dioxide to the base of the O/W cream can only be dispersed, so extra care is needed to avoid sedimentation of the titanium dioxide during storage. To prevent the sedimentation of titanium dioxide and to obtain a stable preparation, formulas with varied amount of triethanolamine.

Based on the observations that have been made, the addition of C. alata leaves extract causes the cream runnier as the concentration increases. This is because C. alata leaves extract solution has a pH that tends to be acidic, emulsifiers in the form of amine soaps such as triethanolamine stearate are not compatible with acids²³. The 10% C. alata leaves extract solution has an acidic pH, which is 4.97±0.04. According the appearance and plasticity of stearic acid as a cream base are determined by the amount of alkalizing agent used. Based on the observations that have been made, the cream base with a pH above 6 is sufficient to prevent sedimentation of titanium dioxide when mechanically tested using a centrifuge. The target pH value for C. alata leaves extract cream is 6 – 6.5 in order to stay within the pH range for topical preparations.

Based on the evaluation results at 0^th week ( Table 3), it can be seen that all formulas meet the requirements for a good topical cream preparation. In the evaluation of pH and viscositymeasurements varied results were obtained due to variations in the concentration of C. alata leaves extract which has an acidic pH and variations in the concentration of triethanolamine which is an alkalizing agent. The triethanolamine variation formula aims to increase the thickness of the cream preparation, in order to prevent the completion of titanium dioxide after centrifugation. The higher the concentration of C. alata leaves extract, the lower the pH value, so the higher the concentration of triethanolamine needed to raise the pH. The concentration of triethanolamine added to each formula was not patterned because the addition of base to the cream preparation containing the C. alata extract leaves would make the color of the preparation darker. Based on the results of the evaluation of pH value measurements, all formulas have entered the range of pH requirements for topical preparations, namely pH close to skin pH 4.5 – 6.5. Topical preparations that are too acidic can irritate the skin, while topical preparations that are too alkaline can make the skin dry¹³.

In the viscosity test results, blank cream has the greatest viscosity value. The addition of C. alata leaves extract causes the viscosity value to decrease. C. alata leaves extract has a pH value that tends to be acidic, the addition of the extract affects the consistency of the preparation. In this formula, stearic acid is used as an emulsifier, Carbopol 940 and glyceryl monostearate as a co-emulsifier, which work because of the interaction with an alkaline, namely triethanolamine. Viscosity values became varied due to the use of variations in the concentration of extract, triethanolamine, and aquadest in formulas. Further research is needed regarding the effect of triethanolamine concentration on the viscosity of the C. alata leaves extract cream.

The cream formulas were tested mechanically by centrifugation to observe whether there was a phase separation from the preparation and the precipitation of titanium dioxide. Centrifugation testing at a speed of 5000rpm for 30 minutes has a centrifugal force effect which is equivalent to the effect of the gravitational force received by the test preparation for a year²⁴. Based on the test results obtained in Table 15, all formulas did not undergo phase separation and there was no titanium dioxide sedimentation. The results of the centrifugation test can visualize that the preparation is stable outside the influence of chemical changes²⁵.

Accelerated Stability Test of C. alata Leaves Extract Cream:

The accelerated stability test aims to accelerate chemical decomposition by storing the sample at high temperature and high humidity²⁵.

In the results that have been tested (Table 4), the preparation did not undergo organoleptic changes after 12 weeks of storage in the climatic chamber. There were no visible color change, rancid odor, titanium dioxide sedimentation, and microbial growth that appeared in the cream preparation. Texturally, all formulas still feel soft when applied to the skin, but they are getting thinner the longer they are stored under these conditions. Physical instability of cream preparations includes the occurrence of creaming, coalescence, and phase inversion²⁶. There were no signs of phase separation in the emulsion or phase inversion. This indicates that the preparation has used the right emulsifier and is physically stable. Storage of all preparations of C. alataleaves extract cream at a temperature of 40°C and RH±75% did not affect the characteristics of the preparation.

Table 4: Results of Evaluation of the Characteristics of the Preparation of the Cream After the Accelerated Stability Test

Formula		0^th Week	2^nd Week	4^th Week	12^th Week
F0	Organoleptic	White color, similar to cetostearyl alcohol odor, and soft
	Homogeneity	Homogeneous
	Emulsion Type	W/O
F1	Organoleptic	Light brown color, typical of C.alata leaves extract odor, and soft
	Homogeneity	Homogeneous
	Emulsion Type	W/O
F2	Organoleptic	Brown color, typical of C.alata leaves extract odor, and soft
	Homogeneity	Homogeneous
	Emulsion Type	W/O
F3	Organoleptic	Dark brown color, typical of C.alata leaves extract odor, and soft
	Homogeneity	Homogeneous
	Emulsion Type	W/O

Based on the test results of pH values obtained after 12 weeks of storage in the climatic chamber (Table 5 and 6), statistically it was found that F0 had a stable pH value. However, cream preparations added with C. alata leaves extract, namely F1, F2, and F3, gave significant results after 12 weeks of storage in the climatic chamber. It can be interpreted that the storage conditions of 40°C and 75% RH significantly affected the pH value of the C. alata leaves extract cream, which resulted in unstable pH of F1, F2, and F3.

Table 5. pH Test Results of Cream Preparations After Accelerated Stability Test

pH*
Formula	0^th Week	2^nd Week	4^th Week	12^th Week	Sig. Value
F0	6,48±0,01	6,47±0,02	6,43±0,06	6,5±0,01	0,111
F1	6,27±0,05	6,02±0,03	5,98±0,04	6,17±0,03	0,000
F2	6,37±0,07	6,09±0,01	5,99±0,02	6,13±0,01	0,016^a
F3	6,12±0,02	5,95±0,06	5,81±0,01	6,01±0,01	0,015^a

Information:

* : mean test value ± standard deviation three times replication

a : Asymp Sig. value data tested using Kruskal-Wallis H Test

Table 6. pH Test Results of C. alata Leaves Extract Solution (at Condition 40°C and 75% RH)

pH of C. alata Leaves Extract Solutions*
Concentration	0^th Week	1^st Week	2^nd Week	Sig. Value
1,25%	5,23±0,02	4,94±0,02	4,99±0,02	0,026^a
2,5%	5,18±0,01	4,86±0,02	4,80±0,01	0,000
5%	5,13±0,01	4,83±0,01	4,76±0,02	0,026^a

Table 1. Viscosity Test Results of Cream Preparations After Accelerated Stability Test

Viscosity Value at 200 rpm Spindle Rate (cps)*
Formula	0^th Week	2^nd Week	4^th Week	12^th Week	Sig. Value
F0	4040± 156	4780± 175	4548± 188	2955± 286	0.000
F1	3457± 364	3492±311	2968± 111	1163± 355	0.009
F2	1912± 99	2625± 116	2260± 213	1157± 56	0.000
F3	2458± 44	2482± 102	2713± 60	1958± 61	0.000

Table 8: Diffusion Test Results of C. alata Leaves Extract Cream Preparations

Sample	Inhibition Zone (mm)*
Sample	*P. acnes*	*S. epidermidis*
1,25% C. alata Leaves Extract Cream	10.67± 0.58	10.33± 1.16
2,5% C. alata Leaves Extract Cream	12.00± 0.00	11.33± 0.58
5% C. alata Leaves Extract Cream	14.67± 0.58	12.67± 0.58
1,25% C. alata Leaves Extract Solution	9.00± 1.00	8.00± 0.00
2,5% C. alata Leaves Extract Solution	12.67± 0.58	10.67± 0.58
5% C. alata Leaves Extract Solution	14.67± 0.58	12.00± 1.00
Blank Cream	0,00±0.00	0.00±0.00
Cream Base + 1% Clindamycin	24.67±1.53	25.00±1.53

Unlike F0, cream preparations F1, F2, and F3 experienced a drastic decrease in pH. In this test, the pH value of the C. alata leaves extract solutions were checked which was stored in the climatic chamber under the same conditions as the preparation for 2 weeks. This has the aim of knowing the possible causes of pH change in the preparation. The pH results of C. alata leaves extract solutions testingcan be seen in Table 7.

Based on the results of the measurement of the pH value, C. alata leaves extract solutions were not statistically stable at storage conditions of 40°C and 75% RH. In other words, the instability of cream preparations F1, F2, and F3 is influenced by the pH value of the ethanol extract of the C. alata leaves extract which decreases every week. The addition of excipients such as compatible buffering agents to the cream base can be considered to help minimize changes in pH during storage. Although the pH value of C. alata leaves extractcreams were statistically unstable, the pH value was still within the range of pH requirements for topical preparations, which was 4.5 – 6.5.

Based on the analysis obtained in Table 7, it can be said that all formulas have a significant difference in changes in the viscosity value in the four measurement time intervals because they have a Sig. value < 0.05. In other words, viscosity of F0, F1, F2, and F3 were statistically unstable during storage in the climatic chamber. F0, F1, F2, and F3 have similar viscosity value ratio curves over time in the 1st month, but drastically down in the 3rd month. After storage in the climatic chamber for 3 months, the flow properties for F0, F1, F2, and F3 consistently remained in the thixotropic pseudoplastic shear thinning type. On display, the curve has a fixed hysteresis loop, which is characteristic of a time-influenced non-Newtonian fluid system. The curves of F0, F1, F2, and F3 during storage in the climatic chamber can be seen in Figure 1and 2.

Figure 2: Rheology Curve at 12^th Week

The results from accelerated testing studies are not always predictive of physical change, so that real-time data is needed for 12 months. The data accelerated testing may be used to determine the storage of preparations and evaluate the impact of short-term excursions outside the label storage conditions such as might occur during shipping. Based on the results of the evaluation of the cream preparation after the accelerated stability test, it can be seen that the storage conditions for the preparation are below 30°C, because relevant changes were seen in the samples after storage at 40°C.

Antibacterial Activity Test of C. alata Leaves Extract Cream:

The well-diffusion method is commonly used to evaluate the antimicrobial activity of plant extracts. The well-diffusion method was used in this study because the sample in the form of a semisolid can be easily interpreted²⁷. The well-diffusion method tends to be easier and more sensitive than the disc-diffusion method to test natural products, especially those that are cationic, because the product may be adsorbed onto the surface of the disc rather than diffuse into the agar medium²⁸. The results of the measurement of the inhibition zone from the diffusion test of C. alata leaves extract can be seen in Table 8.

Previous study ,C. alata leaves extract 1% solution was tested on P. acnes and S. epidermidis shown not have an inhibitory zone against P. acnes and had an inhibitory zone of 8.49±1.60 mm against S. epidermidis.. Despite having the same variety or species, differences in external variables such as environmental conditions, agricultural practices, and post-harvest handling of plant products affect the phytochemical compounds in plants. In the extraction process, such as the selection of extraction methods and solvents, the compounds contained in the extract also have an effect¹⁰..

Based on the results obtained, the cream base did not affect the release of the C. alata leaves extract in the diffusion method. In this test, some of the inhibition zones produced by the C. alata leaves extract solution were smaller than the inhibition zones of the C. alataleaves extract cream. This is because the diameter of the inhibition zone could be affected by the solubility of the tested substance, diffusion range, and evaporation ²⁸. In this study, 1% clindamycin contained in the same base, had a large zone of inhibition against both test bacteria. Clindamycin with an inhibition zone of 21mm against Staphylococcus species is categorized as susceptible. Although C. alata leaves extract cream did not have a larger inhibition zone than the positive control, it can be seen that higher concentration of C. alata leaves extract gave a larger inhibition zone.

The MIC and MBC values of the C. alata leaves extract against S. epidermidis were smaller than P. acnes. However, the inhibition zone of P. acnes was larger than S. epidermidis in the well diffusion test. This is thought to be due to the presence of a less polar component, which usually diffuses slowly in the agar. Therefore, components such as phenolic compounds in the test obtained inconsistent results²⁸. As in the previous discussion, aloe-emodin, a phenolic compound, has an antibacterial activity against S. epidermidis. The inhibition zone obtained in the well-diffusion method is obtained with a smaller diameter. Further research is needed regarding the antibacterial activity of C. alata leaves extract cream with different method.

CONCLUSION:

C. alata leaves extract had antibacterial activity against P. acnes and S. epidermidis. The results of the evaluation showed the characteristics of a good cream preparation. Based on evaluation results from accelerated stability test, the C. alata leaves extract cream preparations were stable in organoleptic, homogeneity, and emulsion type parameters. However, C. alata leaves extract cream gave significant difference in the evaluation of pH and viscosity during 12 weeks of storage in climatic chamber. In testing the antibacterial activity of the C. alata leaves extract cream preparations shown that variations in the concentration of the extract affected the diameter of the resulting inhibition zone. The cream with 5% concentration of C. alata leaves extract had the largest inhibition zone, namely the inhibition zone of 14.67±0.58mm against P. acnes and 12.00±1.00 against S. epidermidis.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank RP ORKES 2023 for funding support in this research

REFERENCES:

1. Fournière M.Latire T.Souak D.Feuilloley MGJ.Bedoux G. Staphylococcus epidermidis and Cutibacterium acnes: Two Major Sentinels of Skin Microbiota and the Influence of Cosmetics. Microorganisms. 2020; 8(11): 1752. doi.org/10.3390/microorganisms8111752

2. Fournière, M., Latire, T., Souak, D., Feuilloley, M. G. J., and Bedoux, G. Staphylococcus epidermidis and Cutibacterium acnes: Two Major Sentinels of Skin Microbiota and the Influence of Cosmetics. Microorganisms. 2020; 8(11); 1752. https://doi.org/10.3390/MICROORGANISMS8111752

3. Nakase K.Nakaminami H.Takenaka Y. Hayashi N. Kawashima M. Noguchi N. Relationship between the Severity of Acne Vulgaris and Antimicrobial Resistance of Bacteria Isolated from Acne Lesions in a Hospital in Japan. J Med Microbiol. 2014; 63(5):721-728. doi.org/10.1099/jmm.0.067611-0

4. Nakase, K., Nakaminami, H., Takenaka, Y., Hayashi, N., Kawashima, M., and Noguchi, N. Relationship between The Severity of Acne Vulgaris and Antimicrobial Resistance of Bacteria Isolated from Acne Lesions in A Hospital in Japan. Journal of Medical Microbiology. 2014; 63(5): 721–728. https://doi.org/10.1099/JMM.0.067611-0/CITE/REFWORKS

5. Shrivastava A. Parveen N. Dewangan G. Verma VS. Bhattacharya A. Sharma M. Evaluation of Anti-Pyretic Potential of Cassia alata Leaves. Asian J. Pharm. Res. 2014; 4(4): 195-197. Retrieved from https://asianjpr.com/AbstractView.aspx?PID=2014-4-4-6

6. Fatmawati S. Yuliana. Purnomo AS. Abu Bakar MF. Chemical Constituents, Usage and Pharmacological Activity of Cassia alata. Heliyon. 2020; 6(7): e04396. doi.org/10.1016/j.heliyon.2020.e04396

7. Fatmawati, S., Yuliana, Purnomo, A. S., and Abu Bakar, M. F. Chemical Constituents, Usage and Pharmacological Activity of Cassia alata. Heliyon. 2020; 6(7): e04396. https://doi.org/10.1016/j.heliyon.2020.e04396

8. Syal S. Pandit V. Ashawat MS. Traditional Herbs to treat Acne Vulgaris. Asian J. Pharm. Res. 2020; 10(3): 195-201. doi.org/10.5958/2231-5691.2020.00034.9

9. Pukumpuang W. Thongwai N. Tragoolpua Y.(Total Phenolic Contents, Antibacterial and Antioxidant Activities of Some Thai Medicinal Plant Extracts. J. Med. Plants Res. 2012; 6(35): 4953–4960. https://doi.org/10.5897/JMPR12.655

10. Shekhar SK. Jose TP. Impact of Advertisement on Buying Behavior of Customers Towards Fairness Cream. Asian J. Management. 2017; 8(3): 429-431.doi.org/10.5958/2321-5763.2017.00068.3

11. Angelina M. Hanafi M. Suyatna FD.Dewi BE. Drug of Action Cassia Alata Leaves Extract as Antiviral to Dengue Virus Serotype-2 In vitro. Pharmacogn J. 2020; 12(4): 864–871. doi.org/10.5530/pj.2020.12.124https://doi.org/http://dx.doi.org/10.5530/pj.2020.12.124

12. Angelina M. Mardhiyah A. Dewi RT. Fajriah S. Muthiah N. Ekapratiwi Y. Dewijanti ID. Sukirno S. Jamilah J.Hartati S. Physicochemical and Phytochemical Standardization, and Antibacterial Evaluation of Cassia alata Leaves from Different Locations in Indonesia. Pharmacia. 2021; 68(4): 947-956. https://doi.org/10.3897/pharmaciaPHARMACIA.68.E76835

13. Sharma A. Prasar B. Formulation and Evaluation of Herbal Cosmetic Cream to Produce Multipurpose Effect on Skin. Res. J. Topical and Cosmetic Sci. 2013; 4(1): 1-4.Retrieved from https://rjtcsonline.com/AbstractView.aspx?PID=2013-4-1-1

14. Bhide R. Madasu D. Patil P. Dayal N. Koppaka O. Ather S. Thakur M. Formulation, Characterization and In Vitro Evaluation of ProShine MBTMGMIHS Herbal Sunscreen Cream containing Flower Extract. Research Journal of Pharmacy and Technology. 2023;16(3):1309-3.doi.org/10.52711/0974-360X.2023.00215

15. Rutuja Bhide, Durga Madasu, Poonam Patil, Navami Dayal, Omkar Koppaka, Shamshad Ather, Mansee Thakur. Formulation, Characterization and In Vitro Evaluation of ProShine MBTMGMIHS Herbal Sunscreen cream containing Flower Extract. Research Journal of Pharmacy and Technology. 2023; 16(3): 1309-3

16. Tranggono RI. Latifah F. Buku Pegangan Ilmu Pengetahuan Kosmetik 2013;21, Gramedia Pustaka Utama.

17. Billany M. Suspensions and Emulsions. In M. E. Aulton (Ed.) Pharmaceutics: The Science of Dosage form Design 2002; 2nd ed. Pp 334–359, Churchill Livingstone.

18. Trilokchandran B. Vijayakumar G. Thippareddy KS. Formulation and Evaluation of Cosmetic Cream from Cabbage Extract. Research J. Pharm. and Tech 2019; 12(8): 3589-3594. doi.org/10.5958/0974-360X.2019.00612.7S.

19. Valarmathi M. Kumar S. Sharma V. Imran M. Mohanasundaram. Formulation and Evaluation of Herbal Face Cream. Research J. Pharm. and Tech. 2020; 13(1): 216-218. doi.org/10.5958/0974-360X.2020.00043.8

20. Bury-Moné, S. Bury-Moné S. Antibacterial Therapeutic Agents: Antibiotics and Bacteriophages. In Reference Module in Biomedical Sciences. 2014. https://doi.org/10.1016/B978-0-12-801238-3.00244-0

21. Promgool T. Pancharoen O. Deachathai S. Antibacterial and Antioxidative Compounds from Cassia alata Linn. Songklanakarin J. Sci. Technol. 2014; 36(4): 459–463. Retrieved from https://www.thaiscience.info/journals/Article/SONG/10968330.pdfhttp://www.sjst.psu.ac.th

22. Li T. Lu Y. Zhang H. Wang L. Beier RC. Jin Y. Wang W. Li H. Hou X. (2021)Antibacterial Activity and Membrane-Targeting Mechanism of Aloe-Emodin Against Staphylococcus Epidermidis. Front Microbiol. 2021; 12. https://doi.org/10.3389/fmicbFMICB.2021.621866/FULL

23. Rowe RC.Sheskey PJ. Quinn ME. Handbook of Pharmaceutical Excipients (6th ed.) 2009; Royal Pharmaceutical Society Publishing.

24. Neelima G. Raul SK. Kranthi J. Sandeep S. Formulation and Evaluation of Sun Protection Factor of Poly Herbal Sunscreen Cream. Research J. Topical and Cosmetic Sci. 2016; 7(1): 9-14. doi.org/10.5958/2321-5844.2016.00002.9

25. Allen LV. The Art, Science, and Technology of Pharmaceutical Compounding, 6th Edition. In The Art, Science, and Technology of Pharmaceutical Compounding, 6th Edition 2016. American Pharmacist Association.https://doi.org/10.21019/9781582123578

26. Balouiri M. Sadiki M. Ibnsouda SK. Methods for in Vitro Evaluating Antimicrobial Activity: A review. J Pharm Anal. 2016; 6(2): 71. https://doi.org/10.1016/j.jphaJ.JPHA.2015.11.005

27. Bubonja-Šonje M.Knezević S.Abram M. (2020) Challenges to Antimicrobial Susceptibility Testing of Plant-derived Polyphenolic Compounds. Arh Hig Rada Toksikol. 2020; 71(4): 300. https://doi.org/10.2478/AIHT-2020-71-3396

28. Hudzicky J.Kirby-Bauer Disk Diffusion Susceptibility Test Protocol. American Society for Microbiology. 2009. Retrieved fromhttps://asm.org/getattachment/2594ce26-bd44-47f6-8287-0657aa9185ad/Kirby-Bauer-Disk-Diffusion-Susceptibility-Test-Protocol-pdf.pdf

29. Pinit N. Temkitthawon P. Lamlerltthon S. Pitaksuteepong T. Screening of Antibacterial Activities of 53 Thai Herbal Extracts Against Acne-Involved Bacteria. J Sci Tech Ubon Ratchatahni University: Suppl Edition. 2017; 85–92. Retrieved from https://www.thaiscience.info/Journals/Article/JSUU/10989951.pdf.https://www.thaiscience.info/Journals/Article/JSUU/10989951.pdf

30. Shrivastava S. Tirkey R. Kujur A. Jangde R. Daharwal SJ. Glimpses of Ethnopharmacological Approaches to Treat Acne. Research J. Topical and Cosmetic Sci. 2017; 8(1): 40-49. doi.org/10.5958/2321-5844.2017.00005.XJan

31. Pattewar SV. Patil DN. Formulation of Herbal Antibacterial Cream by Using Extract from Kalanchoe pinnata Leaves. Research J. Topical and Cosmetic Sci. 2014; 5(1). Retrieved from https://rjtcsonline.com/AbstractView.aspx?PID=2014-5-1-1

Received on 30.03.2023 Modified on 14.08.2023

Research J. Pharm. and Tech 2024; 17(3):1356-1363.

DOI: 10.52711/0974-360X.2024.00214

Evaluation		F0	F1	F2	F3
Organoleptic	Color	White	Light brown	Brown	Dark brown
	Smell	Similar to cetostearyl alcohol	Typical of C.alata leaves extract	Typical of C.alata leaves extract	Typical of C.alata leaves extract
	Texture	Soft	Soft	Soft	Soft
Homogeneity		Homogeneous	Homogeneous	Homogeneous	Homogeneous
pH		6,48±0,01	6,30±0,03	6,37±0,07	6,14±0,02
Emulsion Type		O/W	O/W	O/W	O/W
Viscosity (at 200 rpm)		4040±156 cps	3457±364 cps	1912±99 cps	2458±44 cps
Rheology		Pseudoplastics thixotropic	Pseudoplastics thixotropic	Pseudoplastics thixotropic	Pseudoplastics thixotropic
Centrifugation Test		No separation and sedimentation	No separation and sedimentation	No separation and sedimentation	No separation and sedimentation