Formulation and Antibacterial Essay of Topical Cream Enriched with (Chromolaena odorata) Leaf Extract Originating from Ie Seuem Geothermal Area, District of Aceh Besar, Indonesia

Munira Munira*, Rasidah Rasidah, Muhammad Nasir

¹Department of Pharmacy, Aceh Health Polytechnics, Banda Aceh, (Aceh) Indonesia.

²Department of Biology, Faculty of Mathematics and Natural Science, Universitas Syiah Kuala,

Kopelma Darussalam, Banda Aceh, (Aceh) Indonesia.

*Corresponding Author E-mail: munira.bio@poltekkesaceh.ac.id

ABSTRACT:

Anti-bacterial creams containing herbs and natural products are important because they provide a safe and effective alternative to chemical-based products. C. odorata leaf extract contains numerous active chemicals that can be used to formulate antibacterial cream for topical application. This study aims to formulate an antibacterial topical cream containing active chemicals extracted from C.odorata leaf originating from geothermal area, and evaluate physicochemical properties of the formulated topical cream followed by in-vitro antibacterial activity of the formula. The C.odorata leaf extraction was performed by cold extraction using ethanol and its chemical contents was investigated using GC-MS followed by predicted bioactivity profile using a computer software. Physicochemical evaluation performed include organoleptic evaluation, spread ability, pH values, viscosity measurement, and homogeneity test. GC-MS spectra indicates that the ethanolic extract of C.odorata leaf contains 22 compounds dominated by alcohol derivative compounds and terpenoids where most compounds shows bioactivities when simulated using a computer program. The formulated cream corresponds to the standard of topical cream with good physical stability, homogeneity, and viscosity. The pH of the cream was found to be stable over 28 days after formulation. The antibacterial test against S. aureus indicated that the cream is active to inhibit the bacteria where F3 shows better results compared to other formulations and the positive control. The C.odorata leaf extract formulated as topical cream have shown great activity against S.aureus at the concentration of 10% due to various chemical contents including alcohol derivatives and terpenoids.

KEYWORDS: Anti-bacterial,C. odorata, Geothermal area, S. aureus, Topical cream, Bioactivity, Natural product.

INTRODUCTION:

The perennial shrub Chromolaena odorata (L.) R.M. King & H. Rob., also called Kirinyuh in the native Indonesian tongue, belongs to the plant family Asteraceae. The American native C. odorata L. is an invasive shrub¹. This plant quickly expanded to other nations in southern and western Africa, eastern and southern Asia, and Australia within a short period of time, where it quickly rose to become one of the most prevalent shrub species used in agriculture².

For instance, C. odorata L. is the second most prevalent species of invasive plant in South Africa. The ecological circumstances that C. odorata L. thrives in might vary from those in its native home. Numerous traits of C. odorata L., such as its rapid plant reproduction rates, high nutrient absorption rates, suppressive effects on other plant species, and growth adaptation to varied soil and climatic conditions, are used to evaluate its capacity.

C. odorata L. has long been prized for its numerous medicinal properties^3,4 , even though it may be grown invasively. To cure wounds, fungal infections, coughs, headaches, toothaches, diarrhea, stomach problems, and dysentery, local medical experts utilize the plant. Some literatures reported that this plant has antibacterial⁴, antidepressant⁵, anti-inflammatory⁶, anti-diarrheal, anti-analgesic, anti-cancer, anti-diabetic, antioxidant³, and wound healing properties⁷. Analyzing plant components taken from C. odorata L. has revealed biological traits. The leaves of C. odorata L. include phenolics, flavonoids, saponins, terpenoids, tannins, and steroids. In the column fraction of the ethanol extract of C. odorata L. leaves, researchers also discovered phenolic acids such as protocatechuic acid, ferulic acid, vanillic acid, and a mixture of flavonoid aglycones like sinensetin, rhamnetin, tamarixetin, and kaempferide.

Due to the rich content of active ingredients contained in C. odorata leaf extract, the ethanol fraction of C. odorata leaves can be used as an antibacterial agent⁸. Antibacterial substances can be used as oral administration drugs or as external drugs such as topical creams that can be used to inhibit bacterial infections through the skin^9,10. Topical cream is a drug that is applied directly to the surface of the skin or mucous membranes.Creams are semisolid emulsion, it is lighter than the ointments they are less greasy and easy to apply¹¹. The purpose of administering drugs to the skin and mucous membranes is so that the drugs can enter the body directly through that area so that skin problems can be resolved directly. This drug is usually used to relieve pain, nourish the skin, or protect the skin from certain risks or problems such as exposure to direct sunlight, skin diseases, and so on¹².

In this work, we formulated the topical cream with antibacterial activity containing natural compounds extracted from C. odorata leaf cultivated from geothermal area of Ie Seuem. The bioactivity properties of the compounds were computerized using molinspirations online system. The physical properties including physical appearance, colour, cream opacity, texture, phase separation, and homogeneity of the formulated cream were evaluated. In addition, pH values changes over time, spreadability, and viscosity of the cream were also measured. The antibacterial test was conducted using disk diffusion technique.

MATERIALS AND METHODS:

Materials:

C. odorata leaf was cultivated from the geothermal area of Ie Seuem located in Aceh Besar, Aceh Province, Indonesia. Deionized water and chloramphenicol were obtained from a local pharmacy. Stearic acid, cetyl alcohol, triethanolamine (TEA), methyl paraben, propyl paraben, ethanol, and glycerin were purchased from Sigma-Aldrich, Singapore. The antibacterial test kit was purchased from a local pharmaceutical company (Easytest) equipped with chloramphenicol antibiotic test kit.

General procedure:

Extraction of the plant material:

The extraction of C.odorata leaf was performed according to our previous work¹³. To obtain the extract of C. odorata, the maceration method was used. This involved soaking the sample in ethanol for 72 hours with stirring every 24 hours. The solvents were changed twice and then the mixture was filtered using filter paper. The resulting macerate was collected and concentrated using a rotary evaporator at 40°C and a pressure of 20±0.5 kPa. The remaining filtrate was also evaporated in a water bath using a rotary evaporator from Buchi, Switzerland until a thick extract was obtained.

Topical Cream Preparation:

The oil phase (stearic acid, cetyl alcohol) was melted over a water bath (mass 1). While the water phase (TEA, Methyl paraben, Propyl paraben and glycerin and water) is mixed then heated over a water bath (mass 2). Masses 1 and 2 were mixed little by little then crushed until a creamy mass is formed. Then put in the C. odorata leaf extract and then grind it until smooth and homogeneous then stored it in a container.

Table 1: Antibacterial cream formulation

No	Material	Unit	Concentration (%)
1	Kirinyuh leaf extract	g	0	5	7	10
2	Stearic Acid	g	12.0	12.0	12.0	12.0
3	Cetyl alcohol	g	0.5	0.5	0.5	0.5
4	Triethanolamine (TEA)	mL	1.0	1.0	1.0	1.0
5	Methyl paraben	g	0.1	0.1	0.1	0.1
6	Propyl paraben	g	0.5	0.5	0.5	0.5
7	Glycerin	mL	2.0	2.0	2.0	2.0
8	Deionized water add	mL	100.0	100.0	100.0	100.0

Physical evaluation:

Viscosity Evaluation:

The viscosity of the prepared cream formulations was determined using Oswald viscometer (Pyrex, Germany). The viscosity was measured in milipoises (mps) at ambient temperature (25^oC) This experiment was performed for both the negative and and the medicated formulations.

pH values measurement:

The desired amount of cream was placed in a Beaker glass, and the pH values was measured by immersing the electrode to the cream using pH meter (Smart Meter, PH818). The recording was performed 3 times each week until 4 weeks.

Spreadability test:

Spreadability was measured using the wooden board spreadability apparatus equipped with scale and two glass slides with two pans on both sides mounted on a pulley. The sample was put between the glass slides and 100 g weight was placed on the glass slide to compress the sample to a uniform thickness for five minutes. 250 g weight was then added to the pan. The spreadability of the cream was calculated using the following formula:

Where, m = weight tied on upper slide of the glass (g), l = length of glass slide (cm), and t (s)

In vivo anti-bacterial activity assay:

The disc diffusion technique was used to assess the antibacterial activity of ethanolic extracts of C. odorata leaf on the PCA medium. The procedure was conducted according to the work conducted by⁶. A sterile swab dampened with the bacterial solution was used to disperse the inoculum, which contained 108 CFU/ml of bacteria (S.aureus), across the solid PCA plates. Sterile Whatman filter discs (6 mm in diameter) were formed in the PCA plate using a sterile cork borer (5 mm). Then 100 mL of the extracts, each with a concentration of 5, 7, and 10%, were added to the discs created in the infected plates. The treatments also included 100 ml of ethanol as negative control and chloramphenicol as a positive control. The plates were incubated for 48 hours at ambient temperature (27 °C), and the inhibition zones formed on the discs was measured in millimeters (mm). A minimum of two repetitions of each treatment were made up of three duplicates.

RESULT:

Chemical constituents of ethanolic C.odorata leaf extract:

C. odorata leaf have been known to contain several biologically active compounds such as phenolic, terpenoids, and unsaturated methyl ester. Depending on what solvents are used to extract, the chemicals found in the leaf of plant would vary significantly due to different polarity of the solvent. Furthermore, different instruments used to analyze the sample would also give significantly different compounds due to limitation of each instrument. GC-MS for example could only capable of detecting volatile compounds with maximum retention time up to 120 minutes. In this work, GC coupled with MS was utilized to investigate active compounds in the ethanolic phase of C.odorata leaf resulting in 22 active compounds detection.

Figure 1. GC spectra of C.odorata leaf extract cultivated from Ie Seuem geothermal area

No	Compound Name	RT (minute)	Peak Area (%)
1	1-Tridecanol	11.32	1.84
2	2-Isopropyl-5-methyl-1-heptanol	11.69	1.57
3	alpha-Copaene	12.73	1.80
4	7,10-Pentadecadiynoicacid	13.39	1.75
5	trans-Caryophyllene	13.60	5.99
6	alpha-Guaiene	13.98	1.82
7	alpha-Humulene	14.28	2.07
8	gamma-Muurolene	15.42	1.51
9	delta-Cadinene	15.60	7.83
10	1-Heptacosanol	15.90	4.12
11	1-Octadecanethiol	16.09	1.50
12	1-Dodecanol,2-hexyl-	16.451	2.16
13	Cyclopropanol,1-(3,7-dimethyl-1-octenyl)-	16.670	3.30
14	Cyclopentane ethanol,beta,2,3-trimethyl-	17.276	3.85
15	1-Heptatriacotanol	18.086	26.67
16	Cyclohexane,eicosyl-	19.419	2.83
17	1-Dodecanol,2-hexyl-	19.983	6.63
18	2-Heptene,5-ethyl-2,4-dimethyl-	20.112	2.97
19	1-Dodecanol,2-hexyl-	20.502	6.07
20	Cyclohexane,1,2,3,4,5,6-hexaethyl-	20.628	1.57
21	Iso-citronellol	20.692	3.28
22	Hexadecanoicacid,methylester	22.855	6.70

Figure 2. Chemical compounds contains in ethanolic extract of C. odorata leaf determined using GC-MS, alcohol derivatives (10 compounds) and terpenoid (6 compounds) are dominant in the extract.

Table 2. Drug properties and bioactivity of compounds using molinspirations

No	Compound	Drug properties			Predicted Bioactivity
No	Compound	MW (kDa)	Mi log P	TPSA	GPCR Ligan	Enzyme Inhibitor	ICM	Kinase inhibitor	NRL	Protease inhibitor
1.	1-Tridecanol	200.37	5.67	20.23	-0.42	-0.07	-0.08	-0.57	-0.42	-0.50
2.	2-Isopropyl-5-methyl-1-heptanol	172.31	4.09	20.23	-0.55	-0.29	-0.30	-0.97	-0.73	-0.59
3.	alpha-Copaene	204.36	5.75	0.00	-0.33	0.10	0.17	-0.79	0.02	-0.49
4.	7,10-Pentadecadiynoic acid	234.34	5.02	37.30	0.15	0.37	0.29	-0.27	0.26	0.05
5.	trans-Caryophyllene	204.36	5.17	0.00	-0.34	0.19	0.28	-0.78	0.13	-0.60
6.	alpha-Guaiene	190.33	4.62	0.00	-0.69	-0.36	-0.53	-1.22	-0.22	-0.93
7.	alpha-Humulene	204.36	5.30	0.00	-0.14	0.31	0.02	-0.93	0.34	-0.67
8.	gamma-Muurolene	232.41	6.24	0.00	-0.25	0.46	0.10	-0.79	0.46	-0.29
9.	delta-Cadinene	218.38	6.05	0.00	-0.47	0.36	-0.03	-0.89	0.31	-0.54
10.	1-Heptacosanol	396.74	9.75	20.23	0.07	0.09	0.02	0.01	0.11	0.09
11.	1-Octadecanethiol	286.57	8.76	0.00	-0.11	0.34	-0.07	-0.24	-0.05	0.22
12.	1-Dodecanol, 2-hexyl-	270.50	7.98	20.23	0.06	0.16	0.06	-0.07	0.02	0.03
13.	Cyclopropanol, 1-(3,7-dimethyl-1-octenyl)-	196.33	3.33	20.23	0.15	0.28	0.21	-0.32	-0.28	-0.10
14.	Cyclopentaneethanol, beta,2,3-trimethyl-	156.27	2.69	20.23	-0.77	-0.18	-0.27	-1.12	-0.26	-0.19
15.	1-Heptatriacotanol	537.01	10.38	20.23	0.05	0.05	-0.09	-0.00	0.07	0.06
16.	Cyclohexane, eicosyl-	364.70	9.70	0.00	0.08	0.05	0.01	-0.07	0.07	0.11
17.	1-Dodecanol, 2-hexyl-	270.50	7.98	20.23	0.06	0.16	0.06	-0.07	0.02	0.03
18.	2-Heptene, 5-ethyl-2,4-dimethyl-	154.30	4.86	0.00	-0.87	-0.17	-0.29	-1.50	-0.61	-1.06
19.	1-Dodecanol, 2-hexyl-	270.50	7.98	20.23	0.06	0.16	0.06	-0.07	0.02	0.03
20.	Cyclohexane, 1,2,3,4,5,6-hexaethyl-	252.49	7.67	0.00	-0.16	0.03	0.05	-0.30	-0.10	-0.24
21.	Iso-citronellol	156.27	3.15	20.23	-0.81	-0.12	-0.24	-1.16	-0.61	-0.83
22.	Hexadecanoic acid, methyl ester	270.46	7.37	26.30	-0.11	0.04	-0.05	-0.34	-0.09	-0.13

MW=molecular weight, TPSA= Topological Polar Surface Area, GPCR=G-protein-coupled receptor, ICM=Ion channel modulator, NRL=Nuclear receptor ligand

22 compounds contained in C. odorata leaves based on the GC-MS test have been measured for their chemical properties and bioactivity using molinspiration. The measurement indicates that the compound with the lowest MW of 154.3 g/mol is no. 18, its structure can be seen in Figure 2. While, the compound with the largest MW of 537.01 g/mol is no. 15 which is a long-chain alcohol group. Based on the prediction performed, all compounds show positive miLog P values. The milog P value indicates the level of lipophilicity of a material based on its solubility ratio in the octanol/water partition. The milog p value is a crucial value of a compound when used as a drug, a compound with high lipophilicity (positive milog P) will have higher bioavailability properties so that the dose used can be lower because of the compound's ability to penetrate the cell's semi-permeable membrane more effectively^9,10.

Physical evaluation of formulated cream:

Table-3 shows physicochemical properties of formulated cream containing different concentrations of C. odorata leaf extract. All extracts were physically appeared as smooth and homogeneous ointment without phase separation. F0 was found to be transparent while other formulations were opaque due to the addition of active chemical compounds, including the extract and the positive standard. F0 and F+ were white in color while other formulations were brownish green correspond to the incorporation of the C. odorata leaf extract.

The physicochemical properties of topical creams refer to the physical and chemical characteristics that impact the cream's appearance, texture, and stability^22,23. These properties include color, opacity, homogeneity, physical appearance, and phase separation. The color of topical creams can vary depending on the formulation and ingredients used, while the opacity can affect the penetration of active ingredients. Homogeneity is essential for maintaining a consistent texture and distribution of ingredients. The physical appearance of creams should be smooth, lump-free, and pleasant to the touch. Finally, phase separation, which can occur when two or more phases separate within a cream, can reduce the cream's effectiveness and consistency. Manufacturers must carefully monitor and control these properties to ensure that the creams are aesthetically appealing, stable, and effective. Regular testing of these properties is necessary to ensure consistent product quality over time.

Table 4.pH values of topical cream measured up to 4 weeks

Formulation	pH values (Mean ± SD)
Formulation	Week 1	Week 2	Week 3	Week 4
F0	6.6 ± 0.01	6.7 ± 0.02	6.8 ± 0.01	6.8 ± 0.01
F1	6.2 ± 0.02	6.0 ± 0.02	6.3 ± 0.02	6.6 ± 0.02
F2	5.8 ± 0.01	5.8 ± 0.01	6.5 ± 0.01	6.5 ± 0.02
F3	5.6 ± 0.02	5.6 ± 0.02	6.2 ± 0.01	6.2 ± 0.01

Table-4 shows the measurement values for the pH of the cream formulation for 4 weeks. The data in the table shows that the pH value of the C. odorata leaf cream formula ranges from 5.6 to 6.8. This value is in accordance with the pH standard for cream preparations based on SNI 16-4954-1998. ANOVA analysis showed that the pH value between formulations was significantly different with a p value = 0.014 while when tested by ANOVA the pH value per week found that there was no significant change in the pH value from week first to fourth week p=0.17. This proves that the prepared topical cream formula does not change pH easily and can be considered as a stable cream.An ideal stability indicating method is one that quantifies not only the drug compound alone but also resolves its degradation products²⁴.

The standard value of pH of topical creams can vary depending on the intended use and the specific formulation. However, in general, the pH of topical creams should be in the range of 4.5 to 7.5. This range is considered to be compatible with the natural pH of the skin, which is slightly acidic with a pH of around 5.5. Topical creams with a pH that is too high or too low can disrupt the natural pH of the skin, leading to skin irritation, inflammation, and reduced efficacy of the cream. The optimal pH for a topical cream will depend on the active ingredients, the intended use, and the specific patient population. Manufacturers must carefully control the pH of topical creams during production to ensure that they meet the intended specifications and are safe for use by patients. Regular testing of the pH of topical creams is necessary to ensure consistent product quality over time.

Table 5. Spreadability of the formulated cream over weeks

Formulation	Spreadability (cm)
Formulation	Week 1	Week 2	Week 3	Week 4
F0	7.0	6.0	6.0	5.0
F1	6.0	5.7	5.5	5.0
F2	5.5	5.0	5.0	5.0
F3	5.5	5.0	5.0	5.0

Table-5 shows the spreadability of the cream formulation from the ethanol extract of C. odorata leaves for measurement for 4 weeks. The lowest spreadability value was 5 cm in measurements in the second and third weeks for formulations F2 and F3 and in the fourth week for all formulations. While the highest spreadability occurred at F0 in the first week. The results of the ANOVA test for spreadability per week showed that there was no significant difference in the value of spreadability from the first to the fourth week (p=0.087), as well as the results of the ANOVA test between formulations which did not show a significant difference in terms of spreadability (p= 0.078).

The tolerable spreadability of topical creams over four weeks of observation will depend on several factors, including the specific formulation of the cream, the intended use, and the location on the body where the cream is applied. However, in general, topical creams should maintain their spreadability and consistency over time to ensure effective delivery of active ingredients and patient compliance. The data indicates that the cream was persistent in terms of its sreadability ensuring that the formulation would stand for longer period without any significant change in viscosity and physical properties in general²⁵.

Table 6. Viscosity of formulated cream

Formulation	Viscosity (Poise)
Formulation	Week 1	Week 2	Week 3	Week 4
F0	2,420	2,860	2,879	2,960
F1	4,300	5,280	8,260	8,520
F2	9,600	10,160	11,420	11,860
F3	10,060	13,200	16,680	16,880

Table-6 shows the value of the viscosity of the cream formulation measured every week for four weeks in millipoise units. The data shows that the higher the percentage of extract added the thicker the resulting formulation. Furthermore, the results of the ANOVA test on the viscosity values for four weeks did not show a significant difference (p=0.744). However, the viscosity values between the formulations were significantly different with a significance level of p=0.00.

The standard viscosity of topical creams can vary depending on the intended use and application of the cream. Generally, topical creams have a viscosity ranging from 10,000 to 100,000 centipoise (cP), which is a measure of the cream's resistance to flow. The viscosity of a topical cream is important because it affects the cream's spreadability, consistency, and absorption into the skin. A cream that is too thin or runny may not stay on the skin long enough to be effective, while a cream that is too thick or viscous may be difficult to apply or spread evenly.

The ideal viscosity of a topical cream depends on several factors, including the intended use, the location on the body where the cream will be applied, and the skin type of the patient. For example, creams intended for use on the face or other sensitive areas of the body may have a lower viscosity to ensure ease of application and absorption, while creams intended for use on thicker skin or on the hands and feet may have a higher viscosity to ensure adequate coverage. Regulatory agencies such as the US Food and Drug Administration (FDA) often provide guidance on acceptable viscosity ranges for topical creams based on their intended use. For example, the FDA recommends that topical creams used for wound healing have a viscosity of 10,000 to 25,000 cP, while creams used for sunscreens should have a viscosity of 6,000 to 12,000 cP. Overall, the standard viscosity of topical creams is important to ensure that the cream is easy to apply, effective, and safe for use by patients. Manufacturers must carefully control the viscosity of topical creams during production to ensure that they meet the intended specifications and perform as intended²⁶.

Figure-3 illustrates the inhibition zone exhibit by 5 different formulations of antibacterial cream enriched with C. odorata leaf extract obtained from Ie seuem geothermal area. The graph confirms that the greater the amount of the C. odorata leaf extract the better the activity of the cream against S.Aureus and even better than F+ which was added by a positive commercial antibiotic. Furthermore, analysis of variance (ANOVA) test revealed that the inhibition zone among the group of formula is significantly different with p-value = 0.00.

Antibacterial tests are important for topical creams because they help ensure that the cream is effective in killing or inhibiting the growth of bacteria on the skin's surface²⁷. Bacteria can cause a variety of skin infections, such as acne, impetigo, folliculitis, and cellulitis, and can also worsen existing skin conditions¹². Topical creams that are formulated to treat bacterial infections or prevent bacterial growth usually contain antibacterial agents, such as antibiotics or antiseptics. These agents work by targeting the bacteria's cell walls, enzymes, or DNA, which ultimately leads to the death or growth inhibition of the bacteria. Antibacterial tests can determine the efficacy of the antibacterial agent in the cream by measuring the minimum inhibitory concentration (MIC), which is the lowest concentration of the agent that can prevent bacterial growth²⁸. MIC testing is important because it helps identify the appropriate concentration of the antibacterial agent that should be included in the cream to ensure its effectiveness. In addition to ensuring the cream's effectiveness, antibacterial tests are also important for assessing the cream's safety. Topical creams that are designed for use on the skin need to be safe and non-irritating to avoid causing adverse reactions such as redness, swelling, or itching. By testing the cream for antibacterial efficacy and safety, manufacturers can ensure that the cream is safe and effective for use by consumers^29,30.

CONCLUSION:

The ethanolic extract of C odorata contains 22 active compounds dominated by alcohol derivatives and terpenoids based on GC-MS analysis. The formulated topical cream has meet the standard in terms of physical properties, pH values, and spreadability. The cream was also proven to be active against S.aureus with the most effective concentration of 10%.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors acknowledge Politeknik Kesehatan Aceh for the research fund through the scheme of PDUPT contract number DP.04.03/2291/2023.

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Received on 01.10.2023 Modified on 16.04.2024

Research J. Pharm. and Tech 2024; 17(10):4803-4810.

DOI: 10.52711/0974-360X.2024.00739

Formulation	Physical appearance	Color	Texture	Phase separation	Homogeneity
F0	Transparent	White	Smooth	No	Homogeneous
F1	Opaque	Brownish green	Smooth	No	Homogeneous
F2	Opaque	Brownish green	Smooth	No	Homogeneous
F3	Opaque	Brownish green	Smooth	No	Homogeneous
F+	Opaque	White	Smooth	No	Homogeneous