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1 Formulation and physical stability test of hand sanitizer based on nutmeg oil (Myristica fragrans Houtt)

R D Agustin* and Y M J Taihuttu

Faculty of Medicine, Pattimura University, Ambon Indonesia

Email: rachmawatidwia@gmail.com

Abstract. The hand is one of the pathways for spreading disease through bacterial contamination, so an antibacterial agent must be applied as a handwashing agent. A

practical alternative to washing hands is to use an antiseptic gel. Nutmeg contains active

compounds as antibacterial agents. This research aimed to obtain the most suitable formulation of nutmeg oil antiseptic gel as a hand sanitizer. Nutmeg oil antiseptic gel preparations were made in three different formulations with concentrations of 15% (F1), 20% (F2), and 25% (F3).

The physical properties stability test of gel included freeze-thaw for stability test, pH,

homogeneity, organoleptic, spreadability, and adhesion test. The results showed that the gel was white and had a peculiar smell of nutmeg, the gel form was semisolid, and the pH value was 6. Based on the freeze-thaw test, the preparation is stable in 3 cycles. The results of the spreadability and adhesion tests were influenced by differences in nutmeg oil concentration. That the nutmeg oil concentration of 25% (F3) was found to be the optimal formulation because it meets all the requirements for the physical properties of the gel.

1. Introduction

At this time, hand washing can use an antiseptic gel as a practical alternative to using soap and water.

The use of hand sanitizers among the middle and upper classes has become a way of life [1]. Several

hand sanitizer preparations can be found in the market and usually contain a lot of alcohol

composition, but it can cause irritating defection to the skin [2]. Therefore, the use of an alcohol-

containing antiseptic is inconvenient for repeated use [3]. Therefore, natural antiseptics that are safe

for repated use are being developed that are safe for repeated use, one of which is from the nutmeg seeds. Nutmeg is a tropical plant commonly found in Indonesia, especially in the Banda and Maluku islands. This plant has benefits in all parts, in which the fruit consists of pulp, seeds, and mace. Myristica fragrans Houtt is one of the best-known types of nutmeg in Indonesia [4]. Each part of the nutmeg has an active substance as an antimicrobial. The mace and the extracted nutmeg can inhibit the growth of gram-negative and positive bacteria [5]. Based on research, the bacteria inhibited by nutmeg extracts are Escherichia coli and Staphyloccocusaureus [6]. Nowadays, the production of nutmeg oil is starting to developed. Nutmeg oil can be used as an ingredient in making skin whitening cosmetics. It can use as an additional active ingredient in the manufacture of

antibacterial and fungal-based bath soaps [4]. Nutmeg oil can inhibit bacteria greater than the nutmeg

extract because it has more active compounds than the crude extract. Nutmeg contains essential oils of

about 5-15% and is known to have the power to kill bacteria [7]. Nutmeg oil in this study was formulated into a topical preparation, which can be used as an

antibacterial agent with good physical stability during storage. The selected preparation is a gel hand

sanitizer. The gel is a semi-solid system in the form of a suspension made of small inorganic particles

and large organic molecules mixed in a liquid [8]. The choice of preparation in the form of hand 2 sanitizer is because hand sanitizers are able to kill microorganisms without causing permanent skin damage. For hand sanitizers with good physical stability, it is necessary to add materials such as Carbopol 940, Triethanolamine, Methylparaben, Glycerin, and distilled water solvents. Carbopol 940

was chosen as a gelling agent because it is stable and hygroscopic in semi-solid preparations [9]. Based on the literature above, nutmeg oil can act as a natural antiseptic because of its ability to

inhibit bacteria. The authors are interested in making an antiseptic hand gel preparation formulation

made from nutmeg oil and testing its physical properties' stability to apply to the hands repeatedly.

2. Materials and Methods

2.1 Research instruments The instruments used in this study include a set of distillation devices, Gas Chromatography-Mass

Spectrophotometer (GC-MS) (Shimadzu QP-2010S), autoclaves, mixer, hot plate stirrer, pH-test strips, digital balance, and vortex mixer.

2.2. Materials

The material used includes nutmeg oil (FMIPA, UniversitasPattimura), Carbopol-940 (Brataco),

triethanolamine (Brataco), methyl-paraben (Brataco), glycerin (OneMed), and distilled water.

2.3. Hand sanitizer gel formulation

The hand sanitizer gel formula refers to the modified procedure by Astuti [10]. Preparationwas carried

out by sterilizing the tool that will be used first to kill microbes. The formulation was made by

weighing all the ingredients to be used in accordance with the calculation of the formulation. The preparation of hand antiseptic gel for nutmeg oil (in 10 mL) was carried out by dissolving Carbopol

940 in 5 mL of hot distilled water (70

0C) until completely dissolved, then add methylparaben. The

nutmeg oil was dissolved in glycerin, then put into a Carbopol solution, stirred until homogeneous.

Triethanolaminewas gradually added to the solution by increasing the stirring speed until a

homogeneous gel was formed, the remaining distilled water was added to the solution. The production of the gel was carried out by adding Carbopol-940 in hot distilled water then stirred.

TEA was added drop by drop while stirring, then methylparaben, nutmeg oil, glycerin, and distilled water was added to the desired volume. Commercial hand sanitizer (Antis Brand) was used as a

positive control and bases gel without nutmeg oil as a negative control. The gel hand sanitizer

formulations are presented in Table 1.

Table 1. Hand sanitizer gel formulations

Component Negative control F1 (15%) F2 (20%) F3 (25%)

Nutmeg Oil - 1.5 mL 2 mL 2.5 mL

Carbopol-940 0.1 g 0.1 g 0.1 g 0.1 g

TEA 0.05 mL 0.05 mL 0.05 mL 0.05 mL

ethyl-paraben 0.02 g 0.02 g 0.02 g 0.02 g

Glycerin 1.5 mL 1.5 mL 1.5 mL 1.5 mL

Distilled water 10 mL 10 mL 10 mL 10 mL

2.4 Hand sanitizer gel evaluation

The physical properties and stability of the gel preparations are tested, including organoleptic test, pH

test, spreadibility test, adhesion test, and stability test. 3

2.4.1. Organoleptic test. Physical observations were made on gel preparations including odor, color,

texture, and homogeneity. In the homogeneity test, the gel preparation was smeared on the slide. If the

gel color was evenly distributed, and there were no clumping powders, the gel was declared to meet the homogeneity requirements.

2.4.2. pH test. A pH measurements were carried out using pH Universal indicator paper by smearing

0.1 gram of gel on the pH indicator paper, after which it was observed and the color-matched with a

pH indicator paper comparison.

2.4.3. Spreadibility test. The spreadability test of the gel preparations was carried out by weighing

0.5 grams of the gel, which was placed in the middle of a petri dish spiked with millimeter block

paper, then put another petri dish for 1 minute. Then the average diameter was measured. Then add 50-

200 grams of weight, each left for 1 minute, and calculate the average diameter. Replication was

carried out three times.

2.4.4. Adhesion test. The test for the adhesion of the gel preparations was carried out by placing the

gel on the slide as much as 0.5 grams, then putting other object-glass on top of the gel, then pressing

and giving 1 kg of weights for 5 minutes. Then, the load is lifted,and the time is calculated until the

two slides are separated. Replication was carried out three times.

2.4.5. Stability test. A stability test on the optimum formula was carried out to determine the stability

of the formula during storage. The method used was freeze-thaw. This method was done by storing the preparation in the refrigerator at 4

0C for 24 hours (freeze), then the preparation was stored at room

temperature for 24 hours (thaw) [11]. This treatment was calculated as one freeze and thaw cycle. This

treatment was carried out in 3 cycles.

3. Result and Discussion

The organoleptic test was done by visually observing the smell, color, texture, and homogeneity in

table 2. Organoleptic test results on the three formulas for gel preparations with different nutmeg oil

concentrations showed that the color tended to be white. The color obtained was influenced by the

addition of clear-yellow nutmeg oil mixed with white Carbopol, resulting in a white-colored

preparation. This is because the nutmeg oil concentration given is small so that the effect of the

Carbopol color is stronger. The resulting odor is influenced by the addition of nutmeg oil, which is the

distinctive smell of nutmeg. The higher the concentration of nutmeg oil given, the stronger the

characteristic smell of nutmeg. The resulting texture tends to be thick due to the effect of giving nutmeg oil, which increases the viscosity of the preparation. The higher concentration of nutmeg oil given, the thicker the texture of the preparation. Table 2. The organoleptic test of hand sanitizer based on nutmeg oil

Formula

Organoleptic test

Color Aroma Texture Homogeneity pH

Negative control Clear Odorless Thick Homogeneous 6 ± 0 Positive control Clear Smells of orange Thick Homogeneous 6 ± 0

F1 White Smells of weak

nutmeg Thick Homogeneous 6 ± 0

F2 White Smells of strong

nutmeg Thick Homogeneous 6 ± 0

F3 White Smells of strong

nutmeg Thick Homogeneous 6 ± 0 4 The homogeneity test was carried out to see that the ingredients of the gel were mixed perfectly.

The nutmeg oil hand sanitizer gel has good homogeneity. That is, it does not show any coarse grains in

any preparation formulations. A pH test is carried out to observe the safety of the preparation when used on the skin by equating

the pH of the gel preparation with the skin's pH. The pH of human skin ranges from 4.5 to 6.5[12]. The

pH value of the preparation obtained enters the pH range of the skin so that the preparation is safe for

use on the skin. Nutmeg oil concentration variations did not affect the pH of the preparation (Table 2).

Statistical results showed that the p-value was not significant or equal to 1.00, so it could be concluded

that there was no difference in pH for each preparation. The spreadability test was conducted to determine the ability of the preparation to spread on the

skin. Increasing the concentration of nutmeg oil reduced the dispersibility of the gel. This is because

the higher the oil concentration, the higher the viscosity, thereby reducing the dispersibility of the gel

preparation. The dispersibility of a good gel preparation is between 5-7 cm

2 [13]. Statistical results

showed that the p-value = 0.016 (<0.05 or significant), so it can be concluded that there is a difference

in the spreadability of each preparation due to the influence of the variation in the concentration of

nutmeg oil given. The adhesiveness of preparation is inversely proportional to the dispersion power. The higher the

dispersibility value of preparation, the lower value of the gel preparation. In nutmeg oil gel

preparations, variations in the concentration of nutmeg oil affect the stickiness of the preparation. The

higher the concentration of nutmeg oil given, the greater the value of the gel preparation obtained. Too

strong adhesion will block the skin pores. Likewise, if the adhesion power is too weak, the

effectiveness of the gel preparation to penetrate the skin surface is not good. The longer the gel is

attached to the skin; the effectiveness of the gel is absorbed into the skin better because the absorption

of the active substance increases [10]. Based on statistical results, it shows that the p-value = 0.009

(<0.05 or significant) so it can be concluded that there is a difference in the adhesion of each

preparation, which means that variations in the concentration of nutmeg oil affect the stickiness of the

preparation. In the whole cycle, there was no change in organoleptic and pH. Nevertheless, there was an

increase in adhesion and spreadability from the first cycle to the second cycle, but it fell back down on

the third cycle. The freeze-thaw test results after the third cycle can be seen in table 3. This is because

Carbopol 940 has stable properties as a gelling agent so that the gel preparation obtained has good stability during storage [10]. Table 3. The result of freeze-thaw test after the 3rd cycle

Formula

Organoleptic test Spreadibility

test in 200 g (cm) Adhesion test (s)

Color Aroma Texture Homogeneity pH

Negative

control Clear Odorless Thick Homogeneous 6 ± 0 6.85±0.310 0.35±0.032

Positive

control Clear Smells of orange Thick Homogeneous 6 ± 0 6.825±0.457 0.69±0.015

F1 White

Smells of

weak nutmeg Thick Homogeneous 6 ± 0 6.55±0.122 0.39±0.025

F2 White

Smells of

strong nutmeg Thick Homogeneous 6 ± 0 6.47±0.363 0.47±0.036

F3 White Smells of

strong Thick Homogeneous 6 ± 0 6.36 ± 0.508 0.69± 0.026 5 nutmeg

4. Conclusion

It was concluded that all formulas met the criteria for the stability test of the gel preparation's physical

properties.The concentration of nutmeg oil 25% (F3) is an optimal formulation with the best stability

of physical properties, spreadability, and adhesion ability.

Acknowledgement

The authors would like to thank Faculty of Medicine Pattimura University, who have supported and funded this research and provided a place and facilitating to conduct research.

References

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Materials Science and Engineering 846

[2] Wijana S, Pratama E P, Rahmah N L and Arwani M 2020 Hand sanitizer formulation using orange peel essential oil. IOP Conference Series: Earth and Environmental Science 524 [3] Lee J, Jing J, Yi T P, Bose R J C, Mccarthy J R, Tharmalingam N and Madheswaran T 2020 Hand a review on formulation aspects, adverse effects, and regulations. International Journal of Environmental Research adn Public Health 17(9): 3326 [4] Atmaja T H W, Samingan S and Mudatsir M 2017 Pengaruh konsentrasi ekstrak etanol buah pala (Myristica fragrans) terhadap daya hambat Staphylococcus aureus. Jurnal EduBio

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[5] Arrizqiyani T, Sonjaya N and Asty A 2017 Optimalisasi potensi tanaman pala sebagai antibakteri Escherichia coli menggunakan metode ekstraksi. Prosiding Seminar Nasional Publikasi Hasil-Hasil Penelitian dan Pengabdian Masyarakat 37582 [6] Cui H, Zhang X, Zhou H U I, Zhao C, Xiao Z, Lin L and Li C 2015 Antibacterial properties of nutmeg oil in pork and its possible mechanism. Journal of Food Safety 35(3): 370-7 [7] Kusumaningrum G S, Suranto J and Setyaningsih R 2003 Aktivitas penghambatan minyak atsiri dan ekstrak kasar biji pala (Myristica fragrans Houtt dan Myristica fattua Houtt) terhadap pertumbuhan bakteri Xanthomonas campestris Oammel asal tanaman brokoli (Brassica oleraceava Italica).Jurnal Biofarmasi 1: 204 [8] Syamsuni H A, I E Ella R., Winny and Syarief 2007 Ilmu Resep (Jakarta: Buku Kedokteran EGC) [9] Octavia N 2016 Formulasi sediaan gel hand sanitizer minyak atsiri pala (Myristica fragrans Houtt.): Uji stabilitas fisik dan uji aktivitas antibakteri terhadap bakteri Staphylococcus aureus. Universitas Muhammadiyah Surakarta (Skripsi)

[10] Astuti D P, Husni P and Hartono K 2017 Formulasi dan uji stabilitas fisik sediaan gel antiseptic

tangan minyak atsiri bunga lavender (Lavandula angustifolia Miller). Farmaka 15: 17684 [11] Hidayawati E 2018 Optimasi Sediaan Gel Ekstrak Jahe Merah (Zingiber officinale Roscoe varrubrum) Menggunakan Gelling Agent Carbopol dan Humektan Propilen Glikol dengan Metode Simplex Lattic Design. Universitas Muhammadiyah Surakarta (Skripsi) [12] Noor S U and Nurdyastuti D 2009 Lauret-7-Sitrat sebagai detergensia dan peninngkat busa pada sabun cair wajah Glysinesoja (Sieb.) Zucc. Jurnal ilmu Kefarmasiun Indonesia 7: 3947 [13] Surini S, Amirtha N I and Lestari D C 2018 Formulation and effectiveness of a hand sanitizer gel produced using Salam bark extract. International Journal of Applied Pharmaceutics 10: 21620
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