INTRODUCTION:

Dandruff (also called syntax and fatigue), with the scientific name Pityriasis capitis, is excessive exfoliation of the dead skin on the scalp. Dandruff sufferers usually complain because the scalp becomes more oily, hair becomes brittle and falls out. People who suffer from severe dandruff will show an increase in seborrheic dermatitis with signs of inflammation or inflammation of the skin in the seboreal region (scalp, eyebrows, lips, ears, and groin), which is caused due to the activity of excessive sweat glands^1,2..

Treatment and prevention of dandruff are important because it is not only the itchiness that makes us want to continue to scratch it but the white flakes that pollute the clothes really disturb our appearance. The most commonly used hair care products for men, women, children, and adults are shampoo. One important requirement for anti-dandruff shampoos is that they must contain active germicidal, fungicidal or antiseptic substances that can kill microbial growth and prevent infection after use³. Disorders of the scalp are caused by several factors, namely excessive consumption of fatty foods, hormonal imbalances, and fungal infections. The microorganism that is suspected as the cause of dandruff is Pityrosporumovale or Malassezia furfur, which is a microscopic fungus found on everyone's scalp and is lipophilic. The shape is oval-round or like a bottle, measuring 3-8µm⁴. The wound caused by this fungus is reddish and covered by an oily scalp and feels very itchy. M.furfur will grow very easily on media containing olive oil⁵.

Traditionally cabbage is often used by Indonesian society to overcome the problem of fungus on the scalp, hands, and feet; arthritis; difficult bowel movements; and increase milk production. Its chemical content, fresh cabbage contains water, protein, fat, carbohydrates, fiber, phosphorus, iron, sodium, potassium, vitamins (A, C, E, thiamine, riboflavin, nicotinamide), calcium, and beta carotene. The active ingredient, sulforaphane, and histidine can inhibit tumor growth, prevent colon cancer, and rectum, detoxify harmful chemical compounds, such as excessive cobalt, nickel, and copper in the body, and increase the body's resistance to fight cancer. The content of amino acids in sulfur, also efficacious in reducing high cholesterol levels, calming nerves, and arouse enthusiasm⁶.

Based on the results of previous studies, the antifungal activity test of cabbage leaf ethanol extract shows that ethanol extract has antifungal activity against M.furfur. In this research, an anti-dandruff shampoo formulation was made with the active ingredient of cabbage ethanol extract (Brassica oleracea, var. capitata L.) and its activity test on the M.furfur fungus, so that a dosage form that was practical, stable, effective, and safe in its use.

Today the society is more inclined to return to the era of herbal medicine. Many herbal shampoos are sold commercially, the results of this study add to the presence of shampoos that use herbs

MATERIALS AND METHODS:

Materials:

Simplicia:

The test material used was cabbage obtained from the Vegetable Crops Research Institute (BALITSA) Lembang, West Java and determined in the Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas.

Fungus:

The fungus used was Malassezia furfur obtained from the Mycology Laboratory of Pt. Bio Farma, Bandung, West Java.

Mushroom Growth Media:

The seed medium used was 65 grams of Sabouraud Dextrose Agar (SDA) plus 10 cc of olive oil suspended in 1 liter of distilled water, boiled until completely dissolved, then sterilized in an autoclave at 121⁰C for 15 minute.

Equipment and chemicals:

Analytical balance (Mettler Toledo), Water bath (Memmert), Rotary evaporator (IKA® RV 10 Basic), Spectrophotometer UV-Betrachte (Lamag). Chemicals used such as ammonia (Merck), Dragendroff reagent, Lieberman-Burchard reagent, Mayer reagent, vanillin sulfate, and toluene unless stated otherwise all chemicals were analytical grades.

Methods:

Extraction:

The extraction method used in this study was maceration. The selection of this method was done to prevent the occurrence of damage to the thermolabile chemical compounds contained in the Brassica oleracea. This maceration was done by soaking the sample in the macerator with ethanol 96% for 3x24 hours at room temperature with stirring occasionally^7-9.

Phytochemical investigation:

Phytochemical screening was applied to the viscous extract of B. oleracea based on Farnsworth's method¹⁰ for secondary metabolites.

Determination of Minimum Growth Inhibitory Concentration (MGIC):

Making Test Microbial Suspension: The test fungus was inoculated on Sabouraud Dextrose Agar (SDA) media and incubated for 3-5 days at 32°C. One ose of fungal colonies was suspended in 5mL of sterile physiological NaCl. The concentration of the fungus in the suspension was measured based on turbidity using a spectrophotometer. For Malassezia furfur mushrooms the absorbance value was 0.3 - 0.5.

Procedure for Determination of Minimum Growth Inhibition Concentration (MGIC:

Provide an initial solution consisting of cabbage ethanol extract (100%), Provides 8 sterile test tubes for diluting cabbage extract and 2 tubes for media control and growth control. Conducted the dilution of cabbage ethanol extract for tube 1 by 1mL cabbage extract the initial solution was added with 19mL of media to be solid (SDA), mixed until homogeneous, and poured into sterile Petri dishes allowed to condense (concentration of 5%).Conducted dilutions for tubes 2 to tube 8 using the same method and calculation, to obtain the concentration of cabbage ethanol extract in sterile Petri dishes 10%, 15%, 20%, 25%, 30%, 35%, and 40%. Each dilution concentration was made 3 times (triple). Apply test microbes to solidified agar in each petri dish (8 Petri dishes) and incubated for M.furfur at 32⁰C for 3-5 days. Setting up media controls and growing controls. Media control was agar media with cabbage ethanol extract without being planted with M.furfur. The growth control contained agar medium that was planted with M.furfur without cabbage ethanol extract.

Preparation of Anti-dandruff Shampoo:

The formula is designed as in Table 1.

Observation of Physical Stability of Anti Shampoo Shampoo:

The observation was conducted for Organoleptic Observations, Foam Height Measurement, Measurement of pH, Viscosity measurement, and Surface tension Measurement.

Testing antifungal activity:

A total of 20μL of suspension of each test mushroom was put into a sterile Petri dish then poured SDA which was still liquid at a temperature of 45-50°C as much as 20mL, then the Petri dish was shaken so that the fungus with SDA was mixed homogeneously, and allowed to solidify. After compacting the holes were made using a perforator (3 holes, each for shampoo preparations with cabbage ethanol extract concentrations of 15%, 30%, 45%). Furthermore, using a micropipette into each hole 50ml of shampoo was added. Then incubated for 3-5 days at 32 °C. Shampoo preparation activity could be seen from the diameter of the inhibition which was indicated by the presence of clear areas around the hole and measured using a clipper. Conducting activity tests from blanks, namely shampoo preparations that did not contain cabbage ethanol extract were tested for their antifungal activity in the same manner and time.

Safety Test for Shampoo:

Patch Test:

Anti-dandruff shampoo safety test with various concentrations of cabbage ethanol extract was done by applying shampoo preparations to the rabbit's back skin, fur the location was scraped an area of approximately 25 cm. Shampoo preparation to be tested was made into a 2% solution in water, then only to be applied to the sticky location. The attachment location was left open for 24 hours, and the skin reaction that occurs was observed. Observations were made every day for 3 consecutive days.

Eye irritation test:

As an experimental animal rabbit eyes were used, and as a test preparation was a solution of 10% shampoo in water. As much as 0.1ml of the diluted preparation was dropped into one of the rabbit's eyelids and the other was used as a control.

Observations were made with the help of a flashlight for 1-7 days after dropping, including reactions that occur in the cornea, iris, and eye conjunctiva. Reactions that occurred on the cornea, visible with the turbidity of the iris and changes in pupil size or even bleeding in the iris. While the reaction that occurred in the conjunctiva was the emergence of redness, swelling, and closure of the eyelids.

RESULTS:

Table 1: Phytochemical screening results of B. oleracea extract

S No	Chemical constituents	Ethanol extract
1	Alkaloids	-
2	Polyphenols	-
3	Tannins	-
4	Flavonoids	+
5	Quinone	-
6	Saponins	+
7	Monoterpenoid and sesquiterpenoid	+
8	Steroids	-
9	Triterpenoid	-

Notes: (+): detected, (-): not detected

Table 2: Anti-dandruff formula

Compounds (%)	Anti-dandruff Shampoo Formula with Various Concentrations of Ethanol Cabbage Extract (%)
Compounds (%)	F0	F1	F2	F3
Texapon liquid	10	10	10	10
Cocamide DEA	4	4	4	4
CAB-30	3	3	3	3
Polydimethylsiloxane	1	1	1	1
EDTA₂Na	0,1	0,1	0,1	0,1
Cabbage extract	0	15	30	45
Methyl paraben	0,15	0,15	0,15	0,15
Propyl paraben	0,05	0,05	0,05	0,05
NaCl	3,3	3,3	3,3	3,3
*Mimusops elengi* oil Bahan (%) Formula Sampo Antiketombe dengan Berbagai Konsentrasi Ekstrak Etanol Kubis (%) F0 F1 F2 F3 Texapon cair 10 10 10 10 Cocamide DEA 4 4 4 4 CAB-30 3 3 3 3 Polydimethylsiloxane 1 1 1 1 EDTA2Na 0,1 0,1 0,1 0,1 Ekstrak kubis 0 15 30 45 Methyl paraben 0,15 0,15 0,15 0,15 Propyl paraben 0,05 0,05 0,05 0,05 NaCl 3,3 3,3 3,3 3,3 Minyak tanjung qs qs qs qs Air suling sampai 100mL 100mL 100mL 100mL Bahan (%) Formula Sampo Antiketombe dengan Berbagai Konsentrasi Ekstrak Etanol Kubis (%) F0 F1 F2 F3 Texapon cair 10 10 10 10 Cocamide DEA 4 4 4 4 CAB-30 3 3 3 3 Polydimethylsiloxane 1 1 1 1 EDTA2Na 0,1 0,1 0,1 0,1 Ekstrak kubis 0 15 30 45 Methyl paraben 0,15 0,15 0,15 0,15 Propyl paraben 0,05 0,05 0,05 0,05 NaCl 3,3 3,3 3,3 3,3 Minyak tanjung qs qs qs qs Air suling sampai 100mL 100mL 100mL 100mL	qs	qs	qs	qs
Aqua destillata	100mL	100mL	100mL	100mL

Note: Fo (Shampoo formula without cabbage ethanol extract), F1 (Shampoo formula with cabbage ethanol extract 15% concentration), F2 (Shampoo formula with cabbage ethanol extract 30% concentration), F3 (Shampoo formula with cabbage ethanol extract 45% concentration)

Figure 1: Comparison of Shampoo Activity with Various

Concentrations of Cabbage Ethanol Extract against M.furfur

DISCUSSION:

Plant Leaves Process:

The obtained cabbage was chopped and washed with running water to clean, this process was called wet sorting which aimed to be free of dirt such as dust and soil. Sorting was also done to separate leaves that were moldy, rotten, or which were not suitable. Then the cabbage was dried without sunlight for about one week. Dried cabbage leaves that had been dried were sorted for preventing contamination, separating unused parts in examiners, as well as to get the best quality sample chopped up¹¹. This is done to increase the surface area of the sample so that the penetration of the solvent was spread evenly to all parts so that the withdrawal of secondary metabolites was more optimal in the extraction process.

Extraction:

The extraction method used in this study was maceration using ethanol 96%. The selection of this method was done to prevent the occurrence of damage to the thermolabile chemical compounds contained in the leaf^{11, 12}. The choice of the ethanol as solvent was because ethanol was a safe and non-toxic universal solvent, which could dissolve and attract almost all secondary metabolites in leaves with broad polarity ranging from nonpolar to polar. According to Harborne ¹³, ethanol could also prevent the occurrence of hydrolysis and oxidation in the extraction process by depositing proteins and inhibiting the work of enzymes contained in leaf leaves. The viscous extract yield after rotary evaporated was 5.98 %.

Phytochemical screening:

The method used was based on the Farnsworth method^{10, 14}. The results of phytochemical screening were carried out on the B.oleracea ethanol extract can be seen in Table 1.Samec et.al emphasized that phytochemically cabbage contained health-promoting phytochemicals such as glucosinolates, polyphenols, and vitamins¹⁵. It also reported containing Vitamin C, β-carotene, Lutein, α-tocopherol, and phenolic^16,17. The cabbage was abundant of polyphenolic compounds and contain 15-20 different glucosinolates like compounds¹⁸. Rokayya et.al suggest that these varieties of cabbage heads could contribute as sources of important antioxidant (flavonoid quercetin) and anti-inflammatory (phenolic compounds) related to the prevention of chronic diseases associated to oxidative stress, such as in cancer and coronary artery disease^19.Phytochemical screening results that are slightly different from each researcher are likely due to the origin of the sample used.

Minimum Growth Inhibitory Concentration (MGIC):

The purpose of MGIC was to determine the smallest concentration of cabbage ethanol extract that could still inhibit the growth of M. furfur. M. furfur was used by researchers to determine antifungal activity showing the MIC of a compound^20,23. It was found the MGIC of cabbage ethanol extract against M. furfur was between 10-15% v/v. Because the concentration of cabbage ethanol extract 10% v / v did not provide activity while at 15% v/v did not show the growth of fungi, this meant that the MGIC of ethanol cabbage extract was between concentrations of 10-15% v/v. While cabbage ethanol extract with concentrations between 5-10% v/v showed the growth of fungus M. furfur meaning that cabbage ethanol extract with concentrations of 5-10% v/v did not have antifungal power.

Formulation of Anti-dandruff Shampoo:

Table 2 showed the formula of anti-dandruff shampoo with a variation of cabbage ethanol extract. Formulations were made based on ingredients in Shampoo Formulations. The must-use ingredients was Distilled water (60-80%), Surfactants (15-30%), Foam Boosters/ Stabilizers (1-4%), Thickeners (0.1-5%), Preservatives (0.1-1.0%), pH Stabilizer (As needed)^24,25. As shown in Table 2 could be known the results of the formulation of anti-dandruff shampoo with various concentrations of cabbage ethanol extract. Shampoo was made in the form of thick solution and the addition of cabbage ethanol extract causes a yellowish-brown shampoo because the cabbage contains ethanol extract from flavonoids. The addition of cabbage ethanol extract and Oleum Mimusops elengi to the formula caused the distinctive smell of cabbage and cape flower perfume to the shampoo preparation. The addition of various concentrations of cabbage ethanol extract to the anti-dandruff shampoo formula was determined based on the MGIC value.

Observation of Physical Stability of Anti-dandruff Shampoo:

Organoleptic Observations:

Organoleptic observation aimed to determine the stability of shape, color, and odor of shampoo preparations for 8 weeks of storage. From all the organoleptic observation tables for 8 weeks of storage, it was found that the F1 and F2 shampoo preparations did not change in shape, color, and odor. Whereas in the F3 shampoo the color and odor changes occurred at the 8th week of storage, this was due to the addition of a lot of cabbage ethanol extract which would make the preservative in the shampoo compound compound unable to prevent the growth of microorganisms so that the decomposition components of the shampoo preparation.

Foam Height Measurement:

This test was carried out to determine the foam height of the shampoo preparation. Although the height of the foam was not the most important thing in making shampoo, in general, the public's view of a good shampoo was that a shampoo that can produce a lot of foam will have a good clean power²⁶.From data obtained, the results of the analysis of variance analysis of the Complete Random Block Design (RCBD) subsampling²⁷, it found that the F_count value was greater than F_table (Ho was rejected), meaning that there was a significant difference in the effect of different shampoo formulas on foam height. While from the results of the further analysis with the Newman Keuls test²⁸, it could be concluded that the greater the concentration of cabbage ethanol extract added to the shampoo formula would increase the high foam value in the shampoo preparation. This was because cabbage ethanol extract contains saponin compounds. Foam height requirements generally range from 1.3 - 22 cm. All of the shampoo preparations tested had an average foam height of between 2.4889 - 3.1963. This meant that the shampoo preparations have met the high foam requirements in general. It was found that high stability of the foams of F1, F2, and F3 shampoos during the storage time was quite stable, although experiencing high and high foam but not significant.

pH Measurement:

This test was very important because the pH was not following the requirements could irritate the scalp and eyes, as well as excessive hair loss. Ideally, shampoo should have a pH from 5-7. The higher the pH, the harsher it was on your hair. Human hair, scalp oil, and sebum (oil produced by our skin) had a pH balance between 4.5 and 5.5. It was slightly acidic, which prevents fungus and bacteria in the hair and scalp²⁹. The RCBD subsampling and Newman Keuls test were applied to analyze the effect of pH. From the data obtained and the results of the calculation of the analysis of the variance of the RCBD, it found that the F_count value was greater than the F_table (Ho was rejected), meaning that there was a significant difference in the effect of different shampoo formulas on pH changes. While from the results of the further analysis with the Newman Keuls test, it could be concluded that the greater the concentration of cabbage ethanol extract added to the shampoo formula would reduce the pH value in the shampoo preparation. This was because cabbage ethanol extract contains high levels of vitamin C. The pH requirements in the library were in the range of 3.9 - 9.5 and the best pH of the shampoo was in the range of 6-7. All shampoo preparations tested had an average pH of between 5.3444 - 7.3667. This meant that the shampoo preparations had met the pH requirements of the shampoo in the library.From the graphic image of Fig. 1, it was shown that the pH stability of the shampoo preparations during storage time was quite stable, although it had fluctuated pH but was not significant.

Viscosity measurement:

At low shear rates around 1 1/s the shampoo for men and the shampoo for children (infants) have about the same viscosity of about 12 Pas. The shampoo for women has a considerably lower viscosity of about 7.5 Pas³⁰. This test aimed to determine the extent of the thickness of the shampoo so that during use could be poured properly. From viscosity data obtained using the analysis of the variance analysis of the RCBD subsampling, it found that the F_count value was greater than the F_table (Ho was rejected), meaning that there was a significant difference in the effect of different shampoo formulas on changes in viscosity. While from the results of the further analysis with the Newman Keuls test, it concluded that the greater the concentration of cabbage ethanol extract added to the shampoo formula would increase the viscosity in the shampoo preparation. This was because cabbage ethanol extract contains protein compounds. All of the shampoo preparations tested had an average viscosity between 25.8889 - 81.1852 poise. Although there was no limit to the viscosity range in the shampoo, the thickness of the shampoo was important, so that the shampoo can be poured properly. It was found, however, that there was a significant storage effect to the viscosity stability of the shampoo preparation. This was because the preservative in the shampoo compound was unable to prevent the growth of microorganisms so that the enzymes decomposed in the active ingredients of cabbage ethanol extract which causes the shampoo preparation to change in thickness. This was one of the reasons a shampoo had added a preservative. It is even recommended for commercial shampoos to be stored for a maximum of 12 months to prevent changes in physical properties^{31, 32}.

Surface tension measurement:

The lesser the surface tension, the stronger the cleaning ability of the shampoo. A shampoo was considered to be of good quality if it decreased the surface tension of pure water from 72.28 dyn/cm to about 40 dyn/cm ^{31, 33}. This surface tension test aimed to determine the effectiveness of surfactants to attract, dissolve, and remove impurities from the hair. By using the RCBD subsampling and Newman Keuls test, it was found that the stability of the shampoo surface tension during storage time was quite stable, although it experienced ups and downs in surface tension but was not significant

Test Results of Anti-dandruff Shampoo Formulation Activities M. furfur: The purpose of testing shampoo activity was to find out whether the shampoo could inhibit growth or kill the fungus M.furfur. Fig. 1 revealed formula shampoo activity with various concentrations of cabbage ethanol extract against M.furfur. From the Table 7 and the results of the analysis of variance analysis of the RCBD subsampling, it was found that the F_countvalue was greater than F_table (Ho was rejected), meaning that there was a significant difference in the effect of different shampoo formulas on the potential of the shampoo preparation. While from the results of the further analysis with the Newman Keuls test, it might conclude that the greater the concentration of cabbage ethanol extract added to the shampoo formula would increase antifungal activity in the shampoo preparation. This was because cabbage ethanol extract contains sulfur compounds so that the shampoo formula with a large concentration of cabbage ethanol extract would obtain a large diameter inhibiting antifungal activity. Requirements for inhibition area diameter in the library, ie inhibition areas of more than 3 mm are fungal sensitive areas, whereas if the diameter of inhibitory regions is less than 2 mm, they were called fungal resistant or insensitive areas³⁴. All of the shampoo preparations tested had an average inhibitory diameter between 12.8507 - 18.33007. This meant that the shampoo preparations have met the diameter requirements for inhibition of antifungal activity in the library. From the graphic images of Fig. 1it was shown that there was a significant storage effect on the antifungal activity of the shampoo preparation.

Safety Test Results for Anti-Dandruff Shampoo:

This test was carried out on rabbit skin and eye irritation test. It was found that the formula did not irritate the rabbit's skin and eyes, this was due to the addition of detergent concentration in the preparation only 10%. According to the literature, the use of detergents over 20% would irritate the eyes and scalp³⁵. The shampoo preparations tested have an average pH between 5.3444 - 7.3667, this means that the shampoo preparations were safe to use and would not cause pain in the eyes because they had acidity levels that met the requirements in the literature range. According to the literature, the pH of the shampoo ranges between 3.9 - 9.5 and the best pH value of the shampoo was in the range of 6-7.

CONCLUSION:

From the results of the study, cabbage ethanol extract contained tannins, flavonoids, monoterpenoids, sesquiterpenoids, triterpenoids, and saponins. Testing the stability of the shampoo for 8 weeks of storage showed that the shampoo with a concentration of 15% and 30% cabbage ethanol extract remained stable. Whereas in shampoo preparations with a concentration of cabbage ethanol extract 45% there was an organoleptic change and a decrease in viscosity. The greater the concentration of cabbage ethanol extract added to the shampoo formula decreased the surface tension value of the shampoo preparation and increased the foam height, pH, viscosity and antifungal activity of the shampoo preparation.

From the results of the study showed that the preparation of anti-dandruff shampoos with active ingredients of cabbage ethanol extract had antifungal activity against M.furfur. The greater the concentration of cabbage extract in the shampoo, the greater the inhibitory power produced against M.furfur. Through the safety test, it could be concluded that the anti-dandruff shampoo formula did not irritate the rabbit's skin and eyes, so it was safe for use.

CONFLICTS OF INTEREST:

There is no conflict of interests between the authors.

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Received on 30.09.2019 Modified on 01.12.2019

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DOI: 10.5958/0974-360X.2020.00655.1