FORMULATION AND CHARACTERIZATION OF SERUM COLLAGEN OF SEA CUCUMBER EXTRACT STICHOPUS HORRENS AS AN ANTIOXIDANT

Objective : The study was aimed to extract, formulate and characterize collagen extract of Stichopus horrens into serum preparations and decide antioxidant activity in powders and serum preparations. Methods: The sea cucumber meat was extracted collagen in three stages, namely the pretreatment stage using 0.3 M NaOH solution 1:10 (w/v) for 48 h, the hydrolysis step in the 0.3 M 1:10 acetic acid solution (w/v) for 48 h, and the extraction stage with distilled water 1: 2 (w/v) for 2 h at 45 °C. The collagen extract was freeze-dried to obtain collagen powder. Collagen powder was characterized by HPLC and its antioxidant activity was determined using the DPPH method. Collagen powder formulated with extract variation of 0, 0.5 and 1%. Evaluation of serum included organoleptic, homogeneity, stability of pH and viscosity as well as antioxidant activity. Results: The results showed that collagen powder had a % yield of 0.24%, which consisted of the amino acids glycine, proline, alanine, and glutamic acid as the dominant amino acids. The % Free radical inhibition of collagen powder at concentration of 5000 ppm was 63.23%. IC50 values were obtained at 4045.37 ppm. The stability test resulted in stable serum preparations without significant changes at 4 °C and 27 °C±2 °C storage temperatures. Conclusion: The measurement of DPPH Radical reduction activity in the highest serum preparation was 1% extract with a value of 2.4%.


INTRODUCTION
Currently, the use of medicinal ingredients is not only derived from plants but marine life is also being developed because it has great potential as a source of medicinal ingredients. Sea cucumbers are one of the most potential marine resources. Approximately 53 species of sea cucumbers are found in Indonesia, there are 22 species that can be consumed, and 8 of them have a high market value. The 8 species are sand sea cucumbers (Holothuria scraba), milk sea cucumbers or koro (Holothuria nobilis and Holothuria fuscogilva), rock sea cucumbers (Actinopyga echinites), bilbao sea cucumbers (Actinopyga lecanora), lotong sea cucumbers (Actinopyga miliaris), cat's eye sea cucumber (Bohadschia argus) and pineapple sea cucumber (Theleonata ananas) [1].
Sea cucumbers have complete nutritional content due to often being referred as seabed ginseng and are used as supplements. The results of laboratory analysis showed that the dried sea cucumber extract contained up to 86.8% protein, 80% collagen, minerals, mucopolysaccharides, glucosaminoglycans (GAG), natural antiseptics, chondroitin, omega 3, omega 6, and omega 9, as well as various amino acids [2].
The active ingredients in various types of sea cucumbers have been reported in various publications, including the antibacterial activity of the sea cucumber Cucumaria frondosa was detected in extracts mainly coelomocyte and eggs from Cucumaria frondosa and potential for discovery novel antibiotics [3], isolate from sea cucumber Psolus patogenicus are glycoside and patagonicoside A have the antifungal activity against fungus Cladosporium cucumerinum [4], isolation of arginine kinase enzyme in sea cucumber Stichopus japonicus was success cloned the gene for sea cucumber into an E. coli expression vector purified functional enzyme [5], the activity of serum amyloid A in sea cucumber Holothuria glaberrina can activation of the immune system [6], glycoside structure in sea cucumber Stichopus mollisto classify S. mollis in the new genus Australostichopus levin [7], and isolation of fucan sulfate in sea cucumber Stichopus japonicus as an inhibitor of osteoclastogenesis [8].
Previous research regarding hydrolyzed collagen derived from golden sea cucumbers has an antioxidant effect (IC50 of 5.25±0.15 mg/ml) [9]. Other studies confirm that sea cucumbers are a source of collagen and need further research [10]. Collagen plays an important role in the food, cosmetic and pharmaceutical industries [11]. Collagen which is a connective tissue in bones and skin, can be used for skin beauty and can increase the regeneration of dead cells due to wounds so that it can accelerate healing. Therefore, sea cucumber extract can be used as a cosmetic ingredient and ointment to heal wounds [2].
Collagen is used in the cosmetic field as an active ingredient in skin care products with the function to increase skin moisture, prevent wrinkles, keep the skin from the bad effects of radiation and maintain elasticity. Collagen is a fibrillar protein and is suitable for connective tissues in the human body, both skin, joints, and bones. Due to its abundance in the body, its strength and relationship are directly proportional to skin aging. Collagen fibers are damaged over time, losing thickness and strength, which are closely related to the phenomenon of skin aging and aging [12]. The addition of collagen in cosmetic formulations is intended to replace damaged collagen due to environmental influences and age factors. The specialty of using collagen is related to the physicochemical characteristics of collagen, including non-toxicity, low antigenicity, biocompatible and biodegradable, making collagen the main source in medical applications [13]. Cosmetics have developed rapidly into various dosage forms that function to increase user comfort. One form of cosmetic dosage has been widely developed is topical serum. Serum is a gel with a lower viscosity. Serum has the advantage that it can provide a more comfortable effect and is easier to spread on the surface of the skin because its viscosity is not too high [14]. Based on this description, no research has been found regarding the serum from this sea cucumber using Stichopus horrens collagen as an antioxidant serum. This study aims to extract, formulate and

Collagen extraction
Collagen extraction was carried out in three stages, namely the pretreatment stage using sodium hydroxide solution, the hydrolysis stage in acetic acid solution, and the extraction stage with distilled water. Sea cucumber meat of Stichopus horrens as much as 1000 g was soaked in 0.3M sodium hydroxide with a ratio of meat to solution 1:10 (w/v) for 48 h to remove non-collagen protein then filtered and the filtrate is neutralized with water to a neutral pH. The filtrate soaked in 0.3 M acetic acid with a ratio of meat to solution 1:10 (w/v) for 48 h, followed by the neutralization process again until the pH was neutral. Last step was extraction using distilled water for 2 h at 45 °C with a ratio of 1:2 (w/v) sample to solvent. The results of extraction are obtained in the form of liquid collagen which is then freeze-dried [15].

Characterization using HPLC and FTIR
The amino acid composition was determined by HPLC (AOAC 1995) and FTIR analysis. The HPLC instrument was rinsed with eluent to be used for 2-3 h. The syringe that will be used is rinsed with distilled water. Amino acid analysis using HPLC consisted of 4 stages, namely:

a. Making protein hydrolyzate
The sample was weighed as much as 0.1 grams and crushed, the crushed sample was added with 5-10 ml of 6 N HCl. The solution was heated in an oven at 100 °C for 24 h. This is done to remove gas or air present in the sample so as not to disturb the resulting chromatogram. After heating, the protein hydrolyzate was filtered using a millipore measuring 45 microns.

b. Drying
Drying The filter results were taken as much as 10 µl and added 30 µ� of drying solution. Dryer solution made from a mixture of methanol, sodium acetate, and trimethylamine in a ratio of 2:2:1.
After the sample is dried with a vacuum pump to speed up the process and prevent oxidation.

c. Derivatization
The derivatization solution was prepared from a mixture of methanol, picoiodothiocyanate, and trimethylamine solutions in a ratio of 3:3:4. The derivatization process is carried out so that the detector is easy to detect the compounds present in the sample.
Further dilution was carried out by adding 10 ml of 60% acetonitrile and 1 M sodium acetate and then left for 20 min, filtered again using a millipore measuring 45 microns. 30 µ� of derivatization solution was added to the drying product.

d. Injection
The filter results were taken as much as 20 µ� to be injected into the HPLC FTIR analysis to determine the typical functional groups of collagen. 100 mg KBr and 2 mg of the test sample were ground until smooth and well mixed in a mortar and then molded into a pellet mold. Measurements were made with the FTIR tool at wavenumbers between 4000-500 cm-1.

Antioxidant activity test
Antioxidant activity is decided using the DPPH method. 10 mg of sample was dissolved in methanol p. a then made in series with concentrations 5000, 4000, 3000, 2000, and 1000 ppm. Each concentration series was inserted into the microplate as much as 160 µl, added to the DPPH solution (0.3 mg/ml) each 40 µl. The DPPH solution was prepared by dissolving 3 mg of DPPH into 10 ml of methanol p. a. Sample blanks were made by inserting 160 µl of sample into the well and adding 40 µl of methanol p. a. Negative control was made by adding 160 µl of methanol p. a with 40 µl of DPPH and 200 µl of methanol p. a as a blank. Ascorbic acid was used as a positive control with a concentration series of 4;2;1; and 0.5 ppm. The microplate was incubated at room temperature for 30 min and the absorbance was read using a UV-Vis spectrophotometer at a wavelength 517 nm.

Serum preparation formula and test the antioxidant activity
The serum was formulated by adding Stichopus horrens collagen extract with a concentration of 0.5% and 1% into the serum formula with a Natrosol concentration of 0.75%. Testing of antioxidant activity was carried out using the DPPH method. The DPPH solution was made by weighing 0.002 g of DPPH then dissolved in 100 ml of methanol to obtain 0.002% DPPH. Preparation of a sample solution by weighing 50 mg of sample and dissolving in 10 ml of methanol so that the concentration becomes 5000 ppm and diluted to 4000 ppm. The antioxidant test using the DPPH method, added 5 ml of DPPH stock solution into the sample tube and incubated for 30 min in a dark room. After that, the absorbance was measured using a UV-Vis spectrophotometer at a wavelength 516 nm. The process of making serum preparations is carried out based on the formula design as shown in table 1.

Physical evaluation of Stichopus horrens collagen extract serum preparations
Physical evaluation and stability tests of serum preparations include organoleptic, homogeneity, pH, viscosity. Then the antioxidant activity test was carried out on serum preparations of Stichopus horrens collagen extract using the DPPH method (positive control of vitamin C serum).

Stability test
The stability of the preparation was evaluated at 4 °C±2 °C and 27 °C±2 °C for one month by observing organoleptic, homogeneity, pH, and viscosity measurements.

1) Organoleptic test
Organoleptic tests can be observed by determining changes in color, odor and texture that occur during 1 mo of storage [16].

2) Homogeneity
The serum preparation of Stichopus horrens collagen extract was placed between two slides and then observed for the presence of coarse particles or inhomogeneities under light [16].

3) pH measurement
pH measurement using a digital pH meter.

4) Viscosity
Viscosity measurements were carried out using a rotary viscometer with a rotational viscometer type (NDJ 5S). The preparation is put into a beaker and the appropriate spindle is lowered to the limit of the spindle immersed in the preparation, then the motor and spindle are turned on. The rotational speed is set at 60 rpm. The viscosity number shown on the display screen shows the viscosity value.

RESULTS
The sea cucumber samples used in this study were identified at the SITH ITB Zoological Museum. Based on the results of No. 1272/l1. C02.2/lL/2019 the analysis of the types of sea cucumbers in this sample is Stichopus horrens, family Stichopodidae.
The collagen extract obtained was in the form of a cloudy white collagen solution and a freeze dryer process was carried out so that the solid form was brownish in color with a cotton-like texture with a yield of 0,24 %. The extraction process is carried out by soaking with NaOH solution, causing sea cucumber meat to expand (swelling) so that it can dissolve non-collagen proteins in sea cucumber meat [17]. The addition of acetic acid will break the cross-linked strands in the collagen so that it dissolves the non-crosslinked collagen and the collagen can dissolve completely [18]. Qualitative and quantitative analysis of amino acids using HPLC instruments. The results of the amino acid analysis of Stichopus horrens sea cucumber extract obtained 15 amino acid peaks consisting of 9 types of essential amino acids and 6 types of nonessential amino acids. The essential amino acids found in Stichopus horrens sea cucumbers are isoleucine, leucine, lysine, methionine, phenylalanine, histidine, threonine, valine, and arginine. The nonessential amino acids analyzed were aspartate, serine, glutamate, glycine, alanine, and tyrosine. The basic molecule of collagen is formed from three polypeptide chains that are twisted together to form a triple helix structure with a typical amino acid arrangement, namely Gly-XY, at position X is proline and Y is hydroxyproline. Interpretation peak can be seen in table 2.
Based on table 2 shows that among the 15 amino acids obtained, three types of essential amino acids are dominated. There are arginine 3.76%, leucine 1.27% and threonine 1.51%, besides non-essential amino acids are dominated by glycine 8.29%, proline 3.04%, alanine 3.09% and glutamic acid 4.09%. This study is in accordance with the results of previous research [19] who reported that glycine is an amino acid that is dominant in collagen and all families. Collagen is characterized by the presence of repeats of the amino acid sequence Gly-X-Y. Amino acids in the form of proline and hydroxyproline are important for the structural integrity of collagen because they plays an important role in the bond formation of intramolecular hydrogen [20].    Based on the calculations, the IC50 value is 4045.37 ppm which indicates that the antioxidant activity is very weak. The results of this antioxidant test are better than the results of the IC50 tyrosinase enzyme study that showed that golden sea cucumber collagen has an IC50 value of 5610 ppm [22]. The lower value IC50, showed the higher antioxidant activity. The difference in IC50 value is influenced by the amino acid content. Peptides containing total high hydrophobic amino acids (THAA) have high antioxidant activity to suppress oxidative reactions [23].
The results of the serum preparation of Stichopus horrens collagen extract, formula A produces a transparent color because, without extract, formula B is light brown with 0.5% collagen extract and formula C produces a dark brown serum with a concentration of 1% collagen extract ( fig. 4).
Stability testing was carried out by storing the serum preparation of Stichopus horrens collagen extract at a low temperature of 4 °C and 27 °C at room temperature for 1 mo. Observation of the stability test included organoleptic examination, homogeneity, pH, and viscosity.
The results of organoleptic observations of each serum preparation for one month at each temperature stability test did not show any significant changes in color and odor, only slightly thickened in the last week because it was influenced by storage temperature (table 5).  Homogeneity test is an important factor for uniformity of content and patient comfort in using the preparation. Based on the results of these observations do not appear coarse grains (table 6-9).   Data represented as mean±SD (n=2) *SD= standar deviation In this research, we have succeeded in formulating sea cucumbers into serum preparations that meet SNI 16-4399-1996 based on physical properties test, only for the antioxidant activity is very weak so it needs an increase in the concentration of the active substance and it is necessary to hydrolyze collagen before formulation so that it has the opportunity to have good antioxidant activity.

DISCUSSION
The sea cucumber samples were identified with the aim of proving the correctness of the materials used in the study. Fresh sea cucumber meat Stichopus horrens was cut into small pieces to facilitate the extraction process. The smaller the sample size, the solvent will be easily penetrated into the cells due to the target compound being extracted and dissolved in the solvent. The deproteinization process uses 0.3 N NaOH, which is to remove noncollagen proteins, fats, minerals, and pigments present in sea cucumber meat. During immersion in NaOH allows water to enter and causes non-collagenous proteins trapped in the collagen matrix to be more easily released. After pretreatment, the samples were washed with distilled water until the pH was close to neutral. The function of neutralization will reduce the remaining acid and base so that the resulting pH is close to neutral. The hydrolysis function uses 0.3 N acetic acid to change the structure of the collagen fibers into simpler fragments so as to facilitate the extraction process at a later stage. Soaking with distilled water after being immersed in a solution of NaOH or acetic acid is shown so that collagen has a pH close to neutral (pH=7 In the extract of Stichopus horrens sea cucumber collagen based on the physical properties of collagen, the color of Stichopus horrens sea cucumber collagen is colorless after freeze dry, the color changes to brownish-white and has a cotton-like texture and has a pH of 6 according to the quality requirements of SNI 8076-2014 collagen, which has a pH of 6-7.5. The results of the pH examination of serum preparations showed that the pH value was in the range of 5.5-7.4. Formula 2 and 3 has a lower pH than Formula 1. This is due to the addition of the concentration of the active substance, namely collagen extract. Collagen extract is acidic so it can affect the pH of the preparation serum. Based on the results of pH measurements, it can be observed that the longer storage, the pH decreases. The decrease in pH can be caused by due to the presence of ionic contamination from the materials used in the formulations, either ion positive or negative ions that can affect acidity or basicity preparation. The serum pH in this experiment all formulas met the quality standard of SNI 16-4399-1996 for serum or sunscreen preparations was 4,5-8. The decrease or increase in the value of the viscosity can be caused by the influence of temperature, which causes a change in the polymer structure of the dosage base becomes more tenuous or denser so that more serum preparation is thick from the initial preparation. The effect of adding collagen extract also increases viscosity. The viscosity required by SNI is 2000-5000 cPs, in this experiment, the formula with the addition of 1% active substance meets the viscosity requirements.

CONCLUSION
Collagen powder had a % yield of 0.24%, which consisted of the amino acids glycine, proline, alanine, and glutamic acid as the dominant amino acids. Collagen extracts can be used as serum preparations that meet SNI requirements. Collagen powder has a higher activity % Free radical inhibition at a concentration of 5000 ppm at 63.23% with IC50 value is 4045,37 ppm when compared to serum preparations with an extract concentration of 1% at 2,4%.