Electronic ISSN 2287-0237

VOLUME

EVALUATION OF THE ANTIOXIDANT ACTIVITY, COLLAGEN SYNTHESIS ANDSTABILITY OF BRIGHT AND FIRM HYBRID EMULSION I

SEPTEMBER 2019 - VOL.15 | ORIGINAL ARTICLE

In 2011, Bunman et al., extracted proteoglycan from the head of fishcartilage and developed cream extraction from the head of fish cartilage.Proteoglycan is a hybrid molecule composed of a central core proteinby bonding it with polysaccharides (glycosaminoglycans or GAGs) with acovalent bond, previously called mucopolysaccharide due to its physicalproperty (jelly-like, sticky and viscosity). GAGs can be divided into 5 groups:chrondroitin/chorndroitan sulfate, keratin/keratin sulfate, dermatin/dermatansulfate, heparin/heparan and hyaluronan. Proteoglycan in cartilage plays animportant role in wound healing because the molecular structure is similarto an epidermal growth factor (EGF)-like domain. The bioactive mechanismof proteoglycan is also similar to that of EGF with regard to wound healingthrough promotion of epidermal growth and regeneration of tissues andblood vessels.1 In 2015, this material was added to silver sulfadiazine toaccelerate and facilitate wound healing and to promote collagen synthesis.1After that, they studied liver and kidney toxicity in rats receiving creamcontaining proteoglycan extraction from fish cartilage in 2016.

This result demonstrated that the cream containing proteoglycan extractedfrom fish cartilage accelerated and facilitated wound healing withoutcausing to toxic effects to the liver and kidney in rats in long term use.2 In2018, Bunman et al., formulated a new serum for facial use, which whenapplied promotes collagen synthesis and wrinkle relief. The studydemonstrated that this serum has accelerated collagen synthesis and lowpotential skin sensitization in rat models.3 In 2019, Bunman developed andformulated a new cream for facial use, called “bright and firm hybrid emulsion I”.Bright and firm hybrid emulsion I is composed of many active ingredientssuch as pseudoalteromonas ferment extract, chlamydomonas extract, hamamelis virginiana (witch hazel) extract, and astaxanthin etc. to reducewrinkles, to increase facial whitening and to promote collagen synthesis.However, this product does not have scientific findings of antioxidant activity,collagen synthesis, and stability.

The aim of this study is to evaluate antioxidant activity,collagen synthesis, and stability of bright and firm hybridemulsion I.

Material and chemical

Hydrochloric acid, 2,2′-Diphenyl-1-picrylhydrazyl(DPPH), Distilled water, Ethanol, Methanol, Ascorbic acid,2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid(ABTS), Iron (II) sulfate heptahydrate, Iron (III) chloridehexahydrate, 2,4,6-tri (2-pyridyl)-1, 3, 5-triazine (TPTZ),Acetic acid, Sodium acetate, Manganese dioxide, Potassiumpersulfate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid (Trolox), 96-well microtiter plate, microplate reader,UV-Vis spectrophotometer

Stability evaluation
Determination of pH

The pH meter was calibrated using standard buffer solution.A 0.5 g of the bright and firm hybrid emulsion I was weighedand dissolved in 50 ml of distilled water and its pH wasmeasured.4

Heating and cooling test

The heating and cooling test was used for the stabilitydetermination of bright and firm hybrid emulsion I. Theheating and cooling method was investigated using themodified method from Biskup et al.5 (2013). Bright and firmhybrid emulsion I was kept at a temperature of 4ºC (48 hours)and a temperature of 45ºC (48 hours) for 7 cycles. Thephysical appearances of the bright and firm hybrid emulsionI such as color, smoothness, and pH were observed before andafter testing.

Microbial growth test

The bright and firm hybrid emulsion I formulation wasinoculated on the plate of Muller Hilton agar media by streakplate method. The plates were incubated at 37 ºC for 24, 48,72, 96, 120, 144, and 168 hours. After incubation time, plateswere taken out and checked for microbial growth by comparingit with the control.4

Antioxidant methods

2,2′-Diphenyl-1-picrylhydrazyl (DPPH) method

DPPH (2,2-diphenyl-1-picryl-hydrazyl) method is arapid way to evaluate antioxidants by spectrophotometry. Thisassay measures the ability of antioxidants which will donatehydrogen (H) on DPPH. It is based on changing the DPPHfree radicals which are purple to pale yellow. In brief, 0.22mg/ml DPPH stock solution was prepared by accuratelyweighing 22 mg of DPPH in 100 ml of absolute methanol andallowed to settle overnight at 4 °C. To obtain DPPH workingsolution, the DPPH stock solution was diluted at 1:5 byadding 20 ml stock solution to 80 ml of methanol. To 1.9 ml ofDPPH working solution, 100 μl of the final concentration of theextract and standard solution were added separately. Eachsolution was incubated in the dark at 37 ºC for 30 minutes.After incubation absorbance of the solution, it was measured at517 nm by using UV-Vis spectrophotometer. Ascorbic acid wasused as the reference standard compound. All determinationswere carried out in triplicate.6 Antioxidant capacity is expressedin mg Ascorbic acid equivalent /100g extract.

The percentage inhibition was calculated by using thefollowing equation:

2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid(ABTS) method

ABTS method was evaluated for antioxidant activity. Theexperiment was performed according to Censi et al.(2018).7In brief, ABTS•+ stock solution was prepared by 1:1 chemicalreaction of 15 mM ABTS and 5mM potassium persulfate indark condition, overnight. To obtain ABTS•+ workingsolution, the ABTS•+ stock solution was diluted 1:50 with 5mM phosphate buffer saline, pH 7.4. In the assay, either finalconcentration of the extract (10 μl), Trolox standard solution(10 μl) or ethanol (10 μl) for controls were mixed withABTS•+ working solution (290 μl) in 96-well microplate. Themicroplate was incubated in the dark at 37°C for 6 minutes.All determinations were carried out in triplicate. After incubation,the absorbance of each well was determined at 734 nm byusing a microplate reader. Background absorbance wascorrected by subtracting the absorbance value of blank. Theantioxidant activities are expressed in percentage inhibitionand mg Trolox equivalent/100 g extract. The ABTS percentageinhibition was calculated by using the equation alreadydescribed for the DPPH method. Antioxidant capacity isexpressed in mg Trolox equivalent /100g extract.

Ferric reducing antioxidant power (FRAP) method

The FRAP method measures the antioxidant potential ina sample based on the ability of a compound to reduce thecomplex of ferric ion (Fe3+) and TPTZ to the ferrous form(Fe2+) in acidic condition. The reaction was monitored bydetermining the change of absorbance at 593 nm. Thismethod was investigated using the modified method fromBiskup et al. (2013).5 FRAP working solution was freshlyprepared each time by adding 0.3 M acetate buffer (pH 3.6)to 0.01 M TPTZ (2,4,6-tripyridyl-s-triazine) and 0.02 MFeCl3●6H2O (Iron (III) chloride hexahydrate) in ratio 10:1:1

(v/v/v). This working solution was protected from light andprewarmed to 37°C before used. In the assay, the finalconcentration of the extract and standard solution were addedto 2.25 mL FRAP working solution and 0.225 mL of deionizedwater. FeSO4●7H2O (Iron (II) sulfate heptahydrate) wasdissolved in deionized water for using as standard solution.The reaction mixture was shaken and incubated at 37°C for5 min. Absorbance was measured at 593 nm by using theUV-Vis spectrophotometer. FRAP working solution withdeionized water instead of a sample and was used as a blank.All determinations were determined in triplicate. Results areexpressed in mg Iron (II) sulfate heptahydrate equivalent /100gextract.

Histological analysis

Animals

In this study the histological analysis was investigatedusing the method of Bunman et al. (2015)1, which wasmodified from Chuenwattana et al. (2018).2 Male Wistar rats(age 8 weeks, weighing 250-300 grams) were purchased fromthe national Laboratory Animal Centre, Mahidol University,Salaya, Thailand. Rats were housed in the Laboratory AnimalUnit under standard conditions of temperature 25 ± 2 °C, 50 - 60 %humidity, and a 12 hours/12 hours light/dark cycle. The ratswere kept under laboratory conditions for one week prior tothe start of the experiments and allowed food and water adlibitum. At the end of each experiment, the rats were sacrificedwith carbon dioxide asphyxiation. Animal experiments in thisstudy were carried out in accordance with the EthicalPrinciples and Guidelines for the Use of Animals for ScientificPurposes of the National Research Council of Thailand.

Animal preparation

Male Wistar rats were randomly divided into 2 groups of6 animals, and assigned to receive 0.5 gram of cream base(control), and bright and firm hybrid emulsion I applied to theskin. Histological analysis evaluated collagen synthesis onday 28 post applied.

The Masson’s trichrome examination

Collagen synthesis was proved by skin stained withmasson’s trichrome. This study was investigated using the method of Chuenwattana et al. (2018).3 Skin tissue sectionswere deparaffinized in xylene, hydrated, and stained inWeigert’s iron hematoxylin solution for 10 minutes. Theywere washed and stained in acid fuchsin solution for 3 minutes,rinsed in distilled water, treated with phosphomolybdicphosphotugsticacid solution for 15 minutes, immediatelysubmerged into aniline blue solution for 10 minutes, rinsedin distilled water, treated with acetic solution for 10 minutes,dehydrated in 95% alcohol, cleared twice in xylene, mountedwith a cover slip, and observed under a light microscope.

Statistical analysis

The results are expressed as means ± standard deviation(SD). Stability studies and histological analysis wereanalyzed using descriptive statistics.

Heating and cooling test of bright and firm hybridemulsion I evaluated color, smoothness, and pH. The color ofbright and firm hybrid emulsion I appeared as pearl like color.On 1st to 7th cycle, the color and pH of bright and firm hybridemulsion I was unchanged when compared with the controlgroup (Table 1). On 1st to 7th cycle, the physical of bright andfirm hybrid emulsion I appeared smooth, the physical appearancewas unchanged or no separation phase (Figure 2).

The microbial growth test was observed post incubate 24,48, 72, 96, 120, 144, and 168 hours, respectively. Bright andfirm hybrid emulsion I was not found to have a bacterialcolony or no bacterial growth on media plates when comparedwith the control (Table 2).

Figure 1: Color of bright and firm hybrid emulsion I on 1stto 7th cycles.

 

Table 1: Heating and cooling test of bright and firm hybrid emulsion I

 

Table 2: The result of microbial growth test

 

Table 3: The antioxidant activity of bright and firm hybrid emulsion I.

The antioxidant activity of bright and firm hybrid emulsionI was evaluated by 3 methods as DPPH, ABTS, and FRAPmethods respectively. The result of antioxidant activity ofbright and firm hybrid emulsion I had a percentage ofinhibition 14.93 ± 0.39 ascorbic acid equivalent 0.53 ± 0.01mg/100 g extract by DPPH method, percentage of inhibition59.60 ± 6.21 Trolox equivalent 14.91 ± 1.50 mg/100 g extractby ABTS method, and Iron (II) Sulfate Heptahydrateequivalent 8.32 ± 0.44 mg/100 g extract by FRAP method,respectively (Table 3).

The results from Masson’s trichrome of rat skinsstaining can clearly differentiate important morphologicalkeys for collagen synthesis assessment. Muscle, Hemoglobin,keratin and fiber are stained red color. Adipose tissue andcytoplasm are stained light red or pink. Cell nuclei showdark brown to black and collagen fiber is stained anilineblue. On day 28 post applied with bright and firm hybridemulsion I showed higher levels of collagen fiber whencompared to the control group (Figure 2).

 

Figure 2: Histological appearance on day 28 post applied. Masson’s trichrome stains of rat skins applied with (A) cream base(control), and (B) bright and firm hybrid emulsion I, Bar = 50 μm.

Determination of pH, heating and cooling test, and microbialgrowth test were used for evaluation of the stability of acosmetic creams.8 The general properties of the bright and firmhybrid emulsion I had a pH that ranged between 5.5 and 6.This pH range is necessary and appropriate for skin pH andbecause the preservatives are effective at those levels. Studyof the effect of temperature on the stability of the bright andfirm hybrid emulsion I showed that the temperatures did notaffect the physical appearance of the bright and firm hybridemulsion I through the period of 7 cycles. The temperatureranged between 4°C and 45ºC and is necessary and appropriatefor storage.

The study on the effect of light on the stability of the brightand firm hybrid emulsion I demonstrated that the physicalappearances (color and solidity) of the bright and firm hybridemulsion I were not changed throughout the period of 7 cycles.However, antioxidant ingredients such astaxanthin, ascorbic References1. Bunman S, Aramwit P, Larbcharoensub N, et al. Applicationof proteoglycans from fish cartilage for the acceleration ofburn wound healing. TJPS 2015; 39(3):64-9.2. Bunman S, Dumavibhat N, Larbcharoensub N. Evaluation ofLiver and Kidney Toxicity in Rats Receiving Proteoglycansfrom Fish Cartilage for the Acceleration of Burn WoundHealing. The Bangkok Medical Journal 2016;12:11-6.3. Chuenwattana W, Chatthanawaree W, Bunman S, et al. TheEvaluation of Potential Skin Sensitization and CollagenSynthesis of Rats Receiving Proteoglycan Serum. TheBangkok Medical Journal 2018;14(1):1-4.4. Muthukumarasamy R, Ilyana A, Fithriyaani NA, et al.Formulation and evaluation of natural antioxidant creamcomprising methanolic peel extract of Dimocarpus longan.IJPCR 2016; 8(9):1305-95. Biskup I, Golonka I, Gamian A, et al. Antioxidant activity ofselected phenols estimated by ABTS and FRAP methods.Postepy Hig Med Dosw 2013;67:958-63.6. Parasuraman S, Kumar E, Kumar A, et al. Free radicalscavenging property and diuretic effect of triglize, a polyherbalformulation in experimental models. J PharmacolPharmacother 2010;1(1):38-41.7. Censi R, Vargas Peregrina D, Lacava G, et al. CosmeticFormulation Based on an Açai Extract. Cosmetics 2018;48(5):1-11.8. Panichayupakaranant P, Itsuriya A, Sirikatitham A. Preparationmethod and stability of ellagic acid-rich pomegranate fruitpeel extract. Pharm Biol 2010;48(2):201-5.9. Li P, Yu X, Xu B. Effects of UV-C Light Exposure andRefrigeration on Phenolic and Antioxidant Profiles ofSubtropical Fruits (Litchi, Longan, and Rambutan) inDifferent Fruit Forms. Journal of Food Quality 2017;1-12.10. Moser CL, Meyer BK. Comparison of compendial antimicrobialeffectiveness tests: a review. AAPS Pharm Sci Tech2011;12(1):222-6.11. Abdelaziz AA, Ashour MS, Hefni H, et al. Microbialcontamination of cosmetics and personal care items in Egyptshaving creams and shampoos. J Clin Pharm Ther1989;14(1):29-34.12. Dao H, Lakhani P, Police A, et al. Microbial Stability ofPharmaceutical and Cosmetic Products. AAPS Pharm Sci Tech2018;19(1):60-78.13. Viola P, Viola M. Virgin olive oil as a fundamentalnutritional component and skin protector. Clin Dermatol2009;27(2):159-65.14. Kwansang J, Itthipanichpong C, Limpanasithikul W. Evaluationof wound healing activity of Thunbergia laurifoliasupercritical carbon dioxide extract in rats with second-degreeburn wounds. J Adv Pharm Technol Res 2015;6(3):103-7.15. Papakonstantinou E, Roth M, Karakiulakis G. Hyaluronicacid: A key molecule in skin aging. Dermatoendocrinol2012;4(3):253-8.acid, niacinamide etc. can degrade in light conditions, becausethe antioxidant ingredients are oxidized when they are exposedto light.9 Therefore, bright and firm hybrid emulsion I isrecommended to be kept under dark condition.

The microbial growth test was evaluated for preservativeproperty, which control microbial growth.10-12 Bright and firmhybrid emulsion I formulation added Caprylyl Glycol(1,2-Octanediol), Sodium Benzoate, and Potassium Sorbateas a preservative. The result demonstrated that bright and firmhybrid emulsion I formulation did not have microbial growth.Therefore, this preservative of bright and firm hybrid emulsionI can control microbial growth.

UV radiation from sun light damages the skin by increasinglevels of free radicals. Cigarette smoke, pollution, stress,illnesses, and drugs etc. can increase levels of free radicals inthe physical body.7 For their elimination antioxidants containedin antioxidant creams are helpful.13 The study demonstratedthat bright and firm hybrid emulsion I can reduced free radicalscavenging activity because it contained high active ingredientssuch as pseudoalteromonas ferment extract, chlamydomonasextract, hamamelis virginiana (witch hazel) extract, andastaxanthin etc.

Collagen and hyaluronan are synthesized in the fibroblastcells, which is the main component of connective tissue suchas skin, tendon, etc., giving it strength and flexibility. Thefibroblasts’ proliferation is related to collagen content in skin.14Skin aging is also associated with loss of skin moisture. Thekey molecule involved in skin moisture is a glycosaminoglycan(GAG), hyaluronic acid (HA), or hyaluronan with a uniquecapacity to bind and retain water molecules.15 From the results,the collagen of skin in the treated group on days 28 washigher than the control group. Therefore, bright and firm hybridemulsion I may play an important role in fibroblasts proliferation,which increases collagen and HA synthesis in skin.

This study was able to show that bright and firm hybridemulsion I not only displays cosmetic efficacy as an antioxidanteffect and to accelerate collagen synthesis, but also haspleasant sensorial characteristics for cosmetic use.

The authors declare that they have no conflicts of interest