coden jpbsct ethanolic extract...

Effects of Centella asiatica Ethanolic Extract Encapsulated in Chitosan Nanoparticles on Proliferation Activity of Skin Fibroblasts and Keratinocytes, Type I and III Collagen Synthesis and Aquaporin 3 Expression In Vitro

Received Date: 22 April 2016 – Accepted Date: 14 May 2016 – Published Online: 21 May 2016

Copyright © 2016

INTRODUCTION

Skin aging is a complex process controlled by genetic determination (chrono-logic aging) and under the influence of external factors (extrinsic aging). Skin aging leads to various modifications in cells and tissue1.

A number of studies have shown that aging process results in reduced proliferation activity in both keratinocytes and fibroblasts. Furthermore, cell renewal rate and the synthesis of type I and type III collagen in skin fibro-blasts decreased naturally in skin as the years go by. This loss of regenerating power is both, cause and a measure of skin aging1.

Aging skin is manifested by the diminished water content of stratum cor-neum (SC). Age-related trans-epidermal water loss (TEWL) can be evaluated by removing the superficial epidermal layer by tape stripping. In aged skin,

ORIgINal aRTICle

Linda Yulianti1*, Kusmarinah Bramono2, Etik Mardliyati3, Hans-Joachim Freisleben4,5

1 Biomedical Sciences Doctoral Program, Faculty of Medicine, University of Indonesia, Salemba Jakarta, Indonesia

2 Department of Dermatovenerology, Faculty of Medicine, University of Indonesia, Salemba Jakarta, Indonesia

3 Centre for Pharmaceutical and Medical Technology, Agency for the Assessment and Application of Technology,

4 Medical Research Unit, Faculty of Medicine, University of Indonesia, Salemba Jakarta, Indonesia

5 German–Indonesian Medical Association (DIGM e. V.)

Address reprint requests to: *Dr. Linda Yulianti, MD, Biomedical Sciences Doctoral Program, Faculty of Medicine, University of Indonesia, Salemba Jakarta, Indonesia E-mail: [email protected]

Article citation: Yulianti L, Bramono K, Mardliyati E, Freisleben HJ. Effects of Centella asiatica ethanolic extract encapsulated in chitosan nanoparticles on proliferation activity of skin fibroblasts and keratinocytes, type I and III collagen synthesis and aquaporin 3 expression in vitro. J Pharm Biomed Sci 2016;06(05):315–327.

Available at www.jpbms.info

Statement of originality of work: The manuscript has been read and approved by all the authors, the requirements for authorship have been met, and that each author believes that the manuscript represents honest and original work.

Source of funding: None.

Acknowledgment: The authors wish to thank Dani, IntanRazari, Ita and Labibah for laboratory assistance and Indra Kusuma for the cell cultures of NHDF and NHEK.

Competing interest / Conflict of interest: The author(s) have no competing interests for financial support, publication of this research, patents and royalties through this collaborative research. All authors were equally involved in discussed research work. There is no financial conflict with the subject matter discussed in the manuscript.

Disclaimer: Any views expressed in this paper are those of the authors and do not reflect the official policy or position of the Department of Defense.

NLM Title J Pharm Biomed Sci

CODEN JPBSCT

2230-7885ISSN No

ABSTRACT

Background The activity of skin cell proliferation, collagen synthesis and skin hydration decrease with the process of aging; therefore, the skin looks dull, dry and sagging. Aquaporin 3 (AQP3) is a key protein that plays a major role in skin cell proliferation and skin hydration. Retinoic acid (RA) is still considered for anti-aging treatment, but it fre-quently shows side effects, such as skin irritation. Centella asiatica (CA) formulation in chitosan nanoparticles has a promising potential as anti-aging cosmetic.

aim The aim of this study was to evaluate the effects of CA ethanolic extract encapsu-lated in chitosan nanoparticles (CAEE + CNP) on skin cell proliferation, collagen synthesis and AQP3 expression in vitro, when compared to RA.

Methods Microculture tetrazolium assay was conducted to analyse the proliferation of normal human dermal fibroblasts (NHDF) and normal human epidermal keratinocytes (NHEK) at 24, 48 and 72 h. Type I and III collagen synthesis was evaluated at the same time points using ELISA. Aquaporin 3 expression at 24 h was evaluated using immunocy-tochemistry and measured quantitatively using ImageJ software. All treatments involved several concentrations of CAEE + CNP and RA through serial dilution.

Results Collagen type I and III synthesis of NHDF and NHEK was neither significantly different from untreated controls nor from RA-treated cells. Nevertheless, CAEE + CNP stimulated the proliferation of both NHDF and NHEK. Additionally, AQP3 expression in both cell types was upregulated by CAEE + CNP.

Conclusion CAEE + CNP is a promising formulation for anti-aging activity by inducing skin cell proliferation and AQP3 expression. The clinical trials are still needed to evaluate skin hydration in vivo.

KeywoRdS aquaporin 3, Centella asiatica, chitosan nanoparticles, collagen, fibroblasts, hydration, keratinocytes, proliferation

DOI http://dx.doi.org/10.20936/jpbms/160246

http://dx.doi.org/10.20936/jpbms/160246

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Linda Yulianti

the barrier repair capacity is significantly impaired2. Hydration of the SC determines the appearance and the physical properties of the skin. The aging process reduces skin surface hydration, which results in dry, dull, coarse and saggy appearance with loss of elasticity. Other visi-ble signs include pigmentation and appearance of benign skin tumours. Moreover, the epidermis becomes thinner with age and as a result wrinkles develop2–4.

Cosmeceutical anti-aging formulations contain active ingredients to achieve local therapeutic effects, and should not have harmful systemic side effects2. Well known anti-aging ingredients like vitamin A or retinoic acid (RA) and its derivatives (retinoids), have harmful side effects by irritating the skin and causing dry, flaky skin, which is sensitive to light5. Moreover, usage of these retinoids con-taining formulations is limited, as they may cause contact RA dermatitis and have teratogenic effects5,6. Therefore, it is necessary to develop innovative cosmeceutical anti- aging formulations with active ingredients which are effective and stable, yet safe and nontoxic. Additionally, they should be compatible with other active ingredients and be delivered efficiently to the target cells7,8.

Indonesian biodiversity promises great potential for selection and development of herbal cosmetics and cosmeceuticals, which are effective and safe. Natural products contain a wealth of interesting and possibly beneficial cosmepharmaceutically active compounds. For example, Pereda et al.9 reported that hydroglycolic Piptadenia colubrina extract can improve skin hydration.

Centella asiatica (CA) (gotu kola, Hydrocotyle asiatica) is often found in Indonesia and has been used for vari-ous medicinal purposes since 1,000 years ago, CA has been applied for wound healing and the treatment of asthma, ulcers, leprosy, lupus erythematosus, psoriasis, vein disease and cancer10–13. CA stimulates the synthe-sis of collagen for skin tissue regeneration14–16. The bio-logically active ingredients in CA are triterpenes namely asiatic acid, madecassic acid, asiaticoside and madecassoside12,13. Although CA extracts (CAE) possess high potential of biological activities, their clinical usage is limited due to poor physical stability, with CA extracts having high hygroscopic index. Powder extracts are promptly liq-uefied within a few minutes when exposed to normal environment. Therefore, nanoparticles which entrap the extract and protect it from external moisture should be developed for stabilisation17.

Chitosan is a biopolymer produced from chitin by partial deacetylation. The polysaccharide chitin is the main compound of the exoskeletons in crustaceans and fungal cell walls. Chitin nanofibrils are applied in cos-metics as carriers to increase penetration18,19, as well as active ingredients for anti-aging cosmetics as it helps to maintain the integrity of skin barrier and increases the capacity of corneocytes to store water20. Chitosan consists of acetylated and deacetylated units. Later, they are composed of β-(1,4)-d-glucosamine, whereas, the former units consist of N-acetyl-d-glucosamine. Chitosan is hypoallergenic and has natural antibacterial

properties, it is biocompatible and biodegradable and its low cost warrants biomedical application in wound healing21,22. Furthermore, chitosan is used in anti- aging skin cosmetics, both as penetration enhancer and as active anti-aging compound19.

Nanocarrier systems as innovative drug and cosmetic delivery technology have been widely used23,24. This technology is commonly based on lipid carriers, such as liposomes and solid lipid nanoparticles with 100–300 nm in diameter23–25. Nanoparticles have unique physi-cal properties which make them ideal for various skin care products26. For example, a lipid nanocarrier (LNC) system loaded with tocopheryl acetate (TA) exhibited the ability to enhance skin hydration26. Apart from lip-id-based carriers, polymeric nanoparticles are being devel-oped as novel delivery systems, including those based on chitin/chitosan27,28.

Aquaporins (AQPs) as water channels are the key factors in skin hydration mechanisms29. The most abun-dant AQP in the skin is AQP3, a water/glycerol trans-porting protein. However, its expression in normal human dermal fibroblasts (NHDF) and normal human epidermal keratinocytes (NHEK) decreases with the aging process. Non-expression of AQP3, particularly in the basal layer leads to reduced SC hydration and skin elasticity30. Therefore, AQP3 is a key protein target for future anti-aging treatment to improve durability, tex-ture and quality of the skin surface31. Naturally active compounds that can stimulate AQP3 expression would be effective hydrating agents as emollients in anti-aging cosmetics31.

RA influences keratinocyte differentiation, reduces epidermal cell adhesion and facilitates corneocyte exfo-liation from the surface of epidermis. In the dermis, retinoids regulate fibroblast proliferation, induce angio-genesis and play an essential role in the synthesis of col-lagen and elastin fibrils32. Moreover, it was reported that RA stimulates AQP3 expression in NHEK33.

In this study, we evaluated the effect of CA ethanolic extract (CAEE) encapsulated into chitosan nanoparticles (CAEE + CNP) on the proliferative activity of NHDF and NHEK, on the synthesis of type I and type III collagen in NHDF and determined the ability of CAEE + CNP to increase AQP3 expression in NHDF and NHEK.

MaTeRIalS aND MeTHODS

MaterialsRA was purchased from BASF, Germany and chitosan with a deacetylation degree >75% from Sigma-Aldrich, USA. All other chemicals were obtained from Merck, Germany; the assay kits used were purchased from Sigma-Aldrich, via their subsidiaries in Indonesia or Singapore. CA, from Tawangmangu, was used to prepare the ethanolic extract in the Centre for Pharmaceutical and Medical Technology, Agency for the Assessment and Application of Technology (BPPT PUSPITEK), Serpong.

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Effects of CA ethanolic extract in chitosan nanoparticles

CAEE was encapsulated into chitosan nanoparticles by ionic gelation method34 using Sigma-Aldrich chitosan with a deacetylation degree of >70%.

Serial concentrations of all compounds used were obtained by 1 + 1 dilution steps with the respective sol-vent: CAEE + CNP (100, 50, 25, 12.5, 6.25 and 3.125 mg/mL); RA (10, 5, 2.5, 1.25, 0.625 and 0.3125 mg/mL) and (1, 0.5, and 0.25 mg/mL).

Serial concentrations of all compounds used were obtained by 1 + 1 dilution steps with the respective solvent: CAEE + CNP from 100, 50, 25, 12.5, 6.25 mg/mL down to 3.125 mg/mL; RA from 10, 5, 2.5, 1.25, 0.625 mg/mL down to 0.3125 mg/mL to deter-mine proliferation activity of NHDF and NHEK. RA dilution steps from 1, 0.5 mg/mL to 0.25 mg/mL were used to determine type I and III collagen synthesis and in immunocytochemistry.

Cell cultures of human fibroblasts and keratinocytes In this study, primary NHDF and NHEK cells were derived from male children’s foreskin and cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetalbovine serum (FBS) and 1% penicillin–streptomycin from the Cell Culture Laboratory Faculty of Medicine, University of Gajah Mada, Yogyakarta. NHDF cell culture from 7th passage was used to test the synthesis of type I and III col-lagen as well as for the immunocytochemical test in the Integrated Laboratory of Yarsi University, Jakarta. Meanwhile, NHEK cell culture was used in the same laboratory to test keratinocyte proliferation and immu-nocytochemistry. Early, the cell passages between 2 and 7 were used in all experiments.

MTT assay of fibroblast and keratinocyte proliferationMTT assay (MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-tetrazolium bromide) was adapted to measure fibroblast and keratinocyte cell proliferation. Cells were seeded at a ratio of 1:3 and 1 × 106 cells/mL density, before subjected to treatment with 0.3125, 0.625, 1.25, 2.5, 5 and 10 mg/mL of RA and 6 concentration (3.125, 6.25, 12.5, 25, 50, 100 mg/mL) of CAEE + CNP. The MTT assay was repeated three times. The number of viable cells was evaluated at 24, 48 and 72 h after treat-ment, as an indicator of fibroblast proliferation, fur-thermore at 24 and 48 h for keratinocytes proliferation. Subsequently, obtained values were normalised against untreated control group (100%).

Evaluation on type I and III collagen synthesis using ELISAType I and III collagen synthesis was examined by using designated ELISA kit Alpha 1 from CUSABIO Life science, US.

Fibroblast collagen synthesis activity was evaluated at 24 and 48 h after treatment and compared against untreated NHDF as negative control. Results for collagen synthesis were reported in mg/mL.

Immunocytochemistry testImmunocytochemistry is a specific protein detection technique using a specific antibody (Ab) to recognise the antigen presented on cells. Immunochemical stain-ing was done by using anti-aquaporin 3 Ab, Ab125219. Then, the expression of AQP3 in both NHDF and NHEK was quantitatively analysed by using ImageJ.

STaTISTICal aNalYSIS

The data obtained from this study are presented as images, tables or graphs. To assess significance across the different treatment groups, unpaired t-test and one-way ANOVA test were used, followed by post-hoc Tukey test. Significance level was set at P < 0.05 using SPSS software.

ReSUlTS

Figure 1 shows the transmission electron micrographs (TEM) of CNP and CAEE + CNP.

Particle size distribution of CAEE + CNP analysed with Beckman Particle Size Analyzer CV. Main peak was obtained at 96.3 ± 26.6 nm with a polydispersity index (PI) of 0.308. The cumulative percentage of this peak amounts to 100%, which means that there is only one size population with uniform distribution.

Figure 2 compares cell proliferative activity of cul-tured NHDF after exposure with serial dilutions of CAEE + CNP and RA at 24, 48 and 72 h. At 24 h stim-ulation, fibroblast proliferation is high for 6.25 mg/mL CAEE + CNP with ~160% of control. This is significantly higher as compared to untreated control and RA-treated cells. At 48 h, both compounds demonstrate similar results with maximum effects seen on 3.125 mg/mL concentra-tion. After 72 h, RA shows dominant effects on fibroblast proliferation. The statistical analysis with unpaired t-test revealed no significant difference between the two groups.

Figure 3 compares type I collagen synthesis in cul-tured NHDF after 24, 48 and 72 h of exposure with CAEE + CNP and RA. The different concentrations of CAEE + CNP were obtained by serial dilution from 12.5 mg/mL down to 3.125 mg/mL and with RA by serial dilution from 0.1 mg/mL down to 0.025 mg/mL.

In general, CAEE + CNP and RA exerted similarly positive effects as compared to negative control. After 24 and 48 h of exposure, CAEE + CNP and RA did not differ from control. At 72 h, however, both CAEE + CNP and RA showed greater type I collagen synthesis, as com-pared to control group. Comparing for all time periods (24, 48 and 72 h) through one-way ANOVA test, there were no statistically significant differences between CAEE + CNP, RA and control at all concentrations.

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Fig. 1 Chitosan nanoparticles (CNP) (a) and Centella asiatica ethanolic extract (CAEE) encapsulated into chitosan nanoparticles (CAEE + CNP) (b); magnification ×100,000; black bars = 50.0 nm.

a b

Fig. 2 Comparison of cell proliferative activity of NHDF without treatment (control), after exposure to Centella asiatica ethanolic extract encapsulated into chitosan nanoparticles (CAEE + CNP) and retinoic acid (RA) at various concentrations at 24, 48 and 72 h. The con-centrations of CAEE + CNP were obtained by serial dilution from 100 mg/mL down to 3.125 mg/mL and RA from 10 mg/mL down to 0.3125 mg/mL.

Figure 4 compares type III collagen synthesis in cul-tured human fibroblasts after 24, 48 and 72 h of exposure to CAEE + CNP and RA. The different concentrations of CAEE + CNP were obtained by serial dilution from 12.5 mg/mL down to 3.125 mg/mL and with RA by serial dilution from 1 mg/mL down to 0.25 mg/mL. Type III collagen synthesis generally increases over time (with its highest value at 48 h) and fluctuates slightly more than type I collagen (compare with controls in Figs. 3, 4).

After 24 h, results from CAEE + CNP, RA and con-trols were not significantly different at 3.125 mg/mL (P = 0.151) using one-way ANOVA test.

After 48 h of exposure to CAEE + CNP and RA, the lowest concentration, synthesis of type III collagen pro-vided a higher yield than control. At 72 h, control had the highest value. Overall, it appears that the amount of synthesised collagen does not strictly depend on the time exposure and concentrations of the test compounds.

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Fig. 3 Comparison of type I collagen synthesis in NHDF without treatment (control) and after 24, 48 and 72 h of exposure to Centella asiatica ethanolic extract encapsulated into chitosan nanoparticles (CAEE + CNP) and retinoic acid (RA) at various concentrations for 24, 48 and 72 h. The concentrations of CAEE + CNP were obtained by serial dilution from 12.5 mg/mL down to 3.125 mg/mL and RA from 1 mg/mL down to 0.25 mg/mL.

Fig. 4 Comparison of type III collagen synthesis in NHDF without treatment (control) and after 24, 48 and 72 h of exposure to Centella asiatica ethanolic extract (CAEE) encapsulated into chitosan nanoparticles (CAEE + CNP) and retinoic acid (RA) at various concentrations for 24, 48 and 72 h. The concentrations of CAEE + CNP were obtained by serial dilution from 12.5 mg/mL down to 3.125 mg/mL and with RA by serial dilution from 1 mg/mL down to 0.25 mg/mL.

After 48 h, there is no statistically significant difference at all the concentrations tested between CAEE + CNP, RA and control (P-value >0.05) using one-way ANOVA test. These results are not significantly different when

compared to 24 h exposure, though there is an increase in collagen synthesis with CAEE + CNP and RA.

After 72 h, CAEE + CNP and RA showed no statisti-cally significant differences using one-way ANOVA test,

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although there was a decrease in type III collagen syn-thesis activity with CAEE + CNP and RA compared with 24 and 48 h of exposure.

Figure 5 compares cell proliferative activity of cul-tured NHEK after exposure with CAEE + CNP and RA at serial dilutions for 24 and 48 h.

After 24 h of treatment, RA showed maximum result with 155% of control at a concentration of 5 mg/mL, while CAEE + CNP gave 145% at 3.125 mg/mL. Overall, CAEE + CNP stimulated lower proliferative effect than RA. Unpaired t-test statistical analysis showed no signif-icant difference.

Keratinocyte proliferation at 48 h looked similar for the two compounds tested. CAEE + CNP at all concentra-tions (between 3.125 and 25 mg/mL) and RA (between 0.3125 and 5 mg/mL) stimulated proliferation to 110–120% of control (100%). Interestingly, the two high-est concentrations of CAEE + CNP and the highest RA concentration showed lower proliferative activity than control (<100%).

Figure 6 shows AQP3 expression in NHDF with-out and with exposure to CAEE + CNP for 24 h, as detected by immunocytochemistry. The results showed an increase in protein AQP3 expression in NHDF after exposure for 24 h to CAEE + CNP at 3.125, 6.25 and 12.5 mg/mL. Semi-quantitatively, we were able to obtain optimal results at the highest concentration of 12.5 mg/mL.

Figure 7 shows protein expression of AQP3 in cul-tured NHDF without and with exposure to RA for 24 h, as detected by immunocytochemistry. Similar AQP3 expression was seen at all concentration, although with a reverse trend as compared to CAEE + CNP.

The results showed an increase in AQP3 protein expression in NHDF after exposed to RA at concentration of 0.25, 0.5 and 1 mg/mL. Semi-quantitatively, optimal results were gained at 0.25 mg/mL concentration of RA.

Figure 8 shows AQP3 expression in cultured NHDF before and after treatment with CAEE + CNP and RA for 24 h, as quantitatively analysed with computer software ImageJ.

Quantified particle density from ImageJ analysis demonstrated that CAEE + CNP stimulated the expres-sion of AQP3 protein in a concentration-dependent manner, with optimal concentration at 12.5 mg/mL. In contrast, the expression of AQP3 in dermal fibroblast after treated with RA decreased with increasing concen-tration (0.25–0.5 and 1 mg/mL).

By using one-way ANOVA test, we obtained a sta-tistically significant difference on AQP3 expression in cultured NHDF (P < 0.001) after being exposed with CAEE + CNP and RA at concentration of 3.125 and 0.25 mg/mL, respectively, compared with control. Further-more, with post-hoc Tukey test, there is also a signif-icant difference between CAEE + CNP groups and RA groups (P < 0.001).

Another significant difference on AQP3 expression in NHDF was also obtained by exposing 6.25 mg/mL of CAEE + CNP and 0.5 mg/mL of RA compared with control (P < 0.05). The following post-hoc Tukey test showed a significant difference between CAEE + CNP and RA (P = 0.006).

This study revealed a significant difference of AQP3 expression in NHDF after being exposed to CAEE + CNP and RA at concentration of 12.5 and 1 mg/mL, respec-tively, compared with control (P < 0.05). Another

Fig. 5 Comparison of cell proliferative activity of NHEK without treatment (control) and after exposure to Centella asiatica ethanolic extract encapsulated into chitosan nanoparticles (CAEE + CNP) and retinoic acid (RA) at various concentrations for 24 and 48 h. The concentrations of CAEE + CNP were obtained by serial dilution from 100 mg/mL down to 3.125 mg/mL and RA from 10 mg/mL down to 0.3125 mg/mL.

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statistical analysis was conducted by using post-hoc Tukey test, and there is no significant difference between CAEE + CNP and RA.

Figure 9 shows protein expression of AQP3 in cul-tured NHEK without and with exposure to CAEE + CNP for 24 h, as detected by immunocytochemistry. After 24 h of exposure to CAEE + CNP concentration at 3.125 and 6.25 mg/mL, there was an increased expression of AQP3 protein in the cytoplasm of keratinocytes com-pared to control, while the exposure of 12.5 mg/mL actually decreased AQP3 expression. These results indi-cate that AQP3 expression in cultured NHEK showed inverted concentration dependence upon exposure with CAEE + CNP. Similarly, quantitative analysis was done using ImageJ software.

Figure 10 shows protein expression of AQP3 in cul-tured NHEK without and with exposure to RA for 24 h, as detected by immunocytochemistry. AQP3 expression on the keratinocytes increased after exposure to RA at concentration of 0.025, 0.05 and 0.1 mg/mL. The inten-sity of protein AQP3 expression in keratinocytes increases proportionally with the increasing of RA concentration.

Figure 11 shows AQP3 expression in cultured NHEK as quantitatively analysed using computer software ImageJ.

Qualitative results of immunocytochemistry were anal-ysed by using ImageJ software to obtain quantitative data for further statistical evaluation.

From the immunocytochemistry results on kerati-nocytes, the quantitative analysis based on colour den-sity showed that CAEE + CNP at concentration of 3.125 and 6.25 mg/mL stimulated AQP3 protein expression slightly higher than control. For RA stimulation, how-ever, there is a linear correlation between AQP3 protein expression and the treatment concentration.

Statistical analysis by using one-way ANOVA test showed no significant difference on AQP3 expression in cultured NHEK after being exposed with CAEE + CNP and RA at concentration of 3.125 and 0.25 mg/mL, respectively, compared with control.

After exposure with 6.25 mg/mL of CAEE + CNP and 0.5 mg/mL of RA showed a significant difference on AQP3 expression in NHEK (P < 0.05) compared with control, as statistical analysis was conducted by one-way ANOVA test. Further statistical analysis using post-hoc Tukey test showed no significant difference between CAEE + CNP and RA.

Using one-way ANOVA test, we obtained another significant difference on AQP3 expression in NHEK after

Fig. 6 Aquaporin 3 (AQP3) expression (brown colour, pointed by arrows) in NHDF: (a) without treatment (control); and after 24 h exposed to CAEE + CNP at concentration of (b) 3.125 mg/mL; (c) 6.25 mg/mL; and (d) 12.5 mg/mL; immunochemical staining with anti-aquaporin 3 antibody Ab125219.

a b

c d

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Fig. 7 Aquaporin 3 (AQP3) expression (brown colour, pointed by arrows) in NHDF: (a) without treatment (control); and after 24 h exposed to retinoic acid (RA) at concentrations of: (b) 0.25 mg/mL, (c) 0.5 mg/mL, and (d) 1 mg/mL; immunochemical staining with anti-aquaporin 3 antibody Ab125219.

Fig. 8 Quantitative analysis of AQP3 expression in cultured NHDF by computer software ImageJ. *Statistically significant difference found between 3.125 mg/mL of CAEE + CNP and RA 0.25 mg/mL using one-way ANOVA and post-hoc Tukey tests (P < 0.05). **Statistically significant difference found between 6.25 mg/mL of CAEE + CNP and RA 0.5 mg/ mL using post-hoc Tukey test (P < 0.05). ***Statistically significant difference found between 0.25 mg/mL of RA and control using post-hoc Tukey test (P < 0.05). ****Statistically significant difference found between 0.5 mg/mL of RA and control using post-hoc Tukey test (P < 0.05).

a b

c d

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Fig. 9 Aquaporin 3 (AQP3) expression (brown colour, pointed by arrows) in NHEK: (a) without treatment (control); and after 24 h exposed to CAAE + CNP at concentration of: (b) 3.125 mg/mL; (c) 6.25 mg/mL and (d) 12.5 mg/mL; immunochemical staining with anti-aquaporin 3 antibody Ab125219.

a

c

b

d

Fig. 10 Aquaporin 3 (AQP3) expression (brown colour, pointed by arrows) in NHEK: (a) without treatment (control) and after 24 h exposed to retinoic acid (RA) at concentration of: (b) 0.25 mg/mL; (c) 0.5 mg/mL and (d) 1 mg/mL; immunochemical staining with anti-aquaporin 3 antibody Ab125219.

a

c

b

d

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Fig. 11 Quantitative analysis of AQP3 expression in cultured keratinocytes by computer software ImageJ. *Statistically significant difference found between 12.5 mg/mL of CAEE + CNP and control using one-way ANOVA and post-hoc Tukey tests (P < 0.05). **Statistically sig-nificant difference found between 0.5 mg/mL of RA and control using post-hoc Tukey test (P < 0.05). ***Statistically significant difference found between 1 mg/mL of RA and control using post-hoc Tukey test (P < 0.05). ****Statistically significant difference found between 12.5 mg/mL of CAEE + CNP and control using post-hoc Tukey test (P < 0.05).

being exposed with 12.5 mg/mL of CAEE + CNP and 1 mg/mL of RA compared with control (P < 0.05). The following post-hoc Tukey test showed no significant difference between CAEE + CNP and RA (P < 0.05).

DISCUSSION

RA is a potent stimulator of keratinocyte proliferation and regulator of cell differentiation. It increases the thickness of stratum granulosum and is widely used in cosmetic anti-aging treatment of the skin35. Since RA has harmful side effects, it is interesting to search for alternatives, i.e., other active substances with better pharmacological profile (bioavailability, toxicity) as compared to RA.

In this study, we used CAEE, which had shown better proliferative activity in preliminary experiments than the aqueous extract (data not shown). A study in Malaysia had also proven that ethanolic extracts have better biological effects than aqueous extract of CA36. Chitosan–alginate nanoparticles developed as carriers for CA extracts were considered as promising systems to stabilise the extracts17. Hence, we used our own CNP to encapsulate our CAEE (CAEE + CNP).

Leonida (2012) investigated the activity of chitosan as antimicrobial, wound-healing and anti-aging com-pounds. The authors reported that ‘nano-sizing’ enhanced its effectiveness23. Moreover, when well-known sub-stances are prepared into nanoparticles, they may dis-play completely different properties and can behave in an unpredictable manner. This means that nanoparticles could re-invent the properties of substances currently used in cosmetic dermatology to create novel chemical entities from the old ones8.

In this study, we used chitosan with a degree of deacetylation more than 85% (not consistent with ‘Materials and Methods’ Section). A study by Howling et al. (2001) demonstrated that chitosan with a relatively high degree of deacetylation strongly stimulated fibro-blast proliferation while chitosan with lower levels of deacetylation showed less activity22.

Proliferation of cultured NHDFFrom our results, it can be concluded that the prolifera-tive effect of CAEE + CNP is not strictly concentration- dependent but rather time-dependent (Fig. 3). After 24 h of exposure, CAEE + CNP showed a generally better proliferative effect on NHDF (with at a peak at 6.25 mg/mL = 160% of controls) than RA. After 48 h, there are no significant differences between CAEE + CNP and RA. After 72 h, the effect of RA dominated the picture with a peak of about 180% of controls (Fig. 3).

A study by Wu et al.37 investigated the effect of four triterpenes from CA on the proliferation of NHDF. At low concentrations (1, 3 and 10 µM), all compounds tested did not enhance proliferation compared with the control group. Meanwhile, Song et al.38 showed that madecasso-side, a bioactive compound of CA-inhibited proliferation of keloid fibroblasts in a time and concentration- dependent manner. A study by Lu et al. (2004)39 found that asiaticoside, another compound of CA, strongly induced cell-cycle progression, proliferation and col-lagen synthesis in dermal fibroblasts. Asiaticoside also increased migration of keratinocytes by ~20% in wound healing process)40.

In the dermis, RA was found to regulate fibroblast proliferation and play a role in aging prevention because

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of its inhibitory effects on the expression of metallo- proteinases41. Varani et al.35 demonstrated that the abil-ity of RA to stimulate NHDF proliferation depends on the concentration of Ca2+ in the extracellular environ-ment. Although RA exerts the above effects and has also such clinical activity in dermal tissue, all-trans-RA (ATRA) reduced proliferation in NHDF cultures42. Moreover, retinoids generally decreased NHDF proliferation after 48 h of incubation.

Collagen expression in cultured NHDFType I and III collagens are the major structural and functional components of skin connective tissue. Hence, we were interested to investigate the efficacy of CAEE + CNP on the biosynthesis of these collagens in NHDF compared to RA. We found that the effect of CAEE + CNP on type I collagen synthesis at 24 and 48 h of exposure was not significantly different from RA and controls. At 72 h, control values were slightly decreased, but CAEE + CNP and RA maintained type I collagen synthesis on the same level.

Type III collagen synthesis fluctuated and generally increased over time with its highest control value at 48 h; exposure to CAEE + CNP and RA at their lowest concen-trations slightly stimulated biosynthesis at 48 h. At 72 h, control value was higher than all other values; in other words, all the treatment had inhibitory effects on type III collagen synthesis.

In a study from Malaysia, CA was shown to have stim-ulatory effect on collagen synthesis in a concentration- dependent manner. At 50 mg/mL, their CA extract enhanced 3-fold collagen production, compared to the control. Their data indicated higher proliferative stimu-lation by CA than by vitamin C which was used as a pos-itive control and enhanced collagen synthesis of 2-fold over negative controls43.

Proliferative activity of cultured NHEKOur findings revealed that CAEE + CNP and RA increased proliferation activity at certain concentrations; however, the effect is not strictly concentration-dependent. The picture differed between 24 and 48 h of incubation. The highest results were obtained with each of the two compounds at 24 h: RA (5 mg/mL, 155%) >CAEE + CNP (3.125 mg/mL, 145%). All other results including all results at 48 h were not significantly different from controls.

In literature, exposure of NHEK to RA increases pro-liferative activity; RA has regulatory effects on epidermal keratinisation, keratinocyte differentiation and inflam-mation41. In keratinocytes, RA activates peroxisome pro-liferator-activated receptor (PPAR) β/γ in parallel to the activation of RA receptor (RAR); consequently, PPAR β/γ is central to the maintenance of skin permeability, bar-rier integrity and keratinocyte survival during inflam-mation and wound healing. RA regulates keratinocytes

proliferative activity and differentiation through retinoid receptors of RAR and RXR44.

The expression of AQP3 in NHDFAquaporin 3 (AQP3) is the most common aquaporin in skin. AQP3 is a membrane transporter of water and glycerol expressed in plasma membrane in the basal layer of human epidermis30. The function of AQP3 in NHDF is not totally clear yet, but it may be involved in cell migration which occurs in wound healing process45. AQP3 expression in human skin increased as a response to stress in diseases such as atopic eczema and to various agents such as RA46.

The expression level of AQP3 in NHDF decreases sig-nificantly beyond the age of 60 years. This finding might explain why wound healing process is slower in old age than in youngsters, suggesting that AQP3 may play an important role in the aging of human skin47. Li et al.45 found that the expression of AQP3 protein in sun-pro-tected human skin decreased with skin aging.

The research by Cao et al.48 showed that the expres-sion of AQP3 in NHDF increased after treatment with EGF in concentration- and time-dependent patterns. Ryu et al.49 investigated the effect of AQP3 overexpression in HPMC after incubated with TGF caused fibrosis, but the same effect have not yet found in HDF fibroblast49 Our study demonstrates that CAEE + CNP formulation increases the expression of AQP3 in NHDF after 24 h of incubation in a concentration-dependent pattern. RA also influenced the expression of AQP3 in NHDF, but the result decreased with increasing concentrations (Fig. 8).

The expression of AQP3 in NHEKHydration of the SC is an important determinant of skin appearance. Reduced SC hydration was found in aged skin, making it looked dry, dull and saggy2. The expres-sion of AQP3 in NHEK decreases with aging45.

In skin epidermis, as a water channel, AQP3 facili-tates cell migration in wound healing and as a glycerol transporter enhances keratinocyte proliferation and dif-ferentiation. Therefore, AQP3 is also a key target protein for anti-aging treatment30. The correlation was found among AQP3 expression, epidermal ATP concentration and cell proliferation as AQP3-facilitated glycerol trans-port and lipid synthesis in the epidermis30.

ATRA increases AQP3 expression in NHEK through RAR-γ46. ATRA of 0.05% increased AQP3 expression in human skin explants after application for 24 h33. This is consistent with our finding that RA increased AQP3 expression in keratinocytes at concentrations of 0.25 and 0.5 mg/mL, with optimal concentration at 1 mg/mL. A study by Song et al. showed the AQP3 overexpression induced by ATRA, in which time and dose dependent will be followed by increased water transport, keratino-cyte, skin permeability and TEWL, and caused dry skin. Nicotinamide attenuates AQP3 overexpression induced

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by RA through inhibitor of EGFR in cultured human skin keratinocyte50.

AQP3 is the abundant skin aqua-glycero-porin functioning as a channel for both water and glycerol. Thus, AQP3 has an important role in skin hydration51. The recent discovery of AQP3 functions has led to sev-eral studies of other novel compounds51, motivated by the relation between AQP3 expression, water content and moisturising effect on skin. Several cosmetic com-panies have marketed cosmetics-containing ingredients that are claimed to increase AQP3 expression in skin. The increase of AQP3 expression is claimed to have anti- aging, anti-wrinkle and other beneficial effects on skin appearance31.

Currently, research is under way to investigate com-pounds with the ability to stimulate AQP3 expression, thereby increasing skin hydration endogenously. Recent studies of herbal AQP3 stimulants included P. colubrina9, green Coffea arabica L.52 and extract of Ajugaturga turkesnita (a plant from Central Asia)51. The increase of AQP3 expres-sion in NHEK was claimed to have moisturising effect on skin31. Our research indicates that after 24 h of expo-sure, CAEE + CNP can induce AQP3 expression in kera-tinocytes at concentrations of 3.125 and 6.25 mg/mL.

Our study demonstrates that CAEE + CNP increased the expression of AQP3 in NHEK ~1.6-fold, com-pared to negative control at an optimal concentration of 3.125 mg/mL after incubation for 24 h. This result suggests that CAEE + CNP increases skin hydration and that CAEE + CNP can be used as an active compound for cosmetic skin care/moisturiser.

SUMMaRY aND CONClUSION

CAEE + CNP can be used as an effective formulation to stimulate the proliferation of fibroblasts and keratino-cytes during short 24 h exposure time. The best effect of CAEE + CNP for the proliferation of fibroblasts and keratinocytes was also obtained at low concentrations (6.25 and 3.125 mg/mL, respectively).

Efficacy of CAEE + CNP on stimulating type I and III collagen synthesis in fibroblasts was not signifi-cantly different from RA, after exposure to all concen-trations and time periods tested. CAEE + CNP increase AQP3 expression in fibroblasts and keratinocytes after 24 h of exposure at optimal concentrations of 12.5 and 3.125 mg/mL, respectively.

Thus, CAEE + CNP can be used as an active formu-lation for novel anti-aging cosmetic that regulates skin hydration by increasing skin cell proliferation and AQP3 expression. In the future, pre-clinical and clinical studies are needed for further validation.

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