Broccoli Flower Extract (Brassica oleracea L. var.italica Plenck) Inhibits Photoaging by Increasing Type I Procollagen Expression in Human Skin Fibroblast

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   Broccoli Flower Extract (Brassica oleracea L. var.italica Plenck) Inhibits Photoaging

by Increasing Type I Procollagen Expression in Human Skin Fibroblast

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International Journal of PharmTech Research CODEN (USA): IJPRIF, ISSN: 0974-4304 Vol.9, No.3, pp 114-118, 2016 Brassica oleracea L. var.italica Broccoli Flower Extract (

  

Plenck) Inhibits Photoaging by Increasing Type I

Procollagen Expression in Human Skin Fibroblast

Nelva K. Jusuf*

  

Department of Dermatovenereology Faculty of Medicine University of Sumatera Utara,

Indonesia

Abstract:

  Photoaging of the skin is caused by intrinsic process superimposed with

degenerative changes due to environmental sources such as ultraviolet irradiation. Ultraviolet B

(UVB) reduces type I procollagen level and increases matrix metalloproteinase-1 (MMP-1)

level in human skin which plays a major role in the process of photoaging. Broccoli flower

extract (BFE) is a crucciferae group of vegetables which has multiple antioxidants. It had been

studied, acting as MMP-1 inhibitor agent both at mRNA and protein level on skin photoaging

in vitro. BFE were investigated for their capacity to regulate type I procollagen expression at

₂

protein level in primary human fibroblast culture irradiated by UVB 50 mJ/cm and 100

₂

mJ/cm . Type I procollagen protein expression was quantified by Enzyme Immuno Assay. The

result of studies showed pre-treatment with various concentration of BFE increase type I

procollagen expression. There were significant differences of the mean value of type I

procollagen expression based on irradiation dose (p<0.05) and BFE concentration (p<0.05).

There was also interaction between irradiation dose and BFE concentration (p<0.05). There was

positive correlation between BFE concentration with type I procollagen expression. Therefore

BFE has been proved to increase type I procollagen expression at protein level on UVB

irradiated human skin fibroblast. BFE has a potential effect as antiphotoaging agent in the near

future.

  Introduction The skin aging process can be divided into intrinsic aging and extrinsic aging. Ultraviolet (UV)

irradiation cause premature aging which is called as photoaging. It is caused by intrinsic processes

_1,2,3

superimposed with degenerative changes to solar radiation. UV irradiation disrupts the skin collagen matrix

by stimulating collagen degradation by matrix metalloproteinase (MMP) and inhibiting procollagen production.

The balance of MMP-1 and type I procollagen expression play an important role in photoaged skin. Alterations

and defficiencies of collagen have been suggested to be a cause of the skin wrinkling observed in photoaged

₄

and naturally aged skin. Collagen degradation is mainly regulated by MMPs, while collagen synthesis is

₅

mediated by both transcriptional (gene/mRNA level) and post translational (protein level) processes. Broccoli

  

(Brassica oleracea L.var.italica Plenck) is a cruciferous vegetables which has a great amount of antioxidant. It

has been known that it consists of sulphoraphane, indole, beta carotene, quercetine, kaempferol, gluthatione,

_6,7

vitamin A, C, E, selenium. In dermatology it has been proved as skin antinflammatory in UV- induced

_{8 9}

  

Nelva K. Jusuf / International Journal of PharmTech Research, 2016,9(3),pp 114-118. 115

Experimental Material and methods Plant material

  Broccoli flowers were collected from broccoli field at Berastagi, North Sumatera, Indonesia. After

processed to a standardized simplicia it was extracted with ethanol 96% in the laboratory of Faculty of

Pharmacy University of Sumatera Utara, Indonesia.

  Cell culture The normal human fibroblast cells were aseptically isolated from preputium circumcised skin of healthy

volunteer age 11-12 years old. After the epidermis and dermis were separated mechanically, the dermis was

minced and attached on the surface of tissue culture flask. The cells were grown in Dulbecco’s modified eagle’s

medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin

(GIBCO,Grand Island, NY USA). After 3 passages the fibroblast were used for the experiment.

  Ultraviolet irradiation The UV light source originated from a Philips TL 20 W(12 RS fluorescent sun lamp with an emission ₂

spectrum of 285-350 nm (peak at 310-315 nm). The cells were then exposed to 50 and 100 mJ/cm dose of

  UVB light.

  Treatment with BFE BFE was dissolved in DMEM. The BFE concentration that use for treatment comprise of 25, 50 and

100 mg/ml. For treatment, the cells were maintained in culture media without FBS overnight, followed by

treatment with BFE for 24 hours. The cells were rinsed twice with phosphate buffer saline (PBS) and UVB

irradiation exposure were performed under a thin layer of PBS. Immediately after irradiation, the cells were

incubated in serum-free fresh culture media containing BFE.

  Enzyme Immuno Assay (EIA) The supernatant from the cell culture was collected and the procollagen type I protein expression was

quantified by procollagen type I C peptide EIA kit (Takara Biomedical Inc. Japan) at 450 nm using a

microplate reader.

  Statistical Analysis Data were expressed as the mean value and with Fisher’s least significant difference (LSD) were

analyzed by analysis of variance (Anova) 2 ways, continued by multiple comparison test. Statistical

significance was set a prior at p<0.05. The correlation between BFE concentration and type I procollagen

protein expression were analyzed by Spearman’s correlation test.

  Result Effect of BFE on type I procollagen protein expression To establish effect of BFE on type I procollagen protein expression in photoaging induced by UVB

radiation, EIA analysis were performed. It revealed that the BFE up regulated UVB –induced reduction of type

  

I procollagen expression (Fig.1). Anova two ways showed there were significant differences of mean value of

type I procollagen protein expression between every group based on irradiation dose (p<0,05) and BFE

concentration (p<0,05) (Table 1). Continued by multiple comparison test (LSD) showed significant differences

(p<0,05) between every group based on irradiation dose and BFE concentration. These result indicated that

BFE increased of type I procollagen expression at the protein level in UVB-irradiated human fibrolast.

  

Nelva K. Jusuf / International Journal of PharmTech Research, 2016,9(3),pp 114-118. 116

Figure 1. Effect of BFE on type I procollagen protein expression Table 1. Mean value of type I procollagen protein expression between every group based on irradiation dose and BFE concentration. Sources Sum of squares Df Mean square F p ₁₀ Irradiation dose 34094,525 1 34094,525 1x10 0,0001* ₉ Extract concentration 41852,762 3 13950,921 6x10 0,0001* ₉ Interaction dose * concentration 17483,594 _{-5 -6} 3 5827,865 2x10 0,0001* Error 4,20x10

  18 2,33x10 0,0001* Total 1220951,621 27 0,0001* Corrected Total 157603,336

  26 Correlation between BFE concentration with type I procollagen protein expression By Spearman’s correlation test we found there were significant positive correlation between BFE ₂

concentration with type I procollagen protein expression at 50 mJ/cm UVB irradiation dose (r=0.975, p<0.01)

₂ and at 100 mJ/cm UVB irradiation dose (r=0.973, p< 0.01) (Table 2).

  Table 2.Correlation between BFE concentration with type I procollagen protein expression. n r p Procollagen type I protein (50) 12 0,975 0,0001 Extract concentration Procollagen type I protein (100) 12 0,973 0,0001 Spearman’s correlation test; Correlation is significant at the 0,01 level (2-tailed); 50: UVB irradiation dose 50 mJ cm-2;100: UVB irradiation dose 100 mJ cm-2; r: correlation coefficient.

  Discussion Skin ages with time progress. Both intrinsic aging and photoaging process involve the expression and

degradation of several molecules participating in the metabolism of the connective tissue, including changes in

_11,12

the amount of extracellular matrix components such as collagen and in MMPs activities. The balance of

₅

  

Nelva K. Jusuf / International Journal of PharmTech Research, 2016,9(3),pp 114-118. 117

₁₃

further processed to be a collagen fiber in the extracellular matrix. Photoaging is a process involving alteration

of type I collagen which is the major component of the dermis. Among collagen, type I is the most abundant

and comprises between 85% of the total collagen in the skin. Type I collagen degradation is the major cause of

₁₄

wrinke formation. The use of antioxidant is an effective approach to prevent symptoms related to photo-

induced aging of the skin.Strategies to prevent or at least minimize ROS-induced photoaging necessarily

₁₅ include protection against UV irradiation and antioxidant homeostasis.

  Broccoli flower extract (Brassica oleracea var.italica Plenck) is an antioxidant plant which has been

studied in dermatology. Sulphorapane, the most potent antioxidant in broccoli, has antiinflammatoric effect

₈

which is shown by preventing erythema from sunlight. In our previous study, we reported that BFE decreased

the level of MMP-1 expression both at mRNA and protein level in the process of photoaging induced by UVB

₁₀

irradiated in vitro. Therefore on the other hand we investigated wether BFE also may increase the level of type

  

I procollagen expression. Our results showed that BFE has enhancer activity on type I procollagen protein

expression in UVB irradiated human skin fibroblast. We also found there were interaction between UVB

irradiation dose and BFE concentration to the expression of type I procollagen. This result indicated that both

factors together influence the mean value of type I procollagen expression.We also found there were positive

correlation between BFE concentration with type I procollagen protein expression, thereby the increasing of

BFE concentration leads the type I procollagen expression increased. These results demonstrates the inducer

effect of BFE on type I procollagen expression via protein assay in skin photoaging process in vitro. Therefore

it is suggested that BFE should be viewed as a potential therapeutic agent for preventing and treating skin

photoaging based on decrease the MMP-1 expression and increase procollagen type I expression.

  Conclusion BFE had inhibitory effect on skin photoaging induced by UVB in vitro by increasing type I procollagen

expression at protein level in human skin fibroblast culture. It is suggested further studies on BFE involving

human subjects should be carried out in the near future.

  Acknowledgement Author thanks to Prof.Dr.dr Hardyanto Soebono, Sp.KK(K), Prof.Drs. Sumadio Hadisahputra, Apt.,

Ph.D, dr. Adang Bachtiar, MPH, D.Sc, Dr.dr. Imam Budi Putra, MHA, SpKK for support and cooperation. The

author have no funding or support to report. No conflict of interest.

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• *****