Anti-aging And Tyrosinase Inhibition Effects Of Cassia Fistula Flower Butanolic Extract Part 2

Effect of C. fistula flower extract on fibroblast cells  cytotoxicity

Cytotoxicity testing of C. fistula flower extract on fibroblast cells was measured using SRB assay. Fibroblast cells were treated with or without various concentrations of the flower extract (0–200 μg/mL). After a treatment of 48 h, the flower extract was found to not affect skin fibroblast cell growth (0–200 μg/mL). The IC20  and IC50 of the flower extract were found to be more than 200 μg/mL, which could also be applied in other experiments without toxicity (Fig. 2).

Glycoside of cistanche can also increase the activity of SOD in heart and liver tissues, and significantly reduce the content of lipofuscin and MDA in each tissue, effectively scavenging various reactive oxygen radicals (OH-, H₂O₂, etc.) and protecting against DNA damage caused by OH-radicals. Cistanche phenylethanoid glycosides have a strong scavenging ability of free radicals, a higher reducing ability than vitamin C, improve the activity of SOD in sperm suspension, reduce the content of MDA, and have a certain protective effect on sperm membrane function. Cistanche polysaccharides can enhance the activity of SOD and GSH-Px in erythrocytes and lung tissues of experimentally senescent mice caused by D-galactose, as well as reduce the content of MDA and collagen in lung and plasma, and increase the content of elastin, have a good scavenging effect on DPPH, prolong the time of hypoxia in senescent mice, improve the activity of SOD in serum, and delay the physiological degeneration of lung in experimentally senescent mice With cellular morphological degeneration, experiments have shown that Cistanche has the good antioxidant ability and has the potential to be a drug to prevent and treat skin aging diseases. At the same time, echinacoside in Cistanche has a significant ability to scavenge DPPH free radicals and can scavenge reactive oxygen species, prevent free radical-induced collagen degradation, and also has a good repair effect on thymine free radical anion damage.

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Effect of C. fistula flower extract on Collagen Synthesis in Human Skin Fibroblast Cells

Collagen plays a key role in both skin wound healing and the skin rejuvenation process. Collagen synthesis from skin fibroblast cells was achieved using a Sirius Red/Fast Green Collagen Staining Kit. Collagen synthesis from fibroblast cells was found to have significantly increased in a dose-dependent manner after cells were treated with various concentrations (100–150 μg/mL) of the flower extract (Fig. 3).

Effect of C. fistula flower extract on collagenase activity

Collagenases are the enzymes that digest native collagen in the triple helix region. Therefore, the inhibition of collagenase activity could protect against collagen breakdown. Collagenase activity was measured using fluorogenic DQ™-gelatin assay. Collagenase activity was dramatically decreased in a dose-dependent manner after treating the fibroblasts with the flower extract. At high concentrations, the flower extract (200 μg/mL) could completely inhibit collagenase activity (Fig. 4).

Effect of C. fistula flower extract on MMP-2 activity

MMP-2 is an enzyme that is involved in the breakdown of the extracellular matrix (ECM) and plays an important role in influencing normal homeostasis, aging, and wound healing of the skin. MMP-2 activity was measured using gelatin zymography. It was determined that MMP-2  secreted from skin fibroblast cells could digest gelatin in the gel. However, after the gel was incubated with various concentrations of the flower extract (50–200 μg/mL), the level of MMP-2 activity was significantly reduced in a dose-dependent manner (Fig. 5).

Effect of C. fistula flower extract on HA Synthesis in  human skin fibroblast cells

HA synthesis from skin fibroblast cells was evaluated using an ELISA kit. After the cells were treated with various concentrations of the flower extract for 48 h, HA synthesis was found to have significantly increased in a dose-dependent manner (50–200 μg/mL). After the fibroblasts were treated with flower extracts at 200 μg/  mL, HA synthesis was induced at a level that was fourfold when compared with the non-treated cells (Fig. 6).

Effect of C. fistula flower extract on tyrosinase activity

Tyrosinase is an enzyme that is involved in the rate-limiting step for the control of melanin production. Therefore, the inhibition of tyrosinase activity tends to induce skin whitening due to a reduction of melanin synthesis. When the tyrosinase enzyme was incubated with the flower extract, it could inhibit tyrosinase activity in a dose-dependent manner at a concentration of 50–200 μg/mL (Fig. 7).

Anti-oxidant activity of C. fistula flower extract

The free radical scavenging activity of C. fistula flower extract was then examined by DPPH and ABTS assay. C. fistula flower extract dose-dependently inhibited oxidant activity. Vitamin E and Trolox were used as positive control in each experiment. C. fistula flower extract exhibited scavenging activity (DPPH assay) at a value of 65% at 100 μg/mL. At 25 μg/mL of the flower extract,  the radical scavenging activity was found to still be approximately 33% and the IC50 of the flower extract and vitamin E were recorded at 70 and 72 μg/mL, respectively. These findings indicate that C. fistula flower extract is a potent antioxidant and in this capacity is comparable to vitamin E (Fig. 8a). To confirm the antioxidant activity of the flower extract, the ABTS assay was determined and showed % inhibition of 47% at 4 μg/mL and IC50 of the flower extract and the trolox were 4.8 and 3 μg/mL, respectively, which was by the data acquired from the DPPH assay (Fig. 8b).

Discussion

Extrinsic and/or environmental factors cause skin aging signs which can include wrinkles and pigment spot formations [30]. In previous studies, UV radiation which is known to induce skin aging has been a major topic of research through the focus on the pathogenesis and molecular mechanisms. The generation of ROS can stimulate skin inflammation leading to the activation of transcription factors that regulate the degradation of the skin collagen and the extracellular matrix (ECM) [30]. These events result in a loss of the skin’s ability to resist stretching, which ultimately leads to skin aging.

C. fistula flower extract has been traditionally used for the treatment of skin diseases, abdominal pain, and wound healing [17]. Our results show that the major phytochemicals represented in the C. fistula flower extract were the phenolic compounds and flavonoids. The main phenolic components in the C. fistula flower extract were protocatechuic acid followed by vanillic acid,  chlorogenic acid, and ferulic acid. In addition, Bahorun T, et al have reported that C.fistula flowers contain various types of flavonoids including kaempferol, rhein, fistula, alkaloids, and triterpenes. Among those phytochemical compounds, kaempferol, catechins, ferulic acid, chlorogenic acid, and protocatechuic acid have been proven to exhibit antiaging activities. In this study,  the antiaging activity of the C. fistula flower extract was investigated to determine the effects of the extract on collagen, HA, and melanin production. Our results indicate that high concentrations of C. fistula flower extract (200 μg/ml) did not affect the viability of human skin fibroblast cells. Therefore, C. fistula flower extracts could be safe in applications to human skin.

Collagen synthesis in skin fibroblasts plays a major role in skin rejuvenation. The reduction of types I and III procollagen synthesis is a critical feature of aged skin leading to skin thinning and increased fragility of the skin. [31]. Hence, the inhibition of collagen synthesis or a loss in the function of collagen results in chronologically aged skin. Our results indicate that the C. fistula flower extract significantly induced collagen synthesis from the skin fibroblasts and also dramatically inhibited collagenase activity, which is the enzyme involved in collagen breakdown. Chronologically aged skin induced by UV radiation also occurs through an increase in MMPs production, including, MMP-1, MMP-2, MMP-3, and MMP-9, which causes an imbalance of collagen synthesis by the induction of collagen or by ECM degradation [32]. This is the first report indicating that C. fistula flower extracts significantly inhibit MMP-2 activity in a dose-dependent manner. For applications in cosmetic formulations, the C. fistula extract at a concentration of 50 μg/mL should be considered. These findings suggest that C. fistula flower extract possesses useful booster collagen benefiting the skin via reduced collagen breakdown.

Glycosaminoglycans (GAGs) or hyaluronic acid (HA),  a major component of the extracellular matrix, is induced during wound healing and skin regeneration and keeps skin hydrated [33]. Environmental factors such as UV  radiation induce the type of skin aging that results in a  loss of skin elasticity causing skin to become wrinkled by decreasing HA synthesis [34]. This result indicates that the C. fistula flower extract dramatically increased HA synthesis in a dose-dependent manner. Hence, the flower extract can enhance skin moisture and can result in skin being less dry by increasing HA synthesis.

Hyperpigmentation causes human skin aging and occurs as a result of both internal and external factors including those related to hormones, UV exposure, drugs,  and the presence of various chemicals [4]. Melanin biosynthesis is a pathway that appears in melanocytes. Hyperpigmentation is particularly obvious in darker skin and is often difficult to treat. Cosmetic scientists have conducted various in vivo and in vitro studies on skin-lightening agents. The key enzyme that regulates melanin synthesis is tyrosinase, which is involved in two steps of melanin synthesis, including the hydroxylation of tyrosine to β-3,4-dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to DOPA quinone [4]. Our results indicate that the C. fistula flower extract can successfully reduce tyrosinase activity. This result was similar to that of certain previous studies, which showed that C. fistula pods have displayed skin whitening activity in vitro and in vivo by using tyrosinase activity as an endpoint bioassay [35]. Therefore, it can be concluded that this C. fistula flower extract can reduce hyperpigmentation in human skin. Previous studies have shown that some parts of the C. fistula plant exhibited antioxidant activity [36–38]. The aqueous and methanolic extracts of the C. fistula bark showed the free radical scavenging effect of DPPH in a dose-dependent manner [38]. The hydroalcoholic extract of the C. fistula flower and fruit pulp showed antioxidant activity by inhibiting DPPH and hydroxyl radicals [36, 37]. Additionally, our study on the antioxidant activity of the butanol extract of the C. fistula flower similarly displayed the free radical scavenging effect of DPPH and ABTS in a dose-dependent manner.

Conclusions

These results indicate that C. fistula flower extract displays a  high potential for anti-aging in normal skin fibroblast cells,  both in terms of the inhibition of wrinkles and a decrease in the number of pigment spots. C. fistula flower extract could prevent skin aging via an increase in collagen and HA  production. Moreover, the flower extract also inhibited collagenase, MMP-2, and tyrosinase activity, all of which are involved in skin aging. Therefore, C. fistula extract which has displayed a non-toxic effect might be an alternative ingredient for use in cosmetics or supplements that are being developed for anti-aging applications.

Abbreviations

ABTS: Azino-bis (ethylbenzthiazoline-6-sulfonic acid); DI water: Deionized  water; DMEM: Dulbecco’s Modified Eagle Medium; DPPH: Diphenyl-2-  picrylhydrazyl; ECM: Extracellular matrix; ERK: Extracellular-signal-regulated  kinases; FBS: Fetal bovine serum; g: Gram; GA: Gallic acid; h: Hour; HA: Hyaluronic acid; HPLC: High-performance liquid chromatography; JNK:  c-Jun N-terminal protein kinase; mg: Milligram; min: Minute; mL: Milliliter;  mM: Millimolar; MMP-2: Matrixmelloproteinase-2; MMPs: Metalloproteinases;  nm: Nanometer; PBS: Phosphate buffer saline; rpm: Round per minute; SDS: Sodium dodecyl sulfate; SRB: Sulforhodamine B; TCA: Trichloroacetic  acid; TFA: Trifluoroacetic acid; μg: Microgram; μL: Microliter

Acknowledgments

This research study was granted financial support by the Agricultural Research Development Agency (Public Organization) (ARDA), the National Research Council of Thailand (NRCT), and the Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Thailand.

Funding

This research study was granted financial support by the Agricultural Research Development Agency (Public Organization) (ARDA) and the National Research Council of Thailand (NRCT).

Availability of data and materials

All raw data of this study has been deposited in an appropriate repository. Data are available upon request from the authors.

Authors’ contributions

PL designed all experiments in this study, analyzed and interpreted the data, and wrote the manuscript. SY, WP, and PT experimented with and interpreted the data. JS provided the skin specimens that were processed for the ethical procedure. All authors read and approved the final manuscript for submission.

Competing interests

The authors declare that they have no competing interests.

Consent for publication 

Not applicable.

Ethics approval and consent to participate

This study obtained ethical approval from the Medical Research Ethics Committee, Chiang Mai University (Study code: BIO-2558-035490). Primary human skin fibroblasts were aseptically isolated from an abdominal scar after the surgical procedure involving a cesarean delivery at the surgical ward of CM Maharaj Hospital, Chiang Mai University (Chiang Mai, Thailand).

Author Details

Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. 2 Department of Obstetrics & Gynecology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand.

References

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