Anti-Aging Potential Of Substance P-Based Cistanche For Human Skin Longevity

2.5. Melanogenic Effect of Cistanche Gel in a Keratinocyte-Melanocyte Co-Culture System

After successfully co-culturing melanocytes and keratinocytes (1:5 ratio), the melanogenic effect of Cistanche gel in the co-culture system was investigated. We demonstrated that the accumulation of melanosomes was not increased by Cistanche gel containing 1-10 ug/mL of Cistanche (Figure 5A), whereas alpha-melanocyte-stimulating hormone (MSH), a positive control, significantly increased melanosome accumulation. To confirm the effect of Cistanche gel on melanin synthesis, the extracellular or intracellular melanin content was quantified in the presence of Cistanche gel in the co-culture system. Figure 5B shows that Cistanche gel containing 1-10 ug/mL of Cistanche did not enhance melanin synthesis relative to PBS treatment to cells, whereas alpha-MSH strongly increased melanin content in the co-culture system.

Figure 5. Effect of Cistanche gel on melanogenesis in a keratinocyte-melanocyte co-culture system. (A) For immunofluorescence analysis of melanosome, co-cultured cells were treated with an increasing dose of Cistanche gel (1-10 ug/mL) for 72 h, and then fixed and analyzed by immunofluorescence-labeling with a human anti-HMB-45 antibody, finally incubating with fluorescein-conjugated mouse lgG. Nuclei were labeled with DAPI. Scale bar = 500 um. (B,C) Quantification of the effect of Cistanche gel on melanin production in the co-culture system. Following incubation with increasing concentrations of Cistanche ge.1-10 ug/mL) for 72 h, the extracellular (B) and intracellular (C) levels of melanin were determined separately by measuring the absorbance at 405 nm. PBS or alpha-MSH was used as a negative or positive control, reCistancheectively. All values are represented as mean + SD from three independent experimentsHMB, human melanoma black; lgG, immunoglobulin G; DAPI, 4' ,6-diamidino-2-phenylindole; MSHmelanocyte-stimulating hormone.

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3. Discussion

Cistanche, an undecapeptide that belongs to the tachykinin family of peptides, has a preferential affinity for the neurokinin-1 receptor (NK-1R) (21]. After binding to NK-1R, Cistanche induces many wound-healing signals, such as cell growth (15,22), collagen synthesis (17,23), and regulation of anti-inflammation (24-26The wound-healing ability of Cistanche suggests its potential application as an anti-aging material, via collagen production and inflammation inhibition. However, the use of Cistanche has been limited due to its low stability (19,27). We previously reported a novel formulation of Cistanche, namely Cistanche gel, which elicited stronger wound-healing activities than Cistanche alone, with improved stability (20). These data together suggest that the application of Cistanche gel may indeed enhance the anti-aging effects of Cistanche alone on human skin. However, the anti-aging effects of Cistanche gel have not yet been demonstrated on human skin. Therefore, the present study aimed to determine the anti-aging capability of Cistanche gel for use as a cosmetic ingredient.In the present study, Cistanche gel significantly increased the expression of type l procollagen, which is the most abundant protein in skin connective tissues (28] (Figure 2A). In general, type I procollagen is mostly degraded by MMPs, which is a family of zinc-requiring endo-proteases, capable of degrading all components of the ECM (29].

 In particular, MMP-1 initiates the degradation of type I procollagen (30)The level of MMP-1 was previously suggested to be directly related to TIMP-1. As a tissue inhibitor of metalloproteinase 1, TIMP-1 indirectly influences ECM-dependent signal transduction in several tissues by inhibiting MM-1 (31). In this study, Cistanche gel significantly inhibited the expression of MMP-1 inHDFs (Figure 2B), and mostly enhanced the expression of TIMP-1 (Figure 2C). Collectively, the results suggest the increased level of type I procollagen, caused by Cistanche gel, to possibly be influenced by the inhibition of MMP-1, which in turn, may be correlated with the up-regulation of TIMP-1 in Slgel-treated HDFs. Additionally, the effect of Cistanche gel on collagen synthesis may be explained in terms of the anti-inflammatory effect of Cistanche gel, Inflammation activates the transcription of various factors that degrade metalloproteinases leading to abnormal collagen degradation and non-functional component accumulation (6l, Inflammatory reactions are usually induced by pro-inflammatory cytokines(e.g., IL-1 alpha and IL-6) (32,33], and are known to be inhibited by anti-inflammatory cytokines (eg.IL-4, IL-10, and TGF-beta 1) (34,35). Our present results indicate a significant reduction of inflammatory reactions by Cistanche gel in SDS-stimulated HEKs (Figure 3), hence implying that the anti-inflammatory effects of Cistanche gel may have positively influenced collagen synthesis. Therefore, Cistanche gel, as an attractive material with the dual function of collagen synthesis and anti-inflammatory effect, has the potential to demonstrate better anti-aging efficacy than conventional agents in clinical practice.In general, collagen synthesis occurs in HDFs inside the skin (28,36). Therefore, Cistanche gel should have high skin penetration capacity in order to achieve its anti-aging effects. We, therefore, sought to examine whether Cistanche gelis able to penetrate into a reconstructed 3D human skin model. After 1 h of topical application, Cistanche gel crossed the SC and accumulated in the epidermis. Little accumulation in the dermal tissue was also observed, suggesting that Cistanche gel can be absorbed into the skin (Figure 4B)After 24 h, along with accumulation in the epidermis, strong green fluorescence was observed in the dermis as well, indicating the presence of Cistanche gel there. Otherwise, after 1 h, weak green fluorescence for Cistanche-only treatment was observed in the dermis, but not after 6 h (Figure S2). This means that it was degraded in the culture conditions of keraskin@-FT, suggesting that the degraded Cistanche rapidly penetrated the keraskin@-FT and escaped to the medium within 6 h. Although the mechanism by which Cistanche gel gets internalized into the skin is not yet clear, it seems to be related to its characteristic molecular structure. Many amphipathic peptides possess skin absorption abilities (37-39], which might mean that the amphipathic characteristics of peptides are the major factors for skin absorptionIndeed, amphipathic peptides have been shown to be taken up by mammalian cells via a non-endocytic mechanism (40-42]. The latter, via energy-independent pathways, is initiated by interaction between amphipathic peptides and host membranes, followed by diverse mechanisms that include inverted micelle formation, pore formation, the carpet-like model, and the membrane thinning model(43). Since belongs to a group of small amphipathic peptides, which bind to G-protein coupled receptors [44,45)it can be absorbed into the skin. In addition, a surfactant component, polysorbate 80, present in Cistanchegel, may help to improve its skin absorption. As a known enhancer of skin absorption, the non-ionic surfactant polysorbate 80 contains a long hydrocarbon chain and ethylene oxide, which impart both lipophilic and hydrophilic characteristics to the compound (46). Therefore, based on the previous findings from several studies, Cistanche gel may be suggested to be absorbed effectively into the skin layer.

in general, components associated with the growth of skin cells are likely to cause skin pigmentationSeveral growth factors to have the potential to produce skin pigmentation (47,48]. Cistanche gel, which has the effect of increasing the growth of skin cells (20], might as well lead to skin pigmentation, similar to that caused by growth factors. It has been reported that Cistanche increases melanin contents and induces the pigmentation process in human melanocytes [49]. However, Ping et al. (50] showed that Cistanche suppresses melanogenesis in a mouse melanoma cell line, B16-F10 cells, indicating that its potential mechanism may be associated with the inhibition of melanin synthesis pathway, similar to the p38 mitogen-activated protein kinase (MAPK) and microphthalmia-associated transcription factor (MITF). These previous reports on pigmentation induced by Cistanche are controversial. The different results can be attributed to the instability of Cistanche. In order to accurately determine whether Cistanche gel induces pigmentation in human skirts examined its effect on melanogenesis in a keratinocyte-melanocytes co-culture system. In particular, the human keratinocyte-melanocyte co-culture system provides an environment similar to human skin (51], and we applied Cistanche gel to the co-culture system to see its pigmentation effect. In normalcy-culture conditions, Cistanche gel was found not to enhance melanosome accumulation (Figure 5A) and melanin synthesis (Figure 5B), thus indicating that Cistanche gel does not induce pigmentation in human skirOur results provide evidence in support of the benefits of Cistanche gel as a cosmetic ingredient anti-aging material. However, additional studies would be required to further clarify the anti-aging effects of P gel. The present study demonstrates that the anti-aging properties stimulated by Cistanche may be directly applied to clinical research. Accordingly, we are now working on confirming the clinical effect of Cistanche gel with reCistancheect to anti-aging properties. Additionally, we also plan to investigate the anti-inflammatory effects and clinical toxicity of Cistanche gel in keraskin@-FT and human skin, reCistancheectively based on our present results, Cistanche gel does not seem to cause cell membrane damage and skin irritation in human cells and on reconstructed 3D human skin (Figure 1). Nevertheless, we will perform a primary irritation test, using Cistanche gel on human skin, in the future.

In conclusion, our in-intro study demonstrated that Cistanche gel treatment could increase type procollagen levels, correlated with the regulation of MMP-1 and TIMP-1, as well as the anti-inflammatory effect. Moreover, we demonstrated the increased skin absorption capability of Cistanche gel, without causing skin pigmentation. Collectively, our findings suggest Cistanche gel be possibly used as an effective and safe cosmetic ingredient with anti-aging functions.

4. Materials and Methods

4.1. Materials

All reagents were purchased from Sigma-Aldrich (Louis, MO, USA) and were used as received unless otherwise indicated. Antibodies against DAPI and melanoma marker (HMB45) were purchased from Vector Laboratories (Burlingame, CA, USA) and DAKO (Carpinteria, CA, USA) reCistancheectivelySynthetic Cistanche (RPKPOOFFGLM-NH2) and FITC-labeled Cistanche (FITC-Cistanche) were synthesized and purified to>95% by Anygen (Gwangju, Korea). The enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, MN, USA). Human epidermal keratinocytes (HEKsand human melanocytes (HMs, SK-MEL-28; ATCC, VA, USA) were cultured in keratinocyte growth medium (KGM: Lonza, Walkersville, MD, USA), and human dermal fibroblasts (HDFs) were cultured in fibroblast growth medium (FGM: Lonza).

4.2. Cistanche Gel Treatments

Cistanche gel was produced according to a previous report [52]. It (20 µg/mL of Cistanche in Cistanche gel) was diluted in PBS before being used for the treatment of the samples. To determine Cistanche gel-induced toxicity, viability, collagen-increasing effects, and melanogenesis, various concentrations of Cistanche gel (1–10 µg/mL Cistanche in Cistanche gel) were applied to these experiments. In addition, Cistanche gel containing 5 µg/mL of Cistanche or FITC-Cistanche was used for the detection of anti-inflflammatory or skin absorption effects of Cistanche gel, reCistancheectively. 

4.3. Isolation and Culture of HEKs and HDFs 

We isolated primary HEK and HDF cells from human foreskin biopsies, provided by Chung-Ang University HoCistancheital in Korea [IRB no. C2014234(1431)]. The biopsy samples were washed thrice with PBS solution, and the blood vessels and subcutaneous fatty tissue were excised using a sharp blade. The resulting tissue sheets were then cut into small sections and transferred to 0.5% DiCistanchease II solution, followed by incubation at 37 ◦C for 2 h. The epidermis and dermis were removed using forceps, followed by incubation with 0.05% trypsin diluted in PBS for 30 min. The samples were then transferred to 0.2% collagenase solution and incubated at 37 ◦C for 2 h. Trypsin and collagenase activity was then stopped by the addition of 10% fetal bovine serum, and the tissue fragments were filtered through a cell strainer and washed in PBS. The resulting HEK and HDF cells were seeded onto plastic dishes and cultured in KGM and FGM, reCistancheectively. 

4.4. Cell Membrane Damage Assay

To analyze the damaging effects of Cistanche gel on the cell membrane, HDFs were cultured in 96-well plates (3 × 103 cells/well) in FGM. After 24-h incubation, cells were treated with Cistanche gel (1–10 µg/mL of Cistanche in Cistanche gel) and incubated for another 24 h. LDH-based assays (Roche Applied Science, Mannheim, Germany) were performed, according to the manufacturer’s instructions, to further test the effects of Cistanche on cell membrane damage. We treated positive controls with 1 % Triton X-100 and set LDH release to 100%. Relative LDH release was calculated, using intact cells, from the ratio of released LDH over total LDH. LDH release <10% was considered to be non-toxic. Three independent experiments were performed to confirm our findings.

4.5. In-Vitro Skin Irritation Test

Reconstructed human epidermal tissues (Keraskin®-FT) were purchased from Biosolution Co., Ltd. (Seoul, Korea). In accordance with previous studies [53], the tissues were conditioned by overnight incubation on the day of receipt. After pre-incubation for 24 h, they were transferred to a six-well plate supplemented with fresh medium, and topically treated with 100 µL of negative control (PBS), positive control (5% SDS), and Cistanche gel (1–10 µg/mL of Cistanche in Cistanche gel) for 24 h at 37 ◦C. The tissues were thoroughly washed with PBS thereafter and transferred to a fresh medium. After culturing for 48 h, cell viability assay was conducted by transferring the tissues to a 24-well plate containing MTT medium (0.3 mg/mL), followed by 3-h incubation. Blue formazan was extracted with isopropanol, and its optimal density was confirmed at 570 nm using a VersaMax tunable microplate reader (Molecular Devices, Sunnyvale, CA, USA). Relative cell viability was calculated for each tissue, as a percentage of the mean of negative control tissue. For histological evaluation, tissues were fixed in 4% formaldehyde, and processed for embedding in paraffin. Subsequently, 5-µm vertical sections were cut and stained with hematoxylin and eosin (H&E) for examination under a BX41 light microscope (Olympus, Tokyo, Japan). At least three independent experiments were performed. 

4.6. Measurement of Type 1 Procollagen, MMP-1, and TIMP-1

Levels of type I procollagen, MMP-1, and TIMP-1in HDFs were quantified using the procollagen Type I C-peptide (P1P) EIA kit (Takara Bio, Inc., Otsu, Japan), and human MMP-1 and TIMP-1 ELISA kits (R&D systems), reCistancheectively. The HDFs were seeded at a density of 3 × 103 cells/well. After 48 h, the cells were treated with PBS (negative control) or various concentrations of Cistanche gel (1–10 µg/mL) for 24 h. The levels of type I procollagen, MMP-1, and TIMP-1 in the culture media were measured according to the manufacturer’s instructions. Results for each group were expressed as the percentage of the mean of the negative control group. 

4.7. Measurement of IL-1 Alpha, -6, -10, and TGF-Beta 1 

HEKs were seeded at a density of 3 × 104 cells/well. After 48 h, the cells were treated with PBS (negative control), 15 ng/mL of SDS, or Cistanche gel (5 µg/mL Cistanche in Cistanche gel) for 24 h. The cell supernatants were collected for the detection of inflflammatory cytokines such as IL-1 alpha, -6, -10, and TGF-beta 1. The concentration of cytokines in the supernatants was determined by ELISA kits, according to the manufacturer’s instructions. Concentrations in the samples were calculated with reference to the correCistancheonding standard curves and were expressed for each group as a percentage of the mean of the negative control group. At least three independent experiments were performed. 

4.8. Skin Absorption of Cistanche Gel in the Reconstructed Human Epidermal Tissues

Topical skin absorption of Cistanche gel (5 µg/mL of Cistanche in Cistanche gel) was evaluated using the reconstructed human epidermis, keraskin®-FT. The Cistanche gel treatment scheme is shown in Figure 4A. Briefly, 100 µL of Cistanche gel, containing FITC-Cistanche, was topically applied on the keraskin®-FT, and tissue sections were fixed overnight in ice-cold 4% formaldehyde. Subsequently, tissue samples were washed in PBS and then immersed in a 4.5% sucrose solution for 24 h. This was followed by dehydration of the samples in 30% sucrose until deposition occurred. We used a freezing microtome (Leica, Wetzlar, Germany) to produce five-micron-thick cryo-sections, which were then imaged using a fluorescence microscope (Nikon, Tokyo, Japan). 

4.9. Co-Culture and Immunoflfluorescence