The Dolichos species have been reported to benefit human health with regard to the protection of blood cell progenitors1 and anti-hepatotoxic,2 hypo-lipidemic,3 anti-nephrotoxic and free radical-scavenging activities.4, 5 Dolichos biflorus (D. biflorus), in particular, is a legume crop with high nutritional value in terms of proteins, vitamins and minerals. For this reason, its beans have been used as a dietary source worldwide.
In relation, recent studies have shown that plant cell culture extracts in general are rich sources for compounds including phenols, flavonoids and their derivatives, which impart a wide range of efficacy and benefits for cosmetic formulations such as neutralizing free radical damage.6–9 Compared with plants grown in fields, plant cell cultures prove useful in developing extracts with multiple specific activities on skin cells and desired characteristics, thanks to their totipotency, safety and biosustainability.10, 11
Here, the authors evaluate a D. biflorus cell culture extract for its effects on skin cells. Results indicate the extract provides strong anti-inflammatory, antioxidant and protective properties for skin, and the ability to repair UV-induced damage. Further, the work presented reinforces cell culture systems as efficient biofactories to produce natural and effective compounds.
To investigate the effects of the extract on skin, it is important to first consider mechanisms of the skin aging process. This complex progression is characterized by dramatic morphological changes of the cells and results in wrinkle formation, loss of elasticity and dryness. These effects are mainly determined by chronological aging and photoaging, and amplied by other environmental assaults, such as free radicals, extreme temperatures and pollutants.12, 13 Prolonged and chronic UV exposure can cause alterations and damage to the various skin cell types—i.e., epidermal keratinocytes, melanocytes, dermal fibroblasts and even cells of the immune system, such as macrophages and neutrophils, which can transiently reside in the skin. These alterations lead to an increase of inflammation, connective tissue degradation and oxidative stress, all accompanied by a decrease in cellular metabolism and functionality.14
Keratinocytes, which constitute the outermost skin layer, are the most exposed to UV light and thus the most affected by this type of stress. UV light, particularly UVB, induces keratinocytes to express cytokines,15 which are small proteins that act as inter-cellular messengers and trigger the inflammatory response. During the inflammatory process, blood flow increases and immune cells are attracted by chemical signals to the site of injury, causing skin redness and erythema.16 Moreover, cytokines induce the dermal fibroblasts to secrete matrix metalloproteases (MMPs) and elastase,17, 18 which digest collagen and elastin fibers, respectively. As a consequence, the skin loses fitness and vigor, leading to wrinkle formation and sagging, the most visible signs of skin aging.
UV light also produces damage on a cellular level, inducing biochemical modifications to DNA, organelles, proteins and membranes, thus compromising the functioning of the whole cell. In particular, UV can cause serious damage to mitochondria and their DNA. The mitochondria are the cell’s energy factories, producing energy for the cell in the form of adenosine triphosphate (ATP), but its production may be compromised after prolonged UV irradiation.19 Also, mitochondrial DNA (mtDNA) can be damaged; it has been shown that a particular type of DNA deletion, called common deletion,20 is more frequently found in photodamaged skin than in sun-protected skin.21 This common deletion, or 4977-bp deletion, causes the loss of approximately one-third of the mtDNA genome, which contains important coding information for the enzymes required for ATP synthesis.
Further, UV-induced degradation at the cellular level is the structural modification of proteins and their resulting loss in functionality. Proteasome, a multiple sub-unit enzymatic complex, has the specific role of degrading all damaged proteins in a cell, acting as a cell-detoxifying component. The degradation of UV-damaged proteins by proteasome is doubly essential to the cell; besides preventing the damaged proteins from interacting erroneously with other cell constituents, it yields the amino acids that can be reused to build new proteins. However, UV stress increases the amount of damaged proteins as well as decreases proteasome functionality, resulting in a dangerous accumulation of damaged proteins in the cells.22
To evaluate the impact of the D. biflorus cell culture extract on aging mechanisms such as these, several tests were conducted. These included: Oxygen Radical Absorbance Capacity (ORAC), UVB-induced interleukin expression in keratinocytes, UVA-induced expression of MMP-1 (collagenase-1) and MMP-3 (stromelysin-1), and single cell electrophoresis (comet assay). Note that all tests described were performed in triplicate, and the values were analyzed by t-test and shown to be statistically significant at p < 0.01.
Materials and Methods
Cell culture and extract preparation: To produce calluses, leaves of D. biflorus were surface sterilized in 70% ethanol for 15 min and 1% bleach for an additional 15 min, then washed three times with sterile water. Each leaf explant was cut into 0.5-cm pieces, wounded with a scalpel blade and placed on a mediuma supplemented with 500 mg/L myo-inositol; 30 g/L sucrose; 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D); 0.1 mg/L kinetin; 1 mg/L adenine; and 7.5 mg/L plant agar for callus induction. White and friable callus was obtained after five weeks of cultivation in the dark. The callus cultures were maintained by transferring the tissues onto fresh medium every four weeks.
To initiate stem cell liquid culture, 50 mg of 40/45 day-old callus was resuspended in 25–30 mL liquid mediuma. The culture was incubated at 27°C in the dark under constant orbital stirring (110 rpm). The callus tissue gradually disintegrated and formed single cells or small cell aggregates after 10 days. The suspensions were then transferred into larger volumes, where 50 mL of a dense culture was used as the starting material to inoculate 1 L of culture medium.
After seven days, the cells were collected and filtered through a layer of fabric comprised of rayon-polyester with an acrylic binderb. The drained cells (approx. 300 g) were then resuspended in 300 mL of phosphate buffered saline (PBS) (136 mM NaCl, 2.7 mM KCl, 12 mM NaH2PO4 and 1.76 mM KH2PO4, pH 7.4) and homogenized in a mortar. The resulting lysate was centrifuged at 10,000 rpm to precipitate the particulate fraction and isolate the soluble components.
The supernatant was collected and lyophilized, and the powder obtained was dissolved in water or cell culture medium at the required concentrations. To use it in cosmetic formulas, the lyophilized extract was dissolved in glycerol at a concentration of 0.4%, and the solution was added to formulas at the concentration of 0.5%, which corresponds to the 0.002% concentration tested on cell cultures.
Antioxidant profiling: To determine the antioxidant capability of the extract, chromatographic separation of phenolic compounds was performed using a high performance liquid chromatography (HPLC) apparatus, a UV/Vis detector set at 280 nm, and a 100 Å-column (250 x 4.6 mm, particle size 5 μm). The eluents were 0.2% formic acid in water and acetonitrile/methanol (60:40 v/v). The gradient program was as follows: 20–30% acetonitrile/methanol, 6 min; 30–40% acetonitrile/ methanol, 10 min; 40–50% acetonitrile/methanol, 8 min; 50–90% acetonitrile/methanol, 8 min; 90–90% acetonitrile/ methanol, 3 min; and 90–20% acetonitrile/methanol, 3 min, at a constant flow of 0.8 mL/min.
The LC flow was split, and 0.2 mL/min were sent to the mass spectrometer (MS). The injection volume was 20 μL, and three injections were performed for each sample. The MS and MS/LC analyses of D. biflorus cell extract were performed in multiple reaction monitoring (MRM) by using a MS equipped with an ion source working in the negative or positive mode, depending on the type of compound analyzed. The ionization parameters used were as follows: ionization potential, +5000 V or -4000 V; temperature of drying gas, 400°C; and declustering potential (DP), 60 V.
ORAC assay: The ORAC assay is based on the ability of a test compound to inhibit the oxidation of a fluorophore, generally fluorescein, by the potent oxidant 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH). Twenty-five microliters of D. biflorus cell extract dilutions in phosphate buffer, 75 mM, pH 7.4, were aliquoted into 96-well plates, and 150 µL of fluorescein solution (8.5 nM in phosphate buffer) was added to each sample. After incubation at 37°C for 15 min, 25 µL of AAPH solution (153 mM in phosphate buffer) was pipeted into each well and the progress of the reaction monitored at 535 nm using a fluorescence multi-well reader.
Fluorescence was measured every minute for 40–60 min, and the antioxidant power of the mixture was calculated according to the method described by Huang et al.23 The net area under the curve (AUC) of the samples and standards, as represented by different dilutions of a water-soluble analog of vitamin Ec, was calculated. The standard curve was obtained by plotting the antioxidant concentrations against the average net AUC of the two measurements for each concentration. Net AUC was obtained by subtracting the AUC of the blank control from that of the sample or the standard. ORAC values of the samples were expressed as µmole of the antioxidant equivalents per gram.
Cytokine and MMP expression: For gene expression analysis of the inflammatory cytokine interleukins IL-1 beta, IL-6 and IL-8, and of the metalloproteases MMP-1 and MMP-3, keratinocytes (HaCat) and fibroblasts (HDFn) were used. These cells were incubated with 0.01% and 0.002% D. biflorus cell culture extract, or other reagents, for 20 hr before UV irradiation for inflammatory cytokine analysis. To induce the expression of inflammatory cytokines, the keratinocytes were irradiated with UVB 40 mJ and collected after 4 hr; to induce MMP expression, the fibroblasts were irradiated with UVA 10 mJ, then incubated with D. biflorus cell culture extract or other reagents for 6 hr and finally collected.
The cells were then processed for RNA extraction using an RNA purification kit according to the manufacturer’s instructions. Reverse transcription polymerase chain reaction (RT-PCR) was performed using gene-specific primers and an oligo-nucleotide internal standard kitd. The PCR products obtained were visualized and quantified with an imaging systeme. The amplification band corresponding to the gene analyzed was normalized to the amplification band corresponding to the oligo-nucleotide internal standard kitd. The values obtained were finally converted into percentages by arbitrarily setting the values obtained by the samples of irradiated cells as 100%.
Comet assay: Keratinocytes (HaCaT) were plated in 6-well plates and grown for 24 hr. The cells were then incubated with control compounds or different concentrations of D. biflorus cell culture extract for 16 hr, then irradiated with UVB 20 mJ or treated with 175 µM H2O2. After an additional UV or H2O2 treatment of the cells with the control compounds or extracts, 1 hr for UV irradiation or 2.5 hr for H2O2 treatment, the cells were detached from the plate, washed once with PBS 1X, then immediately dropped onto a Normal Melting Agarose (NMA) pre-coated slide using 80 µL of Low Melting point Agarose (LPMA) 0.5% in PBS. Coverslip slides were placed on the top, without squeezing the cells, and the slides placed on a tray in the refrigerator until the agarose layer hardened (15 min). Coverslips were then gently slid off without scraping the agarose layer containing the cells. The slides were placed in cold lysis solution for at least 2 hr at 4°C, then placed in an electrophoresis tank filled with cold electrophoresis buffer.
Slides were left in alkaline buffer for 10 min to allow DNA unwinding and the expression of alkali-labile damage, then the power supply was turned on to 24 volts. Slides were electrophoresced for 20 min and at the end, placed in cold neutralization buffer for at least 10 min. After drying the slides, cells were stained with a 10 µg/mL solution of ethidium bromide, covered with a coverslip and scored by a fluorescence microscope.
Common deletion assay: Keratinocytes (HaCaT) were plated in 6-well plates and grown for 12 hr. After two days of incubation with test compounds or different concentrations of D. biflorus cell culture extract, the cells were exposed to 1.5 mJ UVB radiation. After 1 hr, the cells were processed for DNA extraction using a DNA purification kitf according to the manufacturer’s instructions. Multiplex PCR was performed using control and test mitochondrial deletion gene-specific primers. These are designed to anneal outside the common deletion region, which gives different amplification product in intact and deleted mtDNA. The PCR products obtained were visualized and quantified with the same imaging systeme described previously. The amplification band corresponding to the deleted mitochondrial DNA was normalized to the amplification band corresponding to control mitochondrial DNA.
ATP assay: Keratinocytes (HaCaT) were seeded in 96-well plates and grown for 20 hr. The cells were then incubated with different concentrations of D. biflorus cell culture extract or the vitamin E analogc for 2.5 hr, and irradiated with UVB at 20 mJ. After 1 hr, to measure the ATP content, an equal volume of detection reagentg was added to each well and the luminescent signal of the samples was measured using a plate readerh. As reference standard, scalar dilutions of ATP were used.
Proteosome activity assay: Keratinocytes (HaCaT) were seeded in 96-well plates and grown for 12 hr. The cells were stressed with 70 mJ of UVB and 10 J of UVA, then treated with different concentrations of D. biflorus cell culture extract. After 6 hr, the cells were washed with PBS 1X and lysed in cold lysis buffer containing 4 mM Suc-LLVY-AMC, an amino methyl coumarin (AMC)-conjugated peptide that is the specific substrate of the beta 5 proteasomal subunit. After 1 hr, the fluorescence was measured by a multi-well plate readerj.
The value obtained by the ORAC assay was 150 μmol of the vitamin E analogc per gram of dried extract, which was higher than other similar plant extracts,24 suggesting the presence of compounds with strong antioxidant power in the D. biflorus cell culture extract. As shown in Figure 1, chemical analysis of the extract revealed the presence of compounds such as genistein, genistin, daidzein, daidzin and their glucosidic derivatives. These entities have reported activity against UV-induced senescence in human skin cells,25 as well as a synergic photoprotective effect on UV-induced DNA damage.26 By MS analysis, the amount of genistein and its glucosidic derivatives was estimated at 2,845 μg/g of dried extract, while that of daidzein and its derivatives was 540.6 μg/g. It is important to note that any effects detected are not given by the genistein alone but by the whole extract, which is a mixture of several compounds.
The presence of compounds with UV protective properties prompted the investigation of D. biflorus cell culture extract to protect and repair UV-induced damage in skin cell cultures, in particular those associated with the inflammatory response. The reactive oxygen species (ROS) generated after UV exposure are the main elements responsible for the activation in epidermal keratinocytes of inflammatory cytokines, such as IL-1β, IL-6 and IL-8.27
D. biflorus cell culture extract significantly decreased the UVB-induced interleukin expression in the keratinocytes after UV treatment. At a concentration of 0.01%, the expression of IL-1β, IL-6 and IL-8 was reduced by 35%, 57% and 47%, respectively. In the case of IL-1β and IL-6, these reductions were more important than those produced by a commercially available drugk used as positive control in the experiment (see Figure 2). Note that this positive control was chosen because typical anti-inflammatory drugs such as aspirin and ibuprofen work by other mechanisms and have a more systemic effect in the body.28 The results indicated that D. biflorus cell culture extract had a good potential anti-inflammatory effect on the epidermis, and by inhibiting the primary events of UV-induced inflammation, could be beneficial to the health and well-being of skin overall.
Dermal Matrix Results
Besides keratinocytes, fibroblasts in the dermis can be seriously damaged by UV radiation. Once exposed to UV light, they over-produce collagenases, which are the main agents responsible for dermal collagen degradation. In the experiments with fibroblasts, UVA instead of UVB rays were used, since they affect fibroblast activity in human skin the most due to their higher penetrating power.
D. biflorus cell culture extract reduced the UVA-induced expression of MMP-1 and MMP-3 enzymes. At the highest tested concentration of 0.01%, the extract reduced MMP-1 and MMP-3 expression by 35% and 50%, respectively (see Figure 3). The effect was higher than that produced by retinoic acid, used as positive control,29 suggesting a good protective effect on the dermis against UVA-induced damage.
Nuclear DNA and Mitochondria Results
On a cellular level, UV-induced damage can be evaluated by looking at DNA integrity and mitochondrial functionality. The comet assay detects nuclear DNA integrity in a single cell by measuring levels of DNA fragmentation. As shown in Figure 4a, treatment with D. biflorus cell culture extract produced a strong protective effect on nuclear DNA after UVB exposure. The comet tail length, an index of DNA breakage, was reduced by 40% in keratinocytes by treatment with the extract, and this effect was even higher than that produced by the analog of vitamin Ec, used at a concentration of 200 μM. An even stronger protective effect was obtained using the D. biflorus cell culture extract on keratinocytes treated with H2O2 as a stressing agent. As shown in Figure 4b, the extract almost completely eliminated nuclear DNA damage, suggesting a stronger effect on indirect damage caused by ROS after UVB exposure.
Mitochondrial protection against UVB-induced damage, as investigated by common deletion assay and ATP production assay, again indicated a level of functionality on the mitochondria after stress insult. Treatment with D. biflorus cell culture extract protected keratinocyte mitochondrial DNA from UVB radiation by completely eliminating the deletion of mitochondrial DNA caused by UV irradiation, as shown in Figure 5. This effect was even stronger than that produced by treatment with vitamin E or coenzyme Q10, known for their protective roles on mitochondrial DNA.30, 31
On the basis of these results, the effect of the D. biflorus cell culture extract on mitochondrial activity, expressed as ATP production, also was investigated. As shown in Figure 6, the total amount of ATP synthesized in the mitochondria, which was reduced upon UVB irradiation, increased in response to D. biflorus cell culture extract treatment, both in the presence and absence of UV stress. At the highest D. biflorus cell culture extract concentration of 0.01%, the amount of ATP increased by 20% with no UV and by 60% following UV exposure, compared to control cells. These results are similar to those observed after treatment with the vitamin E analogc at a concentration of 200 μM. All these results confirmed the capacity of D. biflorus cell culture extract to preserve the integrity of cells against UV insult, acting both as protector of nuclear DNA and as shield for mitochondrial functionality.
Finally, the ability of D. biflorus cell culture extract to preserve the activity of the proteasome, which becomes compromised after UV exposure, was analyzed by the proteasome activity assay. D. biflorus cell culture extract counteracted the UV-induced reduction of proteasome activity at all the tested concentrations. At the highest concentration of 0.01%, it completely eliminated the reduction in activity caused by both UVA and UVB (see Figure 7), suggesting a strong effect on preserving proteasome functionality.
In vivo Results
The ability of D. biflorus cell culture extract to protect skin from UV-induced damage also was evaluated in vivo (n = 10) by an independent lab using creams containing either the extract at 0.002% w/w or a placebo. For two weeks before irradiation, the test or placebo product was applied. Subjects’ skin was then irradiated by a UVA/UVB solar simulator, and erythema levels were assessed; measurements were taken initially and 26 + 4 hr after irradiation in areas treated with the product or placebo, or left untreated. Results showed that the extract inhibited the formation of the UV-induced erythema by 23.94% compared with untreated areas, and by 12.79% compared with placebo-treated areas (see Figure 8).
A second series of tests assessed the soothing effects of the extract on skin after UV irradiation. Erythema values were measured on irradiated areas treated with the test product or placebo at T0 (26 ± 4 hr after irradiation), and at 2 hr and 4 hr after irradiation. Analogous to the previous tests, results indicated that D. biflorus cell culture extract reduced the erythema redness caused by UV radiation by 7.56% after 2 hr and 12.98% after 4 hr (see Figure 9).
In this article, the authors propose a new cell culture extract as active ingredient for skin care and cosmetics. The extract is derived from D. biflorus cell suspension cultures and shows interesting properties for protecting skin cells. The present studies demonstrate that the extract can act on multiple fronts by preventing damage on a cellular level, both in keratinocytes and fibroblasts, and by alleviating inflammation caused by UV light in epidermal and dermal tissues. Chemical analysis also confirmed the presence of important classes of compounds, known for their beneficial effects in promoting cell health by inducing natural mechanisms of repair and protection against UV insult.23, 26, 32
Plant cell cultures have increasing interest in the cosmetic field since they are rich in compounds often found in lower amounts in plant tissues; they also have therapeutic and beneficial features for skin cells.10, 11 In agreement with the data published on other species of legume plants cultivated worldwide,33 D. biflorus cell culture extract also contains good amounts of isoflavones, such as genistein, daidzein and their derivatives, conferring to the extract’s preventive and protective properties against UV damage on skin cells. Moreover, plant cell cultures represent an advantageous and sustainable biotechnological system to develop extracts for skin care products, which then can be safe and effective—two key priorities for today’s market.
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