Active reduces wrinkles and illuminates tired skin

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Australian kakadu plum extract, (Superox-C™ from Lucas Meyer Cosmetics) has been obtained through a proprietary process and shown to be a potent multifunctional active ingredient for global anti-ageing care. This unique ‘superfruit’ is inherently super rich in vitamin C and polyphenols with solid antioxidant activities.

In vitro, kakadu plum extract fights oxidative stress to support skin structural proteins and curbs the production of pro-inflammatory mediators to reduce the consequences of ‘inflamm’ageing’. Kakadu plum extract also rebalances cell defences, for a positive effect on wrinkle reduction and skin radiance, as documented through clinical studies.

Australia, the only country in the world spanning an entire continent, is recognised for its ‘megadiversity’. Australia is home to some of the world’s truly unique plant species. One of these is Terminalia ferdinandiana commonly known as kakadu plum. While the plant naturally occurs over a wide area, spanning from Western Australia to Arnhem Land in the Northern Territory, the plant originally obtained its name from its prevalence in Kakadu National Park. This beautiful reserve is truly one of the wonders of Australia, spreading over almost 20,000 square kilometres in the tropical north, east of Darwin. Kakadu National Park is a timeless place with immense environmental and cultural value that is listed as a UNESCO World Heritage site. The Park is jointly managed by its Aboriginal traditional owners and the Commonwealth Government. 

 Many of the plants in northern Australia have been traditionally used by Aboriginals as bush foods, medicines and weaving materials. Terminalia ferdinandiana (kakadu plum) is one of the most popular among these unique plants. The slender tree flowers in the pre-monsoon season to further mature into small yellow-green fruits in the rainy season. The fruit has a smooth skin, is 1.5-2 cm long with an ovoid shape (Fig. 1) and has a pleasant, acidic taste. Local people reportedly use it as bush food.1,2 Recent studies have highlighted the strong antioxidant, antibacterial and anti-apoptotic activities of kakadu plum extract and suggested it may have chemotherapeutic potential in cancer.3–5

With reported concentrations up to 5300 mg/100 g, kakadu plum has the highest levels of ascorbic acid (vitamin C) of any fruit in the world.6,7 By comparison, this is about 500 times those found in blueberries (10 mg/100 g) and 100 times those found in oranges (53 mg/100 g). Kakadu plum is also rich in other valuable phytochemicals, including various polyphenols (mainly gallic and ellagic acids), as well as vitamin A, vitamin E and folate.8,9 It is believed that the plant has developed such a powerful cocktail of antioxidant molecules as a mean to protect itself from the consequences of the high UV index that prevails in Australia, due to the thinning of the ozone layer. Kakadu plum is a ‘superfruit’ that holds great promise for skin care applications. 

The present paper documents the beneficial effects of kakadu plum extract on skin structure and radiance and reveals its anti-inflamm’ageing possibilities, as supported by in vitro and clinical studies.

Material and methods

Kakadu plum extract
The ripe fruit is sustainably hand-picked, from January to June, by small teams on foot, in Australia. The extract is prepared from the whole fruit through a proprietary aqueous process (Phyto-CrossTM) that maintains the integrity of the botanical active components.

In vitro studies 

  •  Total antioxidant activity 

The free radical scavenging capacity of kakadu plum extract was evaluated using the DPPH (2,2-diphenyl-1-picryl-hydrazylhydrate) method. DPPH is a stable free radical with a deep violet colour in solution. It becomes colourless or pale yellow when neutralised. The resulting colour change is stoichiometric with respect to the number of electrons captured. Colour change can thus be used as a direct measure of the free radical scavenging activity of a given substance. For this experiment, kakadu plum extract (0.05%, 0.1%, and 0.2%) or equivalent concentrations of vitamin C were added to a DPPH solution (10–4 M) and the mixtures were incubated for periods of 30 minutes and 24 hours, at room temperature. Trolox (an analog of vitamin E) served as a positive control. The DPPH solution without any addition served as a negative control. At the end of the incubation period, the absorbance of the solutions was measured at 540 nm. The lower the absorbance, the higher is the scavenging activity. The activity was calculated using the following formula: 

DPPH scavenging activity (%) = (1–[(Xextract –XTrolox)/(Xneg.control –XTrolox)]) x100 

where Xextract is the absorbance in the presence of the extract, Xneg.control is the absorbance of the negative control and XTrolox is the absorbance of the positive Trolox™ control. Statistical analysis of results was done using 2-way ANOVA followed by Bonferroni post-test.

  •  Evaluation of hyaluronic acid synthesis 

Normal human dermal fibroblasts (NHDF) were cultured in complete DMEM for 24 h. The culture medium was then replaced with serum-free DMEM for an additional 24 h to promote a quiescent state. Transforming growth factor beta (TGF-b) (1 ng/mL), used as a positive control, or kakadu plum extract (0.05%, and 0.2%) was then added and the incubation prolonged for 24 h. At the end of the incubation period, supernatants were collected and hyaluronic acid expression levels were quantified using ELISA. In parallel, cell viability was assessed with the MTT assay. Results were expressed as a percentage of basal level, taking into account cell viability.

 

  • Evaluation of pro-collagen I synthesis 

Normal human dermal fibroblasts (NHDF) were cultured in complete DMEM for 24 h. The culture medium was then replaced with serum-free DMEM for an additional 24 h to promote a quiescent state. Transforming growth factor beta (TGF-b) (10 ng/mL), used as a positive control, or kakadu plum extract (0.05%, 0.1%, 0.2% and 0.4%) was then added and the incubation prolonged for 24 h. At the end of the incubation period, supernatants were collected and pro-collagen I expression levels were quantified using ELISA. In parallel, cell viability was assessed with the MTT assay. Results were expressed as a percentage of basal level, taking into account cell viability.

 

  • Evaluation of syndecan-I synthesis

Human keratinocytes (NCTC 2544) were grown under normal conditions in a Lab-Tek chamber slide system which consists of a removable polystyrene media chamber attached to a specially treated standard glass microscope slide. Cells were incubated for 24 h, before addition of kakadu plum extract (0.2% and 0.4%), or TGF-b (10 ng/mL) as a positive control. Incubation was resumed for an additional period of 72 h, at the term of which cells were chemically fixed to their slides.

Following fixation, cells were incubated with a specific syndecan-I monoclonal antibody. Cells were subsequently incubated with an FITC secondary antibody for 1 h. The fluorescence signal (em. 488 nm, exc. 520 nm) was then analysed with an optical microscope and a dedicated software (Image).

 

  • Inhibition of IL-8 production 

Non confluent Human Dermal Fibroblasts (NHDF) derived from adult skin were treated with IL-1a (0.004 ng/mL), a known inducer of IL-8 production, in the presence or absence of kakadu plum extract (0.05%, 0.1% and 0.2%). Dexamethasone (DMS) at 1 mM served as a positive control for IL-8 inhibition. IL-8 levels were quantified using ELISA.

Clinical studies

Anti-wrinkles and fine lines
The study was double-blind and placebocontrolled. Twenty healthy volunteers aged 35 to 55 (mean age 47) were recruited. Each volunteer applied the test product (2%) and the placebo in a split-face fashion, twice daily (morning and evening) for 30 days. No sunscreen was used. The trial took place in October 2013. Measurements of skin topography were performed by Laser Scan VISIO-3D (DermaTOP-V3) on the crow’s feet area at Day 0, Day 15, and Day 30. An automatic system of repositioning guaranteed the precise re-identification of the measurement zone. The technique allows for a direct in vivo analysis of the skin surface. OptoCAT scanning software was used for visualisation, capture, and analysis of results. The cutaneous relief parameters thus obtained include the average roughness (Ra), maximum amplitude (Rt), and average relief (Rz). The Rz parameter presents the advantage of not being much affected by artefacts. Statistical analysis of results was done using the Student’s t-test (intergroup analysis; p<0.005).

Skin radiance
The radiance of skin (gloss value parameter) was also evaluated at Day 0, Day 15, and Day 30, for the group of volunteers described above. Gloss value, defined as the ability of the skin to reflect light, was measured using the CM-700D spectrophotometer/colorimeter (KonikaMinolta). The instrument evaluates colour in accordance with a standardised method validated by the International Commission on Illumination (CIE). Data were analysed statistically by means of intra-group comparison (2 tailed Student’s t-test). Significance was established at p<0.05.

Results and discussion

Antioxidant activity
Kakadu plum is one of the richest natural sources of vitamin C, which is a potent antioxidant molecule.3 In line with this property, the present kakadu plum extract demonstrated strong antioxidant activity when tested using the DPPH free radical scavenging assay. Within 30 minutes, the scavenging activity of the extract reached +54% and increased to +79% at 24 h when tested at a dosage of 0.2% (Fig. 2). The response was dose-dependent. At all concentrations tested, the scavenging activity of kakadu plum extract was superior to what could be observed in the presence of vitamin C alone, suggesting that additional antioxidant molecules within the extract may contribute to its better performance in the assay. Since oxidative stress contributes significantly to both extrinsic and intrinsic skin ageing, such results point to substantial anti-ageing benefits for kakadu plum extract.10

Stimulation of dermal component synthesis
Structural proteins are the backbones of skin. For instance, collagen I and hyaluronic acid are both important structural components that provide tensile strength to the dermis. Collagen is synthesised by fibroblasts as a precursor molecule (pro-collagen). Following its processing into collagen and secretion in the extracellular matrix (ECM), the molecules self-assemble to define the shape and the form of skin tissues.11 For its part, hyaluronic acid is a natural linear polysaccharide, synthesised by fibroblasts and keratinocytes, which assembles in the plasma membrane of cells before being released in the ECM. Hyaluronic acid contributes to maintain skin’s hydration and viscoelastic properties.12 Loss of collagen and hyaluronic acid are observed in ageing skin.13 

 

 

 

 

 

In the present study, kakadu plum extract was able to dose-dependently stimulate the synthesis of pro-collagen I and hyaluronic acid in fibroblasts. Following 24 hours of incubation in the presence of the extract, hyaluronic acid synthesis was increased by +59% at 0.2%, while the rate of pro-collagen I synthesis was increased by +68% at 0.2% and +144% at 0.4% (Figs. 3 & 4). Concurrent stimulation of both proteins synthesis in the presence of TGF-b, used as a positive control, validated the experiments. Supporting the expression of structural proteins such as collagen and hyaluronic acid is known to stimulate fibroblast functions in ageing, thus contributing to skin rejuvenation.14

 

Stimulation of syndecan-I expression
Syndecan-I is a transmembrane heparin sulfate proteoglycan strongly expressed in keratinocytes.15 The molecule has multiple functions: it serves as a co-receptor for growth factors,16 binds to ECM molecules to provide structural support,17 and also modulates the activity of chemokines, cytokines, integrins, and other adhesion molecules which play important roles in the regulation of inflammation.18 Syndecan-I expression is reduced in aged skin.19 

 

 

 

As shown in Figure 5, kakadu plum extract was able to, dose-dependently, stimulate syndecan-I expression in keratinocytes reaching +90% at 0.2% and +104% at 0.4%. Again, concurrent stimulation of the synthesis of the protein in the presence of TGF-b, used as a positive control, validated the experiment. Overexpression of syndecan-I in the epidermis has been associated in the past with the stimulation of epidermal proliferation and a decrease of keratinocyte adhesion which improves desquamation.20 Stimulation of syndecan-I expression in the presence of the extract thus suggests a positive impact on epidermal renewal, a skin property adversely affected by age.21

Anti-inflammatory activity
Under the constant pressure of external and internal challenging factors, the skin tends to accumulate oxidative damages with time, contributing to the development of subclinical chronic inflammatory reactions.22 The phenomenon has been termed ‘inflamm’ageing’ highlighting the fact that such low grade but persistent inflammation is a major driver of tissue ageing. Inflamm’ageing involves the mobilisation of pro-inflammatory cytokines such as IL-1a and IL-8.23 IL-1a is an early initiator of inflammatory responses that induces the release of other cytokines, including IL-8. The latter recruits neutrophils and macrophages from blood vessels and activates dermal fibroblasts to secrete matrix metalloproteinases (MMPs).23

In turn, activated MMPs attack and degrade collagen and elastin fibres weakening the structure of the dermis and leading to the apparition of signs of ageing such as fine lines, wrinkles, and skin sagging.23 Interleukin-driven inflammation is also a major cause of skin redness. 

As expected, treatment of fibroblasts with IL-1a induced an abundant release of the pro-inflammatory cytokine IL-8. The response was inhibited in the presence of kakadu plum extract by –15% and –34% at concentrations of 0.05% and 0.2% respectively (Fig. 6). At 0.2%, the inhibition observed with the extract was superior to that of DMS, a known anti-inflammatory molecule. Kakadu plum extract thus represents a good candidate to fight global inflammatory reactions including inflamm’ageing in the skin.

Anti-wrinkle efficacy
Taken together, in vitro results indicate that kakadu plum extract has a strong potential as an anti-ageing active ingredient. The hypothesis was tested in a clinical trial involving 20 volunteers, looking at antiwrinkle efficacy, in a split-face study against placebo. As reported in Figure 7, the extract significantly (*p<0.05) decreased the appearance of wrinkles in average by 5% at D15 and –11% at D30. Maximal responses of –21% at D15 and –38% at D30 were registered (results not shown). All subjects (100%) achieved skin roughness improvement. A representative result is shown in Figure 7. Reducing the appearance of wrinkles conferred a younger look to the skin of volunteers.

Improvement of skin radiance
Skin radiance (gloss value) was also evaluated by spectrophotometry, for the group described above. As shown in Figure 8, kakadu plum extract significantly (*p<0.05) improved skin radiance on average by +10% at D15 and +17% at D30. Maximal responses of +21% at D15 and +30% at D30 were registered (results not shown). By improving skin radiance kakadu plum extract promoted a younger and healthier appearance in volunteers.

Conclusion

Building on the antioxidant potential of kakadu plum, we demonstrated that kakadu plum extract (more specifically Superox-CTM, a proprietary extract) supports and stimulates the synthesis of structural components of the dermis (collagen I and hyaluronic acid) and the epidermis (syndecan-I). The extract also modulates the production of a pro-inflammatory cytokine (IL-8) involved in inflamm’aging. These properties translated into a reduction of the appearance of wrinkles and an improvement of skin radiance in human volunteers. For the formulator, the extract offers potent anti-ageing benefits and could also be used to dim inflammatory processes that may lead to skin erythema. Kakadu plum extract is a multifunctional active ingredient ideal for use in a wide range of applications including anti-ageing and anti-wrinkle care, radiant care, sun care, and skin protection. 

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