Ameliorating Effects of Delphinol ® , Anthocyanin Standardized Maqui Berry Extract, on Skin Brightness and Redness in Japanese Females: A Randomized Double-Blind Placebo-Controlled Pilot Study

Maqui berries are wildly grown or cultivated in the Patagonian region in Chile. The berries contain large amounts of anthocyanins, especially delphinidin glycosides, which are strong anti-oxidants. We previously reported that standardized maqui berry extract, which contained lots of anthocyanins, has ameliorative effects against dry eyes and visible light-induced photoreceptor cell damage. However, the influence of maqui berry extract on the human skin has not been evaluated even though many skin troubles are related to ac-tive oxygen species. Therefore, we conducted a randomized double-blind placebo-controlled pilot study with Delphinol®, a maqui berry extract standardized for anthocyanins, especially delphinidins and cyanidins, in healthy Japanese female subjects. Capsules containing Delphinol® (60 mg) or placebo were orally administered to subjects aged 27 to 57 years old for eight weeks. Skin conditions [pigmentation, redness, skin tone, wrinkles, texture, oil content, water content, trans epidermal water loss (TEWL), collagen score, dermal thickness, and elasticity] were measured before and after intervention. In the Delphinol® group, the saturation significantly increased after eight weeks and reddish spots significantly decreased after four weeks of intake. The collagen score,


Introduction
Maqui berry (Aristotelia chilensis) is a shrub distributed in Southern Chile, especially in the Patagonia region. The dark purple fruit is high in anthocyanins [1], including delphinidin glycosides and cyanidin glycosides [2], and exhibits excellent anti-oxidant activity [3], which can protect food from oxidation [4]. It is not only consumed as fresh fruit, but also as processed foods including liquors [5], beverages [6], and functional foods [7]. Recently, the amount of exported maqui berry products has increased. In order to prolong the shelf life of anthocyanins in processed foods, trials selecting suitable binders that can stabilize maqui berry juice or extract in powder form [8], and conditioning to increase anthocyanin stability in liquid form [9] have been performed.
Regarding the beneficial health effects of maqui berry, anti-inflammatory [10] [11] [12] [13], anti-diabetic [14], antidepressant [15], anti-obese [16], and memory improving [17] effects have been reported. Moreover, we previously confirmed that maqui berry extract (MBE) possesses excellent ameliorating effects against dry eyes in animal models [18] and clinical trials [19] [20] with Japanese subjects. Moreover, we found that the extract has photoprotective activity in photoreceptor cells in mouse eyes [21]. On the other hand, we developed a MBE named Delphinol®, which has a standardized anthocyanin content including delphinidin glycosides. Delphinol® exhibits potent anti-oxidant [22], hypoglycemic [23] [24], and anti-bone loss [25] activities. However, the effects of MBE on the skin condition have not been examined except for its skin photoprotective activity in model cells [26]. Therefore, we conducted a clinical trial of MBE ingestion regarding facial skin parameters in Japanese female subjects.

Participants
Female employees aged 27 to 57 years were recruited by Oryza Oil & Fat Chemical Co., Ltd. Their work categories are office workers and researchers. They have an employment relationship with Oryza Oil & Fat Chemical Co., Ltd. Exclusion criteria were as follows: 1) Current use of medications for treatment of chronic diseases.
2) Allergy to the test product.

4) Use of oral or topical medications.
Then, 16 subjects were randomly allocated to each of two groups by a third member who was not involved in the study. The average age was 37.3 ± 8.9 years N. Shimizu et al. old in the Delphinol® group and 38.5 ± 10.5 years old in the placebo group. Informed consent was received from all subjects prior to the study. After a full explanation of the study, including the purpose, methods, and test product, in addition to the voluntary nature of participation and no disadvantages from refusing to participate, written consent was received from all participants. The subjects were asked to maintain a regular lifestyle during the study period.

Study Protocol
The study design was a placebo-controlled double-blind comparison of two groups treated concurrently and was carried out at Oryza Oil & Fat Chemical Co., Ltd.
Subjects in the placebo and Delphinol® groups took one capsule daily (placebo or Delphinol®, respectively) with water after breakfast for eight weeks. Each participant was examined at the Oryza Oil & Fat Chemical Co., Ltd. (Aichi, Japan). The temporal scope of the study comprised duration from October 17, to December 12, 2019. Subjects were examined for skin conditions before the start of treatment, and after four and eight weeks of intervention. The outcomes were pigmentation, redness, skin tone, wrinkles, texture, oil content, water content, trans epidermal water loss (TEWL), collagen score, dermal thickness, and elasticity.

Evaluation of Skin Parameters
All skin parameters were measured in an air-conditioned room at 25˚C ± 1˚C and 50% ± 5% RH. Prior to the study, the subject washed her face. After wiping, Skin elasticity was measured at the mid-point of a line between the right eye and right ear by a Triplesense (MORITEX Co., Saitama, Japan). Evaluation of TEWL was performed using a VapoMeter (Delfin Technologies Ltd., Kuopio, Finland). Lastly, the collagen score and dermal thickness were analyzed via DermaLab (Cortex Technology, Hadsund, Denmark).

Ethics, Adherence, and Compliance
This study was performed according to the Declaration of Helsinki (2013 revision, Fortaleza, Brazil) and was carried out in conformity with ethical considerations. The ethics committee of Oryza Oil & Fat Chemical Co., Ltd. was convened to assess the ethicality and appropriateness of the study protocol. The study was implemented according to the protocol approved by the ethics committee, and any substantial protocol deviations required authorization by the committee. All subjects received a full explanation about the purpose and procedures of the study before consenting to participation.

Statistical Analysis
The results are reported as the mean and standard deviation (SD). A two-tailed paired t-test was used for comparisons between before and after the ingestion of capsules. The unpaired t-test was used to compare the placebo group with the Delphinol® group. A probability of less than 5% was considered significant.

Study Performance
None of the subjects took less than 90% of the specified capsules during the study period. Accordingly, data on all subjects were included in the analysis.

Skin Tone (Hue, Saturation, and Brightness)
The skin tone (hue, saturation, and brightness) scores before and after ingestion, and the degree of change after ingestion are shown in Table 1

Skin Redness
The number (Lv. 1, 2, 3) and area (Lv. 1, 2, 3) of redness before and after ingestion, and the degree of change after ingestion are shown in Table 2

Collagen Score
The collagen scores before and after ingestion, and the degree of change after ingestion are summarized in Table 3. There was no significant difference between the placebo group and the Delphinol® group during all periods.
On the other hand, as shown in Figure 2, the collagen score slightly decreased with age (as at baseline). However, after taking Delphinol® for eight weeks, the correlation between age and collagen score slightly increased.

Other Skin Parameters
Comparison of values before and after intervention and between placebo and the Delphinol® groups revealed no significant difference in the items, including pigmentation, number and length of wrinkles, texture, oil content, water content, TEWL, thickness of dermis, and elasticity (Table 4).

Discussion
In the present study, ingestion of Delphinol® for eight weeks improved the skin tone saturation compared with the placebo group. The saturation is presented as a value ranging from 0 to 100, and a higher value means improvement of the skin color tone, termed shining skin tone. Therefore, Delphinol® can improve the saturation, suggesting color tone-up effects. In the three-dimensional space of chromatic colors (hue, saturation, and brightness), a higher skin saturation value is related to higher emotion and attractiveness [27]. Moreover, color saturation is an important optical parameter of skin that determines the visual age of skin in addition to lightness and local light-diffusing ability [28]. Thus, the upregulating effects of Delphinol® on saturation may reflect a positive influence on the facial skin condition.
N. Shimizu et al. Actual scores (upper lines) and changes in the scores (lower lines) are represented as the mean ± SD (n = 8 in both groups).  Delphinol® ingestion for four-weeks significantly reduced the degree and area of redness on the face compared with the placebo group. Facial redness is clinically termed facial erythema or rosacea. It is caused by sensitive skin [29], winter xerosis [29], alcohol ingestion [30], and UV radiation [31]. A red face can cause depression or may result in complex social relations [30]. In addition, skin diseases can induce facial redness indirectly. Withdrawal from steroidal ointment in atopic dermatitis patients sometimes causes redness, known as steroid addiction syndrome [32]. To ameliorate facial redness, the use of cosmetics is dominant, including sun protectors [33] [34]. However, alternative care using herbs and nutritional dietary supplements has been examined. These methods are defined as oral photoprotection [35], in addition to the commonly used supplementation of vitamins C and E [36], and carotenoids [37]. For example, dietary supplements containing rosemary and grapefruit extracts were reported to reduce UV-induced skin redness [38]. Moreover, oral treatment with vitamin D has anti-inflammatory effects and improves skin redness [39]. However, although there are many botanical extracts and their constituents that exhibit photoprotective activity by ingestion, especially polyphenols, only the above ingredients have been reported to improve facial redness. So, the suppression of N. Shimizu et al. skin redness by Delphinol® is a novel observation. The anti-oxidant effects are most likely related, in addition to the photoprotective effects of Delphinol® [26] because anthocyanins can suppress allergic inflammatory responses in mast cells [40]. Released histamine induces skin flare [41]. The anti-oxidant effects of Del-phinol® are involved because oxidative stress induces skin inflammation [42] and redness. Thus, the anti-oxidant effects of Delphinol® can help to suppress inflammation. Kikuchi et al. [43] evaluated the seasonal change in hemoglobin index (one of the indicators of redness) in the face of Japanese women, and reported that facial skin redness increased from autumn to winter. Our present study was conducted from autumn to winter. However, regardless of seasonal factors, improvement of color unevenness due to redness can be expected by Delphinol® supplementation. Kikuchi et al. [44] also reported that Caucasian skin becomes more reddish with age than Asian skin. Therefore, Delphinol® is expected to be useful for improving the color unevenness not only Asian women, but also in Caucasian women.
Regarding the collagen score, although there was no significant difference, the Delphinol® group exhibited a slight increase after eight weeks of ingestion compared with before the ingestion. So that, we analyzed the relationship between collagen score and age. As a result, a positive relationship was observed in the Delphinol® group but not in the placebo group. The collagen score analyzed by ultrasound skin images captured by DermaLab is recently used for non-invasive diagnosis [44]. tra-observer reproducibility [46]. Thus, Delphinol® was suggested to possess some ability to maintain collagen in the dermis. Further studies of Delphinol® on collagen production and collagen decomposition are needed to clarify the exact mechanism. Besides this, some limitations exist in this study. First, since the study subjects were small number, they may be healthier than the general population, causing a selection bias. Second, all subjects were Asian women and worked in Oryza Oil & Fat Chemical Co., Ltd. which is another area of potential bias. But in future, we would like to do more experiments with women in different areas to verify the effects.

Conclusion
In conclusion, we found that ingestion of Delphinol®, anthocyanin-standardized maqui berry extract, increases skin brightness and collagen content, and reduces skin redness. These effects suggest that the maqui berry can maintain a healthy N. Shimizu et al.