Tyrosinase Inhibitory Activities of Fruit-ext, Leaf-ext, Seed-ext and Kojic Acid (a) OD: optical density. Each value represents the mean±S.E. of 3 experiments. Significantly different from control group, i:
During the aging process, morphological changes in the human skin appear most noticeably in areas of frequent exposure to ultraviolet (UV) light from the sun, such as the face and hands. Chronic UV exposure induces photoaging, characterized by pigmented spots and wrinkles in the skin. Gradual destruction of the ozonosphere has raised photoaging risk. This has led to rapid growth of the anti-photoaging cosmetic market, especially among women with young and fair skin. Sunscreen agents are a first choice for protection against photoaging. However, a certain amount of UV irradiation penetrates skin dermis, and adverse effect may occur with use of these agents. Because of this, a current trend is the development of safer cosmetic ingredients that effectively inhibit the UV signaling pathways leading to photoaging .
Therefore, in this chapter we discuss searching for novel cosmetic ingredients from natural resources which prevent the generation of pigmented spots and wrinkles
2. Subtropical plant noni
Noni has been utilized for various reasons in many areas. The root was used as dye in Japan, and the leaves and the seeds, as well as the fruit, were eaten frequently in Southeast Asia and the Pacific islands. Currently, the fruit juice and leaf tea are sold in the functional foods market. Noni is also used as an herbal medicine to promote health and beauty. The whole plant, from the root to fruit, has been used without waste for more than 2000 years as a panacea . Therefore, noni was considered a "gift from God" by generations of Pacific Islanders.
In the last decade, many papers have reported the chemical constituents and biological activities of noni including hypotensive , hypoglycemic , and anticancer activities , which scientifically support traditional claims of noni. Noni has been used traditionally for the treatment of dermatoses such as ringworm, dry skin, acne, pustule, and other skin troubles. Moreover, ripe noni fruit juice has been drunk for cosmetic reasons. In some areas, the dried immature noni fruit, the leaves, or the seeds mixed with coconut oil have been used as an external treatment . But studies reporting potential cosmetic uses of noni, such as inhibition of melanogenesis and reduction of wrinkles, have only recently been completed. Noni fruit contains a large number of seeds throughout its flesh. But during the production of noni fruit juice, the seeds are removed and discarded. Considering the potential utility of all parts of the plant, we investigated extracts from the fruit flesh, leaves, and seeds for active anti-photoaging agents.
3. Skin whitening effect of noni
3.1. Screening tests for melanogenesis inhibitory effect of noni
During photoaging, UV rays trigger melanogenesis and chromatosis. These processes generate pigmented spots by UV activation of melanocyte tyrosinase (a melanin synthesis enzyme) which then converts L-tyrosine to L-DOPA, followed by conversion to dopaquinone. Dopaquinone subsequently forms melanin through several steps, including auto-oxidation. As such, tyrosinase inhibitors may be useful for prevention of pigmented spots.
Initial evaluation of the anti-melanogenesis activity of noni was carried out using an
3.1.1. Tyrosinase inhibitory activities
The results of the
3.1.2. DPPH radical scavenging activities
As oxidative reactions contribute to melanogenesis, the DPPH assay was performed to measure the antioxidant activity of noni. As shown in Table 2, Seed-ext exhibited potent DPPH radical scavenging activity, with an IC50 value of 12 µg/ml. Leaf-ext and Fruit-ext exhibited weaker antioxidant activities, with IC50 values of 113 and 240 µg/ml, respectively .
The results of the two assays reveal that Seed-ext has stronger tyrosinase inhibitory and antioxidant activity than Fruit-ext and Leaf-ext.
|Kojic acid||10 (µM)||207±3i||56|
|Fruit-ext||100 (µg/ml)||759±3i||22||240 (µg/ml)|
|Leaf-ext||50 (µg/ml)||765±4i||22||113 (µg/ml)|
|Seed-ext||5 (µg/ml)||771±2i||21||12 (µg/ml)|
|L-Ascorbic acid||20 (µM)||570±4i||41||23 (µM)|
3.2. Inhibitory effect of noni seeds on melanogenesis and its active compounds
According to the
The tyrosinase inhibitory activity of Seed-ext was not as potent as that of other well known skin whitening agents. But in the B16 melanoma cells culture system, it inhibited melanin production. Thus, noni seed may be useful as an anti-photoaging cosmetic ingredient which prevents pigmented spots by interacting with a different active site than existing general skin whitening agents.
|Kojic acid||100 (µM)||1||13.5±0.4i||107.4±1.9|
As Seed-ext was confirmed to potently inhibit melanogenesis, activity guided isolation of the active compounds was carried out. Two lignans, 3,3'-bisdemethylpinoresinol (
Compared to kojic acid, which is a common skin whitening ingredient,
3.3. Effects of noni compounds; 3,3'-bisdemethylpinoresinol and americanin A on inhibitory melanogenesis activity
As the anti-melanogenesis of Seed-ext may be due to
3.3.1. Effects of 3,3'-bisdemethylpinoresinol and americanin A on tyrosinase expression in B16 melanoma cells
First, the effect of
3.3.2. Inhibition of tyrosinase in B16 melanoma cells by 3,3'-bisdemethyl-pinoresinol and americanin A
With suppressed expression of the enzyme, the activity of tyrosinase in the cell may decrease. Secondly,
As shown in Fig. 2A, the intracellular melanin content of control group increased remarkably after cultivation for 24 to 72 hrs, whereas the content in cells treated with
As just described, lignans
3.3.3. Effect of 3,3'-bisdemethylpinoresinol and americanin A on phosphorylation of p38 MAPK
During melanogenesis in melanocytes, it is known that microphthalmia-associated transcription (MITF) is a transcription factor that regulates expression of the tyrosinase gene, and that melanins are produced by the activation of MITF . Since mitogen-activated protein kinases (MAPKs) pathway is one of the intracellular signals that activates MITF, further research on this pathway was performed. It has been reported that phosphorylation of p38 MAPK activates MITF, whereas that of ERK1/2 and p70 S6K suppress MITF [12, 13]. The effects of
The levels of phosphorylation of p38 MAPK were compared, at 6 and 12 hrs after stimulation of B16 melanoma cells by
As the inhibitory mechanism of melanogenesis in α-MSH-stimulated B16 melanoma cells, These results strongly suggest that the lignans inhibit tyrosinase expression by suppressing p38 MAPK phosphorylation and enhancing ERK1/2, which then prevents activation of MITF.
4. Inhibitory effect of noni on wrinkle formation
It is thought that wrinkles form mainly by aging, but they are also caused by photoaging. The causes are degradation of moisture-retaining property of epidermis and transformation of dermal configuration due to altered elastic or collagen fibers in the cutis. Anti-wrinkle cosmetics are useful for comparatively slight wrinkles and fine lines. The main functions of anti-wrinkle cosmetics included normalization of moisture retention by the cornified layer, acceleration of keratinocyte turnover, and acceleration of collagen and elastin synthesis through proliferation and stimulation of fibroblasts. Recently, retinol and its analogs, so-called retinoids, have received increased attention and utilization as a cosmetic ingredient because they promote fibroblastic collagen production in the cutis and improve skin tension. Also, vitamin A reportedly accelerates turnover, which reduces hyperpigmented skin spots caused by melanin excreted by the epidermis, even though it has no inhibitory effects on activation of tyrosinase and melanogenesis. But retinoids irritate the skin and have adverse effects such as dermatitis. Therefore, safer reducing wrinkle ingredients are needed .
4.1. Wrinkle inhibition screening test of noni
One cause of photoaging wrinkle formation is the degradation of collagen, a main component of corium connective tissue . The degradation of collagen is promoted by release of human leukocyte elastase (HLE) from infiltrated neutrophils into the skin by UV irradiation . HLE cleaves the triple helix structure of type I collagen and degrades elastic fiber in human skin . Thus, HLE inhibitors may be useful for the prevention of wrinkle formation.
4.1.1. Inhibitory effect of noni on HLE and its active compounds
The inhibitory effect of noni on wrinkle formation was investigated by measuring HLE inhibiting activity
As Seed-ext exhibited potent HLE inhibitory activity compared with Fruit-ext and Leaf-ext, noni seeds appear to be the most source of cosmetic ingredients capable of preventing wrinkle formation during photoaging.
The active compounds in Seed-ext were isolated using the HLE inhibition bioassay as a fractionation guide. Ursolic acid (
|Control A c)||946±23|
|Seed-ext||0.1 (mg/ml)||1052±20i||-14||1.0 (mg/ml)|
|Control B d)||925±9|
|PMSF||0.08 (mM)||676±7ii||29||0.14 (mM)|
4.2. Inhibitory effect of noni seeds on matrix metalloproteinase-1 (MMP-1) secretion and its active compounds
Matrix metalloproteinases (MMPs) are matrix degrading enzymes associated with destructive processes including inflammation, tumor invasion and skin aging . More than 20 subtypes of MMPs have been reported . Among these, MMP-1, secreted from human skin fibroblasts, is mainly responsible for the degradation of dermal type I collagen in the photoaging process . Also, HLE activates MMP-1 . Thus, it is expected that MMP-1 and HLE inhibitors may be useful for the prevention of photoaging and subsequent wrinkle formation.
Since Seed-ext displayed strong HLE inhibitory activity, its ability to inhibit MMP-1 secretion was investigated in UV-irradiated normal human dermal fibroblasts (NHDFs). The amount of MMP-1 protein secreted from NHDFs into culture media was analyzed by Western blotting. UVA irradiation (5 J/cm2) enhanced the secretion of MMP-1 from NHDFs in the vehicle control group at 9 to 48 hrs incubation, as compared to the control group without UVA-irradiation (Fig. 4). The group which was treated with Seed-ext (10 µg/ml) after UVA-irradiated NHDFs inhibited the secretion MMP-1 at 24 to 48 hrs, when compared to the vehicle control (Fig. 4) . Seed-ext was not cytotoxic at 3 to 30 µg/ml.
The active compounds responsible for the inhibitory effect of Seed-ext on MMP-1 secretion were searched for by following bioassay guided fractionation. As shown in Fig. 5A and 5B, 3,3'-bisdemethylpinoresinol (
It is clear that ursolic acid (
4.3. Inhibitory effect of 3,3’-bisdemethylpinoresinol on MMP-1 secretion
In UV irradiated skin, MMP-1 is a major collagenolytic enzyme responsible for collagen damage . It has been reported that UV irradiation promotes expression of MMP-1 in NHDFs  and secretion into culture media . It is also known that UV irradiation activates intracellular fibroblast signals, c-Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) cascades, promotes phosphorylation of JNK and p38, enhances expression of c-Jun and c-Fos, followed by activation of activation protein-1 (AP-1), and, in the end, enhances expression of MMP-1 [14, 24]. In order to find the mechanism of inhibition of MMP-1, compound
4.3.1. Effect of 3,3’-bisdemethylpinoresinol (1) on intracellular MMP-1 expression in UVA irradiation NHDFs
First, the effect of
4.3.2. Effect of 3,3’-bisdemethylpinoresinol on MAPKs phosphorylation in UVA-irradiated NHDFs
The effect of
The results suggest that 1 inhibits MMP-1 secretion in UVA-irradiated NHDFs by decreasing JNK and p38 phosphorylation and suppressing c-Jun and c-Fos expression. This, subsequently, inhibits AP-1 and MMP-1 expression, resulting in a reduction in MMP- secretion.
5. Inhibitory effect of noni seeds on poor blood fluidity
In addition to melanogenesis and degradation of collagen by HLE and MMP-1, blood stagnation may also contribute to hyperpigmented spots on the skin. In oriental medicine, the clinical state of blood stasis in the venous system is called "Oketsu" in Japanese. Accumulation of wastes caused by blood stagnation in the skin leads to pigmentation. Degradation of blood flow, leading to dry skin and atrophy, also leads to wrinkle formation. Therefore, improved blood fluidity is likely to prevent formation of pigmented spots and wrinkles. As cosmetic ingredients that improve blood fluidity are desired for use in anti-photoaging products, the effect of noni seeds on poor blood fluidity was investigated.
5.1. Antithrombotic effect of noni seeds in a disseminated intravascular coagulation (DIC) rat model
The effect of noni seeds on excessive increased blood coagulation, accompanying poor blood fluidity, was examined by using a DIC rat model, induced by lipopolysaccharide (LPS) [25, 26].
Each Seed-ext (200 and 500 mg/kg) was administrated orally once a day to the rats for 7 successive days. LPS was injected on Day 7. Four hrs after injection, blood samples were collected from the abdominal great vein (vena cava). Whole blood passage time was measured by a micro channel array flow analyzer (MC-FAN). As shown in Table 6, the vehicle control group (LPS-induced) significantly prolonged blood passage time, in comparison with the control group. However, Seed-ext dose-dependently reduced passage time .
Platelet count, fibrinogen and fibrin degradation products (FDP) were also measured. Seed-ext had no effect on any of these hematological parameters (Table 6) .
The results from the DIC rat model experiments suggest that Seed-ext improves poor blood fluidity. Therefore, noni seeds may help reduce pigmentation associated with blood stagnation.
5.2. Inhibitory effect of noni seeds, and active consituents, on platelet aggregation
Blood fluidity, as measured by using MC-FAN, is influenced by erythroid deformability, leukocytic adherence ability and thrombocytic agglutinability [28, 29]. Blood flow regulating factors in microcirculation have vascular systemic and blood component functions. Circulatory system function deteriorates with platelet aggregation induced coagulation, degradation of erythrocyte deformability, hemagglutination, elevation of plasma viscosity, and by degradation of fibrinolytic system activation [28, 29].
In order to examine the inhibitory effect of Seed-ext on blood coagulation, collagen-induced platelet aggregation  and polybrene-induced erythrocyte aggregation ,
The results suggest that one inhibitory mechanisms behind the effect of Seed-ext on poor blood fluidity in the DIC rat model may be anti-hemaggulatination by 1, 2, and 3.
5.3. Fibrinolytic activity of noni seeds in rats
Activation of the fibrinolytic system improves blood flow by promoting the lysis of thrombi in blood vessel walls. To better understand the fibrinolytic potential of Seed-ext, as it relates to degradation of blood fluidity, the euglobulin lysis time (ELT) assay in normal rats was conducted. ELT is the time required for the disappearance of a fibrin clot produced by the addition of thrombin to the eugloblin fraction obtained from blood samples . A reduction in ELT reveals activation of fibrinolysis activity, whereas an extension in ELT implies reduced activity .
Seed-ext was administrated orally, and 1 hr later, blood samples were collected. Then ELT was measured using eugloblin fractions from the sample. As shown in Table 8, Seed-ext significantly reduced ELT at dosages from 50 and 200 mg/kg in dose-dependent manner. This reveals that Seed-ext may have an enhancing effect on fibrinolysis activity .
|Control A a)||98±2|
|Control B b)||i.v.||97±2|
|Dextran sulphate sodium salt||5 (mg/kg)||i.v.||32±3ii|
Seed-ext has an inhibitory effect on hemagglutination. But it also activates fibrinolysis, suggesting that it may improve blood flow through anti-coagulation and fibrinolysis systems. As such, noni seeds may be a useful supplementary ingredient for the prevention of both pigmented spots and wrinkles caused by venous blood stagnation.
We are the first to investigate and find 4 inhibitory effects—namely tyrosinase, melanogenesis, HLE, and MMP-1—for noni seeds related to prevention of pigmented spots and wrinkles by photoaging. As a desirable anti-photoaging agent that is antagonistic to the UV signaling pathways of photoaging, Seed-ext may be a useful novel cosmetic ingredient for the prevention or treatment for pigmented spots and wrinkles. Since we found Seed-ext may improve blood fluidity, it may also be a useful supplemental ingredient aimed for beauty. However, further research, including clinical trials, is needed.
Noni fruit flesh and leaves have been used as functional foods, but the seeds have been discarded without utilization in most cases. Production of a cosmetic ingredient from noni seeds adds significant value to this largely unused natural resource.
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