Solar radiation is a significant environmental factor in skin damage and can induce skin cancer [14]. UV radiation causes a pro-inflammatory response, extracellular matrix degradation and antioxidant depletion [15, 16]. UV causes formation of reactive oxygen species (ROS) that induce hyperpigmentation and collagenase expression [17, 18]. Our study investigated 14 Thai plants extracted with three different solvents for their potential as anti-wrinkle and skin whitening ingredients. In this study, we used petroleum ether, dichloromethane and ethanol for plant extraction using Soxhlet apparatus. Ardisia elliptica Thunb. had the highest yield in the petroleum ether and ethanol extracts, whereas Garcinia mangostana L. had the highest percent yield from dichloromethane extraction. These solvents are a series of organic solvents with increasing polarities. Variation among the percent yields depended on the plant species, and probably reflected differences in chemical composition of the plants.
Phenolics are the largest group of phytochemicals found in plants and they have various biological activities in animals, including humans [19]. Total phenolic content in the plants was determined by the Folin-Ciocalteu method. Overall, the ethanol fraction had the richest phenolic content, followed by dichloromethane, while petroleum ether with low polarity had the lowest phenolic content compared to the other solvents. In this study, Ardisia elliptica Thunb. had the highest phenolic content in all three types of extracts. In previous studies, dichloromethane leaf extracts of Ardisia elliptica Thunb. have a phenolic content of 101 ± 1.3 mg GAE per g dry plant material, which is more than the content in a twig extract [20]. Moreover, a methanol extract of ripe Ardisia fruit contained 5.64 ± 0.37 g GAE per 100 g extract [21]. Hence, leaves and fruits of Ardisia elliptica Thunb. have a high phenolic content that can be easily extracted with methanol, dichloromethane and ethanol.
Flavonoids are pigments in flowers, leaves, fruits and seeds. These compounds are secondary metabolites of plants and are widely distributed among plant species [22]. Next, the flavonoid content within the Thai plants was evaluated using the aluminium chloride colorimetric assay. Our results showed that the highest flavonoid quantity was found in the ethanol extract from Senna alata (L.) Roxb leaves. In a previous study, high flavonoid content was found in water (4.25 mg QE per 100 g) and methanol fractions (3.97 mg QE per 100 g) of Senna alata (L.) Roxb. [23]. Thus, Senna alata (L.) Roxb preparations have a high flavonoid content when extracted with high polarity solvents including ethanol, methanol and water. Ardisia elliptica Thunb. had the richest flavonoid content in the dichloromethane fraction. Fruit of this plant also has a high flavonoid content 36.91 ± 2.37 mg QE per g extract [24]. Hence, fruit and leaves of Ardisia elliptica Thunb. are rich in flavonoids. Total flavonoid content did not correlate with total phenolic content. However, flavonoids have many biological activities such as UVB protection [25], anti inflammatory [26], anti-hepatotoxicity [27] and anti cancer [28].
Free radical scavenging activity using DPPH and ABTS assay. In the DPPH assay, DPPH receives a hydrogen atom from an antioxidant [29]. We found that Ardisia elliptica Thunb ethanol extract had the highest scavenging activity. Other investigators have also reported that dichloromethane fractions of Ardisia elliptica Thunb. leaves and stems have high levels of antioxidant activity as determined by the DPPH assay, and, hence, this plant is very interesting to investigate further as a herbal treatment [20]. The extracts from the ethanol fraction with high polarity clearly showed better antioxidant activity than fractions with lower polarities containing dichloromethane and petroleum ether. Ethanol extracts contained the highest levels of free radical scavenging activity compared with the other extracts, and all ethanol extracts were active. In the ABTS assay, ABTS is converted to its radical cation by the addition of potassium persulfate. In the presence of an antioxidant, the reactive ABTS cation (or ABTS•+) is converted to its colorless natural form [9]. In agreement with the DPPH assay, ethanol extracts contained the highest levels of scavenger activity as compared with the other extracts. Again, the highest scavenging activities in ethanol, dichloromethane and petroleum ether extracts were from the same plants as shown by the DPPH assay. The results of the DPPH and ABTS assays were highly correlated as expected (Fig. 1f).
However, total flavonoid content of the plant extracts did not correlate with free radical scavenger activity as detected by the DPPH assay (Fig. 1c) or by the ABTS assay (Fig. 1e). Our findings of no significant relationship between flavonoid content and scavenger activity using the ABTS assay is consistent with other investigators’ results [30]. By contrast, total phenolic content of the plant preparation positively correlated with scavenger activity measured by both assays (Fig. 1b and d) in agreement with a previous study [31]. Noticeably, the scavenging activity depended on total phenolic content and solvents with high polarity, such as ethanol and dichloromethane. These results suggest that the phenolic content is the major constituent with antioxidant activity in the 14 Thai plants.
Melanin, the major pigment of skin and hair color, is synthesized by melanocytes within melanosomes. Overproduction and accumulation of melanin in skin may lead to pigmentary disorders and aesthetic problems. Hyperpigmentation occurs in sun-exposed areas of the skin [32]. In the melanogenesis, tyrosinase is the key enzyme in the rate-limiting step in which L-tyrosine is hydroxylated to L-DOPA, which is further oxidized into DOPAquinone. After that, it is converted into DOPAchrome that is a substrate for melanin synthesis [3]. Downregulation of tyrosinase activity has been proposed to be responsible for decreased melanin production. The development of novel whitening phytochemical compounds from natural products has recently become a growing trend. Our finding showed that the ethanol fraction from Rhinacanthus nasutus (L.) Kurz was the most potent tyrosinase inhibitor, followed by the ethanol extracts from Ardisia elliptica Thunb. and Phyllanthus acidus (L.) Skeels. Obviously, 7 plants from 14 plants had the high phenolic content, especially Ardisia elliptica Thunb. and Annona squamosa L.. Moreover, Senna alata (L.) Roxb. had the richest flavonoid content which can inhibit tyrosinase activity. Active compounds from the plants such as arbutin, aloesin, gentisic acid, flavonoids, hesperidin, licorice, niacinamide, yeast derivatives, and polyphenols, can inhibit melanogenesis without cytotoxicity to melanocytes [6].
Collagenase is a transmembrane zinc peptidase that cleaves the X-Gly bond of collagen. Collagen is an abundant structural protein and extracellular matrix component [33]. Decreased collagen and elastin fibers increases with age and damage from UV radiation inducing wrinkled skin [34]. Collagenase inhibition has been proposed to prevent skin aging. Of those causing inhibition in our study, Ardisia elliptica Thunb. exhibited the highest level of collagenase inhibition, followed by Annona squamosa L., Senna alata (L.) Roxb., and Croton sublyratus Kurz in rank order. In a previous study, cocoa pod extract had phenolic acid and flavonoids that inhibited elastase and collagenase activity [35]. Notably, three ethanol extracts (Ardisia elliptica Thunb., Annona squamosa L. and Senna alata (L.) Roxb. inhibited both tyrosinase and collagenase. These plants also had high phenolic and flavonoid levels, and antioxidant activity. Interestingly, these extracts have possible uses as ingredients for cosmetic products.