Part 1: Why Herbal Extracts Can Be Potential Antioxidant, Anti-Aging, Anti-Inflammatory, And Whitening Cosmeceutical Ingredient
Mar 22, 2022
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Wantida Chaiyana, Wannaree Charoensup, Suwannee Sriyab, Chanun Punyoyai, and Waranya Neimkhum
Abstract: The aim of this research was to investigate and compare the antioxidant, anti-tyrosinase, anti-aging, and anti-inflammatory activities of 16 herbal extracts for topical application in cosmetic/cosmeceutical products. Herbal plant materials were extracted by infusion in boiled water for 15 min. The total phenolic content and total flavonoid content of each extract were investigated by the Folin–Ciocalteu and aluminum chloride methods, respectively. Antioxidant activities were investigated using 2,2’-diphenyl-1-picrylhydrazyl and a ferric reducing antioxidant power assay. Anti-tyrosinase and anti-aging activities were investigated using an in vitro enzymatic-spectrophotometric method. Anti-inflammatory activities were investigated using an enzyme-linked immunosorbent assay. The findings show that the Stevia rebaudiana extract has the most significant levels of both phenols and flavonoids (p < 0.05). The S. rebaudiana, Rosa damascene, and Phyllanthus emblica extracts possessed the most significant antioxidant activities (p < 0.05) and a promising whitening effect with moderate anti-tyrosinase activities. Furthermore, the Echinacea purpurea extract possessed the most significant anti-collagenase (78.5 ± 0.0%), anti-elastase (69.0 ± 1.4%), and anti-hyaluronidase activity (64.2 ± 0.3%). The Morus alba extract possessed the most significant anti-inflammatory activity since it could inhibit the secretion of interleukin-6 and tumor necrosis factor-α(p < 0.05). Therefore, these herbal extracts have promising skin benefits and have the potential for use as active ingredients in cosmetic/cosmeceutical products.
Keywords: antioxidant • anti-tyrosinase • collagenase • elastase • hyaluronidase • interleukin-6 • tumor necrosis factor-alpha
Introduction
The skin is the body's outermost layer, protecting the internal organs from losing water, minerals, and dissolved proteins. [1] Additionally, healthy skin defends against the external environment and foreign microorganisms. [2] Aside from its protective barrier features, skin also serves as a sensory organ, regulates body temperature, and reflects perfection and elegance. [3] However, the appearance of skin gradually changes over time. [4] During the human lifetime, a decrease in elasticity and an increase in laxity become clinically noticeable characteristics that appear as a result of aging. [5] Additionally, exposure to ultraviolet irradiation negatively affects the skin by inducing a pigmentation process and extrinsic skin aging. [6] Since most people desire to be forever young and looking youthful, skincare products have recently become an essential part of everyday life to improve an individual’s looks.
Nowadays, the interest in skincare has become more intensive, and the trend of using natural materials in cosmetic products is notably on the rise due to their effectiveness and safety. [7] Generally, various herbal plants have been widely consumed as food, drugs, and complementary or alternative medicines according to their beneficial biological activities. Additionally, natural extracts from herbal plants have been used in a variety of industrial markets, including the food, pharmaceutical, and chemical industries.[8] The diversity of chemical constituents in herbal plants has led to a variety of biological activities. A large number of herbal plants have been evaluated for their chemical constituents and biological activities. However, the biological effects of some herbal plant extracts are still unknown, especially those related to topical applications.
Oxidative stress is a major cause of skin photoaging. The external supply of endogenous antioxidants is hence effective prophylaxis of oxidative stress since they can balance the oxidant/antioxidant equilibrium.[9] Topical antioxidants have been reported to protect the skin from free radical damage, and daily use can actually reverse the previous photodamage.[10] Apart from oxidative stress, skin aging could be characterized by a gradual decrease in the function of skin tissue.[11] Skin structural integrity and skin function are mainly dependent on its extracellular matrix (ECM), which is primarily composed of type I collagen fibrils that are mainly degraded by the collagenase enzyme, especially matrix metalloproteinases-1 (MMP-1). [12] Moreover, the loss of skin elasticity is caused by not only the decrease of elastin production but also the accelerated deterioration of elastin fibers by the elastase enzyme.[13] Therefore, an inhibitor of collagenase and elastase activity would retard collagen atrophy, elastin network destruction, and skin aging. The decrease of hyaluronic acid in the skin caused by hyaluronidase resulted in dry and wrinkled skin, so the hyaluronidase inhibitor was used for anti-aging. [14] Furthermore, the downregulation of tyrosinase, which is a key enzyme catalyzing a rate-limiting step of melanin synthesis, is the most prominent approach for melanogenesis inhibitors that lead to skin whitening effect.[15] Interleukins, which are pro-inflammatory substances, can be released from the dead stratum corneum in response to external stimuli, causing a sequence of cytotoxic modifications in the viable epidermis and an inflammatory response in the dermis.[16] An anti-inflammatory agent is a compound that could protect the strength of the skin.
Therefore, the present study prepared herbal extracts from various herbal plant materials that are edible, and the parts that are used are widely consumed and commonly available in local markets in Thailand. This study also aimed to investigate the total phenolic content, total flavonoid content, and biological activities related to skin applications, including antioxidant, anti-tyrosinase, anti-aging, and anti-inflammatory activities. Although the herbal extracts used were made from edible plants, the aim of this research was to examine the potential of these extracts for cosmetic/cosmeceutical purposes when applied topically to the skin. The topical application not only produces a local effect but also prevents the active compound from being damaged in the gastrointestinal tract and from first-pass metabolism.
Results and Discussion
Plant microscopy
All plant materials (Table 1) have been identified and their microscopic structures are shown in the Supporting Information (Figure S1- S16). Parenchyma, stomata, trichome, vascular strand, and various types of vessels were found in almost all plant materials. On the other hand, some structures were discovered only in certain areas as shown in Figure 1. Chlorenchyma and palisade mesophyll is only presented in the leaves. Phloem parenchyma was only presented in the bark. Pollens, papillae, petals, corolla, anther, ovary wall, and papillose stigma are presented in all flowers and inflorescences. Epicarp, mesocarp, and fibrous sclereids from seed coats were presented in the fruit. Besides, some characteristic structures were discovered as shown in Figure 2. The resinous duct was discovered in safflower (CA). Pappus was only found in the flower of Florist's daisy (CM) and purple coneflower (EP), which were in the family of Asteraceae. Oil cells were observed in cinnamon bark (CV). Calcium oxalate prism sheath was discovered in butterfly pea flower (CT). Starch grains were discovered in cinnamon bark (CV) and sweet tea vine leaves (GP). Tannin granules were discovered in the fruit of Indian gooseberry (PE).
Table 1. Plants and parts used
Herbal extracts
Herbal extracts from various plant materials exhibited different external appearances, as shown in Figure 3. To compare the potential of herbal plant extracts for use as cosmeceutical ingredients, the same weight of 1 g of each plant material was extracted under the same infusion conditions. All herbal plant extracts were tested directly after the infusion, with no lyophilization or solvent removal. Most herbal extracts were yellow, ranging from a light yellow to deep amber. On the contrary, the CT extract was dark blue, whereas the HS extract was dark red. Apart from the different colors, the odors and flavors of the herbal extracts were also varied. Each herbal extract had its own characteristic odor. Most herbal extracts had no taste, but the SR extract was the only one that was sweet. Furthermore, the extracts from AE, GE, GP, MA, and MS had a bitter taste, whereas the HS and PE extracts were sour.
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Total phenolic and flavonoid content
The total phenolic and flavonoid content of each herbal extract is shown in Figure 4. The SR extract contained the outstandingly highest phenolic and flavonoid content with a gallic acid equivalent of 9.4 ± 0.2 mg per mL extract and a quercetin equivalent of 8.1 ± 0.1 mg per mL extract (p < 0.05). It was found that most phenolic compounds of the SR extract were flavonoids. The results were strongly in accordance with the previous study reporting that the major components of the S. rebaudiana aqueous extract were quercetin and quercetin derivatives. [17] Furthermore, the RD extract contained outstanding phenolic content (7.4 ± 0.7 mg gallic acid per mL extract) but low flavonoid content (1.1 ± 0.1 mg quercetin per mL extract). According to previous research, gallic acid (phenolic acid) is the most abundant phenolic compound contained in the methanolic extract of the R. Damascena flower. [18] However, some studies reported quercetin (flavonoid) as a major component of the R. Damascena flower ethanolic extract. [19,20] Since natural phenolic compounds and flavonoids are gaining popularity for their potential use as functional ingredients in cosmetic industries,[21] herbal extracts high in these compounds could be used as the active components in cosmetic products.
Antioxidant activity
The antioxidant activities of the herbal extracts are shown in Figure 5. It was found that PD, RD, and SR possessed significantly outstanding DPPH• scavenging activity and ferric reducing/antioxidant power (p < 0.05). Interestingly, the antioxidant activities of these herbal extracts were comparable to that of ascorbic acid, which has been known worldwide as a potent antioxidant via a radical scavenger and reducing agent, which is widely used in cosmetic preparations (p >0.05). [22,23] The phenolic compounds accompanied with flavonoids were major chemical constituents responsible for antioxidant activities of SR, whereas the antioxidant activities of RD mainly resulted from polyphenols only. On the contrary, PE, which contained the least significant amount of both phenolic and flavonoid content (p < 0.05), possessed strong antioxidant activities. The likely explanation was due to the high ascorbic acid content of P. emblica. [24-26]
Interestingly, the results showed that radical scavenging activities were highly related to their reducing ability in most herbal extracts. The correlation between DPPH radical scavenging activity and EC1 value was at a moderate level with an R 2 of 0.7005 (Figure 6). However, some herbal extracts were prominent in reducing power compared with the radical scavenging abilities, e.g., CA, MS, and PA. The likely explanation for this might be due to an inert or slow reaction of some antioxidants in CA, MS, and PA to DPPH. [27] Additionally, some chemicals exhibited a falsely low antioxidant capacity in a DPPH assay. Eugenol has been reported to have a reversible reaction with DPPH and has led to such false-negative results. [28] Since eugenol has been reported as a major component in C. tinctorius, M, alba, and P. amaryllifolius, [29-31] the non-prominent radical scavenging activities of these herbal extracts were observed. In contrast, some herbal extracts were prominent in radical scavenging abilities compared with reducing power, e.g., AE, CM, CV, GE, and HS. The likely explanations for this were the fact that the FARP assay could not detect some species that acted by radical quenching (H transfer).
As oxidative stress throughout the skin plays a significant role in the aging process and eventually leads to skin wrinkles, [32] natural extracts with antioxidant activity are suggested for use as active ingredients in anti-aging cosmetic/cosmeceutical products. Since there are various mechanisms leading to oxidative stress, antioxidant activities via radical scavenging abilities and ferric reducing/antioxidant power were investigated in the present study. The most commonly published process for natural product antioxidant activity is radical scavenging abilities. [33] In the radical scavenging mechanism, ROS/RNS, e.g., O2•−, •OH, NO• or OONO−, were sacrificially reduced and led to a reduction of more reactive ROS/RNS formation, breaking down the oxidative chain reaction and eventually preventing biomolecular damage.[34] Although the radical-scavenging ability of several compounds is commonly investigated to reveal their antioxidant activity, free radicals (DPPH•) used in the assay have little relevance to those present in biological systems and are not accountable for the iron-binding properties. [33] Therefore, the FRAP assay was also used to reveal the ferric reducing/antioxidant power of herbal extracts. In the human body, cellular reductants (NADH) recycle Fe(III) for reaction with H2O2, resulting in the formation of •OH, which directly damages DNA. [33] Therefore, the compound that could reduce a Fe(III)(TPTZ)2Cl3 complex to a colored Fe(II)(TPTZ)2 is suggested as an antioxidant. [35] PD, RD, and SR, which possessed significantly potent DPPH• scavenging activity and ferric reducing/antioxidant power, are suggested for further use as antioxidants in cosmetic/cosmeceutical products (p < 0.05).
Figure 1. DPPH radical scavenging activity (a) and EC
Anti-tyrosinase activity
The anti-tyrosinase activities of the herbal extracts are shown in Figure 7. It was found that PE was the most potent tyrosinase inhibitor among several herbal extracts (p < 0.05). The tyrosinase inhibitory activity was more prominent when the substrate was L-DOPA (%inhibition = 53.1 ± 7.6), as opposed to L-tyrosine (%inhibition = 5.6 ± 1.4). Since ascorbic acid is widely known as a major component of P. emblica, [36]ascorbic acid is suggested to be the major compound responsible for the anti-tyrosinase activities of PE. The results were well-supported by previous studies reporting that L-ascorbic acid had no direct effect on tyrosinase when L-tyrosine was used as a substrate, whereas it possessed potent anti-tyrosinase activities when L-DOPA was used as a substrate.
Furthermore, GP, RD, and SR also showed promising anti-tyrosinase activities, with inhibition against tyrosinase of 39.9 ± 7.5%, 35.6 ± 5.9%, and 34.3 ± 8.5, respectively. Ginsenosides have been proposed as the active constituent responsible for the anti-tyrosinase activity of GP.[38] Besides anti-tyrosinase activity, GP has been reported to have an ability to control the melanogenesis in B16 melanoma by reducing extracellular melanin aggregation, downregulating different proteins involved in melanogenesis, and decreasing melanin delivery to keratinocytes.[39] Furthermore, several components, e.g., quercetin, kaempferol, and ellagic acid, were responsible for the anti-tyrosinase activity of RD. A previous study reported the potent anti-tyrosinase activities of these compounds, which were approximately 10 times more potent than kojic acid.[40] However, the anti-tyrosinase activity of SR has rarely been reported. This study is the first to highlight its anti-tyrosinase activity.
Since tyrosinase is a rate-limiting enzyme critically associated with melanin synthesis,[41] tyrosinase inhibitors resulted in a skin whitening effect. The present study indicates that the herbal extracts from PE, GP, RD, and SR are a potential active ingredient for skin whitening product development. Nano-delivery systems are proposed as the most effective formulation, since, to properly function, these herbal extracts with anti-tyrosinase activity must penetrate into a deeper skin layer in order to act on melanocytes in the basal layer of the stratum corneum.
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Anti-aging activity
The anti-aging activities of the herbal extracts are shown in Figure 8. The EP extract was outstanding; it possessed the most significant anti-collagenase, anti-elastase, and anti-hyaluronidase activities compared with the other extracts (p < 0.05). Interestingly, the anti-collagenase activity of EP (% inhibition = 78.5 ± 0.0) was significantly higher than that of EGCG (% inhibition = 66.1 ± 1.3) (p < 0.05). Although the anti-elastase and anti-hyaluronidase activities of EP were not as much as the EGCG and oleanolic acid used as standard compounds, EP possessed promising anti-elastase and anti-hyaluronidase activities with inhibition of 69.0 ± 1.4% and 64.2 ± 0.3%, respectively.
Collagen fibers in dermal connective tissue account for the majority of the skin's thickness and are responsible for skin tensile strength.[42] However, a decline in collagen fibers inevitably leads to skin aging, i.e., the depletion of collagen fibers has been identified as the most common histological finding in aged skin. [43] The collagen degradation is initiated by various collagenase enzymes secreted by fibroblasts, granulocytes, and epidermal cells. [44] Collagenase from Clostridium histolyticum, which is known as MMP-1, was used in the present study. The inhibition against collagenase leads to a restraining effect on collagen breakdown. Therefore, a collagenase inhibitor would be a promising component for anti-skin aging products.
Elastin fibers contribute to the elasticity and resilience of the skin. [42] However, elastin fibers were fragmented, degraded, and lysed during the aging process in several tissues, including the dermis. [45] Therefore, elderly people tend to have sagging skin because of such elastin degradation, especially at the superficial region of the dermis. Elastases or elastase-type proteases are proteolytic enzymes capable of destroying elastic fibers. [45] The inhibition against elastases would retard the decline of elastin fiber and maintain the elasticity of the skin.
Additionally, hyaluronate gels also possess elastic properties of the skin. [45] Normally, hyaluronan is localized in the dermis, especially around the skin appendages and below the basement membrane, and in intercellular spaces of the viable epidermis, except the upper granular layer. [46] In general, hyaluronan immobilizes water in skin tissue, altering dermal volume and compressibility. However, hyaluronan is only found in the upper dermis in elderly people. Therefore, dryness and skin wrinkles are seen in aged skin. [47]
Apigenin, a flavonoid present in EP, has been proposed as the main compound responsible for MMP-1 inhibition. [48] Additionally, apigenin was reported to be active in the prevention of interleukin-1-induced matrix metalloproteinase development and UVA/UVB-induced skin carcinogenesis in a previous study.[49] Furthermore, caffeic acid derivatives were suggested to be the compounds responsible for the anti-hyaluronidase activity of EP. [50] However, the anti-elastase activity of EP has rarely been reported. Consequently, EP, which presents significant potent anti-collagenase, anti-elastase, and anti-hyaluronidase activities, is suggested as an anti-aging ingredient in cosmetic/cosmeceutical products. However, herbal extracts with anti-aging activities need to penetrate more deeply into the dermis layer. Therefore, delivery systems that deliver these active compounds to the target site are necessary to fulfill their cosmeceutical properties.
Figure 2. Inhibitory activities against the collagenase
Anti-inflammatory activity
Inflammation is a complex defensive system in which leukocytes migrate from the vasculature into infected tissues to destroy agents that may trigger tissue damage. [51] However, inflammatory responses have been reported to decline in aged skin. [52] Therefore, the skin of elderly people tends to become inflamed more easily. The anti-inflammatory activities of the herbal extracts shown in Figure 9 indicate that these herbal extracts possesses moderate to low anti-inflammatory activities via the inhibition against IL-6 and TNF-α. However, among these herbal extracts, MA possesses the most significant IL-6 and TNF-α inhibition, followed by MS (p < 0.05). Although the inhibition against IL-6 and TNF-α of MA (% inhibition = 50.3 ± 0.4 and 41.5 ± 2.0, respectively) and MS (% inhibition = 41.9 ± 5.1 and 28.0 ± 7.0, respectively) were not comparable to that of dexamethasone (98.4 ± 0.5 and 93.8 ± 0.5, respectively), a well-known steroidal drug used for anti-inflammation (p < 0.05), M. alba could be a promising natural source of anti-inflammatory agents.
Since IL-6 is generated at the site of inflammation and is essential for the acute phase response, inhibition against IL-6 secretion can reduce the clinical and biological features related to inflammation. [51] Similarly, TNF- has a plethora of pro-inflammatory effects on the skin by inducing adhesion molecules and chemokines, which leads to inflammatory cells adhering to vessels, rolling, emigration, and ultimately chemotaxis into the skin, [53] and inhibition against TNF-α secretion also reduces the inflammation response. Therefore, MS and MA, which can inhibit IL-6 and TNF-α secretion, are promising topical anti-inflammatory agents for both normal and aged skin.
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