Biological Activities of Paper Mulberry ( Broussonetia papyrifera ): More than a Skin-Lightening Agent

: Background: Paper mulberry is one of the most common skin-lightening agents in the beauty industry due to its strong anti-tyrosinase activity. This narrative review aims to summarize the chemical composition, biological activities, and applications of paper mulberry in cosmetics. Method: The literature for this article was acquired from the PubMed, Web of Science, and Google Scholar databases before September 2022. The keywords for searching included “paper mulberry”, “ Broussonetia papyrifera ”, “skin-lightening”, “skin-whitening”, “depigmentation”, “pharmacological activity”, and “biological activity”. Results: Paper mulberry consists of various components, including ﬂavonoids, tannins, alkaloids, phenols, saponins, coumarins, glycosides, and polysaccharides, which possess a wide range of pharmacological properties. Apart from its anti-tyrosinase activity, paper mulberry and its compounds exhibited anti-inﬂammatory, antioxidant, antimicrobial, antiviral, anticancer, antidiabetic, anticholinesterase, antigout, antinociceptive, and hepatoprotective effects. Phenols and ﬂavonoids were demonstrated to be the main contributors to the biological activities of paper mulberry. Paper mulberry is widely applied in cosmetics for skin lightening and skin moisturizing purposes and shows potential for application in hair care products due to the hair nourishing effects. The safety of paper mulberry for topical application was proven in clinical studies. Conclusion: The current review provides a better understanding of paper mulberry’s properties and allows us to extend the application of this plant and its bioactive components in cosmetics.


Introduction
Skin lightening is a lucrative industry with a global market size valued at USD 9.96 billion in 2021, and estimated up to USD 16 billion in 2030, according to statistics from Grand View Research [1].Skin lightening, also known as skin whitening or depigmentation, is a cosmetic procedure to lighten dark skin areas and achieve a lighter skin complexion using laser treatment or skin-lightening products [2,3].Dark skin or skin hyperpigmentation is a result of exposure to ultraviolet light or chemical irritants, and also is a manifestation in several skin disorders such as melasma, solar lentigines, or post-inflammatory hyperpigmentation [4].The use of skin-lightening agents aims to reduce the level of melanin, the main pigment in the skin, resulting in a brighter skin tone [5,6].
Melanogenesis, a process of melanin production and distribution by melanocytes, is regulated by several melanogenic enzymes, including tyrosinase, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2) [7].Tyrosinase catalyzes the rate-limiting conversion of L-tyrosine to L-3-4 dihydroxyphenylalanine (L-DOPA), and subsequently to L-dopaquinone and dopachrome [8,9].In the next step, TRP2 (dopachrome tautomerase) converts dopachrome to 5,6-dihydroxyindole-2-carboxylic acid and 5,6dihydroxyindole, which are further converted to eumelanin (black or brown pigment) by tyrosinase or TRP1 [10,11].Different tyrosinase isozymes might play different roles in the regulation of melanin formation.The soluble isozymes T1, T2, and T3 showed blocking activities, while the isozyme T4, the only isozyme found in melanosomes, accelerates the conversion of dopachrome into melanin [12].In the presence of cysteine, L-dopaquinone reacts with cysteine, leading to the formation of cysteinyl-dopa and the production of pheomelanin (red or yellow pigment) [13].TRP1 has been demonstrated to increase the eumelanin/pheomelanin ratio [14].In addition, the precursors of melanin, L-tyrosine and L-DOPA might act as hormone-like bioregulators of melanin pigmentation by binding to their specific receptors or stimulating melanocyte-stimulating hormone receptors to positively regulate melanogenesis [9,15].Melanin has diverse regulatory effects on cellular processes.Eumelanin showed protective effects against radiation and photodamage through antioxidant activity, while pheomelanin was thought to induce oxidative stress and DNA damage, contributing to melanoma progression [16].A previous study suggested that the inhibition of tyrosinase, TRP1, and TRP2 was associated with a reduction in melanin content in human skin cells [17].However, after the dopaquinone formation stage, melanin formation might proceed with the velocity of the reaction regulated by metal cations and pH, instead of enzyme involvement [18,19].Therefore, among these three melanogenic enzymes, tyrosinase might play the most important role in melanin biosynthesis, and skinlightening agents majorly regulate the production and activity of tyrosinase to reduce the melanin content in the skin [5].
Paper mulberry (Broussonetia papyrifera (L.)) is a common plant in the Asia-Pacific region [20].Many parts of the paper mulberry plant, such as the root, bark, leaves, and fruits, have been used in traditional herbal medicines for the treatment of various diseases, including skin disorders and ophthalmic diseases [21].Paper mulberry contains numerous chemical components, such as flavonoids, polyphenols, alkaloids, coumarins, and saponins, which possess a wide range of biological and pharmacological effects [22].Paper mulberry extracts and their constituents showed strong inhibitory effects on the activity of tyrosinase enzyme, and have bene applied in cosmetics as skin-whitening ingredients [23].Apart from its anti-tyrosinase activity, paper mulberry and its derived compounds have been reported to exert various biological effects, including anti-inflammatory, antioxidant, antimicrobial, anticancer, and other activities [24][25][26].This narrative review aims to summarize the chemical composition, biological activities, as well as applications of paper mulberry in cosmetics.

Materials and Methods
This article provides a narrative review of the biological activities of paper mulberry and its application in cosmetics.PubMed, Web of Science, and Google Scholar databases were used for searching the published literature up until September 2022 for this review article.The main keywords included "paper mulberry", "Broussonetia papyrifera", "skinlightening", "skin-whitening", "depigmentation", "pharmacological activity", and "biological activity".Original articles and patents in English were analyzed in this article.

Part
Compound Reference

Antityrosinase Activity
Paper mulberry is one of the most well-known skin-lightening ingredients with tyrosinase inhibitory effects.An ethanolic extract from the leaves of paper mulberry exhibited inhibitory activity in mushroom tyrosinase assays, with a IC50 value of 17.68 ± 5.3 µg/mL.Moreover, the antityrosinase effect of paper mulberry leaf extract was stable over two months at 4 • C or room temperature [63].Another study demonstrated that methanolic extracts of the leaves or bark of paper mulberry inhibited tyrosinase activity by 90-100% at 666.67 µg/mL [64].The antityrosinase effect of paper mulberry might be related to the flavonoids and diterpenes in its composition.Flavonoid derivatives from paper mulberry, including broussoflavonol B/F/H-K, papyriflavonol A, isolicofavonol, glycyrrhiza flavonol, 3,5,7,4 -tetrahydroxy-3 -(2-hydroxy-3-methylbut-3-enyl)flavone, uralenol, and quercetin, showed strong inhibition effects on mushroom tyrosinase with IC50 values less than 100 µM [40,48].Three ent-kaurane diterpenes, broussonetones A-C, from paper mulberry leaves exerted more stable inhibitory activities on mushroom tyrosinase than the positive control, kojic acid [60].Kazinol F, a compound derived from paper mulberry, also inhibited mushroom tyrosinase activity [80].All published studies have reported the inhibitory effects of paper mulberry and its components on mushroom tyrosinase, not on human tyrosinase or human melanocytes.Hence, further studies should be conducted to provide strong scientific evidence for the application of paper mulberry as a depigmentation agent in the beauty industry.

Anti-Inflammatory Activity
The anti-inflammatory effect of paper mulberry and its components has been indicated in numerous studies using both in vitro and in vivo models.Butanol and hexane fractions from the stem bark of paper mulberry showed anti-inflammatory activity by suppressing the production of pro-inflammatory mediators, including nitric oxide (NO), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α), as well as decreasing the expression of inducible NO synthase (iNOS) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages [24,65].Similarly, treatment with oil from paper mulberry fruits also reduced NO production in LPS-activated RAW 264.7 cells [53].A methanolic extract of the root bark of paper mulberry and its main bioactive compounds broussoflavonol B and kazinol J significantly reduced TNF-α-induced inflammation in 3T3-L1 adipocytes and adipose tissues by inhibiting the NF-κB pathway via AMPK activation [66].Flavonoids from the root bark of paper mulberry, including broussochalcone A/C, broussoflavanonol A/B, kazinol V/W, and (2R)-7,3 ,4 -trihydroxy-6-prenylflavanone, reduced the expression of NO, iNOS, and pro-inflammatory cytokine (TNF-α and IL-6) in LPS-induced macrophages [32,67].In a similar study, kazinol M and broussoflavonol A/B inhibited the production of IL-1β and TNF-α by suppressing NF-κB/AP-1 activation in LPS-stimulated THP-1 cells [49].Another study showed that broussoflavonol H decreased IL-2 production in Jurkat induced by PHA and PMA (IC50 = 9.95 µM) [40].Root and fruit extracts from paper mulberry also exerted in vivo anti-inflammatory effects by suppressing carrageenan-induced edema in rats [42].Paper mulberry (B.papyrifera) combined with Lonicera japonica exhibited inhibitory effects on lung inflammation in LPS-treated rats and alveolar macrophages by downregulating the production of NO and pro-inflammatory cytokines [68].Papyriflavonol A, a flavonoid from paper mulberry, showed in vivo anti-inflammatory effects by inhibiting IgE-induced passive cutaneous anaphylaxis in rats [69].The anti-inflammatory effects of paper mulberry and its bioactive components might be effective for the prevention of post-inflammatory hyperpigmentation and other inflammatory diseases.

Antioxidant Activity
Paper mulberry and its bioactive components possess antioxidant activities both in vitro and in vivo.Extracts from paper mulberry leaves exhibited radical-scavenging activities in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2 -azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) assays, and phenols, particularly flavonoids, might be the main contributors to this antioxidant effect [59,61].Another study indicated that the broussonetones A−C, apigenin, and vitexin from the leaves of paper mulberry showed antioxidant effects in SOD-like effect assays [60].Paper mulberry stem bark, wood, fruit, and flower extracts also exerted strong radical scavenging activities due to their high contents of total phenols and flavonoids [26,29,50].A root extract of paper mulberry showed inhibitory effects against H 2 O 2 -induced oxidative stress in SH-SY5Y cells by reducing extracellular peroxide levels and improving the activities of SOD, catalase, glutathione peroxidase, and glutathione reductase [70].Kazinol M, broussoflavonol A, and 5,7,3 ,4 -tetrahydroxy-3methoxy-8,5 -diprenylflavone exhibited strong antioxidant effects in cellular antioxidant activity assays [49].Broussochalcone A exerted a strong radical-scavenging activity in a diphenyl-2-picrylhydrazyl assay system [67].Broussoflavan A, broussoflavonol F/G, and broussoaurone A from paper mulberry roots inhibited the oxidative stress caused by Fe 2+ in rat brains [43].In addition, lignans and lignins from paper mulberry also showed high antioxidant activities in DPPH and ABTS assays [51,71].In vivo, paper mulberry extracts significantly enhanced antioxidant capacities in dairy cows, beef cattle, and piglets [72][73][74].These findings imply the protective effect of paper mulberry against oxidative stress, suggesting potential in the treatment of various diseases.

Anti-Microbial Activity
A previous study indicated that a methanolic extract of paper mulberry leaves showed inhibitory effects against bacteria (Enterococcus faecalis, Vibrio cholera, Bacillus subtilis, Pseudomonas aeruginosa, Klibsella pneumonia) and fungi (Aspergilus niger, A. flavus) [75].The seed oil of paper mulberry exerted antibacterial activities against Staphylococcus aureus, Proteus vulgaris, Bacillus cereus, and Enterobacter aerogenes, but did not affect fungal strains [57].Extracts from the aerial parts of paper mulberries and the derived compounds, daphnegiravan F and 5,7,3 ,4 -tetrahydroxy-3-methoxy-8,5 -diprenylflavone, exhibited an anti-oral microbial effect on both Gram-positive and Gram-negative bacteria [76].Prenylated flavonoids from paper mulberry also possess antimicrobial activities.Papyriflavonol A showed antifungal effects against Candida albicans and Saccharomyces cerevisiae, as well as antibacterial activity against Escherichia coli, Salmonella typhimurium, S. epidermis, and S. aureus [25,44].Kazinol B exerted inhibitory effects on S. cerevisiae, S. epidermis, and S. aureus, while broussochalcone A was only effective on C. albicans [25].The antibacterial effects of these flavonoids might occur through inhibiting bacterial neuraminidase [36].Paper mulberry polysaccharides showed antibacterial activities against E. coli, P. aeruginosa, B. subtilis, and S. aureus in a dose-dependent manner [56].These findings suggest the potential application of paper mulberry and derived compounds for the treatment of diseases caused by bacterial and fungal infections.

Anti-Cholinesterase Activity
Acetylcholinesterase plays a crucial role in cholinergic transmission by catalyzing the hydrolysis reaction of acetylcholine.Acetylcholinesterase and the related enzyme butyrylcholinesterase have been demonstrated to be involved in the pathogenesis of Alzheimer's disease by directly interacting with amyloid-beta (Aβ) and triggering the formation of Aβ plaques [85,86].Paper mulberry ethanol extracts with prenylated flavonoids as the main active components exerted inhibitory effects on both human acetylcholinesterase and butyrylcholinesterase, suggesting their potential for the treatment of Alzheimer's disease [41].

Anti-Gout Activity
An ethanolic extract of paper mulberry root bark showed antigout potential by inhibiting the activity of xanthine oxidase (XOD), an enzyme that synthesizes uric acid from hypoxanthine [34,87].Two phenolic compounds, broussochalcone A and 3,4-dihydroxyisolonchocarpin, were found to be the main contributors to the XOD-inhibitory effects of the paper mulberry extract [34].

Antinociceptive Activity
A previous study demonstrated that the administration of extracts from the roots, stems, leaves, and fruits of paper mulberry (2 g/kg) exerted antinociceptive activity by inhibiting the acetic acid-induced writhing response in rats [42].

Hepatoprotective Activity
Polysaccharides from paper mulberry ameliorated acetaminophen-induced liver damage, reduced liver apoptosis, enhanced antioxidant capacity, and improved the detoxification ability of the liver to acetaminophen by regulating the intestinal microbiota in mice [30].An extract of paper mulberry root significantly suppressed hepatic steatosis in HFD-induced obese mice by decreasing lipogenic gene expression and increasing AMPK phosphorylation in the liver [66].These data suggest the application of paper mulberry for the treatment of hepatic diseases.

Skin Lightening and Moisturizing
Paper mulberry is commonly used as a skin-lightening agent in cosmetics.Paper mulberry might prevent skin hyperpigmentation by inhibiting the activity of tyrosinase and melanin formation [88].Extracts from paper mulberry are included in many skin-whitening compositions for external application [89,90].Paper mulberry combined with Styela clava extract is blended into a facial mask sheet for the whitening purpose [91].A mask pack containing paper mulberry showed moisturizing effects on the skin [92].Paper mulberry combined with white ginseng was incorporated in a cosmetic composition for skin moisturizing and smoothing [93].A study conducted on 24 male participants demonstrated that kazinol F, a compound derived from paper mulberry, showed a significant depigmentation effect against ultraviolet B (UVB) radiation.Even though melanin pigmentation was considered a response of the skin to UV radiation and the inhibition of melanogenesis may increase the skin's vulnerability to the damage [94], the lotion containing kazinol F did not only reduce skin darkness, but it also alleviated UV-induced erythema in human skin.This effect was assessed using three different instruments (Chromameter CR200, Dermaspectrometer, and Mexamter MX16) and was comparable with hydroquinone, a common skin-lightening agent [95].Paper mulberry has been widely applied in the cosmetic industry in Europe and South America; however, there have been limited clinical trials to prove its skin-lightening effects in humans [96].

Hair Protection and Hair Growth
A previous study showed that the application of formulations containing paper mulberry root extract exerted hair-protective effects by improving the tensile strength, optical absorption, and luster of damaged hair [97].Another study on 11 healthy subjects indicated that using a leaf extract of paper mulberry for 12 weeks showed beneficial effects on hair growth, indicated by increased total hair count as compared with the start date of the trial.The underlying mechanism might be through regulating the WNT-β-catenin and STAT6 pathways to promote the proliferation of dermal papilla cells [98].These data suggest the potential application of paper mulberry in hair-care products in cosmetics.

Safety Assessment of Paper Mulberry for Cosmetic Topical Application
The safety of paper mulberry for topical application was assessed in previous studies.A microemulsion formulation containing paper mulberry leaf extract was applied for human skin irritation tests in the form of single-application closed-patch tests in 30 women without any skin symptoms.The results showed that transepidermal water loss and erythema values were not significantly different between microemulsions containing paper mulberry and the placebo, suggesting that paper mulberry did not cause skin irritation in humans [99].Another report indicated that a skin composition containing paper mulberry showed no adverse effects on human skin [89].The application of a lotion product containing kazinol F, a component of paper mulberry, did not show any irritation or sensitization effects on human skin [80].These findings suggest the safety of paper mulberry for application in skin-lightening products.

Conclusions
Paper mulberry is a common skin-lightening ingredient in the cosmetic industry.Extracts from different parts of the paper mulberry contain various bioactive components, such as phenols, flavonoids, alkaloids, and tannins, which possess a wide range of biological activities.Among them, antityrosinase, antioxidant, antimicrobial, and anti-inflammatory effects are considered the typical pharmacological properties, which can be utilized for cosmetic applications.Although paper mulberry is demonstrated to be safe for topical application and has been used in many cosmetic preparations, there is a lack of clinical studies to prove its skin-lightening effects in humans.Hence, more research on paper mulberry as well as the combination of paper mulberry with other agents might be helpful to expand the applications of paper mulberry in cosmetics.

Table 1 .
Chemical composition of paper mulberry.

Table 2 .
Biological activities of paper mulberry.