The Genus Broussonetia: An Updated Review of Phytochemistry, Pharmacology and Applications

The Broussonetia genus (Moraceae), recognized for its value in many Chinese traditional herbs, mainly includes Broussonetia papyrifera (L.) L’Hér. ex Vent. (BP), Broussonetia kazinoki Siebold (BK), and Broussonetia luzonica (Blanco) Bureau (BL). Hitherto, researchers have found 338 compounds isolated from BP, BK, and BL, which included flavonoids, polyphenols, phenylpropanoids, alkaloids, terpenoids, steroids, and others. Moreover, its active compounds and extracts have exhibited a variety of pharmacological effects such as antitumor, antioxidant, anti-inflammatory, antidiabetic, anti-obesity, antibacterial, and antiviral properties, and its use against skin wrinkles. In this review, the phytochemistry and pharmacology of Broussonetia are updated systematically, after its applications are first summarized. In addition, this review also discusses the limitations of investigations and the potential direction of Broussonetia. This review can help to further understand the phytochemistry, pharmacology, and other applications of Broussonetia, which paves the way for future research.


Introduction
Broussonetia is one of the most significant genera in the Moraceae family, a member of the Urticales order. The genus is composed of eleven species, comprising Broussonetia papyrifera (BP) (see Figure 1) [1], Broussonetia kazinoki (BK) (see Figure 2) [2], Broussonetia zeylanica (Thwaites) Corner (BZ) [3], Broussonetia luzonica (Blanco) Bureau (BL) [4], Broussonetia rupicola F.T. Wang and Tang (BR), Broussonetia kurzii (Hook.f.) Corner (BKU), Broussonetia kaempferi Siebold (BKA) [5], Broussonetia integrifolia Buch.-Ham. (BI), Broussonetia harmandii Gagnep. (BHG), Broussonetia × hanjiana M. Kim (BHM), and Broussonetia greveana (Baill.) C.C.Berg (BG) [6]. The various Broussonetia species have been an excellent source of conventional medicine to treat different diseases. Their roots, barks, fruits, and leaves have all been used in conventional medicine. In China, the leaves have been used to treat chronic prostatitis as a folk medicine [7], as well as for bleeding [8]. The bark could be used for special recipes [8]. The fruits have been confirmed to treat impotence and ophthalmic diseases [7], while the hematochrome from the fruits could be used as a foodstuff in history [9]. In the traditions of Tonga, Fiji, and Samoa, BP, a fibrous tree, was the main raw material used to make tapa cloth [10]. Moreover, one of the Broussonetia species was used by Cai Lun to create paper, one of the four great inventions of ancient China. Broussonetia species were also used as woody forage in ancient history [11].  According to the publication, Flora of China [12], the morphology of the genus is described thus: "Trees or shrubs, or vine-like shrubs; there is emulsion, and the winter buds are small. Leaves are alternate, split or non-divided, with serrated margins, basal  According to the publication, Flora of China [12], the morphology of the genus is described thus: "Trees or shrubs, or vine-like shrubs; there is emulsion, and the winter buds are small. Leaves are alternate, split or non-divided, with serrated margins, basal  [53]. In 2010, Zhou et al. [54] reported that compounds 151, 170, 171, 180, 181, and 194 were isolated from BP for the first time; these compounds showed antioxidant activity against H 2 O 2 -induced injury in SY5Y cells. In 2014, four compounds (148, 165, 166, and 178) were isolated from an n-butanol extract of BP, and the structures of these compounds were elucidated on the basis of NMR spectra analysis and chemical evidence [36]. In 2019, Li et al. [38] reported compounds 185-192. In 2020, compounds 162 and 167 were isolated from the CHCl3-soluble part of an ethanolic extract of branches and twigs of BP by Malanik et al. [42]. In 2021, Vu et al. [14,44] isolated compounds 156-161 from the roots of BK. In addition, compounds 145, 146, 147, 152, and 153 were reported by Yadav et al. [45].
All penylpropanoids are summarized in Table 2, and the structures were summarized in Figure S2.

Polyphenols
Compounds with two or more hydroxyl groups that are not flavonoids, phenylpropanoids, terpenes, or alkaloids are classified as polyphenols. The pharmacological effects of polyphenols, apart from skin whitening and anti-wrinkle properties, have been evaluated by researchers, but this is far from the level of research into flavonoids.
In total, 38 polyphenols were isolated from Broussonetia. In 1999, compounds 203 and 226 were isolated from the root bark of BK, and the cytotoxic activity of these compounds was evaluated against several different cell lines [27]. In 2001, Lee et al. [28] reported that compounds 195, 198, 199, 205, 206, 223, and 232 were isolated from the ethyl acetatesoluble extract of the whole plants of BP, and compound 199 exhibited to be active as aromatase inhibitors. In 2009, compounds 204, 207, 224, 225, and 231 were isolated from the methanol extract of BK, and the monophenolase inhibition of compounds 204, 224, and 225 was determined [55]. In 2018, new polyphenols, compounds 221 and 222, and known compounds 196 and 210 were isolated from the twigs of BK; compounds 196 and 222 showed the in vitro inhibition of protein tyrosine phosphatase 1B [37]. In 2019, compounds 219 and 220 were reported by Li et al. [38]. In addition, the structures of compounds 216-218 were elucidated on the basis of spectroscopic data (1D and 2D NMR, MS, MS/MS, and HRMS), and compound 218 exhibited significant inhibitory effects on the NO, iNOS, and pro-inflammatory cytokine production [40]. In 2020, compounds 208, 209, 211, 212, 123, 214, and 215 were reported by Qureshi et al. [43]. In 2021, compounds 197, 200, 201, and 202 were reported by Yadav et al. [45].
All polyphenols are summarized in Table 3, and the structures were summarized in Figure S3.

Alkaloids
Nitrogen-containing organic compounds are classified as alkaloids. Until now, there has been little research on the pharmacological effects of alkaloids.
All alkaloids are summarized in Table 4, and the structures were summarized in Figure S4.

Terpenoids and Steroids
Olefins where the molecular formula is an integer multiple of isoprene are classified as terpenoids. Most of the terpenoids are triterpenes.
Until now, a total of 17 triterpenes have been isolated from Broussonetia. Fang et al. reported that two known terpenoids, compounds 283 and 284, were isolated and characterized from BP in 1994 [24] and 1995 [25] respectively. In 2008, three new ent-kaurane type diterpenes, compounds 279-281, were isolated from leaves of BP; these compounds showed mild inhibition of tyrosinase and significant inhibition of xanthine oxidase [32]. In 2011, four new euphane triterpenes, compounds 274-277, were isolated from the bark of BP, and the structures of these compounds were determined by spectroscopic evidence and chemical methods [69]. In addition, compound 270 was a new tirucallane triterpenoid, and compounds 271-273 were isolated from BP for the first time. In 2019, compounds 268 and 269 were reported by Li et al. [38].
All the terpenoids are summarized in Table 5, and the structures were summarized in Figure S5. All steroids were isolated only from BP. Three compounds (287-289) were reported by Qureshi et al. [43] in 2020, while compounds 285-286 were reported by Yadav et al. [45]. All steroids are summarized in Table 6, and the structures were summarized in Figure S6.

Other Compounds
Apart from the compounds mentioned above, a total of 49 other compounds isolated from Broussonetia species are classified as "others".
Fang et al. [24,25] showed that compounds 329-332 were isolated and characterized from the root bark of BP. In 2007, two new megastigmane O-glucopyranosides, compounds 327 and 328, were isolated from the leaves of BP; the structures of these compounds were established by chemical methods and spectroscopic techniques, including 2D NMR [72]. In 2010, Zhou et al. [54] established that compounds 323-326, isolated from the fruits of BP for the first time, showed antioxidant activity against H 2 O 2 -induced injury in SY5Y cells. In 2011, a novel compound, 322, and a known compound, 321 were isolated from the n-BuOH extract of BP seeds, while their cAMP-regulating activity was evaluated by Mei et al. [73]. In 2014, compounds 316-320 were isolated from the n-butanol extract of BP, and compounds 317, 318, and 320 were found to potently inhibit estrogen biosynthesis in KGN cells [36]. Moreover, compounds 309-314 were reported by Yu et al. [51]. In 2016, compounds 333-338 were isolated from the ethyl acetate leaf extract of BL [71]. In 2019, compounds 293-308 were reported by Li et al. [38]. In 2021, compounds 290 and 291 were reported by Yadav et al. [45].
All these compounds were summarized in Table 7, and the structures were summarized in Figure S7.  309

Pharmacology
Various uses of Broussonetia species have inspired researchers' interest in exploring pharmacological activities by scientific pharmacological assays including in vitro and in vivo. A variety of crude extracts and purified compounds from Broussonetia species have been evaluated for different biological effects, such as their antitumor, antioxidant, anti-inflammation, antidiabetic, anti-obesity, antibacterial, and antiviral properties, as well as skin whitening, anti-wrinkle, and other activities. Despite the extensive bioactivities that have been identified, the targeted clinical trials that are normally used to evaluate safety and effectiveness for humans are currently absent. Perhaps the addition of clinical trials might be a more comprehensive and scientific way to ascertain the medical role of the Broussonetia genus. All these pharmacological activities are summarized in Table 8.

Anti-Tumor
In 2010, the dichloromethane fraction extracted from the stem barks of BP was found to induce apoptosis-related DNA fragmentation; it increased sub-G1 accumulation, increased p53, caspase3, and Bax expression, and inhibited the proliferation of human colon cancer HT-29 cells [74]. The ethanol extract of BP exhibited the inhibition of the growth of human osteosarcoma MG63 cells, affected morphological apoptosis, and induced cell-cycle arrest, as found in 2013 [75]. In 2014, seven alkaloids isolated from the ethyl acetate fraction of BP fruits at dosages of 1, 5, 10, and 50 mg/mL showed high cytotoxic activities on the BEL-7402 and Hela cell lines, with IC 50 values of 6.61-47.41 mg/mL and 5.97-40.17 mg/mL, respectively [66]. Zhu et al. [76] explored the mechanism of gastric carcinoma cell SGC-7901 apoptosis, as induced by CALCBP, and the results showed that apoptosis might be related to oxidative stress in the cell mitochondria via the p38-MAPK and ERK-MAPK signal pathways. In an in vitro assay, polyphenols showed significant apoptotic activities on HepG2 cells in a dose-dependent and time-dependent manner by inducing cell cycle arrest at the G1 phase, unregulating the ratio of Bax/Bcl-2 and inhibiting the expression of PKB/AKT and ERK [77].
In 1999, the pure compounds kazinol Q, kazinol R, kazinol D, kazinol K, and 7,4dihydroxyflavan, isolated from BP root barks, showed strong inhibitory effects on T24, CaSki, PLC/PRF/5, HT3, and SiHa, respectively [27]. In 2011, Wei et al. explored the ability of kazinol Q to induce DNA breakage in the presence of Cu; the results showed that the cell viability of gastric carcinoma SCM-1 cells was significantly decreased [78]. In 2013, (+)-pinoresinol-4 -O-β-D-glucopyranosyl-4 -O-β-D-apiofuranoside, apigenin-6-C-β-D-glucopyranside, and liriodendrin, isolated and purified from BP leaves, exhibited inhibitory effects on HepG-2 cells during the dosage of 100 mmol·L−1; their IC 50 values were 17.19, 14.56, and 19.53 µg/mL, respectively [35]. Moreover, broussoflavonol B restricted the growth of breast cancer SK-BR-3 cells and breast cancer MDA-MB-231 cells at sub-micromolar concentrations via inducing cell-cycle arrest at the G0/G1 and G2/M phases and inducing the differentiation of cells [79]. In the same year, broussoflavonol B and 5,7,3 ,4 -tetrahydroxy-3-methoxy-8,5 -diprenylflavone were prepared from an ethyl acetate-soluble fraction of BP barks, exerting potent antiproliferation activities on the ERpositive MCF-7 cells, with IC 50 values of 4.41 and 4.19 mM, respectively [34]. The two compounds could also inhibit tumor proliferation on BCAP-37 cells in vivo, from a dosage of 0-25 µM [34]. In 2016, kazinol A showed cytotoxicity in T24 and T24R2 cells from a dosage of 0-50 µM via G0/1 arrest, mediated by decreasing cyclin D1 and increasing p21 [80]. In addition, kazinol E was a targeted molecule for breast cancer stem-like cells from a dosage of 0-50 µM, by blocking EGF-induced ERK activity directly [81,82]. Moreover, in 2017, Kim et al. identified that marmesin eliminated mitogen-stimulated proliferation and invasion in both p53 wild-type A549 and p53-deficient H1299 NSCLC cells [83]. In 2018, Park et al. reported that broussochalcone A showed high cytotoxic activities in human hepatoma HepG2 and SK-Hep1 cells, with an IC 50 value of 20 µM from a dosage of 0-40 µM; these activities were due to cell-cycle arrest by increasing FOXO3, regulating the cell cycle, and activating pro-apoptotic proteins [84]. In another study conducted by Shin et al. in 2019, broussochalcone A also exerted strong cytotoxic effects upon colon and liver cancer cells with a dosage of 0-20 µM, by promoting the phosphorylation/ubiquitin-dependent degradation of β-catenin [85]. In 2019, broussoflavonol K showed stronger inhibitory effects on NCI-H1975, MCF-7, and HepG2 than isolicofavonol, with IC 50 values ranging from 0.90 to 2.00 µM, which were due to cyclization between the isoprenyl moiety and the adjacent phenolic hydroxyl group [41]. A recent study in 2020 investigated the anti-tumor effect of broussoflavonol B; the results showed that it significantly repressed the proliferation of human pancreatic cancer PANC-1 cells, by inactivating the ERK/c-Myc/FoxM1 signaling pathway, with a dosage of 0-100 µM [86]. In 2021, Vu et al. studied the inhibitory effects of eriodictyol, apigenin, and kaempferol against HL-60 cells, with IC 50 values ranging from 46.43 to 94.06 µM [14]. In a study in 2022, marmesin also exerted cytotoxicity on esophagus cancer cells via inhibiting the PI3K/Akt pathway [87]. From this evidence, it is clear that AMPK is a major regulator of energy metabolic pathways and plays an important role in the regulation of autophagy. In this work, the active compound kazinol C, isolated from BK whole herbs or root barks, could markedly induce apoptosis in colon cancer cells by activating AMPK phosphorylation [2,88].

Anti-Oxidant Activity
Excessive oxidative stress is harmful to cells, protein, DNA, and others, so antioxidants are important molecules that can protect humans from this danger. Various assays of antioxidant activity have been used to test these properties, such as DPPH, ABTS, CAA, hydrogen peroxide scavenging activity assays, hydroxyl radical scavenging activity assays, FRAP, lipid peroxidation inhibitory activity, mitochondrial swelling assays, chelation of metal ions (Fe 2+ ) assays, xanthin oxidase inhibitory activity assays, hydroxyl radical scavenging activity, superoxide anion free radical scavenging activities, superoxide anion radical scavenging activity assays, ferrous ion chelating capacity assays, and TEAC.
The antioxidant activities of the crude extracts of Broussonetia species were measured via the methods mentioned above, of which the most frequently used methods were DPPH, ABTS, FRAP, and hydroxyl radical scavenging activity assays. In 2014, DPPH and pyrogallol autoxidation assays showed that the hydroxyl radical inhibition rate of the seed oil of BP was 91.21% [89]. In the same year, the ethanol extract of BP fruits was revealed to demonstrate antioxidant activity (0-400 mg/mL), with an IC 50 value of 155.7 µg/mL for lipid peroxidation inhibition on liver homogenate [90]. In 2013, the ethanolic extract of BP fruits showed maximum antioxidant activity by DPPH assay during the dose of 0-600 µg/mL, with an IC 50 value of 156.3 µg/mL [75]. In 2012, Sun et al. indicated that the ethanol extract from BP flowers showed more potent radical scavenging activity than the water extract, which showed 62.88% in the DPPH assay at 5 mg/mL and 61.15% in terms of chelation Fe 2+ -activity at 6 mg/mL [91]. In another experiment, Sun et al. reported that the ethanol and water extracts of BP fruits showed strong DPPH radical-scavenging activity at 87.17 ± 0.18% and 58.11 ± 0.11%, respectively [7,91]. The Fe 2+ -chelating activity was approximately 77.51% and 48.26% from an aqueous extract of 5 mg/mL and an ethanol extract of 5 mg/mL [7,91].
From Table 8, it can be seen that a plethora of investigations revealed that the roots and leaves possessed stronger antioxidant activities than other parts [93]. Moreover, many assays indicated that the antioxidant activities of bark extracts were superior to wood extracts [94].

Anti-Inflammation
In 2001, broussochalcone A, isolated from BP at a dose of 1-20 µM could inhibit NO production in LPS-activated macrophages by inhibiting IkBa phosphorylation, IkBa degradation, nuclear factor-kappa B activation, and iNOS expression, with an IC 50 value of 11.3 mM [92]. In 2003, papyriflavonol A, isolated from BP, was demonstrated to inhibit human group IIA and V sPLA2s dose-dependently and reduce IgE-dependent passive cutaneous anaphylaxis in rats from a dose of 0-250 µM with IC 50 values of 3.9 to 4.5 mM, suggesting that it could be a novel anti-inflammatory drug in the future [95]. In 2019, Huang et al. demonstrated that broussonin E, isolated from the bark of BK, could treat inflammatory diseases by modulating the activation state of macrophages by suppressing ERK and p38 MAPK and enhancing the JAK2-STAT3 signaling pathway [96]. In the same year, flavanone, broussochalcone C, broussoflavanonol A, kazinol V, kazinol W and broussoflavonol B, isolated from root bark in 100% methanol, were shown to have potent anti-inflammatory effects on LPS-stimulated RAW264.7 cell through downregulating iNOS, COX-2, and TNF-α expression within the dose of 1.25-40 µM [40]. In the same year, the anti-inflammatory effect of broussoflavonol H was studied by Tian et al.; the results showed that the compound could significantly suppress the production of IL-2 in Jurkat induced by PHA and PMA, with an IC 50 value of 9.95 µM [41]. Moreover, in 2020, 5,7,3 ,4 -tetrahydroxy-3-methoxy-8,5 -diprenylflavone, kazinol M, broussoflavonol B, broussoflavonol A, and broussofluorenone C, isolated from the branches and twigs of BP, showed anti-inflammatory effects by activating NF-κB/AP-1 [42]. In 2021, eriodictyol, apigenin, and kaempferol reduced LPS-induced iNOS expression within the dosage of 0-30 µM in a dose-dependent manner, with IC 50 values of 11.98, 10.16, and 24.06 µM, respectively [14].
In 2008, the ethanol extract of BP roots was shown to reduce abdominal Evan's blue extravasations, including serotonin and sodium nitroprusside, caused by inflammatory mediators; the effects might be related to inhibiting the vascular permeability via autocrines and NO [97]. In 2010, the anti-inflammatory effect of methanol extract of BP heartwood was investigated in NC/Nga mice induced by an extract of the house-dust mite, Dermatophagoides farina; the results showed that the methanol extract could obviously inhibit AD-like skin lesions by decreasing the levels of IgE and IL-4 and inhibiting the induction of TARC/CCL17, MDC/CCL22, and RANTES/CCL5 in HaCaT cells [98]. Furthermore, it was reported that the n-hexane fraction and n-butanol fraction of the methanol extract of BP stem bark at a dosage of 10-80 µg/mL were found to have significant anti-inflammatory activities in RAW 264.7 cells by inhibiting NO and pro-inflammatory cytokine production [74,97]. In 2014, the ethanol extract of BK leaves within a dose of 200-1000 µg/mL could treat Nc/Nga mice that were predisposed to develop AD-like skin lesions induced by D. farinae extract; further study demonstrated that its mechanism might be related to significantly downregulating the plasma levels of IgE and IL-4, as well as inhibiting hTARC secretion in HaCaT cells by activated TNF-α/IFN-γ [97].
In general, the anti-inflammatory activity of Broussonetia species was mainly studied in a murine macrophage RAW264.7 cell model and in mice stimulated with LPS. Moreover, the mechanism of anti-inflammatory activity was mainly concentrated on inhibiting NO production and iNOS expression. iNOS was primarily found in macrophages induced by LPS or cytokines to produce a high level of NO as a pro-inflammatory mediator [49]; therefore, the inhibition of NO production or iNOS expression was a critical strategy for the treatment of inflammatory diseases.

Anti-Diabetic and Anti-Obesity Effects
Diabetes is a chronic disease that presents as high levels of glucose in the blood, which may be caused by insulin deficiency and insulin resistance. All in vitro and in vivo studies have demonstrated the antidiabetic effects of different extracts and compounds prepared from Broussonetia species.
In 2008, Cha et al. indicated that the ingestion of stem bark powder from BK decreased the serum levels of glucose, fructosamine, triglyceride, and total cholesterol, as well as the activity of ALT in the genetically diabetic OLETF rats; the important regulatory factor would be the increased blood insulin level in the animal model [16]. In 2010, Ryu et al. showed that broussochalcone A, papyriflavonol A, broussochalcone B, kazinol A, kazinol B, and 8-(1,1-dimethylallyl)-5 -(3-methylbut-2-enyl)-3 ,4 ,5,7-tetrahydroxyflanvonol have inhibitory effects against α-glucosidase with a dose of 0.01-1000 µM; the IC 50 values were 5.3, 11.1, 12.0, 26.3, 3.6, and 2.1 µM, respectively [56]. Moreover, kazinol U [99], isokazinol D, and kazinol C [100] showed therapeutic potential in delaying pancreatic β-cell destruction in type 1 diabetes by blocking the NF-kB pathway in pancreatic β-cells and reducing RINm5F cell damage. A report in 2012 indicated the anti-obesity effect of broussonone A, as well as of other isolated phenolic compounds isolated from BK stem barks, the mechanism being related to noncompetitive inhibitory activity on pancreatic lipase, with an IC 50 of 28.4 µM and an inhibitory effect of adipocyte differentiation in 3T3-L1 cells [33]. In 2016, antidiabetic activity was also observed in mouse 3T3-L1 preadipocyte cells and C2C12 myoblast cells. Lee et al. demonstrated that kazinol B, isolated from BK roots, within a dose of 0-20 µM could increase insulin sensitivity via improving glucose uptake through the insulin-Akt signaling pathway, along with AMPK activation [101]. In 2020, treatment with broussoflavonol B and kazinol J in HFD-fed C57BL6 male mice within the dosage of 0-100 µg/mL, showed therapeutic potential in obesity and type 2 diabetes via suppressing pro-inflammatory responses by activating AMPK in 3T3-L1 adipocytes [102]. In addition, an ethanolic extract of BK fruits could treat β-cell damage by preventing STZ-induced oxidative stress and suppressing β-cell apoptosis via inhibiting Erk phosphorylation, as found in mice injected with STZ [15], and it could also treat diabetic nephropathy via the activation of Nrf2 and provide protection against PA-induced lipotoxicity in the mesangial cells in diabetes [103].
In a word, the mechanism of anti-diabetic effects is mainly related to blocking the NF-kB pathway and inhibiting α-glucosidase activity. The generation of NO via iNOS and reactive oxygen species plays an important role in pancreatic β-cell damage. The NF-kB transcription factor was activated by oxidative stress due to reactive oxygen species as well as regulating iNOS expression. Thus, the NF-kB pathway can protect the β-cell from damage [99].

Antibacterial and Antiviral Effects
Some studies have shown that the extracts or pure compounds of Broussonetia species could suppress bacteria. In 2015, N. Naveen Kumar et al. [104] reported that the hexane extract of BP seeds showed high inhibitory activity on Staphylococcus aureus, Proteus vulgaris, Bacillus cereus, and Enterobacter aerogenes, whereas it had no inhibitory effect on fungal strains [104]. In 2017, Park et al. analyzed the antibacterial activity of papyriflavonol A within the dosage of 1-1000 µM; the results showed that the potent inhibitory effect of PLpro, with an IC 50 value of 3.7 µM, along with a further study, showed that it may be a potential anti-COVID-19 agent [105]. Geng et al. [17] indicated that 5,7,3 ,4 -tetrahydroxy-3-methoxy-8,5 -diprenylflavone, isolated from the BP air-dried aerial part, showed more antibacterial activity in suppressing Actinomyces naeslundii and Porphyromonas gingivalis (MIC = 1.95 ppm) than the positive control, triclosan, at a dosage of 0.12-250 ppm. In 2021, Ghosh et al. [18] found that six polyphenols (broussochalcone A, papyriflavonol A, 3 -(3-methylbut-2-enyl-3 ,4 ,7-trihydroxyflavane, broussoflavan A, kazinol F, and kazinol J) showed greater Mpro inhibitory effect than two repurposed drugs (lopinavir and darunavir) and may serve as promising anti-COVID-19 drugs.

Skin Whitening and Anti-Wrinkle Activities
In 2019, Lim et al. [19] reported that kazinol U, a constituent of BK root barks, could attenuate melanogenesis within a dose of 0-20 µM via inhibiting MITF expression, inactivating target genes such as tyrosinase, Tyrp1, and Tyrp2, and activating AMPK and MAPK proteins in both in vitro and in vivo experiments. In the same year, it was reported that collagen is the major structural protein in the extracellular space of the connective tissue of the skin, and BK stem extract could maintain skin collagen content via inactivating the reactive oxygen species and inhibiting collagenase activity [106].

Other Properties
Out of these pharmacological activities displayed above, Broussonetia species also showed the treatment of bone diseases, liver protection, promoting hair growth, antiangiogenic activities, anticholinesterase effects, increasing cAMP, immune-stimulating activity, and antinociceptive.
In 2021, Vu et al. observed the antiosteoclastogenic activity of broussonols F, G, and K; the results showed that the compounds significantly inhibited RANKL-induced osteoclast formation with a dosage of 10-30 µM in RAW264.7 cells and they may be the lead compounds against bone diseases in the future [44]. In 2020, CSZ extract increased liver function and alleviated DILI in rats induced in acetaminophen (APAP) via regulating the TLR3/JNK/c-jun/c-fos/JAK/STAT3 pathway [107]. In 2020, Lee et al. [108] showed that ethanolic extract of BP had the capacity of promoting hair growth by regulating β-Catenin and STAT6 target proteins in human hair follicle dermal papilla (hHFDP) cells within a dose of 0-20 µg/mL. In 2014, the ethanolic extract of BK twigs was found to have anti-angiogenic activities with a dosage of 0.1-10 µg/mL; the mechanisms were the inhibition of VEGF-A, stimulated by the phosphorylation/activation of ERK, Akt, and p70S6K, and the downregulation of VEGFR-2 and MMP-2 in human umbilical vein endothelial cells [109]. In 2012, three prenylated flavonols, 8-(1,1-dimethylallyl)-5 -(3methylbut-2-enyl)-3 ,4 ,5,7-tetrahydroxyflanvonol,papyriflavonol A, and broussoflavonol B, isolated from BP roots, suppressed two human cholinesterases related to Alzheimer's disease (AD) in a dose-dependent manner, with IC 50 values ranging from 0.8 to 3.1 µM and from 0.5 to 24.7 µM against HAChE and BChE, respectively [47]. In the next year, the immune-stimulating activity of mice that were immunized intraperitoneally with OVA/alum (100 µg/200 µg) was tested; the results showed that mice given BK water extract orally for 21 days could enhance the Th1 immune response and showed no cytotoxicity in the model system [110]. In 2010, Lee et al. [50] explored the estrogenic activity of broussonin A, (+)−(2R) kazinol I, tupichinol C and kazinol U, which showed that these compounds could regulate the E2-responsive genes as functional ER ligands such as E2 in the ERsensitive MCF-7 cells at 10 µM. Kazinol P, a natural compound isolated from BK, showed the therapeutic effects of improving muscle regeneration and repair with the mechanism of promoting myogenic differentiation through activating p38MAPK and MyoD transcription activities [111]. In a study in 2003, Kazinol B, an isoprenylated flavan, showed significant inhibitory activities of nitric oxide (NO) in lipopolysaccharide-activated macrophages, with an IC 50 of 21.6 mM [112].   Reducing NO production through downregulating iNOS, COX-2, and TNF-α expression and the expression of iNOS protein. [40]

Supplements to the Diet of Animals
It was reported that a supplement of Broussonetia species in the roughage diet was beneficial to the growth performance, carcass traits, meat quality and color, immune response, digestibility of crude protein, and rumen fermentation in different animals, such as Hu rams and lambs and growing goats [20,119]. Tao et al. [120] demonstrated that beef cattle fed on a diet with 15% BP could enhance their antioxidant functions by decreasing blood 8-OHdG and MDA and increasing blood SOD and TAC; the supplements could strengthen the performance by increasing final BW, ADG, DMI and FCR, improve the meat quality by lowing pH and drip loss and increasing CIE L, and also increase the PUFA and DHA concentrations in meat. A diet supplemented with BP could enhance immune and antioxidant function, as well as increase the polyunsaturated fatty acid concentrations in the milk of Holstein cows [121,122]. A diet supplemented with BP leaf extracts at a certain dosage could increase the growth performance and antioxidant capacity of weaned piglets, enhance immune functions and disease resistance, reduce the occurrence of diarrhea, and affect the composition of fecal microflora [123].

Phytoremediation of Heavy Metal-Contaminated Soil
The effective phytoremediation of heavy metal-contaminated soil requires species with high metal tolerance. Broussonetia species were excellent choices, owing to their adaptation to drought and to a saline-alkali environment [124]. Zeng et al. [125] showed that BP could effectively alleviate the adverse effects of heavy metal-contaminated soil on plant growth by enhancing the antioxidant enzyme activities in leaves and binding heavy metal-contaminated soil with organic acids, carbohydrates, protein, and amino acids in roots. Huang et al. [126] isolated Bacillus cereus HM5 and Bacillus thuringiensis HM7, and explored their potential to improve the effect of remedying Mn pollution by BP; the results showed that the biomass, total root length, surface area, crossings, tips, forks, and root activity of BP with the two strains were higher than BP without the two strains, so the two strains could promote the accumulation of Mn. Luo et al. [127] indicated that BP could be used for the revegetation and phytostabilization of zinc-smelting slag sites because of the high heavy-metal tolerance and low heavy-metal accumulation. Co-planting was also a sustainable approach for the phytoremediation of the heavy metal-contaminated soil. The hyperaccumulator Pteris vittata L., co-planted with BP, could improve the environmental quality of heavy metal-contaminated soil by promoting the growth and uptake of P. vittata L. and improving the comprehensive extraction of metal [128]. Zeng et al. [129] selected Pteris vittata L., Arundo donax L., Morus alba L. and BP for tree-herb co-planting; the results indicated that the four-herb co-planting system positively affected the soil microbes and had stronger impacts on the composition of soil microorganisms.

Combination Using
The pharmacological effects of Broussonetia genus are limited but, when combined with other plants, it can exert more meaningful pharmacological effects. A liquid bandage was made from Styela clava tunics and BK barks cellulose powders; it could accelerate wound-healing in the surgical skin wounds of Sprague-Dawley rats by stimulating re-epithelialization and connective tissue formation, without liver or kidney toxici-ties [23]. A new phytoformula containing BP and Lonicera japonica was revealed to have therapeutic potential for systemic septic inflammation, as well as chronic bronchitis, and against LPS-induced septic inflammation in mice by reducing the induction of some important proinflammatory cytokines, at a dosage of 200-400 mg/kg [22]. Phellinus linteus, cultured with BK, could inhibit melanogenesis by activating the phosphatidylinositol 3kinase/Akt/glycogen synthase kinase-3beta signaling pathway and down-regulating the microphthalmia-associated transcription factor [130].

Papermaking and Cloth-Making
The barks of the Broussonetia species were excellent materials for the production of papers and clothes, which can be attributed to the high fiber content of the phloem. Cai Lun papermaking was one of the four great inventions of ancient China; this great invention used BP barks at that time. Notably, the first banknote in the world was made from BP. Papers made by BP barks were once used for making books and for writing in the Tang dynasty and their use continued during the Ming and Qing dynasties, as evidenced by many Dunhuang and Turfan manuscripts [21]. The bark of BP can also be used to make ancient tapa clothes; in this process, the barks were peeled from the cut stems to obtain a long strip, then the manufacturer removed the inner bark and washed and pounded the fibers to flatten them, felted them together with sheets in the sun, and finally printed them with native dyes to produce the finished traditional tapa cloth [10].

Others
In addition to the applications mentioned above, the remaining applications are classified as "others". A study conducted by Qiu et al. explored their application in cleaning up phosphorus pollution; they showed that BP biochar, when combined with phosphate in the forms of exchangeable phosphorus, Al-bound phosphorus, and Fe-bound phosphorus, could be used to treat eutrophic bodies of water [131]. Zhang et al. [132] measured the ability of BP to resist drought due to the electrophysiological characteristics of the plants; the results showed that the relative tensity of BP and M. alba were 3.965 and 2.624, respectively. The author also demonstrated that the minimal fluorescence efficiency and maximal photochemical efficiency were 5.496 and 7.640 for BP and 6.577 and 5.359 for M. alba, respectively; therefore, the drought-resistance ability of BP was greater than that of M. alba. Mo et al. [133] studied the ability of BP to control air pollution; the results showed that BP was efficient in capturing small particles and showed high levels of PM accumulation.

Conclusions and Further Perspectives
This review mainly summarized the phytochemistry, pharmacology, and applications of the Broussonetia genus. In this work, we present a list of 338 compounds that have been isolated from the herbs of Broussonetia, including 144 flavonoids, 50 phenylpropanoids, 38 polyphenols, 35 alkaloids, 17 terpenoids, 5 steroids, and 49 other metabolites, which indicated that flavonoids were the main constituent in the genus of Broussonetia. A variety of pharmacological activities have been demonstrated in vivo or in vitro assays, including anti-tumor, antioxidant, anti-inflammatory, antidiabetic, anti-obesity, antibacterial, and antiviral properties, as well as skin whitening and anti-wrinkle activities. Nevertheless, some studies of the Broussonetia genus are limited at present.
First, phytochemistry clarified 338 compounds isolated from BP, BK, and BL, but only 5 steroids and 17 terpenoids were involved. Undoubtedly, we can make more effort toward the exploration of steroids and terpenoids by the targeted phytochemical methods. Second, the Broussonetia genus consists of 11 species, but the investigations of phytochemistry, pharmacology and applications were only studied in BP, BK, and BL. Therefore, it is extremely important for researchers to conduct a comprehensive evaluation of other species to extend the available source domain. Third, pharmacological studies have uncovered anti-tumor, antioxidant, anti-inflammatory, anti-diabetic, anti-obesity, antibacterial, and antiviral properties, as well as skin whitening and anti-wrinkle activities. Notably, the relevant bioactive compounds only include flavonoids, penylpropanoids, and polyphenols; therefore, the pharmacological activities of the remaining compounds need to be further explored by researchers. Furthermore, the pharmacological activities of the concrete compounds of crude extracts need to be confirmed in the future, which would be conducive to finding new candidates for corresponding diseases. Fourth, mechanism analysis indicated that active compounds and extracts mainly showed anti-tumor effects by inducing cell apoptosis and triggering cell cycle arrest; they showed anti-inflammatory activity mainly by inhibiting NO production and iNOS expression and showed anti-diabetic effects mainly via blocking the NF-kB pathway and α-glucosidase. Undoubtedly, we can see that mechanism studies of the active compounds and extracts isolated from Broussonetia species were mainly concentrated on the typical targets and pathways. Hence, new targets and pathways should be detected in the future. Fifth, in vitro models were considered to be the main conditions; therefore, more in vivo models should be used to investigate properties in the future. Sixth, although the applications indicated that Broussonetia species could be used to supplement the diet of beef cattle, growing goats, cows, and piglets, and showed multiple beneficial effects on their growth performance, carcass traits, meat quality, and immune response, the concrete clinical safety, toxicity and pharmacokinetics studies for animals and humans were extremely limited; thus, exploring these aspects is the top priority in future research.
Overall, this updated review on the plants of the Broussonetia genus can provide an important and valuable reference for researchers interested in Broussonetia and promote scientific development and utilization of the Broussonetia genus.  Thymus-and-activation regulated chemokine MDC/CCL22 Macrophagederived chemokine RANTES/CCL5 Regulated-onactivation-normal T cell-expressed-and-secreted chemokine IL-4 Interleukin-4 IgE Plasma immunoglobulin E RINm5F cells Rat pancreatic β-cell line HFD Ten-week-high fat diet