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provided by SZTE Publicatio Repozitórium - SZTE - Repository of Publications Phytochem Rev (2018) 17:1031–1045

https://doi.org/10.1007/s11101-018-9565-1 (0123456789().,-volV)(0123456789().,-volV)

Phenolic antioxidants of nigra roots, and antitumor potential of morusin

Zoofishan Zoofishan . Judit Hohmann . Attila Hunyadi

Received: 8 January 2018 / Accepted: 25 March 2018 / Published online: 31 March 2018 Ó Springer Science+Business Media B.V., part of Springer Nature 2018

Abstract Phenolic compounds are of considerable Keywords Anticancer activity of prenylflavone Á biomedical interest due to their antioxidant properties Black mulberry root bark Á Cancer stem cell Á and potential in the prevention and possibly treatment Ethnopharmacology Á In vivo antioxidant activity of many chronic diseases. The , and root bark of Morus nigra (), the black mulberry tree, have a long history of use for various therapeutic purposes in traditional medicine worldwide. The roots Introduction of the are known to be a rich source of phenolic compounds with a particularly high chemical diver- The Morus genus, belonging to the Moraceae family, sity. This mini-review compiles the currently available comprises of twenty-four with one subspecies, knowledge on phenolic compounds reported from and has ca. one hundred known varieties to date Morus nigra roots, and provides a brief overview on (Ercisli and Orhan 2007; Hojjatpanah et al. 2011). the antioxidant activity with a focus on the available Well known as the ‘‘black mulberry tree’’, Morus in vivo evidence. Morusin, a major phenolic antiox- nigra L. has long been valued for its , which has idant of the root bark, has attracted a rapidly increasing also been claimed as a ‘‘super fruit’’ that has various scientific interest for its versatile and potent antitumor health benefits and promotes longevity (Bircher and properties; recent developments in this regard, includ- Bircher 2000). Morus nigra is a wildly growing rustic ing morusin’s promising activity against cancer stem plant that is also present in many gardens and, cells, are also discussed in the paper. similarly to other Morus species and M. alba in particular, it was also used for sericulture (Hanelt et al. 2001; Orwa et al. 2009). Morus nigra often produces leaves of different shapes, and these multilobed leaves can appear on the same branches. The fruit is -shaped and typically black but sometimes & Z. Zoofishan Á J. Hohmann Á A. Hunyadi ( ) dark blue in color, with a sweet taste. Despite popular Faculty of Pharmacy, Institute of Pharmacognosy, University of Szeged, Eo¨tvo¨s u. 6, H-6720 Szeged, belief, the color of the fruit alone does not determine Hungary the mulberry species; instead, the leaves and wood e-mail: [email protected] characteristics should be taken into account for an exact identification (Orwa et al. 2009). J. Hohmann Á A. Hunyadi Interdisciplinary Centre of Natural Products, University of The plant has a long and remarkable history. Likely Szeged, Szeged, Hungary introduced to Europe originally from Persia, its 123 1032 Phytochem Rev (2018) 17:1031–1045 cultivation on the Continent appears to date back and bark of black mulberry are known folk remedy for thousands of years (Huxley and Taylor 1989). The diabetes, arthritis, and hypertension, and as diuretics ancient Greeks dedicated the fruit to Minerva, the (Abbas et al. 2014; Ribeiro et al. 2017). The root bark goddess of wisdom. Black mulberry was abundant in has anthelmintic activity, and it is also used to treat Italy during the Roman era; it was also depicted in the bronchitis (Mohiuddin et al. 2011), as well as a paintings from Pompeii (Georgics ii verse 121; purgative agent (Hanif and Singh 2012). The roots of Schreiber 1958). Mulberry has also been acknowl- M. nigra contain an outstandingly rich variety of edged in the Bible (I Maccabees 6:34; Luke 17:5-6). phenolic compounds, which compound family is Black mulberry leaves were considered as the feed for likely among the most important constituents related Italian silkworms until 1434 when was to the therapeutic benefits associated with traditional introduced from the Levantine lands (Vivarelli and preparations (Manach et al. 1997). Alvisi 1934). During the ‘‘silk revolution’’ in 1608, the The chemistry and pharmacology of Morus species, practice of sericulture was introduced to Britain by and particularly of M. alba, served as the basis of promoting mulberry cultivation (Grieve 1931). several recent reviews (e.g. Chan et al. 2016; Hussain Although its medicinal use was an ancient practice, et al. 2017; Yaun and Zhao 2017), indicating well the it was the growing silk industry that brought this plant very high current scientific interest in these . into the spotlight, resulting in the worldwide spreading Bioactive constituents of Sang-Bai-Pi, root bark of of mulberry trees to satisfy industrial needs for various Morus plants, have recently been reviewed mulberry leaves (Lev 2002). Despite black mulberry from a general point of view (Wei et al. 2016). In the is in fact more nutritive than Morus alba, the white present paper, we aim to set a more specific focus on mulberry (Venkatesh and Chauhan 2008), its impor- (i) the phenolic compounds isolated from the roots of tance has finally been surpassed by that because of its M. nigra, (ii) the in vivo evidence supporting their weaker success in feeding silkworms. The nutritive antioxidant activity, and (iii) the antitumor activity of richness of black mulberry, however, still makes it an morusin, a new ‘‘rising star’’ among the major excellent animal feed that also enhances the milk yield phenolic antioxidants of M. nigra root bark. of dairy animals (Dillard and Bruce German 2000). The root bark of M. nigra also plays an important economical role through its use to prepare ‘artificial Phenolic composition of Morus nigra cotton’ textile fiber, paper and clothes (Hanelt et al. 2001; Lim 2012). Besides its use as livelihood, the All Morus species are known as rich sources of long history of Morus nigra includes its therapeutic phenolic compounds including flavonoids, stilbenes, use in practically every era of traditional medicine, 2-arylbenzopyrans, and a variety of Diels–Alder under different names, and with various, frequently adducts (Nomura 1988; Nomura and Hano 1994), different uses in each system (Leonti et al. 2015). and, according to many comparative studies, M. nigra Ancient Egyptians used the plant preparations for appears to be the richest of all in this regard (Ercisli corroding ulcers and dispersing inflamed tumors, and and Orhan 2007; Wang et al. 2007; Ercisli et al. 2009; the bark juice was an antidote to the venom of deadly Arfan et al. 2012). As an example, a comparison with snakes (Pliny the Elder 2015). Morus nigra has been M. alba revealed that M. nigra has a higher content of used in Ayurveda, one of the oldest holistic healing reduced ascorbic acid, titratable acidity, iron, total systems dating ca. 600 B.C. Many M. nigra formula- flavonoids and total monomeric (Jiang tions are still in use in Ayurvedic medicine, e.g. and Nie 2015). against cancer (Tut-i-aswad, containing the fruits) Flavonoids have a remarkable chemical diversity (Ahmad et al. 1985), and for treating sore throat or not only due to their variability in hydroxylation/ laryngitis (Rub-e-toot siyah, containing the root bark) methoxylation pattern, but mainly due to the many (Khare 2010). The roots of Morus plants were possibilities for being further substituted with , recorded in the Chinese pharmacopeia in 500 B.C. prenyl and geranyl groups, esterified with organic collectively known as sang-Bi-Pi often used for acids, etc., and many of these substituents open treating lung heat, cough, edema, and oliguria (Phar- possibilities to further cyclization. In particular, the macopoeia Committee of P. R. China 2010). The roots root bark of Morus nigra contains a remarkable variety 123 Phytochem Rev (2018) 17:1031–1045 1033 of prenylflavonoids (Ferrari et al. 1999, Wang et al. Diels–Alder type adducts 2007, 2008, 2009, Zheng et al. 2010, De Souza et al. 2000, Simonetti et al. 2017, Zelova et al. 2014), as The [4 ? 2]-Cycloaddition of a conjugated diene and presented in Fig. 1. Interestingly, phenolic compounds a dienophile to form a six-membered ring is a well- isolated from the roots of Morus nigra L. showed known Diels–Alder reaction. Many of these classical remarkable differences as compared to those biosyn- compounds are known to occur in the Morus genus, thesized by cell culture of plant, based on a compar- and an immense variety of Diels–Alder type adducts ative study of Ferrari et al. (1999). From the roots, the have so far been reported from the roots of M. nigra,as flavonoids Kuwanon U, Cyclomorusin, Morusin summarized in Fig. 4. Kuwanol E isolated from the hydroperoxide, Morusin, Kuwanon C, Kuwanon E, roots was found to be potent inhibitor of Mycobac- Morusinol, Norartocarpetin and seven Diels–Alder terium tuberculosis tyrosine phosphatase B type adducts (Albanol B, Kuwanon O, Kuwanon H, (Mascarello et al. 2013, 2016). Recently, a similar Kuwanon G, Albafuran C, Kuwanon L, and Morace- testing of other constituents of Morus nigra roots, nin D) were isolated. In contrast to this, the callus gave namely Cudraflavanone A, Morusin, Oxyresveratrol, 4-hydroxyisocordoin, oxyresveratrol, resveratrol, and Chalcomoracin, Norartocarpetin, Kuwanon L, Kuwa- six Diels–Alder type adducts (Kuwanon V, Mulber- non G, and Kuwanon H revealed the latter two rofuran E, Kuwanon R, Kuwanon J, Chalcomoracin, compounds as particularly potent inhibitors of above and Kuwanol E). Above the remarkable lack of enzyme of Mycobacterium tuberculosis (Mascarello compounds isolated from both sources, significant et al. 2018). differences could be observed in the stereochemistry of the cyclohexene ring of Diels–Alder type adducts: Arylbenzofurans and stilbenes this was different for compounds from the cell culture (cis–trans) and for those from the plant (trans–trans) Morus nigra has widely been acknowledged as a rich (Ferrari et al. 1999). source of bioactive benzofuran derivatives and stilbe- nes, among which the 2-arylbenzofurans are com- Geranylflavonoids monly substituted by prenyl and geranyl groups. (Nguyen et al. 2009). Various bioactivities (e.g. Three geranylflavonoids have been reported from M. antifungal, antibacterial, etc.) associated with benzo- nigra roots, one flavone and two flavanone type, each furan derivatives make these compounds attractive containing the geranyl group in the B-ring, connected subjects for medicinal chemistry studies (Kone et al. to C-50 (Fig. 2); the presence of an isoprenyl/geranyl 2004; Fukai et al. 2005; Zheng et al. 2010). Similarly group at different positions in the B-ring of the to prenylflavonoids, benzofurans can also inhibit nitric flavonoid skeleton significantly affects tyrosinase oxide synthase, thus indirectly inhibiting the release of inhibitory activity (Zheng et al. 2010). a major inflammatory mediator, nitric oxide (Zelova et al. 2014). Constituents of M. nigra roots with Chalcones 2-phenylbenzofuran skeleton are presented in Fig. 5 (Wang et al. 2008; Zheng et al. 2010; Zelova et al. Although it would be expected to contain a larger 2014). Although one more compound, morunigrol D, variety of these compounds as well, up to now only appears in the literature as a constituent of the root two chalchones have been reported from the roots of bark (Zelova et al. 2014), its presence in the roots M. nigra; structures of these compounds are presented cannot unambiguously be declared here since it was in in Fig. 3. These chalcones were found more biolog- fact isolated from the stem bark (Wang et al. 2010). ically potent in comparison with geranyl/prenyl These compounds were reported with their tyrosinase flavonoids concerning tyrosinase inhibitory activity activity that was found to be lower in 2-arylbenzofuran (Zheng et al. 2010). derivatives as compared to stilbene derivatives, sug- gesting that formation of the five-membered ring significantly affects this bioactivity (Zheng et al. 2010).

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Fig. 1 Prenylflavonoids from the roots of Morus nigra

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Fig. 2 Geranylflavonoids from the roots of Morus nigra

Fig. 3 Chalcones from the roots of Morus nigra

A number of stilbenes (Oxyresveratrol and three of of the health benefits attributed to the traditional its O-b-D-glucopyranosides) have also been reported preparations (Pandey and Rizvi 2009). Accordingly, it from the roots of M. nigra; structures of these is not surprising that a very large number of related compounds are presented in Fig. 6. Glycoside moi- in vitro antioxidant studies are available. Strong free eties were suggested to significantly affect the tyrosi- radical scavenging effect was reported on various nase activity of stilbenes, and glycosidation at 2 or 4 extracts of black mulberry fruits, leaves or roots (e.g. position suppresses the activity to a much larger extent Naderi et al. 2004;Pe´rez-Gregorio et al. 2011) and/or than at the 30 or 50 position (Zheng et al. 2010). phenolic constituents in a number of in vitro exper- imental models, and several related papers have been Coumarins published on the inhibition of xanthine oxidase or lipid peroxidation (e.g. Radojkovic et al. 2016; Arfan et al. Five coumarin glycosides, presented in Fig. 7, are 2012; Imran et al. 2010). known to be present in the roots of M. nigra (Zheng A large number of in vitro antioxidant studies on et al. 2010). Among these, mulberroside B is a individual compounds are also available; here we C-glycoside at C-6, while the others are O-glycosides briefly summarize only a few characteristic examples. containing one or two components connected to Morusin, a major constituents of M. nigra roots, the coumarin skeleton. In each of the latter ones, the showed inhibitory effect on superoxide formation from coumarin bears an O-b-D-glucoside at C-7, which can rat neutrophils stimulated with phorbol myristate be further glycosylated with a 6-deoxymannose or an acetate (PMA) (Ko et al. 1999). The benzofurans apiose (Fig. 7). moracin C and N were reported to scavenge superoxide anion radical and inhibit lipid peroxidation (Sharma et al. 2001). In a study aiming at the antioxidant activity Antioxidant activity of Morus nigra roots and their of four aryl benzofuran derivatives, moracin C, P, and constituents M, and mulberroside C, two compounds, moracin C and M were found particularly effective in the inhibition of Recent studies on M. nigra cover a rich variety of malondialdehyde (MDA) production during microso- constituents and a broad spectrum of bioactivities; in mal lipid peroxidation induced by ferrous cysteine (Tan this mini-review, we aim to focus on the antioxidant et al. 2008). The stilbene oxyresveratrol exerted activity. Since the roots of Morus nigra are very rich in considerable superoxide scavenging effects (Oh et al. phenolic compounds, such an effect might reasonably 2002). Among many other compounds in a series of be expected to significantly contribute at least to some studies, albanol B, moracin M, and mulberrofuran G 123 1036 Phytochem Rev (2018) 17:1031–1045

Fig. 4 Bioactive Diels–Alder type adducts from the roots of Morus nigra

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Fig. 5 2-Arylbenzofurans from the roots of Morus nigra

Fig. 6 Stilbenes from the roots of Morus nigra. Glc = b-D-glucopyranoside

were found particularly effective in the two most within the gastrointestinal tract (Dominguez-Avila common in vitro test for free radical scavenging, et al. 2017), weak absorption and extensive metabolism namely the 2,20-azinobis-3-ethylbenzothiazoline-6-sul- (Rothwell et al. 2016), which are all limiting or altering fonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (Lotito et al. 2011) the in vivo bioactivity of orally (DPPH) assays (Cui et al. 2008;Fuetal.2009), while consumed phenolic antioxidants as compared to that for example the prenylflavonoids morusin and kuwanon in vitro. Unfortunately, to the best of our knowledge, no C showed moderate DPPH radical scavenging activity in vivo antioxidant studies are currently available with (Mazimba et al. 2011). preparations from M. nigra roots (or root bark). Despite there are many reports available on the Nevertheless, there are related reports on the fruits in vitro antioxidant activity of M. nigra root extracts and leaves, whose compositions certainly show over- and constituents, vast majority of these have unfortu- laps with that of the roots. Oral administration of a nately little if any in vivo relevance. It is now well crude, 95% ethanol extract of M. nigra fruits to mice understood that there are significant pharmacokinetic resulted in decreasing MDA content in the serum and constraints, including complex interactions already liver, accompanied by an increase of superoxide 123 1038 Phytochem Rev (2018) 17:1031–1045

Fig. 7 Coumarins from the roots of Morus nigra dismutase (SOD), catalase (CAT) and glutathione compounds might have had in the observed activity. peroxidase (GSH-PX) activity (Feng et al. 2015). Accordingly, it is also unclear whether or not a similar Unfortunately, while the authors claimed this extract effect could be expected from root extracts, since the as ‘‘total flavonoids of M. nigra’’, the chemical presence (and certainly the amounts, if any) of the composition remained uninvestigated, and ethanol is above-mentioned phenolic acids in the roots has not a good extracting solvent of not only flavonoids but yet been reported. also a wide variety of other types of secondary Investigating a traditional use widespread in Brazil, metabolites. Accordingly, there might have been the leaves of M. nigra were also studied for their several constituents in the studied extract (Feng et al. antidiabetic and antioxidant activity on streptozo- 2015) that are also present in the roots of the plant. tocin-induced diabetic rats, focusing on the maternal– In another study, the methanol extract of M. nigra fetal outcome. The aqueous extract was used, and leaves was found to protect DNA from UV-induced while it was unable to control blood glucose levels, it damage, and, after oral administration, to protect mice demonstrated a potent in vivo antioxidant activity: from D-galactose induced cerebral oxidative stress. increased SOD activity, decreased MDA levels were This latter effect manifested in a significantly less found, and the offsprings from diabetic dams showed profound cognitive impairment, lower malondialde- reduced incidence of skeletal and visceral anomalies hyde levels in the serum, liver and brain, as well as in upon treatment (Volpatoa et al. 2011). The extract was elevated SOD, GSH-PX, and CAT activities as prepared in the traditional way, i.e. boiling the dried compared to those in the D-galactose treated group. leaves in water, suggesting it to be likely rich in Since the utilized extract contained phenolic acids phenolic acids and other water soluble substances such as vanillic acid and chlorogenic acid as chief unlike similar root extracts. However, these conditions constituents (Turgut et al. 2016), it is hard to judge likely also led to the extraction of a wide variety of less what relevance other accompanying phenolic polar constituents that could also be present in the 123 Phytochem Rev (2018) 17:1031–1045 1039 roots, including further (though perhaps not exten- anticancer potential of a compound (Klaunig et al. sively prenylated) phenolic compounds as well. 2011; Kotecha et al. 2016). Nevertheless, there is Concerning the in vivo antioxidant activity of much related controversy: increased risk of cancer has isolated pure compounds, a number of studies have also been attributed to certain antioxidants in some been published on the stilbene mulberroside A, special cases (for a related review, see e.g. Saeidnia diglycoside of oxyresveratrol, obtained from Morus and Abdollahi2013), demonstrating well the complex- bombycis but also reported from Morus nigra roots ity of the situation. (see Fig. 6). Antioxidant properties of this compound Morusin is a major phenolic antioxidant of M. nigra have been reported in connection with its antidiabetic roots (De souza et al. 2000), whose antitumor prop- and liver protective activity. Oral treatment of STZ- erties have recently become into the focus of intensive induced diabetic rats with 200–800 mg/kg of mulber- studies (Table 2). In connection with its antioxidant roside A led to an improvement in several diabetic activity, morusin was identified as a potent chemo- markers including a slight decrease in blood glucose, preventive agent in vitro, protecting JB6 P? mouse significant decrease in serum aspartate aminotrans- epidermal cells against the ROS-induced malignant ferase (AST), alanine aminotransferase (ALT), and transformations upon phorbol ester treatment. Pre- blood urea nitrogen, and increase in plasma insulin treatment with non-cytotoxic amounts (2.5–7.5 lM) levels. In addition to this, mulberroside A effectively of morusin significantly mitigated the ROS production decreased lipid peroxidation, so that no significant induced by 12-O-tetradecanoylphorbol-13-acetate difference was found in this regard between the (TPA), and consequently suppressed the TPA-induced diabetic control animals and those treated with the transactivation of activator protein 1 (AP-1) and smallest dose, 200 mg/kg (Heo et al. 2007). In nuclear factor-jB (NF-jB). Moreover, TPA-promoted addition to a rather mild in vitro superoxide scaveng- cell growth and malignant up-regulation of cell ing activity (IC50 = 430 lg/mL), the oral administra- surface proteins (N-cadherin and vimentin) were also tion of 100–600 mg/kg of mulberroside A 30 min reduced by morusin (Cheng et al. 2017). before inducing hepatotoxicity with CCl4 to rats Morusin was found to exert in vitro cytotoxicity exerted a similarly strong protective activity as against cancer cell lines of various origin, including silymarin, and significantly decrease AST and ALT e.g. pancreas, cervical and colorectal (Kim et al. 2016; levels. Hepatoprotective activity exerted through an Wang et al. 2013; Lee et al. 2008). Morusin induced antioxidant action was also confirmed by the inhibi- apoptosis in HT-29 colorectal carcinoma cells through tion of in vitro lipid peroxidation on rat liver caspase 8 subsequently leading to the activation of homogenate in the presence of Fe2?/ascorbic acid, caspase 9 and 3, and was suggested to cause a decrease and by the prevention of serious hystopathological in the phosphorylation of PDK1, PI3 K and Akt, changes caused by the CCl4 treatment (Jin et al. leading to the downregulation of IKK-a, IKK-b and 2006, 2007). In vivo antioxidant studies available on IjB-a, hence suppressing NF-jB (Lee et al. 2008). Morus nigra extracts and mulberroside A are summa- Morusin also demonstrated anti-invasive activity on rized in Table 1. SK-Hep1 cells in non-cytotoxic concentrations, and the suppression of the signal transducer and activator of transcription 3 (STAT3; an oncogene with an Morusin is a potent and versatile antitumor important role in cancer cell survival and prolifera- constituent of M. nigra roots tion) and NF-jB signaling was suggested as the two key mechanisms behind. The in vitro results were also Antitumor properties of antioxidants have long been in confirmed in nude mice, whose lung metastasis upon the focus of extensive research, and keep attracting a i.v. injection with SK-Hep1 cells was reduced when popular attention with high expectations. Such com- pre-treated with morusin, and Western blots of the pounds can participate in various redox cycles and, lung tissues also gave consistent results with those depending on the conditions, they can also contribute observed in vitro (Lin et al. 2015). STAT3 inhibition to the oxidative stress, and induce oxidative cell death by morusin was also reported in various pancreatic of cancer cells. Accordingly, strong connection carcinoma cell lines. Interestingly, morusin treatment appears to exist between the antioxidant activity and led to an accumulation of different phases in the cell 123 1040 Phytochem Rev (2018) 17:1031–1045

Table 1 In vivo antioxidant activity of Morus nigra extracts and mulberroside A Extract/compound Compounds Treatment Model Effect and mode of action Reference identified

Hot (boiling) None 400 mg/kg, pregnant, Diabetic dams: no glycemic changes, MDA Volpatoa et al. water extract of per os, STZ- levels ;, cholesterol, triglycerides and (2011) M. nigra leaves 0–20 days of induced VLDL ;; non-diabetic rats: SOD :; pregnancy diabetic fetuses from diabetic rats: smaller rats regardless of treatment, but skeletal and visceral anomalies ; Ethanol extract of None; UV 100–400 mg/ Kunming Serum and liver MDA ; and SOD activity Feng et al. M. nigra fruits absorbance kg, per os, strain :, liver CAT :, blood and liver GSH-PX (2015) correlated with for 30 days normal :; spleen Langhans cells : that of rutin mice Methanol extract Phenolic acids 50 or 100 mg/ D-galactose Serum, brain and liver: MDA level Turgut et al. of M. nigra (VA 632 lg/g, kg, per os, treated augmentation ;, SOD, GSH-PX, and (2016) Leaves CA 555 lg/g) for 60 days mice CAT activity :; improvement of cognitive deficits; DNA damage protection against UV and H2O2 a Mulberroside A n.a. 100–600 mg/ CCl4- Hepatoprotective activity comparable to Jin et al. kg, per os, induced that of silymarin; AST and ALT levels ;; (2006, 2007) 30 min liver prevention of hystopathological changes; 2? before CCl4 damage in Fe /ascorbic acid stimulated lipid rats peroxidation ; in liver homogenates Mulberroside Aa n.a. 200–800 mg/ STZ- Lipid peroxidation ;, and improved islet Heo et al. kg induced morphology in pancreas; diabetic (2007) diabetic markers improved: blood glucose ;, rats serum aspartate aminotransferase (AST) ;, alanine aminotransferase (ALT), and blood urea nitrogen ;, plasma insulin levels :, liver CYP-2E1 mRNA ; VA vanillic acid, CA chlorogenic acid, n.a. not applicable aIsolated from the roots of Morus bombycis, but reported to be present in M. nigra roots

cycle depending on the tested pancreatic cell line, and increased cell survival. Applying the autophagy induced both early and late apoptosis (Kim et al. inhibitor 3-methyladenine, however, greatly increased 2016). Considerable selectivity in the in vitro cyto- the rate of apoptotic cells (Cho et al. 2017). toxic activity of morusin was observed in a variety of Several in vivo studies confirmed the above obser- breast cancer cell lines as compared to immortalized vations on the antitumor potential of morusin. It had a normal breast MCF10A cells. When studying apopto- marked antitumor effect on H22 hepatocarcinoma- sis-related proteins regulated by STAT3, a suppres- transplanted mice and activity of morusin on the sion of the anti-apoptotic protein Survivin and over- expression of p53, Survivin, CyclinB1, and particu- expression of the pro-apoptotic protein B cell lym- larly on that of caspase-3 and NF-jB was identified as phoma 2-associated-x protein (Bax) was observed. possible mechanism (Wan et al. 2014) coherently with The rate of apoptosis, however, was found lower than previous reports. Morusin was most recently reported expected from the corresponding cytotoxicity results, as an anti-angiogenetic agent: in vitro inhibition of the suggesting that the effect of morusin also involves proliferation, migration and tube formation of human necrosis and/or autophagy. (Kang et al. 2017). This umbilical vein endothelial cells (HUVECs) was found, appears to be consistent with parallel results: morusin confirmed by the in vivo inhibition of hepatocellular was found to indeed induce autophagy through HepG2 xenografts’ tumor growth and angiogenesis. activating AMPK and inhibiting mTOR, which Both constitutive and interleukin-6 (IL-6) induced

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Table 2 In vitro and in vivo antitumor studies on morusin Type of Type of cancer Cancer cell line used Concentration of Effect and mode of action Reference study morusin

In vitro Human colorectal HT-29 29 lM Sub-G1 phase :, DNA Lee et al. carcinoma fragmentation, chromatin (2008) condensation, PDK1, PI3 K, IKK-a, IKK-b, and IkB-a phosphorylation ; =[ NF-kB ;; caspase-8, -9 and -3 activation In vitro Human cervical Non-adhesive culture of 1–4 lM Proliferation, tumor sphere Wang cancer stem HeLa formation and migration of et al. cells human cervical CSCs ;, (2013) apoptosis, NF-jBp65 ;, Bcl-2 ;

In vivo Hepatic H22 cells 10–40 mg/kg p53, Survivin, CyclinB1, Wan carcinoma Caspase-3 expression:, NF-jB et al. expression ; (2014) In vitro Human and MCF7, MDA-MB-231, 4–8 lg/mL in vitro, 5 Forcing differentiation of cancer Li et al. and murine breast MCF-10A; murine 4 T1 or 10 mg/kg i.p. in cells and xenograft tissue into (2015) in vivo cancer and EMT6 MCF7 xenografts adipose like cells, accumulation of lipid droplets, C/EBP b and PPARc :, adipsin D, and perilipin mediated adipogenic differentiation and lipoapoptosis In vivo Human hepatic SK-Hep1 0–15 lM Cell matrix adhesion and cell Lin et al. adenocarcinoma motility ;; modulation of EMT (2015) through E-cadherin expression :, vimentin and a2-, a6-, b1- integrin expression ;; MMP-2 and -9 ; through STAT3 and NF-jB ; In vitro Human AsPC-1, BxPC-3, MIA 20 lM Pro-apoptotic and anti-invasive: Kim et al. Pancreatic PaCa-2 and PANC-1 STAT3 phosphorylation ; (2016) adenocarcinoma through SHP-1 and SHP-2, nuclear translocation of p-STAT3 and STAT3 ;, STAT3 DNA binding ;, JAK1/JAK2, and Src activation ;, caspase-3 activation In vitro Human WJ1, GSCs from non- 1–5 lg/mL in vitro; GSCs growth in vitro and in vivo Guo et al. and glioblastoma adhesive culturing; L02 5–20 mg/kg in vivo ;, stemness of GSCs ;: CD133, (2016) in vivo cancer stem nestin, Oct4, and Sox2 ;; cells induction of adipocyte-like trans-differentiation and apoptosis In vitro Human breast MCF7, MDA-MB-231, 0–20 lM Apoptosis through caspase-3 and - Kang cancer MDA-MB-157, MDA- 9 :, B-cell lymphoma et al. MB-453; MCF 10A 2-associated-x protein (Bax) :, (2017) Survivin ;; little effect on normal human breast epithelial cells (MCF10A)

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Table 2 continued Type of Type of cancer Cancer cell line used Concentration of Effect and mode of action Reference study morusin

In vitro Human cervical HEK293T, HeLa, HepG2, 30 lM LC3-II accumulation, p-ULK1 Cho et al. and cancer HCT116, A549, MCF7, (both at Ser317 and Ser 757) : (2017) In vivo Hep2 in HeLa cells, AMPK activity :, mTOR activity ; =[ autophagy; morusin ? autophagy inhibitor =[ antitumor efficacy : In vitro Hepatocellular Hep3B, HepG2, human 4–8 lg/mL in HCC Apoptosis through caspase-3 and Gao et al. and carcinoma normal liver cells cells, 1–3 lg/mL in Bax/Bcl-2 expression ratio :; (2017) in vivo (LO2), human umbilical HUVECs, 5–15 mg/ in vitro proliferation, migration, vein endothelial cells kg in HepG2 and tube formation of HUVECs (HUVECs) xenografts ;, angiogenic proteins in HCC cells and HUVECs ;; in vivo angiogenesis ;; attenuation of the IL-6/STAT3 signaling pathway In vitro Human gastric MKN45 and SGC7901 1–5 mg/L in vitro, CDK2, CDK4, Cyclin D1 and Wang and cancer 50 mg/kg i.p. in Cyclin E1 ;; c-Myc ; and et al. in vivo xenografts c-Myc protein binding to the (2017) E-Box regions ;; over- expressing c-Myc counteracts antitumor activity phosphorylation of STAT3 was inhibited, and VEGF, relapse. Accordingly, it is insufficient to achieve MMP2, MMP9, and VEGFR2 were down-regulated tumor regression but a special attention is needed to both in vitro and in vivo (Gao et al. 2017). Interest- also eradicate the rare CSC population (Wicha et al. ingly, low-dose morusin treatment forced breast 2006). When testing morusin against cervical CSCs, cancer cells and xenograft tissue to differentiate into obtained through a non-adhesive culturing of HeLa adipose like cells accumulating lipid droplets and cells, it was found to dose-dependently suppress their over-expressing transcription factors C/EBP b and proliferation, tumor sphere formation and migration, PPARc, as well as adipogenic proteins adipsin D, and and it was inducing high rates of apoptosis at as low as perilipin (Li et al. 2015). Most recently, morusin was 2–4 lM concentration (Wang et al. 2013). In a most found to inhibit gastric cancer cell proliferation and recent study by the same group, morusin was inves- tumor growth by down-regulating the oncogene tigated for its potential against glioblastoma stem cells protein c-Myc both in vitro and in vivo, and that cells (GSCs). Glioblastoma multiforme is one of the most engineered to over-express c-Myc can bypass the G1 agressive types of cancer with a very poor prognosis, phase cell cycle arrest induced by this compound and GSCs are believed to play a central role in this. (Wang et al. 2017). Morusin was able to target GSCs with a ca. 2-folds selective in vitro cytotoxicity over that exerted on parental glioblastoma cells and ca. 10-folds over that Morusin holds promise against cancer stem cells on normal liver cells, and it could significantly inhibit the growth of in vivo glioblastoma xenografts initiated Cancer stem cells (CSCs) are a cell sub-population by GSCs (Guo et al. 2016). Coherently with the above- within the cancer tissue that seem to play a major role mentioned results of (Li et al. 2015) on breast cancer in the frequent failure in chemotherapy: due to their cells, GSCs could also be differentiated to adipocytes chemo-resistance, self-renewal and tumor initiating by lower, non-cytotoxic, 0.5–2.0 lg/mL doses of capacity, the surviving CSCs can induce tumor morusin, with a significant decrease of stemness

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Food Chem 103:1380–1384 Ercisli S, Tosun M, Duralija B, Voca S, Sengul M, Turan M Mounting in vitro and in vivo evidence suggests its (2009) Phytochemical content of some black (Morus promising activity against various types of cancers, nigra L.) and purple (Morus rubra L.) mulberry geno- and its selective activity against cancer stem cells, types. Food Technol Biotechnol 48(1):102–106 including its capacity to differentiate them, makes it a Feng RZ, Wang Q, Tong WZ, Xiong J, Wei Q, Zhou WH, Yin ZQ, Yin XY, Wang LY, Chen YQ, Lai YH, Huang HY, potentially valuable lead for related drug development Luo QL, Wang L, Jia RY, Song X, Zou YF, Li LX (2015) initiatives. Extraction and antioxidant activity of flavonoids of Morus nigra. Int J Clin Exp Med 8(12):22328–22336 Acknowledgments Financial support from the National Ferrari F, Monacelli B, Messana I (1999) Comparison between Research, Development and Innovation Office, Hungary in vivo and in vitro metabolite production of Morus nigra. (NKFIH; K119770), and from the Grant GINOP 2.3.2-15- Planta Med 65(1):85–87 2016-00012 is acknowledged. A.H. acknowledges the Ja´nos Fu W, Lei YF, Cai YL, Zhou DN, Ruan JL (2009) A new Bolyai fellowship of the Hungarian Academy of Sciences and alkylene dihydrofuran glycoside with antioxidation activ- the Ka´lma´n Sza´sz Prize. ity from the root bark of Morus alba L. Chin Chem Lett 21:821–823

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