Genus Viburnum: Therapeutic Potentialities and Agro-Food-Pharma Applications

The genus Viburnum (Adoxaceae, Dipsacales) is of scientific interest due to the chemical components and diverse biological activities found across species of the genus, which includes more than 230 species of evergreen, semievergreen, or deciduous shrubs and small trees. Although frequently used as an ornament, the Viburnum species show biological properties with health-promoting effects. Fruits, flowers, and barks of certain species are used for pharmaceutical purposes or as cooking ingredients, hence containing biochemical compounds with health-promoting activity such are carotenoids, polyphenols, and flavonoids. However, its taxonomical determination is difficult, due to its wide distribution and frequent hybridizations; therefore, an objective classification would allow us to understand its biological activity based on its phytochemical components. More than sixty phytochemical compounds have been reported, where vibsanin-type diterpenes and their derivatives are the most prevalent. Leaves and twigs of V. dilatatum contain the largest number of phytochemicals among the genus. Through preclinical evidence, this study provides insight regarding antioxidant, antibacterial, anti-inflammatory, cytotoxic, and anticancer activities of genus Viburnum.


Introduction
The genus Viburnum (Adoxaceae, Dipsacales) is comprised of more than 230 species of evergreen, semievergreen, or deciduous shrubs and small trees distributed primarily within the temperate forest regions of the northern hemisphere but also in the mountains of Central and South America, Southeast Asia (Philippines, Malaysia), and southeastern Australia and Tasmania.
Although the species of the genus are generally well adapted to mesic forest environments, particular species have been reported to inhabit both, cold boreal forests, and tropical rainforests, as is the case of Viburnum edule and Viburnum amplificatum, respectively. The regions of highest species diversity are eastern Asia and Latin America [1][2][3][4]. Viburnum was established by Linnaeus, classified in the Caprifoliaceae family [5] and soon after reclassified in its own family Viburnaceae [6]. Based on the Dipsacales phylogeny research, Viburnum was subsequently listed in Adoxaceae, together with Adoxa, Sinoadoxa, Tetradoxa, and Sambucus [7] This classification is widely accepted today [1,4,8]. Nevertheless, certain plant classification systems, e.g., the one from Takhtajan [9], still recognize Viburnum as a member of the Viburnaceae family.
Leaves of the Viburnum species are simple, petiolate, and opposite and rarely whorled. The small hermaphroditic flowers form paniculate or umbellate inflorescences of 15-500 flowers each. Marginal flowers are sometimes sterile, and their purpose is to attract pollinators. The plants are self-incompatible. The androecium consists of five stamens. Filament bases are attached to corolla petals. The gynoecium is of three carpels. Two of the three inferior ovaries are aborted. The fruit is a red to purple black singleseeded drupe. Floral nectaries located at the gynoecium apex additionally differentiate viburnums from another Adoxaceae [10][11][12][13][14].
Many viburnums are of high ornamental value. Fruits, flowers, and barks of certain species are used for pharmaceutical purposes or as cooking ingredients, hence containing biochemical compounds with health-promoting activity such as carotenoids, polyphenols, and flavonoids. Many viburnums are of high ornamental value. Fruits, flowers, and barks of certain species are used for pharmaceutical purposes or as cooking ingredients, hence containing biochemical compounds with health-promoting activity such are carotenoids, polyphenols, and flavonoids. These biochemical components are responsible for the main biological activities of Viburnum plants, and although the antioxidant, antibacterial, antiinflammatory, and cytotoxic activity have been well documented, some current research also links to the chemical components found in Viburnum exerting protection and treatment against diseases. Chronic diseases including diabetes [25], cancer [26], Alzheimer's [27], and all diseases that can be caused by a clot [28].

Leaves and twigs
Antitumor effects [31] V. awabuki   Oxidative Medicine and Cellular Longevity

Leaves and twigs
Antitumor effects V. propinquum -
Studies carried out on fruit juice [100,103,104], aqueous methanolic extracts [98], acidic mixtures of water/methanol [102], and mixtures of methanol/acetone/water [99] have been assessed in V. opulus (Table 2). It is one of the few 11 Oxidative Medicine and Cellular Longevity species where the volatile compounds of its fruits are described [115]. Mass spectrometry analysis has identified nine components in V. opulus juice, using ultra high-performance liquid chromatography (UPLC) coupled to quadruple time-offlight mass spectrometers (QTOF-MS) [100]. Viburnum fruits have been reported to contain lipids, pectins, proteins, lipid compounds (carotenoids, essential oils, steroids, and saponins), tannins, flavonoids, and anthocyanins-type polyphenols. V. opulus fruits have a higher content of carotenoids, polyphenols, flavonoids, steroids, and pectins than V. lantana; the latter species being characterized by a higher content of proteins, saponins, and essential oils. Metabolites in V. opulus fruits have been found in different layers of the pericarp with greater amounts in the skin [21]. Phenolic compounds have been reported in fruit juice via hydrochloric acid analysis [101] while triterpenic compounds have been obtained from seeds after triterpene esters hydrolysis (terpenes of the sterols-I type, triterpenyl alcohols and their derivatives-II) [105]. In a review published in 2010, it was reported that only 3-O-caffeoylquinic acid and 5-O-caffeoylquinic acid were isolated from the V. opulus [31]. The presence of phenolic compounds, anthocyanins, and others constituents ( Table 2) have been reported in leaves and hydrodistillation extracts of dried V. opulus plants [83,97].
In this context, the genus Viburnum contains chemical compounds grouped in diterpenes, triterpenes, iridoids, monoterpenes, sesquiterhytopenes, flavonoids, lignans, phenols, coumarins, lactones, and alkaloids. Among the chemical contents of Viburnum, vibsane-type diterpenoids are characteristic of the genus, as have not been found in other higher plants. Compounds of the type vibsanin A-F, vibsanol (C-F), phenolic glycoside fordioside, fordiane A and B, and their derivatives have also been highlighted ( Figure 2). The base structure of the visanin (A-F) compound group corresponds to a vibsane carbon skeleton, with a 20-carbon structure ( Figure 2).
Another key aspect is the type of solvent used for the phytochemical extraction, where water, methanol, ethanol, and their mixture are used in higher proportion. In some cases, less polar solvents such as ethyl acetate or n-butanol are used, from branches and leaves. Chloroform has been used in leaves, while benzene or petroleum ether for the extraction of phytochemicals from branches. Table 2 shows the main phytochemicals found in the genus Viburnum, as well as the type of extract obtained, the part of the plant used and the biological activity.

Antioxidant Activities of Viburnum Plants (In Vitro
Studies/In Vivo Studies). Many traditionally used medicinal herbs exert a beneficial impact on human health thanks to their antioxidant potential. Phenolic compounds, commonly found in plants, are the largest group of natural antioxidants. Plants produce them to protect their cells from oxidative damage caused by oxygen radicals and molecular excitation [116,117]. Viburnum plant species have been extensively studied in vitro and in vivo assays. Most antioxidant studies relate to Viburnum fruits and juices, being V. opulus the most investigated plant species. According to the numerous published results, it can be said that Viburnum species and their products are exceptional antioxidants finding their place as naturally safe agents.
4.1.1. In Vitro Studies. The fruit juice of V. opulus (from the Eastern Black Sea Region, Turkey) had a prominent activity in the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ferric reducing antioxidant power (FRAP) system in comparison to methanol, acetonitrile, and aqueous extracts of fruits skin and seeds, where the seed extract contained a higher number and quantity of antioxidant compounds. Coumaroyl-quinic acid, chlorogenic acid, procyanidin  The bark aqueous extract displayed the highest antioxidant capacity, followed by flowers and fruits. Strong correlations were found between total phenolic, flavanol, and proanthocyanidin contents with these assays [34]. The same authors investigated the antioxidant capacities of V. opulus fruits, flowers, and bark ethanol extracts by measuring of ABTS-, hydroxyl-(HORS), peroxyl-(ORAC), and superoxide-(SORS) free radicals scavenging and reducing power (FRAP). The antioxidant activity of different parts of the herb was in the following order for HORS, SORS, and ORAC tests: bark>flowers>fruits, and for ABTS and FRAP tests: bark>-fruits>flowers. The dominant compound in V. opulus bark was (+)-catechin, while chlorogenic acid was dominant in flowers and fruits [34]. Andreeva et al. [120] determined the antioxidant potential of Russian V. opulus bark extracts using the cathode voltammetry method measuring the relative decrease in the oxygen electroreduction. Results showed that the ethyl acetate fraction of the 70% ethanol extract expressed a higher antioxidant potential than chloroform and aqueous fractions, 70% and 30% ethanol extract, respectively. Bubulica et al. [121] conducted an antioxidant effects screening across several extracts of in vitro assays of Romanian plants. The authors found that the V. opulus methanol bark extract produced an antiradical effect against DPPH radicals with IC 50 of 0:918 ± 0:46 mg/ml, as well as ferrous ion chelating effects with IC 50 of 1:865 ± 0:05 mg/ml. Additionally, the antioxidant activity of the ethanol extract of the fruits was determined by the ABTS test. After 24 h of refrigerating, a 16% decrease in the antioxidant effect was recorded, followed by a 22% increase in the next 24 h, showing no correlation between the total the antioxidant potential and the phenol content in the extract during storage [122]. Paşayeva et al. [123] suggests that Turkish V. opulus fruits could be used as a neuroprotective agent. Antioxidant properties of both the decoction ethanol extract and fruit juice were used against hydrogen peroxide-induced oxidative stress in human SH-SY5Y neuronal cells. The Polish V. opulus fresh juice and extracts acted as scavenging potential agents toward ABTS and peroxyl radical cations estimated by ABTS and ORAC assays, expressed using Trolox equivalents (TE). The phenolic-rich fraction from fresh juice was compared to the methanol-acetone extract from pomace. The former was the most active in all identified phenol compounds (flavanols, falvonols, hydroxycinnamic acids, and anthocyanins) with values within the range of 2619:59 ± 123:1 and 7810:29 ± 342:3 μg TE/g. Also, the V. opulus extracts had powerful chemopreventive effects against oxidative stress in Caco-2 cells induced by tert-butylhydroperoxide and against DNA damage through the repair induction after cell exposure to hydrogen peroxide and methylnitronitrosoguanidine [99].
Studies have compared the antioxidant effects of different V. opulus genotypes and cultivars. Kraujalytė et al. [101] reported that V. opulus var. sargentii had the highest radical scavenging capacity (77.5%), followed by V. opulus var. americanum, and V. opulus P3. The strongest activity in a FRAP test system was observed in V. opulus var. sargentii juice while the weakest was expressed by the 'Shukshinskaya' cultivar. The V. opulus var. sargentii juice was most effective in the ORAC method with the highest TEAC (Trolox equivalent antioxidant capacity) value, while 'Shukshinskaya' was powerless as observed in the previous test. Moskalets et al. [124] assessed the antioxidant capacity of Ukrainian V. opulus fruit varieties using a Blisar A analyser. The antioxidant activity was expressed through gallic acid in a range between 387 and 540 mg%. The cultivar with the maximal tested effect was ЕF 3-10-2010. Furthermore, approximately 10 genotypes of Turkish V. opulus fruits showed antioxidant potential, marked SIV 1-10 by a FRAP test system. SIV genotypes in the forms of acetone extracts displayed effects in a narrow range from 21:02 ± 2:6 to 34:90 ± 4:5 μmol TE/g, where SIV-10 had the greatest capacity, suggesting the higher synthesis or accumulations of phenolics and others antioxidant compounds in fruits [33]. Scavenging effects of fruit porridge (25%) of V. opulus var. edule cultivars Leningradskaya otbornaya, Souzga, and Taezny rubiny on nitric oxide, superoxide anion, hydroxyl radical, and lipid peroxidation inhibition, were moderate from 11.20 to 28.50%. Fruits' total antioxidant activity was determined by the ABTS and DPPH methods, where Taezny rubiny was most prominent. A high correlation between total phenolics and ascorbic acid content, and antioxidant activity was reported [102]. V. opulus (Latvia) fruits and pomace were subjected to supercritical carbon dioxide extraction (SFE-CO2) with different conditions to obtain the optimal lipophilic fraction. The antioxidant potential of V. opulus SFE-CO2 extracts was assessed by the ORAC method. The antioxidant capacity values of the washed pomace, unwashed pomace, and dried berry extracts, at the highest yield (optimal SFE-CO2 conditions), were 65:3 ± 1:8, 74:3 ± 2:2, and 142:4 ± 3:6 μmol TE/g, respectively. It can be concluded that the dried berry extracts were twice as higher in the antioxidant activity expression in comparison to the pomace extracts [125].
Çanga and Dudak [126] implemented cellulose acetate/gum Arabic fibers loaded with a V. opulus (Turkey) fruit extract. Within the examination of the materials, they tested the antioxidant activity of the loaded fibers and observed high values of DPPH radical inhibitions ranging 56-59% and 55-58% at 4°C and 25°C, respectively.
These free radical inhibitions were more effective than the free V. opulus fruit extract (40% and 34%, respectively). Barak et al. [98] studied the difference in the antioxidant potential of Turkish V. opulus methanol, and water fruit extracts before and after in vitro gastrointestinal human digestion. Antioxidant effects of the extracts were assessed in the following phases: nondigested, postgastric, colon available, and serum available, by different methods (N,Ndimethyl-p-phenylendiamine-DMPD, cupric reducing antioxidant capacity (CUPRAC), DPPH, FRAP, and total antioxidant capacity). The methanol extract was superior in antioxidant expression than the aqueous one, and the antioxidant effect decreased during the in vitro digestion. An interesting investigation was conducted with V. opulus (Turkey) fruit pomace in wheat flower cakes at concentrations of 0, 5, 10, and 15%. Cakes' total phenolic content and antiradical activity (DPPH) increased in correlation to the level of V. opulus fruit pomace addition (from 10:26 ± 1:5 to 76:83 ± 4:5%) [127]. The V. opulus (guelder rose) fruit concentrate (65%) decreased the thiobarbituric acid reactive substance (TBARS) levels in turkey meat samples during storage at concentrations of 5% and 10%, particularly after 10, 15, and 30 days. The addition of 10% concentrate to the meat significantly reduced the TBARS in comparison to control and butylated hydroxytoluene (p < 0:05) both under aerobic and anaerobic conditions [128].
Erdogan-Orhan et al. [129] observed the antioxidant effects of Turkish V. opulus and V. lantana ethyl acetate, methanol, and aqueous extracts from branches, leaves, and fruits. The most powerful antioxidant agents in the ferrous ion chelating capacity test were the V. opulus ethyl acetate leaf extract (44:62 ± 0:02% of inhibition; 2000 μg/ml) and the V. lantana ethyl acetate fruit extract (58:72 ± 1:00% of inhibition; 2000 μg/ml). V. opulus aqueous extracts from branches and V. lantana methanol leaf extracts exhibited the highest effects (3:396 ± 0:01 and 3:401 ± 0:02; 2000 μg/ml, respectively) using the FRAP method with chlorogenic acid as a reference. In the β-carotene bleaching assay, V. opulus ethyl acetate fruit extracts and V. lantana methanol leaf extracts were significant antioxidant agents with coefficients of 60:5 ± 1:36 and 79:50 ± 1:76, respectively, at 2000 μg/ml. The authors noticed that a higher total phenolic content in the tested extracts usually indicates greater antioxidant effects [129]. Both Viburnum species were also studied by Altun et al. [41]. The antioxidant effects of different water extracts (branches, fruits, and leaves) were assessed using the DPPH and superoxide anion scavenging methods. Branch extracts successfully reduced the effects of the superoxide anion (IC 50 = 3:7 and 3.1 mg/ml, respectively). On the contrary, the extracts produced a positive antiradical effect on the DPPH radical with various inhibition values, especially the V. opulus branch extract with IC 50 values at 0.014 mg/ml [41].
The GC-MS data for the methanol soluble fractions of V. sargentii extract (originating from the Republic of Korea) highlights the presence of guanosine, levoglucosan, vitamin E, stigmast-5-en-3-ol, and stigmata-5,24(28)-dien-3-ol. Patil et al. [134] showed a significant antioxidant potential of the extract in a concentration-dependent manner for all three methods. The extract produced a strong antiradical effect in the DPPH assay with an IC 50 value of 15:33 ± 0:58 μg/ml. For both remaining methods, the extract had an electrondonating capacity which reflected its reducing power to change the ferric (Fe 3+) to the ferrous (Fe 2+ ) form and Mo (VI) to Mo (V), respectively [134]. V. nervosum roots, essential oil, and extracts showed strong antiradical effects in DPPH system. Methanol and ethanol extracts (100% and 80%) were more potent with IC 50 = 22:97 ± 0:38-25:65 ± 1:02 μg/ml than essential oil (IC 50 = 33:32 ± 0:67 μg/ml). Additionally, the inhibition capacity of the extracts was greater in a linoleic acid system, where lipoperoxidation inhibition ranged from 47:67 ± 0:87 to 69:25 ± 1:67% in comparison to oil 44:03 ± 0:96% where the dominant compounds were α-eudesmol, caryophyllene oxide, linalool, spathulenol, and ledene Awan et al. [135]. Fu et al. [136] conducted an antioxidant potential screening of edible fruits from South China. They reported that V. sempervirens (in particular V. fordiae) fruits produced great total antioxidant effects estimated by FRAP and TEAC. However, it is important to note that the V. sempervirens nonpolar fruit fraction showed greater effects as compared to V. fordiae, which polar fruit fraction was more active. The authors also noticed strong correlations of antioxidant expression with total phenols [136]. Leiner et al. [137] evaluated the antioxidant capacity of Alaskan berries by the ORAC method and revealed an excellent effect of V. edule fruits (117 mmol of TE/g), after Vaccinium vitis-idaea. Antioxidant potential was also confirmed for V. dilatatum leaves and fruits [138,139]. Iwai et al. [138] from Japan examined V. dilatatum juice (fruit squeezing solution) claiming its strong antioxidant activity.

Oxidative Medicine and Cellular Longevity
The in vitro antioxidant potential of the V. dilatatum sample was measured by the XYZ-dish and electron spin resonance (ESR) method. In the XYZ-dish technique, the tested sample expressed effective activity against OH· (10:163 ± 2:376 units/ml), as opposed to its antiperoxide effect (H 2 O 2 ) (0:529 ± 0:127 units/ml). The activity of the V. dilatatum juice to scavenge OH· radicals, measured by the ESR method and represented as DMSO equivalent concentration, was also strong (0:937 ± 0:176 mM DMSO eq/ml) [138]. Three Indian Viburnum species and their methanol leaf extracts were investigated in a DPPH test, a nitric oxide (NO) scavenging test, and an assay of reduced glutathione and ferrous sulphate-induced lipid peroxidation. Ponnudurai et al. [140] concluded that these extracts could be effective antioxidants in the following order: V. erubescens>V. coriaceum>V. punctatum. V. awabuki (originating from China) and its extracts exhibited exceptional activities. The ethyl acetatesoluble fraction of the crude methanol-chloroform extract was dominant in antioxidant effects in DPPH radical inhibition (1000 μg/ml = 61:88 ± 0:23%) and reducing power activity (1000 μg/ml = absorbance of 0:287 ± 0:006), while the basic crude extract was prominent in the hydroxyl radicalscavenging activity test (100 μg/ml = 71:26 ± 0:38%). On the contrary, other V. awabuki extracts: petroleum ethersoluble fraction, n-butanol-soluble fraction, and aqueous residue, were significantly weaker [141]. Abbasi [142] revealed that V. foetens fruit from the Himalayan region-Pakistan represented a rich source of natural antioxidants possessing significant antioxidant effects. Compared with the water extract, the acetone extract showed higher levels of the total antioxidant capacity (84:67 ± 0:48 μM AAE/100 g), DPPH antiradical capacity (84:62 ± 0:63%), and hydroxyl radical scavenging capacity (75:53 ± 0:95%). They also found significant correlations between the ascorbic acid, phenols, and metal contents with free radical scavenging activity [142]. Nine V. tinus extracts from Turkey were screened in detail for their antioxidant potential. Antioxidant capacities of the ethyl acetate, methanol, and water extracts of leaves, branches, and fruits were tested against DPPH, DMPD, superoxide, and NO radicals. The methanol extracts of leaves, branches, and fruits and leaves' aqueous extract exhibited a remarkable DPPH antiradical activity, over 89%. The fruits' methanol extract was prominent against the DMPD radicals (67:1 ± 0:33%), the branches' aqueous extract in NO radical quenching (near 80%), and the fruits' ethyl acetate extract was the only active in the superoxide radical scavenging test (38:4 ± 1:01%). The fruits' methanol extract displayed the strongest activity in the FRAP and phosphomolybdenum-reducing antioxidant power (PRAP) tests. In the metal-chelation capacity test, the aqueous extracts were predominant with over 60% of the activity [143]. V. tinus from Tunisia and its leaves' acetone extract also produced strong antiradical effects in a DPPH test with high total phenolic, flavonoid, and tannin content [144]. The Indian V. punctatum leaf ethanol extract was tested for its scavenging effects in a DPPH and ABTS system and total antioxidant capacity (reducing power activity) in a phosphomolybdenum assay. The extract proved to be an efficient scavenging agent with IC 50 values of 83.29 μg/ml and 92.04 μg/ml, respectively, with excellent reduction effects of 65:67 ± 0:15% with the maximal concentration of 100 μg/ml [145]. V. grandiflorum, among other wild berries of the Northwestern Himalayan region, demonstrated promising antioxidant properties. Namely, the methanol fruit and leaf extract produced an antiradical effect in a DPPH system with IC 50 values of 294.5 and 125.82 μg/ml, respectively, as well as effective chelating and reducing power [146]. Fruit, leaf, and branch extracts from V. orientale (traditionally used in Turkey -Anatolia) were studied for their antioxidant and neurobiological effects. The fruit and branch methanol extracts (characterized by the highest total phenolic contents) showed the highest inhibition capacity against DPPH radicals and in FRAP and phosphomolibdenum reducing antioxidant power assays. The leaf aqueous extract showed the greatest NO scavenging (75:00 ± 1:22%), DMPD scavenging (33:70 ± 1:13%), and metal-chelating (54:66 ± 3:56 %) at the concentration of 2500 μg/ml [106]. The aqueous extract of the aerial parts of V. punctatum from India showed a significant antiradical effect in a DPPH test system. The extract inhibited from 44:33 ± 0:21% to 93:65 ± 0:56% of free radicals at the concentrations from 20 to 100 μg/ml Susmitha et al. [147]. The antioxidant effects of V. odoratissimum seed extract, from China, were analysed in superoxide radical scavenging, reducing power, and lipid peroxidation inhibition assays. The butanol-soluble fraction of the methanolchloroform crude extract was dominant in the first two tests, which was in accordance with the total phenol and flavonoid contents. The authors observed that the petrol ether-soluble fraction had the highest antilipoperoxidant activity and that the high temperature was not effective for the lipid peroxidation inhibition [148]. V. mullaha (India) acetone fruit extracts showed prominent antioxidant activities in vitro assays: ABTS, DPPH, superoxide anion, and linoleate peroxyl radicals scavenging and ferric reducing except ferrous metal chelating activity. The V. opulus (Turkey) fruit methanol extract showed protective effects against ischemia/reperfusion-(I/R-) induced oxidative stress during lung transplantation in rats, presumably due to its antioxidant effects and ability to neutralize free radicals. Namely, the treatment with the extract (200 mg/kg, intraperitoneally) significantly increased the levels of the antioxidant system (superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and total glutathione) and repaired the total antioxidant plasma status of rats (69:59 ± 8:9 μmol Trolox eq/mg protein) compared to the untreated group (43:02 ± 4:75 μmol Trolox eq/mg protein). In addition, the extract reduced the malonyldialdehyde (MDA) and protein carbonyl levels. It is considered that malic, caffeic, quinic, coumaroyl-quinic, and chlorogenic acid, as well as particular caffeic acid, derivatives are responsible for the antioxidant effects previously described [150]. The impact of V. opulus water fruit extract (also originating from Turkey) on testicular and epididymal rats tissue treated by i.p. injection of taxane-based chemotherapeutics was investigated by measuring the lipid peroxidation level and antioxidant activities. Docetaxel and paclitaxel imbalanced an oxidant/antioxidant system, which was repaired with an oral dose of 100 mg/kg of the extract. MDA levels were significantly lower in rats' testis and epididymis while the levels of superoxide dismutase, glutathione peroxidase, and catalase were increased. The authors identified several compounds in the extract by GC-MS (α-and β-pinene, butanoic acid, DL-limonene, α-terpineol, and germacrene D) [151]. Furthermore, the lyophilized V. opulus juice and the commercial lyophilized V. opulus juice showed a significant antiurolithiatic activity in rats (100 mg/kg) compared to the Cystone standard. Ilhan et al. [152] attributed this effect to their antioxidant and diuretic activity and the inhibitory effects on the oxalate levels. The antioxidant action was estimated by measuring TBARS, total thiols, and glutathione in kidney tissues. TBARS levels were significantly reduced, after administration of V. opulus juices, with increased levels of total thiols and glutathione [152]. V. opulus proanthocyanidins produced gastroduodenoprotective effects against water immersion and restraint stress in rats, improving the levels of antioxidant enzymes' superoxide dismutase, catalase, and gluthatione peroxidase and decreasing the MDA content. Proanthocyanidins, as V. opulus extract, were intragastrically administered in three doses at 25, 50, and 75 mg/kg body weight [40].
V. dilatatum juice showed an inhibitory effect on gastric ulcer formation and oxidative damage caused by water immersion restraint stress in rats. The concentrations of lipid peroxides, assessed by TBARS, in the plasma, liver, and stomach were significantly lower compared with the group treated with water [138]. The same Japanese researchers tested the V. dilatatum crude fruit extract, proving ineffective for preventing oxidative injury induced by water immersion restraint stress. The extract improved the lipid peroxidation in the plasma, liver, and stomach but had weak effects on the enzymatic activities (superoxide dismutase, catalase, and gluthatione peroxidase). However, ferrous ascorbate-induced oxidation in hepatic homogenate of rats was inhibited. According to the results, V. dilatatum fruits cannot induce antioxidant enzymes and its absorbed antioxidant components have a direct effect on the oxidative injury in the body [153]. In addition, the V. dilatatum crude fruit extract was subjected to the experiment with streptozotocin-induced diabetic rats. The animals received V. dilatatum extract solution (16.8 mg/ml) for 10 weeks. TBARS levels in the plasma, erythrocytes, liver, kidney, and pancreas were significantly reduced together with plasma glucose levels. The extract con-tained two cyanidin glycosides, two chlorogenic acids, and quercetin. It is suggested that cyanidin 3-sambubioside is crucial for the physiological effects of V. dilatatum fruit, given the strong antioxidant nature of the compound [70,154].
The leaf methanol extract of V. tinus from Egypt produced a significant effect on serum lipid peroxides (measuring of thiobarbituric acid-reactive substance) and nitric oxide levels (Griess reaction) with the dose of 50 mg/kg i.p. on CCl4-induced hepatotoxicity in rats, although not at lower doses of 25 mg/kg [155]. Studies). During the last two decades, antimicrobial activity of plant species of the genus Viburnum L. has been extensively studied. Antimicrobial activity of the essential oils from the air-dried whole plants of V. opulus, V. lantana, and V. orientala were tested against the bacteria Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, Bacillus cereus, and the fungus Candida tropicalis. The oils were at a maximum concentration of 250, 500, and 1000 μg/ml in hexane, respectively. The activity was tested using the agar dilution MIC assay. The oils of V. lantana and V. opulus showed no activity against the microorganisms tested. The essential oil of V. orientale showed a weak antibacterial activity against Gram-positive bacteria such as E. faecalis, S. aureus, and B. cereus [83]. In another study, the essential oil of V. betulifolium was analysed and tested for antimicrobial activity using microdilution assay of human pathogenic bacteria and yeast. V. betulifolium is an evergreen shrub widely distributed throughout the Yunnan Province and southwestern parts of China. The main essential oil constituents of the species are phytol (9.8%), trans-b-damascenone (5.9%), α-cadinol (5.7%), γ-cadinene (5.6%), Δ-cadinene (5.3%), methyl pentanoate (4.6%), and tetradecanal (3.8%). The oil showed strong antimicrobial activity against both Gram-positive and Gram-negative bacteria and the yeast, the effect being more significant against Gram-positive than Gram-negative bacteria. Positive inhibitory activity was shown by Pseudomonas aeruginosa (MIC 125 μg ml -1 ) and Candida albicans (MIC 62.5 μg ml -1 ) [156]. Awan et al. [135] investigated antimicrobial activities of Viburnum nervosum root essential oil and several methanolic and ethanolic root extracts. V. nervosum is a large deciduous precocious shrub, 2-3 m tall with stiff stout branches. In Kashmir's traditional medicine, it is used as an astringent and emmenagogue, as well as for the treatment of acute furunculosis. Bergenin, a trihydroxybenzoic acid glycoside, was isolated from its roots. This glycoside is also useful in the treatment of hypercholestraemia, kidney stones, fever, diarrhea, and pulmonary infection. The essential oil of the root possesses carminative, insecticidal, antiseptic, and disinfectant properties. The main compounds of the root oil are eudesmol (30.3%), caryophyllene oxide (17.0%), spathulenol (10.7%), and linalool (12.65%). The essential oil and root extracts of V. nervosum were tested against Grampositive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli and Pasteurella multocida), and pathogenic fungi (Aspergillus niger, Aspergillus flavus, Fusarium solani, and Rhizopus solani) with the disc diffusion method. The oil showed maximum activity against B. subtils, A. niger, and R. solani. However, no activity was observed by the extracts Awan et al. [135]. Nonetheless, the chemical composition and antimicrobial activity of Viburnum species essential oils were examined, neither were the antimicrobial activities of the dominant compounds investigated.

Antimicrobial Activities of Viburnum Plants (In Vitro
Bibi et al. [157] tested the antibacterial activity of the methanolic dry extract of Viburnum foetens (20 mg/ml), with an agar well diffusion method, against Bacillus subtilis, Micrococcus leuteus, Salmonella setubal, Salmonella aureus, and Pseudomonas picketii. All bacteria tested were sensitive to the extract. S. setubal was the most sensitive bacteria. The extracts of V. foetens were subject of another study. Awan et al. [158] examined four different extracts of V. nervosum and V. foetens leaves for antibacterial activities against eight different bacteria (Staphylococcus aureus, Bacillus subtilis, Salmonella typhi, Pseudomonas aeruginosa, Klebsiela pneumoniae, Proteus vulgaris, Citrobacter freundii, and Streptococcus pneumoniae) using the disc diffusion method. The study concluded that the ethanolic extract was the most effective, contrary to that of the petroleum ether extract. The ethanolic and methanolic extracts showed prominent activities against all tested bacteria, in comparison to the chloroform extract which had moderate activity. Turker et al. [159] used the same antibacterial activity evaluation method by analysing the antimicrobial activity of the V. lantana extracts obtained from dry and fresh fruits (water and ethanol) against Gram-positive bacteria (Streptococcus pyogenes, Staphylococcus aureus, and Staphylococcus epidermidis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescens, Proteus vulgaris, Enterobacter cloacae, and Klebsiella pneumoniae). Gram-positive bacteria were more susceptible to the inhibitory effects of the plant extracts than the Gramnegative bacteria. The extracts of V. lantana fresh fruits exhibited antibacterial activities. The inhibition capacity of the hot ethanolic extract was greater than the cold ethanolic extract against S. aureus, S. epidermidis, and S. pyogenes. Both S. marcescens and P. aeruginosa were resistant to all examined fruit extracts.
Eryilmaz et al. [160] also studied the antimicrobial activity of V. lantana, together with Viburnum opulus L., V. orientale Pallas, and V. tinus L. against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Candida albicans. The disc diffusion and tube dilution techniques were used to determine the activities of the extracts. Ethanolic and water plant extracts of leaf, stem, and fruit were used in the experiment. Ethanolic extracts from all analysed species showed antimicrobial activity against all the tested microbes. Water extracts were either weak or not effective against tested microorganisms. The antimicrobial activity of the dried fruit methanolic extract of V. opulus was also analysed by another study with an agar diffusion method [119]. The study analysed ten microorganisms (Aeromonas hydrophila, Bacillus cereus, Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, and Yersinia enterocoli-tica). A 15% concentration extract completely inhibited the growth of all analysed bacteria. The same plant species have shown the capacity to reduce the potential of Staphylococcus aureus and S. epidermidis to colonize inert substratum and form biofilms [121]. Similarly, fruit juices and ethanolic extracts of V. opulus genotypes were tested against ten Gram-positive and Gram-negative bacterial cultures and nine yeast strains. The fruit juices showed greater antibacterial activity compared to the ethanol extracts. The most effective antibacterial activity was exhibited by the juices against Salmonella typhimurium, Salmonella agona, and Listeria monocytogenes. The fruit juices and ethanol extracts showed weak or no activity on the yeast strains [161]. Antimicrobial activities of fruit juices of six V. opulus genotypes were evaluated by Česoniene et al., [42], using the agar well diffusion method against ten Gram-positive and Gram-negative bacteria and seven strains of yeast. The juices strongly inhibited the growth of Gram-negative (S. typhimurium and S. agona) and Gram-positive (S. aureus, L. monocytogenes, and Enterococcus faecalis) bacteria. As previously reported, the effect of the juices on the yeast was low or lacking [42]. Up to Česonienė's studies, most Viburnum plant extracts had presented the highest effect on Gram-positive bacteria strains and some yeasts. However, antimicrobial analysis of the juices has shown that they can be used to fight Gram-negative microorganisms. Differences in antimicrobial activity are most likely due to the chemical composition of the extracts and juices. Therefore, further research of Viburnum species should connect the chemical composition with the antimicrobial activity.
Paulauskas et al. [162] went one step further. They analysed the antimicrobial activity of unripe mashed berries and ripe berry juice of V. trilobum Marshall, V. sargentii Koehne, and V. opulus cultivar "Leningradskaya Otbornaja." The unripe berry mass and ripe berry juices both significantly influenced the bacteria. The unripe berry mass manifested greater antibacterial activity, similarly, on both the Grampositive and Gram-negative bacteria. Micrococcus sp. and S. aureus were the most sensitive bacteria to the mashed berries and all analysed juices. Viburnum juice impacted the microscopic fungi the least.
Methanolic extract of V. cotinifolium leaves from Pakistan was tested against four Gram-positive bacteria, five Gram-negative bacteria, and ten fungal strains with the agar diffusion method. The extract demonstrated maximum activity against Aspergilus flavus and A. fumigates. The extract also showed positive antimicrobial activity against A. niger. The V. cotinifolium extract showed the most effective activity against Enterococcus faecalis and Enterobacter coccus. As can be observed, the extract of Viburnum species was also effective against Gram-negative bacteria [163]. This indicates that V. cotinifolium leaf methanolic extract has great potential as a natural antimicrobial agent. Hence, chemical analyses of the extract should be carried out and associated with antimicrobial activity. This connection is supported by the research carried out by Roy [164], which proves that the methanolic extract (and fraction) from the whole plant of V. foetidum exhibit a significant antimicrobial activity against Gram-positive and Gram-negative bacteria strains, as well as significant antifungal activity. The ethyl acetate (EA) fraction from the methanolic extract exhibited the highest antimicrobial potential. Agrobacterium species were most susceptible to the EA fraction of the extract, and clearly, the EA fraction differs in its composition from its counterparts. Unfortunately, detailed chemical analyses of the extracts and/or fractions have currently not been carried out. Inflammatory diseases are usually treated by steroid drugs, nonsteroidal anti-inflammatory drugs, and immunosuppressant. Although the effects of these drugs have been proven, their side effects are not negligible. The usage of these drugs is often associated with bleeding gastrointestinal and peptic ulcers [165]. In search for new harmless drugs, scientists are once again turning to medicinal plants. Among these plants, the species of the genus Viburnum are of interest. For that purpose, anti-inflammatory activities of V. lantana, V. trilobum, V. pichinchense, V. sargentii, V. fordiae, and V. opulus were investigated [78,[166][167][168][169][170].
The bark of this species has been used in Turkish traditional medicine as a rubefacient and analgesic [41]. Viburnum lantana L. leaf water extract was investigated for anti-inflammatory activity, in rats with a carrageenaninduced rat paw edema test. The anti-inflammatory activity of the extract at doses of 100 and 200 mg/kg has been low as compared to indomethacin [169].
Viburnum trilobum Marshall (American highbush cranberry) is widely used in traditional medicine as it displays an anti-inflammatory and antidiabetic effect, sometimes used to improve lipid metabolism. The bark can act as a sedative and pain reliever. Due to the high content of ursolic acid, which expresses anti-inflammatory properties, the bark acts as an anti-inflammatory agent. This effect is proven in a RAW 264.7 macrophage cell system. All fractions of the V. trilobum ethanolic extract significantly inhibited the levels of IL-1β, IL-6, and TNFα [168].
Viburnum pichinchense Benth also displays antiinflammatory properties. The anti-inflammatory effects of the methanol extract were demonstrated using LPSstimulated macrophages and HCl/EtOH-induced gastritis model mice. The extract expresses anti-inflammatory activity by targeting NF-κB and caspase-11 noncanonical inflammasome pathways in macrophage-mediated inflammatory responses [167].
Leaves, stems, and fruits of the plants have been used in traditional folk medicines as therapeutic agents, as styptics and analgesics, to treat boils, rheumatoid arthritis, traumatic injuries, ringworm, skin itching, and coughs [170]. In the last two decades analgesic, anti-inflammatory, and hepatoprotective activities of its methanol extract were confirmed. The butanol fraction of the methanolic extract showed the highest activity on inflammatory reactions [170].
Viburnum fordiae Hance, a small tree widely distributed in the south of China, has been used in traditional Chinese medicine for centuries to treat rheumatic arthralgia and allergic dermatitis. Recent studies have reported a new, unusual ɣ-lactone, obtained from the aerial parts of these plants, capable of expressing an in vitro anti-inflammatory effect vitro [78].
V. opulus is well known as a medicinal and horticultural plant with a dietary value. Its fruits have been used in traditional medicine to cure pulmonary, stomach, cardiovascular, and kidney diseases, as well as for the treatment of cramps, diabetes, bleeding, coughs, and colds. Arginase activity and arterial vasodilation of the plant extract have also been proven [100,101,104,[171][172][173]. The anti-inflammatory activity of V. opulus water leaf extract was conducted in rats by a carrageenan-induced rat paw edema, test at doses of 50, 100, and 200 mg/kg, i.p., proving the extract had no anti-inflammatory effect at these doses [166].

Cytotoxic Activities of Viburnum Plants (In Vitro
Studies). For centuries, herbs and plants have had a role in the treatment of various forms of tumors as have also shown to reduce the risk of cancer development or serve as a treatment for different types of cancer [174]. Viburnum species and their products have been extensively studied for their cytotoxic properties, being promising anticancer agents. To date, the most studied species in this regard is V. opulus.
Sauter and Wolfensberger [175] were the first to report the cytotoxic activity of Viburnum extracts. Aqueous fruit extracts of V. opulus and V. lantana, from Switzerland, showed no cytotoxic activity on BT 20 breast cancer cells within 72 h of incubation. Similarly, further research revealed no cytotoxicity of V. opulus extracts. An Indian aqueous bark extract was tested for its cytotoxic effect using a simple bioassay, brine shrimp lethality test, but no remarkable effect was observed [3]. The same findings were reported for V. opulus seed extracts by Cantrell et al. [176]. Russian V. opulus fruits and its ethanol extract exhibited low cytotoxicity, suppressing cell growth at concentrations above 200 μg/ml [3].
On the contrary, some authors consider V. opulus as an effective cytotoxic agent. Laux et al. [177] investigated the aldehyde fraction of the V. opulus chloroform-methanol fruit extract (Canada) for the cytotoxic effect on human gastric carcinoma cells. The fraction containing (E) 2-hexenal, (Z) 2-decenal, 2,4-decenal, (E) 2-octenal, and 2-undecenal produced a direct antiproliferative effect on the growth of the carcinoma cells with death at a concentration of 27 μM [177]. The methanol and acetone extract, juice, and juice after extraction to the solid phase of V. opulus from Poland showed cytotoxic activity against human breast (MCF-7) and cervical (HeLa) cancer cell lines. The strongest toxic agent towards both cell lines was observed for the juice obtained after purification with IC 50 values of 63.541 and 19.380 μg/ml for HeLa and MCF cell lines, respectively [178]. According to IC 50 values (250-450 μg/ml), the cytotoxicity of the same origin V. opulus extracts against Caco-2 cells, measured with the PrestoBlue assay, were in the following order: phenoli-rich fraction from fresh juice> methanol-acetone extract from pomace>acetone extract from pomace>fresh juice. This effect could be attributed to the highest content of phenolic compounds (flavanols, falvonols, hydroxycinnamic acids, and anthocyanins) [99]. The commercial V. opulus juice from Turkey produced a cytotoxic effect against Caco-2 (human colon adenocarcinoma) and HeLa cells but was not active in a test using A549 (human type II lung epithelium) cells over a 72hour period with the concentrations of 10-80 μg/ml. Futhermore, V. opulus juice caused no significant decrease in the viability of MDCK (Madin Darby Canine Kidney) and HUVEC (Human umbilical vein endothelial cells) normal cell lines [179].
Antitumor activities of water and ethanol extracts prepared using the hot and cold procedures of fresh and dried V. opulus and V. lantana fruits (Turkey) were tested with the potato disc tumor induction method. V. opulus was more active with the inhibition of 61.9-100%. Both water extracts from dried fruits were the most effective in the assay. Among V. lantana extracts, hot water and ethanol extracts were the most effective, 90.5% and 95.2%, respectively [159]. The same technique was used to assess the antitumor activity of aqueous, ethanol, and methanol V. lantana (Turkey) leaf and fruit extracts. The methanol extract was the most effective with 100% of tumor inhibition, followed by the ethanol (90.9%) and the aqueous one (86.4%) [180].
The researchers from Pakistan, Shah et al. [37], conducted an identical assay for cytotoxicity with V. grandiflorum methanol extract and its n-hexane, chloroform, ethyl acetate, and n-butanol fractions. The chloroform extract was more active, expressing a strong ability to kill brine shrimp (EC 50 = 107:45 μg/ml). The methanol extract of V. grandiflorum from China exerted a strong impact on lung cancer cells H1650, HCC827, and H1299 by decreasing their viability in a concentration-and time-dependent manner. The viability of the H1650, HCC827, and H1299 cells decreased to 34%, 31%, and 29%, respectively, after three days of treatment with the extract. A detailed analysis found that the viability of cells was inhibited by the apoptosis activation through a caspase-dependent pathway [181].
The V. foetens (Pakistan) methanol crude extract and fractions showed a significant anticancer effect against the breast cancer cell line MCF-7. The crude extract was active 90.5% at 200 μg/ml, while the fractions were less effective at the same concentrations. The highest MCF-7 cell line inhibition percentage was reported for the methanol (83%), followed by the chloroform 55.5%, the hexane 25.11%, and water (2%) fractions [157]. The crude methanol extract and fractions of V. foetens (Pakistan) were evaluated against MCF-7, MDA-MB-468, and Caco-2 cancer cell lines by the MTT test and NRU (neutral red uptake) assay. The crude extract inhibited the cancerous cell growth in a dosedependent manner. The ethyl acetate fraction significantly reduced Caco-2 cells (93.44%) growth in the MTT test. The methanol and ethyl acetate fractions decreased 99% and 96% cell growth of MCF-7 and Caco-2 cell lines, respectively, in the NRU assay. Also, ethyl acetate fraction of the V. foetens extract exhibited a considerable inhibition of MDA-MB-468 cells in both used assays. Other fractions (chloroform, hexane, and aqueous) produced a weaker effect on cancer cell proliferation [186]. Methanol leaf extract of V. dilatatum (Korea) produced a cytotoxic effect on MCF-7 human breast cancer cells with IC 50 of 139 ± 16 μg/ml. The authors reported that this effect could not be considered strong, according to the screening program of the National Cancer Institute, USA, which recommends IC 50 under 20 μg/ml to be the effective cytotoxic agent [139]. Roy [164] reported significant lethality in a brine shrimp cytotoxicity assay for the V. foetidum crude methanol extract and its petroleum ether and n-hexan fractions with LC50 of 39.81, 25, and 25 μg/ml, respectively, while LC50 for standard vincrinstine sulphate was 10.44 μg/ml. The Colombian V. cornifolium leaf dichlormetan extract showed high cytoxicity tested on the V79 cell line (Chinese hamster lung fibroblasts) with IC 50 of 25 μg/ml [187]. Ponnudurai et al. [188] (India) tested methanol leaf and chloroform root extracts of V. coriaceum and V. erubescens for their bacterial strain-based cytotoxicity (E. coli AB 1157 strain), using the MTT method (MCF-7 breast cancer cell lines and HeLa cervical cell lines). All extracts, except the chloroform root extract of V. erubescens, showed an effect on the bacterial strain-based carcinogenicity. IC 50 values in the MTT test, only determined for the V. coriaceum extracts, indicated a moderate anticancer activity (over 500 μg/ml for MCF-7 cells and 300 μg/ml HeLa cells) [188]. Calderón-Montaño et al. [189] reported cytotoxic effects for the Spanish V. tinus water fruit and leaf extracts in the MTT test. The fruit extract was more potent in the inhibition of proliferation of A549-human lung adenocarcinoma cells and MRC-5-human lung fibroblastic cells with IC 50 of 26:6 ± 6:5 and 65:4 ± 8:6 μg/ml, respectively. Methanol extracts of Chinese V. odoratissimum wood and bark successfully inhibited melanin biosynthesis and cell proliferation of B16 melanoma cells at 100 and 50 μg/ml, respectively [190].

Anticancer Effects of Viburnum Plants In
Vivo. The anticancer effect of V. opulus juice was previously reported on Ehrlich ascites carcinoma cells [26]. However, Ceylan et al. [191] from Turkey investigated V. opulus juice for its antitumor potential in an in vivo experiment with experimental Balb/c mice. To implement tumors to mice, they applied Ehrlich ascite carcinoma (EAC) 1 × 10 6 cells i.p. and lyophilized V. opulus juice at a dose of 1000, 2000, and 4000 mg/kg. The tumor weight significantly decreased in mouse groups treated with the juice compared to the control group. The survival rate of Ehrlich ascites tumor cells was reported to be 88.72%, 69.02%, and 51.87%, respectively. The results of the in vitro assay indicated the cytotoxicity of the juice with the IC 50 value of 199.58 μg/ml [191]. Also, the same authors reported that gilaburu fractions below and above 50 kDa can stop the cell cycle at the G0/G1 stage and slow the cell division of the Ehrlich ascites tumor [123].
Ulger et al. [192] (Turkey) experimented with Balb-c male mice to study the effect of V. opulus juice on colon tumorogenesis induced with 1,2-dimethylhydrazine (DMH). All groups treated with DMH developed colon tumors as observed by histogenesis. However, mice that received the juice showed a reduced number of tumor lesions, as well as the incidence of invasive carcinoma, as compared to untreated mice. The authors concluded that V. opulus juice could be useful at the initiation stage and prevention of colon cancer.

Other Health-Promoting Effects.
A 1000 mg Viburnum opulus dose and diclofenac on-demand were administered, orally, to 53 patients with urethral stones < 10 mm, in comparison to 50 patients receiving only diclofenac on-demand. It was observed that the expulsion of the stones was greater, and the passage time was faster in the treatment with V. opulus. The demand for diclofenac was lower compared to patients who received only diclofenac [193]. There are no clinical trial reports for the premenstrual syndrome [111], only the folk medicine use, as antispasmodic, in menstrual cramps, dysmenorrhea, and miscarriage prevention [194]. No side effects or reports regarding safety were found [194,195].

Conclusions
The genus Viburnum includes about 200 species, distributed mainly on the Asian side Fruits, flowers, and barks of certain species are used in traditional medicine for the treatment of diseases, such as rheumatoid arthritis, cough, diarrhea. They contain a plethora of biochemical compounds with healthpromoting activity, including carotenoids, polyphenols, and flavonoids, which can explain the high antioxidant activity as shown by in vitro studies. Preclinical evidence supports antibacterial, anti-inflammatory, cytotoxic, and anticancer properties of certain species, such as V. opulus.

Conflicts of Interest
The authors declare that they have no conflicts of interest.