A Scoping Review of Genus Viscum: Biological and Chemical Aspects of Alcoholic Extracts

The genus Viscum comprises a large number of semi-parasitic shrubs popularly known as Mistletoe. The Viscum species grow in many countries of Europe, Africa and Asia with different popular uses in ornamentation, foods and medicine. Many studies about Viscum have been done over the last years focusing on biological activities and chemical composition of the aqueous extracts, mainly related to anthroposophical medicines. However, it is known that non-aqueous preparations, as alcoholic extracts, have demonstrated different biological activities that are species—and host tree—dependent. Considering the potential of these alcoholic extracts, a scoping review was conducted using data from three online databases: PubMed, Scopus and Embase. Inclusion criteria consisted of the in vitro, in vivo, ex vivo, clinical and chemical studies of alcoholic extracts from Viscum species. The present review summarized 124 original publications about fourteen Viscum species. Viscum album, Viscum articulatum and Viscum coloratum were the main studied species. Alcoholic extracts demonstrated hypotensive, anticancer, antimicrobial, analgesic and anti-inflammatory capabilities, among other biological activities. Flavonoids, phenolic acids and terpenoids represented 48%, 24% and 11% of the total identified compounds, respectively. This review contributes to the knowledge of alcoholic preparations of the Viscum species and points out the lack of clinical studies concerning these different extracts.


Introduction
The genus Viscum includes 100-150 species distributed between tropical and temperate regions in Europe, Africa and Asia [1,2]. Mistletoe, as it is popularly known, is an evergreen hemiparasite shrub that grows on different host trees, which reflects important features in the biological activities of these species. A root-like organ called haustorium intimately connects the mistletoe to the host trees, and this structure makes it possible for these plants to absorb solutes and water from their hosts. Species of this genus produce a fruit called berry, which has different colors, depending on the Viscum sp., and this characteristic can help differentiate their origin [3].
The use of European mistletoe in medicine is ancient. Hippocrates (460-377 BC) described this plant to treat diseases in the spleen and complaints associated with menstruation [3]. In 150 AD, Platonist Celsus also reported the use of mistletoe against swellings Fourteen species were identified in this review (Figure 2), and the most cited were V. album L. (87 studies), V. articulatum (10 studies) and V. coloratum (8 studies ever, according to the International Plant Names Index (IPNI) [16] and WFO Plant some names for Viscum sp. considered by authors are synonymous, such as V. DC and V. rotundifolium Bory, V. coloratum Nakai which is another name of the var. colaratum and V. articulatum that can be found as V. liquidambaricola Hayata. A pattern in relation to the main species of the genus Viscum studied worldwide w found by Song et al. [6] but with different proportions V. coloratum > V. album > V. tum [6]. The authors did not delimit the solvent extractor and included different ex forms.  , some names for Viscum sp. considered by authors are synonymous, such as V. triflorum DC and V. rotundifolium Bory, V. coloratum Nakai which is another name of the V. album var. colaratum and V. articulatum that can be found as V. liquidambaricola Hayata. A similar pattern in relation to the main species of the genus Viscum studied worldwide was also found by Song et al. [6] but with different proportions V. coloratum > V. album > V. articulatum [6]. The authors did not delimit the solvent extractor and included different extractive forms.   The correct description of the species, subspecies, site of harvesting, as well as the extract solvent and the standardization of the extraction methods are crucial for the quality evaluation of mistletoe herbal products and its biological activities [7]. Additionally, identification errors or uncertainty regarding the plant origin can lead to serious safety problems [18]. In this scenario, the present work highlights a significant number of Viscum species that have not yet been well studied, which opens to new and more innovative research in this area.
Alcoholic solvents are a good strategy for phenolic compounds extraction [19,20], and our results demonstrated that approximately 70% of the chemical compounds identified by authors were flavonoids and phenolic acids ( Figure 4). Maceration accounts to 31% of the included studies as the method for plant extraction. This process has been used over the years considering some aspects such as simplicity and low cost of the operation. It was possible to find articles in which maceration was used as an extraction methodology from 1998 [21] to 2022 [22,23]. However, this method presents some disadvantages such as long extraction time and low extraction efficiency [24]. The second most common approach was the use of Soxhlet apparatus that is characterized by shorter extraction time and less solvent consumption when compared to maceration or percolation processes. However, the use of high temperature in this methodology increases compounds' thermal degradation [24]. Therefore, even though the same species was extracted using the same method, the solvent was not always the same, leading to difficulties for comparison in this review. The correct description of the species, subspecies, site of harvesting, as well as the extract solvent and the standardization of the extraction methods are crucial for the quality evaluation of mistletoe herbal products and its biological activities [7]. Additionally, identification errors or uncertainty regarding the plant origin can lead to serious safety problems [18]. In this scenario, the present work highlights a significant number of Viscum species that have not yet been well studied, which opens to new and more innovative research in this area.
Alcoholic solvents are a good strategy for phenolic compounds extraction [19,20], and our results demonstrated that approximately 70% of the chemical compounds identified by authors were flavonoids and phenolic acids ( Figure 4). Maceration accounts to 31% of the included studies as the method for plant extraction. This process has been used over the years considering some aspects such as simplicity and low cost of the operation. It was possible to find articles in which maceration was used as an extraction methodology from 1998 [21] to 2022 [22,23]. However, this method presents some disadvantages such as long extraction time and low extraction efficiency [24]. The second most common approach was the use of Soxhlet apparatus that is characterized by shorter extraction time and less solvent consumption when compared to maceration or percolation processes. However, the use of high temperature in this methodology increases compounds' thermal degradation [24]. Therefore, even though the same species was extracted using the same method, the solvent was not always the same, leading to difficulties for comparison in this review.      Disc diffusion, agar well diffusion, broth macrodilution and microdilution assays were the major methods employed for the antimicrobial and antiviral activities of Viscum sp. alcoholic extracts. From the 14 studies included, 9 were performed with Viscum album species. The results revealed a promising antibacterial activity of this plant species, mainly using ethanolic and methanolic V. album extracts obtained by maceration and Soxhlet apparatus. A wide variety of bacteria, fungi and yeasts strains were tested: Aspergillus flavus, Bordetella bronchisiptica, Bacillus subtilis, Candida albicans, Escherichia coli, Enterococcus faecium, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas syringae, Staphylococcus aureus, Saccharomyces cerevisiae, Salmonella typhi, Aspergillus niger, Agrobacterium tumefaciens, Bacillus atrophaeus, Bacillus cereus, Candida guilliermondii, Cryptococcus neoformans, Erwinia carotovora, Enterobacter cloacae, Microsporum canis, Proteus mirabilis, Staphylococcus epidermis, Shigella dysenteriae, Salmonella paratyphi and Tricophyton mentagropytes. Hussain et al. showed that the antibacterial potential differed by the part of the plant used to the extract preparation with inhibition zones (IZ) of 15-20 mm to leaves and of 9-24 mm to branches [12]. However, the authors observed that Aspergillus flavus and Saccharomyces cerevisae were not sensitive to ethanolic and methanolic macerated extracts of the leaves and twigs from V. album [12]. Shah et al. also demonstrated the same dependency, with IZ of 15-35 mm to stems, 15-30 mm to leaves and 15-35 mm fruits, according to the microorganism [30]. In this sense, another study using a percolated ethanolic extract of the leaves and stems from V. album verified that the activity against the microorganisms was host tree dependent with minimum inhibitory dilution between 0.04 and 3.13% in agar dilution method [26]. Additionally, the antibacterial potential of V. coloratum ethanolic extract produced with leaves and stems was tested as a preservative agent in uncooked pork patties during refrigerated storage, which decreased the pH and improved the storage period when compared to the control by inhibition of aerobic microbial multiplication [31]. In contrast, a study using a macerated extract of the V. album leaves on quality properties of rainbow trout fillets showed no extended shelf life when compared to control. In this case, both reached up to 20 days [28] ( Table 2). The antiviral potency of the 50% hydroalcoholic extract of the leaves from V. album subsp. album growing on lime trees against parainfluenza virus type 2 in Vero cells was evaluated by Karagöz et al. [32]. The results showed the growth rate and cell viability, in which both were unaffected by the ethanolic extract, and, therefore, it cannot be considered antiviral (Table 2). However, the same study showed that the aqueous extract appeared to have a potent anti-parainfluenza virus potential, demonstrating the influence of the solvent extractor over biological activity. Furthermore, one work demonstrated the antifungal capacity of methanolic extract from V. album against Coniophora puteana. The authors proved that the use of extract at 18.75% in wood reduces its mass loss promoted by fungi proliferation by approximately 7.97% [103]. In this sense, considering all antimicrobial results included in this review, it was not possible to compare the results obtained by the authors due to the various extraction techniques applied, concentration of the extracts and microorganisms used in the analysis. The majority of the studies evaluated only qualitative aspects through screening tests, such as the disc diffusion assay. They did not present the possible mechanisms of action involved in the biological results and did not present the chemical composition that could be correlated with the observed activity. Additionally, they did not present appropriate positive and negative control groups ( Table 2).

Antiparasitic and Insecticide Properties of Viscum sp. Alcoholic Extracts
Three studies evaluated the activity of ethanolic extracts obtained by different extraction methods and were tested in different parasites. V. congolensis presented anthelmintic activity against earthworms Alma emini after 24 h of exposure to the extract, which was compared to albendazole and mebendazole as controls [126]. Despite showing a promising activity, the authors did not demonstrate the parts of the plant used in the study, which could be an obstacle to reproducing the results in future studies. Butanol subfraction extracts prepared with leaves, fruits or berries from V. album subsp. austriacum (host tree pine) were tested against metronidazole-resistant Trichomonas vaginalis. It was observed that 10 mg/mL of butanol subfractions prepared with leaves or berries were more effective in the viability reduction rate of T. vaginalis than those prepared with fruits. The authors suggested that the presence of the 2-methylfuran, aromatic heterocycle, in the extracts could be the possible active substance [33]. These findings demonstrate the importance of the plant part description, which is extremely important to the biological activity. Lastly, an ethanol extract prepared with a mixture of leaves, stems and flowers from V. album presented 60% of toxicity against Thaumetopoae solitaria pupae, which was considered a low insecticide [34] (Table 3). The evaluation of the antiparasitic and insecticide activity of the Viscum alcoholic extracts is still incipient, showing a great field of research for the genus Viscum.

Cytotoxic and Cytostatic In Vitro Activities of Viscum sp. Alcoholic Extracts
The antitumor activity of the mistletoe by its cytotoxicity is dose-dependent [35][36][37]39,40]. Some studies evaluated the influence of the host tree on mistletoe activity. Methanolic extracts from V. album berries on Pinus sylvestris, Tilia cordata and Populus nigra presented IC 50 of 1 mg/mL, 164 µg/mL and 202 µg/mL, respectively, when tested on human colon adenocarcinoma (LS180) by MTT assay [39]. This result demonstrated the influence of the host tree in the V. album cytotoxic activity, and the extracts produced by V. album from Pinus sylvestris presented lower biological activity, probably due to the difference in chemical aspects. In addition, the authors verified through BrdU assay that the extracts' cytotoxicity did not have correlation with alterations in DNA synthesis [39]. In another study, methanolic extracts from V. album leaves growing on Tilia argentea, Acer campestre subsp. campestre and Robinia pseudoacacia decreased the viability measured by MTT assay of human cervical carcinoma (HeLa) with IC 50 of 93 µg/mL, 165 µg/mL and 85 µg/mL, respectively [37]. Holandino et al. showed that ethanolic extract from V. album subsp. album was more cytotoxic to Molt-4 and Yoshida cancer cell lines than extracts from subspecies abietis and austriacum [90]. These results confirmed the importance of the host tree description for the biological application. Methanolic extract of the aerial parts of V. cruciatum at 30 µg/mL exhibited a moderate degree of growth inhibition on human laryngeal carcinoma cells (HEp-2) after 72 h of the incubation when compared to 6-mercaptopurine positive control [127]. The authors associated the result with the presence of β-Amyrin acetate that presented a greater cytostatic action than the control. Yang et al. showed that isolated terpenes, mainly 3-epi-betulinc acid, oleanolic acid and erythrodiol from the V. coloratum ethanolic extracts, produced with leaves and twigs, were more potent on human ovarian carcinoma (HO-8910) and human hepatocarcinoma (SMMC-7721), with lower IC 50 when compared to the full extract [118]. Another isolated compound (hirsutanone) from aerial parts of V. cruciatum methanolic extract possessed cytotoxic activity against human melanoma (UACC-62), renal adenocarcinoma (TK-10) and breast adenocarcinoma (MCF-7) cell lines, with IC 50 values of 4.8, 6.8 and 1.9 µg/mL, respectively [128]. The anticancer activity of the isolated hirsutanone of the V. cruciatum was associated with apoptosis induction. V. album's apoptotic induction was identified by reduction of Hsp 27 and 14-3-3 and induction of caspase-3 proteins expression in rat glioma cells (C6) [36] and by increasing the cells in Sub G0 cycle, which affected the cell cycle in an early apoptotic activity in murine melanoma (B16F10) [35]. Only one study demonstrated the necrotic cytotoxicity of the Viscum album alcoholic extracts [90]. V. coloratum ethanol extracts (100-200 µg/mL) were not cytotoxic to the cell-derived inflammatory mediator (MDIM)-activated cells and human colorectal adenocarcinoma (Caco-2) cells despite having decreased the inflammatory processes in different ways [14,119] (Table 4). Methanolic extract of the Viscum cruciatum from almond tree was able to arrest the cell cycle of the MCF-7 (breast adenocarcinoma) in the G0/G1 phase. According to the authors, 15 flavonoids present in this extract inhibited CDK2, CDK4 and CDK6 proteins that are cell cycle checkpoint proteins in the Cyclin/CDK pathway [23].  The majority of studies evaluated the cytotoxic and cytostatic activity in human and rat cell lines by MTT assay. The findings demonstrated that the cells' response to stimulus depends on the Viscum species, as well as the host tree, plant parts used in the extract production and on the concentration of the extract analyzed. Most of the described active compounds are from the flavonoid and phenolic acid classes.

Cell Migration and Metalloproteinases Inhibition Induced by Viscum sp. Alcoholic Extracts
Metalloproteases (MMP) are elevated in the cartilage tissues and joint fluids of humans with rheumatoid arthritis, which can be responsible for cell migration and cartilage degradation. In this sense, V. coloratum ethanol extract inhibited cell migration of MDIM-stimulated chondrocyte cells (SW1353) and a reduction in secretion and activity of MMP-1, MMP-3 or MMP-13. Thus, these results suggest its anti-osteoarthritic activity [14]. However, in future studies about this important biological activity, it will be necessary to provide information about the parts of the vegetal species used, as well as the plant's harvest season, in order to ensure the reproducibility of results. In another study, EtOH extract prepared with roots of Viscum album inhibited MMP-13 expression in 64.3%, 70.3% and 80.0% at 50, 100 and 200 µg/mL, respectively [95] (Table 5). 2.1.5. Antiplatelet and Antihypertensive Activities of Viscum sp. Alcoholic Extracts The pharmacological activity of phenylpropanoids isolated from leaves and stems of the V. album from Pyrus caucasica inhibited ADP-induced platelet aggregation in a concentration range from 0.001 to 1.0 µM [21]. Another species, V. cruciatum, also inhibited adrenaline and ADP-induced platelet aggregation in a dose-dependent manner by the aerial parts ethanol extract [129]. A dose-dependent inhibition of V. album ethanol extracts prepared with leaves and stems on rabbit platelet aggregation was higher to the plateletactivating factor (PAF) compared to the ADP and arachidonic acid (ARA) pathways, significantly decreasing thromboxane A2 (TXA 2 ) production [41]. Host tree-dependent activity was evaluated, with V. album from olive and almond host trees, in which both presented prolongation of prothrombin time (PT) and activated partial thromboplastin time (aPTT), important indicators of coagulation [25]. Although the first study describes the potential of phenylpropanoids as the main pharmacological compound, the other three did not mention possible chemical classes or compounds responsible for the observed biological activity [25].
In relation to antihypertensive activity by inhibition of the angiotensin-converting enzyme, the V. triflorum ethanol extract from different host trees did not present 50% or more of inhibition activity at 0.33 mg crude extract in 1 mL test solution. However, the authors demonstrated that six aqueous samples of V. triflorum from Acacia heterophylla host tree showed 64-87% of enzyme inhibition [140]. It was possible to observe that the V. album has been the most studied species for platelet aggregation and coagulation, showing a lack of studies with the other alcoholic Viscum species ( Table 6). Despite of the folk description of the alcoholic Viscum extracts to reduce blood pressure [141], this review highlights the necessity of more in vitro studies to better understand the mechanism of action involved in the treatment of hypertension by the genus Viscum and its extractive solvents.  [25] V. album

Inhibition of platelet aggregation and effects on arachidonic acid
Human citrated whole blood EtOH isolated phenylpropanoids (0.001-1.0 µM) inhibited ADP-induced platelet aggregation. Arachidonic acid metabolism and biosynthesis of leukotrienes were not affected. Two di-glycosides (10 µM) inhibited 20-25% leukotriene B4 release. It suggests antitumor activity related to inhibition of protein kinase C by phenylpropanoids.
[21] Many studies have focused on finding new anti-inflammatory medicinal plants since the known anti-inflammatory drugs promote many side effects. Hence, the antiinflammatory potential of ethanol and methanol extract produced with the V. articulatum whole plant was observed, mainly due to the presence of flavonoids and triterpenoids [109]. V. coloratum ethanol extract reduced the inflammatory responses in inflammatory bowel disease (IBD) by suppressing MMP-2 and MMP-9 expression in Caco-2 cells and recovering the expression of zonula occludens-1, a tight junction protein. These results were associated to the high content of flavonoids [119]. Another study with V. coloratum ethanol extract observed strong inhibitory action in ß-hexosaminidase activity and TNF-α, IL-4, PGD2 and LTC4 formation, blocking the expression of COX-2 and the phosphorylation of 5-lypoxigenase, spleen tyrosine kinase, PLCgama1, PKCδ, Akt, JNK, ERK and p38, thus exerting anti-allergic and anti-osteoarthritic actions [14]. In contrast, ethanolic extracts from V. album subsp. album, subsp. abietis and subsp. austriacum exhibited almost no remarkable inhibitory activity on the inflammatory cytokines (IL-1α, IL-1β, TNF-α) at tested concentrations [43] (Table 7). V. album, V. articulatum and V. coloratum were the species better studied for the anti-inflammatory properties. However, considering the six studies included, only three described the parts of the plant used that were very different from each other (leaf, flower and whole plant). In this sense, the results present an interesting pharmacological potential of these species as an anti-inflammatory remedy, but further studies with more details are necessary. Peripheral blood mononuclear cells and neutrophils are crucial immune cells that carry out host defense. Human CD69 is an important antigen responsible for T-cells activation. In this sense, an immunomodulatory effect of the V. album subsp. album methanolic extract (1 mg/mL) was observed by increased CD69 expression in peripheral mononuclear blood cells, which mediates the activation of CD4 + , CD25 + , CD8 + and CD25 + T cells. The extract also increased the phagocytosis of Candida albicans blastospores and intracellular killing function of neutrophils compared to negative control [44]. These extracts can be considered as a good option for new investigations working on immune system activation, but it is necessary to standardize the host tree and the parts of the plant used, as well as include positive controls for better comparison. Moreover, the authors did not present a chemical composition to correlate with the observed activity.
2.1.8. Hypoglycemic/Hypolipidemic Activities of Viscum sp. Alcoholic Extracts V. schimperi decreased advanced glycation end products [137], and V. album rich in phenolic compounds showed a potent anti-glycation activity [45]. V. album subsp. album presented low inhibitory potential on α-amylase and on α-glucosidase activities and thus might ameliorate hyperglycemia in diabetes type 2 [46]. In complement, V. articulatum ethanolic extract was considered moderately suitable for controlling diabetic conditions by its inhibitory activity on α-amylase [110]. An anti-obesity application of V. album was observed by its inhibitory effect on pancreatic lipase [47] (Table 8). Oxidative stress causes damages that trigger many disorders. V. coloratum ethanol extract presented an inhibitory effect on tyrosinase and Superoxide dismutase (SOD)-like stimulation activity [49]. Important enzymes involved in the oxidative stress, SOD-like, catalase (CAT) and glutathione reductase (GR) activities decreased significantly in two dif-ferent studies [13,104]. Chromosomal aberrations and mitotic index were dose-dependent, and malondialdehyde decreased with V. album extract, suggesting the extract's antioxidant, anti-mutagenic and DNA-repairing mechanism-inducing properties [42]. V. album methanolic extracts did not affect the steady-state level of intracellular ROS, but the activity was host tree-dependent. Pre-treatment with V. album from Robinia pseudoacacia and Tilia argentea completely prevented the damage on nuclear and mitochondrial DNA under stress conditions, decreasing ROS formation, while the one from Acer campestre host tree was effective against nuclear DNA but partially for mitochondrial DNA damage [37]. Lastly, pork meat quality was tested by using V. album extract, which was highly effective in maintaining uncooked pork patties by inhibiting lipid oxidation and preventing odor development, which was higher than the control ascorbic acid [31] (Table 9). All studies included in this section evaluated the V. album species, showing the necessity to research the other species, as well as different parts of the plant and season harvest. Table 9. Viscum sp. in vitro activities-cellular antioxidant effect.

V. album
Hydroxyl scavenging activity (HRSA) using deoxyribose method, superoxide radicals (SRSA) using xanthine oxidase, lipid oxidation using thiobarbituric acid reactive substance (TBARS) EtOH extract (0.5 mg/mL) presented 40.19% of inhibitory activity for HRSA and 30.05% for SRSA. The extract reduced TBARS after 14 days of storage. V. album Oxidative stress and intracellular ROS lever by H 2 O 2 induction and DCFH-DA Extracts from Robinia and Tilia host trees completely prevented nuclear and mitochondrial DNA damage under stress conditions, while extract from Acer was completely effective for nuclear DNA damage but only half-effective for mitochondrial DNA damage. [37] V. album Lipid peroxidation using malondialdehyde (MDA) EtOH extract (0.5 µg/mL) had protective effect against lipid peroxidation and DNA repairing. Thus, it is promising as antioxidant, anti-mutagenic and DNA repair-inducing properties [42] V. album

Hypoglicemic Effects of Viscum sp. Alcoholic Extracts
Methanol extracts of the aerial parts of V. schimperi and V. album and ethanol extract of the leaves of V. album reduced the glucose level in a dose and/or time-dependent manner [50,51,138,139]. Furthermore, V. colaratum enhanced insulin secretion 0.82 ± 0.14 ng/mL (control) to 1.07 ± 0.19 ng/mL (ethanolic extract) in partial pancreatectomized rats probably by the increase in β-cell proliferation [52,138]. The effects in glucose level reduction were also dependent on the Viscum album subsp. and host trees. Extracts reduced the blood glucose level in streptozotocin-induced diabetics rats from 329.8 mg/dL (control) to 289 mg/dL, 286 mg/dL and 243 mg/dL to V. album subsp. abietis, album and austriacm, respectively [53] (Table 10).

Investigation Animal Model Intervention Main Results Authors
Antidiabetic and hypoglycemic activity in streptozotocin-induced diabetic rats.
Male Wistar rats 50 mg/kg and 100 mg/kg b.w. of the extract administered for 4 h or 100 mg/kg the extract administered daily for 3 consecutive weeks.
750 mg/kg b.w. decrease fasting blood glucose level in normal as well as in streptozotocin-induced diabetic rats similar with the glibenclamide control. [51] Antidiabetic activity in partial pancreatectomized rats.
Male Sprague-Dawley rats 0.6% of EtOH extract in diet for 8 weeks.
EtOH extract enhanced glucose-stimulated insulin secretion and β-cell proliferation in diabetic partial pancreactomized rats. [52] Antidiabetic activity was assessed through fasting blood glucose level, insulin levels and area under the curve in oral glucose tolerance test.
Male Wistar rats 75 and 150 mg/kg b.w. of the lyophilized extract was administered in an oral dose.
MeOH extract and organic subfractions presented a significant antihyperglycemic activity after 4 weeks of daily doses. Insulin levels increased in a dose dependent manner and all tested fractions produced reduction in AUC of glucose concentration. [138] Antihyperglycemic and hypolipidemic effect by effect of extract on plasma glucose level, oral glucose tolerance test, plasma insulin level, muscle and liver glycogen and plasma lipid profile.
Male Wistar rats 500 mg/kg b.w. of the extract given orally by gavage as single daily treatments for 4 weeks.
Antihyperglycemic activity was observed by maximum reduction in blood glucose level of 37%. [139]

Hypolipemic Effects of Viscum sp. Alcoholic Extracts
V. album subsp. album was able to reduce serum cholesterol, LDL-C and triglyceride more than 50% and increase serum HDL-C in 46.7% in male Swiss albino mice. V. schimperi methanolic extract also decreased total cholesterol, LDL-C and triglyceride around 30% and elevated the HDL-C to 171.5% [54,139]. However, Vadnere et al. did not observe hypolipemic activity of ethanol extract from V. articulatum [111] (Table 11). These results demonstrated that V. album and V. schimperi could be good sources with hypolipemic action.

Anticancer Activities of Viscum sp. Alcoholic Extracts
The number of studies related to the in vivo activity of aqueous Viscum extracts is enormous. Bonamin et al. in a recent review showed that different aqueous preparations of the Viscum were able to reduce tumor growth and tumor cell viability, increase tumor necrosis and also promote tumor angiogenesis reduction [142]. However, in relation to alcoholic extract, few articles were found to highlight the necessity of more studies (Table 12). In the present review an association of V. album ethanol extract with doxorubicin was able to reduce Ehrlich ascites carcinoma volume and catalase and xanthine oxidase activity [55,56]. Methanol extract of V. angulatum and V. articulatum had a significant dose-dependent effect on the urine excretion volume with diuretic index of 2.76 and 3.00, respectively, in relation to control. An increased natriuretic (Na + /K + ) and saluretic (Na + + Cl − ) index were observed for both species, but the V. articulatum extract presented a higher saluretic index of 272 when compared to 168 of the V. angulatum extract [105,112]. Supporting this data, Bachhav et al. 2012 also demonstrated that the methanol extract of V. articulatum prevented the progression of hypertension in rats due to urine volume and Na + rising [113]. In addition, V. album ethanol extracts presented a hypotensive dose-dependent response in rats with maximal reduction at 1 × 10 −3 mg/kg/b.w. The blood pressure reduction was 23.56 mmHg with effect via muscarinic receptors [15] (Table 13). In this sense, V. angulatun, V. articulatum and V. album are the main species on which studies for antihypertensive activity and have promising data that support future clinical trials.

Investigation Animal Model Intervention Main Results Authors
Hypotensive activity on values of arterial blood pressure by direct method in the left carotid artery.
EtOH extracts presented dose dependent response and the maximal reduction was observed at 1 × 10 −3 mg/kg with arterial blood pressure reduction of the 23.56 mmHg with effect via muscarine receptors. [15] Diuretic, saluretic and natriuretic effects by Na + and Cl − excretions.
Male Wistar rats and Swiss albino mice 100, 200 and 400 mg/kg b.w. of the MeOH extract dissolved in water and administered orally.
The extract at 400 mg/kg had a diuretic index (volume in test group/volume in control group) in 24 h of 2.76 and increased saluretic (Na + + Cl − ) at 168 and natriuretic index (Na + /K + ) at 2.20. [105] Diuretic, saluretic and natriuretic effects by Na + and Cl − excretions.
Male Wistar rats and Swiss albino mice 100, 200 and 400 mg/kg b.w. of the MeOH extract dissolved in water and administered orally.
400 mg/kg of the extract had a diuretic index (volume in test group/volume in control group) at 24 h of 3.00 and increased saluretic (Na + + Cl − ) at 272 and natriuretic index (Na + /K + ) at 2.16. [112] Antihypertensive activity against Nω-nitro-L-arginine methyl ester induced hypertension by blood pressure and heart rate; urine volume and urine sodium/potassium; serum creatinine; serum lipid estimation.
Male Wistar rats 200 or 400 mg/kg/day of the extract orally.
MeOH extract prevented progression of hypertension in rats produced by chronic administration of Nω-nitro-L-arginine methyl ester, which may be due to its diuretic, nephroprotective, hypolipemic and antioxidant effects. [113] 2.2.5. Analgesic and Anti-Inflammatory Activities of Viscum sp. Alcoholic Extracts V. monoicum ethanol extract showed a potential increase in pain threshold analgesia by tail immersion test in a dose-dependent manner of the 5.05 ± 0.43 and 5.81 ± 0.23 seconds at 200 and 400 mg/kg/b.w., respectively, as compared to pentazocine (6.29 ± 0.28 seconds) at 10 mg/kg/b.w. (p > 0.05) after 120 minutes [132] (Table 3). V. album methanol extract of the whole plant exhibited significant analgesic activity in tail immersion test with respect to the control group, but none of the doses showed effects comparable to the standard drug [57]. Methanol extract of V. orientale at 500 mg/kg/b.w. inhibited 88.8% of the acetic acid induced writhing, demonstrating analgesia by prostaglandin synthesis inhibition. At the same dose, the extract inhibited paw licking in the formalin induced pain model 56.4% in the early phase and 72.6% in the late phase, respectively [135]. Lin et al. 1994 [133] demonstrated that ethanol extract of V. multinerve (100 and 300 mg/kg/b.w.) promoted anti-inflammatory activity by reduction of carrageenaninduced edema compared to indomethacin. However, the extract was not effective in protecting the liver against CCI 4 -induced damage. V. coloratum ethanol extract demonstrated activity in inflammatory bowel disease since it significantly attenuated enterorrhagia and colonic edema in Dextran Sodium Sulfate (DSS)-induced colitis. This activity was produced in mice by inhibiting the immune cells infiltration in IBD and by decreasing the levels of immunoglobulin E, IL-6 and TNF-α [119] (Table 14). Table 14. Viscum sp. in vivo activities-analgesic and anti-inflammatory.

Investigation Animal Model Intervention Main Results Authors
Anticolitic effect in induced colitis for 8 days by Dextran Sodium Sulfate (DSS).
Extract attenuated the body weight loss, reduced the scores of Disease Activity Index (DAI), suppressed enterorrhagia and colonic oedema in DSS-treated mice. [119] Analgesic activity by tail immersion method and anti-diarrhoeal activity by Castor oil-induced diarrhoea method.

Male and female Swiss-albino mice
For the analgesic activity, the tail immersion test in hot water was performed after treating the animals via a gastric tube. For the anti-diarrhoea activity, the mice were treated orally after being induced to diarrhoea.
EtOH extract of V. monoicum exhibited analgesic activity through central nervous system in dose dependent manner. EtOH extract of V. monoicum showed anti-diarrheal activity decreasing defecation in a dose dependent manner. [132] Anti-inflammatory activity by Carrageenan-induced oedema and liver-protective effects in CCl 4 -induced hepatotoxicity.
Male Wistar albino rats 100 and 300 mg/kg b.w. of the extract subcutaneously.
EtOH extract promoted anti-inflammatory activity reducing the paw oedema to indomethacin. Extract was not effective in protecting the liver against CCI 4 -induced damage. [133] 2.2.6. Neuropharmacological Activities of Viscum sp. Alcoholic Extracts V. capense methanol extract significantly delayed the onset of pentylenetetrazole-and bicuculline-induced tonic seizures. At 100 mg/kg/b.w. intraperitoneal (i.p.) it significantly attenuated the seizures reducing the number of animals convulsing. However, the same extract did not alter N-methyl-DL-aspartic acid-induced tonic seizures [115]. V. album methanol extract reduced rearing and crossings with respect to control in open field test, suggesting central nervous system (CNS) depressant activity [57]. Khatun et al. 2016 [135] also showed that the V. orientale methanol extract exhibited CNS depressant activity by decreasing exploratory behavior in mice, as well as the frequency and amplitude of the movements.
Genus Viscum alcoholic extracts were also able to reduce anxiety, depression and stress in different animal models. V. album methanol extract at 100 mg/kg/b.w. reduced the anxiety in mice by the increased number of entries and time spent in open arms of elevated plus maze (EPM), statistically equivalent to the standard drug. The authors also showed antidepressant activity using despair swim test after acute administration of methanol extract (200 or 400 mg/kg/b.w., p.o.), which significantly reduced the mice immobility time at all doses with respect to control. However, none of the doses showed activity equivalent to the standard drug. Moreover, the methanol extract significantly reduced the time spent by mice in an immobile state in cold swim test with respect to control, showing a mild anti-stress activity that was not equivalent to the standard drug [57]. At higher doses, the methanol extract was able to promote significant hypnotic activity by increasing the duration of sleep in mice in thiopentone sodium-induced sleeping assay [57] (Table 15). Anticonvulsant activity induced in mice with pentylenetetrazole, bicuculline and N methyl-DL-aspartic acid.

Male and female albino mice
Intraperitoneal injection at 50-100 mg/kg b.w. of the extract in a physiological saline solution.
MeOH extract protected the mice against pentylenetetrazole-and bicuculline-induced tonic seizures but did not significantly alter N-methyl-DL-aspartic acid-induced tonic seizures, suggesting its antiepileptic effect. [115] Behavior and antianxiety by open field exploratory, hypnotic/sedative effect by pentobarbitone-induced sleep.
Animals treated with a dose of 400 mg/kg of MeOH extract decreased the number of total crossings. The extract demonstrated a dose dependent increase in pentobarbitone induced sleep. [117] Anti-nociceptive and central nervous system activity through acetic acid and formalin-induced pain models, respectively, and cross and open field test behavior profiles.
Male and female Swiss albino mice 300 or 500 mg/kg b.w. of the extract orally.
In the acetic acid induced writhing test, extract produced 88.8% of writhing inhibition at 500 mg/kg of body weight. Extract has both peripheral and neurogenic anti-nociceptive and CNS depressant activities.
[ 135] 2.2.7. Toxicity Activities of Viscum sp. Alcoholic Extracts V. coloratum crude ethanol extract promoted mice mortality in acute toxicity test by using intragastric administration (LD 50 7.67 g/kg/b.w). However, with the action of the Rhodobacter sphaeroides in the extract, for a fermentation process, it reduced the number of deaths to zero [118]. This result suggests that the chemical composition was changed by fermentation reducing the toxicity of the extract, probably by biotransformation of toxic proteins. Methanol extract of the V. album subsp. album at 250 mg/kg/day showed protective effects against cyclophosphamide-induced cardiotoxicity, urotoxicity and genotoxicity in mice. The authors demonstrated that the extract improved the levels of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase, Glutathione-S-transferases, reduced glutathione) and mitotic activity of bone marrow cells. Furthermore, the pre-treatment with V. album together with cyclophosphamide significantly decreased aberrant cells and chromosome aberrations when compared to cyclophosphamide alone [58]. V. album methanolic extract at 250 mg/kg/b.w. had a protective effect against methotrexate-induced cyto-genotoxicity in mouse bone marrow, decreasing the number of chromosomal aberrations of 96.40 ± 8.25 to 59.20 ± 1.65 in relation to methotrexate. The extract also significantly increased the mitotic index from 33.12 ± 1.79 to 60.12 ± 1.12 in bone-marrow cells that had been suppressed by methotrexate compared to a positive control [59] (Table 16).

Investigation Animal Model Intervention Main Results Authors
Protective effects against cyclophosphamide-induced cardiotoxicity, urotoxicity and genotoxicity through anti-oxidative stress and -inflammation in the heart and bladder and chromosomal damage in the bone marrow.
Male Swiss albino mice 250 mg/kg b.w./day of the extract were administered orally by gastric gavage.
Antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase, Glutathione-S-transferases and mitotic index were restored to near normalcy as compared to the control group. Lipid peroxidation in heart and bladder were reduced by extract when compared to the cyclophosphamide group. [58] Anti-cytogenotoxic effects of pre-treatment with V. album extract on methotrexate-induced chromosomal aberrations.

Male Swiss albino mice
Extract by oral gavage at 250 mg/kg b.w./day for 10 days.
MeOH extract had a protective effect against methotrexate-induced cyto-genotoxicity in mouse bone marrow decreasing chromosomal aberrations of 96.40 ± 8.25 to 59.20 ± 1.65 in relation to the control. [59] Acute toxicity testing according to the oral administration method.
Male Balb/c mice EtOH extract up to 5000 mg/kg body weight orally.
No animal deaths were observed in the study. [94] Protective effect against chlorpyrifos-induced hepatotoxicity.
The extract recovered the antioxidative system parameters and alleviated some histopathological changes caused by chlorpyrifos. [97] Protective effect against tetrachloride (CCl 4 )-induced acute/chronic liver injury.
Male Wistar rats MeOH extract 300 mg/kg b.w. orally in single dose The extract decreased ALT and AST enzymes increased by CCl 4. [101] Acute toxicity testing according to the oral administration method.

Swiss albino mice
MeOH extract at 5-2000 mg/kg b.w. suspended in 0.5% carboxymethyl cellulose The extract was safe up to 2000 mg/kg body weight. [117] Acute toxicity testing according to the intragastric administration method.
Kunming mice of both sexes Ethanolic extract. EtOH extract presented an acute toxicity of the LD 50 7.67 g/kg. [118] 2.2.8. Other Activities of Viscum sp. Alcoholic Extracts Methanol extract of V. coloratum at 125, 250 and 500 mg/kg/b.w. prolonged the bleeding time in transected rat tail, suggesting its use in improving blood circulation [60].
Karagöz et al. [61] showed that methanol extract of V. album subsp. album improved parameters of heart failure in rats, such as left ventricular diameters, ejection fraction, serum NT-proBNP (N-terminal pro b-type natriuretic peptide) levels and histopathological changes. This resulted in a statistically significant attenuation of increased levels of nitric oxide 48.5 ± 2.3 µmol/L to isoproterenol group when compared to the control (19.0 ± 3.3 µmol/L) and V. album group (37.8 ± 3.1 µmol/L). The levels of high-sensitivity C-reactive protein were also found to be lower in the V. album group (0.133 ± 0.023 ng/mL) when compared to the control (0.183 ± 0.034 ng/mL) and isoproterenol group (0.155 ± 0.025 ng/mL) (Table 17). [60] Effects of V. album on cardiac function through the nitric oxide pathway in isoproterenol-induced heart failure rats evaluated by echocardiographic and biochemical evaluation.

Male Wistar albino rats
Concentrated extract reconstituted in 0.9% NaCl and administered 250 mg/kg/day orally.
MeOH extract improved: left ventricular diameters, ejection fraction, serum N-terminal pro b-type natriuretic peptide) levels and histopathological changes. Attenuation of increased levels of nitric oxide and nitric oxide synthase. The levels of high-sensitivity C-reactive protein were lower in the V. album group compared to the controls. [61] Pharmacokinetic studies Male and female Wistar rats coloratum via the tail vein.
Here, 1.5 and 2.5% of extract in diet increased protease activity and serum antioxidant enzyme activities. These concentrations decreased serum activities of hepatic enzymes. The highest serum lysozyme and total Ig values were observed in the 2.5% of the extract in diet. [100]

Ex Vivo Studies with Viscum sp. Alcoholic Extracts
Six studies evaluated the ex vivo vascular, antispasmodic and antidiabetic activities. Ethanol and n-butanol extracts of V. album promoted contraction in noradrenalinecontracted rat aortic rings that were host tree-and dose-dependent [62]. Corroborating with this data, Deliorman et al. also showed that polar subfractions of V. album subsp. album ethanol extract and some isolated compounds (Syringin, Coniferin, 5,7-dimethoxyflavanone-4 -O-[β-D-apiofuranosyl- (12)]-β-D-glucopyranoside) produced contractile responses in a dose-dependent manner in noradrenaline-contracted rat aortic rings [63]. In addition, the vasodilator activity was observed in the less polar subfractions. However, the Kalopanaxin D (hydroxycinnamic acid derivative) displayed a very slight relaxant response. In this context, V. album methanol extract partially inhibited phenylephrine (1 µM) and K + (80 mM)-induced sustained contractions of the rabbit aortic ring through the blockade of Ca ++ . Additionally, considering the action in cardiovascular diseases, Suveren et al. showed that methanolic V. album extract at 5 mg/L mediated the nitric oxide-dependent cardioprotection against myocardial injury originated by ischemia/reperfusion insult, reducing 53.2% in mean infarct size compared to control hearts [64]. Regarding antispasmodic activity, V. album methanol extract relaxed the spontaneous and the K + (80 mM)-induced sustained contractions of rabbit jejunum in a dose-dependent manner with an EC 50 value of 0.31 mg/mL (0.15-0.57) and 0.62 mg/mL (0.3-0.95), respectively, through the blockade of Ca 2+ [65] (Table 18).

Investigation Main Results Authors
Vascular effects in noradrenaline-contracted rat aortic rings. n-BuOH fraction produced a contractile response in noradrenaline-contracted rat aortic rings. [63] Vascular effects on isolated noradrenaline-contracted rat aortic segments.
EtOH extract contained marked vasodilator activity especially from cherry, quince and acacia host trees. [62] Gut inhibitory and stimulatory effects by in rabbit jejunum and guinea-pig ileum respectively.
Spasmogenic effect was observed with a concentration-dependent contractile effect in guinea-pig ileum at 5-10 mg/mL of the extract. Spasmolytic effect was demonstrated to relax the spontaneous and K + (80 mM)-induced contractions of isolated rabbit jejunum, with EC 50 values of 0.66 and 0.55 mg/mL, respectively. [129] Antispasmodic and relaxant activity in smooth muscle evaluated in isolated rabbit jejunum and in rabbit aortic rings.
Crude MeOH extract inhibited spontaneous and high K + -induced contractions in rabbit jejunum. The extract showed a partial relaxation against high K + (80 Mm) and phenylephrine (1 µM)-induced contractions in isolated rabbit aorta rings. [65] Cardioprotective activity in myocardial ischemia and reperfusion injury in rats.
5 mg/L of the MeOH extract reduced 53.2% in mean infarct size compared to control hearts. [64] Similarly, Gilani and coworkers also found a concentration-dependent (0.01-3.0 mg/mL) relaxation of spontaneous and K + (80 mM)-induced contractions of isolated rabbit jejunum by the V. cruciatum ethanol extract with action in Ca 2+ channels [129]. Additionally, a spasmogenic effect was observed with a concentration-dependent contractile effect in guinea-pig ileum at 5-10 mg/mL of the extract [129] (Table 18).

Clinical Trial
One clinical pilot study of V. album mother tincture was evaluated on 41 newly diagnosed hypertensive patients who had not taken any medication [66]. Blood pressure was taken for the following 3 weeks, after which the treatment started and lasted for 12 consecutive weeks (10 drops in 30 mL of water 3 times a day, half an hour after food). Fifty-nine percent of patients were at stage 1 and 33% at stage 2 hypertension, after which the systolic blood pressure decreased from 155.8 mm Hg to 141.5 mm Hg, as well as diastolic pressure from 84.4 mm Hg to 79.5 mm Hg (p < 0.05). Serum cholesterol was not altered, but a reduction in serum triglyceride was significant (p < 0.001). Serum lactate dehydrogenase and serum urea significantly rose, however not higher than the normal level. None of the patients presented any sign of cardiac or musculoskeletal discomforts. Considering these results, V. album mother tincture could be used to regulate blood pressure and to lower serum triglyceride levels [66]. However, this study did not consider the Viscum album subspecies and its host tree. Furthermore, the authors chose a 1-group pretest-posttest study and did not include a control group and a blinding method. Thus, more clinical studies are necessary, in addition to better methodological design.

Chemical Aspects of Viscum sp. Alcoholic Extracts
The chemical composition of natural products is an essential step in the evaluation of their biological potentialities. Studies focused on phytochemical profile and antioxidant activities of the vegetal extracts have been published over the years. V. album has received remarkable attention due to its effectiveness in clinical oncological therapy [143]. The present review showed different chemical classes present in Viscum sp. with antioxidant properties: flavonoids (e.g., flavanones and flavonols), phenolic acids and triterpenes. In addition, the total content of chemical groups such as total pro-anthocyanidin content (TPAC), total flavonoid content (TFC), total carotenoid content (TCC), total triterpene content (TTC) and experiments applied to antioxidant capacity assay in alcoholic extracts of Viscum species are shown in Table S1.
The total phenolic content (TPC) was expressed mainly in gallic acid with some exception in caffeic acid, p-coumaric acid, quercetin, tannic acid or catechin/dry weight or fresh weight. Others, such as the total flavonoid content (TFC), were expressed mainly by quercetin content but also used rutin and kaempferol/dry weight of plant. Following, total pro-anthocyanidin content (TPAC), total carotenoids content (TCC) and total terpenoid content (TTC) used catechin, β-carotene and oleanolic acid, respectively.
Upon the evaluation of these results in relation to the different total contents in each chemical group by species, the quantitative results varied considerably among authors, especially in the V. album, which may be due to the type of cultivation, geographical origin, host tree, climatic conditions and different extraction procedures [3]. The total phenolic content in this species ranged from 0.19 [71] to 1232 [68] mg/g dry extract or fresh weight. Stefanucci and coworkers showed that extracts from Viscum album leaves had more phenolic and flavonoid content than fruits and seeds. The authors also highlighted that the method of extraction promoted differences in these contents [102]. Furthermore, the other species of Viscum also demonstrated the same pattern, with great variability in the phenolic composition. This characteristic can be explained by differences in the site harvest, host tree and parts used of the plant.
Considering that the total content experiments are the preliminary assay to guide authors in antioxidant evaluation, in vitro colorimetric methods were carried out. Some studies used a unique antioxidant assay model that was not conclusive [130,135,137]. Nevertheless, in the case of V. album, 23 different in vitro assay models have been performed, all of them detailed in Table S1. It is difficult to fully compare one method to another because some differences in the reaction mode, procedure, sample, chemical reagent, etc. [144,145]. The in vitro colorimetric methods that were most frequently used and were put in order of decreasing frequency are described as follows: Folin-Ciocalteu reducing capacity assay; DPPH; ABTS •+ (radical cation 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) scavenging activity); SRSA (Superoxide anion radical scavenging activity); FRAP (ferric reducing antioxidant power) assay and others such as TEAC (Trolox Equivalent Antioxidant Capacity); β-Carotene-linoleic acid assay; SOD (Superoxide dismutase assay); ORAC (Oxygen Radical Absorbance Capacity) assay; α-amylase/α-glucosidase inhibitory activity; chelation power on (Fe 2+ ) ions activity assay; FRSA (Free radical scavenging activity) assay; HRSA (Hydroxyl radical scavenging activity) assay; H 2 O 2 Hydrogen peroxide scavenging activity assay; DMPD (N,N-dimethyl-p-phenylenediamine radical scavenging activity) assay; NO (Nitric oxide radical scavenging activity) assay; PRAP (Phosphomolibdenumreducing antioxidant power) assay; AChE, BChE, and Tyrosinase activity assays; TBARS (Thiobarbituric acid reactive species) assay; XOD (Xanthine oxidase) assay; Chemiluminescence; FTM Ferric thiocyanate method (Table S1). V. album was widely studied in in vitro antioxidant assay in comparison to the other species, and DPPH assay was the most common method applied. In addition, considering the DPPH, for the same Viscum sp. some authors used the IC 50 value [68,70,74] to represent the antioxidant capacity, but they used different equivalent of a specific antioxidant standard, such as rutin, quercetin, chlorogenic acid, etc [46,49,91]. This review shows that a standardization of the antioxidant methods, in which their results can be comparable to each other, is very important. Finally, as described above, the antioxidant activity can be obtained by multiple ways using a variety of experimental procedures, making it difficult to compare these experimental data.
It was observed that flavonoids were the most commonly identified substances in alcoholic extracts from Viscum species [35,45,73,75,78,138]. Phytochemical studies in Viscum genus have reported the predominance of subclasses such as flavanone, flavonol, flavone and their respective glycosylated derivatives (Table S6). In addition to flavonoids, a series of phenolic compounds, mainly phenylpropionic and benzoic acids and their glycosylated derivatives, have been detected. Syringenin, syringenin-apiosylglucoside and some lignans, such as eleutheroside E and syringaresinol monoglucoside, were identified in alcoholic mistletoe preparations [21,35,80,82,84,85] (Table S5). Our review shows that flavonoids were the largest group of secondary metabolites, followed by phenolic acids and terpenoids (Figure 4), which provided target pharmacological profiles such as antioxidant, antihypertensive, antidiabetic, anti-inflammatory and others. Regarding the solvent's physicochemical properties, these were vital for the extraction of these chemical groups. Alcohols (EtOH and MeOH) are polar solvents extensively used to extract antioxidant compounds due to their effectivity in extracting phenolic compounds [19,20].
Information about analytical methods is detailed in Tables S3-S8. Instruments such as high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC), coupled to ultraviolet (UV), diode array detectors (DAD) or mass spectrometry (MS), have contributed to the characterization of phenolic acids and flavonoids. In the case of terpenoid compounds, nuclear magnetic resonance spectroscopy (1D and 2D NMR) and gas chromatography (GC) were the most used techniques. Additionally, primary metabolites such as fatty acids and carbohydrates were identified with the aid of instruments, such as LC-MS, NMR and GC-MS. In the last decades, a dramatic acceleration in the development of new technology of separation, detection and preparation of biological samples was achieved, which brought as a benefit the characterization of hundreds of phytochemical constituents in a single analysis. In this sense, this review showed that most of the chemical compounds had been isolated mainly from V. album, suggesting that other studies regarding chemical characterization from other species need to be explored.

Material and Methods
A literature search was performed using PUBMED, EMBASE and SCOPUS databases up to 31 August 2022. A starting period or language filters were not used. The following search strategy was used: (1) Viscum OR mistletoe; (2) alcoholic OR ethanol OR ethanolic OR EtOH OR methanol OR methanolic OR MeOH OR butanol OR butanolic OR BuOH OR tincture OR mother tincture; (3) 1 AND 2. The inclusion criteria considered publications about chemical, in vitro, in vivo, ex vivo and clinical studies for Viscum sp. alcoholic extracts. Publications were excluded if the content was about ethnopharmacological studies, reviews, short lectures or abstracts, in addition to studies that presented a mixture of plants or non-alcoholic Viscum extracts. Furthermore, studies in languages in which the authors were not proficient (Chinese, Japanese, Korean, Persian or Turkish) and articles that did not provide free access were excluded. First, the titles and abstracts were evaluated by two authors through the Rayyan Systems Inc. tool (https://www.rayyan.ai/, accessed on 6 March 2023). Second, the authors screened the selected ones and excluded nonalcoholic Viscum studies. Then, all publications were checked for eligibility. At this point, all relevant publications were checked by two authors independently based on the inclusion criteria. A third author evaluated the publication if disagreements occurred. The main results of included publications were summarized in a new matrix, specified in categories (Tables 1-4 and S1-S8/Supplementary Material).

Conclusions
This review showed that Viscum sp. alcoholic extracts presented positive and promising activity in hypertension, dyslipidemia, inflammation and diabetes, among other health disorders. Fourteen Viscum species were identified, and Viscum album was the main species studied followed by Viscum articulatum, showing a gap in studies with different Viscum sp. alcoholic extracts. Flavonoids, phenolic acids and terpenoids were the most described chemical classes in these species, with great potential for biological applications, such as antioxidant, antimicrobial and hypolipidemic. Only one clinical study with alcoholic extract of the Viscum album was found, which is in opposition to hundreds of clinical studies with aqueous extracts. This big gap needs more attention and research to support the folk use of these ethanolic extracts, especially as a homeopathic remedy. Some studies did not present important aspects of the raw material, such as season and site of harvest, subspecies and detailed extractive method, which is crucial for their pharmacological uses, reducing the quality of the studies and making their reproducibility difficult. Thus, our study contributes to the genus Viscum literature by compiling the state of the art and can be used as a database for planning new research in the scope of alcoholic extracts from Viscum species.