Zimbro (Juniperus communis L.) as a Promising Source of Bioactive Compounds and Biomedical Activities: A Review on Recent Trends

Plant-derived products and their extracted compounds have been used in folk medicine since early times. Zimbro or common juniper (Juniperus communis) is traditionally used to treat renal suppression, acute and chronic cystitis, bladder catarrh, albuminuria, leucorrhea, and amenorrhea. These uses are mainly attributed to its bioactive composition, which is very rich in phenolics, terpenoids, organic acids, alkaloids, and volatile compounds. In the last few years, several studies have analyzed the huge potential of this evergreen shrub, describing a wide range of activities with relevance in different biomedical discipline areas, namely antimicrobial potential against human pathogens and foodborne microorganisms, notorious antioxidant and anti-inflammatory activities, antidiabetic, antihypercholesterolemic and antihyperlipidemic effects, and neuroprotective action, as well as antiproliferative ability against cancer cells and the ability to activate inductive hepato-, renal- and gastroprotective mechanisms. Owing to these promising activities, extracts and bioactive compounds of juniper could be useful for the development of new pharmacological applications in the treatment of several acute and chronic human diseases.


Introduction
Natural products have an important role in the research and development of new drugs. People have always extracted natural products from several natural sources, such as marine organisms, microorganisms, animals, and medicinal plants [1]. The main extracts from natural products come from medicinal plants. Plant-derived products and compounds have been used worldwide since ancient times in folk medicine as remedies for several diseases, such as tinctures, teas, poultices, maintaining high prevalence in public health [1][2][3][4]. Advances in clinical research and quality control have shown a greater value of herbal medicine in the treatment and overcoming of many diseases. Recent works report promising potential regarding the use of plants in the treatment and/or prevention of several hard-to-cure diseases, such as atherosclerosis [5,6], cancer [1- 3,7,8], cardiovascular diseases [9][10][11][12], diabetes [8,13,14], and neurological disorders [4,15,16], among others.
The genus Juniperus includes roughly 68 species and 36 varieties and belongs to the Cupressaceae family [17]. The plant Juniperus communis L., named "zimbro" in Portugal, is a shrub or small evergreen tree; a perennial and long-lived coniferous, woody pioneer and colonizing plant, adapted to low nutrient availability in soil and having one the widest distribution ranges among the different plant species [18]. Its population is spread globally, being the only Juniperus species found in both hemispheres, with reports of this Juniperus (= Oxycedrus) section has a Holarctic distribution reaching the Mediterranean; it is represented by 14 species with acicular leaves, an anchoring point to the stem, and resiniferous cones. The third section, the Sabina section, is mainly found in the northern hemisphere and mountainous areas of the African continent. However, it also has some type of resin, and is distinguished from the other ones since it has decurrent needle-like or scale-shaped leaves and juicy cones [45]. According to these characteristics and with the fossil record, it is thought that the diffusion point of this genus occurred in the eastern Mediterranean region, first colonizing the northern regions of the Eurasian continent, and from there passing to the American continent at least 25 My ago [43].
All juniper species stand out for their high content of essential oils and phenolic compounds and are largely included in the traditional medicine of different cultures throughout the planet, exhibiting a wide range of biological activities and industrial applications [46]. Among them, it is worth highlighting the "zimbro" (J. communis) plant, since it shows the widest distribution, being practically circumboreal [43,47]. Another remarkable characteristic of this species is the ecological plasticity supported by great genetic variability, which translates into a substantially high number of varieties with phenotypes ranging from medium-sized trees (3-4 m high) to small creeping shrubs ( Figure 1) [41,43]. The populations of the Iberian Peninsula are very diverse, and due to their position, their distribution is relegated to mountainous and more humid areas with a territorial occupation in islands, thus scaping from the thermophilic and xeric character of the nonmountainous lands of the Peninsula [44,47]. Indeed, it is believed that many years ago, this territory acted as a glacial refuge for many varieties that are currently found further north; even so, the populations of var. hemisphaerica show a high degree of genetic uniqueness, while the var. alpina (also known as var. nana) is mainly distributed in the upper areas of the mountains of the Iberian Peninsula, such as the Serra da Estrela mountains. These mountains are located in the middle interior of mainland Portugal and display an oromediterranean climatic island, being an isolated population from other populations of the Central System mountains or Cantabric System mountains [21,48]. As well as other plants, J. communis also receives popular names, both in our own and in foreign languages. For example, Havusa or Matsyagandha (Sanskrit); Arar, Abahal oe Habbul (Assamese); Hayusha (Bengali); juniper berry, or common juniper (English); Palash (Gujrati); As well as other plants, J. communis also receives popular names, both in our own and in foreign languages. For example, Havusa or Matsyagandha (Sanskrit); Arar, Abahal oe Habbul (Assamese); Hayusha (Bengali); juniper berry, or common juniper (English); Palash (Gujrati); havuber or havubair (Hindi); zimbro (Portuguese); padma beeja (Kannada); hosh (Marathi); havulber (Punjabi); hapusha, abhal or arar (Urdu) [21,23].
For curiosity, and despite this plant not having a strong presence in ancient mythology, it is considered a symbol of fertility in Syria. On the other hand, in the Old Testament, it is described that the juniper has an angelic presence, which sheltered the prophet Elijah from Queen Jezebel's pursuit. Moreover, a posteriorly biblical tale described that during their flight to Egypt, the infant Jesus and his parents used juniper to hide from King Herod's soldiers [49].

Phytochemical Composition of Juniperus communis L.
As mentioned before, J. communis L. species are composed of a myriad of constituents, including nonessential substances, i.e., phytochemicals [50]. These compounds are secondary metabolites produced by plants to promote their normal cellular metabolism and offer protection against biotic and abiotic factors, and consequent oxidative injury [51]. Additionally, they are considered the key contributors to the organoleptic characteristics (e.g., aroma and color) and health benefits exhibited by plants [52]. They can be divided into five major categories ( Figure 2). Although the plants' genotype mainly influences their quantitative and qualitative composition, their levels also depend on the plant's age, ripeness degree, cultivation techniques, geographical location, and meteorological conditions [53,54].
For curiosity, and despite this plant not having a strong presence in ancient mythology, it is considered a symbol of fertility in Syria. On the other hand, in the Old Testament, it is described that the juniper has an angelic presence, which sheltered the prophet Elijah from Queen Jezebel's pursuit. Moreover, a posteriorly biblical tale described that during their flight to Egypt, the infant Jesus and his parents used juniper to hide from King Herod's soldiers [49].

Phytochemical Composition of Juniperus communis L.
As mentioned before, J. communis L. species are composed of a myriad of constituents, including nonessential substances, i.e., phytochemicals [50]. These compounds are secondary metabolites produced by plants to promote their normal cellular metabolism and offer protection against biotic and abiotic factors, and consequent oxidative injury [51]. Additionally, they are considered the key contributors to the organoleptic characteristics (e.g., aroma and color) and health benefits exhibited by plants [52]. They can be divided into five major categories ( Figure 2). Although the plants' genotype mainly influences their quantitative and qualitative composition, their levels also depend on the plant's age, ripeness degree, cultivation techniques, geographical location, and meteorological conditions [53,54].

Carotenoids and Chlorophylls
Although no studies have specifically reported the chlorophyll content of J. communis L. species, Rabska and colleagues [56] analyzed their total levels in fertilized and nonfertilized in both genders of this plant in autumn and winter (species not specified). The obtained data revealed nonfertilized plants had a lower concentration of total chlorophyll content than the fertilized ones (mean values of 5.0 versus (vs.) 7.4 mg/g in autumn and 3.6 vs. 4.8 mg/g in winter, respectively), and also lower amounts of total carotenoids (mean values of 0.64 and 0.95 mg/g for female and male, respectively, in autumn, and scores of 0.87 against 1.2 mg/g in winter). Focusing on gender, they observed that female plants had lower amounts of total chlorophyll compounds (values of 2.9 and 4.5 mg/g for female plants in autumn and winter, respectively, and 3.7 and 5.2 mg/g in autumn and winter, respectively, for the male ones) and carotenoid levels (values around 0.90 mg/g for female plants and around 1.0 mg/g for male, in autumn and winter, respectively). Without surprises, and regarding all the comparisons made, the authors also concluded that male and fertilized plants presented the highest levels of total chlorophyll and carotenoids (mean values of 4.3 and 1.3 mg/g, respectively).

Carotenoids and Chlorophylls
Although no studies have specifically reported the chlorophyll content of J. communis L. species, Rabska and colleagues [56] analyzed their total levels in fertilized and nonfertilized in both genders of this plant in autumn and winter (species not specified). The obtained data revealed nonfertilized plants had a lower concentration of total chlorophyll content than the fertilized ones (mean values of 5.0 versus (vs.) 7.4 mg/g in autumn and 3.6 vs. 4.8 mg/g in winter, respectively), and also lower amounts of total carotenoids (mean values of 0.64 and 0.95 mg/g for female and male, respectively, in autumn, and scores of 0.87 against 1.2 mg/g in winter). Focusing on gender, they observed that female plants had lower amounts of total chlorophyll compounds (values of 2.9 and 4.5 mg/g for female plants in autumn and winter, respectively, and 3.7 and 5.2 mg/g in autumn and winter, respectively, for the male ones) and carotenoid levels (values around 0.90 mg/g for female plants and around 1.0 mg/g for male, in autumn and winter, respectively). Without surprises, and regarding all the comparisons made, the authors also concluded that male and fertilized plants presented the highest levels of total chlorophyll and carotenoids (mean values of 4.3 and 1.3 mg/g, respectively).
This subclass of phytochemicals, highlighting carotenoids, possesses notable antioxidant potential and the ability to easily activate metabolic detoxification pathways, reducing the risk of appearance of several chronic and degenerative disorders [51,56].

Phenolic Compounds
Phenolics are the most predominant phytochemicals present in nature, and to date, about 10,000 different structures are currently described [57]. They are usually classified in   n.s.: not specified; a mg equivalent of gallic acid (GAE) per g dry weight (dw); b mg quercetin equivalents per g dw; c mg cyanidin 3-glucoside equivalents per g dw; d mg quercetin 3-O-rutinoside equivalents per g dw; * mg of catechin equivalents per g dw.
Focusing on their biological potential, hydroxycinnamic acids present antimicrobial, antioxidant and anti-inflammatory effects, being able to easily interact with detoxification and inflammation-related pathways, preventing the appearance or attenuating the development of many chronic diseases [52,54].
As well as hydroxycinnamic acids, the hydroxybenzoic ones also display antimicrobial and antioxidant properties; however, they are less efficient given the lack of the CH=CH-COOH group and the double bond between carbons 7 and 8 [54].
Although flavones are less effective in diminishing free radicals and reactive species levels due to the lack of the hydroxyl group at carbon 3 than other flavonoids, they display antimicrobial, antioxidant, and anticancer effects, as well as a notable ability to regulate lipid metabolism [54,64].
Given their multiple hydroxyl groups, anthocyanins are potent radical scavengers, and also present notable anti-inflammatory abilities, being able to interact with related pathways, increase antioxidant defences, and diminish proinflammatory biomarkers, and in this way, prevent the occurrence of many oxidative-stress-related disorders [54,59].
The combination of all of these results is evidence that local origin influences the phytochemical profile. Additionally, Gonny et al. [80] determined the VOC profile of J. communis woods and roots of var. alpina. For woods, α-terpinyl acetate (9.10%) and α-terpineol (8.4%) were the predominant ones, while for roots, a high percentage of cedrol (37.70%) and cinnamyl acetate (11.50%) were found.
VOCs have been gaining great interest owing to their remarkable antimicrobial, antioxidant, anti-inflammatory, and anticancer properties, being able to attenuate, or even mitigate, the development of cardiovascular disorders and neuropathologies, and also ameliorate the mental state of individuals [85].

Biological Potential of Juniperus communis Linnaeus
Since ancient times, J. communis parts have been largely used as antiseptics, contraceptives, and diuretics, and as a remedy to treat colds, chest complaints, rheumatism, headaches, dermatological and respiratory ailments, and kidney and urinary infections [38,39,85]. Given the aforementioned, it is not surprising that this plant is a focus of continuous studies to discover its full potential.
To date, several reports have highlighted its antimicrobial, antifungal, antioxidant, antiinflammatory, and antidiabetic potential, as well as its anticarcinogenic, hepatoprotective, neuronal, and renal effects, as described in Figure 5 and Tables 2-4 [34,38,40,66,[86][87][88]. Next, a summary of the main studies already published concerning the health-promoting properties of this plant will be presented.

Antimicrobial, Antifungal, and Antiparasitic Potential
Antimicrobial and antiparasitic activity can be divided according to the nature of the employed extract, i.e., essential oils and phenolic-rich extracts, which in turn influence the different target activity, use, application, and range of microorganisms and parasitics inhibited [35] ( Table 2). The use of essential oils is widespread in ethnobotanical phytotherapy, and for this reason, several works can be found [46]. Filipowicz et al. [89] analyzed the antibiotic capacity of different essential oils extracted from J. communis berries, each one with a specific composition. They concluded that the extract with a more balanced composition in its components (α-pinene, β-pinene, p-cymeno or limonene, among others) showed greater antibiotic effects against multiresistant hospital isolates belonging to the species Staphyllococcus aureus, Serratia marcescens, Enterobacter cloace, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumanii, and Listeria monocytogenes, as well as in Candida albicans. The authors also verified that there effectively existed a synergistic effect between all components of the oil.
On the other hand, essential oils from J. communis needles (var. alpina) are shown to have notable effects in inhibiting the growth of numerous dermatophyte fungi (Epidermophyton floccosum, Microsporum canis, M. gypseum, Trichophyton mentagrophytes, T. mentagrophytes var. interdigitale, T. rubrum, and T. verrucosum), with active concentrations ranging between 0.32 to 2.5 µL/mL (vs. inhibition values of 16 and 128 µg/µL for the antifungal fluconazole) [90]. However, essential oils of leaves and fruits of J. communis (var. communis) from Sardinia exhibited weak antibiotic activity against C. albicans, S. aureus, and P. aeruginosa (Minimum Inhibitory Concentrations (MICs) higher than 1 mg/mL) [91]. Even so, it was observed that the use of pure solutions of juniper essential oils showed lower activity than the solutions diluted in at least 50% ethanol; this evidence is probably due to an improvement in the solubility of essential oils, which in turn increase its effectiveness [36].
These results agree with those obtained in Slovenia using distillates obtained through medium-scale industrial processes. Here, the essential oils of J. communis were able to inhibit the development of S. aureus and C. albicans, both of type strains and clinical isolates, showing in the latter case inhibition halos of 7.00 ± 0.01 mm and 21.33 ± 0.88 mm, respectively [92].
Furthermore, the use of essential oils obtained from J. communis biomass, without differentiating each of its parts, showed remarkable inhibitory activity against Escherichia (E.) coli, at concentrations between 1.25 and 2.5 mg/mL. As expected, there were observed variations regarding the obtained data due to the different collection sites and consequent different edaphoclimatic conditions, which in turn influenced the essential oil extracts' composition [75]. On the other hand, no notable inhibitory activities were observed against other Gram-negative bacteria, such as Proteus mirabilis, K. pneumoniae, P. aeruginosa, and Morganella morganii; however, a slight activity against L. monocytogenes and methicillinresistant S. aureus was observed [75]. Similar results were obtained comparing the activity of commercial J. communis berry essential oils and hydrodistilled berry extracts from wild Portuguese plants, observing a considerable variation in the MIC, minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC). The obtained data were expressed in % v/v between each of the extracts, and even without showing susceptibility to the highest concentrations tested (2.5% v/v), greater susceptibility was seen to Gram-positive species (B. cereus, B. subtilis and S. aureus) than Gram-negative species [81]. The study of the antibiotic capacity of the essential oils of J. communis leaves against 16 species of bacteria and 14 species of fungi, some of them dermatophytes fungus, showed similar results to those already observed for bacteria, where Gram-positive ones had a greater susceptibility than Gram-negative ones, with MIC and MBC varying between 8 and 70% v/v. The MIC results observed for fungi ranged between 0.39 and 10% v/v, while MBC values were between 0.78 and 12.5% v/v [26]. The essential oils from J. communis fruits (var. alpina) showed an outstanding activity against different types of pathogenic fungi, with MIC values ranging between 1.25 and 20 µL/mL; the highest susceptibilities were found against dermatophyte fungi, such as M. canis, T. rubrum, or E. floccosum [84]. These results are similar to those obtained with essential oils of J. communis, with MIC values of 11.11 mg/mL and 13.98 mg/mL for different Aspergillus species and between 11.11 mg/mL and 12.50 mg/mL for species of the genus Penicillium, which are also 2 times higher than those obtained for the commercial antifungal ketoconazole [93].
The analysis of the volatile fraction of J. communis essential oils presented similar results to those observed in essential oils and phenolic-rich extracts, with an intense activity against Gram-positive bacteria such as S. aureus, but not against Gram-negative ones such as E. coli. In fact, a concentration about 3 times lower was necessary to inhibit S. aureus growth (4.75 µg/mL) compared to E. coli (16.8 µg/mL). The comparison between the volatile composition and other constituents of the Juniperus genus showed that there may exist a relationship between the quantity of sesquiterpene hydrocarbons and aromatic oxygenated hydrocarbons, mostly found in the extract of J. communis, and their effectiveness against Gram-positive bacteria [94]. Even so, the use of the volatile fraction extracted from berries of var. alpina, mainly composed of α-pinene and δ-3-carene, showed a lower MIC than the complete extract, at concentrations ranging from 0.16 to 1.25 µL/mL [84]. In this regard, it has been described that the antibiotic capacity of α-pinene depends on the enantiomeric properties of this compound, cultivation origin, and part of the plant (leaves or cones) [95].  In another way, the use of subinhibitory concentrations (1 mg/mL) of juniper-fruit essential oils has been shown to possess a severe effect as an antiadhesion agent in Campylobacter jelunni, preventing the formation of biofilms by up to 100% to concerning the control on plastic surfaces [96]. The use of essential oils of J. communis from the whole plant (leaves and branches) has been shown to have a temperature-dependent growth-inhibitory effect (MIC) for bacteria of the genus Mycobacterium, at concentrations between 0.8 and 3.2 mg/mL. Although the use of subinhibitory concentrations allows limiting the formation of biofilms by up to 49% after 3 days [98], its combination with essential oils extracted from Helichrysum italicum has been shown to have synergistic activity, allowing the concentrations used to be reduced 3-fold to achieve similar effects [97]. It has also been observed that the use of essential oils from J. communis berries is capable of reducing the adhesion of L. monocytogenes cells to HT-29 and HCT116 colon-cancer cells by 62%, thus reducing the capacity of this foodborne pathogen to cause intracellular infections by establishing favorable and competent unions with the host cells [93].
Comparing a total of 72 different plants used in European ethnobotany, the hexane extract of J. communis leaves was one of the most effective in inhibiting pathogenic microorganisms present in the oral microbiota, with higher inhibition halos in diffusion-inhibition tests and a greater range of inhibited microorganisms [99]. On the other hand, the ethanolic extract rich in phenolic compounds from J. communis seeds showed a discrete antibiotic activity, with inhibition halos ranging between 7 and 12 mm at solutions of 100 mg/mL dw, presenting a higher sensitivity against Gram-positive (S. epidermidis, S. aureus, B. subtilis) than Gram-negative (P. aeruginosa, E. coli) and fungal (C. albicans) strains. These data were supported by the obtaining MIC, yielding a result of 3.125 µg/mL for the three Gram-positive species when compared to values that varied between 12.5 µg/mL and 50 µg/mL regarding the other studied species [100]. The comparison between methanolic and aqueous phenolic-rich extracts from leaves of two different J. communis varieties (var. communis and var. saxatilis) revealed that the methanolic extract presents a higher activity, requiring lower concentrations to inhibit the growth of S. aureus (78.12 µg/mL and 39.06 µg/mL, respectively) when compared to the aqueous extracts, which required much higher concentrations (1250 µg/mL and 312.5 µg/mL respectively). Additionally, it was observed that the var. saxatilis was more active than var. communis [101]. Additionally, berries' phenolic extracts from var. communis and var. saxatilis showed that although the var. communis had a higher concentration of phenolic compounds, the MIC and MBL were lower for the var. saxatilis against S. aureus (156.25 µg/mL), S epidermidis (1250 µg/mL), Enterococcus hirae (156.25 µg/mL), and B. subtilis (156.25 µg/mL), compared with var. communis (S. aureus (156.25 µg/mL), S epidermidis (1250 µg/mL), En. hirae (625 µg/mL), and B. subtilis (321.5 µg/mL) [70]. On the other hand, the use of aqueous extract rich in phenolics against different bacteria and fungi showed around 50% more activity in disk-inhibition tests against Gram-positive bacteria (Lactobacillus fermentum-17 mm, S. aureus-15 mm and L. monocytogenes-15 mm) than against Gram-negative (P. aeruginosa-10 mm and Acinetobacter baumannii-11 mm). In the case of fungi, very discrete inhibition activities were observed (C. utilis-3 mm, Aspergillus sp.-6 mm and Fusarium sp.-2 mm) [102].
Another possibility is the combination of these extracts rich in phenolic compounds with routinely used commercial antibiotics such as tetracycline, chloramphenicol, and erythromycin. It was already reported that their combination with alcoholic extracts rich in phenolic compounds from J. communis leaves can effectively improve their efficiency against E. coli, S aureus, and K. pneumoniae by between 2 and 525 times, allowing a considerable reduction in the MIC of the antibiotic [104].
The use of subinhibitory concentrations (1 mg/mL) of ethanolic extracts and essential oils of juniper berries can also have a severe effect as an antiadhesion agent in Campylobacter jeunni, preventing the formation of biofilms by up to 95% on plastic surfaces [96]. In this way, the use of methanolic and aqueous extracts of J. communis var. communis and var. saxatilis branches can inhibit the growth of S. aureus. The methanolic extract showed reductions in initial adhesion after 3 h (22% in var. saxatilis and 44% in var. communis) and in the formation of biofilms 24 h after inoculation (66% in var. saxatilis and 68% in var. communis). Meanwhile, the aqueous extract proved to be more active in controlling biofilm formation, limiting the initial adhesion of bacterial cells to surfaces (25% in var. communis and 50% in var. saxatilis) and the extent of the biofilm formed (81% in var. communis and 84% in var. saxatilis) [103].
Concerning antiparasitic potential, essential oils extracted from leaves and stems of J. communis showed potential to inhibit the growth of two malarial strains different to Plasmodium falciparum, which were chloroquine-resistant (FcBl) and chloroquine-sensitive (Nigerian) strains, exhibiting in both cases an IC 50 value of 1 mg/mL after 24 and 72 h of exposure. No cumulative effects were found over time [105]. This activity is mainly due to the presence of α-pinene, which is one of the main essential oils extracted from this plant. In fact, this terpene has already been shown to possess notable antimalarial activity (IC 50 value of 1.2 µM) [106].
On the other hand, J. communis shoots showed potential to reduce reactive oxygen species and increase the activity of intracellular antioxidant-enzyme superoxide dismutase and catalase [56]. Furthermore, its acetone, ethyl acetate, and ethanol extracts showed inhibitory percentages of 6.05, 22.59, and 12.31%, respectively, at 1 mg/mL regarding metal-chelating potential [107]. In addition, essential oils of its twigs can inhibit peroxyradical-induced oxidation, exhibiting values of around 120 µmol Trolox/gram of essential oil [53]. Ethanolic extracts of hops also displayed ferric-ion-reducing antioxidant power (4.17 mg of ascorbic acid equivalents per g), and the capacity to capture DPPH • and ABTS •+ species (9.26 and 49.54 mg of ascorbic acid equivalents per g, respectively) [68].

In Vivo Studies
The administration of methanolic extracts of this plant (200 mg/kg) for 21 days on chlorpromazine-induced Parkinson's disease in rats also showed increments in reduced glutathione and decreased levels of TBARS as compared to the untreated group [19]. Furthermore, the inhalation of its oil for 60 min daily for 21 days revealed higher levels of superoxide dismutase and catalase enzymes, and glutathione peroxidase activity on rats' hippocampus subjected to amyloid β (1-42)-induced oxidative stress [85].
The remarkable antioxidant abilities showed by J. communis L. species are intimately linked to their phenolic and terpenoid content, in particular the presence of quercetin aglycone and their derivatives [32,67,69,71,79]. This flavan-3-ol possesses several hydroxyl groups in its constitution, which makes it a potent radical scavenger. As evidence, positive correlations (r > 0.80; p < 0.05) were already reported concerning their levels and capacity to neutralize DPPH • [108] and to inhibit lipid peroxidation in human erythrocytes [109,110]. Regarding terpenes' antioxidant activity, Burits and colleagues [111] already reported that α-pinene, ρ-cymene, limonene, and linalool possess notable capacities to block lipid peroxidation (IC 50 values of 0.51, 0.69 and 0.67 µL/mL, respectively) and to avoid deoxyribose degradation (IC 50 scores of 0.78, 0.91 and 0.28 µL/mL, respectively). Similar potential was also reported by Emamia and collaborators [112] concerning β-pinene, cedrol, and sabinene antioxidant potential. Moreover, this property also depends on the extraction solvents applied, usually being higher when water-alcohol mixtures are used, given their great affinity for both lipophilic and hydrophilic bioactive molecules [50,59].

In Vitro Studies
By in vitro studies, it was already mentioned that aqueous extracts of J. communis can inhibit prostaglandins by 55% at 200 µg/mL and platelet-activating factor-induced exocytosis by 78% at 250 µg/mL [25]. Moreover, Schneider et al. [88] also revealed that methylene chloride extracts of its woods and berries, and berry ethyl acetate extract at 100 µg/mL can effectively reduce the production of 12[S]-hydroxy-5,8,10,14eicosatetraenoic acid by 54.0, 66.2, and 76.2%, respectively. Essential oils of its plant material (twigs, leaves, and fruits) from two different Spanish regions also showed potential to inhibit the lipopolysaccharide-induced nitric oxide production on RAW 264.7 murine macrophage cells (IC 50 values of 84.80 and 23.98 µg/mL for the regions of Almazán and Barriomartín, respectively) [75]. From the methylene chloride extract of the wood were extracted cryptojaponol and β-sitosterol, which in turn showed inhibitory activities of 55.4 and 25.0% regarding 12[S]-hydroxy-5,8,10,14-eicosatetraenoic acid production, respectively, at concentrations of 100 µg/mL.

In Vivo Studies
Focusing on in vivo studies, Mascolo and collaborators [113] screened the anti-inflammatory potential of hydroethanolic extracts of 27 plants from different families largely used in Italian folk medicine and reported that J. communis was one of the most effective in reducing the rats' swelling-foot edema induced by carrageenin. Indeed, the obtained data revealed that at doses of 100 and 200 mg/kg and after 7 days of treatment, a reduction was verified regarding carrageenin-foot edema by 60% and 79%, respectively, against 45% shown by positive-control indomethacin (5 mg/kg). Similar results were reported by Akkol et al. [114]. Additionally, Akkol and coworkers [114] also verified that these extracts also revealed anti-inflammatory potential in PGE-2-induced hind-paw edema in a pattern similar as the carrageenin-edema model. More recently, the anti-inflammatory potential of J. communis was assessed using two different inflammation experimental models (dextran and kaolin), and it was verified through plethysmometry that the treatment with hydroethanolic microemulsions of J. communis berries can effectively reduce paw edema in the dextran-induced inflammation model, mainly due to its antihistaminic and antiserotonin activities. On the other hand, in the kaolin-induced inflammation model, the administration of this microemulsion showed potential to significantly downregulate the expression of proinflammatory interleukins (IL)-1β and IL-6, and tumor necrosis factor alfa, owing to its content in phenolics [40].
Beyond what was reported, Banerjee and colleagues [29] revealed that methanolic extracts of J. communis leaves possess analgesic effects. The authors conducted an in vivo study involving different nociceptive assays (acetic acid-induced writhing, formalin, and tail-flick tests) in rodents and verified that the extract administration of 100 mg/kg and 200 mg/kg can significantly inhibit, in a dose-dependent manner, the writhing response and the late phase related with the formalin test as compared to aspirin. Furthermore, it was also verified that this plant can act centrally, since the extract and pethidine effects were blocked by naloxone in the tail-flick test.
Effects of 3-week juniper nebulization (20 min/day) on the respiratory tract of rats which firstly exposed to 2 cigarettes per day, 5 days a week for 6 weeks Bronchodilator effects mediated by nitric oxide [140] Genotoxicity protective effects
Capacity to exhibit genoprotective effects against aberrations and abnormalities induced by ethanol on root-tip cells of Allium cepa L.

In Vitro Studies
Hydroethanolic extracts of J. communis leaves and fruits already displayed, through in vitro assays, the ability to inhibit α-amylase (inhibitory scores of 29.8 (fruit) and 53.6% (leaf) at 3 mg/mL), and α-glucosidase (IC 50 values of 4.4 and 84.3 µg/mL for fruit and leaf respectively) activities [66]. Moreover, the aqueous extracts of this plant at 50 g/L also showed the capacity to significantly decrease glucose diffusion by 6% when compared with the negative control [124].

In Vivo Studies
Concerning in vivo studies, the capacity of J. communis berries (at 1 g/400 mL) revealed the capability to avoid polydipsia and weight losses, and in this way retard the development of diabetes in streptozotocin mice, as reported by Swanston-Flatt and colleagues [125]. Furthermore, decoctions of J. communis berries orally administrated at doses of 250 and 500 mg/kg showed potential to reduce hypoglycemia in normoglycemic rats, reduce blood glucose levels and mortality index, and prevent weight loss in streptozotocin-diabetic rats after 24 days of treatment at a dose of 125 mg/kg [126]. In addition, Banerjee and colleagues [31] verified that the oral administration of J. communis methanolic extracts (100 and 200 mg/kg) can effectively reduce blood glucose levels, total cholesterol, triglycerides, low-density lipoprotein, and very-low-density lipoprotein cholesterols, and increase highdensity lipoprotein cholesterol in streptozotocin-nicotinamide-induced diabetic rats in a dose-dependent manner after 21 days of treatment.
Finally, a herbal preparation from Croatia composed of natural plants, including J. communis, also revealed the capacity to reduce glucose and fructosamine levels in alloxaninduced nonobese diabetic mice at 20 mg/kg after a 7-day treatment [127].
In addition to the mentioned, Akdogan and collaborators [33] conducted a one-month in vivo trial based on the daily administration of J. communis berry oil (dissolved in 0.5% of sodium carboxymethyl cellulose) in albino Wistar rats and verified that this berry showed potential to reduce cholesterol at concentrations of 50, 100, and 200 mg/kg. Particularly, the highest dose significantly increased blood-urea nitrogen and creatinine levels and reduced total cholesterol, oxidized low-density lipoprotein, alanine aminotransferase, and aspartate transaminase levels by 16%, 24%, 8.2%, and 10% when compared to the untreated cholesterol group. No anaemic effects or distinct morphological changes in rat kidneys were observed.
Briefly, these effects are mainly attributed to the capacity of J. communis to interfere with carbohydrate enzymes, increase peripheral glucose consumption, and protect pancreatic β-cells from damage [66,126].

Antiproliferative Effects
Considering the crescent incidence of cancer, it is not surprising that several different efforts are being conducted to discover new approaches and alternatives useful to reduce the development and/or to act as a complementary treatment against this malignancy [141]. Among plants, J. communis species have been intensively studied [71,73].

In Vitro Studies
Until now, this plant has already shown the in vitro capacity to suppress the growth of many cancer cells. For example, Lee and colleagues [128] revealed that berry extracts can induce apoptosis on OECM-1 human gingival squamous cancer cells, exhibiting an IC 50 [39]. Methanolic extracts of its leaves also showed capacity to block the growth and development of C6 rat-brain tumor and HeLa human-cervix carcinoma cells (IC 50 values of 28.43 and 32.96 µg/mL, respectively) [72], PC3 human-prostate cancer cells (IC 50 = 23.8 µg/mL), HCT 116 human-colon cancer cells (IC 50 = 37.6 µg/mL), and MCF7 breast cancer cells (IC 50 = 23.8 µg/mL) after 24 h of exposure [129]. On the other hand, aqueous berry extracts can decrease the growth and invasion of MCF-7/AZ breast cancer cells (IC 50 value of 50 µg/mL after 24 h of treatment) [130]. In addition to the mentioned, Fernandez and coworkers [131] also reported that methanolic extracts of its berries can block the proliferation of Caco-2 human colorectal and HeLa cervical cancer cells, showing IC 50 values of 1383 and 2592 µg/mL, respectively, after 12 h of exposure. Their aqueous extracts also showed potential to inhibit both cancer cells after 12 h, exhibiting IC 50 scores of 1516 (Caco-2 cancer cells) and 2157 µg/mL (HeLa cancer cells) [131]. On the other hand, essential oil and distilled extracts from J. communis berries revealed the potential to suppress A549 human lung adenocarcinoma epithelial-cell growth and development, revealing IC 50 values of 69.4 and 1270 µg/mL, respectively, after 24 h of treatment [71]. Additionally, they also showed the ability to suppress the development of SH-SY5Y human neuroblastoma cells after 24 h of exposure (IC 50 score of 53.7 µg/mL), which is evidence that this plant can penetrate the blood-brain barrier [134,135,142]. The capacity of J. communis plant material (twigs, leaves and berries) to suppress NCI-H460 lung carcinoma, MCF-7, AGS gastric carcinoma, and Caco-2 cell growth was also evaluated, revealing IC 50 values varying depending on the origin [75]. Essential oil and distilled extracts of seed cones from J. communis also reveal the capacity to inhibit the growth of HT-29 (IC 50 values of 125 and 625 µg/mL for essential oil and distilled extracts, respectively) and HCT116 cancer cells (IC 50 values of 62.5 and 1250 µg/mL for essential oil and distilled extracts, respectively) after 24 h of exposure [93].
J. communis distilled extracts also seem to be useful in the prevention of melanoma tumorigenesis, since they already show potential to block B16/F10 melanoma cells growth, displaying IC 50 values of 27 and 44 µg/mL after 24 and 48 h of exposure, respectively [73]. These data are in agreement with in vivo results [73]. Furthermore, this plant also showed potential to inhibit HepG2, Mahlavu, and J5 human hepatocellular carcinoma cell growth, in a dose-and time-dependent manner, revealing IC 50 values of 43.9 µg/mL for HepG2 cells, 59.4 µg/mL for Mahlavu cells, and 53.2 µg/mL for J5 cells after 72 h of treatment [132].

In Vivo Studies
The administration of J. communis distilled extracts (200 mg/kg) for 23 days C57BL/6 mice showed the capacity to reduce tumor size by 45.2% when compared to the untreated group. It was also verified that J. communis treatment resulted in cell-cycle arrest at the G0/G1 phase; lower concentrations of B-cell lymphoma-2 (Bcl-2), procaspases 8 and 9; and higher levels of Bcl-2-associated X protein, apoptosis-inducing factor, cellsurface death receptor Fas, and Fas ligand [73]. On the other hand, the administration of J. communis essential oils (200 mg/kg) in BALB/c nude mice injected with HepG2 cancer cells showed the capacity to reduce tumor growth and extend the lifespan with no or low systemic and pathological toxicity [132]. Similar information was reported by Lai and colleagues [133] and Tsai and collaborators [135] regarding the antitumor effects of this plant against human colorectal adenocarcinoma and glioblastoma.
Remarkably, the antiproliferative and cytotoxic effects shown by this plant are mainly attributed to the capacity of phenolic compounds and terpenes to interact, in different ways, with cell-signaling pathways and cascades, inducing apoptosis and interfering with cell cycle progression [71,73,132,142]. Particularly, imbricatolic acid isolated from the methanolic extract of J. communis fresh ripe berries showed the ability to prevent cell-cycle progression in p53-null human lung cancer Calu-6 cells by inducing the upregulation of cyclin-dependent kinase inhibitors and their accumulation in the G1 phase of the cell cycle, as well as the degradation of cyclins A, D1, and E1 [143]. On the other hand, isocupressic acid and deoxypodophyllotoxin isolated from this plant can induce caspase-dependent apoptosis in malignant MB231 breast cancer cells; additionally, deoxypodophyllotoxin also showed the potential to inhibit cell-survival pathways mediated by MAPK/ERK and NFκB-signaling pathways [144].

Neuronal Effects and Anticataleptic Activity
J. communis parts also show great potential to working memory, and inhibit the activity of some enzymes associated with the progression of neurological pathologies, such as Alzheimer's and Parkinson's disease [19,61,85].

In Vitro Studies
Focusing on in vitro assays, acetylcholinesterase inhibitory percentages ranging from 5.47% (leaf hydroethanolic extracts at concentration of 100 µg/mL) to 32.89% (berries aqueous extracts at 200 µg/mL) were reported. Additionally, and regarding the inhibition of butyrylcholinesterase, scores ranging between 25.33% and 62.01% for leaf aqueous extracts at concentrations of 50 and 200 µg/mL were reported, and from 25.87% to 49.95% regarding aqueous extracts of its ripe berries at the same concentrations mentioned above [61]. Furthermore, ethyl acetate and ethanolic extracts of its shoots revealed the ability to inhibit both enzymes at a concentration of 100 µg/mL (inhibitory percentages of 20.02 and 21.34% for ethyl acetate extract regarding acetylcholinesterase and butyrylcholinesterase inhibition, respectively, and 22.29 and 45.45% for acetylcholinesterase and butyrylcholinesterase inhibition for the ethanolic extract, respectively) [107].

In Vivo Studies
Regarding in vivo assays, Bais et al. [19] reported that the daily administration of 200 mg/kg (i.p.) of J. communis methanolic extracts for 21 days in rats with induced Parkinson's disease by chlorpromazine can effectively decrease motor dysfunctions, including catalepsy and muscle rigidity, and increase locomotor activity when compared to the untreated group. The obtained results are in line with previous data, which showed that the daily injection of similar extract (200 mg/kg, i.p.) can significantly reduce the retention on bar (catalepsy activity) by 75% in rats with induced catalepsy by reserpine [34]. In addition to the mentioned, the daily inhalation of 1% and 3% for 60 min during 21 days of juniper volatile oils extracted from J. communis, mainly composed of α-pinene, sabinene, myrcene, limonene, terpinen-4-ol, and α-thujene, by rats with induced Alzheimer's disease, showed increases in working and long-term memories and decreases in acetylcholinesterase activity [85,136].

Hepatoprotective Effects In Vivo Studies
Ethyl acetate fractions of leaves from J. communis have already been shown to be promising hepatoprotective agents. Rats with hepatic damage caused by paracetamol who ingested these fractions (200 mg/kg body weight) over two weeks showed lower levels of alkaline phosphatase (−57.41%), direct bilirubin (−30.33%) and total bilirubin (−38.41%), serum alanine aminotransferase (−34.17%), and serum aspartate aminotransferase (−27.58%) than the untreated group. Histopathological observations also proved the hepatoprotective effects of these leaves, promoting favorable portal triads and central-vein rearrangements [65]. Using a carbon tetrachloride-induced hepatic damage model, Mavin and Garg [30] revealed similar effects of J. communis stems. Furthermore, Singh et al. [145] reported that the daily ingestion of a combination of ethanolic berry extract of Solanum xanthocarpum (200 mg/kg) and J. communis (200 mg/kg) for 14 days can significantly attenuate liver toxicity induced by paracetamol and azithromycin in Wistar albino rats. In fact, the administration of both showed a capability to reduce altered biochemical parameters, including serum glutamate oxaloacetate transaminase (−65.4%), serum glutamate pyruvate transaminase (−59.3%), alkaline phosphatase (66.8%), and total bilirubin (62.1%), and reverse histopathological alterations, by promoting the liver tissue's normal architecture and diminishing liver inflammation.

Tyrosinase Inhibitory Activity
In Vitro Studies Methanolic extracts of berries from J. communis already showed the capacity to suppress mushroom tyrosine activity by about 50% at concentrations of 100 µg/mL. This data is very promising and can be considered an indicator regarding the potential of this plant to treat skin disorders, since this enzyme is closely involved in the production of melanin. Moreover, some compounds isolated from them also showed similar potential, namely hypolaetin 7-O-β-xylopyranoside, which exhibited an IC 50 value of 45.15 µM, and kojic acid (IC 50 score of 25.03 µM) [137].
4.9. Renal and Antiurolithiasis Effects 4.9.1. In Vitro Studies Relative to antiurolithiasis properties, J. communis berries at concentrations of 500, 1000, and 2000 µg/mL solutions showed potential to dissolve urinary stones brought out from the human kidney, causing reductions of 50, 20, 10, and 20% in urinary stones composed of calcium oxalate, calcium hydrogen phosphate, magnesium ammonium phosphate, and ammonium urate, respectively. The dry-powder weight of stones in normal saline also decreased from 1458 to 1162, 1124, 1136, 1144, 1096, 1126, and 1130 mg after exposure to increasing Juniperus berry concentrations. Furthermore, it was also observed that the ratio of calcium oxalate in normal saline aqueous solution plus stone increased from 70% to 80% after using some fractions of J. communis berry extracts [138].

In Vivo Studies
Different parts of J. communis plants have been largely used since ancient times to treat renal disorders because of their diuretic and urinary antiseptic effects. Indeed, Stanic et al. [86] reported that the daily intake of 10% aqueous infusion, 0.1% of oil (with 0.2% Tween 20 solubilizer) from juniper berries, and 0.01% of terpinen-4-ol (one of the main components of Juniperus plants) in rats at 5 mL/100 g can effectively stimulate diuresis from day 2, increasing urine excretion without loss of electrolytes. Between them, the infusion showed the most prominent diuretic activity (+43% on day two and 44% on day three), which proves that the diuretic activity of juniper berries is due to the combination of essential oil and hydrophilic components, which together can increase the glomerular filtration rate. Even so, recent studies use do not recommend their continuous use due to the presence of terpinen-4-ol, which has already been shown to promote kidney irritation [146].

Gastrointestinal Effects In Vivo Studies
Pramanik and colleagues [87] reported that J. communis leaves can be useful in ameliorating some gastrointestinal ailments. The authors verified that the intraperitoneal administration of the methanolic extract at doses of 50 and 100 mg/kg can effectively inhibit aspirin, serotonin, indomethacin, alcohol, and stress-induced gastric ulcerations in rats, and histamine-induced duodenal lesions in guinea pigs. The treatment with the leaf extract also enhanced the healing rate of acetic acid-induced ulcers in rats. Additionally, the analysis of gastric juice revealed that although the leaf extract did not alter its pH or its peptic activity, this one managed to significantly diminish its volume and total acidity. These benefits shown by J. communis parts are positively linked to their anti-inflammatory and analgesic properties.

In Vivo Studies
The capacity of J. communis aerosols to reverse the vasomotor impairment associated with passive exposure to cigarette smoke was also evaluated in female Sprague Dawley rats. Animals were first exposed to daily passive smoking for 6 weeks. In the last 15 days of the study, one of the groups was also subject to a daily administration of J. communis oil aerosols for 40 min/day. In the end, thoracic aortas were harvested and analyzed, and it was possible to verify that the use of aerosols can significantly reduce acetylcholine endothelialdependent relaxation [139]. Furthermore, Pleşa and colleagues [140] reported that the nebulization with J. communis berry oil (20 min/day per 3 weeks) exerts bronchodilator effects mediated by nitric oxide in the respiratory tract of rats exposed to 2 cigarettes per day, 5 days a week for 6 weeks. This activity is closely linked to the antioxidant effects shown by this plant. Even so, the authors also verified that this aerosol exposure can cause moderate irritation and inflammation along the tracheobronchial tract in nonsmoker rats.

Genotoxicity Protective Effects In Vitro Studies
Recently, J. communis berries displayed capacity to inhibit chromosome aberrations and mitotic abnormalities induced by ethanol on Allium cepa L. root-tip cells, with these properties being intimately linked to their capacity to scavenge radicals and reduce oxidative stress levels [40].

In Vitro Acute Toxicity
Fernandez and colleagues [131] assessed the toxicity of methanolic and aqueous berry extracts of J. communis through Artemia franciscana nauplii lethality assay, and proved their safety once the obtained IC 50 values were higher than 1 mg/mL. Additionally, the toxicity and undesirable side effects of J. communis were evaluated in albino rats based on the oral administration of ethyl acetate fractions of their leaves for 2 weeks. The obtained data revealed no mortality nor any negative change in physiological parameters and appearance until the dose of 2 g/kg [65].

Antiprogestogenic and Abortifacient In Vivo Effects
Pathak and colleagues [147] reported that hydroethanolic extracts (90% ethanol, v/v) of J. communis berries did not show estrogenic nor antiestrogenic effects, but displayed antiprogestational and antifertility activity at doses ranging from 50-450 mg/kg on female rats. In another study, Agrawal et al. [148] found that the oral administration of hydroethanolic extracts (50% ethanol, v/v) from J. communis berries at doses of 300 and 500 mg/kg in albino female rats from day 1 to day 7 of pregnancy exhibited dose-dependent anti-implantation activity. Furthermore, the authors also reported that these extracts at the same concentrations promoted abortifacient effects when administrated on days 14, 15, and 16 of pregnancy. Still, no evidence of teratogenicity effects was found.

Conclusions
The pharmacological effects of the zimbro plant have been known since ancient times, and are mainly attributed to the high concentration of phenolic compounds, in particular the presence of 5-O-caffeoylquinic and quinic acids, catechin, epicatechin, amentoflavone, quercetin, luteolin, apigenin, and naringenin; and VOCs, namely monoterpenes and sesquiterpenoids. In fact, these phytochemicals confer remarkable biological activities, such as important antimicrobial capacity, the ability to modulate biofilm formation, as well as notable antioxidant, hepatoprotective, anticancer, anti-inflammatory, antihypercholesterolemic, neuroprotective, and genotoxic effects. Given this, it is not surprising that its use and incorporation in dietary supplements, nutraceuticals, and pharmaceutical drugs is a hot topic among researchers, considering its potential to attenuate-or even treat-several