Beyond Essential Oils: Diterpenes, Lignans, and Biflavonoids from Juniperus communis L. as a Source of Multi-Target Lead Compounds

Abstract

Common Juniper (Juniperus communis L.) is a gymnosperm that stands out through its fleshy, spherical female cones, often termed simply “berries”. The cone berries and various vegetative parts (leaves, twigs and even roots) are used in traditional phytotherapy, based on the beneficial effects exerted by a variety of secondary metabolites. While the volatile compounds of Juniperus communis are known for their aromatic properties and have been well-researched for their antimicrobial effects, this review shifts focus to non-volatile secondary metabolites—specifically diterpenes, lignans, and biflavonoids. These compounds are of significant biomedical interest due to their notable pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, and anticancer effects. The aim of this review is to offer an up-to-date account of chemical composition of Juniperus communis and related species, with a primary emphasis on the bioactivities of diterpenes, lignans, and biflavonoids. By examining recent preclinical and clinical data, this work assesses the therapeutic potential of these metabolites and their mechanisms of action, underscoring their value in developing new therapeutic options. Additionally, this review addresses the pharmacological efficacy and possible therapeutic applications of Juniperus communis in treating various human diseases, thus supporting its potential role in evidence-based phytotherapy.

1. Introduction

Since ancient times plants have been used as remedies against human ailments according to documents, monuments, and even preserved original plant products [1]; starting with simple pharmaceutical formulations such as infusions and macerations, the use of medicinal plants changed dramatically once active phytochemicals were identified and extracted in the early 19th century [2]. Although the drying process of plants may induce changes in their phytochemical composition [3], studies showed that the use of total extracts usually trigger more complex and longer-lasting biologic activity compared to pure compounds [4].

Currently, most pharmacopoeias contain monographs describing drugs of plant origin with real therapeutic values; herbal pharmacopoeias have also been published [5]. Natural products provide around 50% of modern drugs [6]; in addition, the market of herbal medicines has developed explosively based on traditional medicine, making up an estimated of 425 million USD in 2022 [7]; the use of plant-based products often involves self-medication as such or in combination with synthetic drugs leading to beneficial outcomes but also potentially harmful side effects or pharmacological interactions.

In order to achieve an effective therapy, the identification of plant components in terms of pharmacological effects is just as important as the proper diagnosis of the patient [8]; therefore, rational (evidence-based) phytotherapy has developed consisting in the use of those products whose therapeutic use is based on scientific research that identified active ingredients and effective dosage in both preclinical and clinical settings.

Juniperus genus comprises plants that can be used as source for the cedarwood oil that is used in folk medicine as antimicrobial and antibiofilm agent [9]; the plants have been reported with antimicrobial [10], cytotoxic [11], diuretic [12], anti-inflammatory [13], analgesic [14], hepatoprotective [15], antidiabetic [16] and hypolipidemic [17] activity. The genus Juniperus comprises around 75 species widely distributed mainly in cold and temperate regions but also reaching tropical areas [12]; in Romania, Juniper-based products (infusion, tincture, decoct) exploit the fruits, bark and aerial parts as well as the whole plant in both internal and external applications.

The current review aims to describe the chemical composition of J. communis L., together with the pharmacological effects and therapeutic benefits of extracts and selected pure compounds pertaining to the diterpene, lignan and biflavonoid subclasses. Both preclinical and clinical data are presented, supporting their use in human therapies. For some of the presented compounds (totarol, ferruginol), a large body of experimental evidence accumulated that deserve to be summarized. For other compounds (imbricatolic acid, pimaric acid, sandaracopimaric acid), promising data on the bioactivity are emerging. On the other hand, there has been progress characterizing the bioactivity of Juniper extracts. Studying the properties that individual metabolites contained in these extracts have, enable us a to better understand the effects of Juniper extracts, but also to guide further, more meaningful research on this plant.

2. Botanical Aspects

Juniperus communis, also known as common juniper, is a small evergreen shrub or a small coniferous evergreen tree that belongs to the Cupressaceae family [11,18,19,20,21,22]. It is native to the northern hemisphere, and can be found in various habitats, such as forests, heathlands, and mountains [19,22,23]. Geographical distribution and morphological differences determine the classification of the species into several subspecies and varieties [19]. The Juniper plant grows to a height of 1–3 m and has a dense, conical crown. The bark is reddish-brown with a rough, scaly texture and tends to peel off in thin strips Its leaves are needle-like, sharp at the tip, grouped in whorls of three, with a vibrant green color [12,21].

The female reproductive organs are spherical, consisting of three layered carpel scales that contain eggs. The male reproductive organs are oval, yellow, and feature numerous stamens [21. In Juniperus communis, male and female cones typically grow on separate plants. The female cones are larger and require about 18 months to reach maturity. [18,21]. Juniperus communis cones are a unique feature and frequently employed as a flavoring agent in cooking and gin production. The pseudo-fruits are globose and short-stemmed [12,19].

For centuries, it has been popular to use the essential oil derived from the berries and needles of the Juniper plant due to its aromatic, and therapeutic properties [24,25]. The unique composition, which incorporates a variety of terpenes, gives it a refreshing, woody, and slightly fruity fragrance, which makes it a popular choice for aromatherapy, and perfumery [12,18].

3. General Chemical Composition of the Juniper Plant

The common Juniper plant is known to possess an extensive array of biologically active compounds, including: terpenes (volatile monoterpenes and non-volatile diterpenic derivatives), lignans, flavonoids, organic acids (glycolic, malic, and ascorbic), proteins, fermentable sugars, wax, and other compounds (Figure 1) [23,26,27]. The volatile compounds that make up the Juniper essential oil have received extensive attention [28,29,30,31]. They include monoterpene hydrocarbons (α-pinene, β-pinene, myrcene, sabinene, limonene), oxygenated monoterpene derivatives (terpinen-4-ol, borneol,), and sesquiterpenes (germacrene, β-caryophyllene) [28]. The content and composition of volatiles in Juniper cone berries varies widely from below 0.5% to over 3.5% [32]. In contrast, the current review focuses on the non-volatile secondary metabolites. This article aims to complement and update other excellent reviews on the topic of Juniper. Seca and Silva [33] made a comprehensive inventory of compounds published between 1970 and 2004 from various Juniperus species, continued by a later second review [34]. Gonçalves et al. [35] described the bioactivity of Juniper extracts, volatile organic compounds, phenolic acids, coumarins, anthocyanins, flavan-3-ols, proanthocyanidins, flavonols, flavones, carotenoids, and chlorophylls. Tavares and Seca [36] focused on a selection of diterpenes, flavonoids and one lignan. Other reviews [23,37] classified data according to the type of biologic activity exerted by various natural products from Juniperus communis.

The antimicrobial [10,38], antifungal [39,40,41], antioxidant [15], and anti-inflammatory [42,43] potentials of these compounds have been highlighted in several scientific articles up until now, along with their protective effects on various organs (liver, kidney) and against cancer [12,15,35,44]. Juniper plant extracts, essential oils, biologically active fractions, and individual compounds are viable options for the development of new molecules aimed at tackling acute and chronic human diseases. Our next section will consist of a short presentation on non-volatile bioactive compounds found in the Juniper plant.

3.1. Diterpenes

Diterpenes are a variety of 20-carbon compounds that can be formed by condensing four isoprene units. Plants, animals, and fungi synthesize diterpenes through the 3-hydroxy-3-methyl-glutaryl-coenzymeA (HMG-CoA) reductase pathway, which involves the primary intermediate geranylgeranyl pyrophosphate [45,46,47]. The classification of diterpenes is determined by their core structures, and they can be classified as: (i) linear (phytane); (ii) monocyclic (retinol); (iii) bicyclic (clerodane, halimane, labdane); (iv) tricyclic (abietane, rosane, pimarane, podocarpane, cassane, vouacapane, chinane); (v) tetracyclic (kaurene, gibberellane, trachylobane, scopadulane, aphidicolane, atisane, stemodane, beyerene, stemarane); (vi) macrocyclic (cembrane, jatrophane, taxane, ingenane, daphnane, tigliane) [48,49,50]. Prominent diterpenes from Juniper pertaining to the above-mentioned classes are depicted in Figure 2.

Natural diterpenes and diterpenoids have been shown to have impressive biological activities, which could lead to their development in the pharmaceutical industry. In the next section, we will describe the most significant diterpenes found in juniper.

3.1.1. Totarol

Totarol, also known as b,8,8-trimethyl-1-propan-2-yl-5,6,7,8a,9,10-hexahydrophenanthren-2-ol, is found in a variety of plants, such as Podocarpus and Juniperus spp., which are known to have bioactive properties [51,52]. It has been noted in J. communis roots [53].

Totarol has been shown to exhibit antibacterial activity against a wide range of bacteria, including both Gram-positive and Gram-negative bacteria. It disrupts bacterial cell membranes and inhibits bacterial growth, making it effective against various types of bacteria, including those responsible for common infections [54]

Harkenthal and co-workers investigated the antibacterial activity of totarol against a range of bacteria, including Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The researchers found that totarol was highly effective against these bacteria, with minimum inhibitory concentrations (MICs) ranging from 1–8 μg/mL [55]

Another study has concentrated on molecular targets and the mechanism of action of totarol in Bacillus subtilis. Their quantitative analysis of proteomes showed that diterpene causes changes in 139 protein expression levels. The same study also reports that the main central metabolic dehydrogenases of Bacillus subtilis are suppressed by totarol at the IC₅₀ = 1.5 µM, leading to metabolic arrest in bacteria. [56]

Study conducted by Gordien and co-workers showed that totarol has the best activity against Mycobacterium tuberculosis genotype H37 Rv (MIC of 73.7 µM) and it was also most active against the isoniazid, streptomycin and moxifloxacin-resistant variants (MIC of 38.4, 83.4 and 60 µM) [53].

Several studies have demonstrated the antifungal activity of totarol. For instance, a study by Han and co-workers evaluated the antifungal activity of totarol against various clinical isolates of Candida albicans and found that it exhibited potent activity, with MIC values ranging from 4 to 32 µg/mL [57].

Totarol has demonstrated neuroprotective effects in both in vitro and in vivo models by safeguarding neurons from glutamate-induced cell death and oxygen-glucose deprivation. This protection is attributed to activation of the Akt/GSK-3β pathway, which boosts phosphorylation of Akt (protein kinase B) and GSK-3β (glycogen synthase kinase 3β), and increases Nrf2 (nuclear factor erythroid 2-related factor 2) and HO-1 (Heme oxygenase 1) protein expression, reducing oxidative stress. Totarol also enhances antioxidant defences by raising levels of glutathione (GSH) and superoxide dismutase (SOD), key in protecting cells from oxidative damage [58].

3.1.2. Ferruginol

Abietanes, also known as abietane diterpenes, display a specific structure containing three fused six-membered rings and alkyl functional groups at C₄, C₁₀, and C₁₃. The most relevant examples of abietanes are ferruginol and sugiol [59].

Ferruginol, or (4bS,8aS)-4b,8,8-trimethyl-2-propan-2-yl-5,6,7,8a,9,10-hexahydrophenanthren-3-ol, is a natural diterpene discovered in various parts of 31 species, with the majority of them belonging to the Cupressaceae, Lamiaceae, Podocarpaceae, and other minor families (e.g., Taxaceae, Meliaceae, Martyniaceae, Pedaliaceae, Lauraceae). The bark and root of species are the primary sources of ferruginol [59,60]. The compound has first been identified in J. communis in 1995 [61].

In the study by Thamaraiselvan and co-workers, ferruginol showed anti-inflammatory activity in MCF-7 breast cancer cells, effectively reducing inflammatory markers like TNF-α and IL-6. The IC50 for ferruginol’s anti-inflammatory effect in this model was determined to be 12 µM, highlighting its potential in modulating inflammation in cancer-related settings [62].

Ferruginol demonstrated a notable gastroprotective effect, reducing gastric lesions by 60% at 25 mg/kg, similar to lansoprazole. It also showed significant ulcer healing at 50 mg/kg with a 92.5% curative rate. Additionally, ferruginol inhibited lipid peroxidation with an IC50 of 1.4 µM and promoted cell proliferation, supporting its potential as an anti-ulcer agent [63].

Another study noted ferruginol for its antimicrobial properties, showing significant activity against various pathogens. It achieved an IC50 of 7.4 μg/mL against Staphylococcus aureus and 13.5 μg/mL against Pseudomonas aeruginosa, indicating its potential as a candidate for new antimicrobial development [64].

In the study by Takei and co-workers ferruginol was found to promote the differentiation of dendritic cells from human monocytes and enhance the generation of interleukin-10 (IL-10)-producing regulatory T cells in vitro. While the focus is on its immunological effects, the findings suggest potential neuroprotective activity related to immune regulation in the nervous system [65].

3.1.3. Sugiol

Sugiol, or (4aS,10aS)-6-hydroxy-1,1,4a-trimethyl-7-propan-2-yl-3,4,10,10a-tetrahydro-2H-phenanthren-9-one, has been reported in 26 species, and it is particularly concentrated in Cupressaceae and Lamiaceae families, as well as minor family members such as Taxaceae, Cladoniaceae, and Podocarpaceae [59,66]. It has been isolated from several Juniper species, including J. communis [33].

Sugiol, exhibits significant antioxidant activity, as evidenced by various studies. Specifically, it has been shown to have an IC₅₀ value of 36.32 µM when evaluated using the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay. This indicates that sugiol effectively neutralizes free radicals, thereby contributing to its overall antioxidant capabilities [67].

Sugiol demonstrates significant antiproliferative activity against various cancer cell lines, particularly MCF-7, HeLa, and HCT116, with an IC50 value of about 22.45 µM. This suggests its potential as a therapeutic agent for inhibiting cancer cell growth [59].

Also, sugiol demonstrates strong antioxidant properties with an IC₅₀ value of 36.32 µM, indicating its effectiveness in scavenging free radicals [66].

3.1.4. Pimaric Acid, Sandaracopimaric Acid and Isopimaric Acid

Pimaric acid, or (1R,4aR,4bS,7S,10aR)-7-ethenyl-1,4a,7-trimethyl-3,4,4b,5,6,9,10,10a-octahydro-2H-phenanthrene-1-carboxylic acid, which is similar to abietane, is a type of diterpene resin and plays a significant role in pine oleoresin [68].

Pimaric acid shows strong antibacterial activity against Paenibacillus larvae, a bacterium harmful to honeybee colonies. The MIC of 6.25 µg/mL and zone of inhibition of 10–14 mm in agar diffusion assays causes disruption of the bacterial cell membrane, causing cell leakage, which contributes to its antibacterial effects. [69]

The study by Ishida and co-workers found that pimaric acid reduces vasoconstriction in rat pulmonary arterial smooth muscle by activating BKCa channels (large conductance calcium-activated potassium channels) and inhibiting VDCCs (voltage-gated calcium channels). This action increases potassium currents and lowers calcium influx, leading to muscle relaxation and reduced vasoconstriction. Pimaric acid specifically countered high potassium and endothelin-1-induced vasoconstriction, highlighting its potential as a vasorelaxant for vascular tension-related conditions [70].

Pimaric acid was shown to inhibit matrix metalloproteinase 9 (MMP-9) production in TNF-α-stimulated human aortic smooth muscle cells. This suppression of MMP-9 reduced cell migration, a key factor in inflammation and vascular remodelling. The effect was achieved by downregulating the NF-κB and AP-1 pathways, both critical for regulating inflammation and cell migration genes [71].

Sandaracopimaric acid, or (1R,4aR,4bS,7R,10aR)-7-ethenyl-1,4a,7-trimethyl-3,4,4b,5,6,9,10,10a-octahydro-2H-phenanthrene-1-carboxylic acid, is a tricyclic diterpene resin acid found in the resin of Callitris species [72].

Sandaracopimaric acid was shown to significantly reduce the contraction of pulmonary arteries induced by phenylephrine, indicating its vasodilatory effects. This vasodilation may be mediated by the endothelial nitric oxide synthase (eNOS) path-way and involves increased nitric oxide production in endothelial cells, with the potential role of the PI3K/Akt signalling pathway [73].

Isopimaric acid, or pyrrole-3-carboxaldehyde, is a diterpenoid present in many organisms naturally, such as Boesenbergia rotunda and Picea obovata [72].

These compounds have been pointed out in numerous Juniper species, including J. communis [33].

Isopimaric acid demonstrated significant anticancer activity against breast cancer cell lines such as MDA-MB-231 and MCF-7. The study reported an IC50 value of 6.81 μM, indicating its potency in inhibiting cell proliferation [74]. The antibacterial properties of isopimaric acid extracted from Pinus nigra, highlight its effectiveness against multidrug-resistant and methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentrations (MIC) were found to be between 32 and 64 µg/mL. Interestingly, isopimaric acid did not enhance the activity of antibiotics or show reduced MIC when combined with the efflux pump inhibitor reserpine; in some cases, MIC even increased [75].

3.1.5. Imbricatolic Acid

Imbricatolic acid, or (1S,4aR,5S,8aR)-5-[(3S)-5-hydroxy-3-methylpentyl]-1,4a-dimethyl-6-methylidene-3,4,5,7,8,8a-hexahydro-2H-naphthalene-1-carboxylic acid, is a diterpenoid found in the resin extracted from the large tree Araucaria araucana (Mol.) Koch. It was obtained from the mature cone berries during research of compounds from Juniper that control the progression of the cell cycle [76]. Imbricatolic acid, have shown significant gastroprotective effects in mice, reducing gastric lesions by up to 78% at doses of 100 mg/kg—comparable to the proton pump inhibitor lansoprazole. Additionally, these compounds demonstrated cytotoxic activity, with IC₅₀ values of 52 μM for AGS cells and 72 μM for fibroblasts, surpassing lansoprazole’s efficacy in similar assays [77]. Imbricatolic acid, isolated from Juniperus communis, has been examined for its effects on cell cycle progression in human anaplastic lung cancer CaLu-6 cells, which lack p53. It was found to induce G1 cell cycle arrest by promoting the accumulation of cyclin-dependent kinase inhibitors while degrading cyclins A, D1, and E1. This mechanism effectively halted the progression of the cell cycle, although it did not trigger significant apoptosis in the treated cells [76,78]. Imbricatolic acid has shown cytotoxic effects when subjected to biotransformation by Aspergillus niger and Rhizopus nigricans. Metabolites like 1α-hydroxyimbricatolic acid demonstrated moderate cytotoxicity, with IC₅₀ values of 307μM against AGS human gastric cells and 631 μM against fibroblast cells. In comparison, imbricatolic acid itself exhibited stronger cytotoxicity, with IC₅₀ values of 134 μM for AGS cells and 280 μM for fibroblasts, suggesting enhanced activity in its native form [79].

3.1.6. Agathadiol, Agathic Acid, Dihydroagathic Acid

Labdane-related diterpenes, also known as secondary metabolites, are abundantly present in fungi, insects, higher plants, and marine organisms, and they exhibit a wide range of biological activities due to their high structural diversity. A fused decalin system (C_1-10), with a six-carbon side chain at C₉, composes the basic core structure of labdane diterpenes, and there are generally four carbons added to the decalin system at C₄, C₈, and C₁₀ [80,81]. In labdane diterpenes class, the most representative bioactive molecules are agathic acid, agathadiol and dihydroagathic acid. Agathadiol was isolated from common Juniper by Feliciano et al. [61], while dihydroagathic acid was pointed out in leaves by Basas-Jaumandreu and co-workers [82].

The study by Salamone and co-workers identified that agathadiol extracted from juniper berries, functions as a positive allosteric modulator of the CB1 cannabinoid receptor. This interaction enhances CB1 receptor activity, suggesting agathadiol’s potential use in therapeutic areas like neuroprotection and pain management [83].

3.1.7. Isocupressic Acid

Isocupressic acid, also chemically known as (1S,4aR,5S,8aR)-5-[(E)-5-hydroxy-3-methylpent-3-enyl]-1,4a-dimethyl-6-methylidene-3,4,5,7,8,8a-hexahydro-2H-naphthalene-1-carboxylic acid, is a bioactive compound that is used for abortion, and can be found in the needles of Ponderosa pine (Pinus ponderosa L.) [84,85]. In J. communis, isocupressic acid was proved in leaves [82].

In the study by Wu and co-workers isocupressic acid was shown to significantly inhibit progesterone production in bovine luteal cells by blocking luteinizing hormone (LH) activity, which is essential for progesterone synthesis [84].

3.1.8. Cryptojaponol

Cryptojaponol, or (4aS,10aS)-5-hydroxy-6-methoxy-1,1,4a-trimethyl-7-propan-2-yl-3,4,10,10a-tetrahydro-2H-phenanthren-9-one, is a natural product found in Juniperus formosana and Cryptomeria japonica [86]. In the current plant, it is as well contained [87].

Cryptojaponol possesses cytotoxic properties, particularly against cancer cell lines, where it demonstrates moderate activity. For instance, in studies focusing on pancreatic cancer cells (PANC-1), cryptojaponol was reported to have an EC50 value of approximately 37.9 µM, indicating its potential as a chemotherapeutic agent [88]. Cryptojaponol has demonstrated activity against various bacterial strains, including Staphylococcus aureus and Escherichia coli. The compound was found to inhibit the growth of these bacteria at concentrations ranging from 50 µg/mL to 100 µg/mL, showing a potential for use in combating bacterial infections [89].

3.1.9. Communic Acids

In Cupresaceae species, particularly in genus Juniperus, communic acids are a diterpenic natural product group with a labdane skeleton that contains three double bonds and a carboxyl group at C₁₉ [90,91]. The most representative communic acids in common Juniper are trans-communic acid and cis-communic acid [33].

Communic acids, have shown strong antimicrobial activity against pathogens like Staphylococcus aureus, S. epidermidis, Aspergillus fumigatus, and Candida albicans. The reported LD50 in a brine shrimp bioassay is 0.16 μg/mL, indicating significant cytotoxic effects [90].

3.2. Lignans

Lignans are phenolic dimers with a 2,3-dibenzylbutane structure. It is known that these compounds are minor constituents in multiple plants, and act as building blocks for creating lignin in plant cell walls [92,93,94]. Most of the compounds are present in glycosidic form. Lignans can be divided into eight subgroups based on their cyclization pattern and oxygen incorporation into the skeleton, and each group includes: (i) furofuran; (ii) furan; (iii) dibenzylbutane; (iv) dibenzylbutyrolactone; (v) aryltetralin; (vi) arylnaphthalene; (vii) dibenzocyclooctadiene; and viii) dibenzobutyrolactol (Figure 3) [94,95]. Compound structures in every subgroup have significant differences in terms of the oxidation levels of both aromatic rings and propyl side chains [94].

3.2.1. Deoxypodophyllotoxin and Podophyllotoxin

Among the lignans, cyclolignans present a carbohydrate cycle between the phenylpropane units, created by two single carbon-carbon bonds through the side chains [96,97]. Podophyllotoxin, which has a five-ring structure, is part of a class of compounds that includes several closely related chemical structures, such as podophyllotoxin, deoxypodophyllotoxin, 4′-demethylpodophyllotoxin, and α- and β-peltatins [96,98,99,100].

In Juniperus communis, the aryltetralin derivative deoxypodophyllotoxin is the main lignan [101]. Its content in leaves varies between 78 mg/100 g dry weight in ‘Horstman’ and 37.1 mg/100 g dry weight in ‘Gold cave’ varieties. Conversely, podophyllotoxin contents only span between 12.5 (‘Horstman’) and 2.2 mg/100 g dry weight (‘Depressa Aurea’). These compounds are of particular interest in antineoplastic therapies. The anticancer effects and mechanisms by which deoxypodophyllotoxin exerts them have recently been reviewed and based on research of numerous cell lines, including prostate cancer, cervical carcinoma, breast cancer, colorectal cancer, glioblastoma, oesophageal carcinoma [102]. Both mitochondrial and non-mitochondrial pathways are involved in the pro-apoptotic activity. Pharmacokinetic investigations performed in tumour-bearing mice showed that the compound had higher affinity for the cancer tissues than for plasma, and achieving particularly high concentrations in reproductive organs, fat and lungs [103]. Deoxypodophyllotoxin has as well other properties of medicinal interest including antibacterial, anti-inflammatory and antifertility [104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164] (

Table 1. The main bioactive compounds from juniper plant and their biological activities.

Class	Chemical Compound	Biological Activity
Diterpenes		▪ Significant antibacterial and antifungal activity [90]
		▪ Anti-mycobacterial activity; Inhibits transcription of P450scc, translation of StAR and P450scc through attenuating cAMP-PKA signaling [84]
		▪ Induces cell cycle arrest in CaLu-6 cells, by two possible mechanisms: (i) the accumulation of p21Cip1/Waf1/Sdi1, mediated by PKCδ activation and ERKs phosphorylation; and (ii) the decrease of cyclins A, D1, and E1 levels [76,78]
		▪ Novel chemotype of positive allosteric modulator of CB1R; ▪ Significant cytotoxicity on NUGC and HONE-1 cells [83]
		▪ Shows the potential for anti-inflammatory effects without exhibiting any cytotoxicity side effects [149] ▪ Exhibits anti-microbial activity against Mycobacterium tuberculosis H37 Rv (MIC = 73.7 M); also induced alterations in Bacillus subtilis proteome [56,150]; successfully inhibit Staphylococcus aureus, MICs for S. aureus strains were in the range of 2–4 lg/mL [54] ▪ Shows significant antibacterial effects against a wide range of bacteria towards oral bacteria and inhibition effects towards the development of the oral biofilm [151] ▪ Neuroprotective effects in primary neuronal cultures and in a brain ischemia rodent model, by activating the Akt/HO-1 pathway, thus contributing to a cellular anti-oxidative defense against neuronal injury [58]
		▪ Antiproliferative and apoptotic potential against human gastric cancer cells (SNU-5) [59] ▪ Induces activation of inflammatory signals via TL4-Myd88-MAPK kinase pathway [66] ▪ Active against promastigotes and amastigotes of L. infantum [152,153,154] ▪ Antioxidant, lipid peroxidation inhibitor [155]
		▪ Shows anticancer effects in MCF-7 cells [62] ▪ Gastroprotective effect by increasing the PGs content, protecting the cells against lipid peroxidation [63] ▪ Antimicrobial activity against Staphylococcus aureus including MRSA strains [64] ▪ Neuroprotective effect against MPTP-induced apoptosis and motor dysfunction [65]
		▪ Shows moderate cytotoxicity against PANC-1 cells under nutrient-starved conditions [155]
		▪ Antibacterial activity against Paenibacillus larvae [69] ▪ Activates large-conductance Ca2+-activated K+ (BKCa) channels and inhibit voltage-dependent Ca2+ channels (VDCCs) [70] ▪ Anti-atherosclerotic activity with inhibitory action on MMP-9 production and cell migration in TNF-alpha-induced HASMCs [71]
		▪ Anti-tumor mechanism affects EMT and Wnt signaling pathways by targeting mitochondria oxidative phosphorylation and Ca2+ signaling pathways, and inducing breast cancer cell cycle arrest and apoptosis [74] ▪ Activity against Multidrug-resistant and EMRSA Strains of Staphylococcus aureus [75]
Lignans		▪ Cytotoxic activity on a wide range of cancer cell lines; proapoptotic [102] ▪ Antibacterial activity; analgesic and anti-inflammation activities; antifertility effects [104]
		▪ Antiproliferative activity [107] ▪ Induces mitosis disturbance [108] ▪ Suppresses the accumulation of lipofuscin in keratinocytes [109]
		▪ Anticancer activity via inhibition of histone deacetylase 8 [110] ▪ Suppresses cell progression and migration, triggers apoptosis and mitochondrial dysfunction through MMP loss, and disturbed calcium regulation [111] ▪ Anti-inflammatory and antioxidant effects in sepsis-mediated brain injury by repressing the MAPK and NF-κB pathways through up-regulating AMPK [114]
		▪ Antidiabetic [116] ▪ Inhibits ROS generation in RAW 264.7 cells [117] ▪ Antibacterial activity [118] ▪ Combats drug resistance in bacteria [119] ▪ Anticancer [121,122]
		▪ Protective effects against cardiovascular diseases, diabetes, cancer, and mental stress; antioxidant activity [123] ▪ Reduction of weight gain [124]
		▪ Fungicide [39] ▪ Antibacterial activity [125] ▪ Anti-hyperglycemic activity [126] ▪ Weak cytotoxic effect [127] ▪ Hepatoprotective [129] ▪ Protective against cardiovascular diseases [132]
Biflavones		▪ Anti-inflammatory, anti-oxidation, antimicrobial activities [135] ▪ Antitumor activity; ▪ Antiviral activity; ▪ Anti-hypertrophic scar activity [156] ▪ Metabolism regulation, neuroprotection, musculoskeletal protection and antipsychotic effects [138,157]
		▪ Analgesic and anti-inflammatory effects [158] ▪ Antioxidant, antibacterial, antiviral activities [159]
		▪ Anti-inflammatory [160] ▪ Antiseptic, astringent, and antiproliferative activities [161]
		▪ Apoptotic effects and anticancer activity [144]
		▪ Antiviral, antiparasitic, cytotoxic, neuroprotective, and hepatoprotective activities [145] ▪ Antioxidant, anti-inflammatory [146] ▪ SARS-CoV-2 replication inhibitor [162]
		▪ Downregulates NO production in LPS-induced human colonic epithelial cells (HT-29) [147,163] ▪ Anti-angiogenic and pro-apoptotic effects [164]

).

Furthermore, in frontline cancer chemotherapy, two semisynthetic deoxypodophyllotoxin derivatives, etoposide and teniposide, are currently being used [105]. In addition to deoxypodophyllotoxin and podophyllotoxin, another derivative (β-peltatin-A-methylether) has been identified in the leaves of J. communis varieties ‘Laxa’ and ‘Oblonga pendula’ [106].

3.2.2. Yatein

Yatein is a member of the class of butan-4-olides, or benzodioxoles, which contains 3,4,5-trimethoxybenzyl and 1,3-benzodioxol-5-yl-methyl substituents at positions 3 and 4. It is an important key biosynthetic intermediate of the antitumor lignan podophyllotoxin. The compound has so far been the subject of only a small number of promising investigations, mostly related to anticancer effects. Earlier research showed that compound has a strong inhibitory activity on the proliferation of murine myeloma [107]. The compound destabilizes microtubules and induces cell cycle arrest at the passage G₂/M [108]. Recently, yatein was isolated during a bioactivity guided fractionation from J. communis cone berry water extract. It proved to be one of the active compounds that effectively suppressed the accumulation of lipofuscin in keratinocytes [109]. These findings should encourage further research on this lignan.

3.2.3. Matairesinol

This compound has been identified in the leaves of common Juniper (‘Laxa’ and ‘Oblonga pendula’ varieties [106]. It is a constituent of the cuticular waxes that cover the leaves where it is the second most abundant lignan after deoxypodophyllotoxin, present in amounts of 18 mg/kg dry weight [82]. The compound has been intensively studied. It retained attention mostly due to its potential to fight cancer. It is able to inhibit histone deacetylase 8, an enzyme involved in the proliferation, invasion and metastasis of cancer cells [110]. The compound has been tested on various cell lines (prostate, breast, cervical, and pancreatic cancer); at least one of the mechanisms involves induction of mitochondrial impairment [111]. It synergized with 5-fluorouracil to combat pancreatic cancer [111] and is able to restore chemosensitivity in colorectal cancer cells [112].

Moreover, matairesinol combats oxidative damage and has anti-inflammatory properties. These abilities are involved in its reduction of pathologic cardiac remodelling [113], the improvement of experimentally induces brain injury [114] and suppression of neuroinflammation [115].

3.2.4. Lariciresinol

This metabolite is, like matairesinol and yatein, a member of the dibenzylbutyrolactone lignan group. It is a minor compound found in the epicuticular wax of common Juniper leaves (2.9 mg/kg dry weight) [82]. It has been investigated in its capacity to combat diabetes via enhancement of insulin signalling and alpha-glucosidase inhibition [116], to counteract oxidative stress [117] and to fight pathogenic bacteria (Staphylococcus aureus, Escherichia coli) [118]. The compound was shown to act as an efflux pump inhibitor and combat drug resistant Salmonella typhimurium [119]. The anti-inflammatory properties of lariciresinol proved in a rat model of experimentally induced arthritis make the compound a promising candidate in the treatment of this condition [120]. The effect of this lignan against cancer showed inhibition of tumour growth and angiogenesis in rats bearing MCF-7 breast cancer xenografts [121] and apoptosis induction mediated through mitochondrial pathways in HepG2 cells [122].

3.2.5. Secoisolariciresinol

This lignan, a derivative of 9,9′-dihydroxybenzylbutane, is constituents of leaf cuticles, where it amounts to about 12.9 mg/kg dry leaves [82]. Until the present, the compound as such (aglycone form) has only marginally been studied in comparison to its diglucoside present mainly in flaxseed, considered a major dietary phytoestrogen [123]. Chronic administration of equimolar amounts of secoisolariciresinol and its diglucoside had a similar activity profile, reducing weight gain, and lipid accumulation at hepatic level [124].

3.2.6. Pinoresinol

Pinoresinol is a furanofurane type lignan, occurring in cuticular waxes of Juniper leaves (14.9 mg/kg dry weight) [82]. It displayed an intense fungicide effect against Candida albicans, damaging the plasma membrane of this pathogenic fungus [39]. With regard to an antibacterial effect, research showed the same site for a disruptive effect—the plasma membrane, leading to the leakage of soluble saccharides and proteins [125]. The compound has an anti-hyperglycaemic effect via inhibition of α-glucosidase [126]. Its anticancer activity in breast cancer cells was evaluated in HEK-293 and SkBr3 cell lines. The experimental setting included two other lignans, lariciresinol and podophyllotoxin. The values of half-maximal inhibitory concentrations, at 48h after application of pinoresinol, were 550 µM (HEK-293 cells) and 575 µM (SkBr3 cells), being in the same range as for lariciresinol (475 µM, and 500 µM, respectively, for the two cell lines) but showed a much lower activity than podophyllotoxin (IC₅₀ values of 0.075 µM and 0.175 µM, respectively) [127]. Most research concerning pinoresinol was however performed on its diglucoside, with implications on a cardiovascular level [128], and showing hepatoprotective [129], neuroprotective [130] and anti-osteoporosis [131] effects. Pinoresinol is considered to be an enterolignan precursor and as such offering protection against certain cancer types and cardiovascular diseases after nutritional intake [132].

3.3. Biflavonoids

Biflavonoids are composed of two monoflavonoid residues or flavonoids dimers. As summarized by He and co-workers, they occur naturally in angiosperms, bryophytes, and gymnosperms. Based on the presence or absence of an atom in the linker between the two residues, they can be classified into three groups (e.g., C-C linkages, C-linear fragments-C type, complex biflavonoids) (Figure 4) [133]. These compounds are present in many Juniper species, including common Juniper and contribute to some valuable bioactivities of these plants. Amentoflavone, hinokiflavone, cupressuflavone, and methyl-biflavones were isolated from J. communis cone berries in amounts ranging from 0.14 to 1.38 mg/g of fresh weight [134].

3.3.1. Amentoflavone

Amenoflavone is an active polyphenolic compound, C₃₀H₁₈O₁₀, classified as biflavonoid; while it is practically insoluble in water, it is easily soluble in alcohol due to its high hydrophobicity. In 1971, three plants from the Selaginella species and Ginkgo biloba were used as vegetal sources for its extraction for the first time. Since then, it has been discovered and extracted from more than 120 plants (e.g., Amanoa almerindae, Alchornea glandulosa, Yersonima intermedia, Calophyllum pinetorum, Calophyllum membranaceum, Biota semipervirens, Antidesma bunius, Selaginella bryopteris) [135]. In common Juniper it was discovered as early as 1975 [136], along with podocarpusflavone A and bilobetin. Its multifaceted therapeutic potential made it a subject of comprehensive reviews [137,138].

3.3.2. Hinokiflavone

Hinokiflavone is a hydroxybiflavone with an aromatic ether structure that has been substituted at position 6 with a 4-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-phenoxy group. It has been isolated from Rhus succedanea, Metasequoia glyptostroboides, Garcinia multiflora, and Podocarpus elongatus [139].

3.3.3. Cupressuflavone

Cupressuflavone is a biflavonoid with the structural formula 8-[5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxochromen-8-yl]-5,7-dihydroxy-2-(4-hydroxyphenyl)-chromen-4-one that can be obtained by the oxidative coupling of two molecules of apigenin resulting in a bond between the two C₈ positions of the chromene rings [140]. The isolation was achieved from Cupressus sempervirens and Juniperus occidentalis and isolated from several authors from J. communis [33].

3.3.4. Podocarpusflavone A

Podocarpusflavone A is considered a natural compound that comes from the Podocarpaceae family and is classified as a cyclic nucleotide phosphodiesterase inhibitor [141]. Recently, this compound was isolated from the ethyl acetate fraction of cone berry extracts showing tyrosinase inhibition [142].

3.3.5. Bilobetin

Bilobetin is a flavonoid oligomer acting as a potent inhibitor of the virus polymerase acidic endonuclease [143]. It effectively inhibits mutant PAN-I38T, and also displays anticancer activity against human hepatocellular carcinoma Huh7 and HepG2 cells [143,144].

3.3.6. Agathisflavone

Agathisflavone, 8-[5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxochromen-6-yl]-5,7-dihydroxy-2-(4-hydroxyphenyl)-chromen-4-one, is a dimer of flavonoid apigenin that has been gaining attention due to its varied biological activities [145,146].

3.3.7. Robustaflavone

By oxidating two molecules of apigenin one can obtain robustaflavone which contains both hydroxyphenyl and chromene rings; it has been reported to be isolated from Thuja orientalis, Rhus succedanea, Schrebera trichoclada, and Nandina domestica [147]. The compound was pointed out in common Juniper by [148].

In Table 1, we summarize some of the most important biological activities of the main bioactive compounds found in the juniper plant.

4. Recent Data on the Bioactivity of Juniperus communis Extracts

Many parts of Juniperus communis have been studied extensively following the extraction of their main ingredients; juniper’s medicinal and food preservation effects are attributed to its chemical constituents. Initially, research and marketing focused on using the plant for fragrance and flavouring, followed by its current medicinal uses based on traditional practices. The exploitation of its biologic effects, especially in the food, cosmetic, and health industries, is a result of recent research trends. As part of our review, we will analyse the biological activities of Juniper using recent data.

4.1. Antioxidant Activity

The antioxidant properties of Juniperus species are well documented and occur due to their rich phytochemical composition. Flavonoids, phenolic acids, and terpenes, which were described in previous paragraphs, are among the bioactive compounds found in such species that contribute to their antioxidant ability. Juniperus extracts have been tested for their antioxidant potential using different methods (e.g., DPPH radical-scavenging assays, Trolox equivalent antioxidant capacity assays, ferric reducing antioxidant power assays) to assess their effectiveness in scavenging free radicals, and preventing oxidative processes [165,166]. For example, in a study conducted by Höferl and co-workers it was shown that Juniper essential oil induces several mechanisms that facilitate radical scavenging, prevent radical formation (chelating capacity, inhibitory effect on xanthine oxidase) and protect against lipid peroxidation. The oil’s effects have also been proven in vivo where it blocked the oxidation processes in yeast cells and improved their adaptability to reactive oxidative species [27,167]. In the review on Juniperus species by Majid and co-workers, several studies on antioxidant activity were covered, noting that the high phenolic content in Juniperus extracts contributes to their antioxidant effects. For example, the ethanolic extract of Juniperus communis demonstrated strong antioxidant activity with an IC₅₀ of approximately 28.55 µg/mL in DPPH assays (2,2-diphenyl-1-picrylhydrazyl) [168].

The study by Belov and co-workers explores the antioxidant activities of extracts from Juniperus communis berries, which were obtained using various solvents and extraction methods. Key assays include the DPPH radical scavenging assay and the ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). For the ethanol extract, particularly the one derived through maceration, an IC₅₀ of approximately 18 µg/mL was reported, reflecting a strong antioxidant potential. The acetone extract, processed through ultrasound-assisted maceration, displayed an IC₅₀ around 23 µg/mL, which also shows significant activity but slightly less effective than the ethanol extract under the same conditions [26].

4.2. Antimicrobial Activity

In a study conducted by Zheljazkov and co-workers it was shown that the essential oil from Juniper cone berries, Juniper galbulid, with different phytochemical compositions can be obtained by capturing fractions at different time points during the hydrodistillation process. This study found that the essential oils extracted from juniper were active against S. enterica and Klebsiella pneumonia, among the three tested Gram-negative bacterial strains, along with Clostridium perfringens and Candida glabrata. Also, their results showed that certain essential oil fractions of J. communis have significant antimicrobial activity which could be utilized in various new antiseptic products or other biomedical applications [168]. According to the findings of Dumitrescu and co-workers, juniper’s essential oil caused a gradual decrease in the bacterial density for all tested strains. However, unlike Gram-negative bacteria, Gram-positive pathogens showed a significantly higher sensitivity, with Staphylococcus aureus being the most affected [18,169]. Muftah and co-workers evaluated the in vitro bioactivity of the active ingredient across various antimicrobial magistral drug formulations and plant extracts commonly used in folk medicine, finding similar MIC values ranging from 16 to 32 μg/mL [170].

4.3. Anti-Inflammatory Activity

Han and co-workers investigated the effects of juniper essential oils containing alpha-pinene as primary component, in human dermal fibroblasts designed to imitate chronic inflammation and fibrosis. Their observations revealed that juniper essential oils had a significant inhibitory impact on the production of two proinflammatory chemokines, interferon-induced protein 10 (IP-10) and interferon-induced T-cell alpha-chemoattractant (I-TAC), and also induced a decrease in collagen I, collagen III, and PAI-I [43]. According to experimental findings by Dae-Seung and co-workers, the pinene extracted from juniper species exhibits anti-inflammatory effects by inhibiting the phosphorylation of MAPKs (ERK, JNK), and the NF-B signalling pathway [171]. In a study published by Darwish and co-workers, it was found that Juniper essential oil inhibited the formation of pro-inflammatory cytokines, such as interleukin (IL)-1 and tumour necrosis factor (TNF-α) [172]. Extracts of J. communis have demonstrated the ability to reduce levels of pro-inflammatory cytokines, such as TNF-α and interleukin-1 beta (IL-1β), which are often elevated during inflammatory responses. For instance, an aqueous extract showed significant inhibition of prostaglandin synthesis (55% inhibition at 0.2 mg/mL) and platelet-activating factor (PAF) exocytosis (78% inhibition at 0.25 mg/mL) [23].

A specific study focused on J. communis extract’s protective effects against lipopolysaccharide (LPS)-induced acute kidney injury. It found that the extract activates the adenosine monophosphate-activated protein kinase (AMPK) pathway, which plays a critical role in cellular energy homeostasis and inflammatory responses. This activation contributes to the downregulation of inflammatory markers and protects against oxidative stress [173].

4.4. Anti-Cancer Activity

Tsai and co-workers’ study analysed the possibility of using Juniper communis extract as a treatment for cancer, with tests being conducted on BALB/c nude mice with subcutaneous glioblastoma. After sixteen days of treatment results showed that the tumour volume had significantly decreased. Their research suggests that juniper extract is a safe herbal remedy able to inhibit angiogenesis thus representing a promising therapeutic option against high-grade gliomas [174]. Lee and co-workers revealed that Juniper communis can induce p53 phosphorylation, Rb dephosphorylation, and p21 activation, resulting in a change in the expression of essential cell cycle proteins and triggering G0/G1 cell cycle arrest. Based on their data, it was revealed that juniper is capable of inducing both intrinsic and extrinsic apoptotic pathways thus contributing to cell death in human gingival squamous cancer cells [149]. In another study, Lai and co-workers investigated the in vitro and in vivo anticancer effects of juniper extract in colorectal cancer (CRC) cells. In vivo studies revealed that juniper extract is more effective against CRC cells than normal cells thus displaying selective antitumor effects and can interact with 5-fluorouracil in a synergistic manner. Cell apoptosis is caused by both internal (Bax/Bcl-2/caspase-9) and external (FasL/Fas/caspase-8) mechanisms, and cell cycle arrest is also triggered by regulating p53/p21 and CDK4/cyclin D1. In vivo studies showed that the extract suppressed tumour growth by inhibiting cell proliferation and inducing apoptosis [175]. Huang and co-workers focused on investigating the anticancer effect and underlying mechanism of J. communis extract in hepatocellular carcinoma cells both in vitro and in vivo. According to their results, juniper extract inhibited the expression of VEGF/VEGFR protein in vivo, similar to the findings reported in vitro, thus suggesting its ability to inhibit the autocrine and paracrine signalling pathways. Furthermore, juniper extract inhibited the formation of blood vessels and suppressed the MMP2/MMP9 protein expression in vivo, indicating that the extract has antimetastatic potential [44,176].

5. Future Perspectives

Studies on Juniperus communis showed the presence of valuable secondary metabolites such as diterpenes, lignans and biflavonoids that revealed a plethora of biological effects including antidiabetic, anti-bacterial, antioxidant and anticancer; hence, this species could be a reliable source of active compounds and standardized extracts that may provide therapeutic alternatives for a number of pathologies. As is the case with other phytocompounds, Juniperus phytochemicals may display poor pharmacokinetic profiles which makes them candidates for technological manipulation such as nanoformulations able to optimize their potential as therapeutic tools. To the best of our knowledge, the nanoformulation of Juniperus communis extracts has not been performed yet. Other Juniper species like Juniperus procera [177] and Juniperus excelsa [178,179] have been used to prepare nanoparticles, with the main field of utilization being antibacterial products such as wound dressings [178]. This type of application is meaningful taking into account both the antimicrobial effects of Juniper metabolites pointed out by modern research (and reviewed in this paper) but also the traditional indications of the plant [35]. Another field of interest is the study of natural products from Juniper in the supportive treatment of cancer. At least for the lignan matairesinol, a synergistic effect with 5-fluorouracil has been observed [111]. Furthermore, Juniper compounds contribute with their own cytotoxic effects, and have potent antioxidant and protective properties. In terms of toxicity, Juniper products was reported safe after short-term oral, inhalatory or topic administration; however, its long-term use especially in high doses may cause nephro- and gastrointestinal toxicity. Given the scarcity of toxicological studies published so far future tests should be conducted in order to clearly establish the extent and severity of adverse effects associated with the use of Juniper products via various administration routes. Despite the numerous biologic effects reported for Juniper phytocompounds clinical data in human subjects substantiating such therapeutic effects are still lacking therefore requiring future investigations. Preparation of extracts that are standardized in the main types of active principles (diterpenes, lignans, flavonoids) should be a prerequisite for effective Juniper-based pharmaceutical products.