Thuja occidentalis L. (Cupressaceae): Ethnobotany, Phytochemistry and Biological Activity

Thuja occidentalis L. (Cupressaceae) has its origins in Eastern North America and is cultivated in Europe and Brazil as an ornamental tree, being known as the “tree of life” or “white cedar”. In traditional medicine, it is commonly used to treat liver diseases, bullous bronchitis, psoriasis, enuresis, amenorrhea, cystitis, uterine carcinomas, diarrhea, and rheumatism. The chemical constituents of T. occidentalis have been of research interest for decades, due to their contents of essential oil, coumarins, flavonoids, tannins, and proanthocyanidines. Pharmacology includes antioxidant, anti-inflammatory, antibacterial, antifungal, anticancer, antiviral, protective activity of the gastrointestinal tract, radioprotection, antipyretic, and lipid metabolism regulatory activity. Therefore, the present review represents the synthesis of all the relevant information for T. occidentalis, its ethnobotany, phytochemistry, and a thorough analysis of their pharmacological activities, in order to promote all the biological activities shown so far, rather than the antitumor activity that has promoted it as a medicinal species.


Introduction
The approach of various complementary therapies is becoming an increasingly used method of treatment. In this context, the use of plants for the treatment of various diseases plays a major role [1][2][3]. Folk medicine is widely used in much of the world, even though it is not officially recognized in many countries [4]. Over 80% of the population in Africa and Asia use plants and plant extracts for the treatment of various diseases [5].
The use of phytomedicine began in China during the Xia dynasty and in India during the Vedic times. Herbal remedies are growing in popularity around the world for several reasons: They have long-lasting curative effects and are characterized by effectiveness, safety, and low side effects [6,7]. Currently, ethnopharmacological studies are encouraged worldwide as a method for highlighting species containing molecules or beneficial products that can be used in the pharmaceutical, Table 1. Comparison of the bioactive potential of T. occidentalis with other Thuja genus species.

Ethnobotany
Thuja occidentalis originated in the Eastern North America and is cultivated in Europe and Brazil as an ornamental tree [57][58][59], known as the "tree of life" or "white cedar" (Figure 1) [21]. The present work provides an overview about the state-of-the-art in ethnobotany, phytochemistry, and a detailed analysis of the pharmacological properties of T. occidentalis, such us: antioxidant, anti-inflammatory, antibacterial, antifungal, anticancer, antiviral, protective activity of the gastrointestinal tract, radioprotection, antipyretic, and lipid metabolism regulatory activity, in order to promote all the biological activities shown so far, rather than the antitumor activity that has promoted it as a medicinal species.

Ethnobotany
Thuja occidentalis originated in the Eastern North America and is cultivated in Europe and Brazil as an ornamental tree [57][58][59], known as the "tree of life" or "white cedar" (Figure 1) [21]. In traditional medicine, T. occidentalis has been used in the treatment of liver diseases, bullous bronchitis, psoriasis, enuresis, amenorrhea, cystitis, uterine carcinomas, diarrhea, and rheumatism [58,[60][61][62]. Essential leaf oil was used in the treatment of fungal infections, cancer, and intestinal worms [41]. As a tincture, T. occidentalis has been used in the treatment of warts, papillomas, and condylomas caused by human papilloma virus (HPV) [57,63].
Since historical times, the essential oil of T. occidentalis has been used in folk medicine. T. occidentalis containing thujone was used for hepatoprotection, bronchial catarrh, rheumatism, psoriasis, and even uterine carcinomas.
The mother tincture of T. occidentalis is used in homeopathy against a number of diseases. Homeopathy uses thuja as one of the main remedies for psychotic constitutions, in case of snake bite, small-pox, and vaccination-induced toxicity, respectively the proliferation or pathological vegetation [64].
Thuja preparations with thujone were regularly used by the American Indian traditional healers. The decoct and the tea prepared from the inner bark of the unwoody twigs could relieve constipation and headache [65]. It has also been used for the treatment of polyps, birthmarks, and wounds and as a painkilling and anthelmintic remedy [66,67]. In traditional medicine, T. occidentalis has been used in the treatment of liver diseases, bullous bronchitis, psoriasis, enuresis, amenorrhea, cystitis, uterine carcinomas, diarrhea, and rheumatism [58,[60][61][62]. Essential leaf oil was used in the treatment of fungal infections, cancer, and intestinal worms [41]. As a tincture, T. occidentalis has been used in the treatment of warts, papillomas, and condylomas caused by human papilloma virus (HPV) [57,63].
Since historical times, the essential oil of T. occidentalis has been used in folk medicine. T. occidentalis containing thujone was used for hepatoprotection, bronchial catarrh, rheumatism, psoriasis, and even uterine carcinomas.
The mother tincture of T. occidentalis is used in homeopathy against a number of diseases. Homeopathy uses thuja as one of the main remedies for psychotic constitutions, in case of snake bite, small-pox, and vaccination-induced toxicity, respectively the proliferation or pathological vegetation [64].
Thuja preparations with thujone were regularly used by the American Indian traditional healers. The decoct and the tea prepared from the inner bark of the unwoody twigs could relieve constipation and headache [65]. It has also been used for the treatment of polyps, birthmarks, and wounds and as a painkilling and anthelmintic remedy [66,67].
Moreover, high molecular weight glycoproteins or polysaccharides have been shown to be particularly relevant for plant activity [72].
In a previous study, we found, in an ethanolic extract made from fresh leaves, 21.13 µg/mL thujone, 2.16 mg/mL total phenolic acids expressed in caffeic acid, 0.36 mg/mL total flavonoids expressed in rutoside, respectively [37].
However, to date, the genetic regulation of thujone synthesis pathway has been studied in two species: Salvia officinalis and Thuja plicata. Geranyldiphosphate (GPP) and neryl-diphosphate are the general precursors' starting points and follow a four-step pathway. Sabinene is the first monoterpene in this path, being catalyzed by the enzyme sabinene-synthase (SS). It has been showed that sabinene is the precursor of thujone in S. officinalis, A. absinthium, and T. vulgare. Next, in three consecutive steps, thujone is formed from sabinene. The next intermediary in thujones' synthesis is sabinone, from which begins the thujone synthesis. There are some corresponding enzymes of this transformation which have not yet been clarified and also the formation of the two isomers of thujone (α and β). There are some species who get both isomers, while other plants accumulate just one of them [67].

Antioxidant Activity
The antioxidant capability is the best described property of phenolic compounds toward free radicals which are produced by cells metabolism [88].
In our previous studies, we showed a high antioxidant activity of a mother tincture of Thuja occidentalis by 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) containing (3.9 mg GAE/g d.w.) polyphenols, as well as its in vitro antioxidant capacity on Caco-2 cells exposed to oxidative stress induced by H 2 O 2 , by MDA-and GSH-level assessments [37]. Nazir et al. (2016) showed that the antioxidant potential of the Thuja occidentalis methanolic extract was assumed to high DPPH radical scavenging activities, ABTS, NO and lipid peroxidation assays [36].
In their study, Mighri et al. (2010) examined the antioxidant and antimicrobial activity of four essential oil types, whose major constituents were β-thujone, α-thujone, α-thujone/β-thujone, and 1,8-cineole/camphor/α-thujone/β-thujone, and the composition was investigated by using capillary GC and GC/MS technique. The results of the study showed that all examined oils had a great antimicrobial potential, and, in addition, they assessed the antioxidant capacity by different in vitro tests [89]. Moreover, in a study, Mahomoodally et al. (2019) assessed the antioxidant potential, antiglycation, and the total phenolics content of essential oils extracted from 19 medicinal plants [90].
The antioxidant potential of T. occidentalis L. cones was assessed in raw chicken ground meat during refrigerated storage in a study made by Yogesh et al. (2014). DPPH free-radical scavenging activity method was used to estimate the antioxidant activity of thuja cones (T. occidentalis) and peach seeds (Prunus persica). They also estimated the total phenolics, flavonoids, and reducing power in these extracts. Thuja cones extract and peach seeds extract had a total phenolics of 7.80 ± 0.04 and 1.92 ± 0.04 mg TAE/g d.w., respectively [39].

Anti-Inflammatory Activity
Inflammation is a primary protective reaction that is given to return the damaged tissue due to injurious stimuli. This response involves extravasation of neutrophils through the capillary network and then activation of macrophages, which generate different proinflammatory cytokines, such as TNF-α, interleukins, and interferons, which participate in the regulation of inflammatory reactions. There are not many studies that have evaluated the anti-inflammatory activity of T. occidentalis components. The study made by Silva et al. (2017) highlighted the anti-inflammatory activity of the aqueous extract and the polysaccharide fraction obtained from T. occidentalis in experimental models of acute inflammation. The doses used were 3, 10, and 30 mg/kg, administered intraperitoneally. They acted through mechanisms that involve modulating mediators, such as histamine, serotonin, PGE2, and bradykinin, and diminishing vascular permeability and neutrophil migration to the affected site. The aqueous extract and the polysaccharide fraction of T. occidentalis reduced production of pro-inflammatory cytokines (TNF-α and IL-6), diminished both COX-2 and iNOS activity, and diminished oxidative stress. High doses of aqueous extract and fraction of polysaccharides obtained from T. occidentalis of 300 mg/kg did not result in gastric toxicity [21].
Polysaccharides obtained from thuja leaf extract have been shown to reduce mice-induced inflammation. They have the ability to prevent metastasis by diminishing inflammatory cytokines, such as IL-1β, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), and TNF-α. Moreover, these polysaccharides stimulated the activity of natural killer (NK) cells, cell mediated antibody-dependent cytotoxicity (ADCC) and complement-mediated cytotoxicity (ACC) and stimulated the activity of antitumor factors, IL-2, and TIMP [43].

Antibacterial and Antifungal Activity
T. occidentalis has been shown to have antibacterial properties against a significant number of species, such as Salmonella sp., Enterobacter cloacae, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Shigella flexenari, Candida albicans, Proteus vulgaris, Entercoccus faecalis, and Staphylococcus [79,80]. They observed that the two components, α-thujone and β-thujone, showed protective effect against Gram-negative bacteria such as Pseudomonas aeruginosa and Klebsiella pneumonia and a mild protective effect against Staphylococcus aureus, Escherichia coli, and Candida albicans [80]. Thuja occidentalis also showed significant antibacterial activity against bacteria and fungi [79]. The antimicrobial profile of essential oil of Thuja occidentalis was also evidenced by Tsiri et al. (2009) [26].

Antiviral Activity
Polysaccharides isolated from T. occidentalis have been shown to have antiviral and immunostimulating effect, having the ability to inhibit HIV-1 and influenza A [34,91]. Thuja polysaccharides (TPS) inhibited HIV at a concentration of 625 µg/mL. They have been shown not to be toxic to MT-4 cells and have inhibited the expression of HIV-1 specific antigen in newly infected MT-2 cells [91]. Later, Gohla et al. (1992) highlighted the property of the high-molecular-weight polysaccharide fraction from T. occidentalis on HIV-1 [34]. An isolated fraction of T. occidentalis was shown to increase the number of cells producing antibodies in an in vitro study [86].

Anticancer Activity
In homeopathy, T. occidentalis is used in the treatment of cancer, but its mechanism of action is not known. Torres et al. (2016) studied the effect of α/β-thujone on glioblastoma, using in vitro and in vivo models. They have observed that α/β-thujone has the ability to diminish cell viability and has antiproliferative, proapoptotic, and antiangiogenic properties in vitro. In in vivo studies, α/β-thujone has been reported to induce regression of neoplasia and inhibited angiogenic markers of VEGF, Ang-4, and CD31 inhibitors in the tumor [42]. The antitumoral effect of the extract obtained from thuja leaves has been evaluated on numerous cancer cell lines [41,44,63,84].
The in vivo study performed by Siveen and Kuttan (2011b) demonstrated the antitumor effect of thujone in the malignant ascites lymphoma model (Dalton). Thujone, a monoterpene naturally found in T. occidentalis, has been shown to increase the number of leukocytes and bone marrow cells. This increased the proliferation of splenocytes and thymocytes, both in the presence and absence of specific mitogenes. Thujone stimulated cell mediated immune response and production of IL-2 and IFN-γ [92]. Thujone also showed the ability to inhibit metastasis in melanoma in vivo [83]. Thujone obtained from T. occidentalis ethanolic extract has been shown to have anticancer properties on the malignant melanoma cell line A375. In the same study, thujone was shown to have an antiproliferative effect and the ability to induce apoptosis [41].
Thuja's antitumoral activity has been highlighted in breast cancer [85]. Polysaccharides obtained from T. occidentalis L. leaf extract have been shown to reduce mice-induced inflammation. They have the ability to prevent metastasis by diminishing inflammatory cytokines, such as IL-1β, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), and TNF-α. Moreover, these polysaccharides stimulated the activity of natural killer (NK) cells, cell-mediated antibody-dependent cytotoxicity (ADCC), and complement-mediated cytotoxicity (ACC) and stimulated the activity of antitumor factors, IL-2, and TIMP [43].

Protective Activity of the Gastrointestinal Tract
According to Dubey and Batra (2008b), the ethanol fraction of T. occidentalis showed a hepato-protective effect in acute and chronic liver-induced HCV. The same researchers revealed that the ethanolic fraction of T. occidentalis provides an important effect against gastric lesions [46]. Saeed et al. (2014) evaluated the effect of low dose of T. occidentalis on rabbit, for three months. They carried out an experiment on rabbits, on treated groups with and without tetrachloride. They analyzed the heart, liver, stomach, and kidney tissues histopathologically, on both control groups and T. occidentalis treated groups. They also tested the CCl 4 injected group by doing the liver function test. Their study showed that there were minor harmful effects in the liver and kidney tissues which were treated with carbon tetrachloride, but there was no major toxicity due to the antioxidant effect of active constituents from T. occidentalis [93].
The methanolic extract obtained from T. occidentalis orally administered at 200 mg and 400 mg/kg body weight has been shown to have gastroprotective effect in rats, comparable to omeprazole. This extract reduced gastric acid production by 45% and 69%, respectively, and favored a significant regeneration of the gastric epithelium at a dose of 400 mg/kg body weight. The antiulcer action of this extract is due to its antioxidant properties [45].
Our previous results show that orally administration of T. occidentalis mother tincture by gavage, for one week, to mice with experimentally induced ulcerative colitis, succeeded in inhibiting the inflammatory process induced by TNBS in the intestine, and normalized the structure and ultrastructure of the intestinal mucosa [37].

Lipid Metabolism Regulation
The ethanolic fraction of T. occidentalis has been shown to have hypoglycemic properties in rats with aloxan-induced diabetes, at a dose of 200 mg/kg, without significant impact on body weight. It has also improved lipid profile and has been shown to have a protective effect against oxidative stress by increasing glutathione level in blood [47].
In the study conducted by Dubey and Batra (2009a), the T. occidentalis ethanolic fraction administered at doses of 200 mg and 400 mg/kg body weight showed 77-92% decrease in serum cholesterol, with 53-84% LDL-cholesterol and 27-46% of triglycerides. Antiatherosclerotic activity was marked by the increase in HDL-cholesterol and the reduction in the atherogenic index. Thuja occidentalis showed a significant free-radical neutralization effect due to its ability to interfere with the absorption, degradation, and excretion of cholesterol [48].
In the study made by Sunila and Kuttan (2005), T. occidentalis showed a protective effect against gamma-induced toxicity in Swiss albino mice. Thus, the Thuja occidentalis alcoholic extract reduced levels of alkaline phosphatase, pyruvate transferase, and lipid peroxidation [49].

Antipyretic Activity
The methanolic extract of Thuja occidentalis showed antipyretic activity in rabbits. It reduces fever and normalizes body temperature at doses of 100 mg and 200 mg/kg body, comparable to paracetamol [50].
Funding: This research received no external funding.

Conflicts of Interest:
The authors declare no conflict of interest.