Natural Sources and Bioactivities of 2,4-Di-Tert-Butylphenol and Its Analogs

2,4-Di-tert-butylphenol or 2,4-bis(1,1-dimethylethyl)-phenol (2,4-DTBP) is a common toxic secondary metabolite produced by various groups of organisms. The biosources and bioactivities of 2,4-DTBP have been well investigated, but the phenol has not been systematically reviewed. This article provides a comprehensive review of 2,4-DTBP and its analogs with emphasis on natural sources and bioactivities. 2,4-DTBP has been found in at least 169 species of bacteria (16 species, 10 families), fungi (11 species, eight families), diatom (one species, one family), liverwort (one species, one family), pteridiphyta (two species, two families), gymnosperms (four species, one family), dicots (107 species, 58 families), monocots (22 species, eight families), and animals (five species, five families). 2,4-DTBP is often a major component of violate or essential oils and it exhibits potent toxicity against almost all testing organisms, including the producers; however, it is not clear why organisms produce autotoxic 2,4-DTBP and its analogs. The accumulating evidence indicates that the endocidal regulation seems to be the primary function of the phenols in the producing organisms.


Introduction
2,4-Di-tert-butylphenol or 2,4-bis(1,1-dimethylethyl)-phenol (2,4-DTBP) is a common natural product that exhibits potent toxicity against almost all testing organisms, including the producing species. The phenol has been well investigated in terms of its natural sources and bioactivities, but it has not been systematically reviewed. A basic question has never been addressed: why does an organism produces autotoxic 2,4-DTBP? This review has summarized the available references in both English and Chinese to date. It will provide some basic information to better understand the physiological and evolutionary roles of 2,4-DTBP in the producing organisms.
2,4-DTBP was also reported in different groups of plants, such as diatom Phaeodactylum tricornutum Bohlin [25], liverwort Marchantia polymorpha L [26], and ferns Osmunda regalis L. [27] and Adiantum venustum D. Don [28] 2,4-DTBP commonly occurs in the violate or essential oils of many seed plant species. GC-MS analysis showed that 2,4-DTBP occurs in the dichloromethane extracts of the bark via distillation and methanol extracts of the cones and bark of Pinus yunnanensis Franch. [29,30], an in n-hexane extracts of the cones of Pinus kesiya var. langbianensis (A. Chev.) Gaussen ex Bui [31]. The analysis also reported that 2,4-DTBP is a major component in the water extracts of fresh needles of Pinus tabulaeformis Carr., but not in the fallen leaves or decomposed leaves of the pine [32]. The phenol had a low or non-detectable presence in the rhizosphere soils of a new plantation of Masson's pine (Pinus massoniana Lamb.); however, it became a major compound in the rhizosphere soils of the continuous pine plantation [33].
The phenol is often found in the essential oils of flowering plants, including dicots (107 species, 58 families) and monocots (22 species, eight families) . In jiangxiang huangtan (Dalbergia odorifera T. Chen), it was found that 2,4-DTBP primarily accumulated in the transition tissues between the heartwood and sapwood as the major component in the ethyl acetate extracts (9.64% based on the dry weight) [70]. The concentration of the compound in the slow-growth heartwood is about 0.83% but is not detected in the fast-growth sapwood when using GC-MS [70].
The antioxidant activity of BHT was about twice as great as that of 2,4-DTBP because two terbutyl groups in BHT protect the aromatic hydroxyl group, which forms a phenoxyl radical and donating a hydrogen atom that could quench active free radicals and stop the propagation of lipid peroxidation [98]. The additional ter-butyl group in BHT may also decrease the toxicity. As a result, BHT is one of most commonly used antioxidants for preserving food and feed, and is also listed as an antioxidant food additive by The U.S. Food and Drug Administration (FDA) and the European Union (EU) [99,100]. As an active ingredient from royal jelly, BHT can eliminate 75.86% of ultraoxygen free radicals at 600 mg/L and 84.47% of the hydroxyl free radicals at 500 mg/L [101]. BHT decreased the Malondiadehyde (MDA) content and increased the superoxide dismutase (SOD) and glutathioneperoxidase (GSH-Px) content in rat liver and serum [101]. The antioxidant activity of BHT can be enhanced in combination use with synthetic 2-ter-butyl-4-methoxyphenol (BHA) and 2,4,6-triter-butylphenl (TBP) [102]. BHT and BHA are fairly heat-stable, [1] but they have been found to exert a dual pro-oxidant and antioxidant action under certain conditions [102]. BHA can stimulate the

Anti-Inflammatory Activities
Lipopolysaccharide (LPS), the endotoxin found in the cell walls of Gram-negative bacteria, triggers inflammation by activating mononuclear phagocytes (monocytes and macrophages) and results in the production of various pro-inflammatory cytokines. LPS administration was observed to increase the expression of tumor necrosis factor alpha (TNF-α) interleukin IL-6 and IL-1b genes significantly, while 2,4-DTBP treatments were found to decrease the expression of all three genes in the RAW264.7 mouse macrophage cell line [103]. BHT has shown a slight anti-inflammatory activity on the expression of cyclooxygenase-2 (Cox2) and TNF-α genes upon stimulation with Porphyomonas gingivalis (Pg) fimbriae [102]. The combination of BHT and BHA at a molar ratio of 0.5-2 provides potent anti-inflammatory activity, as tested by gene-expression systems for Cox2 and TNF-α in RAW264.7 cells [102]. The anti-inflammatory activity may be attributable to complex synergistic antioxidant activity [102].  The antioxidant activity of BHT was about twice as great as that of 2,4-DTBP because two ter-butyl groups in BHT protect the aromatic hydroxyl group, which forms a phenoxyl radical and donating a hydrogen atom that could quench active free radicals and stop the propagation of lipid peroxidation [98]. The additional ter-butyl group in BHT may also decrease the toxicity. As a result, BHT is one of most commonly used antioxidants for preserving food and feed, and is also listed as an antioxidant food additive by The U.S. Food and Drug Administration (FDA) and the European Union (EU) [99,100]. As an active ingredient from royal jelly, BHT can eliminate 75.86% of ultra-oxygen free radicals at 600 mg/L and 84.47% of the hydroxyl free radicals at 500 mg/L [101]. BHT decreased the Malondiadehyde (MDA) content and increased the superoxide dismutase (SOD) and glutathioneperoxidase (GSH-Px) content in rat liver and serum [101]. The antioxidant activity of BHT can be enhanced in combination use with synthetic 2-ter-butyl-4-methoxyphenol (BHA) and 2,4,6-tri-ter-butylphenl (TBP) [102]. BHT and BHA are fairly heat-stable, [1] but they have been found to exert a dual pro-oxidant and antioxidant action under certain conditions [102]. BHA can stimulate the peroxidase-dependent oxidation of BHT to form the potentially toxic BHT-quinone methide. Among several BHT metabolites, BHT-quinone methide (BHT-QM), 2,6-di-tert-butyl-4-hydroperoxyl-4-methyl-2,5-cyclohexadienone (BHT-OOH), and 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHT-CHO) have been reported to induce peroxides [102].

Anti-Inflammatory Activities
Lipopolysaccharide (LPS), the endotoxin found in the cell walls of Gram-negative bacteria, triggers inflammation by activating mononuclear phagocytes (monocytes and macrophages) and results in the production of various pro-inflammatory cytokines. LPS administration was observed to increase the expression of tumor necrosis factor alpha (TNF-α) interleukin IL-6 and IL-1b genes significantly, while 2,4-DTBP treatments were found to decrease the expression of all three genes in the RAW264.7 mouse macrophage cell line [103]. BHT has shown a slight anti-inflammatory activity on the expression of cyclooxygenase-2 (Cox2) and TNF-α genes upon stimulation with Porphyomonas gingivalis (Pg) fimbriae [102]. The combination of BHT and BHA at a molar ratio of 0.5-2 provides potent anti-inflammatory activity, as tested by gene-expression systems for Cox2 and TNF-α in RAW264.7 cells [102]. The anti-inflammatory activity may be attributable to complex synergistic antioxidant activity [102].

Cytotoxicities
2,4-DTBP showed a remarkable cytotoxicity against HeLa cells with an IC50 value of 10 µg/mL [6]. 2,4-DTBP exhibited superior effect in the induction of apoptotic genes in cancer cell lines, as did the standard drug Cisplatin [103]. 2,4-DTBP was found to significantly increase the expression of P53 and caspase 7 in both MCF-7 and A431 cell lines, and exhibited significantly higher activation of the P53 gene in MCF-7. Effect of 2,4-DTBP on caspase 7 gene expression was significantly greater in A431, while the effect appeared to be less pronounced in MCF-7 [103].
Based on hepatic and renal toxicity (histopathological changes and an increase in organ weight with blood biochemical changes) in rats, the respective no-observed-adverse-effect levels (NOAELs) for 2,4-DTBP were concluded to be 5 and 20 mg/kg/day [104]. Histologically, there were no obvious changes in uteri and vagina ovariectomized (OVX) CD1 mice between the 2,4-DTBP treatment and the control, and the uterotrophic effect of 2,4-DTBP was not observed in the range of 10 to 250 mg/kg using an oral gavage [105].
It has been reported that long-term and high quantities usage of BHT can induce liver tumors [106]. Due to their pro-oxidant activity, BHT-quinone and BHT-OOH have been reported to result in internucleosomal DNA fragmentation, which is the characteristic of apoptosis [107]. BHT-OOH was found through oxidative DNA damage directly, whereas BHT-quinone was found via DNA damage through H 2 O 2 generation [107]. After an injection treatment, BHT can considerably increase the number of mitoses in epithelial cell populations from various parts of small intestinal crypts of mice [108]. The effect may be explained by the influence of BHT on the reserve pool of cells and the longevity of individual stages of the mitotic cycle [108]. The BHA/BHT combination (molar ratio 1:1) has inhibited the expression of manganese superoxide dismutase (MnSOD) mRNA in HL60 cells and reversed the transcriptase-polymerase chain reaction (PCR)-activating caspases 3, 8, and 9 [109]. It may contribute to the synergistically antioxidant activity of the BHA/BHT combination and radical-induced formation of intermediates, such as quinone methide [109].

Insecticidal and Nematicidal Activities
2,4-DTBP exhibited significantly adulticidal, larvicidal, ovicidal, repellent, and ovipositiondeterrent activities against the spider mite Tetranychus cinnabarinus [73]. The mites exhibited the highest run-off rate on bean leaf surfaces sprayed with 2,4-DTBP when applied at sublethal doses and moved toward surfaces that had not been sprayed with the compound, according to Pearson's v2 test. The compound also showed nematicidal activity against Caenorhabditis elegans during fumigation or soil treatment at temperatures higher than 25 • C [110].

Antibacterial Activities
Extracellular polymeric substances (EPS) play crucial roles in biofilm formation and biocorrosion, resulting in heavy economic loss in an industrial setup. 2,4-DTBP can modulate the secreted EPS of Serratia marcescens, which in turn could facilitate the disruption of biofilms, as well as favoring the diffusion of antimicrobials into the cell aggregates, resulting in the eradication of persistent biofilms [116]. 2,4-DTBP can be used to enhance the efficacy of conventional antibiotics. Intercellular communication in bacteria (quorum sensing (QS)) is an important phenomenon in disease dissemination and pathogenesis that controls biofilm formation. 2,4-DTBP controls QS-mediated biofilm formation and simultaneously increases the hydration of the cell wall, which results in reduced biofilm formation [13].
2,4-DTBP isolated from thermophilic Bacillus licheniformis in an Algerian hot spring showed bioactivity against two multidrug resistance bacteria Pseudomonas aeruginosa and Staphylococcus aureus in pure and mixed cultures that were investigated using a radial diffusion assay at 55 • C [2]. The phenol from Bacillus, in association with seaweed, was reported to exhibit a dose-dependent antibiofilm activity against group A Streptococcus bacterium [3].

Antifungal Activities
2,4-DTBP was found to be effective against an agriculturally important root-rot fungus Fusarium oxysporum by inhibiting spore germination and hyphal growth [10]. During the fungal spore germination, 2,4-DTBP completely inhibited the germination by preventing the emergence of a normal germ tube and led to the abnormal branching and swelling of hyphae. In such a case, 2,4-DTBP may be binding with β-tubulin in microtubules, inhibiting their proliferation and suppressing their dynamic instability as the microtubules are the cytoskeletal polymers in eukaryotic cells and the loss of microtubules should negatively affect the growth rate of spore germination, with an expected reduction in fungal growth in vitro. [10] 2,4-DTBP distinctly reduced the mycelial growth of Phytophthora capsici by approximately 50% at 100 µg/mL relative to the control [8]. The germinated seeds of pepper treated with 2,4-DTBP significantly reduced radicle infection by P. capsici without radicle growth inhibition [8].
2,4-DTBP had a significant inhibition effect on the mycelium growth at the early stage of culturing tomato leaf mold (Cladosporium fulvum) and 0.1 mmol/L of 2,4-DTBP had the best inhibition effect when the mycelium had grown for seven days [118].
2,4-DTBP can be produced in some species of Aspergillus [18], Penicillium [20,21], and Fusarium [23], but experiments showed the phenol could inhibit the growth of these fungi. Disc diffusion assays showed that 2,4-DTBP (2 mg/25 mL) prevented the fungal mycelial growth of Aspergillus niger, F. oxysporum, and Penicillium chrysogenum on wheat grains [6]. 2,4-DTBP produced from environmental bacterium Shewanella algae strain YM8 significantly reduced the mycelial growth and conidial germination in mold Aspergillus [11]. 2,4-DTBP could inhibit Aspergillus flavus mycelial growth 7 dpi on potatodextrose agar (PDA) medium at a 5 µg/L concentration and complete inhibition of mycelial growth was observed at 100 µg/L. At 200 µg/L, the compound completely inhibited the germination of conidia. The antimicrobial activity of 2,4-DTBP appeared to correlate with its antioxidative activity because it was able to inhibit the reactive oxygen species (ROS) production in both Aspergillus and Phytophthora cinnamomi [120]. Thus, the phenol has potential in the development of biopreservatives and dietary antioxidants for food applications.
2,4-DTBP exhibited fungicidal potential at higher concentrations where fluconazole failed to act completely. Various antibiofilm assays and morphological observations revealed that 2,4-DTBP inhibited and disrupted biofilms of Candida albicans via the possible inhibition of hyphal development [101]. It also inhibited the production of hemolysins and phospholipases, and secreted aspartyl proteinase, which are the crucial virulence factors required for the invasion of C. albicans [121].
2,4-DTBP extracted from the rhizome of cogongrass (Imperata cylindrical (L.) P. Beauv.) was found to have allelopathic effects on the germination and seedling growth of weedy plants under soilless conditions; for instance, 2,4-DTBP at 0.1 mg/mL showed a 78-95% inhibition of root and shoot growth of beggar ticks (Bidens pilosa L.), leucaena (Leucaena leucocaphala L. de Wit), and barnyardgrass (Echinochloa crus-galli (L.) Beauv) [123]. Lab assays showed that leachates of cogongrass are toxic to ryegrass and lettuce, but not toxic to cogongrass [124]. However, another report showed that boiling water extracts of cogongrass rhizomes that contain catechol, chlorogenic acid, isochlorogenic acid, neochlorogenic acid, p-coumaric acid, p-hydroxybenzaldehyde, scopolin, and scopoletin not only significantly inhibited the seedling growth of five other plant species, but also suppressed cogongrass growth [125]. A later investigation indicated that 2,4-DTBP inhibited 100% of the seed germination and growth of cogongrass at the concentration of 0.1 mg/mL [123].
The phenol also showed toxicity on the root and leaf tissues of the grassy weed Leptochloa chinensis (L.) Nees and broadleaf weed Hedyotis verticillata (L.) Lam [126] The phytotoxic effect of 2,4-DTBP on these two weeds became apparent at seven days and 14 days after treatment with symptoms of lamina wilting and necrosis, respectively [126]. After a 2,4-DTBP treatment, both had abnormal and much shorter root hairs compared to those of untreated plants. 2,4-DTBP reduced the shoot biomass growth of L. chinensis and H. verticillata by 50% when applied at concentrations of 50 and 200 µg/mL, respectively [122]. Chuah et al. found that 2,4-DTBP isolated from Napier grass (Pennisetum purpureum) exhibited potent herbicidal activity, whereby it completely prevented the root growth of L. chinensis in soil at an application rate as low as 0.60 kg a.i. ha −1 [127]. 2,4-DTBP induces oxidative stress through the enhanced generation of reactive oxygen species, which cause lipid peroxidation, membrane damage, and the activation of antioxidant enzyme systems, and thus cause a great reduction in chlorophyll content, thereby decreasing chlorophyll fluorescence, transpiration, and the net photosynthetic rate in the leaf tissues [121]. 2,4-DTBP has potent herbicidal properties that can alter the chloroplast ultrastructure, thereby reducing physiological activity of these weedy plants [128]. The present findings imply that 2,4-DTBP may potentially be developed as a soil-applied natural herbicide for the control of L. chinensis and perhaps other weeds in an aerobic rice system [127,129].
It was reported that 2,4-DTBP from P. massoniana significantly inhibited the seed germination, seed viability, hypocotyl and radicle growth, and seedling growth of Masson's pine at 0.25-1.0 mg/mL [33]. Another autotoxic study found that 2,4-DTBP had a toxic effect on microorganisms in the rhizosphere soil of hop (Hamulus lupulus L.) and affected the photosynthesis and growth of hop seedlings [130,131]. 2,4-DTBP had a significant inhibitory effect on the plant immune system and seed germination of Atractylodes macrocephala [132]. 2,4-DTBP from root exudates of chilli pepper showed a medium inhibition against the seed germination and seedling growth of chilli pepper at more than 2 mmol/L [133]. The growth of eggplants was stunted at high concentrations (0.10-1.00 mmol L −1 ) [104]. 2.5-DTBP is one of the compounds responsible for soil sickness in the field of Boehmeria nivea [77]. The results of a pot experiment indicated that 2,4-DTBP first significantly decreased and then increased the abundance of culturable bacteria, fungi, and actinomycetes of the rhizosphere soil after treatment [90,91]. 2,4-DTBP from the bulb of Lilium davidii var. willmottiae and Fusarium display a synergetic effect on the Fusarium wilt in the lily [134]. The antioxidant and anti-inflammatory activities of 2,4-DTBP have been emphasized in many publications. More importantly, however, the phenol exhibited a broad toxicity in all testing organisms, including the producers; for example, cytotoxicity in human cells and animals, insecticidal and nematicidal activities, antimicrobial activities, and phytotoxicities. However, the available data could not explain why an organism produces such toxic 2,4-DTBP. The endocide theory hypothesizes that an organism is more sensitive to its own endogenous metabolites than external molecules and thus an endocidal compound commonly occurring in different species has a broad spectrum of toxicity or low selective activity [135]. 2,4-DTBP provides a good example. This phenol commonly occurs in diversified organisms and has a potent toxicity against almost all testing organisms.
7.5 and 15 mmol/m 2 Photosynthesis and growth of hop seedlings [130,131] Seed and seedling of of Brassica napus L., Echinochloa crus-galli (L.) Beauv 0.1 mg/mL Root and shoot growth [123] Seed and seedling of of Brassica napus L. 0-0.10 mmol/L Seed germination and seedling growth [63] Seed and seedling chilli pepper More than 2 mmol/L Seed germination and seedling growth [133] Seedling of eggplant 0.10-1.00 mmol/L Seedling growth [104] Bulb of Fusarium Fusarium wilt in the lily [134] 2,5-DTBP Boehmeria nivea Soil sickness in the field [77] Author Contributions: Conceptualization, S.L. and F.Z.; methodology and literature, F.Z., P.W., Z.S.; manuscript writing, all authors. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

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