Theacrine From Camellia kucha and Its Health Beneficial Effects

Theacrine, i.e., 1,3,7,9-tetramethyluric acid, is one of the major purine alkaloids found in leaf of a wild tea plant species Camellia kucha Hung T. Chang. Theacrine has been attracted great attentions academically owing to its diverse health benefits. Present review examines the advances in the research on the health beneficial effects of theacrine, including antioxidant effect, anti-inflammatory effect, locomotor activation and reducing fatigue effects, improving cognitive effect, hypnotic effect, ameliorating lipid metabolism and inhibiting breast cancer cell metastasis effect. The inconsistent results in this research field and further expectations were also discussed.

Theacrine has been attracted great attentions academically owing to its diverse healthy benefits (1-3), including reducing inflammation (12), providing stimulatory effects without adaptation or habituation (13), antioxidant effect (14), enhancing sustained energy (15), promoting positive mood elevation, and mental clarity (15)(16)(17) and etc. However, these researches lack systematic summarization and some important mechanisms remain to be further investigated. In this review, we summarized the achievements in the study on health benefits of theacrine and the possible mechanisms behind the observed associations ( Table 1), so as to encourage the scientific community to pay more attention to the nutraceutical properties of theacrine.

TOXICOLOGICAL EVALUATION OF THEACRINE
Acute toxicity test (14 d) on mice using extracts of Kucha showed that the semi-lethal dose (LD 50 ) of Kucha extracts and theacrine was 5,600 mg/kg bw/day (12) and 810.6 mg/kg bw/day (22), respectively. Four-week oral administration of pure theacrine by rats at dose up to 150 mg/kg bw/day, showed that the appearance and behavior, body weights, organ coefficient, hematological and biochemical parameters of the tested rats showed as normal as control group. Pathological examination showed no changes induced by the drug toxicity, and no delayed toxic reaction was observed after stopped giving drug (40). A 90-day oral toxicological evaluation showed that the no observed adverse effect level (NOAEL) of theacrine was 180 mg/kg bw/day, as at this dose there were no toxicologically relevant treatment-related findings in male or female animals (41).
Oral administration test on sixty healthy men and women using TeaCrine R , a nature-identical, chemically equivalent bioactive version of theacrine, confirmed that daily supplementation theacrine up to 300 mg/kg bw/day for more than 8 weeks was clinically safe, without habituating neuroenergetic effects (13). Tests on one-hundred twenty-five men and women (mean age 23.0 years, height 169.7 cm, body mass 72.1 kg; n = 25 persons/group) showed that oral consumption of a mixture containing methylliberine (100-150 mg) and theacrine (25-50 mg) showed no negative effect on the health over 4 weeks of continuous oral administration (42).

Antioxidant Effects
The leaf of C. kucha containing 2.86% (W/W) theacrine and 2.13% (W/W) caffeine had stronger antioxidant activity than Camellia sinensis (Longjing tea) containing 4.64% (W/W) caffeine but no theacrine (14). The IC 50 value (inhibitory concentration in mg/mL of tea necessary to reduce the absorbance by 50%) of C. kucha in scavenging DPPH radicals was 0.79 ± 0.022 mg/mL, being lower than that of C. sinensis (0.91 ± 0.015 mg/mL). The TEAC value (Trolox equivalent antioxidant capacity, TEAC (mg/g) = IC 50(trolox) /IC 50(sample) × 1,000) of C. kucha in scavenging DPPH radicals (436.78 ± 12.44 mg/g) was higher than that of C. sinensis (376.12 ± 5.81 mg/g) (14). The TEAC value of C. kucha in FRAP assay (493.67 ± 8.02 mg/g) was also higher than that of C. sinensis (438.05 ± 9.54 mg/g) (14). These suggest that C. kucha containing theacrine had stronger DPPH and FRAP radical scavenging activities than C. sinensis without theacrine. Purine alkaloids including caffeine, theophylline, theobromine and theacrine, which are detected in C. kucha, showed comparatively lower antioxidant activities in oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity assay (CAA) than Trolox. The ORAC values of theabromine, theophylline, caffeine and theacrine were ∼65, ∼37, ∼35, and ∼25% of that of Trolox (18). Though theacrine showed lower activity in ORAC and CAA than theophylline and theobromine, it could significantly improve vessel density on chorioallantoic membrane and myocardial apoptosis in chick embryo model (18). These properties are related to its upregulating expression of sirtuin which is a group of highly conserved NAD-dependent histone deacetylases in mammals and has regulatory effects on cell survival, proliferation, metabolism, death and aging, as well as longevity of organs. Theacrine upregualted the expression of sirtuin family member SIRT3, which in turn activated the SIRT3/FOXO3/SOD2 signaling pathway, resulting in improvement of the intervertebral disc degeneration (19) and the cardiac function of ovariectomized mice with myocardial infarction (20) by alleviating oxidant stress. Furthermore, the antioxidant capacity of theacrine was also due to its strengthening the antioxidant system in vivo, such as elevating activities and gene expressions of superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px), as well as reducing activity of xanthine oxidase, resulting in significant decrease in the content of malondialdehyde (MDA) and increase in ORAC level in plasma and liver of oxidant-stressed mice (21).

Anti-inflammatory Effects
Oral administration of theacrine by mice (8-32 mg/kg bw/day) significantly inhibited the ear edema induced by xylene and hind paw edema induced by λ-carrageenan in a dose dependent manner (22). Oral administration of theacrine showed potent anti-inflammatory activities. Theacrine inhibited the xyleneinduced ear edema, in which the inhibition percentages were 10.1, 12.9, and 45.2% at doses 8, 16, and 32 mg/kg, respectively, compared with the control. Maximal inhibitory rates of antiedema effect of theacrine at doses 8, 16 and 32 mg/kg were observed at the 2 h, with inhibitory rates being 14.1, 16.6, and 20.1% (P < 0.05), respectively, compared with 18.8% of indomethacin (22). Theacrine showed inflammatory cell infiltration and focal necrosis of hepatocytes. Oral administration of theacrine (10, 20, 30 mg/kg DW/day) for 7 consecutive days was found to reduce hepatic mRNA levels of inflammatory mediators (IL-6, IL-1β, TNF α, and IFN γ), decreased the levels of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and reversed the histologic damages in liver of restraint stressed mice (43). Oral administration of theacrine (12.5-25.0 mg/kg bw/day) showed therapeutic effect on arthritis in SD rat (43). An aqueous formula consisting of bioactive form of theacrine, liberine and methylliberine was used as dietary supplement for reducing inflammation, pain, depression, and alleviating deleterious effects of hyperglycemia and sleep deprivation (43).
Theacrine shows anti-inflammation effect by interfering with the actions of inflammatory mediators such as histamine, serotonin, and bradykinin (22). The inflammatory mediators histamine, serotonin, and bradykinin induce edema by promoting vasodilatation and increasing vascular permeability (44). λ-Carrageenan-induced edema is a biphasic event, in which the early phase (90-180 min) of the inflammation is due to the release of histamine, serotonin and similar substances, and the later phase (270-360 min) is associated with the activation of kinin-like substances (45). Theacrine functions on the first phase of inflammation by interacting with histamine and serotonins and other similar mediators. Furthermore, theacrine has a membrane stabilizing effect, results in reduction of capillary permeability (22). The anti-inflammation mechanism of theacrine is also involved in its enhancement of transforming growth factor-β (TGF-β) mediated shifts via TGF-β/SMAD pathway (23). Biological signals for TGF-β are transduced through transmembrane serine/threonine kinase receptors to a family of intracellular mediators known as SMADs. Theacrine upregulated the expression of RNA and protein of SMAD3, p-ERK and p-p38, but downregulated the expression of nuclear factor-kB (NF-kB) and interleukin-6 (IL-6) in Freund's incomplete adjuvant (FIA)-induced SD rats (23).
Anti-inflammation Mouse Orally 8-32 mg/kg Theacrine significantly inhibited the ear edema induced by xylene and hind paw edema induced by λ-carrageenan.
Hypnotic and antidepressant effects Male ICR mice and male Sprague Dawley rats (n = 5) 3, 10, 30 mg/kg, i.g. Theacrine significantly enhanced pentobarbital-induced sleep in mice by shortening wake time and increasing NREMS time, but it had no effect on REMS. Theacrine significantly reversed the decrease in sleeping time in mice pretreated with the adenosine A1 receptor antagonist DPCPX.
Anti-virus MDCK cells 50 or 100 µM Theacrine at 50 µM significantly inhibited the expression of influenza virus A NS1 protein at 12 hpi; theacrine at 100 µM significantly reduced the yield of viral progenies.
No effect Men (n = 10) and women (n-10) Orally TheaTrim TM containing 150 mg theacrine Theacrine did not result in a statistically significant improvement in cognitive performance, but might favorably impact multiple subjective feelings related to energy and mood.

Locomotor Activation and Reducing Fatigue
There were many studies showing that theacrine activated locomotor and reduced fatigue. In comparison to placebo under double-blind conditions, oral administration of TeaCrine R at a dose equivalent 200 mg theacrine resulted in more energy, less fatigue, better concentration and mood (15). Dietary supplement with 5-850 mg theacrine would improve mood, energy and focus, accompanying with reduction of anxiety or fatigue (25). A formula containing theacrine and caffeine with weight ratio 2:1 to 4:1 induced an increase in energy of at least 8%, accompanying with a decrease in fatigue at least 6% (26). A pre-workout supplement of 10 mg theacrine improved selective attention (27). 27-38% improvements in time-toexhaustion were observed in participants with supplement of 275 mg theacrine, leading athletes to sustain greater focus under fatigue for long periods (16). Sense of energy was significantly increased from baseline to 2 h post-ingestion in treatments of caffeine and combination of theacrine + caffeine; meanwhile, oral administration of 125 mg theacrine produced a significant rise in energy in 3 h post-ingestion, with little impact on heart rate (HR) and blood pressure (BP) (17). The mechanism of theacrine activating locomotor and reducing fatigue is likely involved in its action as an adenosine receptor antagonist. There was study showing that pre-treatment with theacrine (

Improving Cognitive Performance
Test on mice showed that oral administration of theacrine (5, 10, 15 mg/kg) significantly reversed learning and memory impairment caused by central fatigue (28). Combination of theacrine (125 mg) and caffeine (150 mg) moderately improved cognitive performance as assessed by trail making test (TMT) (17) and showed modest cognitive benefits during complex decision making, potentially due to overlapping peak concentrations or enhanced bioavailability (26,27). The improved cognitive accuracy at end-of-game in all conditions may indicate a training effect in highly skilled players for allocation of resources (16). The cognitive improving effect of theacrine was associated with its regulating brain glucose metabolism, inhibiting phosphodiesterases and restoring levels of fatigue-related neurotransmitters in the brains, such as 5-hydroxytryptamine (5-HTP) and dopamine as well as their metabolites (28).
Parkinson's disease (PD) is a long-term degenerative disorder of the central nervous system that mainly affects the motor system, affecting about 1% of adults older than 60 years (28). Oxidative damage of dopaminergic neurons is the fundamental causes of PD. Tests using multiple animal models of PD (6-OHDA-treated rats and in 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP)-treated mice and zebrafish) and 1-methyl-4-phenylpyridinium (MPP + )-treated SH-SY5Y cells showed that theacrine had the potency to relieve PD by retrieving the apoptosis of dopaminergic neurons and activating SIRT3 which deacetylating SOD2 and restoring mitochondrial functions (46).

Hypnotic and Antidepressant Effects
Theacrine is structurally similar to caffeine (Figure 2), but in fact its physiological effects are quite different from the latter. Theacrine is shown to be stimulatory when used in higher doses, however, it can actually have a sedative effect when used in low doses such as the amount consumed through tea leaves of C. kucha. Oral administration of theacrine (10 and 30 mg/kg) could significantly prolong the sleeping time induced by pentobarbital (p < 0.01), while caffeine and theobromine exhibited an inverted effect on mice. This suggests that theacrine possesses potent sedative and hypnotic properties and its central nervous system effects is different from those of caffeine and theobromine (29). Furthermore, caffeine has been demonstrated to show signs of tolerance build-up in as little as 4 days, while theacrine shows no such adaptation (13).

Ameliorating Lipid Metabolism
An in vitro test showed that theacrine up-regulated SIRT3, LCAD, FABP4 and LPL gene expression (32). In vivo test showed that theacrine (10 mg/kg) up-regulated SIRT3 gene expression in liver, but 40 mg/kg of theacrine showed inhibition on SIRT3. Low-dose theacrine (10 and 20 mg/kg) up-regulated hepatic LCAD, FABP4, HTGL, and ATP5c mRNA expression, while high-dose theacrine (40 mg/kg) down-regulated their levels. It shows that theacrine has an effect on fatty acid metabolism through regulating fatty acid metabolism related genes, and the mechanism may be related with its up-regulation of SIRT3 expression (32). Extract (50-200 µg/mL) of C. kucha with 1.44% (W/W/) theacrine showed significant suppressive effect on lipid droplet accumulation and triglyceride (TG) level in 3T3-L1 cells in a dose dependent manner by suppressing the expression of genes involving in lipid metabolism, including FAS, FAT, SCD-1, LPL, SREBP-1c, and ACC (33). Test on C57B/6J mice showed that theacrine significantly decreased hepatic TG content (p < 0.01) and ameliorated hepatic steatosis in mice (34). Acylcarnitine metabolism disorder contributes significantly to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Theacrine ameliorated acylcarnitine metabolism disorder in high-fat diet (HFD)-fed mice, resulting in suppression of hepatic steatosis and liver inflammation and improvement of energy expenditure through the SIRT3/LCAD signaling pathway (35).
The underlying mechanism that theacrine ameliorates high fat diet induced hepatic steatosis in mice involves in SIRT3/AMPactivated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway, during which the activation of ACC by theacrine depends on the phosphorylation of AMPK, but the activation of SIRT3 by theacrine is independent of the phosphorylation of AMPK (34). Theacrine promotes acylcarnitine metabolism in non-alcoholic fatty liver disease (NAFLD) through the SIRT3/LCAD signaling pathway, in which the target of theacrine's activities on NAFLD is identified as SIRT3 (35). Theacrine upregulated the expression of SIRT3 as well as the phosphorylation of AMPK and ACC (28). Theacrine activated the mitochondrial deacetylase SIRT3 and consequently, the increased activity of long-chain acyl coenzyme A (CoA) dehydrogenase (LCAD) through deacetylation (32,35). Peroxisome proliferatoractivated receptor γ (PPAR γ) and CCAAT/enhancer-binding protein α (C/EBP α) are considered to be the key regulators of adipogenesis (47,48). Theacrine suppressed the lipid droplet accumulation in 3T3-L1 adipocytes by downregulation of the expression of major transcription factors of adipogenesis pathway including PPAR γ and C/EBP α (33). Furthermore, theacrine also decreased the mRNA and protein levels of fatty acid synthase, fatty acid translocase, steroylcoenzyme A desaturase-1, lipoprotein lipase and acetyl-CoA carboxylase-1 (33).

Inhibiting Breast Cancer Cell Metastasis
Theacrine also inhibited transforming growth factor-β (TGF-β) induced cell adhesion, migration, and invasion, showing antimetastatic potential implications for disease management in breast cancer (36). Epithelial-to-mesenchymal transition (EMT), whereby epithelial cells transform into mesenchymal cells, is shown to be related to cancer and cancer cell metastasis. The EMT process is coordinated by the loss of expression of epithelial marker such as Occludin and E-cadherin, accompanying with gain of mesenchymal markers such as Vimentin, Fibronectin and N-cadherin, which promote tumor progression, cell invasion, and metastasis (49). The EMT markers expression is associated with poor prognosis of breast cancer patients (50,51). TGF-β-induced EMT has been recognized as an emerging mechanism underlying its metastasis-promoting function (52). The regulators of EMT have become attractive targets for developing anti-metastasis therapies. Theacrine reversed EMT, resulting in downregulation of mesenchymal markers including Fibronectin, Vimentin, N-cadherin, Twist, and Snail and upregulation of epithelial markers such as Occludin and E-cadherin in human breast cancer MDA-MB-231 cells. Also, theacrine attenuated TGF-β-induced EMT, cell adhesion, migration, and invasion in the MDA-MB-231 cells. These suggest that theacrine shows suppressive effects on TGF-β-induced progression and breast cancer cell metastasis by reversing the EMT process (36).
Furthermore, there was study revealing that theacrine showed anti-virus activity. Theacrine at 50 µM dosage significantly inhibited the expression of NS1 protein in human influenza virus A/Puerto Rico/8/34 at 12 hpi, and 100 µM theacrine significantly suppressed the virus progeny production (37).

CONCLUSIONS AND FUTURE EXPECTATIONS
Theacrine is a purine alkaloid found in Camellia kucha and it shows diverse health benefits. Theacrine upregulates the expression of SIRT3 and activates the SIRT3/FOXO3/SOD2 signaling pathway, resulting in antioxidant efficiency. Theacrine upregulates the expression of RNA and protein of SMAD3, p-ERK and p-p38 and downregulates the expression of NF-kB and IL-6, showing anti-inflammatory effects. Theacrine acts as an adenosine receptor antagonist to play a role in the locomotor activation and fatigue reduction. Theacrine regulates brain glucose metabolism, inhibits phosphodiesterases and restores the levels of 5-HTP and dopamine to improve cognitive capacity. Theacrine possesses potent sedative and hypnotic properties and its central nervous system effects through mediating the adenosine system. Theacrine ameliorates lipid metabolism by activating SIRT3/AMPK/ACC pathway and downregulating the mRNA and protein levels of fatty acid synthase, fatty acid translocase, steroylcoenzyme A desaturase-1, lipoprotein lipase and acetyl-CoA carboxylase-1. Theacrine shows suppressive FIGURE 3 | Bioactivities of theacrine. The end of each direction shows one bioactivity of theacrine, in which the intermediates show the actions of theacrine or signaling pathways. CAT, Catalase; EMT, Epithelial-to-mesenchymal transition; FOXO3, Forkhead Box O3, a protein coding gene, including DNA-binding transcription factor activity and protein kinase binding; GSH-Px, Glutathione peroxidase; IL-6, interleukin-6; LCAD, Lightweight chemical agent detector; NF-kB, Nuclear factor-kb; ORAC, oxygen radical absorbance capacity; PD, Parkinson's disease; p-ERK, phosphorylated extracellular regulated protein kinases; p-p38 MARK, phosphorylated mitogen-activated protein kinases; MDA, malondialdehyde; SIRT3, Sirtuin-3, a major mitochondria NAD + -dependent deacetylase; SMAD, the proteins are required for the transmission of the TGF-β signal to the nucleus; SOD, Superoxide dismutase; TGF-β, Transforming growth factor-β. The red up arrow means increase in the indicators and the blue down arrow means decrease in the indicators. effects on TGF-β-induced progression and breast cancer cell metastasis by reversing EMT, which in turn leads to the downregulation of mesenchymal markers and upregulation of epithelial markers, and also attenuating TGF-β-induced EMT, cell adhesion, migration, and invasion. The bioactivities of theacrine are shown in Figure 3.
However, there is still a long way to go to the clinical application of theacrine. First, the mechanism underlying the physiological activities of theacrine remains to be further explored. Theacrine has similar chemical structure as caffeine, but how their physiological activities are differentiated greatly? The mechanism why theacrine was stimulatory at higher doses, but it could actually have a sedative effect at low doses remains to be clarified (30). Further studies need to elucidate the mechanisms by which theacrine downregulated the EMT markers (36). Second, drug-drug interaction between theacrine and other compounds needs to be further investigated. It was found that co-administration of theacrine and caffeine resulted in a clinically significant pharmacokinetic interaction, viz., caffeine co-administration increased maximum plasma concentration and the area under the curve of theacrine without altering theacrine half-life in comparison with the corresponding pharmacokinetic parameters when theacrine is administered alone (53). There was pharmacokinetic interaction between methylxanthines and theacrine in humans when theacrine (50 mg), methylliberine (100 mg) and caffeine (150 mg) were orally administered (54). More work needs to probe how the enhanced oral bioavailability was happened. Third, some inconsistent results remain to be reconfirmed. It was reported that acute intake of the theacrine-containing dietary supplement did not improve cognitive performance statistically though it favorably impacted multiple subjective feelings related to energy and mood (38). There was study showing that neither theacrine (300 mg), caffeine (300 mg) and nor combination of both theacrine (150 mg) and caffeine (150 mg) improved muscular strength, power, or endurance performance in resistancetrained men when consumed 90 min pre-exercise (39). Caffeine treatment led to a marked increase in the ambulatory activity accompanied with decreasing of the immobility time in forced swimming test at both 10 and 30 mg/kg. Under the same conditions, neither theacrine nor theobromine showed obvious excited efficacy (29,55). The clarification of these issues would be greatly helpful for developing clinical applications of theacrine.

AUTHOR CONTRIBUTIONS
Y-RL, Y-YS, and X-QZ conceived the project and references collection. Y-YS introduction. JX toxicological evaluation of theacrine. Z-SW and JJ anti-oxidant effects. Y-QW antiinflammatory effects. Q-SL and Z-SW locomotor activation and reducing fatigue. Z-TF and DL improving cognitive performance. J-LL and J-HY ameliorating lipid metabolism. Y-RL inhibiting breast cancer cell metastasis and abstract. X-QZ conclusions and future expectations. All authors contributed to the article and approved the submitted version.