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The alkaloids in tea plants are mainly purine alkaloids, with a small amount of pyrimidine base. Purine alkaloids are the secondary metabolite of purine nucleotide[2], including methylxanthine and methyluric acid. Their chemical structures are based on the skeleton of xanthine and uric acid[3], differing by the number and position of methyl groups on the purine ring. The three main purine alkaloids in tea plants are caffeine, theobromine and theacrine.
Caffeine
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1,3,7-trimethylxanthine, its chemical formula is C8H10N4O2 (Fig. 1c). Caffeine is the most abundant purine alkaloid in tea, generally accounting for 2%−5% of the dry matter content[4]. It is one of the most important quality and functional components in tea. Chen & Zhou[5] analyzed the caffeine content of 596 accession tea germplasms preserved in China National Germplasm Hangzhou Tea Repository (CNGTR), which basically represent the genetic diversity of Chinese tea plants. The caffeine content varied from 1.2% to 5.9%, averaging 4.2%.
Theobromine
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3,7-dimethylxanthine, the chemical formula is C7H8N4O2 (Fig. 1b). Theobromine, a precursor for caffeine synthesis, lacks a methyl group at the N-1 position compared to caffeine[6]. Theobromine is the main alkaloid of the cocoa plant. Generally, theobromine content is less in tea plants, and the theobromine content in young leaves is 0.05%−0.80%[7]. Among the tea plants in Sect. Thea (L.) Dyer Camellia L., C. ptilophylla Chang discovered in the 1980s is a new tea plant resource with high theobromine (4.7%) and no caffeine[8]. Additionally, C. gymnogyna Chang[9], C. irrawadiensis P. K. Barua, C. crassicolumna Chang, C. purpurea Chang et Chen[10], Hongyacha[11] are all naturally rich in theobromine, no or low caffeine tea genetic resources, respectively.
Theacrine
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1,3,7,9-tetramethyluric acid, the chemical formula is C9H12N4O3 (Fig. 1d), the structure is similar to caffeine, the difference is that the C8 position is increased by a carbonyl group and the N9 position is increased by a methyl group[12]. Theacrine is a special purine alkaloid contained in Kucha, a special tasting bitter tea plant. Kucha once was classified into C. kucha (Chang et Wang) Chang[13], C. sinensis var. kucha and C. assamica var. kucha, but actually it has no significant morphological difference with C. sinensis. Ye et al. isolated and identified a high content of 1,3,7,9-tetramethyluric acid (1.29%) in the young shoots of Kucha for the first time, and they found a new distribution pattern of purine alkaloids in tea plants[14]. The theacrine content in the tender shoots with 'two and a bud' of Kucha was 1.3%−3.4%. The pure theacrine could be obtained from Kucha leaf by high-speed countercurrent chromatography[15].
In addition, the purine alkaloids found in tea also include theophylline (1,3-dimethylxanthine) (Fig. 1a), which is the isomer of theobromine, and the content in tea is much lower, generally about 0.05%. It occurs naturally in trace amounts in coffee and cocoa (Table 1).
Table 1. Purine alkaloids in tea, coffee and cacao (%).
Purine alkaloids Tea Coffee Cacao Arabica Robusta Caffeine 2.0−5.0 1−1.5 2−2.7 1.5 Theobromine 0.06−1.0 Trace Trace 1.8 Theacrine 0.5−3.6 (rare) / / / Theophylline 0.05 Trace Trace Trace Physiological function
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Purine alkaloids not only affect the flavor of tea, but also have important biological activities and physiological functions. Although several major purine alkaloids in tea have similar structures, their functions are quite different.
Caffeine
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At present, the role of purine alkaloids in plants has not been fully determined, there are two hypotheses to explain the physiological role of caffeine in plants, the 'chemical defense' and 'allelopathic function' theories[16]. The 'chemical defense' proposed that high concentrations of caffeine in young tissues and fruits of Coffea arabica and Camellia sinensis have a defensive effect, protecting young shoots from pathogens and reducing herbivore feeding. And the 'allelopathic theory' proposes that the release of caffeine in the seed coat and fallen leaves into the soil can inhibit the growth of competitive plants and regulate the plant spacing[17]. Caffeine in plants has long been thought to have a defensive effect, acting as a natural pesticide and antifeedant. But a recent study found that caffeine, which is naturally present in nectar of plants such as Citrus and Coffea, has no repellent effect on bees. Instead, it may affect the behavior of pollinators to enhance pollination and protect the normal reproduction of plants[18].
Caffeine occurs naturally in many foods and beverages, and most caffeine is consumed as coffee and tea. In long-term consumption, it has been found that caffeine has significant effects on cognitive, physical and ocupational performance[19]. Current research suggests that low to moderate (approximately 40−300 mg or ~0.5−4.0 mg kg−1) doses of caffeine improve cognition, which is mainly manifested in improving alertness, learning and memory[19]. Caffeine is structurally similar to adenosine, and exerts central nervous system (CNS) effects by antagonizing adenosine receptors (ARs)[20], affecting brain function. Epidemiological studies have shown that caffeine, as a non-selective adenosine receptors antagonist[21], has the potential to treat cognitive disorders, including Alzheimer's disease, Parkinson's disease, depression, and schizophrenia[20]. It has been confirmed in many studies involving animals and humans[22]. By studying a transgenic mouse model for Alzheimer's disease (AD), it was found that caffeine can rapidly reduce the level of abnormal protein (amyloid-β; Aβ) in the plasma of AD mice and humans (the core of AD pathogenesis)[23], and reverse AD-induced cognitive impairment in aged mice[24]. Decreased levels of Aβ protein may be the mechanism of action of caffeine to improve cognition[25]. In addition, studies have shown that caffeine also has pharmacological effects such as anti-obesity[26], analgesia[27], diuresis[28], reducing the risk of type II diabetes[29] and cardiovascular mortality[30].
Low doses of caffeine had positive effects on cognition, while higher doses improved exercise performance. Doses > 200 mg (~3 mg kg−1) of caffeine showed ergogenic properties on endurance, muscle strength, and high-intensity exercise[19]. Special occupational groups, especially athletes, should control the time and dosage of caffeine intake.
Theobromine
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Compared to caffeine, theobromine's effects are not well studied. Caffeine is a central nervous system stimulant, while theobromine is a smooth muscle stimulant, promoting vasodilation[31]. The hypotensive effect of cocoa are well established[32], theobromine seems to be partially responsible for this effect. A randomized, double-blind crossover trial demonstrated that high-dose theobromine lowers central systolic blood pressure and is beneficial for cardiovascular health in humans[33]. In addition, theobromine may have hypoglycemic effects[34]. In many cases, theobromine is considered a natural compound with therapeutic properties. An in vitro study have shown that theobromine inhibits uric acid crystallization and has high clinical potential in the treatment of uric acid nephrolithiasis[35]. Usmani et al. demonstrated for the first time that theobromine suppresses cough without CNS side effects. It can be used clinically to treat acute and chronic cough[36]. Similar to caffeine, theobromine has potential obesity therapeutic[37] and neuroprotective effects[38]. Notably, theobromine is less stimulatory to the CNS and may have a higher potential than caffeine. In addition, theobromine also has special effects of protecting tooth enamel[39], immune regulation[40], and anti-inflammation.
Theacrine
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Sheng et al.[41] comprehensively reviewed the beneficial health effects of theacrine, including antioxidant effect, anti-inflammatory effect, reducing fatigue effects, improving cognitive effect and hypnotic effect. Theacrine has been identified as non-toxic by toxicological tests, and a clinical study in which subjects took theacrine supplementation for 8 weeks also concluded that theacrine is safe for humans[42]. Its potential medicinal functions have been studied in recent years. A behavioral test showed that oral theacrine significantly reversed central fatigue-induced learning and memory impairments[43]. There are many studies proving that theacrine can prevent Parkinson's disease by directly activating the Sirt3 signaling pathway, restoring mitochondrial function and inhibiting the apoptosis of dopaminergic neurons[44]. Unlike caffeine, theacrine does not stimulate central excitation and is now considered a new antidepressant purine alkaloid drug[45].
Theophylline
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Theophylline occurs naturally in tea and cocoa beans. Although the content is extremely low, it has a high medicinal value and is an important alkaloid in tea plants. In 1985, theophylline was first extracted from tea and chemically synthesized. It was found that theophylline has diuretic and bronchodilating effects. Recent studies have shown that low-dose theophylline helps treat asthma and chronic obstructive pulmonary disease (COPD), with multiple anti-inflammatory effects. Clinically, it can be administered intravenously or orally. It is worth noting that high-dose theophylline has side effects, such as headache, vomiting and arrhythmia, clinical use should pay attention to the recommended dose[46].
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Purine alkaloids are a class of substances that exist in large quantities in non-alcoholic beverages, of which caffeine is the most important component. Beverages containing caffeine, such as coffee, tea, and functional drinks, have a large global consumption. Appropriate intake of caffeine is beneficial to human health, but excessive intake of caffeine or special populations' intake of caffeine may cause negative effects of anxiety and insomnia. Some consumers are naturally sensitive to caffeine intake. At present, the market demand for low-caffeine or decaffeinated products is increasing rapidly. To decaffeinate tea, manufacturers often treat it with supercritical carbon dioxide or hot water treatments. However, these methods can affect the flavor of the brew and destroy compounds in the tea associated with health benefits. Since Kato et al. successfully cloned the caffeine synthase gene in 2000, scientists began to explore the possibility of obtaining natural low-caffeine coffee or tea through genetic engineering. Using the identified gene encoding caffeine synthase, transgenic plants with reduced caffeine content can be produced by RNA interference. It has been reported that coffee seedlings with a 70% reduction in caffeine content have been obtained through genetic engineering[64], but there has been no relevant reports on tea.
Mohanpuria et al.[79] tried to use RNAi technology to produce low-caffeine content tea plants. Transgenic plants showed a significant suppression of Caffeine Synthesis transcript expression and also showed a reduction of 44%−61% in caffeine and 46%−67% in theobromine content as compared to controls[79]. These results suggest that the RNAi construct developed using a single partial fragment of CS gene reduced the expression of the targeted endogenous gene significantly[79]. It might be a potential molecular technique for generating low-caffeine tea plants.
Application of low caffeine TCS1 alleles
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Allelic variations of TCS1 play an important role in caffeine biosynthesis of tea plants. There are two molecular mechanisms controlling the caffeine biosynthesis in low-caffeine-accumulating tea germplasms. TCS1 natural variant reduces transcript level or its encoded protein has only theobromine synthase activity. Allelic variations of TCS1 can be used for the selection of low-caffeine tea plant varieties. Low caffeine individuals might be selected from progenies resulting from crosses between specific accessions containing TCS1b, TCS1c, or TCS1f, TCS1g with low caffeine biosynthetic activity[55]. Ogino et al. believed that the caffeine-free phenotype was closely related to the TCS1, and used a three-nucleotide insertion TTC in TCS1, developed a DNA molecular marker 'CafLess-TCS1', and confirmed that 'CafLess-TCS1' is an effective selection marker for breeding of caffeine-less tea cultivars[80].
Application of cocoa tea and Kucha
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Among hypernormal tea germplasms, although the health benefits of cocoa tea and Kucha have long been known, relevant scientific study is still very limited. In recent years, scientists have paid more attention to the discovery and utilization of natural tea germplasm rich in theobromine or theacrine. Hypernormal germplasms not only have different morphological and biochemical characteristics, but may also have unique regulatory mechanisms and candidate genes. Ye et al. screened out a batch of low-caffeine, high-theobromine tea germplasms from the wild population, and established the first natural caffeine-free tea germplasm base in China. In the later stage, the screening of excellent individuals of wild cocoa tea will be strengthened to further determine the pharmacological and physiological functions of cocoa tea. Several recent studies on Kucha have identified highly expressed TCS genes (TEA010054, TEA022559) at the transcriptional level[81], which are helpful to reveal the metabolic mechanism of purine alkaloids at the molecular level. This will help in identification and tea plant breeding rich in theobromine or theacrine.
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About this article
Cite this article
Zhang S, Jin J, Chen J, Ercisli S, Chen L. 2022. Purine alkaloids in tea plants: component, biosynthetic mechanism and genetic variation. Beverage Plant Research 2:13 doi: 10.48130/BPR-2022-0013
Purine alkaloids in tea plants: component, biosynthetic mechanism and genetic variation
- Received: 04 July 2022
- Accepted: 29 July 2022
- Published online: 18 August 2022
Abstract: The purine alkaloids, including caffeine, theobromine, and theacrine, are one of the most important quality and functional components of tea plants and commercial teas. In this paper, we review the component, biosynthetic mechanism and genetic variation of purine alkaloids in tea plants. The content of caffeine and theobromine in 403 accessions Chinese tea germplasms core collection preserved in the National Germplasm Hangzhou Tea Repository were analyzed using High Performance Liquid Chromatography (HPLC). The purine alkaloid profiles of different tea varieties, germplasm types, geographical origin, seasons, were highlighted. Some naturally low caffeine or caffeine-free, high theobromine or high theacrine germplasms were identified and the possible biosynthetic mechanism of hypernormal purine alkaloid content in tea plants were partially revealed. Some Cleaved Amplified Polymorphic Sequences (CAPS) DNA markers were developed for the identification of purine alkaloid content. The potential application of genetic engineering and DNA markers developed based on the low caffeine TCS1 (tea caffeine synthesis gene) alleles for marker assisted selection (MAS), and cocoa tea and kucha in low caffeine/high theacrine tea cultivar breeding programs were also reviewed.
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Key words:
- Alleles /
- Genetic variation /
- Purine alkaloids /
- Tea caffeine synthesis gene /
- Tea plants