Ruonn Yn, Chi-Tng Ho*, Xin Zhng,*

a Department of Food Science and Engineering, Ningbo University, Ningbo 315211, China

b Department of Food Science, Rutgers, The State University of New Jersey, NJ 08901, USA

Keywords:

Tea

Polyphenols

Intestinal microbiota

Circadian rhythm

A B S T R A C T

Tea is a widespread functional plant resource.Phytochemicals such as tea polyphenols (TP) can interact with the intestinal flora and participate in regulating the expression and rhythm of biological clock genes.Circadian rhythm controls a variety of behaviors and physiological processes, and circadian misalignment has been found to be closely related to multiple metabolic diseases.Interestingly, the gut microbiota also has diurnal fluctuations, which can be affected by diet composition and feeding rhythm, and play a role in maintaining the host’s circadian rhythm.The two-way relationship between the host’s circadian rhythm and intestinal microbiota confirms the possibility that prebiotics or probiotic can be used to adjust the intestinal environment and microbiome composition to improve the host health.This article reviews the relationship between the host’s circadian rhythm and microbiota and its influence on metabolic diseases.The beneficial effects of the interaction between TP and gut microbiota on diseases related to rhythm disorders are emphasized to improve the theories of disease prevention and treatment.

1.Introduction

Drinking tea has a long history as part of the daily life for millions of people.It is the most commonly consumed beverage in the world, second only to water [1].The habit of tea drinking can improve work performance and reduce fatigue.Tea has been found to increase the negative effects of evening recovery, and also increase morning mood and daytime concentration [2].There are more and more evidence related to health benefits of tea drinking,and the relationship between active compounds in tea (such as tea polyphenols (TP)) and intestinal health is gradually strengthening [3].Recent research shows that TP can prevent and treat diseases related to circadian rhythm.The crucial role of TP in host health through the gut-brain axis (GBA) has attracted more and more attention [4](Fig.1).

Fig.1 Circadian rhythm, tea, microbiota-gut-brain axis and metabolism.

The circadian rhythm is an endogenous oscillation of approximately 24 h, involved in regulating various changes in the host’s physiological and biochemical processes.In mammals, the circadian rhythm is controlled by the central clock located in the suprachiasmatic nucleus (SCN) of the brain and the peripheral clocks composed of liver, intestine and adipose tissues [5].As the main regulator of circadian rhythm, SCN can send signals to the peripheral cells of the whole body, so that the rhythm can be transmitted to appropriate cells and tissues [6].It has been reported that long-term exposure to irregular light/dark cycles, eating at the wrong time(such as late at night), staying up late on weekends (or social jet lag), and shift work can cause disturbances in the circadian rhythm,induce metabolic changes, and increase the risk of cancers, metabolic syndrome and cardiovascular diseases [7].

The composition of the microbiota is critical to the health of the host, gut health depends on maintaining a balanced microbial community in the highly dynamic environment of the gut [8].Many factors can affect the distribution of the gut microbiota, including genes, environment and diet.More and more pre-clinical studies have shown that the flora in the intestine changes throughout the day,and the relative abundance of microbial species and the metabolism of microbes all show a circadian rhythm [9].The diversity and composition of gut microbes are closely related to the use of antibiotics, obesity, allergies, inflammatory diseases and metabolic conditions (such as type 2 diabetes and cardiovascular diseases).Disturbances in the intestinal environment will affect the health of the host, and in turn the emergence of these diseases will also affect the intestinal ecosystem [10,11].The host provides important habitats and nutrients for the microbiome.The intestinal flora supports the development of the metabolic system and the maturation of the intestinal immune system by providing beneficial nutrients, such as the synthesis of vitamins and short-chain fatty acids (SCFAs) [12].Recent study has shown that the intestinal microbiota not only acts on the host’s digestion, metabolism and immune function, but also modulates the host’s sleep and mental state [13].These are achieved through the microbiota-gut-brain axis, and closely interact with emotions, physiological stress, and circadian rhythms.Additionally,the response of gut microbiota to immunization, the success rate of eradication ofHelicobacter pylori, and metabolic syndrome are related to obesity, liver cancer, and neurodegenerative diseases such as autism [14].The purpose of this review is to focus on the effects of tea metabolism in the gut microbes on the host’s circadian rhythm and disease, the causes of host metabolic diseases, and how the intestinal microbiota and circadian rhythm disorders are related to the development of diseases.These will help us better explore the mechanisms by which tea and its functional active substances participate in regulating host health.

2.In fluence of disturbances to either the circadian rhythm or the gut microbiota on host physiology

2.1 Importance of host circadian rhythmicity on the state of health via the perspective of the GBA

The cyclic pattern of circadian protein expression and other rhythm elements depend on many external conditions or timing factors,including light, eating patterns, exercise and others [15].The central biological clock controls many aspects of mammalian physiology,including sleep, metabolism, and immune system regulation [16].It is reported that these factors can assist the host to synchronize the oscillating expression of up to 10% of the genes, thereby contributing to maintaining the health of the host or improving the metabolic process [17].It is worth noting that emotion-related nervous systems,such as the limbic brain area, monoamine neurotransmitters and the hypothalamic-pituitary-adrenal (HPA) axis, are all under the modulation of circadian rhythms [18].The biological clock system of mammals is cell autonomous, and the circadian rhythm control of these systems partly depends on transcriptional regulation.Recently, there is evidence that genome-wide regulation of the clock is carried out through circadian chromosomal organization [19].The circadian rhythm is also regulated after transcription.The clock directly controls metabolism by driving transcription programs that drive certain metabolic pathways.For example, CRY1 regulates cAMP/CREB signal, rhythmically inhibits glucocorticoid receptor genes, inhibits nuclear FOXO1, and then down-regulates gluconeogenesis, thereby inhibiting liver gluconeogenesis during fasting [20].Past study has shown that if the host becomes abnormal about one of the circadian rhythms in the feeding cycle and sleepwake cycle, the host may encounter health problems to a large extent.For example, shift workers who change working hours are more likely to suffer from metabolic diseases such as obesity, hypertension,and diabetes [21].In a new study, some researchers speculate whether the gut microbiota may be involved in regulating the biological clock in the intestine, and thus may be indirectly involved in weight gain.It was confirmed that the gut microbiota turned on the expression of histone deacetylase 3 (HDAC3) in epithelial cells located in the inner wall of the small intestine.This in turn causes the intestinal flora to participate in the synchronous oscillation of gene expression, which is related to lipid metabolism and nutrient transportation.In contrast,mice with sterile intestines did not show this rhythmic regulation.They also found that HDAC3 plays a role in promoting lipid absorption and leading to obesity [22].

Generally speaking, the intestinal flora acts on the brain through the nervous system (intestinal cranial nerve anatomical pathway),and also acts on the brain through the endocrine system, immune system and metabolic system which indirectly affects cognition,sleep, academic ability and emotion.Therefore, the two-way communication between the gut and the brain is always called as GBA [23].The gut microbiota is directly involved in the production of a variety of neurotransmitters, cytokines, and metabolites, such as 5-hydroxytryptamine (5-HT), dopamine, gamma-aminobutyric acid(GABA), SCFAs, and melatonin.These metabolites can directly act on the enteric nervous system and the vagus nerve, and affect the activity of the central nervous system (Fig.2) [13].The treatment ofLactobacillus rhamnosusreduces stress-induced anxiety and depression-related behaviors through the vagus nerve-dependent pathway, and its improvement is associated with the increase of(GABA)Aα1mRNA expression and the decrease of (GABA)Aα1expression in the hippocampus [24].

Fig.2 Two-way communication and function between the intestine and brain via various systems.

2.2 The mediating role of gut microbiota in maintaining host health

Early colonization of certain intestinal types can have a lasting impact on the health of the host.For example, infants with a higher abundance ofBifidobacteriumandCollinsellaat 6 months have a lower incidence of obesity at 18 months [25].In another study, the abundance ofBifidobacteriumandBacteroidesin obese subjects was relatively reduced [26].Throughout the life cycle of the host,the composition and structure of the gut microbiota are not fixed,but due to the influence of external factors such as diet, environment and disease in the host’s daily life, these changes are difficult to summarize.The intestinal flora participates in the digestion and decomposition of food in the intestine, and produces SCFAs to provide energy for the host and stabilize the internal environment.It also produces various metabolites such as ammonia, amines, thiols, phenols and indole [27].In healthy intestines, Bacteroides and Firmicutes account for more than 90% of the total number of bacteria [28].Diet is the most studied and most promising factor to change the intestinal flora.Some dietary interventions (such as increasing fiber intake) can quickly change the level of nutrients, thereby changing the composition of the microbiota and promoting the abundance and diversity of microbes [29].A systematic study showed that various metabolic diseases, such as arthritis, atherosclerosis, inflammatory bowel disease, as well as diabetes and obesity, showed significant changes in patients not only in the composition and structure of the host gut microbiota, changes are also reflected in the homeostasis and metabolism of the host’s intestinal tract [28].In essence, the intestinal microbiota can influence and even promote various metabolic processes, such as regulating the metabolism of heterogeneous organisms and energy production.As discussed earlier in this review,external factors such as diet and environment play important roles in maintaining the homeostasis of the gut microbiota [30].

Recent study has shown that there is a connection between the gut microbiota and the central nervous system, and the imbalance of the central nervous system may be a possible cause of depression and other mental illnesses.Gastrointestinal tract dysbiosis can also affect metabolic disorders, obesity, heart diseases and type 2 diabetes [31].In patients with autism, schizophrenia, Alzheimer’s disease (AD)and Parkinson’s disease (PD), changes in the gut microbiota can be observed.Interestingly, when the gut microbiota of patients with these diseases was transferred to germ-free animals, many symptoms began to appear [32].For instance, sterile animals receiving the gut microbiota of patients with depression showed increased levels of anxiety-related and depressive behaviors.Similarly, germfree animals that received the gut microbiota of patients with PD developed dyskinesia and neuroinflammation [33].In a study,changing from a low-fat diet to a high-fat, high-sugar diet changed the structure of the microbial community within a day, changing the metabolic pathways and gene expression in the microbiome [28].In addition to changes in the composition of the intestinal microbiota,the metabolic potential of the intestinal microbiota has gradually been identified as an influencing factor in the occurrence of diseases.In reality, the function of the intestinal flora is similar to an endocrine organ, which can produce biologically active metabolites and affect the physiological function of the host.Microorganisms interact with the host through a variety of pathways, including trimethylamine(TMA)/trimethylamineN-oxide (TMAO) pathway, SCFAs pathway,and primary and secondary bile acid pathways [27].A recent study showed that volatile fatty acids such as isovaleric acid are increased in the stool of patients with depression.Isovaleric acid can pass through the blood-brain barrier and affect the release of neurotransmitters in the central nervous system, which may aggravate depression symptoms [13].In general, the microbiota has a fundamental impact on the host’s metabolism, and a healthy gut microbiota plays an important role in the overall health of the host.

3.Diseases associated with dysfunction of both circadian rhythms and the gut microbiota

Due to changes in daily life and environmental factors, including adopting unhealthy eating patterns and sleeping habits, multiple metabolic diseases may occur [34].In the past few decades, the prevalence of mental health disorders has increased substantially,including depression, anxiety, cognitive function, and sleep disorders.In addition, it is worth noting that depression and anxiety are often associated with human mental conditions and related to other non-communicable diseases, such as cardiovascular disease, dementia,and cancer [35].

The core circadian rhythm genes manage the 24 h molecular clock through a linked transcription-translation feedback loop, thereby generating circadian rhythm regulation for important functions of the body, such as hormone release (melatonin, cortisol, etc.), body temperature, heartbeat and sleep [36].Disturbed circadian rhythms caused by jet lag or reduced sleep can cause mood swings and sleep disturbances [13].Patients with major depression show significant disturbances in the circadian rhythms of multiple brain regions and the phase shift of core clock genes [37].The destruction of the core molecular clock is both a consequence and a contributing factor of neurodegenerative diseases.For example, the aggregation ofβ-amyloid (Aβ), which is a hallmark of AD, can cause the degradation ofBmal1, leading to interruption of the molecular clock [38].In addition, the imbalance of the circadian rhythm disrupts the internal synchronization of the HPA axis.In the case of circadian rhythm disorders, external pressure continues to stimulate the HPA axis, which may cause damage to the mental health of shift workers.Stimulating the (desynchronized) HPA axis activates the fear system,causing an abnormal response to stress [18].

Biological clock imbalance, lack of sleep and shift work can change the expression of circadian rhythm genes and the structure of microbial community [39].Study has found that the abundance of the two main components of the intestinal microbiota, Bacteroides and Firmicutes, changes periodically with the circadian rhythm [40].The number of mouse intestinal microbiota and the abundance of special species such asClostridiumspp.oscillate in the light-dark cycle [41].In addition to the role of circadian rhythm in mental illness, many cardiovascular functions such as blood pressure, heart rate and thrombosis are affected by circadian rhythm.It is worth noting that the occurrence of acute myocardial infarction, stroke and arrhythmia is also related to the biological clock [42].Most neurodegenerative diseases such as AD and PD are manifested by the presence of plaques of different proteins in the brain, neuronal cell death, etc.,and the host is accompanied by circadian rhythm disorders [43].Experiment has confirmed that AD patients show a severely atrophied SCN, accompanied by a significant decrease in melatonin levels and clock expression, suggesting the importance of circadian rhythm in the disease.At the same time, this may also indicate that pathological indicators in AD patients may reduce the effectiveness of circadian rhythm modulation [44].In addition to the effects of circadian rhythms, researchers have also found that the role of gut microbiota in PD also has a considerable impact.The count of Enterobacteriaceae is higher in PD patients and the count ofPrevotellais lower [45].

Research has confirmed that intestinal problems in children early in life may lead to autism.Disorders related to the nervous system,such as attention deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD), exhibit abnormal behaviors in social and/or group activities [46].Compared with the control group, a group of ASD children with a history of gastrointestinal disease had changes in the metabolism of the gut microbiota.The levels of 5-aminovalerate(considered a weak GABA agonist) and 3-(3-hydroxyphenyl)propionic acid (an antioxidant) in the ASD group were reduced [47].And compared with the healthy control group, the relative abundance ofProteobacteriaandBacteroidesin ASD children is higher, while the relative abundance of Firmicutes andBifidobacteriumis lower [48].Hsaio et al.[46]found that by changing the response of the HPA axis to stress, supplemental intake ofBacteroides fragilliscan improve intestinal permeability, change the microbial composition, and also improve behavioral defects such as communication disorders, anxiety and slow response.

4.Dietary supplement of tea and TP ameliorate circadianrelated metabolic problems

4.1 Tea and gut microbiota: regulation of sleep and mood

Sleep is a basic physiological requirement, and many patients with mental illness show interrupted sleep patterns and changes in the gut microbiota [13].It has been reported that people spend about 30% of their lives sleeping or trying to sleep, and patients with insomnia are more prone to emotional distress and recurring unhealthy symptoms [49].Study has confirmed that sleep deprivation of healthy people will have a great impact, such as distraction, cognitive decline, memory decline, emotional irritability, and others.These all indicate that sleep is crucial to humans from different aspects [50].Disturbed circadian rhythms caused by jet lag or reduced sleep can cause mood swings and sleep disturbances.Sleep is not only necessary for body growth and disease recovery, but also for a series of brain functions such as nerve cell growth, synapse formation and memory function [13].Sleep and circadian rhythm disturbance are the core features of mood disorders [51].A summary study showed that black tea, oolong tea,dark tea can adjust the microbial diversity and the ratio of Firmicutes and Bacteroides.These findings seem to support the hypothesis that drinking tea can beneficially regulate the distribution of intestinal microbiota and help suppress intestinal dysbiosis caused by obesity or a high-fat diet [52].(-)-Epigallocatechin-3-gallate (EGCG) in green tea can counteract the excitement caused by caffeine.While drinking tea daily has become a habit, drinking tea is also used by many people as a way to relieve stress.EGCG can suppress hyperactivity symptoms and irritability caused by caffeine.Observing the slow wave or fast wave ratio of electroencephalograph in animal experiments shows that EGCG has anti-dopaminergic properties [53,54].Supplementing the host with TP has been proven to improve hippocampal learning and memory impairment caused by chronic sleep deprivation through antioxidant effects [55].Interestingly, research has found that 300 mg of EGCG of diurnal life can help relieve patients’ irritability and stress.Compared with some people who have never had the habit of drinking tea, the risk of depression gradually decreases with the increase in tea consumption [56,57].

4.2 Gut microbiota-derived metabolites and SCFAs on host health

Certain endogenous factors or external environmental factors can destroy the structure of intestinal flora, leading to irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), diarrhea and some other gastrointestinal diseases [58].Though the mechanisms of how the gut microbiota interacts with the circadian rhythm are still unclear, it can be partly understood as the oscillation of microbial metabolites.A large number of microbial metabolites to be explored are likely to have a significant impact on human physiology and the development of diseases.Their mechanism of action may not be limited to the intestinal environment, it may extend to surrounding tissues (Fig.3) [59].The intestinal microbiota participates in normal digestion, can affect the energy obtained from the diet and the energy storage in the host, and ferment unusable energy substrates such as dietary fiber into SCFAs [60].Under the action of intestinal microbes,TP can be converted into small molecules of phenolic acids and then be methylated, glucuronated, sulfated or cyclically fissioned into the blood, and then show more effective physiological activities than themselves [61].Previous study has indicated that the bioavailability of polyphenols and other components in tea may be increased by intestinal microorganisms converted into small molecules.Their microbial metabolites can reach a concentration of μmol/L or even mmol/L in the colon, and nmol/L or even μmol/L in the systemic circulation.These metabolites have multiple functions such as antioxidant function, reducing inflammation, protecting intestinal mucosa, and reducing liver fat content [62].

Fig.3 Effects of intestinal microbiota and its metabolites on host health.

SCFAs are fatty acids in the distal part of the large intestine that are produced by bacterial fermentation of insoluble fiber from dietary plant materials, which escape digestion in the upper digestive tract and enter the colon.SCFAs are the center of colon physiology and metabolism.Parasitic bacteria can also metabolize dietary carcinogens, synthesize vitamins, and help absorb various molecules.Most SCFAs (90%-95%) present in the colon are composed of acetate(60%), propionate (25%) and butyrate (15%).Butyrate is considered to be the main energy source of colonic epithelium.SCFAs are related to improving the metabolic function of type 2 diabetes,including controlling blood sugar levels and insulin resistance [63,64].Akkermansia muciniphilais a common mucus-degrading bacterium.A.muciniphilacan produce SCFAs by breaking down mucin, which stimulates goblet cells to produce more mucus, thereby supplementing or maintaining the integrity of the intestinal barrier.In addition,A.muciniphilamay reduce the abundance ofFirmicutes andClostridium, thereby promoting intestinal homeostasis and having a positive impact on host health [65].Previous study indicated that polyphenols seem to have the ability to change the balance of the intestinal microbial environment, and fecal microbial metabolism of polyphenols and its effects on human gut microbiota have been emphasized [66].

Diet has a significant impact on the changes in the composition of the gut microbiota through the process of biotransformation in the intestine [4].Recent study has shown that the intestinal microbiota produces a large number of metabolites, some of which are absorbed into the systemic circulation, while others are further metabolized by host enzymes and act as mediators for microorganisms to affect the host [27].For example, in anin vitroliver organoid model, the addition of butyrate (a more significant change) or acetate causes the difference in circadian gene expressions [67].SCFAs are the products of colonic microorganisms fermenting indigestible fibers.Due to their health benefits, they are currently the most studied microbial products [68].SCFAs can not only promote the division and differentiation of intestinal epithelial cells, but also modulate and maintain the host’s mineral balance and promote the absorption of iron, calcium, and magnesium [69].Supplementing SCFA butyrate in the diet has been shown to prevent weight gain and improve insulin sensitivity by increasing energy consumption [70].At the same time, butyrate and propionate have been shown to induce intestinal gluconeogenesis and improve glucose production in the body through the gut-brain pathway [71].

4.3 Through the interaction of TP and intestinal microbiota to improve the diseases associated with rhythm disturbance

4.3.1 The modulation of TP on host circadian rhythm

Evidence shows that polyphenols in the diet, such as tea catechins,resveratrol and quercetin, can change the cycle, amplitude or phase of the biological clock, and alleviate the symptom caused by circadian rhythm disorders [72].Particularly, it is reported that TP can reverse the abnormal expression of circadian clock genes and alleviate neural redox imbalance and abnormal mitochondrial function by participating in the mechanism of the key circadian rhythm regulatorBmal1[73].EGCG reversed glucosamine-induced lighter daily oscillations of clock gene transcription and protein expression in HepG2 cells [74].In our recent study, when the mice were placed in a constant dark environment, we observed that the relative abundance of the oscillating microbiota in the mouse intestine was reduced, and some circadian genes lost their normal rhythmic expression, and then supplemented with Oolong tea polyphenols (OTP) can obviously alleviate this situation.The data showed that after continuously feeding OTP to mice under dark conditions, the mRNA expressions ofClockandBmal1were similar to those in the control group (normal light-dark cycle) group, indicating that OTP effectively alleviated the effects of darkness on mice at the genetic level [75].

4.3.2 TP interacts with intestinal microbiota to improve diseases associated with host rhythm disorders

Now, more and more studies have proved the various potential health benefits of drinking tea and supplementing its functional ingredients.Drinking tea has many benefits, including lowering cholesterol, anti-aging, anti-bacterial and promoting metabolism [52,58].In addition, tea in the traditional sense has the effects of refreshing and relieving fatigue.Recent studies have shown that TP is beneficial to sleep, improves various problems caused by the nervous system,and participates in regulating the homeostasis of the host’s intestinal microbiota [76].It has also been found that TP can improve health in different ways, such as improving cardiovascular and cerebrovascular diseases and neurodegenerative diseases through the blood-brain barrier [4].In the intestine,LactobacillusandBifidobacteriumhave been found to inhibit a variety of pathogens, improve the digestion of lactose in the host, enhance the intestinal barrier function, and regulate the structure and function of the intestinal microbiota [77].In our previous study, it was found that after supplementation of(-)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3”Me) to obese mice induced by a high-fat diet, the relative abundance of some dominant microbiota changed significantly at the levels of phylum, family and genus.The abundance of Bacteroidetes increased significantly, while Firmicutes decreased [78].

The gut microbiota is of great significance to the homeostasis of the host’s circadian rhythm.It is reported that the interruption of the circadian rhythm will reduce the species abundance of the intestinal microbiota and change the composition and function of the microbiota [67,79].Meanwhile, the gut microbiota is a key factor that mediates the physiological functions of dietary polyphenols.Monomeric polyphenol compounds, such as flavonoids; oligomeric polyphenol compounds, such as proanthocyanidins, are frequently converted to lower molecular weight derivatives by the intestinal microbiota in the colon.These catabolites may play an important role in the physiological functions of dietary polyphenols.They may also affect the microbiota and promote health by activating SCFAs excretion and intestinal immune function [80].Polyphenols, including flavonoids, are often used as antioxidants because of the phenolic groups in their structure that can donate electrons.Some studies have explored their antioxidant function in preventing oxidative stress-related cell and extracellular damage, and found that these have an important role in promoting vascular health and preventing atherosclerosis [81].The antioxidant properties of TP against free radicals allow it to provide protection against cancer stages by preventing inflammation and tumor development, and by inducing cell apoptosis and cell cycle arrest [82].A large amount of evidence fromin vivoandin vitrostudies shows that TP has beneficial effects on a variety of diseases including cancers, diabetes and cardiovascular diseases [83].EGCG is the main catechin in tea.A cup of green tea contains up to 200 mg of EGCG, and has been shown to have cancer prevention and/or chemotherapy effects [84,85].

5.Conclusions

The intestinal flora is generally considered to be related to the destruction of the blood-brain barrier and the pathogenesis of various metabolic diseases.The interaction between TP and the intestinal flora can regulate metabolic diseases related to the circadian rhythm through the microbe-gut-brain axis, thereby contributing to the health of the host.In addition, attentions should be paid to the effects of TP in circadian rhythms regulation as well as their effective dose.In the future, we need to better understand the absorption and metabolism process of TP, the interaction between TP and intestinal microorganisms as well as the signal transduction effect of their metabolites transmitted to the brain through the intestine.

Acknowledgements

This work was sponsored by Zhejiang Provincial Natural Science Foundation of China (LY19C200006), the Ningbo Natural Science Foundation (2019A610433), Zhejiang Provincial Key Research and Development Program (2020C02037) and People-benefit Project of Ningbo (202002N3078).

Conflicts of interest

The authors have declared no conflict of interest.