Shiming Li*, Ling Zhng, Xiochun Wn, Jinfeng Zhn Chi-Tng Ho*

a Hubei Key Laboratory of EFGIR, Huanggang Normal University, Huanggang 438000, China

b State Key Laboratory of Tea Plant Biology and Utilization, School of Tea & Food Science, Anhui Agricultural University, Hefei 230036, China c Department of Food Science, Rutgers University, NJ 08901, USA

Keywords:

Tea chemistry

Flavonoalkaloids

RCS adducts

Aminated tea polyphenols

AGEs

A B S T R A C T

Myriad evidence attests to the health-promoting benefits of tea drinking.While there are multiple factors of tea influencing the effective biological properties, tea polyphenols are the most significant and valuable components.The chemical characterization and physical characteristics of tea polyphenols have been comprehensively studied over the previous years.Still the emergence of new chemistry in tea, particularly the property of scavenging reactive carbonyl species (RCS) and the newly discovered flavoalkaloid compounds,has drawn increasing attention.In this review, we summarize recent findings of a new class of compounds in tea- flavonoid alkaloids ( flavoalkaloids), which exist in fresh tea leaves and can be generated during the process of post-harvesting, and also postulate the formation mechanism of flavoalkaloids between catechins and theanine-derived Strecker aldehyde.Additionally, we detail the up-to-date research results of tea polyphenols regarding their ability to trap RCS and their in vivo aminated metabolites to suppress advanced glycation ends products (AGEs).We further raise questions to be addressed in the near future, including the synthetic pathways for the generation of flavoalkaloids and AGEs in fresh tea leaves before processing and the concentrations of tea polyphenols that affect their RCS scavenging capability due to their pro-oxidant nature.More intensive research is warranted to elucidate the mechanisms of action underlying the biological activity of flavoalkaloids and the pharmacological application of tea polyphenols in scavenging RCS and impeding detrimental AGEs.

1.Introduction

Tea drinking has become a worldwide culture of fashion and a symbol of nutrition.The tasting and nutrition of tea is filled with enjoyment and scientific mystery that have been studying for ages,yet many aspects of tea are to be explored and comprehended.Simply put, tea is the leaves of the plantCamellia sinensis, but its variety and the state-of-the-art postharvest process bring the multitude and polymorphism of science and attraction to the people around the globe.Among many fields of tea research such as plantation, botany,process, nutrition, and biochemistry, the chemistry of tea plays a pivotal role in revealing the nature of tea.

The categories of tea based on the degree of fermentation include unfermented green tea, white tea (tea buds with a couple of young leaves), yellow tea (slower denature of the oxidizing enzyme), partially fermented oolong tea, fully fermented black tea, and post-fermented dark tea [1,2].In some specific regions,however, the plants are recorded as white tea [3]or yellow tea [4],minutely different species from green tea within theC.sinensisfamily.From decades of research, many tea phytochemicals have been uncovered and correlated with tea’s taste and nutrition,including amino acids, abundant polyphenols, theanine, caffeine,polysaccharides, and essential oils, among others.Like other plants,the multi-molecular composition of tea is responsible for the unique flavor and nutrition [5,6].Characteristic polyphenols in each tea category have been identified and characterized, including catechins in green, white, yellow tea, and others [6], theasinensins in oolong tea [7], and theaflavins, thearubigins, and theabrowins in black tea(Fig.1) [8].These tea polyphenols have demonstrated a significant contribution to tea’s sensory and health-promoting properties [9],which unambiguously attests that the polyphenolic compounds are one of the most critical components in tea.Recently, with the assistance of cutting-edge technology, a series of new flavoalkaloid compounds ( flavonoid alkaloids) from various teas have been isolated and evaluated for taste and biological properties.A representative example isN-ethyl-2-pyrrolidinones substitutedflavan-3-ols,also called flavoalkaloids, from tea sources and tea processes(Table 1).A new era of dark tea research has yielded the same or similar flavoalkaloid compounds from the microbial process after harvesting and certain degrees of process.Also, several studies on the interactions between tea polyphenols and model biological systems have identified the reactive carbonyl species (RCS)-trapping activity of tea polyphenols.These interactions lead to the generation of novel polyphenol derivatives characterized as 6- and 8-substituted flavan-3-ols,for example, the interaction between (-)-epigallocatechin-3-gallate(EGCG), a tea polyphenol, and methylglyoxal, one of the main RCS [10].In this review, we first introduce recent achievements in tea chemistry,mainly focusing on novel carbonyl-catechin reactions and new flavan-3-ol conjugates isolated from tea.Then, we will discuss the near future research in tea-related chemistry, particularly the newly added polyphenolic conjugates as markers of tea and biomarkers in the biofunctionality of tea on health related studies.

Fig.1 Representative structures of tea polyphenols.

Table 1N-ethyl-2-pyrrolidinone substituted flavan-3-ols detected in a variety of teas.

2.Formation of flavoalkaloids in tea

As illustrated in Fig.1, there are 4 major tea catechins, i.e.(?)-epicatechin (EC), (?)-epigallocatechin (EGC), (?)-epicatechin gallate (ECG) and EGCG.EGCG is the richest catechin, followed by ECG, EGC, and EC in most varieties of teas except fully fermented pu-erh and post-fermented dark teas.All catechins have a C6-C3-C6 flavan-3-ol skeleton.The substitution at A-ring with 5,7-dihydroxyl,9-alkoxy and 10-alkyl groups attributes the 6- and 8-positions of A-ring to carry heavy electron density, translating to strong nucleophilicity.On the other hand, some substances from tea, such asL-theanine and ascorbic acid, bear an electron-deficient carbonyl group that is a strong electrophile.Also known asN5-ethyl-L-glutamine,L-theanine is recognized as a non-protein amino acid and a characteristic ingredient of tea, accounting for approximately half of the total amount of free amino acids in tea.L-theanine, resembling the structure of glutamate that is a crucial neurotransmitter, can cross the blood–brain barrier and plays significant roles in the central nervous system such as stress management, anxiety relief, and anti-depression [11-13].

During tea processing, the content ofL-theanine was observed to consistently decrease by more than half in the withering step alone,while the content of caffeine and other amino acids were increasing [14].It has been demonstrated that the loss of more than half ofL-theanine is due to the conversion fromL-theanine to an aldehyde through Strecker degradation, which was observed in the manufacture of black tea [15]and the fermentation of pu-erh tea preparation [16].Recent studies have demonstrated that the aldehyde formed fromL-theanine could be attacked by 6- or 8-carbon atoms on catechins to generateN-ethylpyrrolidinone through spontaneous cyclization after the nucleophilic addition.Therefore, flavoalkaloids (i.e.6-C or 8-C-N-ethyl-2-pyrrolidinone-substituted flavan-3-ols) are formed between the aldehyde fromL-theanine and catechins on C-6 or C-8 position of the A-ring (Fig.2) [15-17].As illustrated in Fig.2, the detailed mechanism could be postulated to start from the oxidation of catechins (1) to yield 1,2-dicarbonyl quinones (2), which are then attacked by theα-amino group ofL-theanine to form the imino-linked complex (3) between catechin quinone andL-theanine.The resulted complex goes through Strecker degradation to yieldL-theanine-derived aldehyde, which is in a state of equilibrium between the free aldehyde and the cyclizedN-ethyl-5-hydroxy-2-pyrrolidinone (4).Later, theL-theanine-derived aldehyde and catechins undergo a nucleophilic addition at 6- and 8-position of catechins to yield 6- and 8-substituted catechins (5 & 6), which would undergo intra-molecular dehydration to form the 6- and 8-N-ethyl-2-pyrrolidinone- flavonoids (7 & 8).

Fig.2 Proposed mechanism of flavoalkaloids formation from green tea in post-harvesting process.

The initial discovery of 6- and 8-N-ethyl-2-pyrrolidinoneflavonoids, or flavoalkaloids, in the processed tea has led to the identification of more flavoalkaloids in a variety of teas.Recent isolation and characterization of flavoalkaloids was from various teas ranging from white tea [18-21], yellow tea [22], green tea [17,19],black tea [15,23], and dark tea [16,24].Table 1 summarized the existence of flavoalkaloid compounds in various teas, including(R)- and (S)-8-C-N-ethyl-2-pyrrolidinone-EC, -C, -EGC, -ECG and -EGCG from fresh tea leaves [3,21]; (R)- and (S)-8-C-N-ethyl-2-pyrrolidinone-EC, -EGC and -EGCG and (R)-8-C-N-ethyl-2-pyrrolidinone-ECG from green tea [3,17]; (R)- and (S)-6-C-N-ethyl-2-pyrrolidinone-ECG, (R)- and (S)-8-C-N-ethyl-2-pyrrolidinone-EC and-ECG, (S)-6-C-N-ethyl-2-pyrrolidinone-epiafzelechin, (R)- and (S)-8-C-N-ethyl-2-pyrrolidinone-epiafzelechin-3-O-gallate, and 8-C-N-ethyl-2-pyrrolidinone-theasinensin A from black tea [14,15,23,24];(R)- and (S)-6-C-N-ethyl-2-pyrrolidinone-EGCG, (R)-8-C-N-ethyl-2-pyrrolidinone-ECG, (R)- and (S)-8-C-N-ethyl-2-pyrrolidinone-EC,-EGC, and -EGCG from white tea [18-21]; (R)- and (S)-8-C-N-ethyl-2-pyrrolidinone-EC, -C, -EGC, and -GC from dark tea [16,24]; andN-ethyl-2-pyrrolidinone-EC, -ECG, -EGC and -EGCG from yellow tea [22].

The formation of flavoalkaloids was initially believed to be derived from various processes such as withering, drying,fermentation, manufacturing, and storage after harvesting.For example, 8-C-N-ethylpyrrolidinonyl theasinensin A was suggested that it was mainly produced during heating procedure of black tea [15].In green and white teas, the contents ofN-ethyl-2-pyrrolidinonesubstituted flavan-3-ols increased with extended storage [3,17-21].The formation of flavoalkaloids in Chinese dark tea was attributed to the microbial effects of fungi such asAspergillus nigerin the process of post-fermentation [16,25].The direct syntheses of flavoalkaloids using theanine and catechins without enzyme catalysis provided solid evidence that theanine-formed aldehyde can undergo a nucleophilic reaction with catechins [15,21].Recently, it has also been found that flavoalkaloids exist in various teas, including fresh tea leaves [3,21], which implies that there is at least a biosynthetic pathway of flavoalkaloids in the tea plants [20].The recent finding of flavoalkaloid compounds in various teas prompted a proposal thatN-ethyl-2-pyrrolidone-substituted flavan-3-ols could be used as markers for long-term storage of white tea [21,23].Since that flavoalkaloids are found in many teas, they can be used as marker compounds in monitoring fresh tea leaves and post-processes of teas, such as storage, enzymatic processing, fermentation, and postmicrobial fermentation.

Interestingly, it has been revealed that these newly found flavoalkaloids exhibit several biological activities while more study is ongoing.N-ethyl-2-pyrrolidinone-substituted flavan-3-ols showed antioxidant activity by protecting the injury of human microvascular endothelial cells induced by hydrogen peroxide, similar to catechins in a parallel study [3], and also by scavenging free radicals of DPPH and ABTS with significantly stronger activity than ascorbic acid and Trolox [16].Additionally,N-ethyl-2-pyrrolidinone-substituted flavan-3-ols exerted inhibitory effects on acetylcholinesterase, similar to that of theanine and catechins [3,24], and also on the formation of AGEs with a 10-20 times stronger inhibition than aminoguanidine [20].Furthermore, at concentrations of 1.0 and 10 μmol/L,N-ethyl-2-pyrrolidinone-substituted flavan-3-ols protected human umbilical vein endothelial cells against high glucose-induced cell senescence [19].Moreover,N-ethyl-2-pyrrolidinone-substituted flavan-3-ols was anti-inflammatory in LPS-challenged macrophages by inhibiting the expression, phosphorylation, and nuclear translocation of nuclear factor kappa-B (NF-κB) p65 [17].Also,N-ethyl-2-pyrrolidinonesubstituted flavan-3-ols inhibited the activity ofα-glucosidase [24].A recentin vivostudy has demonstrated that C-8-N-ethyl-2-pyrrolidinone-substituted flavan-3-ols downregulates the aggregation of ubiquinated proteins and inhibits the Aβmetabolic pathway in senescence-accelerated mouse prone 8 (SAMP8) mice at a dose of 10 mg/kg, more effective thanL-theanine [25].It is worth noting that the evaluation of flavoalkaloids’ biological activities has just embarked, and much more in-depth research is warranted to merit the health-beneficial effects of flavoalkaloids for human consumption.

3.Adduct formation of tea polyphenols with reactive carbonyl species

Advanced glycation end products (AGEs) are derived from nonenzymatic glycation bothin vitroandin vivo.Accumulated evidence has attested that AGEs and their cell surface receptor (RAGE) play a central role in the pathogenesis of many age-related diseases,including diabetes complications, cardiovascular disease, cancer, microbiome-associated illness, liver disease, and neurodegenerative diseases, among others [26].The reported mechanisms of AGERAGE-stimulated pathological conditions include: 1) the induction of oxidative stress by generating reactive oxygen species (ROS) that in turn aggravate more AGE production and induce inflammation [26],2) promotion of inflammatory response such as elevated NF-κB [26],3) activation of fibrogenic signaling pathways [27], 4) contribution to unfavorable transplantation environment to stem cell engraftment and survival [28], 5) increasing RAGE expression and interaction with AGEs to induce cardiomyocyte apoptosis via the activation of MAPK pathway [29], and 6) stimulation of other mechanisms related to elevated circulating AGEs [26].AGEs can be formed bothin vitroandin vivo.Endogenous formation and accumulation of AGEs are associated with the body’s natural ageing process, particularly under oxidative stress, inflammation, hyperglycemia, and other metabolic syndromes [30].Exogenous AGEs are predominantly from diet, including foods prepared before consumption and beverages containing high fructose corn syrup.Reactive carbonyl species (RCS),i.e., 1,2-dicarbonyl compounds such as glyoxal (GO), methylglyoxal(MGO), and 3-deoxyglucosone from Maillard reaction during food processing, and malondialdehyde are the precursors responsible for AGE generation.Hence, scavenging RCS is regarded as the most effective way to reduce or eliminate the formation of AGEsin vivoand to prevent and control the development of AGE-related pathological conditions.In searching for effective RCS scavengers,polyphenolic compounds are the most often evaluated because synthetic RCS scavengers, though efficient in some cases such as aminoguanidine, are often accompanied by severe side effects [31].

The potent antioxidant activity of tea polyphenols is due to their electron-rich conjugated system and electron-donating phenolic groups.By the same token, they are strong nucleophiles that can attack electron-deficient RCS to form adducts, making tea polyphenols as efficient RCS scavengers.The mechanisms of RCS trapping by tea polyphenols have been elucidated in past decades.Both catechins and thea flavins demonstrated very efficient scavenging activity on MGO,and the adducts of the major catechin of tea, EGCG, with MGO were characterized as 8-MGO-substituted EGCG (9), 6-MGO-substituted EGCG (11), or 6,8-di-MGO-substituted EGCG (13) (Fig.3) [32],reminiscent of 6-mono, 8-mono or 6,8-dipyrrolidinone-substituted catechins mentioned above (Fig.2).Slight alkaline conditions favored the formation of both mono-MGO adducts and di-MGO adducts of MGO with EGCG [10].In addition to EGCG, other catechins also demonstrated RCS scavenging effects.In a model system of bovine serum albumin (BSA) and fructose, ECG was shown to successfully suppress the carbonylation of proteins and inhibited the formation of AGEs [33].This study showed that ECG at 44.2 μg/mL trapped 61.6% of MGO, thus protecting protein glycation.LCMS analyses have characterized the adducts between ECG and MGO as 8- or 6-mono-MGO- or 6,8-di-MGO-substituted ECG, same addition position as that of EGCG (compounds 10, 12, 14 in Fig.3) [33].

Fig.3 Chemical structures of the adducts formed between tea catechins and metabolites with MGO, malondialdehyde, and trans-4-hydroxy-2-nonenal.

The metabolite of EGCG from microbiota was identified as 4’-aminated EGCG (15), in which the 4’-OH group was replaced with an amino group.Notably, this metabolite demonstrated the same RCS scavenging effects as that of EGCG, and the resulting adduct 16-18 are illustrated in Fig.3 [34].However, an imine type Schiff base could be formed between EGCG and MGO (19).The structures of 10, 12, 14, and 16-24 in Fig.3 were proposed based on LCMS results[33,34].NMR data are required for further structural confirmation.In the same study, EGCG was also found to scavenge malondialdehyde(20) andtrans-4-hydroxy-2-nonenal (23).The corresponding adducts of 4’-aminated EGCG with malondialdehyde (21, 22) andtrans-4-hydroxy-2-nonenal (24) after trapping reaction were also proposed [34].

Theaflavins from black tea were also revealed to exert MGO-trapping activity [10], but the adduct structure was not proposed until last year [35].There were three mono-MGO-substituted thea flavins found based on MS/MS analyses.The positions of addition were assigned to the A-rings of thea flavin moiety (26, 27) (Fig.4), but the precise linkage between MGO and one of the C-6 or C-8 positions of two A-rings has not been identified.It requires further isolation and characterization of MGO glycation adducts of theaflavins by NMR analyses to define the structures of these adducts.The metabolite of black tea thea flavin from CD-1 mice was identified by MS/MS and NMR analyses as mono-aminated theaflavin (28)(Fig.4), where the 8’-OH on the B-ring (benzotropolone core) of theaflavin moiety was replaced with an amino group.The monoaminated thea flavin demonstrated similar scavenging activity of MGO to that of thea flavin.The four MGO adducts (29, 30) were identified based on MS/MS [35], but these structures remain to be confirmed by NMR analyses.

Fig.4 Structures of thea flavin and MGO, amino-thea flavin and the adducts of thea flavin with MGO.

The trapping targets of tea polyphenols are not limited to RCS like GO and MGO.In general, the strong tendency to engage in a nucleophilic reaction is the nature of electron-rich polyphenols.Previously, we have summarized the reaction of EGCG with dehydroascorbic acid, an oxidized derivative of ascorbic acid, to produce C-6- or C-8-ascorbyl-EGCG adducts, leading to preventing the formation of AGEs between dehydroascorbic acid and proteins [1,36].Besides GO and MGO, other electron-deficient agents related to human health are found to be scavenged by tea polyphenols and their metabolites as well.Recently, it has been demonstrated that EGCG and itsin vivometabolites trap acrolein bothin vitro[37]andin vivo[38].Thein vivotrapping of acrolein by EGCG resulted in forming adducts to another pyran ring fused with the A-ring benzene (33)(Fig.5), as revealed by MS/MS while requiring confirmation by NMR [38].Intriguingly, the methylated metabolites of EGCG(31, 32) (Fig.5) also underwent the same path to scavenge endogenous acrolein in mice to yield benzopyran adducts of methylated EGCG(34, 35) (Fig.5).Therefore, ingested EGCG can scavenge endogenous acrolein by direct trapping or by its methylated metabolites [38].

Fig.5 EGCG and its in vivo metabolites trap acrolein.

Other explorations in the inhibitory effects of tea polyphenols on the formation of AGEs include common AGE formation,media change from water to aqueous alcohol, kinetic study, adduct formation, and others.Regarding inhibition of AGE formation, all four major catechins, namely, EC, ECG, EGC, and EGCG, were shown to be more effective in an aqueous alcohol (20%,V/V) media than in water, where EC was most effective than other catechins [39].Also, catechins were found to suppress the glycation of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine by trapping GO and MGO [40].MS analyses further revealed that the adducts contained various ratios of catechin to GO or MGO: EC and GO formed products in ratios of 1:1, 2:1, and 2:2; EC and MGO in ratios of 1;1, 1:2, 2:1, and 2:2; EGC and GO in ratios of 1:1 and 2:1; EGC and MGO in ratios of 1:1, 2:1, and 2:2; ECG and GO in a ratio of only 2:1; ECG and MGO in ratios of 1:1, 1:2, and 2:1; whereas EGCG and GO or MGO only had 1:1 adduct.The sequence of inhibitory effect on AGE formation was EC ＞ ECG ＞ EGC ≈ EGCG, with EC being the strongest inhibitor [40].The preservation of milk flavor stored in ultrahigh temperature was attributed to catechins’ trapping effects on GO, MGO, and other aldehydes produced in the thermal process.The structures of adducts between EC and aldehydes were already elucidated by MS [41].(+)-Catechin effectively inhibited the formation of histone H1N?-carboxymethyllysine induced by MGO at low concentration (12.5-100 μmol/L) but not at high concentration(200-800 μmol/L), potentially due to the increased production of hydrogen peroxide at high concentrations of (+)-catechin [42].

4.Perspectives

It is clear that tea becomes an associated part of daily human life for both taste enjoyment and high nutrition value.However, the understanding of the health-promoting mechanism of tea remains elusive and requires continued exploration.On top of flavor and biology evaluation, understanding the chemistry of tea is a prerequisite.The phytochemical dissection of tea has contributed to the classification of several categories of components, including polyphenols/flavonols, amino acids/proteins, alkaloids/xanthines,and sugar/polysaccharides among others.There is a relatively good knowledge of each category in terms of chemical and physical characteristics, biological function, and process for enrichment or reduction.However, the understanding of interactions between two phytochemical groups of tea or with other health-related agents such as RCS is limited except for the antioxidation reaction.

The recent finding of flavoalkaloids resulting fromL-theanine Strecker derivative-catechins interaction is an example of an interaction between different groups of phytochemicals in tea chemistry previously unimagined.Moreover, new findings have revealed that there are at least two resources of flavoalkaloids:existing in natural tea leaves and being formed during the process of post-harvesting.More studies are demanded to explore the species of tea leaves that contain natural flavoalkaloids, the content and amount of flavoalkaloids, and the mechanisms of flavoalkaloid formation in plant biology.Simultaneously, the generation of flavoalkaloids after harvesting in the tea process also requires attended research to identify synthetic pathways under the processing conditions and methods to control flavoalkaloid formation in a favorable direction.Also,the evaluation of the biological functionality of flavonoid alkaloids is of great value to fully comprehend the biological properties of tea.Therefore, a perceived new wave of research in the field of tea flavoalkaloids will bring more understanding of tea.

Accumulating evidence has highlighted AGEs as detrimental factors for human health.Suppression of AGE formation has been an ultimate goal in the protection of many pathological conditions.Tea polyphenols have been revealed as excellent RCS scavengers to inhibit the formation of AGEs bothin vitroandin vivo, though there is insufficient convincing data from human studies.Despite the adduct structures between tea polyphenols and RCS such as MGO and GO have been explored, the chemistry of tea polyphenolsmediated RCS trapping is still preliminary due to the complex nature of various polyphenolic compounds, multiple attacking locations of each polyphenol, high reactivity of many existing RCS, and the changing media.More characterizations of derived compounds should be performed to establish standard agents for potential identification of products formed from RCS trapping reactions by tea polyphenols.Furthermore, some AGEs formed in fresh tea leaves or during the withering/drying process should be paid special attention as well [43].In the near future, elucidating the biochemical pathways responsible for AGE formation in natural tea leaves or during tea processing will help find methods and technology to reduce the AGE formation and prevent the consumption of AGEs from tea drinking.Certainly it will be an important discovery to understand the mechanisms of AGE formation in the polyphenol-rich environment of tea leaves.

Tea polyphenols are also found as pro-oxidants.The initial interaction of tea polyphenols with aqueous media at physiological conditions generates quinones and releases hydrogen peroxides(H2O2).Low concentrations of H2O2might be health-beneficial under certain conditions, but high concentrations of H2O2might be likely to trigger oxidative stress and more RCS release.On the other hand,tea polyphenols are known as effective RCS scavengers and potent inhibitors of AGE formation at low but not high concentrations [42].More comprehensive studies are warranted to elucidate the mechanisms and the potential influence of tea polyphenols’ prooxidant nature on the biological activities of tea.

In summary, our knowledge of tea chemistry has greatly advanced in recent years.Findings of flavoalkaloids as a new class of compounds in tea and flavonoid-adducts due to RCS trapping by tea polyphenols have prompted a new era of tea research.Yet, more questions are raised regarding the pathways to form flavoalkaloids in fresh tea leaves and during processing, the biofunctional roles of these newly found tea compounds, and the ways to control flavoalkaloid formation.Challenges in exploring RCS trapping by tea polyphenols include the characterization of adducts and the elucidation of the mechanisms underlying product formation,particularly the suppression of AGE formation by tea polyphenols in fresh tea leaves and during tea processing to avoid AGE consumption.Morein vivostudy even human clinical trials should be conducted to prove the efficacy of tea polyphenols in RCS scavenging and disease prevention.

Conflict of interest

The authors declare that there is no conflict of interests.

Acknowledgements

This work was supported by Hubei Science and Technology Plan Key Project (G2019ABA100).