Ting Hu, Peng Wu, Jinfeng Zhn, Weixin Wng, Junfeng Shen,Meiyn Wng*, Chi-Tng Ho, Shiming Li,*

a Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive Utilization,Huanggang Normal University, Huanggang 438000, China

b School of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China

c Department of Food Science, Rutgers University, NJ 08901, USA

Keywords:

Tea polysaccharides

Structure variety

Bioactivities

Relationship

In fluencing factors

A B S T R A C T

Tea polysaccharides (TPSs), one of the major bioactive ingredients in tea, have been widely studied due to their variety of biological activities, including antioxidant, cancer prevention, hypoglycemia, anti-fatigue,anti-coagulant, anti-obesity and immunomodulatory effect.The biological effectiveness of TPSs has direct relation with their structures such as monosaccharide composition, molecular weight, glycosidic linkages,conformation and others, which can be influenced by tea materials, processing methods, extraction and purification procedures among others.Comparing to the study of tea polyphenols, the exploration of TPSs in structural elucidation and biofunctionality is very preliminary.Yet several factors affecting the structural change of TPSs have been studied and identified.Consequently, the variation of some TPS biological activity brought by the change of TPS structures has been evaluated and preliminary correlation of structure activity relationship of TPSs has been performed.Therefore, this review aims to serve as a summary research report regarding the influencing factors on TPSs structures and consequential effects on the biological activities of TPSs.We hope to provide updated information and systematic references for future study and functional food development of TPSs.

1.Introduction

Tea has more than five thousand years of history in dietary and medicinal application in Asian countries, such as China, Japan, India,and Thailand [1].Nowadays, tea is one of the most widely consumed beverages around the world, and possesses health-promoting effects such as hypoglycemic, antioxidant, anticancer, immunomodulatory,anti-inflammatory, and chemo-preventive activities because of its chemical constituents including polysaccharides, polyphenols,proteins, and alkaloids among others [2-6].Since the late 1980s,tea polysaccharides (TPSs), a group of main phytochemicals in tea,have attracted more and more attention from researchers.The study of TPSs has been mainly focused on extraction process, structure elucidation and biological activity.According to a large number of literature reports, the structures of TPSs are quite complex, because of the variety of combinations with more than ten monosaccharides such as rhamnose (Rha), arabinose (Ara), glucose (Glc), galactose (Gal),xylose (Xyl), mannose (Man), fucose (Fuc), ribose (Rib), galacturonic acid (GalA) and glucuronic acid (GluA), and also with multiple glycosidic linkages such as (1→2), (1→3), (1→4), (1→6) among the monosaccharides, leading to a wide range of molecular weight distribution (MWD) from 1.02 kDa to 4 940 kDa [2,7-14].Moreover,the elucidation of configuration and conformation of individual TPSs also faces great challenge due to the limited available research results of fine and high-level structures of TPSs.For instance, TPSs could present as sphere-like, random coil or ordered helix-coil conformation in solution [9,15,16].

TPSs possess various biological activities including ant ioxidant [17],anti-coagulant [13], hypo glycemic [18], bactericidal [19],anticancer [20], anti-fatigue [21], prevention of obesity [22]and immunomodulatory effects [23]among others [3,6].The biological activities of TPSs are closely associated with their chemical components, molecular weight (Mw), primary structure and structural conformation [1,24].It is also reported that the bioactivities of TPSs are proportional to the contents of uronic acid and can be enhanced by combining with inorganic elements (e.g.Se) and proteins [3].

Some studies on the structure of TPSs have been performed, but in general, the elucidation of TPSs structures, particularly the highlevel structure, is primarily exploratory due to the nature of high complexity, influenced by multiple factors such as various tea sources,different processing procedures and isolation methods among others.High diversity of TPSs structures has multiple and multitude impacts on the biological property of TPSs, hence, a predication of bioactivity relying on a TPSs structure is unrealistic based on current available data.For example, TPSs of smallerMwdemonstrated stronger hydroxyl radical scavenging activity than that of largerMw[3,25],whereas in other studies, TPSs with largerMware reported to have better antioxidant activity than TPSs with smallerMw[26,27].

The polymorphism of TPSs structures and its delicate influences on biological property of TPSs has been recognized and approached to understand from different aspects of TPSs with limited available data [1,3,6].In this review, we have summarized major influencing factors on the structures of TPSs including tea sources, processing methods and separation technologies employed, and addressed the consequent effects of these structure characteristics on the biological property, such as chemical compositions, monosaccharide components,Mw, MWD and other factors.The aim of this review is to provide a comprehensive summary of TPSs diverse structures, the factors leading to the diversity and its influential effects on biological properties of TPSs.

2.Structural characteristics and influential factors of TPSs

The properties of TPS structures include monosaccharide composition,Mw, sequence of monosaccharides, location of glycosidic linkages, degree of branches, configuration and conformation of the entire molecule.Due to the differences in tea species, processing technologies of tea, isolation and purification methods of TPS, more than 120 TPSs with different chemical characteristics have been reported [1,3,6].

2.1 Characteristics of TPSs structure

As illustrated in Fig.1, the chemical composition of TPSs mainly consists of carbohydrates (neutral/acidic polysaccharide),also contains protein, uronic acid, polyphenol and inorganic elements (selenium, iron and manganese etc.) among others [3,6,28].Sometimes, the purified TPSs are a protein-bounded polysaccharide complex consisted of glycan, peptides, uronic acids and others [29].The monosaccharide composition of TPSs can have approximately 2-10 kinds of monosaccharides such as Rha, Ara, Glc, Gal, Xyl, Man,Fuc, Rib, GalA and GluA with different molar ratios [3].The averageMwor MWD of TPSs ranges from 1.02 kDa to 4 940 kDa according to the reported research results [3], which is broad and unpredictable without a series of structural characterization.There are several determining factors in the high order structure of TPSs, including configuration, type and position of glycosidic linkages, sequence of monosaccharides, number and location of appended noncarbohydrate groups as well as molecular chain conformation [3].The main glycosidic linkages of TPSs include (1→2), (1→3), (1→4) and (1→6)(Fig.2).The molecular chain conformation of TPSs in solution has been characterized as sphere-like, random coil, and/or ordered helixcoil shapes [3,9,15,16,30].

Fig.1 Chemical characteristics of TPSs.

Fig.2 Structures of TPSs (A [31]; B [31]; C [2]; D [8]; E [23]).

2.2 Tea materials and TPSs structure

Tea sources have great influences on TPSs structure.TPSs extracted from different species of tea in the same category such as green tea, had differentMwand composition of monosaccharides.For example, monosaccharide composition from three green teas, i.e.Xihu Longjing (XTPS), Chawentianxia (CTPS) and Huizhoulvcha(HTPS), were different.XTPS and CTPS were mainly composed of Rha, Ara, Gal, Glc, Xyl, Man and GalA, whereas HTPS was composed of Rha, Ara, Gal, Glc, Xyl, Man, Rib and GalA with different molar ratios [32].Despite two TPSs from oolong tea of Fenhuangdanzong and Tieguanyin had same monosaccharide composition of Rha, Ara, Xyl, Man, Gal and Glu, the molar ratio was different, demonstrating different TPSs composition between two oolong teas [32].

TheMwof TPSs was also influenced notably with tea species.As an example, theMwof XTPS, CTPS and HTPS were different: XTPS was mainly consisted of 3 kinds of TPSs with theMwof 810, 54.5 and 1.26 kDa; CTPS had 4 major TPSs with theMwof 805, 138, 19 and 12 kDa; and HTPS exhibited 4 kinds of polysaccharides with theMwof 771, 137, 11 and 1.2 kDa [32].Hence tea species contributed the variety of TPSs composition andMwwithin the same tea category,almost the complete contradictory to small phytochemicals such as tea catechins, where the only predictable difference among tea varieties is the content as a group.

In addition to tea species, TPSs are different in different parts of a tea plant, that is, the TPSs profiles in the leave, seed and flower from a same tea plant are different.The monosaccharide compositions of TPSs among the leaves, flowers and seeds of tea were different.TPS in the leaves was consisted of Gal, Fuc, Rha, Ara, Xyl, Man, Rib, GalA and GlcA in a molar ratio of 1.00:0.29:0.87:1.27:1.77:0.07:0.11:0.3:2.54:0.24; TPS in the flower had Gal, Rha, Ara, Glc, Xyl, Man, GalA and GlcA in the molar ratio of 1.00:0.42:0.97:0.36:0.11:0.17:0.71:0.08; and TPS in the seeds was composed of Gal, Glc, Rha, Xyl, Ara,GalA and GlcA in the molar ratio of 1.00:1.95:0.35:0.15:0.95:0.23:0.07 [26].The MWD of TPSs from leaves, flowers and seeds were ranged from 3.67 kDa to 758 kDa, 2.56 kDa to 1 460 kDa, 3.66 kDa to 961 kDa, respectively, indicating that theMwof polysaccharide from tea flowers was the highest [26].

2.3 Impact of processing methods on TPSs structure

In the process and separation of TPSs, there are options to select from number of methods and technologies.However,different processing methods can yield TPSs with varied structural characteristics.The evaluation and report of the biological activity of TPSs must be accompanied with the information of methods to obtain the TPSs.The reported influential processing factors include extraction, fermentation, aging and metal interference.

In the extraction of TPSs, water (hot or cold) and alkali solution are the most used extracting solvents and sodium chloride is often the modifier.However, the TPSs yielded from these variations are often different in compositions,Mwor MWD.For example, tea polysaccharides extracted with hot water and alkali solution from a green tea were composed of 7 same monosaccharides, namely Rha,Fuc, Ara, Xyl, Man, Glc and Gal, but with different molar ratios of 8.74:4.69:29.04:0.42:7.11:14.10:35.89 for water extracts and 13.81:1.43:36.07:5.24:4.89:6.28:32.27 for alkali extracts, respectively [33].Moreover, the TPS of water extract had 3 homogeneous components with theMwof 4.55 × 106(37.38%), 4.85 × 104(6.54%) and 6.62 × 103(56.07%) Da, respectively; whereas that of alkali extract was consisted of 4 homogeneous components with theMwof 4.94 × 106(68.57%), 6.77 × 104(2.86%), 1.12 × 104(11.43%) and 4.13 × 103(17.14%) Da, respectively, indicating that the alkali solution tended to extract higherMwof TPSs than water [33].

Changes in fermentation condition also yield different TPSs.It was reported that the composition and content of monosaccharides of TPSs isolated from green tea (no fermentation), oolong tea (partial fermentation), and black tea (full fermentation) were vastly different.Green tea TPS containedD-Rha,L-Ara,D-Xyl,D-Man,D-Gal, andD-Glc in the molecular ratio of 7.8:41.8:7.1:7.3:18.7:17.0; oolong TPS (OTPS) was composed of 4 monosaccharides,D-Rha,L-Ara,D-Gal, andD-Glc in the molecular ratio of 16.2:43.7:18.0:21.9;and black tea TPS also had 4 monosaccharides as in OTPS , but the molecular ratio was 14.4:36.4:19.7:29.4 forD-Rha,L-Ara,D-Gal,andD-Glc [34].The MWD of the three TPSs were also different and ranged from 9.2 kDa to 251.5 kDa, 5.3 kDa to 100.9 kDa and 3.8 kDa to 32.7 kDa, respectively, for the TPSs obtained from green, oolong and black teas, demonstrating a decreased pattern ofMwwith the increase of the fermentation degree [34].

The length of aging time is also an influencing factor for the structure of TPSs.To reveal the impact of the aging time on the structural changes of pu-erh TPSs (PTPSs), Xu’s group [35]investigated the potential connection between the monosaccharide composition andMwof TPSs and the length of aging years.The monosaccharide composition of PTPSs with aging length for 1 year(PTPS-1), 3 years (PTPS-3), and 5 years (PTPS-5) were consisted ofL-Rha,L-Ara,D-Xyl,D-Man,D-Gal,D-Glc, andD-Fuc in a different molar ratio of 5.34:21.86:4.04:21.59:26.93:16.52:3.64, 6.82:26.22:0.35:13.83:39.34:10.23:3.21, and 15.98:20.84:0.15:15.29:40.33:6.08:1.68, respectively [35].TheMwof these 3 aged PTPSs were also different.TheMwof PTPS-1 was 2.7 × 106Da with a major fraction accounting for 92% content; PTPS-3 had two major fractions withMwof 1.93 × 106(47%) and 6.31 × 105Da (52%), respectively; PTPS-5 also contained 2 major fractions withMwof 3.9 × 106(33%) and 1.16 ×1 06Da (60%), respectively [35].Hence the aging time had a notable influence on both composition andMwof PTPSs.

Other factors that affect the structural change of TPSs include extraction process, extracting solvent used such as ethanol content [36], sodium chloride concentration [2], drying method [37]and selenium enrichment of TPSs [31].In summary, due to the influence of multiple factors stated above, the structures of TPSs obtained from varying conditions are differentiated by composition and molar ratio of monosaccharides,Mwand MWD, possible conformation and configuration.

As a consequence, structural changes of TPSs post great impact on their biological activity.Therefore, the correlation between TPSs chemical structure and biological activity should trace back to the influencing factors that resulting in the change of TPSs structure.Owing to the polymorphism of TPSs structural characteristics, the concept of TPSs bioactivity should be specifically connected with the TPSs structure that in turn directly related to the source and process of tea interested.

3.In fluence of key structural factors of TPSs on biological activity

TPSs are a group of bioactive constituents in tea with multiple health beneficial bioactivities, among which the hypoglycemic and antioxidant potential of TPS has attracted the most attention.Different bioactivities of TPSs are directly related to their structural features including chemical composition, configuration and position of glycosidic linkages,Mw, MWD and chain conformation, which are unambiguously and strongly affected by tea materials, processing technologies, extraction methods and modification.Therefore, the relationship study between the structure and biological activity of TPSs not only can perceive the intercorrelation and revelation of the structural functionality of TPSs, but also can provide a guidance in selecting tea resources and processing methods to obtain TPSs with targeted biological activity, which is of great significance for the research and development of tea and its related functional foods.However, due to the large variation and complex nature of TPSs structures, the biological properties of TPSs are generally affected by multi-structural factors, rather than one single factor, leading to more challenging in the elucidation of the structure-activity relationship(SAR) of TPSs.Herein, we summarize the main factors contributing to the variety of biological activities associated with the structural characteristics of TPSs, i.e.composition of monosaccharides,molecular weight, spatial structure among others (Table 1), and the correlations between structural features of TPSs and their bioactivities based on available data have been summarized in Fig.3.

Fig.3 Summary of correlations between structural features of TPSs and their bioactivities based on available data.

Table 1Effects of chemical characteristics on bioactivities of TPSs.

Table 1 (Continued)

3.1 Chemical composition

The biological activities of TPSs may be affected by monosaccharide composition and other different chemical components such as the contents of proteins, neutral polysaccharides, uronic acid, polyphenols and other phytochemicals in tea.The following is a summary of how TPSs composition influenced the biological activities such as antioxidant and inhibition of hyperglycemia.

3.1.1 Monosaccharide composition.

TPSs are consisted of variety of monosaccharides in term of numbers and types, hence the multiplicity of monosaccharides as basic building blocks plays essential roles in the characteristics and biological activity of TPSs.In two TPS fractions isolated from the crude polysaccharides of a post fermented tea (Qingzhuan brick tea),TPS-1 and TPS-2, TPS-2 exhibited better antioxidant activity in DPPH, ABTS+, FRAP and ORAC assays than TPS-1 and the crude polysaccharides prior to fractionation.Composition analysis indicated that TPS-2 had less content of carbohydrate and uronic acid, but larger proportion of protein and polyphenolic compounds, indicating that more content of polysaccharides had less antioxidant activity [38].However, this result is very preliminary and ambiguous regarding the antioxidant activity of polysaccharides.Further exploration should be performed to reveal the antioxidant effects of the carbohydrates in the crude and two fractions of the polysaccharides.

The crude polysaccharides extracted from a green tea flower(TFPS) with heated 85% aqueous ethanol were fractionated with a diethylaminoethyl cellulose column to three fractions (TFPS-1,TFPS-2 and TFPS-3) and tested for antioxidant and inhibitory effects of human gastric cancer BGC-823 cells [37].Results showed that the order of sequence for the scavenging activity on DPPH radicals were TFPS-1 > TFPS > TFPS-2 > TFPS-3, indicating that the fraction TFPS-1 had much stronger antioxidant property than other two fractions.Composition analysis indicated that TFPS-1 was majorly composed of Ara, Fuc, Xyl, Man, Glu and Gal, and the main monosaccharide was Glu, reached to more than 45.4%,whereas TFPS-2 and TFPS-3 had Rha, Ara and Gal with Ara as the major monosaccharide, 55.2% and 53.3% for TFPS-2 and TFPS-3,respectfully [39].In addition, TFPS-1 had higher content of carbohydrate (83.7%) and sulfate (2.6%) than other two fractions.TFPS-1 was neutral TPSs verses acidic TFPS-2 and TFPS-3.Therefore, the composition, content of sulfate and neutrality of TFPS-1 contributed the overall effective antioxidant effects comparing to other two fractions and the crude TFPS.In addition, TFPS-1 and TFPS-3 had relative stronger inhibitory activity against the growth of BGC-823 cells than crude TFPS and TFPS-2, which could be attributed to the higher sulfate and lower uronic acid and galactose content in a more detailed comparison of composition among the four green tea flower polysaccharides [39].However, the in-depth correlation between the composition and bioactivity of the tea flower polysaccharide and its fractions is again very preliminary.Further study is crucially needed to investigate the effect of detailed composition and other structural characteristics of the fractionated green tea flower polysaccharides on the antioxidant, anticarcinogenic and other biological properties, potentially leading to an SAR elucidation.

Contradictory to small molecules, drying methods can have great impact on the composition and conformation of TPSs, even though drying is non-chemical processing.The change of composition and conformation of TPSs regardless of degree is often a cause of TPSs structural change and consequently leads to different biological activities.The antioxidant and inhibitory effects onα-glucosidase andα-amylase of TPSs obtained by different drying methods such as freeze-drying (TPS-F), vacuum-drying (TPS-V), spray-drying(TPS-S) and microwave-vacuum drying (TPS-M) were different [37].TPS-F from lyophilization exhibited higher inhibition ability onα-glucosidase andα-amylase and highest antioxidant capacity than other TPSs, which might be related to differences in monosaccharide proportion and chemical compositions like uronic acid of TPSs from different drying methods [37].Lyophilization is usually regarded as the most non-harmful method for the stability of chemically labile molecules such as polysaccharides, peptides and proteins.Hence it can be postulated that the TPSs yielded from lyophilization represent the original TPSs, which demonstrated the most active ability toward antioxidant and inhibition ofα-glucosidase andα-amylase.

There are number of studies in the composition and conformation of TPSs and the correlation with different biological properties.However, the study is so far very preliminary and still in exploration stages.The influence of more detailed composition and conformation of TPSs, such as types and number of monosaccharides and defined configuration of polysaccharide chain on antioxidant and other bioactivities are explored scarcely.Although it may face enormous difficulties, one of the near future efforts should focus on a systematic exploration on the influence of composition of TPSs-types and numbers of monosaccharides on TPSs biological activity such as antioxidant property or other well established bioactivity models to connect the composition and conformation of TPSs with their bioactivities.

3.1.2 Protein content

Similar to other protein-bounded phytochemicals such as polyphenols, the protein-bounding of TPSs was found to have great influence on biological properties of TPSs.For instance, among 3 OTPSs isolated from Tieguanyin (TTPS), Fenghuangdancong (FTPS)and Dahongpao (DTPS), DTPS yielded from the highest degree of fermentation had the strongest antioxidant and inhibitory activity againstα-glucosidase, followed by FTPS from the intermediate fermentation and then TTPS from the lightest fermentation, indicating that the increase of antioxidant andα-glucosidase inhibition activity of OTPSs was positively related to the degree of increased fermentation of oolong tea, owning to the higher fermentation, the higher content of protein-bound uronic acid heteropolysaccharides in OTPS [40].Also in Chen’s study [41], 3 fractions (TPC-1, TPC-2 and TPC-3) of water-soluble tea polysaccharide conjugates (TPC) from low-grade green tea were screened for antioxidant activity and found that TPC-3 had the highest antioxidant activity in deoxyribose assay (IC50of 182 μg/mL) and NBT photoreduction assay (93 μg/mL), remarkably higher than other two fractions, which was positively correlated to the highest protein content in this fraction of TPS conjugates.Moreover,the antioxidant activity of 3 TPS fractions increased with the protein content increase, manifesting the conjugated protein in the TPSs fractions could play a critical role against oxidative stress.

3.1.3 Neutral polysaccharides

Due to different composition of monosaccharides, there are neutral polysaccharides and charged (or ionic) polysaccharides.Polysaccharides that do not carry charged groups in the molecules are defined as neutral polysaccharides.Both neutral and ionic polysaccharides have great impact on their biological activity [2].In a comparison study, researchers found that among 5 subfractions (F0,F0.1, F0.2, F0.3, and F0.4) of TPSs obtained from a Chinese tea (Zhongcha 108) by eluting with distilled water, 0.1, 0.2, 0.3, and 0.4 mol/L NaCl aqueous solution, the acidic polysaccharide F0exhibited significantly elevating effect on the proliferation of HeLa cells, whereas neutral polysaccharides F0.1-F0.3showed markedly inhibitory effects at a higher concentration, suggesting that neutral polysaccharides with relatively smaller molecular weights and more homogeneous structure in TPSs demonstrated anticancer properties [2].Wei et al.[42]also demonstrated that neutral polysaccharides exhibited stronger inhibition againstα-glucosidase than other polysaccharides.They found that the inhibitory effects of TPSs extracted by different extraction methods againstα-glucosidase were determined in the order of boiling water extraction ＞ enzyme extraction ＞ water extraction, an increased inhibition with the increased content of neutral polysaccharides in TPSs.

3.1.4 Complex with other components

There are several functional groups of bioactive phytochemicals in tea.Each group exhibits its specific biological property with certain mechanisms of action.The biological activity of combined complex between two groups of tea is rarely studied owing to the nature of complexity.In particular, the combinatorial study of TPSs and other tea components encounters more difficulty than that of a combination among small tea molecules such as catechins and alkaloids.Researchers at Monobe’s group [43]compared the immunostimulating activity of crude TPSs from immature and mature tea leaves, and found that TPSs from immature leaf exhibited higher immunostimulating activity than TPSs from mature leaves, but the potency of crude TPSs was dependent on the content of strictinin, a hydrolyzable tannin.Furthermore, the immunostimulating activity was not enhanced by the removal of the tannin, suggesting that the catechin-polysaccharide complex is an important combination responsible for the immunomodulating activity.Subsequently, they found that TPSs from immature tea leaves included many RNAs showed higher immunostimulating activity than that from mature tea leaves [44].Another research group also observed the same phenomenon that a purified polysaccharide fraction containing homogalacturonan pectins from green tea showed a phagocytosisenhancing activity in HL-60 cells [23].Ageing process of tea also causes changes of the chemical composition in TPSs, which in turn demonstrate discrepancies of biological activity.The anti-αglycosidase activities of PTPS increased with the aging year, and the longer the aging year, the higher the anti-α-glycosidase activity,which may be attributed to composition changes, such as the contents of protein and uronic acid as well as proportion of lowMwfraction in PTPS [35].

3.2 Molecular weight

Polysaccharides are polymers of monosaccharides connected through multiple glycosidic bonds.The molecular weight of polysaccharides is widely dispersed, hence there are usually three different ways to describe polysaccharide molecular weight, i.e.weight average molecular weight (Mw), number average molecular weight (Mn) and zeta average molecular weight (Mz).Mwis most common to describe the molecular weight of a polysaccharide.TheMwof TPS plays an important role in its antioxidant and other properties.In general, the largerMwof a TPS, the lower antioxidant activity.Polysaccharides with largerMwshowed high viscosity and poor water solubility, leading to difficulties to pass through membrane barriers and enter the interior of the cell, which in turn limits TPS biological activity.In another study, the antioxidant activity of ten TPSs samples from five categories of tea were analyzed, and found that TPSs from the same type of tea with smaller Mwexhibited stronger antioxidant activity than those with largerMw[26].Three fractions (TPS1, TPS2, and TPS3) from crude TPSs with differentMwwere extracted from coarse green tea leaves by ultra filtration.It was found that TPSs with lowerMwshowed higher radical scavenging activity [27].In Chen’s work [41], the fraction with the lowestMw(4.2 kDa) exhibited the highest antioxidant activities when comparing with other two fractions with larger (26.8 kDa and 11.8 kDa).Likewise, the polysaccharide fraction with smallerMw(10.1 kDa)showed stronger antioxidant activities than the fraction with largerMw(167.5 kDa) [45].By the same analogy with antioxidant property,theMwof TPSs can also affect their anticarcinogenic activities.The fraction with lowerMw(232 kDa) from a crude green tea polysaccharide conjugates demonstrated higher inhibitory effect on HepG2 cells than the crude TPSs, which was postulated that the lowMwfraction of TPSs was easier to pass through the cell membrane to exert inhibitory effects [46], but more evidence is required to merit the conclusion.Yang and Park also found that a homogeneous polysaccharide isolated from green tea with aMwof 70 kDa, possessed antitumor effects on prostate cancer cells [47].A polysaccharide with aMwof 9 kDa purified from mature leaves of green tea also exerted antitumor and antimetastatic activities [48].

However, an opposite observation on relationship betweenMwof TPSs with antioxidant activity was raised by some researchers.For example, the antioxidant ability of three kinds of polysaccharides from tea leaf, tea seed, and tea flower showed different antioxidant capacity.The polysaccharides from leaf and flower with higherMwhad higher antioxidant activity than the TPSs from seed with lowerMw[26].Mwis an important marker to depict structure characteristics of TPSs and it has significant influence on biological activity.Current available data have demonstrated a valid comparison and SAR correlation of TPSs subfractions from a crude TPSs, representing similar composition of monosaccharides, TPSs bounding proteins and TPSs-polyphenol conjugates and possible similar glycosidic linkages.However, the trend of comparison or SAR pattern could possibly be reversed and unpredictable for TPSs origin of different sources.

3.3 Spatial structure

In addition to the primary structure, the spatial structure of TPSs containing secondary structure and above will also affect their bioactivities.For example, an acidic green tea polysaccharide (TPSA)possessed a hyperbranched structure with a sphere-like conformation in 0.15 mol/L NaCl aqueous solution, showing goodα-amylase inhibitory activity.The reason was that the branched chain segments of TPSA exhibited preferential binding toα-amylase through hydrogen bonding interactions, acting as a potential competitor for starch substrates to inhibitα-amylase activity [30].Wang et al.[49]compared the antioxidant activities of native yellow TPSs (YTPS-3N, YTPS-5N and YTPS-7N) and ultrasound-treated yellow TPSs(YTPS-3U, YTPS-5U and YTPS-7U), and found that ultrasound irradiation could increase the antioxidant capacity, which was ascribed to the changes in spatial structure before and after ultrasound.Ultrasonication altered the smooth surface structure of native yellow TPSs to a structure with porous surface, providing more reactive sites with free radicals [49].Qin et al.[50]found that TPSs with a looser spatial structure containing some pores had better anticoagulant activity and bile acid binding capacity for these polysaccharides that could provide more active sites for thrombin and bile acid binding.

3.4 Combination of multiple structural factors

The bioactivities of TPS are affected by two or more structural factors including chemical components, monosaccharide composition,Mw, and conformation.Three TPSs, isolated from green tea (GPTS),oolong tea (OTPS), and black tea (BTPS), respectively, demonstrated distinguished differences on theα-glucosidase inhibitory properties and antioxidant activities in the assays of scavenging hydroxyl and DPPH radicals.BPTS showed the highest antioxidant activity and inhibitory effects againstα-glucosidase [32].The composition of monosaccharides in three TPSs hadD-Rha,L-Ara,D-Gal andD-Glu,but GTPS had extra two monosaccharides-Xyl and Man.BTPS also had lower molecular weight distribution than OTPS and GTPS.Therefore, both composition and MWD of black tea TPSs contributed to its higher bioactivity comparing to green and oolong tea TPSs [34].The DPPH radical scavenging activity of two TPSs extracted from extrusion treated tea at conditions of 4% moisture content at 160oC and 12% moisture content at 100oC were higher than that of TPSs isolated from untreated tea, suggesting that extrusion processing enhanced the antioxidant activities of TPSs due to structure change of TPSs [17].The molar ratios of monosaccharide composition, MWD,and morphological appearance of TPSs may have great contribution to the antioxidant activities of TPSs [17].

3.5 Other influencing factors

3.5.1 Modification

The immunostimulatory effect of TPSs could be improved by enzymatic modification.Yu et al.[51]prepared enzymatic modified TPSs (ETPS) from green tea by glycosidase and compared the immunostimulating activities of ETPS and non-enzymatic treated TPSs using immunosuppressant mice.It was found that ETPS could significantly enhance the immunostimulating activity compared with TPSs.For instance, the humor immune function in immunosuppressant mice was greatly improved by ETPS,indicated by HC50of 43.63 (300 mg/kg) and 34.78 (100 mg/kg) vs the HC50value of 31.07 (300 kg/kg) in the TPS-treated mice.It is speculated that the enzymatic modification induced the hydrolysis of glycosidic bonds and impacted the structural conformation of TPS and consequent immunoregulatory property.Chemical modification also improved the hypoglycemic effects of TPSs.In Wang’s study,animal experiments showed that modified TPSs with rare earth elements (REE) could significantly reduce the content of blood glucose in mice, and the higher the content of REE, the better the reducing activity of blood glucose [52].Two kinds of TPSs, namely neutral polysaccharide (NTPS) and acid polysaccharide (ATPS),were sulfated by pyridine-sulfonic acid method to afford ATPS-S and NTPS-S.Their hypoglycemic effects were determined as:NTPS-S ≈ ATPS-S ＞ ATPS ＞ NTPS, suggesting markedly improvement of hypoglycemic activity due to the sulfation of TPSs [53].

3.5.2 Selenium element

It has been reported that Se-containing polysaccharide generally showed higher bioactivity than its ordinary polysaccharide.For instance, Se-polysaccharides (Se-TPS, Se-TPS1, Se-TPS2, and Se-TPS3) showed higher antioxidant activities in comparison with their regular TPSs counterparts (ATPS1, ATPS2, and ATPS3) [54].The inhibitory activity of selenium-containing TPSs (Se-GTPS) from a selenium-enriched Ziyang green tea against human MCF-7 breast cancer cells was investigated, and found that Se-GTPS exhibited a significant inhibitory effect on the growth of MCF-7 cells [55].Wang et al.[56]also found that polysaccharide from Se-enriched Ziyang green tea TPSs had significant inhibition effect on the proliferation of human osteosarcoma U-2 OS cellsin vitroandin vivo.Cheng et al.[57]compared the antitumor activity of Se-TPS on sarcoma 180in vitroandin vivowith its original TPSs and dietary supplement Se-yeast,and found that Se-TPS had higher potency in antitumor activity than original TPSs and Se-yeast at the same dose, which was due to the synergistic effect of Se and TPSs.Moreover, the element Se could change the morphology of TPSs, leading to that the inhibitory activity of TPSs extracted from artificially selenium-enriched tea leaves onα-glucosidase was markedly higher than TPS extracted from common tea leaves [58].It was also found that the higher content of Se, the higher bioactivity.For example, in Gu’s study [15], two novel selenium polysaccharide fractions (SeTPS-1 and SeTPS-2) were isolated from Se-enriched tea, and the results revealed that the SeTPS-1with more content of Se had higher antioxidant activity than SeTPS-2.

3.5.3 Metal ion

Metal ions in polysaccharides may cause the structure change and further affect the bioactivities of TPSs.In an investigation, total tea polysaccharides (TTPS) extracted from pruning tea leaves of two teas,Fuan Baicha and Pingyang Tezaocha, were fractionated into neutral tea polysaccharides (TPSI) and acidic tea polysaccharides (TPSII).TTPS, TPSI and TPSII showed different inhibitory effects againstα-glucosidase before and after removing the metal ions.But the inhibitory effects of TPSI onα-glucosidase had been improved after removing the metal ions, suggesting that the glycosidase inhibitory activity was impeded by the metal bounding of TPSs [59].

In addition to these influencing factors mentioned above, the bioactivities of TPSs may have correlations with the type and position of glycosidic linkages,α/β-configuration, degree of branching, and chain conformation (e.g., random coil, compact coil, triple helix) [60-64].Therefore, more study is demanded on the SAR between these structural features of TPSs and their bioactivities.

4.Conclusions

Biological activities of TPSs are strongly related to their structural features including chemical composition, configuration and position of glycosidic linkages,Mw, and space conformation, which are affected by tea material, processing technologies, extraction methods and modification.In general, the lower theMwof TPSs, the higher the antioxidant activities, but with exceptions that TPS with higherMwshowed stronger antioxidant activity than that with lowerMw.Tea resources and fermentation/ageing processes have a great impact on the structures of TPSs, leading to their diverse biological activities,yet there is no in-depth mechanism study.The space conformation of TPSs plays an important role in their bioactivities, but there are very few studies on the relationship among them.In addition, the bioactivities of TPSs are affected by a variety of structural factors,hence more in-depth studies need to be performed to find out which structural factors contributes the most to the bioactivities of TPSs.Due to the multiple influencing factors discussed in this review and the complex structures of TPSs, the relationship between structures and bioactivities remains unclear.Thus, a large number of experiments are warranted to identify the complete structures of TPSs and establish their structure-bioactivity relationship.Future studies should tackle the complexity of TPSs structures and other influencing factors to explore the efficacious effects of TPSs.

Declaration of competing interest

The authors declare that there is no conflict of interest.

Acknowledgements

This work was supported by Hubei Science and Technology Plan Key Project (G2019ABA100), Assessment and Comprehensive Utilization of Characteristic Biological Resources in Dabie Mountains (4022019006), Natural Science Fund of Hubei Province(2019CFB395).