Before discussing the biosynthetic pathway to GTs, it appears indispensable to insert a few comments on the structural principles and conventional definitions used in this field. First of all, it should be legible with regard to the chemical structure of GTs. Structurally surrounded by several galloyl units, GTs are with a polyol as the core, most often glucose. On the other hand, GTs are identified as polygalloyl esters of glucose. Apart from glucose, diverse polyol, catechin, or triterpenoid cores could be bounded to the galloyl unit (17). Such GTs, containing glucitol core, were identified from Red maple (Acer rubrum) (18). Unnatural GTs, taking 2-C-(hydroxymethyl)-branched aldoses as the central polyol, were designed and synthesized (19). Employing 1,5-anhydroalditol and inositol as the cyclic polyol cores, Machida et al. designed and obtained 14 unnatural gallotannin derivatives (20).

According to Niemeta and Gross (21), GTs could further be subdivided into “simple” and “complex” galloylglucoses. As a natural product, 1-O-galloyl-β-D-glucopyranose (compound 1, Figure 1), the simplest member of GTs, had been isolated and characterized more than a century ago. According to the number of galloyl groups, there are MoGG, one galloyl group; DiGG, two galloyl groups; TriGG, three galloyl groups; and TeGG, four galloyl groups. Fully substitution of galloyl to glucose would lead to 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG) (compound 2, Figure 1) (22), which seemed as the prototypical “complex” galloylglucoses (23). Sequentially, gallyol units can be grafted through depside bonds to form “complex” galloylglucoses with high molecular weight (MW). 2-O-digalloyl-1,3,4,6-tetra-O-galloyl-β-D-glucose (compound 3, hexagalloylglucose), the typical representative of these “complex” galloylglucoses, is given in Figure 1. One point to be emphasized was that gallic acid now combined with phenolic hydroxyls via a depsidic bond. As known, chemical properties are significantly different between the aliphatic OH-groups of the glucose and phenolic hydroxyl groups. It had been proved that the depside bonds between galloyl units are considerably easier to broken than the core ester linkages (6). In wake of the presence of an esteric linkage between two galloy moieties, GTs are also considered to be depsidic metabolites.

The degradation of GTs consists of a series of enzymatic catalyzed reactions. These enzymes refer to tannase, decarboxylase, pyrogallol 1,2-dioxygenase, pyrogallol-phloroglucinol isomerase, phloroglucinol reductase, dihydrophloroglucinol hydrolase, phenol oxidase, etc. First, cleavage of depside bonds takes place. Following, hydrolysis of the ester bond was undergone to yield gallic acid. Then, catalyzed by gallic acid decarboxylase, decarboxylation would be taken place on gallic acid to form pyrogallol. Ulteriorly, pyrogallol would convert to open loop secondary metabolites, e.g., pyruvic acid, and finally enters the Krebs cycle (24).

Also, GTs could be easily degraded by bacteria, fungi, and yeasts (25, 26). As reported, microorganisms, present in the rumen and the distal portion of the monogastric intestine of the ruminants, were involved in the hydrolysis of ester and depside linkages of GTs. During the procedure, glucose and gallic acid were released. Furthermore, gallic acid would continue degrade to pyrogallol and phloroglucinol and, ultimately, to acetate and butyrate. In the light of the present situation, biodegradation of GTs is still in an incipient stage. For the treatment with large-scale applications, especially in food, fodder, medicine, and tannery effluent, there is still a long way to go.

When it turns to the synthesis of GTs, biosynthesis would be first discussed. Derived from an intermediate of 5-dehydroshikimate, formation of gallic acid was reported to be through a shikimate pathway (27). Catalyzed by enzyme extracts from oak leaves, esterification between gallic acid and glucose was conducted to form β-glucogallin (1-O-galloyl-β-D-glucose) (28). During this procedure, uridine diphosphate (UDP) glucose served as an activated substrate. In association with the biosynthetic pathway, β-glucogallin is presumed to be the first intermediate and a key metabolite in the synthesis of GTs. Enzyme studies conducted by Grundhofer et al. indicated that β-glucogallin was required as a principal acyl donor for the biosynthesis of GTs (29). Without any other cofactors, the transformation of β-glucogallin, formed in situ, to di- and trigalloylglucose, was catalyzed by these enzymes. β-glucogallin was deemed to function not only as acyl acceptor, but also as efficient acyl donor (24). Demonstrated by immunohistochemical studies, leaf mesophyll cell walls were the typical site of origin and deposition of hydrolyzable tannins.

With extreme regularity, substitution of glucose hydroxyls was not randomly distributed in these conversions. Thus, the metabolic sequence followed the following order: β-glucogallin → 1,6-digalloylglucose → 1,2,6-trigalloylglucose → 1,2,3,6-tetragalloylglucose → 1,2,3,4,6-pentagalloylglucose. Accompanied with the formation of PGG, generally considered to be a simple galloyl glucose ester, the first stage for the biosynthetic pathway of GTs is completed. In all the plants, the pathway to PGG had been regarded to be identical. The transition from “simple” galloylglucoses to “complex” GTs is marked by the attachment of further galloyl residues to PGG to obtain the meta-depside groups (30). Taking PGG as the core, further galloylation reactions were conducted at the phenolic hydroxyl groups. During the second step, high-molecular metabolites containing up to 10 galloyl residues were formed (31).

On the other hand, for the reason of natural materials in short supply and difficulties in the isolation and purification, study work and the practical utilization of GTs were limited. Because of this, chemical synthesis of GTs was documented unremittingly. The first total chemical syntheses of two GTs, hexagalloylglucose and decagalloylglucose, was reported by Sylla et al. (32). Employing commercially 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose as the starting material, the first galloyl moiety was introduced at the O3 position of the glucose unit. Under mild non-acidic Steglich-type conditions, successive galloylation reaction was taken place to give the corresponding 3-O-galloylated D-glucofuranose. Mediated by palladium in tetrahydrofuran (THF) at room temperature for 24 h, the hexagalloyl-β-glucopyranoses, PGG, would be obtained after the final hydrogenolysis of the fully protected hexagalloyl-D-glucopyranoses (32). Employing benzyl-protected gallic acid chloride as the substrate, reaction conditions that lead to high anomeric selectivity for the synthesis of GT were investigated. Accordingly, there is a significant correlation between solvent for the reaction and α:β selectivity. After screening, the highest selectivity was found when acetonitrile (CH₃CN) was used as a solvent. Meanwhile, dichloromethane (CH₂Cl₂) was demonstrated to accelerate the rate of mutarotation relative to the rate of condensation to the ester (33). Starting from slight modification of methyl gallate, coupling with galloylation sequentially, alkyl GTs, which are structurally related with natural compounds, were synthesized (34). And then, protective effects were investigated.

Various galloyl-glucopyranoses occur naturally in lots of plants. Representatively, occurrence of GTs was found in the extracts of Chinese galls (Rhus semialata) and Turkish galls (Gallae turcicea). Also, presence of GTs in plants, such as Paeonia (23) and Schinus terebinthifolius, the leaves of Sicillian sumac (Rhus coriora L.) and the common smoke tree (Cotinus coggygria Scop.) were at sufficient levels to allow direct isolation and utilization (17). Among 42 edible beans, red sword bean (Canavalia gladiata) was found to have the highest content of GTs (35). Also, first report of the presence of GTs in nutshell of camellia tree (Camellia oleifera C. Abel) was disclosed by He et al. (36). From the byproducts of mango, barks, kernels, leaves, etc., GTs were identified and quantitated (37).

As to the refined structure, the various sources resulted in GTs with different chemical structures. As well, the concentration of GTs varies with plant genotype, tissue developmental stage, and environmental conditions (38). Several inconsistent theories had been developed regarding the chemical composition of GTs distributed in different plants. Although the contents varied greatly, the presence of GTs was fairly consistent across a range of cultivars. Under the circumstances, general assays were much preferred to elucidate specific structures of GTs. Chelation with metal ions and the formation of complexes guaranteed the detection of GTs by UV–Vis spectroscopy. Advances in describing GTs structural variation using high-performance liquid chromatography–mass spectrometry (HPLC–MS) had been made (39). Undoubtedly, simple colorimetric assays for the measurement of tannins and other phenolics facilitated many ecological studies of GTs, whereas the appropriate standard was crucial for the assay. Quantitatively, huge errors might be obtained if an inappropriate standard was used, for example, the commonly used quebracho tannin. Stumbling blocks for controlled studies on specific GTs were still existed due to the lack of commercially available standards. It is delightful, with the extraction of crude tannin and additional purification, a growing number of standards had been prepared by more and more researchers in a related field. Taking the degree of hydrolysis into consideration, amounts of free gallic acids were also indeterminate in quantity. According to the report, ratio of glucose to gallic acid was 1–9 or 10 in oriental sumac GTs in contrast to 1–5 or 6 in Turkish gall nuts (39).

Certainly, extraction would make a big difference in analysis of GTs. Despite the lack of commercially available standards, pioneer articles related to extraction, optimization, identification, and quantification of GTs were documented constantly. In respect to separation of GTs from mango kernels, extract process comprised of degreasing of seeds, extraction with aqueous acetone [80% (v/v)], and use of high-speed countercurrent chromatography for purification (40). With the crude extract in hand, subsequent purification was applied by being partitioned against ethyl acetate. According to the degree of galloylation (tetra-O-galloylglucose to deca-O-galloylglucose), solvent system employing hexane/ethyl acetate/methanol/water [0.5:5:1:5 (v/v/v/v)] was used in the head-to-tail mode to elute tannins. It had been proved that the using of hexane for the extraction of mango kernel fat would not affect GTs in respect to the profile and contents (41). In contrast, when methanol was used, significant changes were observed, which might result from the methanolysis of GTs to PGG. Methyl gallate and PGG would be yielded due to simultaneous extraction and methanolytic conversion of GTs. Degradation of higher GTs took place when methanol was used merely for the extraction of mango seeds. All these constituted important facets with respect to the utilization of GTs present in byproducts derived from mango fruit processing.

The rapid development of instruments as well as the innovation in analytical methods facilitates the discovery of new novel GTs. Meanwhile, improved detection accuracy guaranteed the detection of GTs in a wider range of plant species. Based on the flow-injection analysis–electrospray ionization–ion trap–tandem mass spectrometry (FIA-ESI-IT-MS-MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), a rapid and reliable analytical approach was proposed to investigate the full file of HTs present in the extracts of Astronium species and evidenced the existence of GTs in Astronium genus first (42). According to Boulekbache-Makhlouf, 18 GTs were present in fruit of Eucalyptus globulus growing in Algeria (43). By means of high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI-MS) method, assisted by diode array detection, 14 GTs could be isolated from birch (Betula pubescens) leaves (44). With the occurrence of absorption maxima between 275 and 280 nm on UV spectra, LC-MS molecular ion and fragment ion peaks, combining with NMR survey to the proton and carbon signals, five GTs were identified from Sapria himalayana f. albovinosa in Myanmar (45). Study result revealed that monogalloyl to hexagalloyl hexosides comprised the GTs film. Characterized by MALDI-TOF-MS, relying on the alteration of cationization reagents, Cs⁺, K⁺, and Na⁺, a different spectrum of Chinese GTs was given (46).

There is one consensus to be emphasized that GT structural details are fateful for many of their biological effects. Equipped with the amount of hydroxyl groups (-OH) as well as the high molecular complexity, GTs were documented with several pro- and antiphysiological effects, such as antioxidant (47), antidiabetic (9), precipitating proteins (48), and antimicrobial (49). Theoretically, cooperative effects of hydrogen bonding and hydrophobic association were responsible for the GT–protein and GT–phospholipid interactions (48). Likewise, hydrogen bonding was pointed to be the predominant effect in the interactions between GTs and sugars.

Despite the diverse physiological activities as well as the extensive application, especially in food industry, double-edged sword of GTs should not be neglected. Intake of GTs might lead to damage of intestine and digestion, especially in susceptible species, e.g., sheep (75). In severe case, failure of kidney and liver would be resulted. Experimental data revealed that synthetic GTs, methyl 2,3,4,6-tetra-O-galloyl-α-D-glucoside (G4Glc), methyl 2,3,4,6-tetra-O-galloyl-α-Dmannoside (G4Man), and methyl 2,3,4-tri-O-galloyl-α-L-rhamnoside (G3Rham), possessed significant antioxidant activities (24). Whereas, very low genotoxic effect on human peripheral blood mononuclear cells (PBMCs) was manifested. Moreover, profiting from the protection of these compounds, damage induced by hydrogen peroxide or Fe²⁺ on DNA would be alleviated considerably. Based on these results, it could be concluded that synthetic GTs represent non-toxic agents, which implied the potential biomedical applications of GTs. At another level, it would be useful for the subsequent design of new antioxidants. Because of the binding with protein of GTs, bioavailability of both the proteins and polyphenols would be reduced. Previous studies suggested that vegetable tannins possess a high binding affinity to proline-rich proteins (76). Hydrophobic stacking of the planar phenolic ring against the pyrrolidine ring of the proline was pointed out to be the primary between PGG and proline-rich peptides. The molecular weight as well as structural flexibility of GTs was related to the ability of precipitation of protein. Depending on GTs concentration and nutrient levels, it would be beneficial for mammals for the ability to bind with proteins in the guts (41).

Due to the limited bioavailability of GTs, intake of GT-containing products orally can influence immune response at the level of the gastrointestinal tract. Modulating effects on the gut microbiota composition would be affected as well. More accurate delivery to affected areas as well as more efficient absorption and utilization is still in front of the urgent need to solve the problem.

At the level of action mechanism, structure–function relationship indicated among polyphenolated templates, naturally occurring GTs may be not the optimal protein recognition agents in fact (77). For this reason, modification of the structure of GTs with the help of chemical synthesis was desired. From another perspective, assays for detection of GTs require the access to standard monomeric substance and chemical synthesis would be a powerful means. Development of the chemical synthetic methodology to construct GTs awaits to be optimized.

With the advantage of analytical instruments, it becomes possible for the determination of GTs with more accuracy and faster. Taking ultramodern instruments and technique into application, characterization of monomers and analogs of GTs remains to be clarified systemically. Also, content of trace in new resources would be no longer the obstacle for verification. As a result, it is worth looking forward to see the increase of the membership of GTs family.