Ellagitannin Digestion in Moth Larvae and a New Dimeric Ellagitannin from the Leaves of Platycarya strobilacea

Ellagitannins (ETs) are plant polyphenols with various health benefits. Recent studies have indicated that the biological activities of ETs are attributable to their degradation products, including ellagic acid and its gut microflora metabolites, such as urolithins. Insect tea produced in the Guangxi region, China, is made from the frass of moth larvae that feed on the ET-rich leaves of Platycarya strobilacea. Chromatographic separation of the Guangxi insect tea showed that the major phenolic constituents are ellagic acid, brevifolin carboxylic acid, gallic acid, brevifolin, and polymeric polyphenols. Chemical investigation of the feed of the larvae, the fresh leaves of P. strobilacea, showed that the major polyphenols are ETs including pedunculagin, casuarictin, strictinin, and a new ET named platycaryanin E. The new ET was confirmed as a dimer of strictinin having a tergalloyl group. The insect tea and the leaves of P. strobilacea contained polymeric polyphenols, both of which were shown to be composed of ETs and proanthocyanidins by acid hydrolysis and thiol degradation. This study clarified that Guangxi insect tea contains ET metabolites produced in the digestive tract of moth larvae, and the metabolites probably have higher bioavailabilities than the original large-molecular ETs of the leaves of P. strobilacea.


Introduction
Tannins are a class of plant polyphenols that precipitate proteins [1] and are attracting increasing attention as food constituents with potential health benefits [2]. Tannins are chemically grouped into two major classes: condensed tannins (also called proanthocyanidins) and hydrolyzable tannins, and the hydrolyzable tannins include gallotannins and ellagitannins (ETs). ETs are esters of hexahydroxydiphenoyl (HHDP) or its biogenetically related acyl groups typically bounded to glucose [3][4][5]. Compared to the condensed tannins and gallotannins, ETs exhibit a large structural diversity associated with structures and location of the acyl groups as well as their molecular sizes. Similar to other tannins, interaction of ETs with salivary proteins by hydrophobic association causes unfavorable bitter and astringent taste [6]. The tannin-protein interaction also causes inhibition of digestive enzymes of herbivores, reducing the nutritional value of plants consumed as food [7]; therefore, ETs are thought to be plant defense substances, and the ecological significance of ETs and condensed tannins has long been discussed [8][9][10]. Common foods that contain ETs include raspberry, strawberry, pomegranate, and walnut, and recent studies have suggested that many of the biological activities of these foods are attributable to ETs. Oak wood is also rich in ETs; therefore, wine, whiskey, brandy, and other alcoholic beverages that are aged in oak barrels also contain ETs or their degradation products that originate from the oak wood [11,12]. ETs are large molecules (larger than 500 Da), and some are larger than 2000 Da [1, [3][4][5]. Thus, ETs are not directly absorbed from the gastrointestinal tract. However, ETs show biological activities by oral administration, and it is now commonly accepted that the biological activities of ETs are attributable to their metabolites produced by intestinal microbiota [13,14], as with the case of tea catechins and proanthocyanidins [15]. Initial degradation of ETs by intestinal bacteria probably occurs via hydrolysis of the HHDP groups to give ellagic acid (2). Subsequent reductive metabolism of 2 by intestinal bacteria generates urolithins. Urolithin A (3,8-dihydroxy-6Hdibenzo[b,d]pyran-6-one, i.e., castoreum pigment) was originally found in exudate from beavers [16] and in renal calculi of sheep [17], suggesting that ET metabolism is common in herbivorous animals. Recent studies have confirmed the same metabolism of ETs in human intestinal microflora [13,14], and this has had a considerable impact on biological studies of ETs. From the viewpoint of chemical ecology, degradation of ETs by insects are also important; however, details of the degradation products are not sufficiently clarified [18,19]. In this study, we investigated insect tea produced in Southwest China that is made from the frass of moth larvae. Although there are several types of insect teas in China, the insect tea of the Guangxi region is made from the frass of larvae of Hydrillodes morose or Nodaria niphona fed on the ET-rich leaves of Platycarya strobilacea [20]. The purpose of this study was to reveal the ET-derived molecules in the insect tea and to clarify the metabolism of ETs in the insect digestive tract by comparing the constituents of the insect tea with the polyphenols in the larvae feed.

Constituents of Insect Tea
Aqueous acetonitrile (CH 3 CN) extract of insect tea was analyzed by high-performance liquid chromatography (HPLC) with photodiode array detection ( Figure 1). The chromatogram showed three prominent peaks, and the two at 8.1 min and 30.8 min were identified as gallic acid (1) and ellagic acid (2), respectively, by comparison of retention times and ultraviolet (UV) absorptions. To identify the remaining major compound observed at 20.8 min and other minor constituents, the aqueous acetone extract was separated on a large scale, and a comparison of nuclear magnetic resonance (NMR) data showed that the major compound detected at 20.8 min was brevifolin carboxylic acid (3) [21,22]. In addition, brevifolin (4) [23], kaempferol 3-O-glucuronide (5) [24], quercetin 3-O-rhamnoside (6) [25], and myricitrin 3-O-rhamnoside (7) [25] were isolated as the minor constituents (see Figure 2). The contents of 1-3 in the insect tea were estimated to be 1.6, 4.3, and 2.16 mg/g, respectively, by HPLC analysis. Furthermore, polymeric polyphenols detected at the origin on thin-layer chromatography (TLC) and observed as a broad hump on the HPLC baseline were obtained by size-exclusion column chromatography using Sephadex LH-20 with 7 M urea in acetone as the elution solvent [26].
The presence of 1 and 2 in the insect tea suggested that ETs in the feed (leaves of P. strobilacea) were degraded to acyl components in the gut of moth caterpillars. Ellagic acid (2) is derived from HHDP esters by hydrolysis. Production of 3 suggested the oxidative degradation of HHDP groups. It is known that the pH of midgut fluids of insect caterpillars is alkaline [18,19,27,28], and our previous study demonstrated that dehydrohexahydroxydiphenoyl (DHHDP) esters, the oxidized form of HHDP esters, decompose and generate 3 under weakly alkaline conditions [19,21].
Compound 13 was isolated as a brown amorphous powder and showed dark blue coloration after spraying with spraying iron(III) chloride (FeCl 3 ) reagent on TLC. The 13 C NMR spectrum showed signals arising from six pyrogallol-type aromatic rings accompanied by six ester carbonyl carbons. The dimeric nature of 13 was apparent from observation of two sets of 1 H and 13 C NMR signals of hexoaldoses, and it was confirmed by high-resolution fast-atom bombardment mass spectrometry (HRFABMS), which showed the [M+Na] + peak at m/z 1289.1352 indicating the molecular formula of C 54 H 42 O 36 (calcd. for C 54 H 42 O 36 Na: 1289.1348). The large coupling constants (8.2-9.9 Hz) of the aldose ring protons observed in the 1 H NMR and 1 H-1 H COSY spectra revealed that both of the two aldoses were β-glucoses. The chemical shifts of the anomeric protons (δ H 5.53 (glc-1) and 5.75 (glc-1')), two C-4 methine protons (δ H 4.79 (glc-4) and 4.94 (glc-4')), and two-sets of C-6 methylene protons (δ H 5.14 and 3.68 (glc-6), 5.14 and 3.78 (glc-6 )) demonstrated acylation of hydroxy groups at these positions of both glucoses. Furthermore, large differences of the chemical shifts of C-6 methylene proton signals were similar to those observed for 11, suggesting formation of macrocyclic rings by acylation of HHDP groups at the glucose C-4 and C-6 hydroxy groups of glucopyranoses I and II [36]. This was supported by HMBC correlations of ester carbonyl carbons with aromatic protons and glucose ring protons ( Figure 4).
As for the acyl groups, the presence of a galloyl group is apparent from the two-proton singlet at δ H 7.18 in the 1 H NMR spectrum and carbon signals at δ C 110.2, 120.6, 139.4, 146.0, and 165.3 in the 13 C NMR spectrum. Similarly, comparison of the NMR signals with those of 11 revealed the presence of a HHDP group (δ H 6.67 (s, H-6) and 6.52 (s, H-6')). Connection of the galloyl group to C-1 of glucose II and the HHDP group to glucose C-4 and C-6 of glucose I was shown by observation of the HMBC correlations between pyranose ring protons and aromatic singlets to corresponding ester carbonyls (Figure 4). The remaining acyl groups that connected glucoses I and II were suggested to be a gallic acid trimer with three isolated aromatic protons (δ H 7.00 (s, H-6"), 6.72 (s, H-6), and 6.51 (s, H-6')). There are three acyl groups composed of three galloyl components; they are tergalloyl [31], valoneoyl [37], and macaranoyl groups [38], which differ in the oxygen atom of the HHDP moiety where a galloyl group forms an ether linkage (tergalloyl, B-ring C-4'; valoneoyl, C-5'; and macaranoyl, C-3'). Among them, the galloyl group of 13 was determined to be the tergalloyl group based on appearance of the carbon signals at δ C 131.6 (C-1'), 148.1 (C-5'), and 148.9 (C-3') characteristic to the tergalloyl B-ring (Figure 4). In particular, the strong low-field shift of C-1' at δ C 131.6 as compared with those of the tergalloyl ring A (δ C 125.5) and the HHDP esters (δ C 126.6 and 127.0) was conclusive evidence for the identification. The location of the tergalloyl group was determined based on the HMBC correlations of sugar protons and aromatic protons to ester carbonyl carbons ( Figure 4). In addition, a large positive Cotton effect at 238 nm and a negative Cotton effect at 261 nm indicated that the axial chirality of the two biphenyl bonds of the tergalloyl and HHDP groups are in S configuration [39]. These spectroscopic results allowed us to determine the structure of 13 as shown in Figure 3, and this compound was named platycaryanin E.  As for the acyl groups, the presence of a galloyl group is apparent from the twoproton singlet at δH 7.18 in the 1 H NMR spectrum and carbon signals at δC 110.2, 120.6, 139.4, 146.0, and 165.3 in the 13 C NMR spectrum. Similarly, comparison of the NMR signals with those of 11 revealed the presence of a HHDP group (δH 6.67 (s, H-6) and 6.52 (s, H-6')). Connection of the galloyl group to C-1 of glucose II and the HHDP group to glucose C-4 and C-6 of glucose I was shown by observation of the HMBC correlations between pyranose ring protons and aromatic singlets to corresponding ester carbonyls (Figure 4). The remaining acyl groups that connected glucoses I and II were suggested to be a gallic acid trimer with three isolated aromatic protons (δH 7.00 (s, H-6"), 6.72 (s, H-6), and 6.51 (s, H-6')). There are three acyl groups composed of three galloyl components; they are tergalloyl [31], valoneoyl [37], and macaranoyl groups [38], which differ in the oxygen atom of the HHDP moiety where a galloyl group forms an ether linkage (tergalloyl, Bring C-4'; valoneoyl, C-5'; and macaranoyl, C-3'). Among them, the galloyl group of 13 was determined to be the tergalloyl group based on appearance of the carbon signals at δC 131.6 (C-1'), 148.1 (C-5'), and 148.9 (C-3') characteristic to the tergalloyl B-ring ( Figure  4). In particular, the strong low-field shift of C-1' at δC 131.6 as compared with those of the tergalloyl ring A (δC 125.5) and the HHDP esters (δC 126.6 and 127.0) was conclusive evidence for the identification. The location of the tergalloyl group was determined based on the HMBC correlations of sugar protons and aromatic protons to ester carbonyl carbons (Figure 4). In addition, a large positive Cotton effect at 238 nm and a negative Cotton effect at 261 nm indicated that the axial chirality of the two biphenyl bonds of the tergalloyl and HHDP groups are in S configuration [39]. These spectroscopic results allowed us to deter-

Polymeric Polyphenols
The Guangxi insect tea contained polymeric polyphenols detected as a dark-blue spot at the origin of the silica gel TLC with FeCl 3 reagent. By HPLC analysis, the polymer was observed as a broad hump on the chromatogram baseline. Similar polymeric polyphenols were also found in the fresh leaves of P. strobilacea. The 13 C NMR spectra of these polymeric polyphenols ( Figure S1, Supplementary Materials) were related to each other and showed broad signals, suggesting the presence of procyanidins and ellagitannins. Signals in the range of δ C 150-160 were assignable to proanthocyanidin A-ring C-5, 7, and 8a, and other aromatic carbon signals were attributed to pyrogallol-and catechol-type rings. In addition, ester carbon signals were observed in the range of δ C 162-170, and the presence of sugar moieties was indicated by anomeric carbon signals at δ C 95 and aliphatic carbon signals in the range of δ C 62-78. To characterize the polymeric polyphenols chemically, thiol degradation for proanthocyanidins and acid hydrolysis for ellagitannins were applied to both samples. The thiol degradation of proanthocyanidins using mercaptoethanol produced thioethers of flavan-3-ol units by nucleophilic substitution at C-4 positions ( Figure S2) [40]. The HPLC analysis of the thiol degradation products of polymeric polyphenols from P. strobilacea leaves showed prominent peaks attributable to thioethers of epigallocatechin, epicatechin, and their galloyl esters ( Figure S3D). However, the chromatogram of polymeric polyphenols of insect tea showed only small peaks of thioethers and a large part of the broad hump arising from the polymers remained ( Figure S3B). This result suggested that the original proanthocyanidins in the feed were converted to crosslinked substances in the digestive tract of the larvae and did not afford the usual thiol degradation products. However, acid hydrolysis of the two polymeric polyphenols yielded similar products and the major products were identified as gallic acid (1) and ellagic acid (2) ( Figure S4). Therefore, the major components of the polymeric polyphenols of insect tea are considered to originate from the ETs in the leaves, and the polymers retain HHDP and galloyl groups.

Decomposition of Ellagitannins in the Digestive Tract of Moth Larvae
The results described above indicate that the galloyl and HHDP esters of the ETs of the feed are hydrolyzed to gallic acid (1) and ellagic acid (2) in the digestive tract of moth larvae. In addition, production of brevifolin carboxylic acid (3) and its decarboxylation product brevifolin (4) suggest that a part of the HHDP esters is oxidatively degraded in the larval gut, since the production mechanism of 3 from HHDP esters include the oxidative process [21]. Previously, Vihakas et al. showed that phenolic profiles of the frass of lepidopteran larvae fed on leaves containing ETs were similar to those of the alkali-treated extracts of the leaves, and they suggested that ETs were modified by the alkaline pH of the larval gut [19]. Because tannins are susceptible to autoxidation under alkaline conditions, 3 in the insect tea may by generated by autoxidation. More detailed investigations are necessary to clarify the production mechanism of 3 in the digestive tract.

Materials
Commercial insect tea produced from excrements of Hydrillodes morose fed on P. strobilacea in Liuzhou, Guangxi Zhuang Autonomous Region, China, was purchased in Guilin. The leaves of Platycarya strobilacea were collected in Hagi city, Yamaguchi Prefecture, Japan, in July 2020. The insect tea and voucher specimen of P. strobilacea were deposited at the Nagasaki University Graduate School of Biomedical Sciences.

Platycarya strobilacea
Fresh leaves of P. strobilacea (450 g) were extracted with 70% aqueous acetone (3 L × 2). The extract was concentrated, and the resulting insoluble precipitates were removed by filtration. The filtrate was applied to a column of Diaion HP20SS (4 cm i.d. × 30 cm) with 0-100% MeOH (10% stepwise gradient elution, each 500 mL) to give two fractions: fr. 1 The presence of gallic acid (1), ellagic acid (2) and brevifolin carboxylic acid (3) in the excrements showed occurrence of ester hydrolysis of ellagitannin acyl groups and further oxidative modification in the digestive tract of the moth larvae. Although further studies on the production mechanism of 3 are necessary, the findings of this study have implications for understanding the fate of ETs in nature. In addition, a new ellagitannin named platycaryanin E (13) was isolated from the leaves of P. strobilacea, and the structure was determined to be a dimer of strictinin (11) having a tergalloyl ester group.