Insight into Medicinal Chemistry Behind Traditional Chinese Medicines: p-Hydroxybenzyl Alcohol-Derived Dimers and Trimers from Gastrodia elata

Graphic Abstract From an aqueous extract of “tian ma” (the steamed and dried rhizomes of Gastrodia elata), ten new compounds gastrodibenzins A−D (1−4) and gastrotribenzins A−F (5−10), along with known analogues (11−20), having structure features coupling between two and three p-hydroxybenzyl-derived units via carbon- and/or ether-bonds, were isolated and characterized by spectroscopic data analysis. Meanwhile, the new compounds 5a, 6a, 8a, 22, and 23, as well as the known derivatives 13a, 14a, 15, 17−21, 24, 25, and p-hydroxybenzyl aldehyde were isolated and identified from a refluxed aqueous solution of p-hydroxybenzyl alcohol. Methylation of 5a and 6a in methanol and ethylation of 6a, 8a, 13a, and 14a in ethanol produced 5 and 6 and 7, 8, 13, and 14, respectively. using ultra-performance liquid chromatography high-resolution electrospray ionization mass spectrometry (UPLC-HRESIMS) analysis of the refluxed solutions of p-hydroxybenzyl alcohol and the refluxed extracts of the fresh G. elata rhizome and “tian ma” extracts indicated consistent production and variation of the dimeric and trimeric derivatives of p-hydroxybenzyl alcohol upon extracting solvents and refluxing time. In various assays, the dimeric and trimeric derivatives showed more potent activities than p-hydroxybenzyl alcohol itself and gastrodin, which are the main known active constituents of “tian ma”. These results revealed for the first time that the more effective dimers and trimers can be produced through condensation of the co-occurring p-hydroxybenzyl alcohol during processing and decocting of the G. elata rhizomes, demonstrating insights into medicinal chemistry behind application protocols of traditional Chinese medicines. Electronic supplementary material The online version of this article (10.1007/s13659-020-00258-w) contains supplementary material, which is available to authorized users.

According to the theory of traditional Chinese medicines (TCM), the drug materials are commonly processed and/or decocted to detoxify and/or to enhance effects of the herbal medicines. Chemical reaction must take place during processing and/or decocting to alter the chemical compositions of final decoctions used for the treatment of patients. This suggests that the aqueous decoction of the processed drug material might contain more benefit components for patients. Thus, there are important secrets of medicinal chemistry hiding behind processing and/or decocting protocols in TCM though these are yet to be confirmed in many cases including "tian ma" [50][51][52][53][54][55][56][57][58]. Because the previous phytochemical studies of "tian ma" were performed mostly by extracting the drug material with ethanol or methanol [2,[5][6][7][8], the extracting protocol completely differed from that of conventional application by decocting with water. Therefore, an aqueous extract of "tian ma" (the steamed and dried G. elata rhizomes [50][51][52][53][54][55][56][57][58]) was investigated as part of our project to investigate chemical diversity and biological activities of several commonly used TCM [59][60][61][62][63][64][65][66][67][68][69][70]. Previously we reported 27 new and 40 known chemical constituents of the aqueous extract, along with their bioassays and pharmacological activities [71][72][73][74][75][76][77][78][79]. Especially we found that several p-hydroxybenzyl-modified gastrodins from the extract could be produced from a coupling reaction of the co-occurring p-hydroxybenzyl alcohol and gastrodin in H 2 O under refluxing [80]. This unraveled production of the new components during processing and decocting of "tian ma" in the classical application protocol. A further investigation resulted in characterization of ten new compounds gastrodibenzins A−D ( 1−4) and gastrotribenzins A−F (5−10) as well as ten known derivatives (11−20) ( Fig. 1) from the remaining subfractions of the extract. Viewing the structures of 4−20, these compounds may be derived from condensations of two or three p-hydroxybenzyl alcohol units at different positions via carbonand/or ether-bonds, followed by etherification with the solvents MeOH or EtOH (4 −14). With the speculation, a refluxed H 2 O solution of p-hydroxybenzyl alcohol was isolated to yield 5a, 6a, 8a, 13a, 14a, 15, 17−19, 21−25 (Fig. 1), and p-hydroxybenzaldehyde. UPLC-HRESIMS analysis of the refluxed methanol solutions of 5a and 6a and ethanol solutions of 6a, 8a, 13a, and 14a confirmed production of 5 and 6 and 7, 8, 13, and 14, respectively. Subsequent UPLC-HRESIMS analysis of the refluxed H 2 O, MeOH, and EtOH solutions of p-hydroxybenzyl alcohol and the extracts of the fresh G. elata rhizomes and "tian ma" provides insights into medicinal chemistry behind the processing and decocting protocols of TCM. Herein described are details.

Isolation and Structure Elucidation of 1−20
The pulverized "tian ma" (the steamed and air-dried G. elata rhizomes) was extracted by ultrasonicating with H 2 O. After concentrated, the aqueous extract was chromatographed over macroporous adsorbent resin, eluting with a gradient increasing EtOH in H 2 O to give fractions A−D. Fraction C was chromatographed over MCI gel, with successive elution using H 2 O, 30% EtOH, 50% EtOH, 95% EtOH, and Me 2 CO, to yield subfractions C1−C5. Further separation of the subfractions by column chromatography (CC) over Sephadex LH-20 and normal phase silica gel, middle-pressure liquid chromatography (MPLC) over reversed phase (C 18 ) silica gel, and reversed phase highperformance liquid chromatography (RP-HPLC) afforded compounds 1−20 (see 'Experimental' section).
Compound 2 was obtained as a yellowish amorphous powder. Its molecular formula was determined as C 14 (Table 1) indicated the presence of a 2-subustituted 4,5-dihydroxybenzaldehyde unit, in addition to the p-hydroxybenzyl identical to that in 1. This was verified by the HMBC correlations from H-3 to C-1 and C-5; from H-6 to C-2, C-4, and C-7; from H-7 to C-2 and C-6; from 4-OH to C-3 and C-5; and from 5-OH to C-4 and C-6, in combination with their chemical shifts. Meanwhile, the connection between the two units was demonstrated by the HMBC correlations from H-3 to C-7′ and from H 2 -7′ to C-1, C-2′/C-6′, and C-3. Therefore, the structure of 2 was determined as 4,5-dihydroxy-2-(4′hydroxybenzyl)benzaldehyde and named gastrodibenzin B.
Compound 3, a brownish amorphous powder, is an isomer of 2 as indicated by its spectroscopic data (Experimental and Table 1). Comparison of the NMR spectroscopic data between 3 and 2 suggested that the p-hydroxybenzyl was at C-3 of the 4,5-dihydroxybenzaldehyde unit in 3 instead of at C-2 in 2. The suggestion was confirmed by 2D NMR data analysis of 3, particularly by the HMBC correlations ( Fig. 2) from H-2 to C-4, C-6, C-7, and C-7′; from H-6 to C-2, C-4, and C-7; from H-7 to C-2 and C-6; and from H 2 -7′ to C-2, C-2′/6′, and C-4, together with their chemical shifts. Therefore, the structure of compound 3 was determined as 4,5-dihydroxy-3-(4′-hydroxybenzyl) benzaldehyde and named gastrodibenzin C.
Compound 4, a white amorphous powder, has the molecular formula of C 19 (Table 1) indicated the presence of two inequivalent p-oxybenzyloxys, two inequivalent ethoxys, and an isolated dioxymethylene. In the HMBC spectrum of 4 ( Fig. 2), the correlations from H 2 -7 to C-1 and C-2/C-6, from H-2/6 to C-4, and from H 2 -7′ to C-1′, C-2′/C-6′, and C-4, together with their chemical shifts demonstrated a head-tail connection of the two p-oxybenzyloxys via an ether bond between C-4 and C-7′. The HMBC correlations from H 2 -7 to the methylene carbon of one ethoxy unambiguously positioned the ethoxy at C-7. Moreover, the HMBC spectrum displayed the correlations from the dioxymethylene protons to C-4′ and the methylene carbon of the remaining ethoxy, indicating that an ethoxymethoxy unit 7 was located at C-4′ of 4. Accordingly, the structure of compound 4 was determined as ethyl 4-[4′-(ethoxymethoxy)benzyloxy]benzyl ether and named gastrodibenzin D.
Compound 6, a white amorphous powder, is an isomer of 5 as indicated by the IR, HRESIMS (see 'Experimental' section) and NMR spectroscopic data ( Table 2). Comparison of the NMR spectroscopic data of the two compounds indicated the presence of two equivalent p-hydroxybenzyls, a symmetrically disubstituted p-hydroxybenzyl, and an methoxy group in 5. This suggested that the terminal 4-hydroxybenzyl moiety at C-3′ in 5 was migrated to C-5 in 6. which was confirmed by 2D NMR data analysis. Especially, the HMBC correlations from OH-4 and H 2 -7′/ H 2 -7″ to C-4 and from OCH 3 and H-2/H-6 to C-7 demonstrated that the two equivalent p-hydroxybenzyls were substituted at C-3 and C-5 of the methyl 4-hydroxybenzyl ether unit to give a symmetric structure. Therefore, the structure of compound 6 was determined as methyl 4-hydroxy-3,5-di-(4′-hydroxybenzyl)benzyl ether and named gastrotribenzin B. Compound 7 was isolated as a white amorphous powder. Comparison of the spectroscopic data between 7 and 6 revealed replacement of the methyl group in 6 by an ethyl group [δ H 3.41 (2H, q, J = 7.2 Hz, OCH 2 CH 3 ) and 1.10 (3H, t, J = 7.2 Hz, OCH 2 CH 3 ), and δ C 65.6 (OCH 2 CH 3 ) and 15.5 (OCH 2 CH 3 )] in 7. This was further confirmed by the correlations of OCH 2 CH 3 /C-7 and H 2 -7/OCH 2 CH 3 in the HMBC spectrum of 7 (Fig. 2). Thus, the structure of compound 7 was determined as ethyl 4-hydroxy-3,5-di-(4′hydroxybenzyl)benzyl ether and named gastrotribenzin C.
The spectroscopic data of compound 10 indicated that it was one more isomer of 7−9. The HMBC spectrum of 10 exhibited the correlations (Fig. 2) from H 2 -7 to C-2, C-6, and OCH 2 CH 3 ; from H 2 -7′ to C-2, C-2′/6′, C-3, and C-4; from H 2 -7″ to C-2″/6″ and C-4′; and from OCH 2 CH 3 to C-7. These correlations, together with their chemical shifts, indicated the connection of C-3 to C-7′ as well as the ether-bond linkages of C-4′ to C-7″ and C-7 to the ethyl group in 10. Therefore, the structure of compound 10 was determined as  [8], gastrol A (20) [85]. The structure of 11 was previously determined only by UPLC/Q-TOF MS analysis [81] and identified in this study by comprehensive analysis of the spectroscopic data including 2D NMR experiments (Fig. 2), for which the detailed physical-chemical properties are reported ('Experimental' section).

Products from a Refluxed Aqueous Solution of p-Hydroxybenzyl Alcohol and Their Etherification with MeOH and EtOH
Among the 20 isolates, compounds 1−3 and 16 contain p-hydroxybenzyl and vanillyl alcohol (1 and 16) or protocatechualdehyde units (2 and 3), while the others are analogues of p-hydroxybenzyl-derived dimers (4, 12−15 and 17) and trimers (5−11 and 18−20). Because p-hydroxybenzyl alcohol, which abundantly occurs in G. elata [50][51][52][53][54][55][56][57][58], is highly reactive to produce quinone methide and complex derivative via self-condensation or inter-condensation with other reactants under various conditions [86][87][88][89], this suggests that, (a) p-hydroxybenzyl alcohol is an origin of the p-hydroxybenzyl unit in the p-hydroxybenzyl-containing chemical constituents of "tian ma"; (b) alcoholic forms of the p-hydroxybenzyl-derived dimers and trimers are generable from p-hydroxybenzyl alcohol during processing and/or extracting of the drug material; and (c) the ethyl and methyl ethers were formed in the subsequent isolation procedure through contacting to the solvents EtOH and MeOH. To verify the suggestions, following experiments were performed: (a) an aqueous solution of p-hydroxybenzyl alcohol was refluxed, from which the products were isolated and structurally identified; (b) methylation and ethylation of the p-hydroxybenzyl alcohol-generating products were examined by UPLC-HRESIMS after refluxing of their MeOH and EtOH solutions; (c) the refluxed solutions of p-hydroxybenzyl alcohol in H 2 O, MeOH, and EtOH were compared by UPLC-HRESIMS analysis using the identified pure compounds as references.
UPLC-HRESIMS analysis proved that 5 and 6 were produced respectively by refluxing of 5a and 6a or p-hydroxybenzyl alcohol in methanol (Fig. 3), while 7, 8, 13, and 14 were yielded by refluxing of 6a, 8a, 13a, and 14a, of which only 14 was undetectable from the refluxed EtOH solution of p-hydroxybenzyl alcohol (Figs. 4 and 5). All the isolated compounds from the refluxed H 2 O solution of p-hydroxybenzyl alcohol were detectable in the refluxed MeOH and EtOH solutions (Supporting Information Figs. S178−S183). However, except for compounds 8, 9, and 20, the phenolic ethers 10−12 and 19 were undetectable in the refluxed EtOH solution of p-hydroxybenzyl alcohol (Figs. 4, 5, and S178−S213), while the corresponding alcoholic forms of 9−12 as well as compounds 19 and 20 were not obtained from the refluxed H 2 O solution of p-hydroxybenzyl alcohol. This may be due to a structural instability of the phenolic ethers and/or their relative low abundance, which was preliminarily supported by interconversion of 9 and 10 in CH 3 CN (Fig. 4). In addition, compositions and abundances of the isomeric dimers and trimers were significantly varied in the sonicated and refluxed solutions (Figs. S184−S213). With increase of the refluxing time, the relative abundances of the p-hydroxybenzyl ethers 15 and 21 were significantly decreased in the H 2 O solution, whereas the corresponding p-hydroxybenzyl-substituted p-hydroxybenzyl alcohols 5a/6a and 13a were significantly increased. Meanwhile, the relative contents of 8a, 14a, 22, and 23 were decreased also with increase of the refluxing time, and the contentdecreased compounds had higher relative abundances in the sonicated H 2 O solution without refluxing (Supporting Information Figs. S190, S191, and S193). However, relative content variations of the isomeric trimers 5a/6a, 8a, and 21−23 in the refluxed MeOH and EtOH solutions of p-hydroxybenzyl alcohol (Figs. S198, S199, S205, and S206) were insignificant as compared with the refluxed H 2 O solution (Figs. S190 and S191), while the relative content variations of the isomeric dimers 13a, 14a, and 15 in the refluxed MeOH solution (Fig. S201)    (Figs. S223, S224, and S227) was unexpected and of interesting, which was confirmed by the paralleled and repeated experiments. Because the ethyl unit to form 7 and 13 was highly suspected to be producible in the reaction system, its ambient ethanol origin should not be excluded.
The above experiments demonstrated that the composition of the refluxed solutions of p-hydroxybenzyl alcohol are highly dependent upon the solvent and refluxing time. Because of the abundant occurrence of p-hydroxybenzyl alcohol in G. elata [50][51][52][53][54][55][56][57][58] (Fig. S233), the p-hydroxybenzyl-derived dimers and trimers from the "tian ma" extracts must be formed at least partially by refluxing of the drug materials in H 2 O, MeOH, or EtOH. Meanwhile, some dimers and trimers can be converted and/or transformed each other. This supports that indeed the chemical reactions take place during the processing and decocting of "tian ma" to produce the compounds and to modify the chemical composition.

UPLC/HRESIMS Analysis of H 2 O, MeOH, and EtOH Extracts of the G. elata rhizomes
To confirm the chemical reactions during processing and decocting of the herbal medicine [80], the fresh G. elata rhizomes were collected at the same field of "tian ma" (the steamed and dried rhizomes) and the extracts were prepared by sonicating of the fresh G. elata rhizomes and "tian ma" in H 2 O, MeOH, and EtOH for 0.5 h, respectively, followed by refluxing (sampling time: 0.5 h, 1.0 h, 1.5 h, 2.0 h, 4.0 h, and 6.0 h). The extract samples were analyzed by UPLC-HRESIMS using the aforementioned pure compounds as the references, showed that the composition and relative content of the extracts were varied with the extracting solvent and refluxing time. The precursor p-hydroxybenzyl alcohol and compounds 5a/6a, 13a, 15, 17, 18, 21, and 25 were detectable in the extracts obtained by sonicating of the fresh G. elata rhizomes and "tian ma" in H 2 O, MeOH, and EtOH, respectively (Supporting Information Figs. S233−S239), except that the trimers 5a/6a, 18, and 21 were undetectable in the H 2 O extract (Figs. S234 and S235). In addition, the relative content of the isomers 13a and 15 in the fresh rhizome extracts were reversed in the "tian ma" extracts ( Fig. S236), suggesting that 13a was generated at least partially during processing of the drug material.    (Fig. S249). Among the ethers, compound 13 was detectable only in the EtOH extracts of the fresh G. elata rhizomes (Fig. S257), indicating that this compound was formed from contacting with EtOH in the experimental procedure. When compared with the refluxed H 2 O extracts (Figs. S240 and S243), with increase of the refluxing time, the relative contents of 5a/6a and 13a were decreased in the MeOH and EtOH extracts (Figs. S247, S250, S254, and S258) while 13 was relatively increased in the EtOH extracts (Fig. S257). This demonstrated that 5a/6a and 13a were reacted with the solvents to be transformed into the corresponding methyl and ethyl ethers during refluxing, though compounds 5−7 were undetectable in the fresh G. elata rhizome extracts possibly due to low content. As compared with extracts of the fresh G. elata rhizomes, more compounds were detectable in the "tian ma" extracts (Figs. S262−S289). Compounds 2 and 3 appeared in all the refluxed "tian ma" extracts (Figs. S262, S270, and S280) and 1 in the EtOH extracts after refluxed for 2 h and 6 h ( Fig. S279). Particularly the methyl ethers 5 and 6 appeared only in the refluxed MeOH extracts (Fig. 8 and Supporting  Information Fig. S271) and the ethyl ethers 7, 9, and 14 in the refluxed EtOH extracts (Figs. 8, S284, and S285). This further supports that the methyl and ethyl ethers were produced from reaction of the corresponding dimeric and trimeric benzyl alcohols (such as 5a/6a, 13a, and 14a) with the solvents.
The isolated minor compounds 4, 8, [9][10][11][12]19, and 20 were undetectable in the extracts of either the fresh G. elata rhizomes or "tian ma", this may be explained by their low contents, since 4, 10−12, and 19 were also undetectable in the refluxed EtOH solutions of p-hydroxybenzyl alcohol and since relative low peak intensities of 8, 9, and 20 were observed in chromatograms of the refluxed EtOH and/or H 2 O solutions of p-hydroxybenzyl alcohol. The detectable main compounds in the extracts were completely identical to the main products from the refluxed solutions of p-hydroxybenzyl alcohol. Particularly the dimeric analogues 13a, 15, 17, 21, and 25 were readily detectable in all the sonicated extracts of the fresh G. elata rhizomes and "tian ma" as well as the refluxed solutions of p-hydroxybenzyl alcohol.

Activities of the Purified Compounds
Because previous studies revealed neuronal protection, anti-inflammatory, and antioxidant played important roles in the neurological effects of the extracts and chemical constituents of G. elata [92], the purified compounds from the aqueous extract were assayed preliminarily on the corresponding cell-based models [72,79]. At a concentration of 10 μmol/L, as compared with the blank control, compounds 5  −19, and the positive control glutathione inhibited Fe 2+ -cystine-induced rat liver microsomal lipid peroxidation with inhibition rates of 74%, 59%, 82%, 62%, 93%, 67%, 89%, 78%, and 49%. The results indicated that compound 17 was active in all the four assays while 5, 7, and 19 were active in the three assays. The remaining compounds including gastrodin and p-hydroxybenzyl alcohol were inactive at the same concentration. The previous study on the isolated guinea-pig ileum smooth muscle showed that 17 and the alcohol form of 11 had inhibitory effects on neurotransmitter release induced by stimulation of nicotine, serotonin, and vanilloid receptors, while 18 and 21 affected acetylcholine-induced contraction more directly [90] . Compounds 13, 14, 17, 18, and 21 were activators of melatonin receptors [7]. In addition, 15, 17, and the methyl ether analogue of 11 exhibited significant inhibitory effects on collagen, epinephrine, arachidonic acid, U46619 induced platelet aggregation [93], 17 had vasodilatory effect [94], and 18 was found to be a heat shock factor 1 (HSF1) inhibitor [95]. The studies also demonstrated   f EtOH solution of p-hydroxybenzyl alcohol was sonicated for 0.5 h then refluxed for 1.0 h that the main components gastrodin and p-hydroxybenzyl alcohol were less active or inactive as compared with the "tian ma" extracts as well as the dimers and trimers [90,93,94]. Thus, the p-hydroxybenzyl alcohol-derived dimers and trimers, which are the modified and recombined components during processing and decocting of the drug material, have important contributions to the clinic effects of the "tian ma" decoction.

Conclusions
Ten new p-hydroxybenzyl-derived dimers and trimers, gastrodibenzins A−D and gastrotribenzins A−F, together with ten known analogues, were isolated from an aqueous extract of "tian ma". Compounds 2 and 3 represents the first examples of p-hydroxybenzyl-coupled protocatechualdehydes. From the refluxed aqueous solution of p-hydroxybenzyl alcohol, isolation and identification of 5a, 6a, 8a, 13a, 14a, 15, 17−19, 21, 24−25, and p-hydroxybenzaldehyde, in combination with UPLC-HRESIMS analysis, unraveled that: (a) the p-hydroxybenzyl unit in the "tian ma" chemical constituents were originated from p-hydroxybenzyl alcohol through self-condensation (4−15 and 17 −20) and inter-condensation with other molecules (1−3 and 16), which could be produced, modified, and recombined during processing and decocting of the drug material; (b) the p-hydroxybenzylderived methyl and ethyl ethers (such as 4−14) could readily be formed by contacting to the solvents MeOH and EtOH in the experimental procedure, respectively; (c) the chemical constituents of 'tian ma" extracts were highly dependent upon processing and extracting protocols including the solvents and refluxing time. This study, together with our previous results 71−80 , provides valuable insights into medicinal chemistry behind the processing and decocting protocols of TCM. This unravels that the composition and content of the diverse p-hydroxybenzyl-derived constituents of "tian ma" and their contributions to the pharmacological effects are modified and recombined by the processing and decocting. The processing and decocting protocols of TCM indeed enhance the medicinal values of the herbal medicine and deserve to be further investigated and to be in deep validated for more complex formulations.

General experimental procedures
See Supporting Information.

Preparation and UPLC/HR-ESI-MS Analysis of the Rresh G. elata Rhizomes and "tian ma" Extracts
The fresh G. elata rhizomes and "tian ma" were cut into small pieces, respectively. The pieces of plant materials (each 12.0 g) were ultrasonicated (280 W) in round-bottom flasks with 30 mL of H 2 O, MeOH, and EtOH for 0.5 h, respectively, followed by heating in a liquid alloy bath to reflux. Two parallel experiments were set for each the plant material and solvent. The extracts (each 400 μL) were sampled after ultrasonicated and at refluxing times of 0. with MeOH and EtOH to 1.0 mL, respectively. The diluted samples were individually filtrated and the filtrates were analyzed by UPLC-HRESIMS under the above described conditions, respectively.

Inhibitory Assay Against LPS-induced NO Production in Mouse Peritoneal Macrophages
See Ref. [97].