Identification of Novel Parishin Compounds from the Twig of Maclura tricuspidata and Comparative Analysis of Parishin Derivatives in Different Parts

Parishin compounds are rare polyphenolic glucosides mainly found in the rhizome of the traditional Chinese medicinal plant, Gastrodia elata. These constituents are reported to have several biological and pharmacological activities. In the present study, two novel parishin derivatives not previously reported as plant-based phytochemicals were identified from a twig of Maclura tricuspidata (MT) and two new compounds were elucidated as 1-(4-(β-d-glucopyranosyloxy)benzyl)-3-hydroxy-3-methylpentane-1,5-dioate (named macluraparishin E) and 1,3-bis(4-(β-d-glucopyranosyloxy)benzyl)-3-hydroxy-3-methylpentane- 1,5-dioate (macluraparishin C), based on the experimental data obtained by UV–Visible (UV–Vis) spectroscopy, high performance liquid chromatography–quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) and nuclear magnetic resonance (NMR) spectroscopy. Additionally, gastrodin, parishin A and parishin B were positively identified by spectroscopic evidence and the comparison of HPLC retention time with the corresponding authentic standards. Gastrodin, parishin A and parishin B, macluraparishin E and macluraparishin C were found to be the most abundant constituents in the MT twig. The compositions and contents of these constituents were found to vary depending on the different parts of the MT plant. In particular, the contents of parishin A, parishin B, macluraparishin C and macluraparishin E were higher in the twig, bark and root than in the leaves, xylem and fruit.


Introduction
Maclura tricuspidata (carr.) Bur, belonging to the Moraceae family, is a thorny deciduous tree distributed throughout the East Asian region including Korea, China and Japan. In the Korean traditional medical books such as Donguibogam (1613 A.D. Joseon Dynasty), most parts of this plant have been utilized as folk medicine for the treatment of various disorders such as neuritis, arthritis, mumps, tuberculosis, inflammation, jaundice and hepatitis [1,2]. Recent research results have also reported that MT extract has several beneficial health effects, including anticancer [3,4], anti-inflammatory [5], antioxidant [6,7], antiobesity and antidiabetic effects [8].
Several bioactive prenylated xanthones, phenolic acids and flavonoids have been previously identified from the stem, stem bark, root, leaves or fruit of MT [2,9,10]. These compounds were also reported to have antibacterial, antifungal [11], antitumor [12,13], antioxidant [14][15][16], neuroprotective [17], cytotoxic [14,18,19], anti-inflammatory [2,20,21], hepatoprotective [22], gastroprotective [20] and α-glucosidase inhibition activities [23]. However, the screening of natural products, particularly those with new chemical structures has been recognized as an important process for the discovery of new biologically and pharmacologically active constituents. In our previous study [24], we reported that MT fruit contains several parishin-related compounds (gastrodin, parishins A, B, C and E) based on tentative identification results using HPLC-QTOF-MS. This finding incited us to conduct further studies to identify the more detailed chemical structures of the parishin-related compounds in the MT plant. Parishin compounds are well-known bioactive principles in the rhizome of G. elata Blume. The G. elata rhizome has traditionally been used for the prevention and treatment of central nervous system (CNS) diseases such as dizziness, insomnia, stroke, spasm, amnesia, sedative, hypnotic, headaches and convulsions [25][26][27] in traditional Korean medicine. It has been reported that various bioactivities of G. elata rhizome are mainly due to the presence of parishin and its derivatives. Parishins A, B, C and E are tri-di-or monoester compounds of citric acid and gastrodin (4-β-D-(glucopyranosyloxy)benzyl alcohol). Parishin and its derivatives can be metabolized into intermediates or final metabolites such as gastrodin and 4-HBA in vitro and in vivo systems [25,28]. Gastrodin and 4-HBA possess a wide range of beneficial health effects for disorders of the central nervous system such as epilepsy, Alzheimer s and Parkinson s diseases, cognitive impairment and cerebral ischemia [25,[27][28][29]. Nevertheless, little is known about the parishin-related compounds in the MT twig and other different parts of this plant. In this study, we report on the isolation and identification of novel parishin derivatives, which have never been reported previously, as plant-based phytochemicals with four known compounds in the MT twig and their composition and contents in different parts of the MT plant.

HPLC-QTOF-MS Analysis of MT Twig Extract
Although HPLC coupled with photodiode array detector (PDA) is the most common method for the detection of polyphenolic compounds, these compound groups have very different maximum absorption wavelengths (λ max ) according to their characteristic chromophores [30]. Therefore, the proper wavelength should be set to detect targeted polyphenolic compounds in the matrix. Figure 1 shows HPLC chromatograms at 280 and 220 nm of 70% methanol extract of MT twig, respectively. The chromatogram monitored at 220 nm ( Figure 1B) showed several large peaks, whereas the chromatogram at 280 nm ( Figure 1A) showed only two large peaks with a few small peaks. These results indicate that the desirable wavelength for the analysis of polyphenolic compounds in TM twig extract is 220 nm rather than 280 nm. Plant-based secondary metabolites can be analyzed with various methods. Among them, The QTOF-MS method coupled with HPLC or ultraperformance liquid chromatography (UPLC) is being widely applied to identify trace constituents which are not detectable by the classical methods due to its high resolution, accurate mass measurement and high sensitivity [31]. In this study, the MT twig extract was analyzed by HPLC-QTOF-MS in negative electrospray ionization (ESI) mode, whereby the detected constituents were characterized by the interpretation of their mass spectra and the searching of library databases.
The total ion current (TIC) chromatogram obtained by HPLC-QTOF-MS coupled with PDA (at 220 nm) of the MT twig extract is shown in Figure 2. The characteristics of the identified compounds are listed in Table 1 with their molecular formula, molecular mass and error values (ppm) calculated by the software. As phenolic compounds, peak 2 yielded a deprotonated molecular ion peak at m/z 465.    Seven compounds (peaks 1, 3 and 7-11) can be considered as parishin-related compounds (λ max 268 nm) because their UV profiles were very similar to those of parishin A (λ max 268 nm). Peaks 1 and 3 were identified as gastrodin (4-(β-D-glucopyranosyloxy)benzyl alcohol) and parishin E (2- respectively, and the molecular masses of peaks 7 and 11 were 30 amu less than those of parishin E and parishin B or C, respectively. The UV spectra (λ max 223.0 and 268.9 nm) of both compounds were very similar to those of parishins A, B and C (221.8 and 268.9 nm). These results indicate that the two compounds have chromophores that are similar to those of parishins A, B and C. Therefore, we attempted chromatographic isolation of the parishin-related compounds, including two novel compounds (7, 11), for a definite structural identification based on spectroscopic data.

Isolation and Structure Elucidation of Parishin-Related Compounds
The 70% MeOH extract of MT twig was suspended in water and was successively partitioned with n-hexane, ethyl acetate, water-saturated n-butanol and water fractions. Each solution was evaporated to obtain the concentrates from n-hexane, ethyl acetate, butanol and water fractions. The HPLC chromatograms of 70% MeOH extract and four concentrates are shown in Figure 3. Thirteen compounds were labelled in the range of retention time, 8-30 min of the chromatogram recorded at 220 nm. Among the detected compounds, the main compounds, taxifolin-7-β-D-glucoside (peak 2) and dihydrokaempferol-7-β-Dglucoside (peak 5) were mainly distributed in the ethyl acetate and butanol fractions. In contrast, parishin-related compounds including gastrodin (peak 1), unknown-1 (peak 7), parishin B (peak 8), parishin A (peak 10) and unknown-2 (peak 11) were mainly detected in the water fraction as shown in Figure 3E. Polarity-driven partition methods have been widely applied in the extraction and separation of compounds from plant matrices. The distribution of these compounds in solvents of different polarity is related to the electronegativity of the chemical compounds [36]. Alcohols and acids are readily soluble in hydrophilic solvents as these compounds have high polar properties due the presence of an electronegative oxygen atom in their function groups [37]. The constituents in the water fractions were isolated by a combination of Amberlite XAD-2, silica gel, Toyopearl HW-40S and Sephadex LH-20 column chromatography, as shown in Scheme 1.
These signals are due to the presence of 4-HBA as the aglycone part of gastrodin. These results indicate that compound 11 contained two β-D-glucopyranosyl units. Significant correlation was also observed between H-6/C-2, H-6/C-3 and H-6/C-4 in the HMBC spectrum ( Figure 5). These results suggest that the carboxyl group at the C-3 position of the acid moiety is substituted by a methyl group. The observation of the signals of two ester carbon at δ C 172.4 (×2, C-1, 5), one oxygenated-methine at δ C 70.9 (C-3), two methylene (×2, δ C 46.3; δ H 2.67, 4H, d, J = 6.0 Hz, C-2, 4) and one methyl (δ C 28.0; δ H 1.32, 3H, s, C-6) are due to the presence of the acid moiety. The correlation was observed between the two primary alcohols (H-7 , 7 ) of 4-HBA and the two carboxyl groups (C-1, 5) in the HMBC spectrum. These results indicate that the two primary alcohols (C-7 , C-7 ) of 4-HBA were combined with two carboxyl groups (C-1, C-5) of the acid moiety. The evidence that the two glucopyranosyl groups are bound to the phenolic hydroxyl groups of two 4-HBA are due to the correlation between the anomeric proton signals (H-1 , 1 ) of the glucosyl moieties with the phenolic hydroxyl groups (C-4 , C-4 ) of two 4-HBA in the HMBC spectrum. From these results, compound 11 was determined to be 1,3-bis [4-β-D-glucopyranosyloxy)benzyl]-3-hydroxy-3-methyl-pentane-1,5-dioate (named macluraparishin C). To the best of our knowledge, macluraparishins E and C are identified as plant-based phytochemicals in this study for the first time. Parishin A is an ester compound formed by the condensation of gastrodin moieties of three moles with citric acid, and parishins B, C and E have one or two less moieties of gastrodin than parishin A. Parishin A is metabolized into gastrodin via parishins B, C or E, to finally be processed into 4-HBA, glucose and citric acid in vitro and in vivo systems [48][49][50]. Two novel parishins (macluraparishins E and C) are ester compounds formed by the condensation of gastrodin moieties of one or two moles with 3-hydroxy-3-methyl-pentane-1,5-dioic acid. Therefore, macluraparishins E and C may also be metabolized into 4-HBA, glucose and 3-hydroxy-3methyl-pentane-1,5-dioic acid. The substance 3-Hydroxy-3-methyl-pentane-1,5-dioic acid is known to be a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase and strongly reduces cholesterol, triglycerides, serum β-lipoproteins and phospholipids in vitro and in vivo systems [51,52].

Comparison of Parishin Compounds in Different Parts of MT
The compositions and contents of plant-based phytochemicals vary significantly between different parts of the plants (i.e., bark, root, leaves, stem, fruit and seed), thereby having their own characteristic bioactivities in in vitro and in vivo systems. The MT also has different bioactivities and chemical compositions depending on the parts of the plant [6]. Six parishins and the metabolite (gastrodin) from the different parts (i.e., twig, bark, root, leaves, xylem and fruit) of MT were quantified for the first time based on the peak areas of the chromatograms recorded at 220 nm. The contents of individual constituents are presented in Table 3. Peak numbers refer to Figure 3A. The values (µg/g, DW) are mean ± standard deviation (n = 3).

Plant Materials
Amounts of 3 kg each of hot-air dried (65 • C) samples (twig, leaves, root and stem) and fresh frozen fruit as different parts of MT plant were directly purchased from a local farm in Milyang city, gyeongsangnam-do, South Korea, in late March 2021. The samples were authenticated by Professor Byung-Kil Choo (Department of Crop Agriculture and Life Science, Jeonbuk National University, Jeonbuk, Republic of Korea). Voucher specimens (FL-202101−FL-202105) were stored in the Laboratory of Fermentation Technology (Professor Myung-Kon Kim, Jeonbuk National University, Jeonbuk, Republic of Korea). The stem was peeled manually and divided into bark and xylem parts. The frozen fruit was washed with tap water, followed by freeze-drying. The samples were pulverized using a household grinder (Hanil SFM-700SS, Yeongdeungpo-gu, Seoul, Republic of Korea). The pulverized samples were kept in air-tight plastic containers and stored in a cold room (4 • C) until use.

Isolation of Parishin Compounds in MT Twig
The powdered MT twig (1.0 kg) was extracted with 3 L of 70% aqueous MeOH with an ultrasonicator (Hwashin Instrument Co., Seoul, Korea) at room temperature for 20 min, followed by centrifugation at 4500 rpm for 15 min. The residue was further extracted twice with 2 L each of 70% aqueous methanol followed by centrifugation as above. The supernatants were combined and evaporated under reduced pressure to obtain MeOH extract (105 g; yield 10.5%). The MeOH extract (100 g) was suspended in water (500 mL) and sequentially extracted with n-hexane, ethyl acetate and water-saturated n-butanol (each 500 mL × 3). Each fraction was evaporated under reduced pressure to obtain n-hexane (6.0 g), ethyl acetate (8.5 g), n-butanol (13.0 g) and water fractions, respectively. The water fraction was passed through preconditioned Amberlite XAD-2 column (50 × 5 cm i.d.) at a rate of 10 mL/min and was washed with distilled water (1.5 L) to remove sugars and organic acids. Parishin and its derivatives were eluted from the column with MeOH (1.5 L). The eluate was evaporated under reduced pressure to obtain the water fraction containing mainly parishin and its derivatives (14.5 g). The water fraction (14 g) was separated by column chromatography on silica gel (300 g) and stepwise elution with a mixture of