UFLC-PDA-MS/MS Profiling of Seven Uncaria Species Integrated with Melatonin/5-Hydroxytryptamine Receptors Agonistic Assay

Abstract Uncariae Ramulus Cum Uncis (Gou-Teng), the dried hook-bearing stems of several Uncaria plants (Rubiaceae), is a well-known herbal medicine in China. The clinical application of Gou-Teng is bewildered for the morphological and chemical similarity between different species. In order to discern their chemical and biological difference, an ultra-fast liquid chromatography equipped with ion trap time-of-flight mass spectrometry (UFLC-IT/TOF-MS) combining with melatonin (MT1 and MT2) and 5-hydroxytryptamine (5-HT1A and 5-HT2C) receptors agonistic assay in vitro was conducted on seven Uncaria species. As a result, 57 compounds including 35 indole alkaloids, ten flavonoids, five triterpenoids, five chlorogenic analogues, and two other compounds were characterized based on their MS/MS patterns and UV absorptions. Specifically, cadambine-type and corynanthein-type alkaloids were exclusively present in U.rhynchophylla and U.scandens, whereas corynoxine-type alkaloids were commonly detected in all the seven Uncaria plants. Three Uncaria species, U. rhynchophylla, U. macrophylla, and U. yunnanensis showed obviously agnostic activity on four neurotransmitter receptors (MT1, MT2, 5-HT1A, and 5-HT2C). This first-time UFLCMS-IT-TOF analyses integrated with biological assay on seven Uncaria plants will provide scientific viewpoints for the clinical application of Gou-Teng. Graphic Abstract Electronic supplementary material The online version of this article (10.1007/s13659-020-00230-8) contains supplementary material, which is available to authorized users.


Indole Alkaloids
Indole alkaloids are the characteristic constituents in Uncaria plants with high response in positive mode MS. In this investigation, a number of 35 indole alkaloids were described and divided into six subclasses including cadambine-type (19,21,23,26,47), vinsosamide-type (15), D-seco-type ( (39,54). In accordance with the previous investigation [17], D-seco alkaloids commonly generated the characteristic fragmentation ions ascribed to the loss of 17 Da (NH 3 ) in the MS 2 experiment; the indole and oxindole alkaloids could be differentiated from their respective maximal UV absorptions around 280 nm (indole) or 240 nm (oxindole); the numbers and types of glycosyl moieties were determined by the mass defects between the parent and fragment ions.  4 ), corresponding to the sequential loss of glycosyl and MeOH moieties [18]. Peak 19 showed the loss of 17 Da from 565 to 548, and the loss of 162 Da from 548 to 386, which was characteristic for the hydrated derivative of cadambine [18]. Peaks 23, 26, and 47 possessed the same molecular formula of C 27 H 34 N 2 O 10 with two more hydrogens than 21. In the MS 2 spectra, the identical fragmentation at m/z 385 (C 21 H 24 N 2 O 5 ) and 367 (C 21 H 22 N 2 O 4 ) suggested closely related structures. In accordance with the previous reports, 3α-dihydrocadambine, 3β-dihydrocadambine, and 3β-isodihydrocadambine were reasonably suggested [19]. Peak 15 showing a molecular formula of C 38 H 50 N 2 O 19 was deduced from the [M+H] + ion at m/z 839.3054. In the positive MS 2 experiment, the sequential losses of three glycosyl moieties (C 6 H 10 O 5 , 162 Da) suggested the presence of three glucosyl in the structure. Finally, this compound was isolated under the guidance of LCMS analysis, and identified to be 2′-O-[β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl]-11hydroxyvincosamide based on rigid 1D and 2D NMR spectroscopic data [20].

D-seco Indole Alkaloids D-seco indole alkaloids
can be well recognized from the diagnostic MS 2 ions attributed to the neutral loss of 17 Da (NH 3 ) from the precursor ions. Peaks 33 and 38 were assigned with the same molecular formula of C 27 H 34 N 2 O 9 from the [M+H] + ion at m/z 531. Their similar MS 2 fragmentations at m/z 514 (C 27 H 31 NO 9 ) and 352 (C 21 H 21 NO 4 ) indicated a pair of isomers, which were generated from the cleavage of 3-epi-strictosidine and strictosidine [21]. Peak 18 with a molecular weight of 516 was deduced to be the demethylated derivative of 38, owing to a CH 2 (14 Da) less in the molecular formula. The MS 2 fragmentation ion at m/z 338.1568 implied the successive loss of 17 Da (NH 3 ) and 162 Da (C 6 H 10 O 5 ), by which this compound was assigned as strictosidinic acid [22]. The molecular formula of 25 was determined as C 28

Corynoxine-Type
Alkaloids The spirocyclic corynoxine-type alkaloids account for the largest number of indole alkaloids within Uncaria genus. Generally, this type of alkaloids can be well recognized by their UV maximum absorption at about 240 nm [17]. Peaks 34, 37, and 42 were isomers with the equal molecular formula of C 22 H 28 N 2 O 4 , which were determined by the [M+H] + ion at m/z 385. The MS 2 fragments at m/z 353 and 321 were attributed to the consecutive losses of methoxyl groups. The ion at m/z 267 indicated the loss of the C 5 -side chain. By comparing their relative retention time on octadecylsilyl (ODS) column, they were deduced as isorhynchophylline, corynoxine, and rhynchophylline [26]. Peaks 27 and 31 occupied the same molecular weight of 384, corresponding to the molecular formula of C 21 H 24 N 2 O 5 . Their MS 2 fragments at m/z 367, 351, and 335 accounting for the lost H 2 O and two additional oxygen atoms indicated an oxygenated derivative of rhynchophyllic acid. Likewise, peaks 24 and 35 were deduced as dehydro-derivatives of rhychophylline, and peak 11 was proposed as the demethylated derivative of rhychophylline [27].  [30]. Similarly, peaks 45 and 51 were tentatively deduced to be the dihydroxy and dihydro derivatives of corynantheine [17].

Flavonoids
Flavonoids display characteristic UV absorptions at 220-280 (band II) and 300-400 (band I) nm, by which they can be easily characterized [33]. Peaks 4 and 8 with UV maximum absorption at 280 nm were designated with the molecular formula of C 30  indicating flavonoids dimers. Their relative retention time on ODS column were in accordance with procyanidin b1 (4) and procyanidin b2 (8) [34]. Peaks 5 and 10 were a pair of isomers with identical molecular formula of C 15 H 14 O 6 . The MS 2 ion at m/z 139 (C 7 H 6 O 3 ) was ascribed to the A 1,3 retrocyclization fragment on ring C. Taking their UV absorptions at 280 nm and retention time into consideration, peaks 5 and 10 were reasonably determined as catechin (5) and epicatechin (10) [12]. Peaks 12 and 14 were isomers with the same molecular formula of C 30  ) were attributed to fisetinidol and catechin moieties. From the above analyses, they were tentatively deduced to be fisetinidol-(4α → 8)-epicatechin and fisetinidol-(4β → 8)-epicatechin [35]. Peak 13 with a formula of C 21 H 24 O 12 showed MS 2 information at m/z 317.0994 (C 16 H 12 O 7 ), corresponding to the loss of a C 5 part from the C-glycosyl moiety. From the above analyses, this peak was defined as gallocatechol C-glucoside [36,37]. Peak 16 was designed with the molecular formula of C 27 H 30 O 16 with an additional C 6 H 10 O 4 part than 17 (C 21 H 20 O 12 ). In the MS 2 experiment, the same fragments at m/z 303 in positive mode and 301 in negative mode suggested the same aglycone in 16 and 17. By retrieving the database, they were deduced as rutin (16) and hyperoside (17) 6 ) in negative mode were indicative for the sequential loss of rhamnosyl and formaldehyde moieties. From the above analyses, this peak was deduced as quercetin 3-rhamnoside [38].

Chlorogenic Acids
Chlorogenic acid analogues are a type of caffeoyl quinic acids widely present in plants. In the UV spectrum, the maximum absorption at around 325 nm was due to the presence of caffeoyl group. In the MS 2 experiment, the product ions at m/z 163 (C 9 H 6 O 3 ) in positive mode and 191 (C 7 H 12 O 6 ) in negative mode were indicative for caffeic acid and quinic acid moieties. In this study, four isomers, namely, neochlorogenic acid (3), chlorogenic acid (6), cryptochlorogenic acid (7), and isochlorogenic acid (9) with identical formula of C 16 H 18 O 9 were detected and tentatively characterized by their retention time on ODS column [39]. Peak 30 was assigned with the molecular formula of C 25 H 24 O 12 with an additional quinoyl moiety compared to chlorogenic acid. This deduction was verified by the MS 2 ions at m/z 353.0882 (C 16 H 18 O 9 ) and 173.0401 (C 7 H 10 O 5 ) in negative mode. Thus, peak 30 was delineated as dicaffeoylquinic acid [40].

Triterpenoids
Peak 56 showing terminal absorption in UV spectrum was revealed with the molecular formula of C 30  and 423 (C 29 H 42 O 2 ) were in accordance with quinovic acid [41]. Peaks 49 and 52 were deduced to be diglycoside and triglycoside derivatives of quinovic acid by the additional two and three glycosyls which were verified by the sequential loss of C 6 H 10 O 5 parts in the MS 2 experiments. Thus, quinovic acid diglycoside and quinovic acid triglycoside were respectively determined [42].

Other Compounds.
Peak 1 was assigned as sucrose which was widely present in plants by the characteristic [M+K] + ion at m/z 381.0792. Peak 2 had a molecular formula of C 16

Chemical Comparison
As shown in Figs. 2 and 3, a temporal and spatial distribution of chemical constituents in seven Uncaria plants provided a visual overview of their difference. The chemical profiles of U. rhynchophylla and U. scandens were similar in terms of either indole alkaloids or other types of compounds. Indole alkaloids as the characteristic constituents were more prolific in U. rhynchophylla and U. scandens when comparing to other Uncaria plants. Cadambine-type and corynanthein-type alkaloids were the characteristic constituents in U. rhynchophylla and U. scandens, whereas corynoxine-type alkaloids were widely distributed in all the seven Uncaria plants. Besides alkaloids, flavonoids were another type of constituent in Uncaria plants, which were mainly distributed in U. rhynchophylla, U. macrophylla, and U. yunnanensis. For the triterpenoids, U. hirsuta and U. laevigata showed more prolific than other plants.

LCMS Analyses
LCMS analyses were performed on a Shimadzu UFLC/ MS-IT-TOF apparatus (Shimadzu, Kyoto, Japan) equipped with a Welch Ultimate XB-C 18 column (2.1 × 100 mm, i.d., 1.8 μm). The mobile phase for LCMS consisted of water (0.05% formic acid, A) and acetonitrile (0.05% formic acid, B) with the flow rate of 0.2 mL/min. A binary gradient elution was performed as follows: linear gradient (B%) from 10 to 35% in 35 min, and fast increased to 100% in one min and maintained for three min. Re-equilibration duration was five min between individual runs. The injection volume was 2 μL for each LCMS analysis. The detailed MS parameters were set as previously reported [44]. The PDA profiles were recorded from 190 to 400 nm. The Shimadzu Composition Formula Predictor was used to speculate the molecular formula.

Agonistic Activities on MT 1/2 and 5-HT 1A/2C Receptors
Bioassay for agonistic activities on melatonin and 5-hydroxytryptamine receptors was performed in accordance with the previous reports [20,45]. In brief, HEK293 cells stably expressing human melatonin (MT 1 and MT 2 ) and 5-hydroxytryptamine (5-HT 1A and 5-HT 2C ) receptors were maintained in DMEM containing 10% FBS. Cells were seeded at a density of 4 × 10 4 cells/well in pre-matrigel-coated 96-well black wall/clear bottom plates. After overnight incubation at 37 °C with 5% CO 2 , the cells were dyed with 100 μL of HDB Wash Free Fluo-8 Calcium Assay kit at 37 °C. An hour later, the cells were transferred into FlexStation3 Benchtop Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, California, United States) for bioassay. The raw data from time sequence recording were normalized as percentage responses to melatonin and 5-hydroxytryptamine as the positive controls, and analyzed to fit the four-parameter logistic equation to assess the agonistic rates.

Statistical Analyses
All experiments were carried out in triplicate. Data were expressed as mean ± standard error of mean (Mean ± SEM). Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA) and Origin 2018 (OriginLab Corporation, Wellesley Hills, MA) software.
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