Accepted Manuscript Title: Phytochemical and analytical characterization of constituents in Urceola rosea (Hook. and Arn.) D.J. Middleton leaves Authors: Hieu Nguyen Ngoc, Duc Trong Nghiem, Thi Linh Giang Pham, Hermann Stuppner, Markus Ganzera PII: DOI: Reference: S0731-7085(17)32315-4 https://doi.org/10.1016/j.jpba.2017.10.031 PBA 11562 To appear in: Journal of Pharmaceutical and Biomedical Analysis Received date: Revised date: Accepted date: 13-9-2017 25-10-2017 26-10-2017 Please cite this article as: Hieu Nguyen Ngoc, Duc Trong Nghiem, Thi Linh Giang Pham, Hermann Stuppner, Markus Ganzera, Phytochemical and analytical characterization of constituents in Urceola rosea (Hook.and Arn.) D.J.Middleton leaves, Journal of Pharmaceutical and Biomedical Analysis https://doi.org/10.1016/j.jpba.2017.10.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Phytochemical and analytical characterization of constituents in Urceola rosea (Hook. & Arn.) D.J. Middleton leaves Hieu Nguyen Ngoc1, Duc Trong Nghiem2, Thi Linh Giang Pham2, Hermann Stuppner1, Markus Ganzera1,* 1 Institute of Pharmacy, Pharmacognosy, Center for Molecular Biosciences (CMBI), University of Innsbruck, 6020 Innsbruck, Austria. 2 Department of Botany, Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hoan Kiem, Hanoi 100000, Vietnam. *Corresponding author: Assoc. Prof. Dr. Markus Ganzera Institute of Pharmacy, Pharmacognosy, Center for Molecular Biosciences (CMBI), University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria Phone: 0043-512-507 58406 (fax: 58499), E-mail: markus.ganzera@uibk.ac.at 1 Graphical abstract Highlights  Urceola rosea leaves were studied phytochemically in detail  Thirteen compounds, mainly flavonoids, were identified  Major compounds were quantified by a validated HPLC method  The composition of samples showed to be variable Abstract In Vietnam and China the leaves of Urceola rosea are widely used as herbal remedy and food. However, in contrast to the plants stem, little information was available on major constituents. In this study, the first in-depth phytochemical 2 investigation of U. rosea leaves is described, which resulted in the isolation of thirteen compounds, mainly flavonoids (kaempferol and quercetin derivatives) and triterpenes. Furthermore, an analytical procedure for the quantification of five major compounds was developed. The HPLC separation was performed on a Synergi MAX-RP column using acetonitrile and 0.1% formic acid as mobile phase. Method validation confirmed that the assay shows good linearity (R2 ≥ 0.997), precision (intra-day R.S.D ≤ 4.31%, inter-day R.S.D ≤ 3.52%) and accuracy (recovery rates ranged from 96.8 to 102.6%). Detection limits were always lower than 0.07 µg/mL. The analysis of several plant samples revealed distinct differences, as for example the content of total flavonoids varied from 0.44 to 1.73%. Keywords Urceola rosea; Ecdysanthera rosea; isolation; flavonoid; HPLC; quantification 1. Introduction Urceola rosea (Hook. & Arn.) D.J. Middleton, an up to 20 m long climbing liana of the Apocynaceae family, is widely distributed in south-east Asia. The plant is also known under the synonym Ecdysanthera rosea [1] or as “La lom” (Vietnam) and “Songhei” (China). Local population utilizes its leaves not only as vegetable because of their sour taste [2] but also as traditional medicine. For example, the Dao minority in 3 Vietnam prepares a decoction as anti-inflammatory and anti-infectious remedy, while the Kinh use the leaves to treat inflammations of the excretory tract or kidney stones [3]. In China, the whole plant is traditionally used against infections of the endosteum, injury, and rheumatism [4]. Despite of these interesting uses as food and medicinal plant, little is known about the constituents in U. rosea. Nearly all of the few, previously reported phytochemical studies focused on the stem, describing the isolation of pregnane glycosides [5, 6], triterpenoids [7], and lignans [8]. For leaves only the occurrence of an uncommon pentacyclic triterpene [9], together with lupeol, amyrin, oleanolic acid and the flavonoids kaempferol rhamnoside, ayanin and casticin [10] was reported. In order to provide deeper insights in the composition of U. rosea leaves, the current study was performed. After phytochemical investigations an analytical study followed, which should clarify whether the metabolite patterns in the plant are consistent and if the amount of quantified compounds possibly can explain its traditional uses. 2. Materials and methods 2.1. Plant material and solvents Three samples of Urceola rosea leaves were collected in northern Vietnam, specifically Ban Thi, Cho Don, Bac Kan (UR-1, September 2016), Ba Vi, Hanoi (UR-2, November 2016), and Ba Vi, Hanoi (UR-3, May 2017). They were taxonomically authenticated by one of the authors (D. T. Nghiem) and voucher specimens are 4 deposited at the Institute of Pharmacy, Pharmacognosy, University of Innsbruck, Austria. All solvents required for extraction and isolation were purchased from VWR (Vienna, Austria), those for analytical experiments had at least pro analysis (p.a.) quality and were obtained from Merck (Darmstadt, Germany). Ultrapure water was produced by a Sartorius arium® 611 UV (Göttingen, Germany) purification system. 2.2. Instrumentation NMR experiments were conducted on a Bruker Avance II 600 spectrometer (Karlsruhe, Germany) operating at 600.19 (1H) and 150.91 MHz (13C). The isolated compounds were dissolved in suitable deuterated solvents from Euriso-Top (Saint Aubin, France) using tetramethylsilane (TMS) as internal standard. LC-ESI-MS studies were performed on an Esquire 3000 ion trap mass spectrometer from Bruker (Bremen, Germany) coupled to an Agilent 1100 HPLC (Santa Clara, USA). For the purification of compounds, a Reveleris® flash chromatography system (Büchi, Flawil, Switzerland) or a semi-preparative HPLC from Dionex (Thermo Fisher, Waltham, USA), comprising a P580 pump, an ASI 100 automated sample injector, an UVD 170 U detector and a fraction collector, were used. Analytical HPLC experiments were performed on an Agilent 1200 system, equipped with binary pump, autosampler, column oven and diode array detector. 5 2.3. Isolation of compounds Finely ground Urceola rosea leaves (sample UR-1, 200 g) were extracted with MeOH by sonication (500 mL × 1 h × 3 times; Bandelin Sonorex 35 KHz, Berlin, Germany). The combined extracts were evaporated under reduced pressure, the crude extract (34.2 g) suspended in distilled water, and then partitioned with EtOAc and nBuOH. The EtOAc portion (14.2 g) was fractionated on silica gel (40-63 µm particle size) using petroleum ether and acetone as eluent. Three of the resulting 18 fractions (FE1-18) were further purified by flash chromatography on RP-18 material and a water / methanol gradient. From fractions FE4 (0.5 g), FE11 (0.3 g), and FE18 (3.2 g) the pure compounds 10 (5.0 mg), 13 (10.0 mg), and 11 (8.2 mg) were obtained, respectively. The n-BuOH portion (10.3 g) was separated in 14 fractions (FB1-14) on silica gel using a dichloromethane/methanol/water gradient. FB10 (2.1 g) was rechromatographed with the same solvents but under isocratic conditions (6/1/0.1) to obtain 12 subfractions (FB10.1-12). FB10.1 (50 mg) was purified by semi-preparative HPLC using a Phenomenex Synergi 4u Polar-RP 80A (250 × 10 mm, 4 µm) column and 30% ACN in 0.1% formic acid as eluent to obtain compounds 6 (11.0 mg) and 7 (4.0 mg). By reducing the ACN content to 23% compound 9 (6.0 mg) could be isolated from fraction FB10.3 (75 mg), with 27% ACN compounds 2 (13.0 mg), 4 (11.2 mg), and 5 (3.8 mg) were obtained from F10.4 (160 mg). From the methanolic solution of FB10.9 (250 mg) crystals precipitated, which were filtered and washed with 50% aqueous methanol (compound 12, 20.0 mg). The major subfractions FB10.10 (309 mg) and FB10.12 (403 mg) were subjected to size-exclusion chromatography on Sephadex LH20 material (Sigma, St. Louis, USA) with MeOH as eluent; this yielded in compounds 1 6 (18.0 mg) and 3 (13.0 mg), respectively. Finally, following the same strategy compound 8 (20.2 mg) was directly obtained from FB14 (300 mg). 2.4. HPLC conditions The best separation of the five standards selected for quantification (1-4, 8) was possible on a Synergi MAX-RP 80A (150 × 4.60 mm, 4 µm particle size) column from Phenomenex (Torrance, USA), protected by a 0.2 µm guard filter from Waters. The mobile phase comprised 0.1% formic acid (A) and acetonitrile (B). The gradient started with 14B/86A (v/v), followed by 19B/81A in 15 min, and 23B/77A in another 10 min; this composition was kept for additional 5 min (total run time 30 min). The column was then washed with pure acetonitrile and re-equilibrated with the initial solvent system for 10 min before the next analysis. The injected sample volume was 10 µL, the flow rate 0.8 mL/ min, and the column temperature set to 40 C. The compounds of interest were detected at a wavelength of 260 nm. 2.5. HPLC sample preparation Powdered plant material (300 mg) was extracted with methanol (1.5 mL × 10 min × 3 times) by sonication. After centrifugation (1400 g, 10 min), the supernatants were combined in a 5 mL volumetric flask and the flask filled to volume. Before analysis, each sample solution was membrane filtered (0.45 µm, cellulose acetate, Minisart, Sartorius, Göttingen, Germany). Samples were stable for at least 2 weeks if stored at 4 C. 7 2.6. Method validation All in-house isolated reference compounds had a purity ≥ 95% as determined by HPLC and NMR. Compounds 1-4 and commercially available chlorogenic acid (8; purity ≥ 95%, Sigma) were used to construct calibration curves. For this purpose, a stock solution containing 0.6 mg of each compound per mL methanol was prepared, and serially diluted with methanol in the ration of 1:1. Respective solutions also were used to confirm linearity and to determine the limits of detection (LOD) and quantification (LOQ). The latter two were visually evaluated corresponding to signal to noise ratios of 3 and 10, respectively. Selectivity was assured by using the peak-purity option in the operating software (Agilent Chemstation version RevB.04.04-Sp2) and by LC-MS. Intra- and interday precision were evaluated on three consecutive days by independently preparing and analyzing five sample solutions of UR-2 per day. Accuracy was confirmed by spiking UR-2 with three different concentrations (high spike: 1.0 mg; medium spike: 0.75 mg; low spike: 0.40 mg) of standard compounds 1, 2 and 8 prior to extraction. 3. Results and discussion 3.1. Identification of isolated compounds The phytochemical investigation of Urceola rosea leaves resulted in the isolation of 13 compounds (1-13, Fig.1). Their structures were determined by NMR, MS, and by comparison with published data. None of the isolated compounds showed to be a new 8 natural product, however, with the exemption of 4 they have not been reported as constituents of U. rosea leaves before. They showed to be quercetin 3-O-β-ᴅglucopyranosyl (1→2)-α-L-rhamnopyranoside (1; [11]), quercetin 3-O-α-Lrhamnopyranoside (2; [12]), kaempferol 3-O-β-ᴅ-glucopyranosyl (1→2)-α-Lrhamnopyranoside (3; [11]), kaempferol 3-O-α-L-rhamnopyranoside (4; [13]), kaempferol 3-O- β-ᴅ-xylopyranoside (5; [14]), quercetin (6; [15]), kaempferol (7; [16]), chlorogenic acid (8; [17]), chlorogenic acid methyl ester (9; [18]), β-sitosterol (10; [19]), daucosterol (11; [20]), ᴅ-(-)bornesitol (12; [21]), and ursolic acid (13; [22]). NMR and MS data were in good agreement with reported values and original NMR-spectra are available from the authors upon request. 3.2. HPLC method development Because they also might be relevant for bioactivity, our analytical investigation focused on compounds that are present in significant amount in U. rosea leaves. Accordingly, the optimized separation of 1-4 and 8 is shown in Fig. 2. Key issues noticed during method development were the use of a C-12 stationary phase and an acidic eluent. On more common C-18 materials (Luna C-18, Zorbax extend-C18, etc.) the standards also could be resolved; however, when analyzing plant extracts the coelution of 3 and a minor, adjacent signal was observed. Concerning the optimum mobile phase an acidic pH was advantageous to overall improve peak shape, and 0.1% formic acid was selected due to its compatibility with LC-MS. Acetonitrile was preferred over methanol as organic solvent because it resulted in a better resolution of 2 and 3, at simultaneously reduced analysis time and column backpressure. Considering that 9 flavonoids as well as a phenolic acid should be simultaneously monitored, selecting a wavelength of 260 nm for detection showed to be a good compromise in terms of sensitivity. 3.3. Method validation Following ICH guidelines [23] the developed HPLC method was validated for linearity, selectivity, LOD and LOQ, accuracy and precision; all respective results are summarized in Table 1. Linearity was confirmed by determination coefficients ≥ 0.9997 within a concentration range of approx. 5-600 µg/mL. The relative standard deviation of the calibration curves slope was always smaller than 1% (n=3). The method permitted a sensitive quantification of all analytes as LOD and LOQ values were found to be below 0.06 and 0.17 µg/mL. Selectivity was assured by no visible co-elutions (shoulders) and very consistent DAD-spectra, which were evaluated using the peak purity option in the operating software at a threshold value of 950. LC-MS experiments (data shown as supplementary information) performed in alternating ESI mode also confirmed this estimation. Accuracy was determined by spiking sample UR-2 with three compounds (1, 2 and 8), which were available in sufficient amount, at three concentration ranges. After extraction and analysis, the observed recovery rates varied from 96.8 (compound 8, low spike) to 102.6% (compound 1, high spike). The assays intermediate precision was confirmed by a relative standard deviation below 4.31% (intra-day, n=5) and 3.52% (inter-day, n=3) for repeatedly analyzing sample UR-2 as proposed (incl. extraction). 10 3.4. Analysis of samples All three U. rosea samples analyzed in this study differed in their harvest time; however, UR-2 and UR-3 were collected at exactly the same place. Interestingly, only in UR-1 and UR-3, all five standards could be assigned, with diglycosidic flavones (1, 3) dominating, followed by their monoglycosidic counterparts (2, 4). The assignment of compounds (see Fig. 2 for typical chromatograms) was easily possible by matching retention times, UV-spectra and LC-MS (supplementary information). In sample UR-2 collected in November 2016 only 2 and 4 were present in addition to chlorogenic acid (8), which was found in all specimens. That these variations are an effect of ßglucosidase can be excluded because the plant material was immediately dried after harvest in an oven at 60 C. However, even if only three specimens were analyzed it can be speculated that the collecting season might be a crucial factor for the flavonoid composition in U. rosea. Before the target compounds were actually quantified, the extraction protocol was verified. After completing the proposed procedure for UR-1 the remaining plant material was extracted once more. As the resulting solution did not contain any quantifiable signals of the five standards the applied procedure was considered exhaustive. The quantitative results obtained are summarized in Table 2. As can be seen, the highest amount of 1 (0.51%) was determined in UR-1, in UR-3 it was slightly lower (0.43%); sample UR-2 contained equal percentages (0.15%) of 2 and 4. In terms 11 of total flavonoid content, the samples varied from 0.44% (UR-2) to 1.73% (UR-3), the concentration of chlorogenic acid also differed more than 4-fold (0.14-0.66%). 4. Conclusions For the first time, the chemical composition of U. rosea leaves was studied extensively, revealing that kaempferol and quercetin derivatives together with chlorogenic acid are the dominating phenolic constituents. Their composition and concentration was found to be quite variable, yet with a total content of up to 1.7 percent, it is likely that phenols contribute to the anti-inflammatory properties of the plant. In order to obtain reliable data, a suitable HPLC method for their quantification was developed and validated, so that together with other minor compounds identified (triterpenes, bornesitol) the knowledge on Urceola rosea has been extended significantly. Considering that the plants leaves are not only used for medical purposes but are also consumed in large quantities as vegetable this seemed to be overdue actually. Acknowledgements This study is part of the project “China-TCM cluster” and was financially supported by the Austrian Federal Ministry of Health and the Austrian Federal Ministry of Science, Research and Economy (BMWFW-402.000/0016-WF/V/6/2016). 12 Supplementary information The assignment of individual compounds by LC-MS and the chromatogram of sample UR-1 recorded at different wavelengths are shown as supplementary material. Conflict of Interest The authors declare no conflict of interest. References [1] D.J. Middleton, New combinations in Urceola (Apocynaceae), Novon 4 (1994) 151. [2] Y.K. Xu, G.D. Tao, H.M. Liu, K.L. Yan, X.S. Dao, Wild vegetable resources and market survey in Xishuangbanna, Southwest China, Econ. Bot. 58 (2004) 647667. [3] The Asia Foundation, Guide to medicinal plants of the Daos in Ba Vi. https://asiafoundation.org/resources/pdfs/MedicinalPlantIndexoftheDaosinBaVi.p df, 2012 (accessed 11.9.17). [4] eFloras, Urceola rosea. http://www.efloras.org/florataxon.aspx?flora_ id=3&taxon_id=210002267, 2008 (accessed 11.9.17). [5] C.W. Song, P.K. Lunga, X.J. Qin, G.G. Cheng, J.L. Gu, Y.P. Liu, X.D. Luo, New antimicrobial pregnane glycosides from the stem of Ecdysanthera rosea, Fitoterapia 99 (2014) 267-275. 13 [6] X. Zhu, G. Wu, J. Xiang, H. Luo, S. Luo, H. Zhu, Y. Wang, New pregnane saponins from Ecdysanthera rosea and their cytotoxicity, Fitoterapia 82 (2011) 632-636. [7] C.W. Song, P.K. Lunga, X.J. Qin, G.G. Cheng, Y.P. Liu, X.D. Luo, Chemical constituents from the stems of Ecdysanthera rosea, Nat. Prod. Bioprospect. 4 (2014) 319-323. [8] X. Zhu, Q. Zhang, L. Kong, F. Wang, S. Luo, New hydroquinone diglycoside acyl esters and sesquiterpene and apocarotenoid from Ecdysanthera rosea, Fitoterapia 81 (2010) 906-909. [9] K.F. Huang, M.L. Sy, J.S. Lai, A new pentacyclic triterpene from Ecdysanthera rosea, J. Chin. Chem. Soc. 37 (1990) 187-189. [10] X. Zhu, Q. Zhang, F. Wang, Y. Ye, Chemical constituents of Ecdysanthera rosea, Zhongcaoyao, 42 (2011) 237-240. [11] A. Hasler, G.A. Gross, B. Meier, O. Sticher, Complex flavonol glycosides from the leaves of Ginkgo biloba, Phytochemistry 31 (1992) 1391-1394. [12] Y. Zhang, D. Wang, L. Yang, D. Zhou, J. Zhang, Purification and characterization of flavonoids from the leaves of Zanthoxylum bungeanum and correlation between their structure and antioxidant activity, PLoS One 9 (2014) e105725. [13] S.K. Kim, H.J. Kim, S.E. Choi, K.H. Park, H.K. Choi, M.W. Lee, Anti-oxidative and inhibitory activities on nitric oxide (NO) and prostaglandin E2 (COX-2) production of flavonoids from seeds of Prunus tomentosa Thunberg, Arch. Pharm. Res. 31 (2008) 424-428. [14] M. Olszewska, M. Wolbis, Flavonoids from the flowers of Prunus spinosa L., Acta Pol. Pharm. 58 (2011) 367-372. [15] H. Liu, Y. Mou, J. Zhao, J. Wang, L. Zhou, M. Wang, D. Wang, J. Han, Z. Yu, F. Yang, Flavonoids from Halostachys caspica and their antimicrobial and antioxidant activities, Molecules 15 (2010) 7933. 14 [16] I. Wawer, A. Zielinska, 13C CP/MAS NMR studies of flavonoids, Magn. Reson. Chem. 39 (2001) 374-380. [17] Y. Sun, L. Liu, Y. Peng, B. Liu, D. Lin, L. Li, S. Song, Metabolites characterization of timosaponin AIII in vivo and in vitro by using liquid chromatography-mass spectrometry, J. Chromatogr. B 997 (2015) 236-243. [18] X. Zhu, X. Dong, Y. Wang, P. Ju, S. Luo, Phenolic compounds from Viburnum cylindricum, Helv. Chim. Acta 88 (2005) 339-342. [19] V.S.P. Chaturvedula, I. Prakash, Isolation of stigmasterol and β-sitosterol from the dichloromethane extract of Rubus suavissimus, Int. Curr. Pharm. J. 1 (2012) 239-242. [20] J. Yoo, E. Ahn, M. Bang, M. Song, H. Yang, D. Kim, D. Lee, H. Chung, T. Jeong, K. Lee, M. Choi, N. Baek, Steroids from the aerial parts of Artemisia princeps Pampanini, Korean J. Med. Crop Sci. 14 (2006) 273-277. [21] R.L. Obendorf, C.E. McInnis, M. Horbowicz, I. Keresztes, L.B. Lahuta, Molecular structure of lathyritol, a galactosylbornesitol from Lathyrus odoratus seeds, by NMR, Carbohyd. Res. 340 (2005) 1441-1446. [22] R. Raza, Z. Ilyas, S. Ali, M. Nisar, M.J. Khokhar, I. Jamshed, Identification of highly potent and selective α-glucosidase inhibitors with antiglycation potential, isolated from Rhododendron arboreum, Rec. Nat. Prod. 9 (2015) 262-266. [23] ICH Harmonized Tripartite Guideline - validation of analytical procedures Q2(R1). http://www.ich.org/products/guidelines/quality/quality-single/article/validation-ofanalytical-procedures-text-and-methodology.html, 2005 (accessed 11.9.17). 15 Legend for figures Fig. 1: Structures of compounds isolated from Urceola rosea leaves (1-13). R1 R2 1 OH Glc (1→2) Rha 2 OH Rha 3 H Glc (1→2) Rha 4 H Rha 5 H Xyl 6 OH H 7 H H R 8 H 9 CH3 R 10 H 11 Glc 16 17 Fig. 2: HPLC separation of a standard mixture (A) and two sample solutions (B: UR-2, C: UR-3) under optimized conditions (column: Synergi MAX-RP 80A, 150 × 4.60 mm, 4 µm; mobile phase: 0.1% formic acid (A) and ACN (B); gradient: in 15 min from 14% B to 19% B, in 10 min to 23% B, and kept at this composition for 5 min; temperature: 40 C; injection volume: 10 µL; flow rate 0.8 mL/ min; detection: 260 nm); peak assignment is according to Fig. 1. 18 Table 1. Results of method validation. 1 2 3 4 8 Regression equation y= y= y= y= y= 20252x+17.68 19930x+8.83 18422x+15.97 19362x+13.09 7925.3x+8.67 rel of slope 0.61 0.65 0.63 0.62 0.62 R2 0.9998 0.9997 0.9997 0.9998 0.9997 Range (µg/mL) 630 - 4.92 600 – 4.69 590 - 4.61 560 – 4.37 590 – 4.61 LOD (µg/mL) 0.02 0.02 0.02 0.02 0.06 LOQ (µg/mL) 0.06 0.06 0.06 0.05 0.17 102.6 98.1 - - 96.8 medium 97.9 spike 97.6 - - 97.3 low spike 102.3 102.5 - - 102.5 intraday - 3.82 - 4.31 3.50 interday - 3.52 - 2.61 2.45 Accuracy1 high spike Precision2 1 expressed as recovery rates in percent (sample: UR-2) 2 maximum relative standard deviation (peak area) within one and three consecutive days (n = 5; sample: UR-2) Table 2. Content (weight percent) of compounds 1-4 and 8 in different Urceola rosea samples with relative standard deviation in parentheses (n=3). 19 compound / sample UR-1 UR-2 UR-3 1 0.51 (0.38%) - 0.43 (1.11%) 2 0.11 (0.54%) 0.15 (0.88%) 0.19 (1.20%) 3 0.38 (0.46%) - 0.30 (1.24%) 4 0.05 (0.39%) 0.15 (0.70%) 0.15 (0.62%) 8 0.24 (0.77%) 0.14 (0.55%) 0.66 (1.03%) 20