Qualitative and Quantitative Analysis of Secondary Metabolites in Morphological Parts of Paulownia Clon In Vitro 112® and Their Anticoagulant Properties in Whole Human Blood

It is not easy to find data in the scientific literature on the quantitative content of individual phytochemicals. It is possible to find groups of compounds and even individual compounds rather easily, but it is not known what their concentration is in cultivated or wild plants. Therefore, the subject of this study was to determine the content of individual compounds in the new Paulownia species, Oxytree, developed in a biotechnology laboratory in 2008 at La Mancha University in Spain. Six secondary metabolites were isolated, and their chemical structure was confirmed by spectral methods. An analytical method was developed, which was then used to determine the content of individual compounds in leaves, twigs, flowers and fruits of Paulownia Clon in Vitro 112®. No flavonoids were found in twigs and fruits of Oxytree, while the highest phenylethanoid glycosides were found in twigs. In this study, we also focused on biological properties (anticoagulant or procoagulant) of extract and four fractions (A–D) of different chemical composition from Paulownia Clon in Vitro 112 leaves using whole human blood. These properties were determined based on the thrombus-formation analysis system (T-TAS), which imitates in vivo conditions to assess whole blood thrombogenecity. We observed that three fractions (A, C and D) from leaves decrease AUC10 measured by T-TAS. In addition, fraction D rich in triterpenoids showed the strongest anticoagulant activity. However, in order to clarify the exact mechanism of action of the active substances present in this plant, studies closer to physiological conditions, i.e., in vivo studies, should be performed, which will also allow to determine the effects of their long-term effects.

Iridoid-catalpol shows radioprotective activity [26] and neuroprotective [36] and cardioprotective effects on the heart muscle (Human); it also increases glucose utilization by increased secretion of β-endorphin from the adrenal gland [37]. Aucubin had an antioxidant and protective effect on the pancreas and can alleviate obesity-induced atherosclerosis [38].
Phytosterols are potentially useful for the treatment of Alzheimer's disease [39]. They show a cytotoxic effect against K562 and K562/ADR human chronic myeloid leukemia, human HL60 and HL60/ADR acute myeloid leukemia cancer cells and neoplastic cell human colon cancer cell lines SW480 and SW620 [40]. They have anti-inflammatory [41,42], antimalarial [43] and anti-diabetic effects [44], reduce blood pressure and are useful in the treatment of traumatic cerebral ischemia [45].
In the present study, a targeted identification and quantification of the secondary metabolites, present in Paulownia Clon in Vitro 112, was achieved by UPLC-MS/MS and UPLC-DAD. Our earlier results demonstrated that extract and four fractions (A-D) from leaves of Paulownia Clon in Vitro 112 have antioxidant and anti-platelet potential in vitro. In these studies, we used human plasma and washed human blood platelets [46,47]. Therefore, in this study, we also focused on biological properties (anticoagulant or procoagulant) of extract and four fractions (A-D) of different chemical composition from Paulownia Clon in Vitro 112 leaves using whole human blood. These properties were determined based on the thrombus-formation analysis system (T-TAS), which imitates in vivo conditions to assess whole blood thrombogenecity.

Plant Material
For the extraction of compounds, the leaves collected on the private plantation were used in the first year after its establishment, early autumn (September), on the fluvisol developed on Vistula river deposits, class I, in the village Łęka, Lublin Province, Poland (21 • The owner-Anna Stochmal-has a certificate for the cultivation of trees. She bought seedlings from the official representative in Poland: Oxytree S.A., ul.Życzliwa 25/2, 53-030 Wrocław.
Oxytree were identified by Sales Director Dariusz Tyma.
Qualitative and quantitative analyses of the morphological parts-leaves, twigs, flowers and fruits-were performed on plant material collected on a 5-year-old plantation located on sandy land, class IV, Września-Wielkopolska-Poland (52 • 20 40.7 N 17 • 36 28.6 E).
Plants after harvest were frozen and freeze-dried (Gamma 2-16 LSC, Christ, Germany). Reference samples are stored at the Institute of Soil Science and Plant Cultivation.
The sample voucher for this material has been deposited in the institute's collection under the deposit number 22/2017/IUNG.

Extraction and Isolation of Compounds
A sequential extraction process was used to prepare an extract of Paulownia leaves. The ground plant material was extracted with 5% methanol (v/v) in an ultrasonic bath at room temperature for 30 min and macerated with a magnetic stirrer at room temperature for half an hour. The contents were centrifuged at 4000 G for 10 min and filtered. The pellet was re-extracted with 30% methanol (v/v) and the third extraction was done with 70% methanol. The supernatants were combined and concentrated under reduced pressure and lyophilized. The extraction yield was 41.7%.
The crude methanol extract was purified stepwise using various chromatographic methods. First, the extract was applied to a pre-conditioned RP-C18 column (80 × 70 mm, 140 µm; Cosmosil C18-PREP; Nacalai Tesque, Inc., Kyoto, Japan), which was then washed with 1% methanol (v/v) to remove sugars. Active metabolites were eluted with 80% methanol (v/v) to give fraction A. The yield at this stage was 44.1%.
In the next step fraction A was separated by flash chromatography on a reverse phase column (140 × 12 mm, 40 µm; Cosmosil C18-PREP, Nacalai Tesque, Inc., Kyoto, Japan) connected to a Gilson HPLC apparatus. A linear gradient of an aqueous acetonitrile solution (2-30% v/v) containing 0.1% formic acid over 140 min was used as the mobile phase at a flow rate of 8 mL min −1 at room temperature. Subsequently, from fraction A, sub-fractions B, C and D were obtained, which, respectively, accounted for 23.4%, 33.6% and 36.3% of fraction A.
Isolation of compounds from subfractions was made on column Atlantis Prep T3, C18 5 µm, 10 × 250 mm (Waters) by preparation chromatography using isocratically different concentration of methanol. From subfraction B two compounds were purified, from C three compounds, while from D one compound. A more detailed description of preparation and the extract and four fractions (A-D) from leaves can be found in the paper of Adach et al. [46].

Analytical Method UHPLC-ESI-MS/MS
Extracts and fractions of Paulownia leaves, flowers, fruit and twigs were prepared by the same method described above (Section 2.3) and next were analyzed by UHPLC-ESI-MS/MS, using a Thermo Ultimate 3000RS (Thermo Fischer Scientific, MS, Carlsbad, CA, USA) UHPLC system, equipped with a charged aerosol detector (CAD) and a diode array detector, and hyphenated with a Bruker Impact II (Bruker Daltonics GmbH, Bremen, Germany) Q-TOF mass spectrometer. Samples were chromatographed on an ACQUITY HSS T3 column (2.1 × 150 mm, 1.8 µm; Waters, MA, USA) maintained at 55 • C; the injection volume was 2.0 µL. A 22 min gradient elution program was applied, from 2 to 45% of solvent B (0.1% formic acid in acetonitrile) in solvent A (0.1% formic acid in Milli-Q water, Sartorius, Gottingen, Germany); the flow rate was 0.500 mL min −1 . The applied MS settings were similar to those described previously [50,51], with small modifications. UHPLC-MS/MS analysis was performed in negative ion mode, the scanning range was from m/z 80 to 2000. The following settings were applied: Capillary voltage was 3 kV, dry gas flow was 6 L min −1 , dry gas temperature was 200 • C, nebulizer pressure was 0.7 bar, collision RF was 750 V, transfer time was 100 ms, prepulse storage time was 10 ms. Two precursor ions of intensity over 2000 counts were fragmented in each scan. Depending on the m/z of a fragmented ion, the value of collision energy was set automatically in the range from 2.5 to 80 eV. The internal calibration of the acquired data was performed with sodium formate, introduced to the ion source via a 20 µL loop at the beginning of a separation. Constituents of the extracts were determined by external calibration, using authentic standards when possible. The UV detection at λ = 330 nm was used for quantitation and semi-quantitation of phenolic compounds. The content of verbascoside was determined on the basis of the following standard curve (y = 4.90599x − 1.97683; R 2 = 0.9999). The same standard curve was also used for semi-quantitation of other phenylethanoids, and hydroxycinnamic acid derivatives. Flavone glycosides were determined on the basis of a standard curve of apigenin-7-O-glucuronopyranosyl-(1→2)-glucurono-pyranoside (y = 6.89810x − 19.60755; R 2 = 0.9997). Apart from phenolics, two iridoids were additionally quantified. 7-hydroxytomentoside was determined on the basis of UV chromatograms (λ = 248 nm), with a standard curve y = 4.02230x − 1.07988; R 2 = 0.9995. Extracted ion chromatograms (EIC) were used for quantitative analyses of catalpol, on the basis of the following standard curve y = −41.329553x 2 + 17210.064761x + 12019.481295; R 2 = 0.9972).

Preparation of Stock Solutions (Extract and Four Fractions (A-D) from Leaves) for Bioassay-T-TAS
The extract and the four fractions (A-D) from leaves were dissolved in 1 mL 50% DMSO (as a universal solvent for many different plant substances), giving final concentrations of 5 µL/mL and 50 µL/mL (in blood samples). The final concentration of DMSO in the tested blood samples was below 0.05% (v/v). The addition of a low concentration of DMSO to human blood has no effect on coagulation parameters (data not presented).

The Samples of Blood
Fresh human blood was collected in the L. Rydygier hospital in Lodz, Poland. All donors were healthy volunteers; none were smokers or reported taking drugs. Blood was collected in tubes with CPDA anticoagulant (citrate/phosphate/dextrose/adenine; 8.5:1; v/v; blood/CPDA). The analysis of blood samples was performed according to the guidelines of the Helsinki Declaration for Human Research. The blood used in T-TAS assays was incubated (30 min, at 37 • C) with extract and four fractions (A-D) from leaves at final concentrations of 5 and 50 µg/mL. The experiments were conducted with the consent of the Bioethics Committee at the University of Łódź (number 11/KBBN-UŁ/I/2019).

Total Thrombus-Formation Analysis System (T-TAS)
The plate plug formation process was determined using a real-time hydrodynamic model. Whole blood (400 µL) was incubated with the tested extract and four fractions (A-D) (30 min, 37 • C). Next, 350 µL of blood was drawn for analysis. The results were obtained as AUC 10 (Area Under the Curve) using a PL chip. The AUC 10 parameter-that is an area under the pressure curve from the start of the test to a time of 10 min-was studied. This parameter determines the growth, intensity, and stability of thrombus formation. A more detailed description of this method can be found in Hosokawa et al. [52].

Data Analysis
Statistical analysis was performed using Statistica 10 (StatSoft 13.3, TIBCO Software Inc., Palo Alto, CA, USA). Normal distribution of data was checked by normal probability plots and the homogeneity of variance by the Brown-Forsythe test. Differences within and between groups were assessed using one-way ANOVA followed by a multicomparison Duncan's test. Results are presented as means ± SD. Significance was considered at p ≤ 0.05. Dixon's Q-test was used to eliminate uncertain data.

Results
The LC-QTOF-MS analysis of the extract from Paulownia Clon in Vitro 112 leaves, grown in Łęka, shows that the main secondary metabolite was the verbascoside (acteoside), which belongs to the phenylethanoid glycosides. It contained also other phenylethanoides. Iridoids such as catalpol, 7-hydroxytomentoside, as well as flavonoids: Glycosides of luteolin, and apigenin, were also found ( Figure 1).  The UHPLC-ESI-MS/MS method was used to determine the content of individual compounds in leaves, twigs, flowers and fruits of Paulownia Clon in Vitro from Września. All verbascoside derivatives, including cistanoside F, were determined as equivalent of verbascoside, while all flavonoids were determined to be apigenin-7-O-glucuronopyranosyl(1→2)glucuronopyranoside equivalent ( Table 1). The concentrations of the individual compounds were determined according to the calibration curves of standards (Table 1), and classes of the individual compounds in Table 2 were created by summing up the concentrations of individual compounds.
The results demonstrated alterations in AUC 10 measured by T-TAS in the presence of all tested plant preparations from leaves ( Figure 3). Tested extract and fractions (A-D) from leaves (at concentrations 5 and 50 µg/mL) decreased AUC 10 relative to control, but these changes were not always statistically significant (Figure 3). However, for three fractions (A, C and D; at the highest used concentration-50 µg/mL) AUC 10 decreased in a statistically significant manner. In addition, fraction D (50 µg/mL) had the strongest effect on this parameter (Figure 3). Figure 4 demonstrates selected diagrams analysed by T-TAS for tested extract (50 µg/mL) and four used fractions (A-D, 50 µg/mL) in whole blood samples. We have calculated AUC 10 for each diagram.
Inflammation, which is the process of faster aging of tissues, including skin tissue, caused by constant exposure to factors causing inflammation is a recently noticed and investigated problem. Counteracting the progressive effects of inflammation may consist in avoiding and reducing the frequency of exposure to external factors, causing inflammatory microstates, such as, for example, ultraviolet radiation, exhaust fumes, cigarette smoke and detergents, although it is not a simple task. Scientists place their hope in plants rich in certain specific glycosides, such as verbascoside, with anti-inflammatory and antioxidant effects. Such plants include: Plantain leaves with the amount of 3.5% of dry weight of verbascoside, verbena herb 3.7%, 12.9% of dry leaves in the fourth stage of Sesamum indicum L., and 27.7% of Cymbaria daurica L., which is one of the highest percentages reported in plants from nature. By comparison, the content of this compound and its derivatives in leaves, twigs, flowers and fruits of Oxytree are 9.47, 22.93, 2.31 and 17.91 mg/g DM, respectively.
In Oxytree, cistanoside F, which is also phenylpropanoid glycoside, was identified. The first time it was isolated and its structure was determined in the herbs of Cistanche deserticola Ma. was in 1985 by Kobayashi et al. [27] The concentration of this compound in Sesamum indicum L. was 0.26%, in lemon verbena 2%, and in Oxytree from 0.47% in twigs to 0.02% in flowers.
Cistanoside F shows vasorelaxant and antioxidative effects; it shows a strong free radical scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and xanthine/xanthine oxidase (XOD) generated superoxide anion radical (O 2 −· ). Since cistanoside F contains only one phenolic ring in the caffeoyl moiety, it has lower antioxidant activity that compounds with two aromatic rings. The highest content of this compound-4.28 mg/g DM-was found in twigs, 1.92 mg/g was found in fruit and 1.22 mg/g in leaves, and the least-0.54 mg/g DM-in Oxytree flowers.
Clerodendrin is used in the manufacture of a medicament for the treatment of degenerative changes in the retina. Clerodendrin showed a hypotensive activity in rats by measurement of the tail volume pressure in preliminary pharmacological tests. This compound belongs to the flavone glycosides with the structure of apigenin7-O-digiucuronide and for the first time has been isolated from the leaves of Clerodendron trichotomum Thunb. It is possible to obtain this compound from Scutellaria barbata and Glechoma longituba (Nakai) Kupr. A method for producing clerodendrin isolated from Clerodendron trichotomum Folium and a pharmaceutical composition containing the above compound for the prevention and treatment of gastritis and reflux esophagitis has been patented (PCT/KR2003/001037). Clerodendrin was identified in trace amounts, while other flavonoids-apigenin glucuronide, apigenin-7-Oglucuronopyranosyl(1→2)-glucuronopyranoside and luteolin-diglucuronide-were in total determined in small amounts only in leaves and flowers: 1.61 and 1.99 mg/g DM.
The third and last group of organic chemicals that belong to terpenes described in Oxytree are the iridoids. The representative of this group with the highest concentration is catalpol-simply monoterpene with a glucose molecule attached. It was primarily obtained from the root of Rehmannia glutinosa Libosch. (1-10% depending on the variety) and also was isolated from the Cymbaria daurica L. (11.4%), Plantago lanceolate, Buddleia species, Radix scrophulariae and Lancea tibetica. Pharmacological effects include analgesic, sedative, hepatoprotective, laxative, anti-inflammatory, antibacterial, anti-tumor and anti-apoptotic effects. In Oxytree, this compound is present in all examined morphological parts, i.e., in leaves, twigs, flowers and fruits, in concentrations 0. 65, 9.96, 3.08 and 7.36 mg/g DM.
Another iridoid is 7-hydroxytomentoside, about which there is very little information in the literature. It was first isolated from Paulownia tomentosa and described in 1993 by Damtoft and Jensen [62]. It was then isolated from Paulownia coreana Uyeki leaves. The 7-hydroxytomentoside has anti-inflammatory effects on skin with irritations, atopy and acne. It is used to lighten the skin and reduce the signs of aging. From all the morphological parts of Oxytree, 7-hydroxytomentoside is found only in leaves at a rate of 6 mg/g DM.
Even if the concentrations of the described compounds in Oxytree are not spectacular, due to the fact that the number of leaves, twigs and even flowers on the trees is so high and the tree density in the plantation is 600 pcs/ha, they can be an excellent source of biologically active natural phytochemicals used in medicine and cosmetology.
So far, only a few studies focusing on biological activity of preparations derived from organs of Paulownia Clon in Vitro 112 have been carried out; therefore, the aim of this study was to investigate the effect of the extract and its four fractions (A-D) derived from Paulownia Clon in Vitro 112 leaves concerning the process of thrombus formation using the T-TAS technique in whole blood collected from healthy volunteers. The first time we have demonstrated that all tested preparations showed anticoagulant activity. The tested extract and four fractions (A-D) decreased AUC10 relative to control (blood without extract/fraction); however, these effects were not always statistically significant. Only three fractions (A, C and D) at the highest concentration-50 µg/mL-induced changes which were statistically significant. In addition, the fraction D (50 µg/mL), rich in triterpenoids, showed the strongest activity. Such a strong effect may be associated with the presence of triterpenoids, including maslinic, pomoleic, ursolic and 3-epiurolic acid. For example, triterpenoids and acylated triterpenoids isolated from sea buckthorn had a greater impact on the coagulation system than phenolic compounds [63]. Results of Babalola et al. [64] also demonstrated that naturally occurring triterpenoids-oleanolic, ursolic, betulinoic and maslinic acids-inhibit platelet aggregation. Three platelet agonists, i.e., thrombin, ADP or epinephrine, were used to assess the anti-aggregation activity of these triterpenes in a rat model. Inhibition of platelet aggregation stimulated by thrombin and ADP was most demonstrated by oleanolic acid (approximately 80%). In other studies, Kim et al. [65] observed that ursolic and oleanolic acids enhanced platelet aggregation induced by thrombin and ADP in a dose-dependent manner-the strongest effect was observed for the concentration of 50 µM (for both used acids). However, after the activation of platelets by collagen, the acids had no effect on their aggregation. Incubation of whole blood with thrombin, in the presence of tested acids, also showed proaggregation activity compared to the control sample. In addition, measurement of prothrombin time and the activated partial thromboplastin time showed that these compounds did not affect plasma coagulation. Results of Kim et al. [23] may indicate that blood platelets, not coagulation factors, are the main target for ursolic and olenolic acid. Moreover, our present results suggest that triterpenoids, including ursolic from used fraction D, may determine its anticoagulant potential. It is interesting that the concentration of this acid in tested fraction D is lower (<50 µg/mL) than its concentration (50 µM) used by Kim et al. [65]. In addition, the differences in the anti-coagulant potential of tested extract and fractions (observed in thrombus formation) might be correlated with their different chemical profile. Our present results indicate that fraction D has stronger anti-coagulant properties than extract (rich in verbascoside) in whole blood. On the other hand, when we used washed blood platelets, this extract had stronger anti-platelet activity than various fractions (A-D) [47]. Campo et al. [66] also observed that verabascocide inhibits platelet aggregation stimulated by ADP and arachidonic acid in vitro.
On the basis of the conducted research, it has been shown that the chemical compounds produced by Paulownia Clon in Vitro 112 leaves, especially presented in fraction D, are promising candidates for natural preparations with anticoagulant potential. However, in order to clarify the exact mechanism of action of the active substances present in this plant, studies closer to physiological conditions, i.e., in vivo studies, should be performed, which will also allow to determine the effects of their long-term effects. Moreover, its high calorific value makes Oxytree a good raw material to be used as a renewable carbon source. This species is ideal for establishing commercial forests.