Antioxidant, Anti-Inflammatory, and Cytotoxic Properties and Chemical Compositions of Filipendula palmata (Pall.) Maxim.

Filipendula palmata (Pall.) Maxim. remains unexplored and underutilized resources with a high potential to improve human health. In this study, a new ursane-type triterpenoid, namely, 2α, 3β-dihydroxyurs-12-en-28-aldehyde (compound 10), and other 23 known compounds were isolated. 5 triterpenoids (compounds 6, 8, and 10–12), 11 flavonoids (compounds 13–15 and 17–24), 6 phenolic compounds (compounds 1, 2, 4, 5, 9, and 16), 2 sterols (compounds 3 and 7) were isolated from the aqueous solution extract of the aerial parts of F. palmata. The structures of all compounds were elucidated by the use of extensive spectroscopic methods such as infrared spectroscopy (IR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), 1H-NMR, and 13C-NMR. The solvent extractions of ethyl acetate fraction were evaluated for antioxidant activities using DPPH (2, 2-diphenyl-1-picrylhydrazyl) and ABTS+ (2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) methods. The anti-inflammatory effects of the compounds were evaluated in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. The extract cytotoxicity on the cancer cell lines MCF-7, HeLa, 4T1, and A549 was determined by MTT assay. As a result, compounds 10, 11, and 12 exhibited better antioxidant activity compared to the other compounds. Compounds 8–24 had different inhibitory effects on the release of NO, TNF-α, and IL-6 in LPS-stimulated RAW 264.7 cells. The new compound has shown a significant inhibiting effect on cancer cells, and the cell inhibition rate increased in a dose-dependent manner. Further research to elucidate the chemical compositions and pharmacological effects of F. palmata is of major importance towards the development and foundation of clinical application of the species.


Introduction
With the improvement of people's health awareness, different kinds of healthcare products are gradually developed. Herbal tea, one of the popular beverages consumed worldwide, has the widest applicability, second only to drinking water. e human uses of herbal teas are believed to have originated around thousands years ago in China, and its efficacy and taste are closely related to the specific components of plants. Nowadays, various kinds of herbal tea have become the most common beverage and food in people's daily life. Meadowsweet tea is one of the most popular herbal tea products, and its raw material comes from the genus Filipendula. e use of some Filipendula genus as herbal medicines is known, and the aerial parts and roots have a significant pharmaceutical interest, being used by daily consumption in Russia and other Siberia countries as wild tea. Filipendula is one of the most popular perennial herbaceous plant genera for herbal tea preparation, and it includes many significant plants of the family Rosaceae and more than 20 varieties are distributed all over the world (http://www.ncbi.nlm.nih.gov/taxonomy/). ese herbs are used due to the specific honey-like fragrance of the flowers and the pleasant taste of water decoctions. Among them, the most studied species are Filipendula ulmaria (L.) Maxim. (meadowsweet) and Filipendula vulgaris Moench (dropwort), which are officinal plant species in many countries. In these species, researchers have found different classes of bioactive constituents including salicylates, phenolic acids, flavonoids and flavonoid glycosides, and tannins [1][2][3][4][5][6][7][8][9]. Recently, Katanic [10] showed that F. ulmaria extracts were effective in reducing kidney oxidative stress and mitigating tissue damage. e same extracts attenuated the genotoxicity of cisplatin in a reverse dose-dependent manner and did not demonstrate any in vitro cytotoxic activity at all the applied concentrations. Bespalov [11] showed that meadowsweet (F. ulmaria) decoction was able to inhibit colorectal carcinogenesis induced by the methylnitrosourea in rats. e chemical composition of meadowsweet had a statistically significant decrease in the overall tumor incidence and multiplicity by 1.4 and 2.9 times. Samardzic et al. [12] reported that meadowsweet and dropwort were rich in polyphenols that belong to the classes of flavonol glycosides, phenolic acids, and hydrolysable tannins as a folk medicine for their antirheumatic, antipyretic, and antiulcer properties.
F. palmata belongs to Filipendula genus, which is widely distributed in Northeast China, usually called by Siberian meadowsweet. F. palmata was the northeast genuine drug in China, species commonly used to treat gout, rheumatism, epilepsy, frostbite, burn, and gynecologic hemostasis. Recently, F. palmata had been considerably less studied plant, its chemical compositions had been reported only with essential oils; moreover, the biological activity of F. palmata had not yet been reported. e objectives of this study were to verify the pharmacological activity of drugs and to find a new substitute with better efficacy in Filipendula genus.  13 C-NMR), TMS was used as an international standard and DMSO-d6 as a solvent. High-performance liquid chromatography (HPLC) was performed using an Agilent 1260 Series HPLC system (Agilent, USA) equipped with four pumps with an in-line degasser, autosampler, oven, and ultraviolet detector (UVD). HR-ESI-MS was measured on IonSpec 7.0 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS) spectrometer (Bruker, USA). Column chromatography was performed with silica gel (200-300 mesh) (Qingdao Marine Chemical Factory, China).

Cell Lines.
Human cervical cancer cells HeLa, human breast adenocarcinoma cells MCF-7, mouse breast cancer cells 4T1, and human lung cancer cells A549 were purchased from American Type Culture Collection (Manassas, VA) and cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 2 mmol/L L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C and in an atmosphere of 5% CO 2 and 95% humidity. e culture medium containing 0.1% DMSO was used as the samples (specimens and controls). e mice macrophage RAW264.7 (American Type Culture Collection, Manassas, VA, USA) cell line was maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin G (100 units/mL), streptomycin (100 mg/mL), and L-glutamine (2 mM). e cells were grown in a humidified atmosphere containing 5% CO 2 at 37°C.

Extraction and Isolation.
e air-dried aerial parts of F. palmata (50 kg) were powdered and macerated with distilled water (1 : 14 w/v) for 12 hours at room temperature and then extracted three times (1.5 h each) with distilled water. After filtration, filtrates were merged, aqueous extracts were evaporated on a rotary evaporator (vacuum rotary evaporator), obtained aqueous extracts (1105 g) were then mixed with diatomite, and successively partitioned with petroleum ether, chloroform, ethyl acetate, and ethanol to obtain the petroleum ether fraction (289 g), chloroform fraction (183 g), ethyl acetate fraction (121 g), and ethanol fraction (309 g). e petroleum ether was removed, and the extraction part of petroleum ether becomes a thick paste, which used the sublimation method and was subjected to LH-20 eluting with MeOH to give compounds 1 and 2. e chloroform soluble fraction was chromatographed on silica gel column eluting with CHCl 3 /MeOH in gradient (40 : 1 to 1 : 1) and then detected by TLC and the same components were merged to obtain 5 fractions (C. 1-C. 5); C. 2 and C.4 fractions were chromatographed on silica gel column eluting with CHCl 3 /MeOH and detected by TLC, and the same components were merged, which were separated by Sephadex LH-20 column to give compounds 3-7. e ethyl acetate soluble fraction was dissolved in methanol. After filtration, fractions were separated by a LH-20 gel and eluted with methanol and the same components were combined by TLC detection, to give 6 fractions (E.1-E.6); E.2 fraction was separated by octadecylsilyl (ODS) reverse-column chromatography and eluted with a gradient of methanol-water (50 : 50-90 : 10) to give compounds 8-12 and E.6 fraction was repeated by silica gel column chromatography and gel LH-20 purification to give compounds 13-24. e ethanol fraction was reserved for reserve ( Figure 1).   e Liebermann-Burchard test showed positive, purple red color by 10% purple vitriol spray test. It has the same Rf value in a TLC test compared with a reference substance.   e Rf value was consistent with the standard product of caffeic acid, and the melting point does not decrease after mixing with a standard product.   e magnesium hydrochloride reaction showed rose-red color (positive), and the FeCl 3 reaction showed dark green. e Rf value was consistent with the standard product of quercetin, and the melting point does not decrease after mixing with a standard product.

Antioxidant Activity
(1) DPPH assay. e methods for determining DPPH free radical scavenging activity were analyzed by Hu's method with slight modifications [13]. About 0.2 mL of tested compounds at various concentrations were added to 2 mL DPPH solution (21.4 µg/mL ethanol solution), respectively. e mixture, protected from light, was reacted for 30 min. e decrease in absorbance was monitored at 517 nm, to obtain absorbance A i . e control was the DPPH solution. e sample solution was replaced with 70% ethanol solution, to obtain A 0 , and the DPPH was used to obtain A j in the same method. % radical scavenging activity � 1−[(A i -A j )/A 0 ] × 100% A 0 : absorption of 2 mL DPPH 70% ethanol solution and 2 mL 70% ethanol solution. A i : absorption of 2 mL DPPH 70% ethanol solution and 2 mL sample. A j : absorption of 2 mL 70% ethanol solution and 2 mL sample.
(2) ABTS + assay. e methods for determining ABTS + free radical scavenging activity were analyzed by Biao's method with slight modifications [14]. About 0.2 mL of tested compounds at various concentrations was added to 2 mL ABTS + solution, respectively. e mixture, protected from light, was reacted for 30 min. e decrease in absorbance was monitored at 734 nm. e control was 0.2 mL of distilled water and 2 mL of ABTS + solution.
e same method was used in vitamin C (Vc). e half-maximal inhibitory concentration (IC 50 ) was used to evaluate the ABTS + free radical scavenging activity and the DPPH free radical scavenging activity.

Cytotoxicity Assay.
Cell proliferation was measured using the colorimetric MTT method for HeLa, 4T1, A549, and MCF-7 cells [16]. Compounds 8-24 were dissolved in DMEM culture media containing 0.1% DMSO at final concentrations of 0-400 μg/mL. e HeLa, 4T1, A549, and MCF-7 cells were grown in 96-well plates at 9 × 10 3 cells per well, incubated at 37°C for 24 h, and then treated with various concentrations of compound 10 for 48 h. e control cells were exposed to culture media containing 0.1% DMSO.
en, 50 mL of MTT solution (5 mg/mL) was added to each well. Cells were incubated for three additional hours. Finally, 150 mL of DMSO was added to dissolve the formed crystals. e absorbance at 570 nm was measured by scanning with a microplate reader. e experiment was repeated 3 times. Calculation of the impact of drugs on cell growth inhibition rate and IC 50 values is performed with the following equation: where D 0 is the OD value of the control wells and D 1 is the OD value of the sample wells.

Statistical Analysis.
Statistical analyses were performed using SPSS 21.0 for the variance of the experimental data. e results were expressed by X ± s. P < 0.05 was considered to have statistical significance.
Characteristics of compound 10 are as follows: It is a white amorphous powder, its IR spectrum exhibited absorption bands due to -OH at 3406 cm   13 C-NMR, and DEPT spectra's data suggested that an ursane-type triterpenoid moiety existed in the structure of compound 10. Compared with the reference which published a compound methyl, 2α, 3β-dihydroxyurs-12-en-28-oate, it was showed that all data except C-28 were consistent. Based on the above information, it was confirmed that C-28 was an aldehyde group. In summary, the structure of compounds is inferred to be 2α, 3β-dihydroxyurs-12-en-28-aldehyde.

Antioxidant Activity.
e ability of plants to show antioxidant activity is owing to their composition and a mixture of different antioxidants, mainly polyphenolic compounds with different action mechanisms. Because of their synergistic interactions, it is indispensable to use several methods in order to determine the in vitro antioxidant capacity of plant extracts [37]. DPPH and ABTS + scavenging capacity are the most commonly used methods for determining antioxidation performance in vitro. e DPPH and ABTS + radical scavenging activities were performed to evaluate the antioxidant capacities of the different active fractions compared with ascorbic acid (vitamin C), which was served as a control. e semi-inhibitory concentration (IC 50 ) of compounds 8-24 for ABTS + and DPPH· free radical scavenging activity is shown in Table 3. Compared with other active components, compound 10 has better antioxidant activity, which is related to the structure of ursane-type compounds [38]. Another possibility is that the compound containing aldehyde group was more likely to undergo oxidation reactions and have better antioxidant capacity [39]. Previous studies have shown that the

Anti-Inflammatory Activity.
Macrophages are necessary to maintain the body's immune balance and play an important role in the host's resistance to pathogen infection [41]. In the process of regulating immunity, stimulating activated macrophages can release some immune regulatory factors. Proinflammatory cytokines have been used as biomarkers for the development and progression of inflammation in macrophage models, such as NO, IL-6, and TNF-α. Lipopolysaccharide (LPS) is a component of the cell wall of Gram-negative bacteria. It is an endotoxin. When it acts on macrophages, it stimulates the Toll-like receptors on the cell membrane of the host cell to secrete inflammatory factors. e mechanism is to change the expression of NO, TNF-α, and IL-6 via the NF-κB pathway, which was induced by lipopolysaccharides [42,43]. erefore, we evaluated the effects of different compounds on mouse macrophages (RAW 264.7) induced by LPS. In the inflammatory response of RAW264.7 cells stimulated by LPS, compounds 8-24 had different inhibitory effects on the release of NO, TNF-α, and IL-6 in LPS-stimulated RAW 264.7 cells and showed good anti-inflammatory activity in vitro, in which compound 10 was effective against inflammatory factors. e results are shown in Table 4. Among plants of the same genus, meadowsweet and dropwort have been confirmed to have good antihyperlipidemia and antiedema activity in the rat inflammation model induced by carrageenan injection, and both displayed good safety profiles [44]. In a word, F. palmata exerts its anti-inflammatory activity in RAW264.7 cells stimulated by lipopolysaccharide by inhibiting the production of nitric oxide and proinflammatory cytokines.

Cytotoxic
Activity. Compounds 8-24 isolated from ethyl acetate fraction were examined for their antiproliferative activity towards four cancer cell lines: MCF-7, HeLa, 4T1, and A549 cell lines. e MTT colorimetric assay was adopted to assess the antiproliferative activity as described by Mosmann [16]. Doxorubicin was used as a control in this assay. As shown in Table 5, the results were expressed as median growth inhibitory concentration (IC 50 ) values that represent the compound concentration required to produce a 50% inhibition of cell growth after 48 h of incubation.
e results of the MTT assay showed that compounds 10, 11, and 12 can significantly inhibit the   Figure 4, after 48 h of compound 10 treatments, the proliferation of HeLa, 4T1, A549, and MCF-7 cells was obviously inhibited and the inhibition rate was increased dose-dependently within the concentration ranges tested, and when the concentration of compound 10 reached 100 μg/mL, it led to a significant increase in MCF-7, 4T1, A549 and HeLa cell growth inhibitions, which were 19.85%, 6.55%, 58.21%, and 10.22%. A large number of studies had reported that triterpenoids have potential antitumor activity, including examples of lupane-, oleanane-, and ursane-type triterpenoids. Previous studies had confirmed that ursane-type triterpenoids have extensive anti-inflammatory, antibacterial, antifungal, and antitumor activities [45][46][47][48]. However, the antitumor effect of ursane-type triterpenoids from the F. palmata had not been reported, especially the anticervical cancer effect of ursane-type triterpenoid compound 10. In this experiment, the effect of ursane-type triterpenoid compound 10 on human cervical cancer HeLa cells and human breast adenocarcinoma MCF-7 cells had been further confirmed, suggesting that the compound has a certain antihuman cervical cancer potential. On the other hand, it is speculated that F. palmata is often used to repel mosquitoes in summer in Northeast China, which may be related to the chemical

Conclusion
In this study, we use F. palmata as our research object. We found a new ursane-type triterpenoid compound, after extraction and separation by column chromatography, namely, 2α, 3β-dihydroxyurs-12-en-28-aldehyde (compound 10), and other 23 compounds, based on the spectroscopic analysis. Most of the compounds from F. palmata possessed antioxidant, anti-inflammatory, and antitumor effects. In addition to the new compounds found, compounds 11, 12, and 24 were first isolated from this genus and confirmed that each had significant biological activity by detection. e results suggested that F. palmata extraction was a potent natural antioxidant and antineoplastic, it was a new natural functional plant source with high content of healthy ingredients, and it would become a new beverage source of plant raw materials or a more efficient substitute for herbal tea. Further study on isolation and identification of more bioactive compounds from F. palmata will be helpful to understand this important herbal medicine.

Data Availability
e data used to support the findings of this study are included within the article, and the data used to support the findings of this study are available from the corresponding author upon request.

Authors' Contributions
Hongyin Zhang and Rongxin Han undertook the extraction, isolation, and structure identification process and prepared the manuscript. Rongrong Zhang, Miao Wang, and Xintong Ma carried out biological activity assay. e manuscript was revised by Mingming Yan, Shuai Shao, and Guangzhe Li. e experiment protocol was reviewed and approved by Daqing Zhao. Hongyin Zhang and Guangzhe Li contributed equally to this work.