Phytochemical screening of Pulsatilla species and investigation of their biological activities

The present study aimed to identify biologically active secondary metabolites from the rare plant species, Pulsatilla patens subsp. patens and the cultivated P. vulgaris subsp. vulgaris. Chromatographic fractionation of the ethanolic extract of the roots of P. patens subsp. patens resulted in the isolation of two oleanane-type glycosides identified as hederagenin 3-O-β-d-glucopyranoside (2.7 mg) and hederagenin 3-O-β-d-galactopyranosyl-(1→2)-β-d-glucopyranoside (3.3 mg, patensin). HPLC analysis of the methanolic extract of the crude root of P. patens subsp. patens and P. vulgaris subsp. vulgaris revealed the presence of Pulsatilla saponin D (hederagenin 3-O-α-l-rhamnopyranosyl(1→2)-[β-d-glucopyranosyl(1→4)]-α-l-arabinopyranoside). Chromatographic analysis using GC-MS of the silylated methanolic extracts from the leaves and roots of these species identified the presence of carboxylic acids, such as benzoic, caffeic, malic, and succinic acids. The extracts from Pulsatilla species were tested for their antifungal, antimicrobial, and antimalarial activities, and cytotoxicity to mammalian cell lines. Both P. patens subsp. patens and P. vulgaris subsp. vulgaris were active against the fungus Candida glabrata with the half-maximal inhibitory concentration (IC50) values of 9.37 μg/mL and 11 μg/mL, respectively. The IC50 values for cytotoxicity evaluation were in the range of 32–38 μg/mL for P. patens subsp. patens and 35–57 μg/mL for P. vulgaris subsp. vulgaris for each cell line, indicating general cytotoxic activity throughout the panel of evaluated cancer and noncancer cells.


Introduction
Plants are known to produce different biologically active substances, including secondary metabolites. It had been reported that plants from the Pulsatilla genus contain ranunculin, anemonin, protoanemonin, triterpenes, and saponins (9%) of mainly the oleanane and lupane-type [1,2]. These metabolites were also subjected to analyses in order to determine the contents of polyphenol compounds, flavonoids, and anthocyanidins active protection [39]. On the territory of Poland, P. patens subsp. patens reaches the western border of its range and the number of its sites clearly decreases towards the west and south [40] (Fig. 1B).
Pulsatilla vulgaris subsp. vulgaris is a species occurring across Central Europe, ranging from France in the south to Sweden at its northern limit [31] (Fig. 2A) and is listed as "near threatened" by the International Union for Conservation of Nature [41]. Currently, P. vulgaris subsp. vulgaris is listed as vulnerable (VU category) in Ukraine, Slovakia [42], Sweden [43], and the United Kingdom [44]. This species is also classified as endangered (EN category) in Germany [45] and Switzerland [46] and as critically endangered (CR category) in Austria [47]. In Poland, it is listed as an extinct (RE category) species [48] and mainly cultivated. Pulsatilla vulgaris subsp. vulgaris is currently extinct in Poland on natural sites [49]. It was only reported at four sites before 1930 [40] ( Fig. 2B) on the habitats of the pine forests of the All. Dicrano-Pinion.
The aim of this study was the identification of biologically active secondary metabolites from the rare plant species Pulsatilla patens subsp. patens and the cultivated P. vulgaris subsp. vulgaris. In the course of our study, we evaluated the extracts of leaves and roots of these plant species for their antifungal, antimicrobial, and antimalarial activities, and cytotoxicity to mammalian cell lines.

Material collection
The leaves and roots of P. patens subsp. patens were collected in May 2013-2015 at the Knyszynska Forest near Bialystok City, NE Poland, while P. vulgaris subsp. vulgaris were obtained from cultivation at the Herbarium "The Herbal Corner" in May 2015, Podlaskie Province, NE Poland. Plant material was identified by Prof. Grażyna Łaska from the Bialystok University of Technology, Faculty of Civil and Environmental Engineering, Poland.
Extraction and chromatographical analysis TLC/CC analysis. For thin layer chromatography (TLC) and column chromatography (CC), ethanolic extracts were prepared from roots of P. patens subsp. patens (K 2 ). Dried roots of P. patens subsp. patens (10 g) were extracted with 1 L of 50% aqueous EtOH at room temperature for 12 h and then filtered. The extraction was repeated three times. The combined filtrates were concentrated in a vacuum evaporator to obtain a syrupy brown residue (0.95 g). Then 5 mg of ethanolic extract from roots of P. patens subsp. patens (K 2 ) was transferred to normal and reverse phase (RP) TLC on silica gel with fluorescence (stationary phase). The following solvent system was used for normal phase TLC: hexanes-ethyl acetate (C 6 -H 14 (Fig. 3). In the next step, an ethanolic extract of the roots of P. patens subsp. patens was fractionated by CC (0.9 g) using CHCl 3 -MeOH gradient (90:10). Compounds were eluted by reverse-phase C 18 column using the H 2 O-MeOH (30:70) solvent system (Fig. 4). The structures of the isolated compounds were determined by 1 H and 13 C-NMR spectroscopy.
HPLC analysis. The fraction which exhibited the most potent activity was again chromatographed by high performance liquid chromatography (HPLC) using Agilent Technologies 1260 Infinity system. The HPLC profiles were determined at 35°C on a COSMOSIL Packed Column 5C-18-MS-II (4.6 μm × 250 mm) (Nacalai, USA) eluted with methanol (A) and water (B) as mobile phases in a linear gradient elution with the flow rate of 1 mL/min. The elution program was as follows: 0-8 min, 90% A to 77%  A, 8-25 min, changed to 71% A, 25-40 min, to 60% A, 40-50 min, to 50% A, 50-75 min, to 10% A, 75-80 min, to 0% A [50]. The volume of sample injected was 35 µL. Fluorescence was monitored by an Agilent 1260 Infinity Fluorescence Light Detector (Agilent Technologies, USA; 210 nm excitation and 460 nm emission wavelength). The analytical data were integrated using the Agilent OpenLAB software. The fraction was purified by HPLC to give Pulsatilla saponin D. The peak of Pulsatilla saponin D was identified based on retention time with respect to the reference standard (purchased from Toronto Research Chemicals) previously run in an analytical HPLC.
GC-MS analysis. The pressurized solvent extractions were performed using a Büchi E-916 Speed Extractor. The raw materials were extracted with methanol (leaves 32.3 g, and roots, 23.6 g of P. patens subsp. patens; leaves, 21.8 g and roots, 56.1 g of P. vulgaris subsp. vulgaris). The extraction time was 30 min, temperature 100°C and pressure 1.2 × 10 7 Pa. Three extracts were prepared from each raw material. Extracts were filtered through paper filters. The methanol was dried using a rotor evaporator.
Each dry residue (10 mg) was redissolved with 1 mL of pyridine and 200 µL BSTFA [Ν,Ο-bis-(trimethylsilyl)-trifluoroacetamide] added. Next, the mixture was heated at 60°C for 30 min. The solution was analyzed by gas chromatography / mass spectrometry (GC-MS) using an Agilent 7890A gas chromatograph equipped with an Agilent 5975C mass selective detector. The injection of a 1-µL sample was performed using an Agilent 7693A autosampler. The separation was performed on an HP-5MS (30 m × 0.25 mm × 0.25 μm film thickness) fused silica column at a helium flow rate of 1 mL/min. The injector worked in split mode (1:5), and the injector temperature was 280°C. The initial column temperature was 50°C, rising to 310°C at 3°C/min and the final temperature was held for 10 min. The ion source and quadrupole temperatures were 230°C and 150°C, respectively. The ionization energy (EIMS) was 70 eV. The detection was performed in full scan mode from 41 to 600 amu.
After integration, the percentage of each component in the total ion current (TIC) was calculated. Mass spectral data and calculated retention indices were used to identify the compounds. The mass spectrometric identification was carried out with an automated system of GC-MS data processing supplied by the National Institute of Standards and Technology (NIST). Retention indices of the analytes were determined, taking into account C 10 -C 40 n-alkanes retention times and comparing them with the NIST database.

Antimicrobial assay
For the antimicrobial assay, we used the fungi Candida albicans ATCC 90028, C.  [55] was used. All samples were diluted in 20% DMSO/saline and then transferred in duplicate to microplates. In order to obtain final target inocula, microbial inocula were prepared by correcting the optical density at 630 nm (OD 630 ) of the microbe suspensions in the incubation broth. In each assay, drug controls were used, with ciprofloxacin (ICN Biomedicals, USA) for the bacteria and amphotericin B (ICN Biomedicals, Ohio) for the fungi. The readings were made for all organisms before and after incubation at either 530 nm using the Biotek Powerwave XS plate reader (Bio-Tek Instruments, USA) or 544 excitation / 590 emission, (M. intracellulare, A. fumigatus) using the Polarstar Galaxy Plate Reader (BMG LabTechnologies, Germany). To determine the minimum fungicidal or bactericidal concentrations required, 5 µL was removed from each clear well, transferred to agar, and incubated. The MFC/MBC (the lowest test concentration that kills the organism) was also established.

Antimalarial assay
In the antimalarial assay, the chloroquine sensitive (D6) strain of Plasmodium falciparum was used and plasmodial LDH activity was measured using the procedure devised by Makler and Hinrichs [56]. To the wells of a 96-well plate containing 10 µL of diluted sample, a suspension of red blood cells infected with P. falciparum of 200 µL was introduced, together with 2% parasitemia and 2% hematocrit in RPMI 1640 medium supplemented with 10% human serum and 60 µg/mL Amikacin. Then, the plate was incubated at 37°C for 72 h with 90% N 2 , 5% O 2 , and 5% CO 2 using a modular incubation chamber. In order to determine the parasitic LDH activity, 20 µL of the incubation mixture was mixed with 100 µL of the Malstat reagent (Flow Inc., USA) and incubated at room temperature for 30 min. Then, 20 µL of a 1:1 mixture of NBT/PES (Sigma, USA) were added and the plate was incubated in the dark for 1 h. To stop the reaction, 100 µL of a 5% acetic acid solution was added and the absorbance was read at 650 nm. The drug controls were artemisinin and chloroquine. The half-maximal inhibitory concentration (IC 50 ) values were calculated on the basis of the dose response curves of growth inhibition using XLfit 4.2 (IDBS, USA).

Cytotoxicity assay
In the cytotoxicity assay, the root extract was tested against a panel of cancer and noncancer cell lines. In this assay, we used 96-well tissue culture-treated plates. The cells were seeded at a density of 25,000 cells/well and grown for 24 h. Next, samples of the extract at different concentrations were added and cells were incubated for 48 h. The neutral red method [57] was used to determine the cell viability. The IC 50 values were obtained on the basis of dose response curves. Doxorubicin was used as the control drug.

Results
Chromatographic fractionation of the ethanolic extract (0.9 g) of the roots of P. patens subsp. patens resulted in the isolation of two oleanane-type glycosides identified as hed-  HPLC analysis of the silylated methanolic extract of the crude root of P. patens subsp. patens and P. vulgaris subsp. vulgaris revealed the presence of Pulsatilla saponin D -hederagenin 3-O-α-l-rhamnopyranosyl(1→2)-[β-d-glucopyranosyl(1→4)]-α-larabinopyranoside (Fig. 6). Analysis of the chromatographic spectra of the extracts from P. patens subsp. patens and P. vulgaris subsp. vulgaris showed similar spectra to the standard solution chromatogram of Pulsatilla saponin D. The other chromatographic peaks for the extracts of P. patens subsp. patens and P. vulgaris subsp. vulgaris also showed the presence of triterpenoid saponins, which will need to be investigated further. The Pulsatilla saponin D has not been isolated in Pulsatilla medicinal plants in Poland until now, with this type of study being conducted here for the first time. Pulsatilla saponin D, C 47 H 76 O 17 , has multiple demonstrated biological properties including antifungal, antimicrobial, and cytotoxic activities.  Carbohydrates were the main components of the extracts from P. patens subsp. patens and P. vulgaris subsp. vulgaris. The content of carbohydrates in each extract from P. patens subsp. patens was over 60% while those from P. vulgaris subsp. vulgaris were over 90% carbohydrates. The analyzed extracts that were the most abundant were saccharose, glucopyranose and fructofuranose. The extracts from the leaves of these species revealed the presence of carboxylic acids, such as benzoic, caffeic, malic, and succinic acids. Furthermore, glyceric, quinic, and protocatechuic acids were identified in the leaf extracts of P. patens subsp. patens, while the roots extracts from P. vulgaris subsp. vulgaris contained the smallest amount of carboxylic acids (Tab. 1).
Microbiological assays of the root extracts showed activity against the fungal pathogen Candida glabrata, indicated by the IC 50 value of 9.37 μg/mL for P. patens subsp. patens and 11 µg/mL for P. vulgaris subsp. vulgaris (Tab. 2, Tab. 3). This result is particularly important as with the increasing use of immunosuppressants, the number of cases of inflammation of mucous membranes and particular systems by C. glabrata have significantly increased, especially in people with human immunodeficiency virus. Candida glabrata has become the second most important agent responsible for surface (oral cavity, esophagus, vaginal, or urinary duct) or systemic inflammation. The main problem with the inflammation caused by C. glabrata is their innate resistance to azole antibiotics that are very effective against other Candida species. Treatment of C. glabratacaused inflammation often require the use of amphotericin B that is toxic for humans and can trigger undesired side effects [58]. It has been experimentally shown that the root extracts form Pulsatilla species can contribute to new, safer drugs in antifungal therapy against C. glabrata. The extracts from the leaves and roots of this plant species showed lower ability to inhibit the growth of the four different fungi (Candida albicans, C. krusei, Aspergillus fumigatus, and Cryptococcus neoformans) and other bacteria (Staphylococcus aureus, MRSA, Escherichia coli, and Pseudomonas aeruginosa) pathogenic to humans (Tab. 2, Tab. 3). As far as C. albicans, A. fumigatus, E. coli, and P. aeruginosa are concerned, the leaf and root extracts have been found to show biological activity. Although their activity is rather low, the extracts are still highly valuable as C. albicans is a frequent fungus pathogen in humans. This species is responsible for the painful inflammation of mucous membranes, e.g., vagina inflammation in women or oral-pharyngeal thrush in AIDS patients. In certain groups of sensitive patients, this species is responsible for serious and life threatening bloodstream inflammation and subsequent inflammation of internal organs [59]. The extracts from Pulsatilla species are also attractive in fighting A. fumigatus, which is one of the most common saprophytic fungi in the air. With an increasing number of patients treated with immunosuppressants, the incidence of serious and usually lethal invasive aspergillosis has also considerably increased worldwide [60]. Extracts from Pulsatilla species are also promising agents for the treatment of E. coli caused inflammation. This bacteria strain is the dominant facultative anaerobic  [61]. The Pulsatilla extracts should also be considered in fighting P. aeruginosa, the opportunistic pathogen responsible for hospital infections in patients with immunodeficiency. This pathogen causes inflammation of the lungs related to the use of a respirator, inflammation of the urinary tract, wounds in patients with serious burns, as well as sepsis [62]. Unfortunately, the extracts considered are inactive against M. intracellulare. In our antimalarial assays, the extracts from the leaves and roots showed low activity against the protozoan (27% of inhibition for P. patens subsp. patens and 1-8% for P. vulgaris subsp. vulgaris), while the antimalarial drug chloroquine (positive control) showed 94-98% inhibition (Tab. 4). The root extracts showed cytotoxicity to all the cell lines included in the assay. As shown in Tab. 5, the IC 50 for cytotoxicity of P. patens subsp. patens was in the range of 32-38 μg/mL and for P. vulgaris subsp. vulgaris, 35-57 µg/mL for each cell line, indicating general cytotoxic activity throughout the panel of cancer and noncancer cells.

Discussion
The extract of the roots of P. patens subsp. patens revealed the presence of two oleananetype glycosides identified as hederagenin 3-O-β-d-glucopyranoside and hederagenin 3-O-β-d-galactopyranosyl-(1→2)-β-d-glucopyranoside (patensin). Structures of both compounds were established via comparing 1 H and 13 C-NMR spectral data on compounds reported earlier by other authors [63,64]. Patensin, C 42 H 68 O 14 , a triterpenoid glycoside, was also isolated from the ethanolic extract of roots of P. patens subsp. multifida [63] and five oleanane-type glycosides and two triterpene saponins were also isolated from the roots of P. patens subsp. multifida [64]. Phytochemical studies of new secondary metabolites from P. patens subsp. multifida have revealed the presence of a hitherto unknown saponin [12]. This saponin isolated from the methanolic extract of this plant has been found to inhibit the growth of skin cancer. Roots from P. patens subsp. multifida have been used historically in traditional Chinese medicine [26] as the agent shows anticancer, antimalarial, and antibacterial properties. At present, it is used as a homeopathic drug for the treatment of the ear conditions, exanthema, rheumatism, bronchitis, coughing, asthma, as well as an agent for alleviating stress and anxiety.
The triterpene acid and triterpene glycosides (saponins Pulsatilla A and B) were isolated from the plant material of P. chinensis [65]. The triterpene acid shows high cytotoxic activity against the malignant cells of lung cancer. The triterpene saponins are used for the treatment of inflammatory conditions [66] and reveal high antiprotozoal Tab. 4 The test results of the antimalarial activity of leaf and root extracts of Pulsatilla patens subsp. patens and P. vulgaris subsp. vulgaris.
Analysis of HPLC fingerprints of different species of Pulsatilla (P. koreana, P. chinensis, P. ambigua, P. dahurica, P. turczaninovii) showed significant differences between their HPLC fingerprints, but with 10 common peaks (Fig. 7B). These common peaks not only can be used to classify and authenticate Pulsatilla species, but also provide important  [78], and 10 common peaks of different Pulsatilla species on HPLC fingerprint study (B) [50]. references for HPLC fingerprints and quality control of Pulsatilla medicinal plants [50]. These studies confirmed chromatographic spectra can show differences depending on the Pulsatilla species. Significant differences between P. koreana and P. dahurica, and between P. turczaninovii and P. ambigua were observed. Slight differences in the chromatographic spectra were also found for P. patens subsp. patens and P. vulgaris subsp. vulgaris; however, the presence of the triterpenoid saponins and the biological activity of their plant extracts were demonstrated for the first time in this work. This is very important because the triterpenoid saponins, such as Pulsatilla saponin D (C 47 H 76 O 17 ), have demonstrated multiple biological properties including antifungal, antimicrobial, and cytotoxic activities. These compounds are also produced from the root of several Pulsatilla species, including P. koreana [1], P. chinensis [79], and P. cernua [80]. In particular, Pulsatilla saponin D shows high cytotoxic activity against cancer cells. Pulsatilla saponin D isolated from the root of P. koreana showed potent inhibition rate of tumor growth (IR, 82%) at the dose of 6.4 mg/kg on BDF1 mice bearing Lewis lung carcinoma (LLC) cells [1]. This compound is also proving to be promising for the treatment of Alzheimer's disease [15].
It has been reported that the extracts from other species containing triterpenoid saponins, with hederagenin being the active ingredient, showed marked inhibition of serotonin (5-HT), norepinephrine (NE), and dopamine (DA) transporters [81]. Monoamine transporter inhibition studies on pure isolated compounds have not been reported yet, hence the importance of performing CNS studies on isolated compounds from Pulsatilla species. In future, we will focus on pure isolated compounds from P. patens subsp. patens and P. vulgaris subsp. vulgaris and submit them to the Psychoactive Drug Screening Program (PDSP), Chapel Hill, NC for monoamine transporter inhibition studies for extracellular monoamines including serotonin (5-HT), norepinephrine (NE), and dopamine (DA) transporters. The two oleanane-type glycosides have been sent to the National Center for the Natural Product Research (NCNPR) at the University of Mississippi to evaluate their antimicrobial activity.
Currently, this preparation, which is called SB31, is undergoing phase II clinical studies in Korea. It was previously reported that SB31 exhibited cytotoxic activity against some human cancer cell lines and potent antitumor activity on a mouse tumor model. An active antitumor constituent from the roots of P. koreana was isolated and the main component was found to be deoxypodophyllotoxin (DPT), which exhibited antiangiogenic activity and good antitumor activity against mice bearing LLC cells. However, the DPT content of Pulsatillae Radix, which is an active constituent of SB31, is too low for discernible antitumor activity. This suggests that DPT is not the major constituent responsible for the antitumor activity of P. koreana roots.
The extracts from P. cernua have been found to contain anthocyanidins, as well as triterpene saponins and phytosterols [82]. The natural dyes isolated from P. cernua show antiallergic, antifungal, anticancer, and antiulcer activity. Moreover, they improve blood flow in coronary arteries and reduce blood pressure so are attractive substances for the treatment of atherosclerosis. The anthocyanidins isolated from the extracts have been used in medicine as perspiration-promoting agents in the treatment of colds and in the treatment of inflammation of the stomach and intestines.
The flavonoids and tannins isolated from the methanolic extract from P. nigricans show considerable antianxiety effect [83]. Pulsatilla nigricans is one of the species used in traditional medicine for the treatment of nervous exhaustion and anxiety. Therefore, their high activity permits their application for the treatment of depression.
The confirmation of our results shows the extracts from P. nigricans can also be used as antifungal agents and to alleviate the weakness of an organism.
Phytochemical studies of the extracts from P. montana subsp. balcana, P. halleri subsp. rhodopaea, and P. slaviankae have shown polyphenolic compounds, flavonoids, and anthocyanidins [13]. The highest contents of these substances were established in the extracts from P. montana subsp. balcana and P. halleri subsp. rhodopaea. The antioxidative properties of P. slaviankae were a consequence of the presence of astragalin, isoquercetin, quercetin, kaempferol, caffeic acid, and isorhamnetin in the aboveground shoots of this species [84]. The natural biologically active substances isolated from this species may play an important role in the protection of humans against breast cancer, colon cancer, prostate cancer, and leukemia. Phytochemical studies of plant material from the species P. montana subsp. balcana have also revealed the presence of caffeic acid, chlorogenic acid, gallic acid, methylgallic acid, ferulic acid, and ellagic acid [85]. Our results also identified caffeic acid, and so are in some accordance with the observation of these previous studies. The therapeutic effects related to the presence of these polyphenolic compounds include diastole of blood vessels, and assistance in stabilizing the blood pressure of patients with heart disease and hypertension.
The chemical composition of extracts from the leaves and roots of P. patens subsp. patens and P. vulgaris subsp. vulgaris were significantly different. The leaf extracts, in contrast to the root extracts, contained up to 22% carboxylic acid for P. patens subsp. patens and 2% for those of P. vulgaris subsp. vulgaris, whereas the leaf extracts showed lower biological activity. Chemical composition of extracts from dry and raw materials from this species were slightly different (Łaska and Sienkiewicz, unpublished data), while the extracts from fresh material were the most rich with compounds. Upon drying, some compounds were lost through evaporation or destruction.
However, phytochemical analysis of P. patens subsp. patens and P. vulgaris subsp. vulgaris showed nearly similar results due to the presence of phytochemical constituents. A previous study on the chemotaxonomic characteristics of Pulsatilla species revealed that P. patens subsp. patens, P. vulgaris subsp. vulgaris, and P. grandis Wenderoth contain only delphinidin glycosides, whereas the remaining species [P. halleri subsp. rhodopaea (Stoj. & Stef.) K. Krause, P. montana subsp. balcana (Vel.) Zamels, P. pratensis (L.) Mill., P. rubra (Lamk.) Delarbre, and P. vulgaris subsp. vulgaris var. touranginiana E. G. Camus] contain pelargonidin and delphinidin glycosides [86]. Our studies revealed the presence of the triterpenoid saponins and the biological activity of the extracts from P. patens subsp. patens and P. vulgaris subsp. vulgaris. Therefore, isolated saponins have chemotaxonomic features that may be recognized as specific to Pulsatilla species.