Bioassay-Guided Fractionation with Antimalarial and Antimicrobial Activities of Paeonia officinalis

Bioassay-guided fractionation technique of roots of Paeonia officinalis led to isolation and structure elucidation of seven known compounds, including four monoterpene glycosides: lactiflorin (1), paeoniflorin (4), galloyl paeoniflorin (5), and (Z)-(1S,5R)-β-pinen-10-yl β-vicianoside (7); two phenolics: benzoic acid (2) and methyl gallate (3); and one sterol glycoside: β-sitosterol 3-O-β-D-glucopyranoside (6). The different fractions and the isolated compounds were evaluated for their antimicrobial and antimalarial activities. Fraction II and III showed antifungal activity against Candida neoformans with IC50 values of 28.11 and 74.37 µg/mL, respectively, compared with the standard fluconazole (IC50 = 4.68 µg/mL), and antibacterial potential against Pseudomonas aeruginosa (IC50 = 20.27 and 24.82 µg/mL, respectively) and Klebsiella pneumoniae (IC50 = 43.21 and 94.4 µg/mL, respectively), compared with the standard meropenem (IC50 = 28.67 and 43.94 µg/mL, respectively). Compounds 3 and 5 showed antimalarial activity against Plasmodium falciparum D6 with IC50 values of 1.57 and 4.72 µg/mL and P. falciparum W2 with IC50 values of 0.61 and 2.91 µg/mL, respectively, compared with the standard chloroquine (IC50 = 0.026 and 0.14 µg/mL, respectively).


Introduction
Malaria is one of the major global life-threatening diseases, especially among children and pregnant women [1]. Malaria is caused by five protozoan species of genus Plasmodium, called Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi [2]. The diversity and ability of P. falciparum to evade the host immune responses make it the most deadly form of malaria [2,3], and, therefore, the WHO recognizes the importance of malaria as a major cause of morbidity and mortality, particularly in sub-Saharan Africa [4]. P. falciparum spread is caused by Anopheles gambiae and An. Funestus, which are the highly prevalent mosquitoes [1]. The natural products application in therapeutic management against microorganism-caused diseases presents advantages that place them above drugs derived from synthetic sources. Owing to the comparably minimal side effects of these drugs, their toxicological and pharmacological activity, and likewise gastroenterology and bacteriology, revealed remarkable interest in the natural products' pharmacological activities against different infectious agents. Therefore, the natural products are capable of aiding the world health system in the discovery of new effective drugs to overcome the emergency of microbial resistance to available drugs [5,6].
Paeonia genus (family Paeoniaceae) consists of approximately of 33 species, which are mainly distributed in warm temperate regions of Europe and Asia, among them a total of
The root extracts of P. officinalis have been utilized in Indian and Chinese medicine for a long time due to their pharmacological effects, which include neuroprotection, anti- The antimalarial and antimicrobial evaluation of the identified compounds revealed the potency of compounds 3 and 5 against Plasmodium falciparum D6 with IC 50 values of 1.57 and 4.72 µg/mL and P. falciparum W2 with IC 50 values of 0.61 and 2.91 µg/mL, respectively, comparing with the standard chloroquine (IC 50 = 0.026 and 0.14 µg/mL, respectively), with no exhibition of antimicrobial activity.
The root extracts of P. officinalis have been utilized in Indian and Chinese medicine for a long time due to their pharmacological effects, which include neuroprotection, antihypertensive, and anti-ulcer [11]. Ethanol extracts of P. officinalis petals demonstrated antioxidant and antibacterial activities toward Staphylococcus aureus and Escherichia coli [23]. In the present study, fractions II and III showed antifungal activity against C. neoformans and antibacterial potential against P. aeruginosa and K. pneumoniae. The isolated compounds were inactive against the assayed microorganisms at the tested concentration.
In the antimalarial assays, fractions II and III demonstrated low activity against P. falciparum D6 and W2. Methyl gallate (3) and galloyl paeoniflorin (5) showed remarkable antiplasmodial activity against the P. falciparum D6 and W2 ( Table 2). The antimalarial potency of the isolated compounds, was attributable to the presence of the number of phenolic hydroxyl groups in the galloyl moiety, indicated its role in the inhibition of Plasmodium. Fractions II and III and the isolated compounds did not exhibit cytotoxicity toward mammalian kidney fibroblasts (Vero).
Thin layer chromatography (TLC) analyses were carried out using pre-coated silica G plates w/UV254 (Sorbent Technologies, Norcross, GA, USA; 20 × 20 cm, 200 µm in thickness). Ultraviolet lamp (UVP, LLC, Spectroline, Westbury, NY, USA) was used for visualization of spots on thin layer chromatograms at 254 and/or 365 nm. Spots were visualized by spraying with 2% vanillin-sulfuric acid in ethanol followed by heating at 110 • C on a hot plate.

Plant Material
The roots of P. officinalis were collected in January 2021 from public nurseries, Minia governorate, Egypt. Authentication of the plant was identified by Prof. Dr. Nasser Barakat, Department of Botany, Faculty of Science, Minia University, Minia, Egypt. A voucher specimen (Mn-ph-Cog-062) has been deposited in the Herbarium of Pharmacognosy Department, Faculty of Pharmacy, Minia University, Minia, Egypt.

Extraction and Isolation
The air-dried roots of P. officinalis (650 g) were powdered and extracted through maceration with 95% ethanol at room temperature and concentrated under reduced pressure, affording 60 g of solvent-free residue. The residue was suspended in 50 mL of distilled water to perform liquid-liquid fractionation, and then defatted with pet. ether, followed by partitioning with EtOAc (100 mL each × 6). The solvents were separately evaporated under vacuum, affording 4.0 g of pet. ether fraction and 25 g of EtOAc fraction. Finally, the remaining mother liquor was concentrated under reduced pressure to afford aqueous fraction.
The EtOAc fraction of P. Officinalis roots was fractionated by using VLC technique, in which it is eluted initially with DCM and then the polarity was increased gradually in 10% by MeOH until DCM-MeOH was 70:30. Each polarity was collected and concentrated under reduced pressure, affording four subfractions (E-1:E-4).

Evaluation of Antimicrobial Activity
The microorganism strains that were employed to test the antimicrobial activity were Candida albicans Pinh, C. neoformans Pinh, Aspergillus fumigatus Pinh, methicillin-resistant Staphylococcus aureus Pinh (MRS), E. coli Pinh, Pseudomonas aeruginosa Pinh, Klebsiella pneumoniae Pinh, and Vancomycin-resistant Enterococci (VRE) Pinh. The strains were bought from the American Type Culture Collection. A modified version of the CLSI methodology was used for the susceptibility testing. A final DMSO concentration of 1% was maintained in the assay by serially diluting all samples in 20% DMSO/saline and transferring them in duplicate to 384 well flat-bottom microplates. Following the McFarland standard, inocula were created by adjusting the OD 630 of microbe suspensions in incubation broth. C. albicans was cultured in RPMI 1640 broth (2% dextrose, 0.03% glutamine, and MOPS at pH 6); C. neoformans in Sabouraud Dextrose; MRS, VRE, E. coli, K. pneumoniae, and P. aeruginosa in cation-adjusted Mueller-Hinton at pH 7.3, and A. fumigatus in RPMI 1640 broth (2% dextrose, 0.03% glutamine, buffered with 0.165 M MOPS at pH 7). Five percent Alamar Blue was added in A. fumigatus, VRE, and MRS. In every assay, there were drug controls for bacteria and fungi. A. fumigatus and C. albicans were incubated at 35 • C for 48 h, while C. neoformans was cultured at 35 • C for 68 to 72 h. MRS, VRE, E. coli, K. pneumoniae, and P. aeruginosa were also incubated at this temperature for 18 to 24 h at 35 • C. A Bio-Tek plate reader was used to measure the optical density (530 nm) or fluorescence (544ex/590em) before and after incubation [24,25].

Evaluation of Antimalarial Activity
The assay is based on the measurement of plasmodial LDH activity. The test was conducted using a suspension of red blood cells infected with D6 or W2 strains of P. falciparum (200 µL, with 2% parasitemia, and 2% hematocrit in RPMI 1640 medium supplemented with 10% human serum and 60 µg/mL Amikacin) into the wells of a 96-well plate containing 10 µL of test samples diluted in medium at various concentrations. The plate was put in a modular incubation chamber (Billups-Rothenberg, San Diego, CA, USA); flushed with a gas mixture consisting of 90% N 2 , 5% O 2 , and 5% CO 2 ; and incubated for 72 h at 37 • C. Parasitic LDH activity was measured by utilizing Malstat reagent (Flow Inc., Portland, OR, USA). Briefly, 20 µL of the incubation mixture was combined with 100 µL of the Malstat reagent and incubated at room temperature for 30 min. Then, twenty microliters of a 1:1 mixture of NBT/PES (Sigma, St. Louis, MO, USA) was added and the plate was further incubated in the dark for 1 h. The reaction was then stopped by the addition of 100 µL of a 5% acetic acid solution. The plate was read at 650 nm using the EL-340 Biokinetics Reader (Bio-Tek Instruments, Winooski, VT, USA). The dose-response curves were used to calculate IC 50 values. Chloroquine was used in each experiment as the reference drug. Dimethyl sulfoxide (0.25%) was used as vehicle control [26].

Conclusions
The biological-based fractionation of P. officinalis roots resulted in the isolation and structure elucidation of seven compounds, including four monoterpene glycosides, two phenolics, and one sterol glycoside, in which compounds 3 and 5 showed antimalarial activity against Plasmodium falciparum D6 and P. falciparum W2. On the other hand, no other compound showed either antifungal or antibacterial activity. Accordingly, P. officinalis is considered to be a dietary supplement that exhibited antimicrobial and antimalarial activities with no apparent cytotoxicity against mammalian cells.