The tests of total phenolic content and flavonoids of crude extract of the roots of Paeonia algeriensis prove that there are medicinally active constituents. Furthermore, the CG/Ms analysis was used in order to investigate phytoconstituants contain in crude extract reveal the presence of three majors molecule constitute 88, 38% of the total detected molecules. Benzoic acids (42.70%) is a C6–C1 aromatic carboxylic acids that serve as precursors for a wide variety of essential compounds and natural products playing crucial roles in plant fitness [26]. 5-Hydroxymethylfurfural (HMF) is the second majors molecule (28.75%) a multifunctional molecule because it is at the same time an aromatic aldehyde, an aromatic alcohol and a furan ring system. HMF is a versatile intermediate that can be further transformed into a high value-added [27]. The compound 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) consists flavonoid fractions has received much attention for its antimicrobial activity [28]. It has been reported as a major contributor to the anticancer effects [29], mutagen antimicrobial, anti-inflammatory, and antioxidant capacity [30]. Others molecules at a low concentration were detected like Furaneol (1.0748%) a flavour compound, Propanoic acid, 2-oxo-, methyl ester or Pyruvic acid (1.1522%), an important organic acid, has been widely used in the food, agrochemical, and medicine fields [31] [32].
Antioxidants from natural products detoxify the toxins, removes excessive ROS, actively involves in anti-lipid peroxidation and scavenging of free radicals [33].The antioxidant activities of Crude extract of the roots of Paeonia algeriensis have been examined against DPPH radicals, the reducing power and lipid peroxidation tests. It might be connected to the chemical composition which includes bioactive phytochemicals.
The DPPH method was used for these studies because the DPPH free radical is stable and unaffected by certain side reactions of polyphenols, its direct measurement of inhibition, and simplicity and quick analysis [34] [35], in this study the obtained results indicate a powerful substance better than standard explained by the presence of flavonoids and polyphenols considered as a good source of antioxidant. Moreover HMF showed an important DPPH radical scavenging activities [36].
The antioxidant activity was further proved by measuring the reduction process of ferric (Fe+ 3) to ferrous (Fe+ 2) under the action of extracts that transformed the yellow colour of test solution to green [37]. Reducing power assay indicate an important results but still lower than standard due to the identified phytoconstituants like DDMP as a strong antioxidant [38] and HMF [36]. However, in case of Benzoic acids, Carboxyl group is an electron-withdrawing group, which does not benefit radical scavenging [39], may explain the low capacity to reduce (Fe+ 3) to ferrous (Fe+ 2).
Lipid peroxidation has been reported to be a marker of oxidative stress [40] [41]. It alters the organization of the membrane by inducing changes in fluidity and permeability [42]. Lipid peroxidation of biological membranes can cause alterations in fluidity, reductions in membrane potential, increased permeability to H+ and other ions, and eventual membrane rupture leading to release of cell and organelle contents. Cytotoxic aldehydes resulting from lipid peroxidation can block macrophage action, inhibit protein synthesis, inactivate enzymes, cross-link proteins, and can lead to the generation of thrombin [43]. In our study MDA concentration was dependent manner in standards where maximum of concentration reach 3,97 ± 0,11mM at 10 µg/mL then decrease to 1,20 ± 0,16 mM at 60 µg/mL. However, the extract show a value inferior to 1 mM whatever the concentration (maximum of 0,93 ± 0,03 mM*** at 20 µg/mL) indicate a powerful capacity to eliminate reactive molecules such as MDA and 4-hydroxynonenal (HNE). Oxidative processes related to lipid peroxidation might have occurred in parallel with a severe depletion on lipophilic antioxidants, mainly due to carotenoids [44], and others molecules related to phytochemicals detected by CG/Ms analysis.
The result of antimicrobial activity has shown strong activity against MRSA and Klebsiella pneumonia. Using agar disc-diffusion method, the extract was selective against both gram-positive bacteria and gram-negative bacteria. According to the 2019 CDC report, MRSA is one of the most common antibiotic-resistant bacterial pathogens and is estimated to cause more than 323,000 cases and 10,600 deaths annually in the United States alone [45]. MRSA often results from the horizontal acquisition of mecA gene by the methicillin-sensitive Staphylococcus aureus (MSSA). The gene is encoded for penicillin-binding protein (PBP2a) that confers resistance to all b-lactam antibiotics [46]. However, Klebsiella pneumonia is a gram-negative bacterium responsible for pneumonia, high morbidity and mortality due to excessive neutrophil and macrophage infiltration and severe lung injury [47]. The resistance of this bacterium to antibiotic is threatening to human health [48]. Due to the complex pathogenicity of the diseases, present vaccines and treatments available for Klebsiella infections are currently unsuccessful [49]. Hence, Paeonia algeriensis potentially acts against these two publicly dangerous strains by their phytoconstituents specially DDMP [28] [30].
The main action of anti-inflammatory agents is the inhibition of cyclooxygenase enzymes which are responsible for the conversion of arachidonic acid to prostaglandins. Since human red blood cell (HRBC) membranes are similar to these lysosomal membrane components, the prevention of Hypotonicity induced HRBC membrane lysis was taken as a measure in estimating the anti-inflammatory property of various extracts [50]. Thence, the studied extract prove an anti-inflammatory activity but no so powerful as the standard. The extract was concentration dependent manner until the concentration of 100 µg/mL, where only 13,09 ± 2,15%* of haemolysis was registered beyond which the extract become pro-inflammatory substance. This result is related to molecular composition of the extract, like 5-HMF’s which can modulate the innate immune responses through anti-inflammatory or anti-allergic effects [51] [52]. Further, DDMP reveal anti-inflammatory power [30]. Thus, may explain the antihaemolytic effect of the extract.
The In-vivo study of the healing wounds power of Paeonia algeriensis roots crude extract was performed to find the percentage of wound closure. The wound healing is a complex process that involves the synchronization and activation of coagulatory and inflammatory events, epithelialization, fibrous tissue accretion, deposition of collagen, wound contraction, tissue granulation and remodelling [53]. During healing, contraction plays a crucial role as it decreases the dimension of the wound and hence shortens the healing time. Moreover, contraction reduces the extracellular matrix amount needed to repair the defect and helps re-epithelisation by reducing the distance travelled by migrating keratinocytes [54]. Our results indicate a remarkable wounds contraction in control group during the first three days explained probably by animals’ saliva by liking. However, this process slow down during the period between 4 and 15 days of experiment. Healing process occurs by immunological activities of victim itself, but various risk factors such as infection and week immunity may cause delay in healing has brought attention to promote this process [55] [56]. Thus may explain the result of the control group.
Topical applications of the standard and Paeonia algeriensis ointments present an important results. In which, the extract show 100% of contraction after 13 days compared to the standard which still unhealed until the end. Applying the extract directly on the affected wound cannot bring the desired effect as it does not stay longer on the wounded skin of the experimental animals. Ointment is necessary to achieve a sustained drug release at the application sites [57]. In the present study, the phytochemical play an important role in healing process due to their antioxidant (powerful scavenging effect) and antimicrobial activities (bacterial mutagenic and anti-inflammatory effects of DDMP [30]). Moreover, the lipid peroxidation is an important process in several types of injuries like burns, infected wound, skin ulcer etc. Hence, any drug that inhibit lipid peroxidation is believed to increase the viability of collagen fibrils, which in turn results in an increase in the strength of collagen fibre by increasing the circulation, preventing the cell damage and promoting the DNA synthesis [58]. Thus, may explain the capacity Paeonia algeriensis roots crude extract to heal wounds.