Medicinal and molecular profiling of selected rutaceous members with a focus on Citrus L

Abstract

From the earliest time, plants as a whole or their important parts have been utilized by humans for their well­being as well as the remedy for different ailments. In the recent era, the medicinal properties of plants are meticulously been investigated concerning their bioactivities and phytochemical constituents. Medicinal plants are the core of traditional medicine. Several medicinal plants have been identified, studied, and extensively employed for their therapeutic usage from the very beginning of civilization. More than 3000 plant species from 200 families were studied and identified to have medicinal properties in the region of the Eastern Himalayas, Western Ghats, and Andaman & Nicobar Island of India. Citrus is a genus of perennial shrubs and trees in the rue family, Rutaceae. The genus Citnis is native to East Asia, South Asia, Southeast Asia, Australia, and Melanesia. Numerous citrus species have beeo utilized and domesticated by indigenous cultures in these areas since time immemorial. From these areas its cultivation spread into Polynesia and Micronesia by the Austronesian expansion (c.3000-l 5 0 0 BCE); and t 0 the Mediterranean and the Middle East (c.1200 BCE) via the incense trade route, and onwards to the Americas and Europe. Moreover, the medicinal properties of citrus and other Rutaceous members are attributed to their Phyto-constituents which are believed to cure diverse ailments. Even though, ethnobotanically, they have various medicinal properties, their experimental validations are largely indecipherable. Thence, the present study was carried out to evaluate some of the medicinal properties and connected pharmacological potentialities of selected members of the Rutaceae, The elementary screening was done by assessing the qualitative, and qu antitative est imations and antimicrobial activities. Antioxidant activity was checked against the Human HEPG2 cell line. In vivo, Anti -inflammatory activity was studied in the carrageenan-induced mouse model, and in vitr o, An ti­inflammatory activity was checked on the hRBC membrane of the blood of the murine model. Nanoparticles were synthesized from selected Rutaceous members and their antimicrobial activity was carried out on gram­positive bacteria Staphylococcus aureus and gram-negative bacteria E. coli. Anti covid activity was detennined via in silica approach in Citms macroptera. A comprehensive phytochemical analysis was also executed to elucidate the major bioactive components in the genus citrus. In addition, a venture was made to access the genetic relationship among the IO species of Rutaceae members. ln the Qualitative assay, all the Rutaceous members respond more or less in all the different nine non-polar to polar solvents. All six members of Rutaceae exhibited potent results rn qualitative tests in the solvent of Methanol followed by Acetone, Ethanol, and Water. Among the six members ( C. unshiu, C. reshni, C. limon, C. macroptera, C. reticulata, and Z. budrunga) C. macroptera showed good result (in methanol) in the case of all the assays implemented here. The six members didn't respond well to Hexane, Benzene, and Chloroform extract. However, the Chloroform extract of citrus reshni gave a positive result in the benedict test. Chloroform extract of some varieties also gave positive results in coumarin, diterpene, steroid, and tannin tests. In the quantitative assay, all extracts showed the best result in methanol extract followed by acetone and ethanol extract. After that, all experiments were done in methanol extract. Among the six extracts, C. macroptera showed the highest amount of scavenging activity with a scavenging percentage of 90.61±0.1 followed by C. reshni which was 89.95±0.08, C. unshiu which was 86.95±0.08, scavenging percentage of Zanthoxylum budrunga was 86.42±0.1 and C. reticulata was 62.85±0.1 as compared to their standard ascorbic acid which was 95.77±0.1. In the Ferric reducing power assay C. macroptera showed the highest amount of reducing activity with an absorbance of 3.21±0.02 followed by C. reshni which was 2.34±0.1, Zanthoxylum budrunga 3.14±0.01, C. unshiu 2.44±0.002 and C. reticulata 2.23±0.004. The hydroxyl radical scavenging percentage in the case of Citrus reshni was found highest i.e. 66.88±0.8 followed by citrus macroptera which was 59.06±0.6, citrus unsbiu which was 55.68±0.5, Zanthoxylum which was 53.94±0.5 and Gl ycosmis pentaphylla 47.44±0.06. In case of NO radical scavenging assay, The highest scavenging percentage was seen m case of Citrus reshni (73.24±0.8) followed by Citrus macroptera (71.95 ±1.6), Citrus sinensis (63.96±1.8), Citrus reticulum (63.75±1.7) as compared to their standard Ascorbic acid (88.21± 1.0). Amoog the six extracts of Citrus, C. unshiu contained the highest phenolic, and C. macroptera contained the highest flavonoid. These contents deployed crucial antioxidant scavenging potentialities in all the plants which unveiled potent medicinal activity. To scrutinize the probable cytotoxic effects of experimental extracts, a good deal of evaluation was carried out. In brief, a significant percentage of erythrocyte membrane protection was detected in all extracts while a negligible amount of haemolytic effect towards human erythrocytes was observed in C. reshni, and C. sinensis extract indicating a non-detrimental effect on erythrocytes. Acute toxicity was also checked m case of C. macroptera extract. C. macroptera extract was orally administered and zero mortality was observed in the experimental animals at a 2000 mg/kg dose. Therefore, 1140th i.e. 50 mg/kg, and 1/10th i.e. 200 mg/kg of the maximum dose was contemplated safe for the in-vivo studies. Therefore, the ex tracts are we) I treated as consumable bio-safety stuft: making them acceptable for the preparation of various drugs involved in the treatment of various ailments. The human liver cancer cell line (Hep-02) were used to examine the effects of CML and Zanthoxylum budrunga under oxidative stress. H202 increases the oxidative stress levels in the liver tissue and based upon that study, it is hypothesized that H202 may induce the oxidative stress in Hep-G2 cells. After that, Hep-G2 cells were treated with H202 for 0-24 h, and intracellular oxidative levels were measured using the dichlorofluorescein assay. Oxidative tress is very much associated with increased levels of ROS production in living cells. H2DCFDA was used as an indicator to distinguish the production of intracellular ROS generation. H2DCFDA recognized hydrogen peroxide by exhibiting fluorescence on the liver cell line exposed to H202 • as it induced oxidative stress. The experimental results were completely following our hypothesis on the scavenging activity of CML and Zanthoxylum extracts and there were no harmful effects on the viability of cells. With these results, it can be concluded that increasing the production of hydrogen peroxide due to oxidation generates an excess amount of ROS and causes hepatic damage. Thus, it has been evident that CML and Zantboxyllum extracts possess both in-vivo and in-vitro antioxidant activity and might have effective functions to prevent different types of oxidative stress-related disorders. The methanolic extract of C. macroptera, C. reslmi, C. anshi11, C aurantifolia, Zmztho.rylum budrunga. and G(yco.mzis pe11caphylla showed antibacterial activity (zone of 1nh1bition, mm) against two pathogenic gram-positive bacteria Bacillus subtilis, Staphylococcus aureus and two pathogenic gram­negative bacteria Escherichia coli and Klebsie/la p11eumoniae at a concentration of 0.1, 0.25, 0.5 and 1.0 mg/ml. Among the six extracts, C. macroptera showed the highest inhibition zone followed by C. reshni. C.unshiu, and Z. budrunga. MIC value varied from 0.1 mg/ml to 0.5 mg/ml concentration. Among the five concentrations srudied here, CML extract showed MIC 0.5 mg/ml in the case of Staphylococcus aureus and 2 mg/ml in the case of Klebsiella pneumoniae. The anti-covid activity of C. macroptera was evaluated against long-haul covid complications via molecular docking and molecular simulation study. A total of six prospective compounds were selected for this study. Amidst them, three compounds namely Limonene, proximadiol, and Stigrnasterol showed the most promising results. Their physicochemical properties supported their candidature as drug molecules. Moreover, citrus plant parts contained everal flavonoids that might have to act synergistically with limonene, proximadiol, and stigmasterol to escalate their potency. Thus phenols and flavonoids along with the above­mentioned phytocompounds showed a combining effect where the whole crude extract of the plant can be more commanding than independent compounds. The COVID-19 condition was relatively new and so was the post -Covid scenario. In these situations, searching for a new interactive site on inflammation-related proteins will unlock a new direction to solve this riddle. ln this experiment, the active sites of the target proteins were already. It has been reported that NSAID aspirin interacts with ser530, Ile523, Argl 20, Leu533, Val509, and Tyr385 residues of COX-2; Ibuprofen interacts with Ala I 07, Glu 110, lso 111, Phe 176; However, the interacting amino acids of VCAM-1 and NMDAR with naproxen is not well characterized. As per a report, Piroxicam can hinder NMDAR and Ibuprofen can impede with VCAM-l. Hence blind docking ofNMDAR with Piroxicam and VCAM-1 with Ibuprofen both was performed. Both the results were further compared with the blind docking results of C. macroptera-derived Phyto­compounds. The elite binding energy scores for Cox-2 receptor were found to be -7.2 kcaVmol for Stigmasterol, - 7.0 kcal/mo! for proximadiol, -6.8 kcal/mo! for copaene and -6.6 kcal/ mol for Alpha gurjunene respectively. In the case of the NMDAR receptor, four compounds were found to display binding energy scores i.e. Proximadiol (-7 .1 kcal/mo I), Alpha gurjunene(-6.8 kcal/mo!), Limonene (-6.6 kcal/mol), and Copaene(-6.5 kcal/mol) respectively than the conventional drugs such as Aspirin (-5.0 kcal/mol), Piroxicam (-6. 9 kcal/mo I), fbuprofen (-5.2 kcal/mo]), and Naproxen (-4.5 kcal/mol). In the case of VCAM I, Alpha gurjunene, Copaene, and Limonene displayed the highest binding energy potential with scores of -6.5 kcal/mo!, -6.9 kcal/mol, and - 10.3 kcal/mol. Among the three phytocompounds, Limonene which had showed the highest negative binding potential among the total 79 studied phytocompounds was noticed to interact with the residues Ile 97(8) and Asn 49 (A). Besides it is a proven fact that d-Limonene exerts significant anti-inflammatory effects in dennal inflammation of rnurine. Lirnonene also exerts potent antiintlammatory effects mediating multiple pathways and conciliators of inflammation. including inhibition of adhesion molecules, pro-inflammatory cytokines, and chemokines along with the prevention of macrophage infiltration and neutrophil-endothelial cells interaction, which might establish a significant approach to neutralize the cytokine storm, which is generally a serious consequence of mortality seen in patients suffering from COVID-19. Nanotechnology is emerging as a rapidly growing field with its numerous application in science and Technology. The utilization of biological organisms such as plant extracts, and microorganisms could be a possible substitute for physical and chemical methods for the production of nanoparticles in an eco-friendly method. A green avenue for the rapid synthesis of silver and zinc nanoparticles employing an extract of wild orange (C. macroptera), Za nt hoxylum budrunga. and Glycosmis pentaphyl/a has been established in this study. The phytochemicals present in the above­mentioned plants act as both a reducing as well as a capping agent. The generation of silver nanoparticles synthesized from C. macroptera, Za nrhoxylu m budr unga , and G/ycosmis pentaphylla leaf extract was initially screened by the change in color of the reaction mixture. Structural analysis using UV, SEM, EDX, and XRD was also employed. Antimicrobial activity was also detem1ined against synthesized C. macropte ra-AgNPs and Zanthoxylum­A GNP s were found to show antimicrobial properties against Staphylococcus aureus, E. coli, and Bacillus subtilis. The plant(leaf) extract itself showed antimicrobial activity against tbe gram-positive strains and showed minimum activity against the gram-negative ones. Moreover, the antimicrobial activity was escalated with the formation of AgNPs (silver nanoparticles) and znNPs (zinc nanoparticles) using the C. mac roptera extract.