Exploring in vitro and in silico Biological Activities of Calligonum Comosum and Rumex Vesicarius: Implications on Anticancer and Antibacterial Therapeutics.

Introduction: The adverse effects of clinically used anti-cancer medication and the rise in resistive micro-organisms have limited therapeutic options. Multiple anti-cancer drugs are derived from medicinal herbs which also have shown anti-bacterial effects. This study aimed to identify the optimal extraction solvent for detecting the cytotoxic and anti-bacterial effects of Calligonum comosum (C. Comosum) and Rumex vesicarius (R. Vesicarius) extracts. Additionally, the study aimed to identify active metabolites and assess their potential as future drug candidates for anti-cancer and anti-bacterial therapeutics. Methods: Leaves from both plants were extracted using ethanol, ethyl acetate, chloroform, and water. The cytotoxic effects of the extracts were tested on liver, colon, and breast cancer cell lines. Apoptosis was assessed using High Content Imaging (HCI) and the ApoTox triplex Glo assay. The anti-bacterial effects were determined using agar-well diffusion. Liquid chromatography-mass spectrometry (LC-MS) was used to tentatively identify the secondary metabolites. In silico computational studies were conducted to determine the metabolites' mode of action, safety, and pharmacokinetic properties. Results: The ethanolic extract of C. Comosum exhibited potent cytotoxicity on breast cancer cell lines, with IC50 values of 54.97 µg/mL and 58 µg/mL for KAIMRC2 and MDA-MB-231, respectively. It also induced apoptosis in colon and breast cancer cell lines. All tested extracts of C. Comosum and R. Vesicarius demonstrated anti-bacterial activity against Staphylococcus aureus and Escherichia coli. Seven active metabolites were identified, one of which is Kaempferol 3-O-Glucoside-7-O-Rhamnoside, which showed strong (predicted) anti-cancer activity. Kaempferol 3-O-Glucoside-7-O-Rhamnoside and Quercetin-3-O-Glucuronide also exhibited potential anti-bacterial effects on gram-positive and negative bacteria. Conclusion: Ethanol extraction of C. Comosum solubilizes active metabolites with potential therapeutic applications in cancer treatment and bacterial infections. Kaempferol 3-O-Glucoside-7-O-Rhamnoside, in particular, shows promise as a dual therapeutic drug candidate for further research and development to improve its efficacy, safety, and pharmacokinetic profile.

Anti-ulcerative colitis effect of Calligonum comosum L'Hér. using rat model: Chemical, pharmacological and histopathological evidences.

Calligonum comosum is a perennial shrub growing and widely used in traditional medicinal system in Saudi Arabia. The total phenolic content and in vitro antioxidant activity were compared between the water extract (WE) and methanol extract (ME). The protective potential against acetic acid (AA) induced ulcerative colitis (UC) was also evaluated in rats. The obtained results showed that the total phenolic content of the WE and ME were 8.378 ± 0.738 and 33.819 ± 0.488 µg/mL. The antioxidant properties of the two extracts were directly influenced by their total phenolic contents. The ME with higher phenolic contents and stronger antioxidant power was more effective than the WE in protection against AA-induced colitis. Phytochemical study of the ME led to the identification of three flavonoid derivatives: (-)-epi-catechin, quercetin-3-O-α-l-arabinofuranoside (Avicularin) and quercetin-3-O-ß-d-glucuronide-6″-methyl ester by various spectroscopic methods. (-)-Epi-catechin was the major component while the other two compounds were obtained in minute quantities. The anti-ulcerative colitis effect of the ME can be explained by the presence of the antioxidant flavonoids since AA-induced colitis featured by imbalance between oxidant and antioxidant substances. Further support of such explanation was provided by HPLC quantification of (-)-epi-catechin in the ME and WE. The percentage in ME was higher than the WE but the difference was higher in term of Total Phenolic Content (TPC). These results support the traditional use of C. comosum as anti-ulcerative colitis.

Protective effect of Calligonum comosum on haloperidol-induced oxidative stress in rat.

The aqueous and methanolic extracts of Calligonum comosum were investigated for their antioxidant and dopaminergic effects on haloperidol (HL)-induced neuro- and hepatotoxicities in male albino rat model. The total phenolics, flavonoid content and free radical-scavenging activity of the extracts were determined. The results showed that the antioxidant activity of the methanolic extract was higher than the aqueous one. HL significantly reduced GSH and increased MDA in brain and liver tissues. These values were nearly normalized, in the examined tissues, on concomitant administration of C. comosum methanolic extract with HL. Superoxide dismutase activity in the examined tissues was significantly decreased by HL administration that was normalized by the coadministration of the methanolic extract and, to a less extent, the water extract. Determination of the brain neurotransmitter contents revealed a marked decrease in norepinephrine, dopamine and serotonin, which were restored to near control values by concomitant administration of both C. comosum extracts with HL. The results of this study showed that C. comosum methanolic and aqueous extracts ameliorated HL-induced neuro- and hepatotoxicities in rats.

Apoptosis-mediated anti-proliferative activity of Calligonum comosum against human breast cancer cells, and molecular docking of its major polyphenolics to Caspase-3.

Due to poor diagnosis breast cancer in women has emerged as the most common cause of death disease in developing countries. Medicinal plants have been used for thousands of years and can be useful in healthcare, especially in developing countries. Ethanol extracts of leaves of fire bush or arta (Calligonum comosum; EECC), exhibited significant anticancer potencies against two breast cancer cell lines, MCF-7 and MDA 231. These in vitro effects of EECC indicated potential anticancer activities that were determined to be specific since minimal toxicity was recorded against MCF-12, a non-cancerous breast cell line used as a reference. EECC also induced cell cycle arrest in MCF-7 and MDA 231 as revealed by the increased proportions of sub-G1 cells. Fluorescence-activated cell sorter analysis (FACS), utilizing double staining by annexin V-FITC/propidium iodide, revealed that the observed cytotoxic effects were mediated via apoptosis and necrosis. FACS measurement of thegreater in fluorescence intensity, linked with oxidation of DCFH to DCF, revealed that apoptosis was attributable to production of free radicals. EECC-mediated apoptosis was further validated by observation of up-regulation in the "executioner" enzyme, caspase 3. The current findings reveal that EECC exhibits significant, selective cytotoxicity to breast cancer cells, that proceeds via the generation of ROS, which culminates in apoptosis. The anti-proliferative effects of EECC weres further verified by use of a structure-based, virtual screening between its major bioactive polyphenolic constituents and the apoptosis executioner marker enzyme, caspase-3. Based on their glide score values against the active site of caspase 3, some phyto-constituents present in EECC, such as DL-alpha-tocopherol and campesterol, exhibited distinctive, drug-like potential with no predicted toxicity to non-target cells. Taken together, the usefulness of natural phenolic and flavonoid compounds contained in Calligonum comosum were suggested to be potent anticancer agents.

Potent antioxidant and anticancer activities of the methanolic extract of Calligonum comosum (L'Her) fruit hairs against human hepatocarcinoma cells.

Cancer is a devastating and aggressive disease that is globally ranked as the second-leading cause of deaths despite the relentless efforts being directed towards the discovery of novel chemotherapeutic drugs. Plants naturally produce a plethora of secondary metabolites that play a crucial role as effective therapeutic agents. Cancer treatment rely primarily on chemo- and radio-therapeutic strategies that suffers from known side effects. Recently, the strategy of controlling cancer progression by use of plant-derived natural products have extensively attracted research interests. In this study, the antioxidant and anticancer activities of the methanolic extract of Calligonum comosum (MeCc) fruit hairs were investigated. According to DPPH and ABTS assays, MeCc exhibited potent antioxidant capacity as it displayed significant free-radical scavenging activity. Results of the MTT cytotoxicity assay revealed that the MeCc exhibited potent anti-proliferation activity (IC50 = 10.4 µg/ml) that is specific against human hepatocarcinoma cells (HepG2), as only marginally harmful effect against non-cancerous control BJ-1 cells was detected. Results of the RT-qPCR gene expression analyses indicated that MeCc resulted in significant overexpression of mRNA transcript levels of the pro-apoptotic genes p53, caspase-3 and Bax, while downregulating the level of Bcl-2, an anti-apoptotic marker gene. Immunoblotting of the protein expression levels for the same markers showed similar pattern to that observed in RT-qPCR profiling. While the levels of p53, caspase-3 and Bax proteins exhibited significant increase, the protein level of Bcl-2 was significantly reduced. In conclusion, it is proposed that the observed specific anticancer activity of MeCc against HepG2 cells takes place via the engagement of apoptosis. This highlights the value of C. comosum as a source of potent natural anticancer agents and warrants further investigation to identify the active principals involved.

Critical discovery and synthesis of novel antibacterial and resistance-modifying agents inspired by plant phytochemical defense mechanisms.

Antimicrobial resistance is at increasing risk worldwide since it is threatening the ability to control common infectious diseases, resulting in prolonged illness, disability, and death. Herein, we inspired by the effective plant phytochemical mechanisms evolved to overcome microbial pathogenesis and evolved resistance. Cuminaldehyde is previously reported as the main antibacterial component in Calligonum comosum essential oil. The toxicity of cuminaldehyde limits its medical application for human use. On the other hand, compared to cuminaldehyde, the plant total extract showed similar antibacterial activities, while maintained lower toxicity, although it contains 22 times less cuminaldehyde. Thus, we assumed that other components in the plant extracts specifically affect bacteria but not mammalian cells. Bioassay-guided fractionations combined with comparative metabolomics analysis of different plant extracts were employed. The results revealed the presence of bacterial species-specific phytochemicals. Cinnamyl linoleate and linoleic acid enhanced the antibacterial activities of cuminaldehyde and ampicillin against S. aureus including MRSA, while decanal and cinnamyl linoleate enhanced the activities against E. coli. Computational modeling and enzyme inhibition assays indicated that cinnamyl linoleate selectively bind to bacterial ribosomal RNA methyltransferase, an important enzyme involved in the virulence and resistance of multidrug resistant bacteria. The results obtained can be employed for the future preparation of pharmaceutical formula containing cinnamyl linoleate in order to overcome evolved multidrug resistance behaviors by microbes.

Macro and micro-elements concentrations in Calligonum comosum wild grazing plant through its growth period.

In this study, the change in the content of the macro and micro elements in the growing wild grazing plant of Calligonum comosum was tracked at the Research and Training Station of King Faisal University in Al-Hassa Governorate, Kingdom of Saudi Arabia. Mineral elements were estimated in aerial parts (plant as a whole, leaves and stem) from January-April 2020. The results showed that the concentration of nitrogen, phosphorus and potassium in the plant as a whole plant > leaves > roots, while the concentrations of calcium, magnesium, manganese, zinc and copper elements in the leaves was higher than other parts whereas the concentrations of these elements of whole plant were higher than the concentrations in roots. The results showed that the plant contents of nitrogen, potassium and zinc were the highest in March, while the concentrations of phosphorus, calcium, iron and copper were in February. The concentrations of magnesium, manganese and copper was the highest in January and April respectively. The values ​​of nitrogen, phosphorous, potassium, calcium, magnesium, iron, manganese, zinc and copper ranged from 11.1 to 18.4 g kg-1, 4.17-2.33 g kg-1, 13.73-18.97 g kg-1, 24.50-28.90 g kg-1, 10.40-12.30 gkg-1, 1500-1677 mg kg-1, 45.45-49.29 mg kg-1, 70.70-177.23 mg kg-1, 16.78-73.46 mg kg-1, respectively. Furthermore, the results exhibited that the lowest values of the elements appeared in the plant roots in April. As well as, the distribution of the elements followed the normal life curve from January to April. Besides that, the evaluated elements satisfy the needs of the grazing animals' life in which this type of plant grows.

Biogenic synthesis of silver nanoparticles: Antibacterial and cytotoxic potential.

In green chemistry, the application of a biogenic material as a mediator in nanoparticles formation is an innovative nanotechnology. Our current investigation aimed at testing the cytotoxic potential and antimicrobial ability of silver nanoparticles (AgNPs) that were prepared using Calligonum comosum roots and Azadirachta indica leaf extracts as stabilizing and reducing agents. An agar well diffusion technique was employed to detect synthesized AgNPs antibacterial ability on Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus bacterial strains. Furthermore, their cytotoxic capability against LoVo, MDA-MB231 and HepG2 ca cells was investigated. For phyto-chemical detection in the biogenic AgNPs the Fourier-transform infrared spectroscopy (FT-IR) was considered. Zeta sizer, TEM (Transmission Electron Microscope) and FE-SEM (Field Emission Scanning Electron Microscope) were used to detect biogenic AgNPs' size and morphology. The current results showed the capability of tested plant extract for conversion of Ag ions to AgNPs with a mean size ranging between 90.8 ± 0.8 and 183.2 ± 0.7 nm in diameter. Furthermore, prepared AgNPs exhibited apoptotic potential against HepG2, LoVo, and MDA-MB 231cell with IC50 ranging between 10.9 and 21.4 µg/ml and antibacterial ability in the range of 16.0 ± 0.1 to 22.0 ± 1.8 mm diameter. Activation of caspases in AgNPs treated cells could be the main indicator for their positive effect causing apoptosis. The current investigation suggested that the green production of AgNPs could be a suitable substitute to large-scale production of AgNPs, since stable and active nanoparticles could be obtained.

Anti-Proliferative and Pro-Apoptotic Effects of Calligonum comosum (L'Her.) Methanolic Extract in Human Triple-Negative MDA-MB-231 Breast Cancer Cells.

Triple-negative breast cancer (TNBC), the most aggressive subtype, does not respond to targeted therapy due to the lack of hormone receptors. There is an urgent need for alternative therapies, including natural product-based anti-cancer drugs, at lower cost. We investigated the impact of a Calligonum comosum L'Hér. methanolic extract (CcME) on the TNBC MDA-MB-231 cell line proliferation and related cell death mechanisms performing cell viability and cytotoxicity assays, flow cytometry to detect apoptosis and cell cycle analysis. The apoptosis-related protein array and cellular reactive oxygen species (ROS) assay were also carried out. We showed that the CcME inhibited the TNBC cell viability, in a dose-dependent manner, with low cytotoxic effects. The CcME-treated TNBC cells underwent apoptosis, associated with a concomitant increase of apoptosis-related protein expression, including cytochrome c, cleaved caspase-3, cyclin-dependent kinase inhibitor p21, and the anti-oxidant enzyme catalase, compared with the untreated cells. The CcME also enhanced the mitochondrial transition pore opening activity and induced G0/G1 cell growth arrest, which confirmed the cytochrome c release and the increase of the p21 expression detected in the CcME-treated TNBC cells. The CcME-treated TNBC cells resulted in intracellular ROS production, which, when blocked with a ROS scavenger, did not reduce the CcME-induced apoptosis. In conclusion, CcME exerts anti-proliferative effects against TNBC cells through the induction of apoptosis and cell growth arrest. In vivo studies are justified to verify the CcME anti-proliferative activities and to investigate any potential anti-metastatic activities of CcME against TNBC development and progression.

Calligonum comosum (Escanbil) extract exerts anti-angiogenic, anti-proliferative and anti-inflammatory effects on endometriotic lesions.

ETHNOPHARMACOLOGICAL RELEVANCE: Calligonum comosum is a desert plant that is applied in traditional folkloric medicine for the treatment of abnormally heavy or prolonged menstruation and menstrual cramps. Moreover, it has been suggested for the treatment of infertility-causing conditions. Its bioactive chemical constituents inhibit multiple processes, such as angiogenesis, inflammation and invasive tissue growth, which may be beneficial in the therapy of endometriosis. AIM OF THE STUDY: We investigated the effects of Calligonum comosum on the development of endometriotic lesions. MATERIALS AND METHODS: We evaluated the anti-angiogenic activity of Calligonum comosum ethyl acetate fraction (CCEAF) in different in vitro angiogenesis assays. Moreover, we surgically induced endometriotic lesions in BALB/c mice, which received 50 mg/kg Calligonum comosum total extract (CCTE) or vehicle (control) over 4 weeks. The growth, cyst formation, vascularization and immune cell infiltration of the lesions were assessed with high-resolution ultrasound imaging, caliper measurements, histology and immunohistochemistry. RESULTS: CCEAF doses of up to 10 µg/mL did not impair the viability of human dermal microvascular endothelial cells (HDMEC), but dose-dependently suppressed their migration, tube formation and sprouting, indicating a substantial anti-angiogenic effect of CCEAF. Furthermore, CCTE significantly inhibited the growth and cyst formation of developing murine endometriotic lesions when compared to vehicle-treated controls. This was associated with a reduced vascularization, cell proliferation and immune cell infiltration. CONCLUSIONS: Our findings show that Calligonum comosum targets multiple, fundamental processes in the pathogenesis of endometriosis, which may be beneficial for the treatment of this common gynecological disorder.

Calligonum comosum extract inhibits diethylnitrosamine-induced hepatocarcinogenesis in rats.

Calligonum comosum (C. comosum) is an Egyptian desert plant that contains polyphenol antioxidants. The present study examined the chemopreventive effect of an extract of C. comosum in a rat model of hepatocarcinogenesis. Male Wistar rats (n=40) were administered 100 mg/kg diethylnitrosamine (DEN) by intraperitoneal (i.p.) injection once a week for 3 weeks. Subsequently, depending on whether the rats received further administration of 0.8 mg/kg carbon tetrachloride (CCl4) i.p. once a week for 7 weeks and 100 mg/kg C. comosum extract in their diet for 7 weeks, the rats were divided into four groups as follows: Group 1, treatment with DEN alone; group 2, treatment with DEN and C. comosum extract; group 3, treatment with DEN and CCl4; and group 4, treatment with DEN, CCl4 and C. comosum extract. The supplementation of C. comosum extract significantly suppressed the elevation in serum liver enzyme levels, including aspartate aminotransferase, alanine transaminase and γ-glutamyl transferase, and reduced the degree of oval cell proliferation induced by DEN and CCl4. In addition, C. comosum extract significantly decreased the number and area of glutathione S-transferase placental form-positive preneoplastic hepatic foci induced by DEN, with or without CCl4 treatment. To the best of our knowledge, the present study is the first to provide definitive evidence of the hepatoprotective and chemopreventive effects of C. comosum.

Detection and extraction of anti-Listerial compounds from Calligonum comosum, a medicinal plant from arid regions of Tunisia.

Calligonum comosum, a Tunisian plant from arid regions, is traditionally used in folk medicine to treat rural population microbial infections. The plant was investigated in vitro for its ability to inhibit the growth of Listeria ivanovii. Various aqueous and organic extracts were prepared from different plant tissues. Results indicated that ethanolic, methanolic and acetonic extracts from whole plant tissues except seeds, exhibited significant antibacterial activity with growth inhibition zones (9 - 18mm) as shown by the agar-well diffusion method. Minimum Inhibitory Concentration (MIC) of 0.65mg/ml was obtained in acetonic extract generated from C. comosum roots. Preliminary phytochemical analysis based on heat and protease treatments showed that bioactive extracts were stable up to 10m in heating at 100°C and that they resist protease digestion. Based on these latter results, the activity of organic extracts may be related to the presence of sterols, terpenoids, and/or phenolics. Overall, these results indicate that C. comosum organic extracts are probably useful in the control of food contamination by listerial species.