Combinatorial Chemistry & High Throughput Screening - Volume 24, Issue 10, 2021

Background: Alzheimer’s disease is an irreversible, progressive brain disorder manifested with symptoms like loss of memory (known as dementia), personality changes, loss of cognition, impaired movement, confusion, deteriorated planning and thought process. Neurodegeneration in Alzheimer’s disease is the result of the deposition of protein beta-amyloid that forms plaques and another protein called tau, forming tangles that prevent the proper functioning of nerve cells in the brain. Methods: The goal of the review was to comprehensively study the utilization of nanotechnology and the role that carbon nanotubes can play as a drug delivery system for the amelioration of Alzheimer’s disease. Results: Nanotechnology is one of the most researched domains of modern science. It contributes significantly to therapeutics by facilitating drug therapy to reach the target sites, which are otherwise difficult to reach with conventional drug delivery systems. Carbon nanotubes are the allotropes of carbon in which several carbon atoms bind with each other to form a cylindrical or a tube-like structure. The carbon nanotubes possess several unique qualities, which confer them with a high potential of being utilized as an efficient drug delivery system. They offer high drug loading and can readily cross the toughest biological barriers like the BBB. Carbon nanotubes also facilitate the passage of drugs to the brain via the olfactory route, which further helps in restoring normal autophagy, thus preventing the elimination of autophagic chemicals. They can carry a vast range of cargos, including drugs, antigens, genetic materials, and biological macromolecules. Conclusion: Carbon nanotubes are a highly promising drug delivery system for anti-Alzheimer’s drugs. They have the potential of overcoming the various biological barriers like the BBB. However, more extensive research is required so as to set up a firm base for the development of advanced commercial products based on carbon nanotubes for the treatment of Alzheimer’s disease.

Neurodegenerative disorders are a result of continuous deterioration of the structure and function of neurons, involving the death of neurons. Neurodegenerative disorders affect millions of people around the world. Several conventional drug delivery strategies are available to treat neurological disorders. However, they are unable to provide adequate cytoarchitecture restoration and connection patterns due to lower solubility, poor bioavailability, drug resistance and incapability to traverse the blood-brain barrier (BBB). Therefore, designing and developing new and efficient delivery systems, that can bring drugs to CNS and have good bioavailability in the brain is very important. Nano drug delivery system acts as a ray of hope in the diagnosis and treatment of neurological disorders. The use of nanomedicines encapsulating therapeutic molecules may enhance transport of drug across BBB, absorption of drug and its controlled release in the human body with minimum adverse effects. This review article includes a comprehensive overview of the BBB, recent developments and application of nanomedicines, including liposomes, nanoparticles, nanomicelles, carbon nanotubes, etc., to the management as well as clinical therapy of various neurodegenerative disorders.

Background: Virus nanoparticles have been extensively studied over the past decades for theranostics applications. Viruses are well-characterized, naturally occurring nanoparticles that can be produced in high quantity with a high degree of similarity in both structure and composition. Objectives: The plant virus Cowpea Mosaic Virus (CPMV) has been innovatively used as a nanoscaffold. Utilization of the internal cavity of empty Virus-Like Particles (VLPs) for the inclusion of therapeutics within the capsid has opened many opportunities in drug delivery and imaging applications. Methods: The encapsidation of magnetic materials and anticancer drugs was achieved. ^SuperscriptCPMV denotes molecules attached to the external surface of CPMV and CPMVSubscript denotes molecules within the interior of the capsid. Results: Here, the generation of novel VLPs incorporating iron-platinum nanoparticles TCPMVFePt and cisplatin (Cis) (TCPMVCis) is reported. TCPMVCis exhibited a cytotoxic IC50 of TCPMVCis on both A549 and MDA-MB-231 cell lines of 1.8 μM and 3.9 μM, respectively after 72 hours of incubation. The TCPMVFePt were prepared as potential MRI contrast agents. Conclusion: Cisplatin loaded VLP (TCPMVCis) is shown to enhance cisplatin cytotoxicity in cancer cell lines with its potency increased by 2.3-folds.

Herbal medicines play an important role in treating various ailments due to their potentially high therapeutic values and acceptability by patients with different health complications. The practice of herbal medicine involves the use of a part of a plant, the entire plant, or a selective isolated phytoconstituent. The search for new drugs during the scientific era revived the interest in the discovery of herbal drugs from different natural resources. The present modern healthcare system involves the utilization of drugs and 50% of them are of natural origin. In recent years, there had been an upsurge in the interest for search of novel herbal drugs by the pharmaceutical industry. However, the discovery of such new novel phytomedicines involves various challenges in the identification of active constituents; their characterization, pharmacological activity, toxicity/adverse effects, drug interactions, and, most importantly, their regulatory requirements. The present review is mainly focused on the history of herbal medicine, current clinical perspective, and pharmaceutical and regulatory challenges facing herbal drugs. Moreover, problems encountered in drug discovery from herbal resources and their possible solutions have been delineated.

Sleep is considered as one of the most important aspects for maintaining a healthy life. For a person to function normally, at least 6-8 hours of sleep daily is necessary. Sleep not only affects our mood, but also regulates the efficiency of work done. Many complications arise due to inadequacy of sleep. The unhealthy food and lifestyle choices have made us more prone to sleep disorders. The medications used for the treatment of sleep disorders are mainly habit forming and have tendencies of withdrawal symptoms. This inadequacy in medication has lead to search for newer, better options. The field of nutraceuticals fits apt for treating such disorders. The quality of being non-toxic, non-habit forming, and being practically more efficient has had made it an excellent option. Nutraceuticals make use of food or part of food for the treatment or to prevent any disease. Remarkable positive effects of nutraceuticals like Caffeine, Chamomile, Kava kava, Cherries and Cherry juice, L tryptophan, Valerian, Vitamin D, Marijuana, melatonin, Lemon balm had been mentioned in the treatment of sleep disorders. The present review gives a general overview of nutraceuticals and discusses their use in sleep disorders.

Background: Lead (Pb) remains a common contaminant in the environment in many parts of the world. Pb exposure adversely affects many human organs, including the gonads, via oxidant and inflammatory marker propagation in affected tissues. Moringa oleifera leaf extract (MOE) is a rich source of antioxidants, reported to have robust anti-inflammatory properties. Aims: This investigation assessed whether MOE could mitigate testicular damage caused by Pb acetate treatment in rats. Methods: Four experimental groups were used: control animals (saline only), MOE (MOE only), PbAc (Pb acetate injection only), and MOE+PbAc. All treatments were administered for two weeks, after which animals were sacrificed, and tissues and serum were examined. To confirm the potential antioxidant effect of MOE, the total polyphenolic (TP) and flavonoid (TF) concentrations were determined. Results: The obtained results revealed that the TP concentration was 17.4 mg gallic acid equivalents per gram MOE dried weight and the TF concentration was 5.6 mg of quercetin equivalents per gram MOE dried weight. Moreover, MOE partially restored levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and testosterone and significantly attenuated oxidative stress biomarkers, malondialdehyde (MDA) and nitric oxide (NO), compared to levels observed in the PbAc-only group. MOE significantly increased the enzymatic and non-enzymatic antioxidant molecules superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), and glutathione (GSH). Testicular levels of inflammatory cytokines tumor necrosis factor-alpha (TNFα) and interleukin-1beta (IL-1β) were significantly decreased in MOE+PbAc compared to PbAc. MOE also significantly decreased pro-apoptotic Bax and caspase- 3 mRNA and protein levels and increased anti-apoptotic Bcl-2 mRNA and protein levels. Conclusion: MOE extract was associated with significant antioxidant, anti-inflammatory, and anti- apoptotic activity that ameliorated testicular damage induced by Pb acetate. MOE is proposed as a favorable adjuvant to existing treatments for Pb-induced toxicity.

Background: Eimeriosis is a parasitic intestinal infection that affects the poultry industry; it is responsible for economic losses on a global scale. Objective: This study investigated the protective role of Morus nigra leaf extracts (ME) against Eimeria papillata infection in mice. Methods: C57Bl/6 mice were divided into six groups. The first group was gavaged daily with 100 μL 0.9% NaCl. The second group was treated daily by oral gavage with 100 μL M. nigra leaf extracts (ME) (200 mg/kg), while the third, fourth, fifth, and sixth groups were orally infected with 1000 E. papillata oocysts. The last three groups were daily treated for 5 days with 200, 400, and 800 mg/kg of ME, respectively. Samples of jejunum were obtained for examining the histopathological changes as well as changes in the nitric oxide and catalase levels. Results: Infrared spectroscopy of ME showed the presence of several expected active classes of phytochemical compounds. ME was able to reduce the E. papillata oocysts in mice feces by about 80% and decrease the infection-induced jejunal histopathological injuries. This was quantified using the histological injury score. In addition, ME significantly recovered the changes in nitric oxide and catalase levels. Conclusion: Our findings showed that the M. nigra extract has an antioxidant activity which protects against murine eimeriosis. Further studies are required to determine the exact active compounds in ME and their mode of action.

Background: Hepatotoxicity remains an important clinical challenge. Hepatotoxicity observed in response to toxins and hazardous chemicals may be alleviated by delivery of the curcumin in silver nanoparticles (AgNPs-curcumin). In this study, we examined the impact of AgNPs-curcumin in a mouse model of carbon tetrachloride (CCl4)-induced hepatic injury. Methods: Male C57BL/6 mice were divided into three groups (n=8 per group). Mice in group 1 were treated with vehicle control alone, while mice in Group 2 received a single intraperitoneal injection of 1 ml/kg CCl4 in liquid paraffin (1:1 v/v). Mice in group 3 were treated with 2.5 mg/kg AgNPs-curcumin twice per week for three weeks after the CCl4 challenge. Results: Administration of CCL4 resulted in oxidative dysregulation, including significant reductions in reduced glutathione and concomitant elevations in the level of malondialdehyde (MDA). CCL4 challenge also resulted in elevated levels of serum aspartate transaminase (AST) and alanine transaminase (ALT); these findings were associated with the destruction of hepatic tissues. Treatment with AgNPs-curcumin prevented oxidative imbalance, hepatic dysfunction, and tissue destruction. A comet assay revealed that the CCl4 challenge resulted in significant DNA damage as documented by a 70% increase in nuclear DNA tail-length; treatment with AgNPscurcumin inhibited the CCL4-mediated increase in nuclear DNA tail-length by 34%. Conclusion: Administration of AgNPs-curcumin resulted in significant anti-oxidant activity in vivo. This agent has the potential to prevent hepatic tissue destruction and DNA damage that results from direct exposure to CCL4.

Objectives: The issue of food-additive-toxicity causing several health hazards needs to be therapeutically managed with an immediate effect. Alloxan, a food additive, is used for whitening and shining flour. It is capable of inducing genotoxicity, diabetes, and associated mitochondrial dysfunction. Therefore, to explore a non-toxic, phyto-based compound that can delay the onset of diabetes and prevent the multitude of damage associated, Chlorophyllin (CHL) was selected for our study, having been reported to exhibit anti-cancer, anti-diabetes, and antiinflammatory responses. Therefore, the objective of the present study is to evaluate the protective role of CHL in controlling genotoxicity, glucose imbalance, and associated cytochrome c mediated mitochondrial signaling dysfunction against food-additive-induced genotoxicity, diabetic state, and its complexities in mice model in vivo. Methods: Mice were pre-treated with CHL through oral gavage before they were exposed to alloxan. Diabetic markers, anti-oxidant enzyme profile, chromosomal study, mitochondrial functioning factors, and expression of proteins were checked against food-additive injected mice. Results: The results revealed that CHL pre-treatment could delay the onset of diabetes, restrict alloxan-induced elevation of blood glucose, reduce DNA-damage and chromosomal aberration, optimize enzymatic profile (glucokinase, pyruvate, insulin), and modulates protein expression (insulin, IRS1, IRS2, GLUT2). Further, CHL-pre-treatment could stabilize mitochondrial-membrane-potential, intracellular calcium ion, ATP/ADP ratio, ATPase activity, thereby maintaining optimum functioning of cytochrome-c, bcl2, and caspase3 mitochondrial protein. Conclusion: Therefore, the present study reports, for the first time, the screening of phytobased bioactive CHL for preventing/limiting the extent of food-additive-induced genotoxicity and mitochondrial dysfunction and serves as an advanced therapeutic tool in the management of diabetes.

Objective: The present investigation aimed to prepare metronidazole (MTZ) topical bigel for the effective delivery of MTZ and to study the effect of applied variables as per statistical design. The study also signifies the implementation of the statistical method using the Quality by Design technique for MTZ bigel. Methods: The MTZ bigels were prepared as per the runs suggested by Box Behnken design (BBD) using statistical software. A total of 28 runs were suggested by the BBD, considering sodium carboxymethylcellulose (Na CMC), guar gum, hydrogel and RPM as independent variables. The prepared bigels were evaluated for organoleptic properties, percentage drug content, spreadability, viscosity, percentage in-vitro drug release, and antimicrobial efficacy. Model selectivity was ascertained by p-value considering responses along with predicted R² and adjusted R² values.The fitting of model was ascertained by F-value as well as “lack of fit” was carried out to find out the suitability of the experimental design. Furthermore, the characteristic distribution of data was ascertained by the “normal plot of residual” method. The compatibility of MTZ and excipients in bigels was confirmed by FTIR and the crystalline nature of MTZ in formulations was studied by DSC and XRD studies. Furthermore, the dispersion of bigel was assessed by the SEM study. Results: The effect of independent variables on spreadability (mm), viscosity (cp), pH, drug release in 6 hours (%)and drug content (%) was evaluated. The optimized formulation was selected and evaluated by a polynomial equation while considering the p-value. These variables showed a significant effect on responses. A less significant difference was observed (6.37, 14463, 6.97, 86.29, and 67.47, respectively, for spreadability, viscosity, pH, and percentage drug release and % drug content) between the observed and predicted values indicating the model’s suitability. The prepared bigels were found to be compatible and globules uniformly dispersed throughout the bigel. Conclusion: The 3D response surface design ascertained the optimal MTZ bigel at 1.25g of NaCMC, 0.5g of guargum, 37.5g hydrogel, and 1000 RPM. The selected bigel showed good antimicrobial efficacy against S. Aureus and may be considered an effective delivery vehicle for MTZ.

Thymoquinone (TQ), the bioactive constituent of Nigella Sativa seeds, is a well-known natural compound for the management of several types of cancers. The anti-cancer properties of thymoquinone are thought to be operated via intervening with various oncogenic pathways, prevention of inflammation and oxidative stress, inhibition of angiogenesis and metastasis, and induction of apoptosis, as well as up-regulation and down-regulation of specific tumor suppressor genes and tumor promoting genes, respectively. The proliferation of various tumor cells is inhibited by TQ via induction of cell cycle arrest, disruption of the microtubule organization, and downregulating cell survival protein expression. TQ induces G1 phase cell cycle arrest in human breast cancer, colon cancer and osteosarcoma cells through inhibiting the activation of cyclin E or cyclin D and up-regulating p27 and p21, a cyclin dependent kinase (Cdk) inhibitor. TQ concentration is a significant factor in targeting a particular cell cycle phase. While high concentration of TQ induces G2 phase arrest in human breast cancer (MCF-7) cells, low concentration causes S phase arrest. This review article provides mechanistic insights into the anticancer properties of thymoquinone.

Background: Natural Phytoestrogens present in plants are effective hormonal replacement therapy. They are converted to estrogenic substances in the gastrointestinal tract, which is considered as the natural alternative to estrogen substitute treatment for postmenopausal women. Aims and Objective: Salvia officinalis, a herb traditionally used to ameliorate postmenopausal complications, can provide a safe alternative to synthetic pharmaceuticals for the treatment of menopause. Therefore, it is conceivable to detect the possible estrogenic effect of Salvia Officinalis extract as an estrogen replacement therapy in female mice. Methods: Phytochemical, pharmacological, and immune histopathological techniques are adopted in this study. HPLC is used for the identification of extracted constituents of sage herb. The uterotrophic activity of the extract was determined in immature female mice. Moreover, the mean thickness and luminal epithelium and the photomicrographs of the luminal epithelium of the uterus were also studied. Results: HPLC revealed that quercetin is the major extracted constituent (28.6%) of the total components. Saliva officinalis extract produced a significant increase in the uterine dry weight of equal potency to estrogen. The uterus exhibited a significant increase in luminal epithelial cell height (43.3 ± 6.1μm and 36.5 ± 2.5μm) for estradiol and sage extract, respectively, compared with the control group (18.2 ± 3.5μm). Furthermore, the endometrium showed the lining epithelium formed of a single layer of columnar epithelium. The stroma seemed more voluminous with dilated vasculature. Conversely, the myometrium within the uterus was not affected in any of the experimental groups. Conclusion: The sage herbs induced proliferative changes in the uteri of treated mice, which suggest possible estrogenic properties. Saliva officinalis extract can be used as a hormonal replacement for women during menopause and could be further explored for contraceptive use.

Aim and Objective: This study aimed at investigating the gastro-protective effects of Algerian Sahara (Sidr) honey from Apis mellifera intermissa against HCl/Ethanol-induced gastric ulcers in rats. Materials and Methods: Total phenolic content, antioxidant activity and phenolic compounds were determined. Then, three groups of rats (control, HCl/ Ethanol-induced ulcer, and orally administered honey) were used for the determination of gastro-protective effect of Sidr honey. Results: Total phenolic content, total flavonoid content, and DPPH activity of the honey sample were determined as 47.35±3.35 mg GAE/ 100 g, 2.13±0.17 mg QE/ 100 g, and 229.24±0.02 mg/mL, respectively. Oral pretreatment of rats with honey (1.2 g/Kg body weight orally at an interval of 2 days) protected gastric mucosa against HCl/Ethanol-induced damage by decreasing ulcer score, the volume and acidity of gastric juice and increasing pH. Conclusion: These results were confirmed by the histological assessment, which demonstrated a significant gastro-protective activity of Saharian (Sidr) honey against HCl/Ethanol-induced stomach ulcer. Plasma tumor necrosis factor-α, IL-6 and PGE2 were also measured. Sahara honey significantly decreased the plasma TNF-α, PGE2, and IL-6 concentrations.

Aim and Objective: Currently, the use of ingredients from natural sources has gained great attention in the cosmetic field, especially for the development of new photoprotective formulations. Therefore, the present study aimed to evaluate the cosmetic potential of the crude methanol extract and the ethyl acetate fraction of the medicinal halophyte Tamarix gallica L. (Tg) growing in the area of Tebessa in the eastern part of Algeria, by assessing their phenolic and flavonoid contents, photoprotective and antioxidant activities. Methods: The research approach consisted of determining phenolic and flavonoid contents of aerial parts via Folin-Ciocalteu and aluminum chloride methods, respectively. The antioxidant activity was measured through two in vitro methods, DPPH radical scavenging activity and total antioxidant capacity test (TAC). The in vitro photoprotective effect was evaluated according to the parameter SPF (Sun Protection Factor) by using the UV spectroscopic method in the UV-B region (290-320 nm). Results: The methanol extract (Tg-MeOH) and ethyl acetate (Tg-EtOAc) fraction showed good antioxidant activity with IC50 values of 14.05±0.66, 27.58±1.98 μg/mL respectively in the DPPH test. Furthermore, both extracts displayed strong total antioxidant capacity (287.01±7.85, 246.7±1.12 mg AAE/g, respectively) in the TAC test. Both extracts exhibited high photoprotective activity, with sun protection factor (SPF) values 37.03±0.22 and 36.08±0.03. The antioxidant and photoprotective activities of these extracts were probably related to polyphenols content (190.27±0.74 mg AGE /g and 121.77±1.29 mg AGE /g, respectively) and flavonoids (78.75±0.06 mg QE /g and 58.67±1.19mg/g). Conclusion: Our findings show that extracts of Tamarix gallica L. could be a promising source to be mixed as a natural sunscreen and antioxidant agents into photoprotective cosmetic formulations.

Background: Propolis is a natural resinous material produced by honeybees. The biological activity and phenolic profile of propolis were largely studied all over the world. However, only a few investigations have been carried out on Algerian and Turkish propolis. The aim of the present study was to compare the phenolic content, antioxidant, and antibacterial activity of propolis samples collected from different localities of Algeria and Turkey. Methods: Propolis extracts were performed using maceration in ethanol 80%. Total phenolic and flavonoid contents were determined. Antioxidant and antibacterial activities were evaluated using FRAP assay and the MIC was determined against four bacterial strains (S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853 and K. pneumonia). Results: TP varied from 19.51 ± 0.86 to 219.66 ± 1.23 mg GAE/g. Whereas, TF varied from 5.27± 0.07 to 74.57 ± 1.03 QE/g. All samples showed good ferric reducing antioxidant power ranging from 267.30 ± 4.77 to 2387.30 ± 44.15 μmol Trolox eq./g. All Algerian propolis samples displayed a more pronounced activity against S. aureus ATCC 25923 with MIC values ranging from 0,04 ± 0.00 mg/mL to 0.30±0.06 mg/Ml, with an activity 30 times more powerful than Anatolian propolis. While, Anatolian propolis samples were most active against P. aeruginosa ATCC 27853 with MIC values ranging from 0.20±0.00 mg/mL to 0.60±0.00 mg/Ml, with an activity 5 to 10 times more powerful than Algerian propolis. Conclusion: Algerian and Anatolian propolis possessed considerable phenolic and flavonoids contents. In addition, they exhibited interesting antioxidant and antibacterial activities. Our findings suggest that both propolis could be useful in the food and pharmaceutical industries.

Background: Pollen and propolis are two bee products with highly health promoting properties. But there are some limitations of raw propolis usage not only in daily consumption but also in putting it in food formulations. Propolis should be extracted to convert it into consumable form and ethanol is the first choice as a solvent. But ethanol consumption, either in health-wise or religious aspect, is one of the factors limiting the usage of propolis extract. The strong taste and strong smell of propolis are other factors. The immobilization of propolis active compounds could be a tool for overcoming either all or some of these factors. Objective: This study was aimed at the immobilization of propolis active constituents on the surface of whole pollen grains. Methods: Chemical composition of raw propolis was determined by using GC-MS technique. Total phenolic content and antioxidant activity of the samples were measured spectrophotometrically. The release property of the beads was determined. Results: Immobilization efficiency was 53%. Total phenolic and flavonoid content of pollen, propolis and pollen-propolis beads were measured. It was determined that pollen-propolis beads contain more phenolics than pollen and propolis itself. Ferric reducing activity of the samples was also investigated and pollen-propolis beads showed better activity. Release behavior of pollen and pollen-propolis beads was studied in simulated digestive systems. Better release properties of pollen-propolis beads were achieved in all tested systems as well. These findings support the immobilization of propolis active compounds on the surface of whole pollen grains. Conclusion: It could be concluded that the product obtained, pollen-propolis beads, could be considered as a more valuable healthy product since the synergistic action of pollen and propolis.

The appropriate selection of initial receptor structure has been the "cornerstone" or foundation of successful structure-based virtual screening (SBVS), and plagued the structure-based design with a significant practical problem to determine the major physiological states or important transition states of receptors (e.g. proteins with multiple low-energy conformations and liganddependent conformational dynamics). It is well known that current SBVS methods lack the capacity to capture and characterize the intrinsic receptor flexibility with ideal cost-effectiveness. In recent years, cryoelectron microscopy (cryo-EM) has been routinely applied in the determination of biomolecular assemblies within the physiological state. In this work, we review the roles of cryo-EM and ensemble docking methods to present the intrinsically dynamic behavior of biomacromolecules, as well as the ever-improving estimation of ligand binding affinities and receptor-ligand thermodynamics. Finally, we also provide a viewpoint for further research works on modeling receptor dynamics.

Background: 1,8-diaminonaphthalen (1,8-DAN) with special organic structure was applied in organic synthesis to provide efficient complex scaffolds, through the two or fourcomponent fashion. This review highlights its recent application in organic reactions under different conditions and heterogynous catalysts to produce various molecules, which were used as medicines, sensors, and dyes. Objective: 1,8-diaminonaphthalene (1,8-DAN) is a bicyclic compound with two amino groups which has received much attention in organic chemistry due to their medicinal activities such as antifungal, antimicrobial, antiulcer, antitumor, oxidation dyestuff, hair color, fluorescent, and chemo-sensors. In continuation of our studies, herin, recent application of 1,8-DAN in organic synthesis was reviewed. Conclusion: In conclusion, the application of 1,8-DAN 1 in different reactions was reviewd through the various conditions to yield the target compounds with high medicinal activities, and potential for sensitive detection of different ions. The target compounds including 1,8-DAN 1 with various structures were provided such as perimidines, perimidinones, aminonaphthalenes which were produced through different methods as brought for further study in this review.