Combinatorial Chemistry & High Throughput Screening - Volume 24, Issue 8, 2021

Background and Objective: Respiratory Syncytial Virus (RSV) is the leading cause of infant lower respiratory tract infections with no mature vaccines and medicines available. Pneumonia caused by RSV kills many infants every year. There are unique advantages of Traditional Chinese Medicine (TCM) to fight against the virus. Houttuynia cordata Thunb is a commonly used antivirus medicine in TCM, but its mechanism has not been investigated. The current study explores the anti-RSV mechanism of H. cordata Thunb by means of network pharmacology and bioinformatics. Methods: The candidate compounds of H. cordata Thunb and the potential targets were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), PubMed, CNKI, PubChem Database, and Swiss Target Prediction database. Then the potential targets and pathways of H. cordata Thunb against RSV were screened by GeneCards, GenCLiP 3, and NCBI Database. We developed a Protein-Protein Interactions (PPI) Network and Compound-Target-Pathway Network through the Cytoscape software. Furthermore, core targets were preliminary verified by Gene Expression Omnibus (GEO) database by bioinformatics methods. At last, the first six pathways were screened out to draw a map of the main target signal pathways. Results: A total of 12 potentially active compounds and 47 potential interaction targets were screened. PPI Network and data from GEO showed that IL-6, STAT3, TNF, AKT1, PTGS2, SRC, and MAPK3 may play a core role in the antivirus process. KEGG enrichment pathway analysis predicted that H. cordata Thunb exerted its anti-RSV effect by regulating TNF, Rap1, HIF-1, PI3KAkt, MAPK, and VEGF signaling pathways. Conclusion: This study preliminarily predicted the main active compounds, targets and related pathways of H. cordata Thunb in the treatment of RSV-induced diseases, which laid a good foundation for further revealing its mechanism.

Background: Osteoarthritis (OA) is a degenerative joint disease that seriously affects the quality of life of elderly. Regrettably, the pathological mechanism for OA has not yet been fully elucidated. Methods: This study is committed to distinguishing key genes and the underlying mechanisms for OA. Raw data was acquired from the Gene Expression Omnibus (GEO) database. We identified differentially expressed genes (DEGs), hub genes, and key genes through bioinformatics analysis. Subsequently, we predicted the microRNAs (miRNAs) and circular RNAs (circRNAs) associated with these key genes that may play key roles in OA using web tools. We also constructed a protein- drug network and found potentially effective drugs by analyzing the relationships between the drugs and the key genes. Results: The analysis revealed 360 DEGs, 24 hub genes, and 15 key genes enriched in many categories potentially related to the pathological mechanism of OA. hsa-miR-29a-3p, hsa-miR-29b-3p, and hsa-miR-29c-3p were predicted to be important miRNAs for OA, while hsa_circ_0025119, hsa_circ_0025113, hsa_circ_0009897, and hsa_circ_0002447 were predicted to be the most important circRNAs. Further studies indicated that Ocriplasmin and Collagenase clostridium histolyticum may be effective drugs for the treatment of OA. Finally, CD34 and VWF were inferred to be the most meaningful biomarkers for OA. Conclusion: In conclusion, we determined the underlying key genes, miRNAs, and circRNAs for OA, predicted potentially effective drugs, and identified the most meaningful biomarkers for the disease. Our findings may provide insight into the pathological mechanism of OA and guide future research.

Objective: Lingzhu San (LZS) is a traditional Chinese medicine (TCM) prescription that can be effective in treating febrile seizures (FS) and there are few research works conducted on its mechanisms. In order to better guide the clinical use of LZS, we used the research ideas and methods of network pharmacology to find the potential core compounds, targets and pathways of LZS in the complex TCM system for the treatment of FS, and predict the mechanism. Materials and Methods: Databases such as BATMAN, TCMSP, TCMID, and SWISS TARGET are used to mine the active compounds and targets of LZS, and the target information of FS is obtained through GENECARDS and OMIM. Using Venny2.1.0 and Cytoscape software, the potential core compounds and targets of FS were obtained. The R language and ClusterProfiler software package are adopted to enrich and analyze the KEGG and GO pathways of the core targets and the biological processes and potential mechanisms of the core targets are also revealed. Results: 187 active compounds and 2113 target proteins of LZS were collected. And 38 potential core compounds, 35 core targets and 775 metabolic and functional pathways were screened, which were involved in mediating FS. Finally, the role of the core compounds, targets and pivotal pathways of LZS in regulating FS in the pathogenesis and the therapeutic mechanism of FS were discussed and clarified. Conclusion: In this paper, the multi-compounds, multi-targets and multi-pathways mechanism of LZS in the treatment of FS were preliminarily screened through the analysis of network pharmacology data, which are consistent with the principle of multi-compounds’ compatibility with TCM prescriptions and a unified treatment of diseases from multiple aspects, and it provides a new way for TCM to treat complex diseases caused by multiple factors.

Background: Bladder cancer (BC) is the 10th most common cancer worldwide with significantly varied prognosis in different pathological subtypes. MMPs, a group of enzymes, could involve in the invasion and metastasis of numerous malignancies. The function of MMPs in BC is partly reported in several studies but with great conflicts; hence, a systematic analysis of expression levels and prognostic values of these MMP genes are still to be determined. Methods: Firstly, differentially expressed genes (DEGs) of MMPs were identified in ONCOMINE, GEPIA, and UALCAN databases, and these DEGs were also detected by real-time RT-qPCR. More importantly, we investigated the clinical significance of these DEGs in BC patients via Kaplan- Meier (KM) Plotter, UALCAN, and cBioPortal databases. Results: The study found that the mRNA expression of MMP1/11 in BC samples was significantly higher than that in normal bladder tissues, and MMP2/3 was lower in the former than in the latter. The expression level of MMP1/2/7/9/11/13/23B was significantly related to the tumor stages. Furthermore, the prognostic analysis suggested that the high transcription levels of MMP7 and low transcription levels of MMP23A were correlated with favorable relapse-free survival and overall survival in the patients with BC, respectively. Notably, high MMP11/13 expression levels indicated poor overall survival (OS) and relapse-free survival (RFS) in patients with BC. Conclusion: This study revealed that MMP1/2/3/7/9/11/13/23A/23B are possible prognostic biomarkers and clinical therapeutic targets for patients with BC.

Aim and Objective: Fatty acid desaturase 1 (FADS1) has been reported to be a potential biomarker in various cancers. However, no study has explored the relationship between FADS1 expression and bladder cancer. Our study aimed to investigate the role of FADS1 in bladder cancer prognosis via The Cancer Genome Atlas (TCGA). Materials and Methods: RNA-Seq expression of 414 tumor tissues and 19 paired normal tissues, as well as corresponding clinical data, were downloaded from the TCGA database. Two cancer cases were excluded due to a lack of clinical information. The association between FADS1 and the clinicopathological features of bladder cancer was analyzed. This study was conducted in October 2019 in China. Results: The high expression of FADS1 in bladder cancer was significantly related to histological grade (OR = 0.155 for low vs. high), clinical stage (OR=2.074 for III or IV vs. I or II), T classification (OR=2.326 for T3 or T4 vs. T1 or T2), lymphatic metastasis (OR=1.923 for N1 or N2 or N3 vs. N0) and distant metastasis (OR=4.883 for yes vs. no) (all p-values <0.05). Bladder cancer with high FADS1 levels was related to a worse prognosis than bladder cancer with low FADS1 levels (p= 1.626*10^-5), according to median expression value 3.622. FADS1 was an independent factor of overall survival in bladder cancer, with a hazard ratio of 1.048 (95%CI: 1.020–1.077, p = 0.001). Conclusion: Increased FADS1 expression in bladder cancer is associated with advanced clinicopathological features and may be a potential biomarker for poor prognosis.

Background: Colon cancer is one of the most common cancers worldwide and has a poor prognosis. Through the analysis of transcriptome and clinical data of colon cancer, an immune gene-set signature was identified by single sample enrichment analysis (ssGSEA) scoring to predict patient survival and discover new therapeutic targets. Objective: To study the role of immune gene-set signature in colon cancer. Methods: First, RNASeq and clinical follow-up information were downloaded from The Cancer Genome Atlas (TCGA). Immune gene-related gene sets were collected from the ImmPort database. Genes and immunological pathways related to prognosis were screened in the training set and integrated for feature selection using random forest. The immune gene-related prognosis model was verified in the entire TCGA test set and GEO validation set and compared with immune cells scores and matrix score. Results: A total of 1650 prognostic genes and 13 immunological pathways were identified. These genes and pathways are closely related to the development of tumors. 13-immune gene-set signature was established, which is an independent prognostic factor for patients with colon cancer. Risk stratification of samples could be carried out in the training set, test set, and external validation set. The AUC of five-year survival in the training set and validation set is greater than 0.6. Immunosuppression occurs in high-risk samples and compared with published models, riskScore has a better prediction effect. Conclusion: This study constructed a 13-immune gene-set signature as a new prognostic marker to predict the survival of patients with colon cancer, and provided new diagnostic/prognostic biomarkers and therapeutic targets for colon cancer.

Background: Sirtuin 1 (Sirt1) and sirtuin 2 (Sirt2) are NAD+-dependent histone deacetylases which play important functional roles in the removal of the acetyl group of acetyllysine substrates. Considering the dysregulation of Sirt1 and Sirt2 as etiological causes of diseases, Sirt1 and Sirt2 are lucrative target proteins for treatment, thus there has been great interest in the development of Sirt1 and Sirt2 inhibitors. Objective: This study compiled the bioactivity data of Sirt1 and Sirt2 for the construction of quantitative structure-activity relationship (QSAR) models in accordance with the OECD principles. Methods: Simplified molecular-input line-entry system (SMILES)-based molecular descriptors were used to characterize the molecular features of inhibitors while the Monte Carlo method of the CORAL software was employed for multivariate analysis. The dataset was subjected to 3 random splits in which each split separated the data into 4 subsets consisting of training, invisible training, calibration, and external sets. Results: Statistical indices for the evaluation of QSAR models suggested the good statistical quality of models of Sirt1 and Sirt2 inhibitors. Furthermore, mechanistic interpretation of molecular substructures that are responsible for modulating the bioactivity (i.e., promoters of increase or decrease of bioactivity) was extracted via the analysis of correlation weights. It exhibited molecular features involved in Sirt1 and Sirt2 inhibitors. Conclusion: It is anticipated that QSAR models presented herein can be useful as guidelines in the rational design of potential Sirt1 and Sirt2 inhibitors for the treatment of Sirtuin-related diseases.

Objective: Osteopontin (OPN), a phosphorylated sialoprotein, has been shown to overexpress in a variety of cancers and to contribute tumor progression and metastasis by increasing cell migration and adhesion. In the current study, we aimed to investigate the prognostic and predictive role of OPN in patients with advanced gastric cancer. Methods: A total of 42 consecutive chemonaive patients with advanced gastric cancer and 29 healthy controls were included. The patients were treated with a modified DCF regimen. The blood samples were obtained before each chemotherapy cycle from the patients and once from the healthy controls. The plasma OPN is measured by ELISA. Results: The overall response and disease stabilization rates were 25% and 72%, respectively. The median serum OPN level of the patient group was significantly higher compared to healthy controls (176.9 ng/ml (range: 41.5 -521.4) vs 64.3 ng/ml (range 42.1-105.3 ng/ml), p<0.0001). The median overall survival time was 7.0 ± 1.1 (95% CI: 4.9–9.2) months and 1-year overall survival rate was 20.8%. The patients who responded to mDCF had lower plasma OPN levels than the non-responding ones (110.7±29.3 ng/mL, 211.9±24.4 ng/mL respectively, p=0.002). The performance status and the plasma OPN levels were found to be significant factors for overall survival in the multivariate analysis (p=0.004 and 0.016, respectively). Conclusion: The serum OPN seems to be a novel significant prognostic and predictive factor in patients with advanced gastric cancer who were treated with DCF regimen.

Background: Various effects of Astaxanthin were shown in the studies, including its antioxidant, anti-inflammatory, anti-tumor and immunoregulatory effects. Objective: The aim of this study was to evaluate the beneficial effects of Astaxanthin on renovascular occlusion induced renal injury and to investigate the possible mechanisms. Methods: The rats were randomly assigned into three groups as follows: Group 1: control group (n=12), Group 2: renal ischemia-reperfusion injury group (n=12), Group 3: renal ischemia-reperfusion + asthaxantine treated group (n=12). The control group and the renal ischemia-reperfusion group were given 2cc/kg/g olive oil for 7 days before establishing ischemia to renal tissue. Astaxanthin dissolved in olive oil was given orally to the renal ischemia+astaxanthin group for 7 days before inducing renal ischemia. Caspase-(3, 8, 9), GSH, SOD, Total Thiol, TNF-α, IL-6, 8-OHdG were evaluated in each group. Results: Renal IRI was verified by analysing the pathological changes of renal tissues and the renal functions after renal reperfusion. Much less renal tubular damage was determined in the IRI+ASX group in comparison to the IRI group. Caspase-8, -9 and -3 immunoreactivity was observed to be minimal in the control group. Apoptosis was observed to be significantly reduced in the IRI + ASX group with respect to the IRI group and close to the level of the control group (p <0.05). Caspase-3 levels of tissue samples were significantly increased in the IRI group compared to the other groups, but significantly lower in the IRI+ASX group with respect to the IRI group (p<0.05). The TOS and OSI levels, indicating increased oxidative stress, were significantly lower in the IRI+ASX group with respect to the IRI group (p <0.001), but still higher than the control group (p <0.001). In addition to GSH, SOD and Total Thiol levels, TAS levels were also significantly higher in the IRI + ASX group in comparison to the IRI group (p <0.05). TNF-α, IL-6, lipid hydroperoxide, AOPP and 8-OHdG levels were lower in the IRI+ASX group than the IRI group (p <0.001). MPO, IL-6, TNF-α levels, representing the parameters indicating neutrophil infiltration and inflammation of the renal tissues, significantly increased in the IRI group with respect to the other groups (p <0.005). Conclusion: When all the data obtained in our study were evaluated, ASX was determined to prevent renal damage due to renovascular occlusion to a great extent, through complex mechanisms involving antioxidant, anti-inflammatory and antiapoptotic effects. Biochemical, histological and oxidative stress parameters were improved due to ASX.

Background/Aim: This study aimed to ascertain the effects of astaxanthin on the lungs of rat pups with bronchopulmonary dysplasia (BPD) induced by hyperoxia and lipopolysaccharide (LPS). Materials and Methods: Forty-two newborn Wistar rats, born to spontaneous pregnant rats, were divided into three groups: Hyperoxia (95% O2) + lipopolysaccharide (LPS) group, hyperoxia + LPS + astaxhantin group, and control: no treatment group (21% O2). Pups in the hyperoxia + LPS + astaxanthin group were given 100 mg/kg/day oral astaxanthin from the first day to the fifth day. Histopathologic and biochemical evaluations, including glutathione (GSH), total anti-oxidant status (TAS), total oxidant status (TOS), lipid hydroperoxide (LPO), 8-hydroxydeoxyguanosine (8-OHdG), advanced oxidation protein products (AOPP), myeloperoxidase (MPO), total thiol, tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β), and caspase-3 activities, were performed. Results: Better survival rates and weight gain were demonstrated in the hyperoxia + LPS + astaxanthin group (p <0.001). In the histopathologic evaluation, the severity of lung damage was significantly reduced in the hyperoxia+LPS+astaxanthin group, as well as decreased apoptosis (ELİSA for caspase-3) (p <0.001). The biochemical analyses of lung tissues showed that TAS, GSH, and Total thiol levels were significantly higher in the astaxanthin treated group compared to the hyperoxia + LPS group (p <0.05) while TOS, AOPP, LPO, 8-OHdG, MPO levels were significantly lower (p <0.001). In addition, unlike the hyperoxia + LPS group, TNF-α and IL-1β levels in lung tissue were significantly lower in the astaxanthin-treated group (p <0.001). Conclusion: Astaxanthin was shown to reduce lung damage caused by inflammation and hyperoxia with its anti-inflammatory, anti-oxidant, anti-apoptotic properties, and to protect the lung from severe destruction.

Aim and Objective: Nano-2-[N',N'-dimethyl-N'-(silica-n-propyl)ethanaminium chloride]- N,N-dimethylaminium bisulfate (nano-[DSPECDA][HSO4]) was used as a highly effective and heterogeneous silica-based nanostructured catalyst for the synthesis of 1-thioamidoalkyl-2-- naphthols and substituted tetrahydropyridines. Material and Methods: The expected products were prepared in mild conditions. In this work, three novel 1-thioamidoalkyl-2-naphthols and two new tetrahydropyridine derivatives were synthesized and characterized by IR, ¹H and ¹³C NMR and Mass spectroscopy. Results: One-pot multi-component condensation of 2-naphthol with arylaldehydes and thioacetamide catalyzed by nano-[DSPECDA][HSO4] under green, mild and solvent-free conditions led to 1-thioamidoalkyl-2-naphthols in high yields. The nanocatalyst was also used for the preparation of functionalized tetrahydropyridines by the one-pot multi-component reaction of anilines, arylaldehydes and ethylacetoacetate under solvent-free and mild conditions. Conclusion: The reaction results were found to be better compared to the literature in terms of one or more of these factors: yield, time, and the reaction media. All the products were purified by recrystallization from EtOH, and without column chromatography, being in good agreement with the green chemistry protocols.

Aims and Objective: In this work, 1,3-oxazoles were generated using the multicomponent reaction of α-bromo ketones, alkyl (aryl) isothiocyanates, sodium hydride and Fe3O4 MNPs in the water at room temperature in good yields. The nanoparticles generated via the biosynthesis method have potential value in different purposes, such as organic synthesis. Materials and Methods: To study the antioxidant ability of some synthesized thioxo-1,3-oxazoles, diphenyl-picrylhydrazine (DPPH) radical trapping and power of ferric reduction testes are employed. Among the studied thioxo-1,3-oxazoles, compound 4b have good power for radical trapping and reduction activity than the standard antioxidants such as BHT and TBHQ. In addition, the antimicrobial activity of some thioxo-1,3-oxazoles was studied, employing the disk diffusion test on Gram-positive bacteria and Gram-negative bacteria. The results of the disk diffusion test showed that compounds 4a, 4b, 4d and 4f prevented bacterial growth. Results: Without employing catalyst, these reactions have low yield and busy mixture. The synthesis of compound 4a as sample reaction has a similar yield in the presence of ZnO-NPs and Fe3O4 MNPs (entry 20 and entry 30), but the removal of catalyst from the mixture of reaction after the completion of the reaction is comfortable in the presence of Fe3O4 MNPs. Structures of compound 4a–4i are confirmed by IR, ¹H NMR and ¹³C NMR mass spectra. Conclusion: The reaction of α-bromo ketones, isothiocyanate and sodium hydride in the presence of the catalytic amount of Fe3O4 MNPs in water generates 1,3-oxazole derivatives in good yields. Some of the advantages of performing these reactions with the present procedure are to carry out these reactions in water as a green solvent and simple removal of catalyst.

Background: Novel coronavirus is a type of enveloped viruses with a single-stranded RNA enclosing helical nucleocapsid. The envelope consists of spikes on the surface which are made up of proteins through which virus enters into human cells. Until now, there is no specific drug or vaccine available to treat COVID-19 infection. In this scenario, reposting of drug or active molecules may provide rapid solution to fight against this deadly disease. Objective: We selected 30 phytoconstituents from the different plants which are reported for antiviral activities against coronavirus (CoVs) and performed in silico screening to find out phytoconstituents which have potency to inhibit specific target of the novel coronavirus. Methods: We performed molecular docking studies on three different proteins of novel coronavirus, namely COVID-19 main protease (3CL pro), papain-like protease (PL pro) and spike protein (S) attached to ACE2 binding domain. The screening of the phytoconstituents on the basis of binding affinity compared to standard drugs. The validations of screened compounds were done using ADMET and bioactivity prediction. Results: We screened five compounds biscoclaurine, norreticuline, amentoflavone, licoricidin and myricetin, using in silico approach. All compounds were found safe in In silico toxicity studies. Bioactivity prediction reveals that these compounds may act through protease or enzyme inhibition. Results of compound biscoclaurine norreticuline were more interesting as this biscoclaurine had higher binding affinity for the target 3CLpro and PLpro targets and norreticuline had a higher binding affinity for the target PLpro and Spike protein. Conclusion: Our study concludes that these compounds could be further explored rapidly as it may have potential to fight against COVID-19.

Background: The quantitative structure-activity relationship (QSAR) approach is most widely used for the prediction of biological activity of potential medicinal compounds. A QSAR model is developed by correlating the information obtained from chemical structures (numerical descriptors/ independent variables) with the experimental response values (the dependent variable). Methods: In the current study, we have developed a QSAR model to predict the inhibitory activity of small molecule carboxamides against severe acute respiratory syndrome coronavirus (SARS-- CoV) 3CLpro enzyme. Due to the structural similarity of this enzyme with SARS-CoV-2, the causative organism of the recent pandemic, the former may be used for the development of therapies against coronavirus disease 19 (COVID-19). Results: The final multiple linear regression (MLR) model was based on four two-dimensional descriptors with definite physicochemical meaning. The model was strictly validated using different internal and external quality metrics. The model showed significant statistical quality in terms of determination coefficient (R²=0.748, adjusted R² or R²adj = 0.700), cross-validated leave-one-out Q² (Q²=0.628) and external predicted variance R²pred = 0.723. The final validated model was used for the prediction of external set compounds as well as to virtually design a new library of small molecules. We have also performed a docking analysis of the most active and least active compounds present in the dataset for comparative analysis and to explain the features obtained from the 2D-QSAR model. Conclusion: The derived model may be useful to predict the inhibitory activity of small molecules within the applicability domain of the model only based on the chemical structure information prior to their synthesis and testing.

Aims and Objective: Copious proinflammatory cytokines including TNF-α and IL-1β are involved in progression of inflammation in human body. Inhibition of signaling mediated by proinflammatory cytokines offer effective in the treatment of inflammatory diseases. The treatment of dreadful infectious disease mycobacterium tuberculosis still remains a challenge owing to resistance to multiple drugs hence an urgent need for newer drugs. Pyrazolo[3,4-d]pyrimidines have been disclosed to possess numerous pharmacological activities including anti-inflammatory, antimicrobial and antitubercular activities. Here in we report the synthesis of pyrazolo[3,4-d]pyrimidines for anti-inflammatory and antitubercular activities. Materials and Methods: The targeted compounds having pyrazolo[3,4-d]pyrimidine scaffold 8a-m were synthesized in three step reactions with the formation of key intermediate 5-amino-4-- cyno-1-phenyl pyrazole which upon cyclization resulted in 4-amino pyrazolo[3,4-d]pyrimidine for subsequent benzoylation with substituted benzoyl chlorides to form 8a-m. Antiinflammatory activity of 8a-m was assessed at 25 mg/Kg dose and minimum inhibitory concentration against gram positive, gram negative and mycobacteria was also performed. Binding interactions were also measured in binding pocket of p38 kinase. Results: Four compounds 8a, 8b, 8e and 8i significant anti-inflammatory activity in rat paw edema model induced by carrageenan and among all 8b was potent with 80.6% activity. Numerous compounds exhibited potent activity against fungal strains than bacterial strains, compound 8k was most potent against gram negative bacteria Klebsiella pneumoniae. Compounds 8d, 8e and 8f exhibited antitubercular activity with MIC value of 6.25 μg/mL. Conclusion: Substituted N-benzoylated amino pyrazolo[3,4-d]pyrimidines endowed significant and potent anti-inflammatory and antimicrobial activities. Molecular docking studies also revealed favorable interactions in active site of p38 kinase.

Background: Boswellic acids are the main constituents of Boswellia serrata gum. These comprise of four pentacyclic triterpenes, 11-keto-β-boswellic acid (KBA) being one of them. Objectives: Comparing the extraction efficiency of KBA through microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE) followed by optimizing the extraction process using response surface methodology (RSM) and validation of HPTLC. Methods: UAE and MAE of KBA were carried out employing methanol as an extracting solvent. To figure out the better mode of extraction, single factorial experiments were conducted for further optimization. Design expert software was used for optimization purposes where solvent to drug ratio, extraction temperature and extraction time were taken as input variables. Quantification of KBA in each extract was done through High-Performance Thin Layer Chromatography (HPTLC), and the method was validated as per International Council for Harmonization (ICH) guidelines. Results: UAE stood out to be a better mode of extraction for KBA. Solvent to drug ratio of 20.42 mL/gram, extraction temperature of 44.01°C and extraction time of 11.54 minutes were established as optimum conditions, which yielded 8.44%w/w of KBA. Regarding HPTLC, the Rf value of KBA was measured, and the correlation coefficient was calculated from the standard curve. Accuracy, precision and recovery were found within limits. Conclusion: From this study, it was concluded that a non-thermal method is a better choice of extraction for KBA. All the input variables significantly affected KBA content, which was confirmed by model fitting. Moreover, the HPTLC method was developed for the quantification of KBA, which was found to be accurate, reliable and highly sensitive.