A green, facile, and practical preparation of capsaicin derivatives with thiourea structure.

Capsaicin derivatives with thiourea structure (CDTS) is highly noteworthy owing to its higher analgesic potency in rodent models and higher agonism in vitro. However, the direct synthesis of CDTS remains t one or more shortcomings. In this study, we present reported a green, facile, and practical synthetic method of capsaicin derivatives with thiourea structure is developed by using an automated synthetic system, leading to a series of capsaicin derivatives with various electronic properties and functionalities in good to excellent yields.

Theoretical exploration of the phenolic compounds' inhibition mechanism of heterocyclic aromatic amines in roasted beef patties by density functional theory.

The ability of spices (bay leaf, star anise, and red pepper) and their characteristic phenolic compounds (quercetin, kaempferol, and capsaicin) to inhibit Heterocyclic aromatic amines (HAAs) in roasted beef patties were compared. Density functional theory (DFT) was used to reveal phenolic compounds interacting with HAAs-related intermediates and free radicals to explore possible inhibitory mechanisms for HAAs. 3 % red chili and 0.03 % capsaicin reduced the total HAAs content by 57.09 % and 68.79 %, respectively. DFT demonstrated that this was due to the stronger interaction between capsaicin and the ß-carboline HAAs intermediate (Ebind = -32.95 kcal/mol). The interaction between quercetin and phenylacetaldehyde was found to be the strongest (Ebind = -17.47 kcal/mol). Additionally, DFT indicated that capsaicin reduced the carbonyl content by transferring hydrogen atoms (HAT) to eliminate HO·, HOO·, and carbon-centered alkyl radicals. This study provided a reference for the development of DFT in the control of HAAs.

Discovery of N-(1,4-Benzoxazin-3-one) urea analogs as Mode-Selective TRPV1 antagonists.

A series of 1,4-benzoxazin-3-one analogs were investigated to discover mode-selective TRPV1 antagonists, since such antagonists are predicted to minimize target-based adverse effects. Using the high-affinity antagonist 2 as the lead structure, the structure activity relationship was studied by modifying the A-region through incorporation of a polar side chain on the benzoxazine and then by changing the C-region with a variety of substituted pyridine, pyrazole and thiazole moieties. The t-butyl pyrazole and thiazole C-region analogs provided high potency as well as mode-selectivity. Among them, antagonist 36 displayed potent and capsaicin-selective antagonism with IC50 = 2.31 nM for blocking capsaicin activation and only 47.5 % inhibition at 3 µM concentration toward proton activation, indicating that more than a 1000-fold higher concentration of 36 was required to inhibit proton activation than was required to inhibit capsaicin activation. The molecular modeling study of 36 with our homology model indicated that two π-π interactions with the Tyr511 and Phe591 residues by the A- and C-region and hydrogen bonding with the Thr550 residue by the B-region were critical for maintaining balanced and stable binding. Systemic optimization of antagonist 2, which has high-affinity but full antagonism for activators of all modes, led to the mode-selective antagonist 36 which represents a promising step in the development of clinical TRPV1 antagonists minimizing side effects such as hyperthermia and impaired heat sensation.

Enhancing biofouling resistance in microfiltration membranes through capsaicin-derivative functionalization.

The primary focal point in the fabrication of microfiltration membranes revolves around mitigating issues of low permeability stemming from the initial design as well as countering biofouling tendencies. This work aimed to address these issues by synthesizing an antibacterial capsaicin derivative (CD), which was then grafted to the poly(vinylidene fluoride-co-chlorotrifluoroethylene)-g-polymethacrylic acid (P(VDF-CTFE)-g-PMAA) matrix polymer, resulting in an antibacterial polymer (PD). Notably, both CD and PD demonstrated low cytotoxicities. Utilizing PD, a microfiltration membrane (MA) was successfully prepared through non-solvent-induced phase inversion. The pore sizes of the MA membrane were mainly concentrated at around 436 nm, while the pure water flux of MA reached an impressive value of 62 ± 0.17 Lm-2 h-1 at 0.01 MPa. MA exhibited remarkable efficacy in eradicating both Gram-negative (E. coli) and Gram-positive bacteria (Bacillus subtilis) from its surface. Compared with M1 prepared from P(VDF-CTFE), MA exhibited a lower flux decay rate (41.00% vs. 76.03%) and a higher flux recovery rate (84.95% vs. 46.54%) after three cycles. Overall, this research represents a significant step towards the development of a microfiltration membrane with inherent stable anti-biofouling capability to enhance filtration.

Electronic Tongue Based on ZnO/ITO@glass for Electrochemical Monitoring of Spiciness Levels.

Capsaicin, a chemical compound present in chili peppers, is widely acknowledged as the main contributor to the spicy and hot sensations encountered during consumption. Elevated levels of capsaicin can result in meals being excessively spicy, potentially leading to health issues, such as skin burning, irritation, increased heart rate and circulation, and discomfort in the gastrointestinal system and even inducing nausea or diarrhea. The level of spiciness that individuals can tolerate may vary, so what may be considered incredibly hot for one person could be mild for another. To ensure food safety, human healthcare, regulatory compliance, and quality control in spicy food products, capsaicin levels must be measured. For these purposes, a reliable and stable sensor is required to quantify the capsaicin level. To leverage the effect of zinc oxide (ZnO), herein, we demonstrated the one-step fabrication process of an electronic tongue (E-Tongue) based on an electrochemical biosensor for the determination of capsaicin. ZnO was electrodeposited on the indium tin oxide (ITO) surface. The biosensor demonstrated the two notable linear ranges from 0.01 to 50 µM and from 50 to 500 µM with a limit of detection (LOD) of 2.1 nM. The present study also included the analysis of real samples, such as green chilis, red chili powder, and dried red chilis, to evaluate their spiciness levels. Furthermore, the E-Tongue exhibited notable degrees of sensitivity, selectivity, and long-term stability for a duration of more than a month. The development of an E-Tongue for capsaicin real-time monitoring as a point-of-care (POC) device has the potential to impact various industries and improve safety, product quality, and healthcare outcomes.

Arvanil induces ferroptosis of hepatocellular carcinoma by binding to MICU1.

Hepatocellular carcinoma (HCC) is a primary liver cancer with a high mortality rate that requires research and improved treatment strategies. Chemotherapy is still one of the main methods of HCC treatment, but it may lead to drug resistance and damage to normal organs. Capsaicin, a naturally occurring active ingredient in chili peppers, has demonstrated anticancer properties in a variety of malignant tumor cell lines. However, the anti-cancer mechanism of capsaicin needs to be further explored in HCC. In this study, we utilized Arvanil, a non-stimulating synthetic capsaicin analog, in place of capsaicin. We found that Arvanil induced high mitochondrial calcium flow, which contributed to a decrease in mitochondrial membrane permeability transition pore (mPTP) opening and oxidative phosphorylation levels, ultimately triggering cellular ferroptosis by live cells in real time with a high content screening (HCS) platform and confocal microscopy. It was further confirmed by vina molecular docking and point mutation experiments that Arvanil directly binds to two amino acid sites of mitochondrial calcium uptake protein 1 (MICU1), namely Ser47 and Phe128, to trigger this process, which in turn inhibits the growth of HCC cells. In addition, it was confirmed that Arvanil enhances cisplatin chemosensitivity by inducing HCC cellular ferroptosis in vivo. In conclusion, our study suggests that Arvanil induces ferroptosis in HCC cells and is a candidate drug for the treatment of HCC.

3D-Printed Capsaicin-Loaded Injectable Implants for Targeted Delivery in Obese Patients.

Diet-induced obesity and hyperlipidemia are a growing public health concern leading to various metabolic disorders. Capsaicin, a major bioactive compound obtained from natural chili peppers, has demonstrated its numerous beneficial roles in treating obesity and weight loss. Current treatment involves either administration of antiobesity drugs or surgical procedures such as Roux-en-Y-gastric bypass or sleeve gastrectomy, both of which are associated with serious side effects and poor patient acceptance. Capsaicin, a pungent molecule, has low oral bioavailability. Therefore, there is a need for the development of site-specific drug delivery system for capsaicin. The present study is aimed at preparing and characterizing 3D-printed capsaicin-loaded rod-shaped implants by thermoplastic extrusion-based 3D printing technology. The implants were printed with capsaicin-loaded into a biodegradable polymer, polycaprolactone, at different drug loadings and infill densities. The surface morphology revealed a smooth and uniform external surface without any capsaicin crystals. DSC thermograms showed no significant changes/exothermic events among the blends suggesting no drug polymer interactions. The in vitro release studies showed a biphasic release profile for capsaicin, and the release was sustained for more than three months (~ 85% released) irrespective of drug loading and infill densities. The HPLC method was stability-indicating and showed good resolution for its analogs, dihydrocapsaicin and nordihydrocapsaicin. The implants were stable for three months at accelerated conditions (40°C) without any significant decrease in the assay of capsaicin. Therefore, capsaicin-loaded implants can serve as a long-acting injectable formulation for targeting the adipose tissue region in obese patients.

Binding interactions of fatty acyl lipid mediators within the vanilloid pocket of TRPV1: A molecular dynamics study.

The transient receptor potential vanilloid 1 (TRPV1) channel is a ligand-gated, nonselective cation channel expressed in primary sensory neurons, which has a role in nociception. The channel is activated by noxious heat, pH, capsaicin and other endogenous vanilloids, including lipid mediators (LMs) enzymatically derived from polyunsaturated fatty acids (PUFA). Although capsaicin binding to TRPV1 has been well characterized, the molecular mechanism by which endogenous LM ligands bind the channel is not well understood. In this study, we characterized the binding interactions for 13 endogenous LM ligands, within the vanilloid pocket of TRPV1 using a molecular dynamics (MD) approach. We observed that LM ligands can be grouped based on their structure and affinity for the vanilloid pocket. Furthermore, the position as well as the number of the polar groups on the LM ligand directly impact binding stability through various polar interactions with the protein. As an additional control we performed docking experiments of the PUFA precursor molecules linoleic acid and arachidonic acid which failed to form stable interactions within the vanilloid pocket. While LM ligands with similar structures displayed similar binding interactions, there were notable exceptions in the case of 20-HETE, 9-HODE, and 9,10-DiHOME. Our study offers new insights into the mechanisms involved in TRPV1 activation by endogenous LM ligands. The observed binding interactions may assist in the interpretation of in vivo and in vitro pharmacodynamics studies.

Cytotoxicity of capsaicin and its analogs in vitro.

Capsaicin (CAP) is the main compound responsible for the spicy flavor of Capsicum plants. However, its application can be inhibited due to its pungency and toxicity. This study aimed to evaluate and compare the cytotoxic effect of CAP and its analogs N-benzylbutanamide (AN1), N-(3-methoxybenzyl) butanamide (AN2), N-(4-hydroxy-3-methoxybenzyl) butanamide (AN3), N-(4-hydroxy-3-methoxybenzyl) hexanamide (AN4) and N-(4-hydroxy-3-methoxybenzyl) tetradecanamide (AN5) on the hepatoma cells of Rattus norvegicus using the MTT test. The results showed cytotoxicity of CAP at concentrations of 100, 150, 175, and 200 µM (24 hours), AN1 at 150 and 175 µM (48 hours), AN2 at 50 µM (24 hours) and 10, 25, 50, and 75 µM (48 hours), AN4 at 175 µM (24 hours), and AN5 at 50 µM (48 hours). Removing the hydroxyl radical from the vanillyl group of capsaicin, together with reducing the acyl chain to 3 carbons, which is the case of AN2, resulted in the best biological activity. Increasing the carbon chain in the acyl group of the capsaicin molecule, which is the case of AN5, also showed evident cytotoxic effects. The present study proves that the chemical modifications of capsaicin changed its biological activity.

Dispersive magnetic solid-phase extraction for capsaicinoid compounds in human serum using LC-HRMS: targeted and non-targeted approaches.

A new analytical method based on the use of dispersive magnetic solid-phase extraction (DMSPE) is described for the preconcentration of capsaicin (CAP), dihydrocapsaicin (DCAP), and N-vanillylnonanamide (PCAP) from human serum samples. The influence of several experimental factors affecting the adsorption (nature and amount of magnetic material, adsorption time, and pH) and desorption (nature of solvent, its volume and desorption time) steps was studied. Among seven different nanomaterials studied, the best results were obtained using magnetic multiwalled carbon nanotubes, which were characterized by means of spectrometry- and microscopy-based techniques. Analyses were performed by ultra-high-performance liquid chromatography with quadrupole-time-of-flight mass spectrometry using electrospray ionization in positive mode (UHPLC-ESI-Q-TOF-MS). The developed method was validated by obtaining several parameters, including linearity (0.3-300 µg L-1 range), and limits of detection which were 0.1, 0.15, and 0.17 µg L-1 for CAP, DCAP, and PCAP, respectively. The repeatability of the method, expressed as relative standard deviation (RSD, n = 7), varied from 3.4 to 11%. The serum samples were also studied through a non-targeted approach in a search for capsaicinoid metabolites and related compounds. With this objective, the fragmentation pathway of this family of compounds was initially studied and a strategy was established for the identification of novel or less studied capsaicinoid-derived compounds.

Genetic analysis of pungency deficiency in Japanese chili pepper 'Shishito' (Capsicum annuum) revealed its unique heredity and brought the discovery of two genetic loci involved with the reduction of pungency.

The sensation of pungency generated by capsaicinoids is a characteristic trait of chili peppers (Capsicum spp.), and the presence or absence of pungency is central in determining its usage as a spice or a vegetable. In the present study, we aimed to clarify the heredity and genetic factors involved in the deficiency of pungency (quite low pungency) that is uniquely observed in the Japanese chili pepper 'Shishito' (Capsicum annuum). First, the F2 population ('Shishito' × pungent variety 'Takanotsume') was used for segregation analysis, and pungency level was investigated using capsaicinoid quantification with high-performance liquid chromatography. Also, restriction site associated DNA sequencing of the F2 population was performed, and genetic map construction and quantitative trait locus (QTL) mapping were implemented. The results indicated that the F2 population showed varying capsaicinoid content and two major QTLs were detected, Shql3 and Shql7, which explained 39.8 and 19.7% of the genetic variance, respectively. According to these results, the quite low pungency of 'Shishito' was a quantitative trait that involved at least the two loci. Further, this trait was completely separate from general non-pungent traits controlled by individual recessive genes, as described in previous studies. The present study is the first report to investigate the genetic mechanism of pungency deficiency in Japanese chili peppers, and our results provide new insights into the genetic regulation of pungency in chili pepper.

Intervention effects of multilayer core-shell particles on colitis amelioration mechanisms of capsaicin.

The intervention effects of delivery systems on the digestion and adsorption profiles and, thus, the pharmacological effects of bioactive compounds represent an intriguing scientific hypothesis that can be proven with research case studies. Delivery systems with tailor-made structures fabricating from the same building materials offer a new research strategy for deciphering the modulating effects of the digestive fate on the therapeutic efficacy of encapsulated bioactive compounds. Herein, we developed capsaicin-loaded core-shell nanoparticles (Cap NPs), microparticles (Cap MPs) and nano-in-micro particles (Cap NPs in MPs) and investigated their regulatory effects on the digestive fate and colitis-alleviating mechanisms of capsaicin. Results suggested that the small intestine dominant absorption of Cap NPs differed significantly with the colorectal dominated accumulation of Cap MPs and Cap NPs in MPs in terms of the colitis alleviating mechanisms. Cap NPs alleviated colitis mainly through promoting the colonization of short-chain fatty acid-producing bacteria, maintaining intestinal barrier homeostasis and partially inhibiting the activation of the NF-κB pro-inflammatory pathway. Whereas, better dietary intervention effects were achieved from Cap NPs in MPs via promoting the proliferation of mucus-related bacteria and enhanced triggering efficiency on the TRPV1-mucus-microbiotas cyclic cascade. This work confirmed that rationally designed biomaterial-based delivery vehicles can flexibly interfere with the therapeutic mechanisms of encapsulated cargos, representing a new horizon in the field of precise nutrition.

Acyclic cucurbit[n]urils-based supramolecular encapsulation for enhancing the protective effect of capsaicin on gastric mucosa and reducing irritation.

Capsaicin (CAP) is an alkaloid isolated from pepper fruit, which possesses various pharmacological activities including antioxidant, anti-inflammatory, antibacterial and gastric mucosa protection. However, its inherent poor aqueous solubility and strong irritation impede the further clinical application. In our study, acyclic cucurbit[n]urils (ACBs, M1, M2 and M3) were rationally utilized to prepare a series of CAP inclusion complexes to improve the bioavailability and reduce stimulation. Their properties and inclusion behaviors were further investigated by multiple characterization methods, the data indicated that the inclusion complexes of ACBs/CAP were formed by a stoichiometric ratio of 2:1 with strong binding interaction. After complexation, the solubility of CAP was significantly increased by 12,076 times and its antioxidant activity also increased. Moreover, the anti-inflammatory activity and the ability to prevent gastric mucosal injury were both significantly improved, and the inhibition rate of nitric oxide (NO) and interleukin-1ß (IL-1ß) has been effectively improved while cytotoxicity against human normal hepatocytes cell (LO2), human lung fibroblasts cell (HLF) and the human gastric mucosal cell (GES-1) was greatly attenuated. Confocal laser scanning microscope (CLSM) images indicated that the complexes could be efficiently internalized by GES-1 cells and primarily located in cytoplasm. In vivo model of mouse, our complexes exhibited excellent biosafety. In summary, our study may provide a promising new strategy for the further clinical application of CAP.

Beneficial effects of capsaicin and dihydrocapsaicin on endothelial inflammation, nitric oxide production and antioxidant activity.

Capsaicin and dihydrocapsaicin (DHC) are major pungent capsaicinoids produced in chili peppers. Capsaicin has been previously shown to promote vascular health by increasing nitric oxide (NO) production and reducing inflammatory responses. While capsaicin has been extensively studied, whether DHC exerts cardiovascular benefits through similar mechanisms remains unclear. The current study aimed to investigate the direct effects of DHC on endothelial inflammation, NO release, and free radical scavenging properties. DHC at concentrations up to 50 µM did not affect cell viability, while concentrations of 100 and 500 µM of DHC led to endothelial cytotoxicity. Capsaicin decreased cell viability at concentration of 500 µM. To investigate the effects of capsaicinoids on endothelial activation, we first demonstrated that TNFα induced Ser536 phosphorylation of p65 NFκB, expressions of adhesion molecules, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1, and IL-6 production in primary human endothelial cells. These effects were robustly abrogated by DHC. Consistently, DHC treatment led to a marked reduction in TNFα-mediated monocyte adhesion to endothelial cells. Additionally, NO production was significantly induced by DHC and capsaicin compared to vehicle control. Similar to capsaicin and vitamin C, DHC scavenged DPPH (1,1-diphenyl-2-picrylhydrazyl) free radicals in vitro. Our present study highlights the benefits of DHC and capsaicin treatment on human endothelial cells and provides evidence to support cardiovascular benefits from capsicum consumption.

A new era for the design of TRPV1 antagonists and agonists with the use of structural information and molecular docking of capsaicin-like compounds.

The design of TRPV1 antagonists and agonists has reached a new era since TRPV1 structures at near-atomic resolution are available. Today, the ligand-binding forms of several classical antagonists and agonists are known; therefore, the specific role of key TRPV1's residues in binding of ligands can be elucidated. It is possible to place the well-defined pharmacophore of TRPV1 ligands, conformed by head, neck, and tail groups, in the right pocket regions of TRPV1. It will allow a more thorough use of molecular modelling methods to conduct more effective rational drug design protocols. In this work, important points about the interactions between TRPV1 and capsaicin-like compounds are spelled out, based on the known pharmacophore of the ligands and the already available TRPV1 structures. These points must be addressed to generate reliable poses of novel candidates and should be considered during the design of novel TRPV1 antagonists and agonists.

A Potential Route of Capsaicin to Its Binding Site in the TRPV1 Ion Channel.

Transient receptor potential (TRP) ion channels are important pharmacological targets because of their role in the perception of pain, and so, understanding their chemical regulation is essential for the development of analgesic drugs. Among the currently known TRP channel chemical agonists, capsaicin, the active compound of chili pepper, is probably the most exhaustively studied. The availability of the three-dimensional structure of the vanilloid receptor 1 (TRPV1) has fueled computational studies revealing the molecular details of capsaicin binding modes. Although this is a significant step, a comprehensible binding mechanism or pathway is invaluable for targeting TRP channels in modern pharmacology. In the present work, free-energy and enhanced sampling techniques have been used to explore a possible membrane-mediated pathway for capsaicin to enter the TRPV1 binding pocket where capsaicin accesses the protein starting at the extracellular milieu through the outer leaflet and into its binding site in the protein. The main states visited along this route have been characterized and include (i) a bound state in agreement with the binding mode "head-down, tail-up" and (ii) an alternative state corresponding to a "head-up, tail-down" binding mode. In agreement with previous reports, binding is mediated by both hydrogen bonds and van der Waals interactions, and residue Y511 is crucial for stabilizing the bound state and during the binding process. Together, these results provide a foundation to further understand TRPV channels, and they could be used to guide therapeutic design of selective inhibitors potentially leading to novel avenues for pharmacological applications targeting the TRPV1 channel.

Towards engineered yeast as production platform for capsaicinoids.

Capsaicinoids are bioactive alkaloids produced by the chili pepper fruit and are known to be the most potent agonists of the human pain receptor TRPV1 (Transient Receptor Potential Cation Channel Subfamily V Member 1). They are currently produced by extraction from chili pepper fruit or by chemical synthesis. Transfer of the biosynthetic route to a microbial host could enable more efficient capsaicinoid production by fermentation and may also enable the use of synthetic biology to create a diversity of new compounds with potentially improved properties. This review summarises the current state of the art on the biosynthesis of capsaicinoid precursors in baker's yeast, Saccharomyces cerevisiae, and discusses bioengineering strategies for achieving total synthesis from sugar.

Preparation, characterization and evaluation of capsaicin-loaded indica rice starch nanoparticles.

Capsaicin (CAP) is an alkaloid with multiple physiological effects, but its application is difficult. In this research, indica rice starch nanoparticles (IRSNPs) based nanocarrier was used to load CAP to obtain capsaicin-loaded indica rice starch nanoparticles (CAP-IRSNPs). The microstructure, characteristics and in vitro release behaviors of CAP-IRSNPs were analyzed. CAP-IRSNPs presented average particle sizes of 617.84 ± 6.38 nm, encapsulation efficiency of 70.05 ± 1.78% and loading capacity of 13.41 ± 0.18%. Fourier-transform infrared spectroscopy confirmed that CAP-IRSNPs might be formed by hydrogen-bonding action. Differential scanning calorimetry and X-ray diffraction showed that IRSNPs influenced the crystallization and melting temperatures of CAP. In in vitro release study, CAP-IRSNPs exhibited a sustained release. The CAP concentration, CAP diffusion from matrix and matrix erosion might be the potentially possible mechanisms for capsaicin release from CAP-IRSNPs. These new results concluded that IRSNPs may be a promising nanocarrier for CAP or other hydrophobic bioactive ingredients.

Development and Validation of an HPLC Method for Simultaneous Determination of Capsaicinoids and Camphor in Over-the-Counter Medication for Topical Use.

A reverse-phase high-performance liquid chromatography method was developed to determine and quantify capsaicin (trans-8-methyl-N-vanillyl-6- nonenamid), dihydrocapsaicin (8-methyl-N-vanillylnonanamide), and camphor (trimethylbicyclo[2.2.1]heptan-2-one). It is applicable in analyses of over-the-counter (OTC) medications for topical use and raw materials such as chili pepper oleoresin. Chromatographic separation was carried out on a C18 column using an isocratic flow of the mobile phase containing acetonitrile and ultrapure water in a ratio of 2:3, with pH adjusted to 3.2 using glacial acetic acid, and a flow rate of 1.5 mL/min. The concentration of the eluting compounds was monitored by a diode-array detector at a wavelength of 281 nm. The method was evaluated for several validation parameters, including selectivity, accuracy (confidence intervals < 0.05%), repeatability, and intermediate precision. The limit of detection (LOD) was determined to be 0.070 µg/mL for capsaicin, 0.211 µg/mL for dihydrocapsaicin, and 0.060 µg/mL for camphor. The limit of quantification (LOQ) was determined to be 0.212 µg/mL for capsaicin, 0.640 µg/mL for dihydrocapsaicin, and 0.320 µg/mL for camphor. Linearity was set in the range of 2.5-200 µg/mL for capsaicin and dihydrocapsaicin and 25-2000 µg/mL for camphor. The suggested analytical method can be used for quality control of formulated pharmaceutical products containing capsaicinoids, camphor, and propolis.

The effects of mutation on the drug binding affinity of Neuraminidase: case study of Capsaicin using steered molecular dynamics simulation.

The influenza virus is an important respiratory pathogen that causes many incidences of diseases and even death each year. One of the primary factors of this virus is the Neuraminidase surface protein, which causes the virus to leave the host cell and spread to new target cells. The main antiviral medication for influenza is designed as a protein inhibitor ligand that prevents further spread of the disease, and eventually relieves the emerged symptoms. The effectiveness of such inhibitory drugs is highly associated with their binding affinity. In this paper, the binding affinity of an herbal ligand of Capsaicin bound to Neuraminidase of the influenza virus is investigated using steered molecular dynamics (SMD) simulation. Since mutations of the virus directly impact the binding affinity of the inhibitory drugs, different mutations were generated by using Mutagenesis module. The rapid spread of infection during the avian influenza A/H5N1 epidemic has raised concerns about far more dangerous consequences if the virus becomes resistant to current drugs. Currently, oseltamivir (Tamiflu), zanamivir (Relenza), pramivir (Rapivab), and laninamivir (Inavir) are increasingly used to treat the flu. However, with the rapid evolution of the virus, some drug-resistant strains are emerging. Therefore, it is very important to seek alternative therapies and identify the roots of drug resistance. Obtained results demonstrated a reduced binding affinity for the applied mutations. This reduction in binding affinity will cause the virus mutation to become resistant to the drug, which will spread the disease and make it more difficult to treat. From a molecular prospect, this decrease in binding affinity is due to the loss of a number of effective bonds between the ligand and the receptor, which occurs with mutations of the wild-type (WT) species. The results of the present study can be used in the rational design of novel drugs that are compatible with specific mutations.