Andrographolide Research Articles

Andrographolide Encapsulation in Metal‐organic Frameworks (MOFs) via Solvent‐free Process at High Pressure

Andrographolide (ADG) encapsulation was carried out on MOFs MIL‐53(Al) and ZIF‐8 by high‐pressure (0.3 GPa) contact. This methodology is not only environment‐friendly but also energy/time‐saving and gives rise to ADG‐MOFs with physical features equivalent to those of materials obtained by common liquid phase encapsulation. The loaded MOFs were characterized through TEM, SEM, XRD, TGA, FT‐IR, BET, and NMR. The observed decrease in the intensity of ADG XRD peaks is due to the adsorption of ADG into the MOFs. TGA showed the decomposition step of ADG in the range of 200‐300 °C in both loaded MOFs. FT‐IR also showed intense signals of the ADG in the synthesized materials. The dissolution profile of ADG in MIL‐53(Al) in PBS (pH = 7.4) was carried out showing that the drug was released up to 96% after 75 h. NMR confirmed the interactions between ADG molecules and ZIF‐8 groups and the formation of a hydrogen bond between the carboxylic group of ADG and the hydroxyl group of MIL‐53(Al). Coefficient partition studies determined that both MOFs did not improve the hydrophilicity of the ADG, due to the loading of the drug preferably occurring by interactions in the hydrophobic areas within the pores of the MOFs.

Effects of drying conditions on physical properties, bioactive compounds and antioxidant activity of Andrographis paniculata leaves

Andrographis paniculata (Burm. f.) Wall. ex Nees (AP) is a medicinal plant widely used in the tropical area of Asia. The effects of drying temperature and time on the andrographolide (AG) content, total phenolic content (TPC), 2,2-diphenyl-1- picrylhydrazyl (DPPH) radical scavenging activity and ferric ion reducing antioxidant power (FRAP) activity were investigated. A 3×4 factorial completely randomized design was performed. Sun drying and hot-air drying at different temperatures (50℃, 60℃ and 70℃) and different drying times (5 hrs, 6 hrs and 7 hrs) were investigated. The results demonstrated that a higher drying temperature and a longer time decreased the AG content, the TPC, the DPPH radical scavenging activity and the FRAP activity. Dried AP leaves at 50℃ for 5 hrs had the highest AG content. However, sun drying for 5 hrs resulted in the greatest TPC, DPPH radical scavenging activity and FRAP activity. Therefore, the recommended options for the optimum drying of AP to maintain active ingredients were 50℃ for 5 hrs, while sun drying was recommended to maintain antioxidant activity. These results provided the basis for further development of dried Andrographis paniculata leaf products containing high contents of bioactive and antioxidant activity as functional or nutraceutical foods in the future.

Targeting TLR4 and regulating the Keap1/Nrf2 pathway with andrographolide to suppress inflammation and ferroptosis in LPS-induced acute lung injury

Acute lung injury (ALI) is a severe inflammatory condition with a high mortality rate, often precipitated by sepsis. The pathophysiology of ALI involves complex mechanisms, including inflammation, oxidative stress, and ferroptosis, a novel form of regulated cell death. This study explores the therapeutic potential of andrographolide (AG), a bioactive compound derived from Andrographis, in mitigating Lipopolysaccharide (LPS)-induced inflammation and ferroptosis. Our research employed in vitro experiments with RAW264.7 macrophage cells and in vivo studies using a murine model of LPS-induced ALI. The results indicate that AG significantly suppresses the production of pro-inflammatory cytokines and inhibits ferroptosis in LPS-stimulated RAW264.7 cells. In vivo, AG treatment markedly reduces lung edema, decreases inflammatory cell infiltration, and mitigates ferroptosis in lung tissues of LPS-induced ALI mice. These protective effects are mediated via the modulation of the Toll-like receptor 4 (TLR4)/Kelch-like ECH-associated protein 1(Keap1)/Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. Molecular docking simulations identified the binding sites of AG on the TLR4 protein (Kd value: -33.5 kcal·mol-1), and these interactions were further corroborated by Cellular Thermal Shift Assay (CETSA) and SPR assays. Collectively, our findings demonstrate that AG exerts potent anti-inflammatory and anti-ferroptosis effects in LPS-induced ALI by targeting TLR4 and modulating the Keap1/Nrf2 pathway. This study underscores AG's potential as a therapeutic agent for ALI and provides new insights into its underlying mechanisms of action.

Gut microbiota-mediated C-sulfonate metabolism impairs the bioavailability and anti-cholestatic efficacy of andrographolide

ABSTRACT Cholestatic liver injury results from the accumulation of toxic bile acids in the liver, presenting a therapeutic challenge with no effective treatment available to date. Andrographolide (AP) has exhibited potential as a treatment for cholestatic liver disease. However, its limited oral bioavailability poses a significant obstacle to harnessing its potent therapeutic properties and restricts its clinical utility. This limitation is potentially attributed to the involvement of gut microbiota in AP metabolism. In our study, employing pseudo-germ-free, germ-free and strain colonization animal models, along with 16S rRNA and shotgun metagenomic sequencing analysis, we elucidate the pivotal role played by gut microbiota in the C-sulfonate metabolism of AP, a process profoundly affecting its bioavailability and anti-cholestatic efficacy. Subsequent investigations pinpoint a specific enzyme, adenosine-5’-phosphosulfate (APS) reductase, predominantly produced by Desulfovibrio piger, which catalyzes the reduction of SO4 2- to HSO3 −. HSO3 − subsequently interacts with AP, targeting its C=C unsaturated double bond, resulting in the formation of the C-sulfonate metabolite, 14-deoxy-12(R)-sulfo andrographolide (APM). Inhibition of APS reductase leads to a notable enhancement in AP bioavailability and anti-cholestatic efficacy. Furthermore, employing RNA sequencing analysis and farnesoid X receptor (FXR) knockout mice, our findings suggest that AP may exert its anti-cholestatic effects by activating the FXR pathway to promote bile acid efflux. In summary, our study unveils the significant involvement of gut microbiota in the C-sulfonate metabolism of AP and highlights the potential benefits of inhibiting APS reductase to enhance its therapeutic effects. These discoveries provide valuable insights into enhancing the clinical applicability of AP as a promising treatment for cholestatic liver injury.

Andrographolide sensitizes glioma to temozolomide by inhibiting DKK1 expression.

Temozolomide (TMZ) is the first-line chemotherapeutic drug for gliomas treatment. However, the clinical efficacy of TMZ in glioma patients was very limited. Therefore, it is urgently needed to discover a novel approach to increase the sensitivity of glioma cells to TMZ. Western blot, immunohistochemical staining, and qRT-PCR assays were used to explore the mechanisms underlying TMZ promoting DKK1 expression and andrographolide (AND) inhibiting DKK1 expression. HPLC was used to detect the ability of andrographolide (AND) to penetrate the blood-brain barrier. MTT assay, bioluminescence images, magnetic resonance imaging (MRI) and H&E staining were employed to measure the proliferative activity of glioma cells and the growth of intracranial tumors. TMZ can promote DKK1 expression in glioma cells and brain tumors of an orthotopic model of glioma. DKK1 could promote glioma cell proliferation and tumor growth in an orthotopic model of glioma. Mechanistically, TMZ increased EGFR expression and subsequently induced the activation of its downstream MEK-ERK and PI3K-Akt pathways, thereby promoting DKK1 expression in glioma cells. Andrographolide inhibited TMZ-induced DKK1 expression through inactivating MEK-ERK and PI3K-Akt pathways. Andrographolide can cross the blood-brain barrier, the combination of TMZ and andrographolide not only improved the anti-tumor effects of TMZ but also showed a survival benefit in an orthotopic model of glioma. Andrographolide can enhance anti-tumor activity of TMZ against glioma by inhibiting DKK1 expression.

RETRACTED: Andrographolide inhibits porcine epidemic diarrhea virus by inhibiting the JAK2-STAT3 pathway and promoting apoptosis

Porcine epidemic diarrhea (PED) is an acute, virulent, and highly contagious disease caused by the porcine epidemic diarrhea virus (PEDV). The high mutation rate of PEDV makes it difficult to effectively control using traditional vaccines, emphasizing the need for novel anti-PEDV-specific drugs. Therefore, this study aimed to investigate the activity and mechanism of action of andrographolide (AND) against PEDV in vitro and in vivo. In vitro experiments showed that AND treatment significantly inhibited PEDV replication in a cell model. The mechanism is that AND treatment significantly suppressed PEDV-induced activation of the JAK2-STAT3 pathway, which promoted apoptosis and inhibited the proliferation of the virus. Moreover, PEDV-infected 3-day-old piglets were treated with AND, and clinical symptoms, intestinal morphology, and viral load were examined. In vivo experiments showed that AND treatment reduced clinical symptoms, ameliorated intestinal damage, and increased the survival rate of infected piglets by 16.7 %. Conclusively, this study contributes to the field of PEDV antiviral drug development and provides new directions for PED prevention and treatment.

Fabrication and Characterization of Andrographolide-loaded Microsponges to Enhance Oral Bioavailability of Drug against Colon Cancer Using HT29 Cells

Background: The current research aimed to determine ways to improve the bioavailability of andrographolide (AGP) for use in colon cancer treatment by developing and evaluating microsponges loaded with the drug. Methods: Utilising the quasi-emulsion solvent diffusion approach, microsponges containing AGP were synthesised. A total of ten formulations were prepared using different concentrations of drug, polymer and other excipients. Particle size, shape, and differential scanning calorimetry (DSC) were used to characterise the microsponges that were created. To find out the rate at which the microsponges would expel their contents, researchers measured their release dynamics. In vitro anticancer activity of formulation was determined using HT29 cells Results: Results showed that the percentage yield of the formulations ranged from 10.85-41.03%. The highest drug concentration was achieved in formulation F8 with a particle size of 33.7 nm. SEM analysis demonstrated that the particles were round and possessed a rough and porous surface. Increasing the ratio of ethyl cellulose to AGP reduces surface roughness. The microsponge's DSC difractogram reveals prominent peaks at 18°, 24°, and 38° (2 θ) with reduced intensity, suggesting that the microsponges' crystalline character has diminished. In vitro drug release study showed 93.85% release upto 12 hours. Mathematical models showed normal release of the formulations with “n” values greater than 0.90 of all the formulations. Formulation F8 decreased the HT-29 cells' ability to survive. The percentage of cell cytotoxicity was 75.54 at 100μg/ml. Since AGP microsponges had a detrimental effect on the survival of colorectal cancer cells. Conclusion: It can be concluded from the study that prepared formulations possess anticancer properties against cancerous cells and can be used as an alternative anticancer drug.

Analytical method development and validation for simultaneous estimation of seven markers in polyherbal formulation JKC by using RP-HPLC

BackgroundThe RP-HPLC method has been established to simultaneous estimation of seven markers in polyherbal formulation JKC using the C18 (25 × 0.46 cm, i.d,5 µm) column. The mobile phase consisted of methanol: water (80:20) at a flow rate of 1.0 mL/min and observed retention time at 2 to 11 min with sharp points. The marker compounds viz. Andrographolide (AG), Piperine (PP), Picroside-I (P-I), Picroside-II (P-II), α-Cyprone (AC), 6-Shogaol (6S), and 6-Gingerol (6G) were quantified in JKC formulations by HPLC method. Detection was performed at the wavelength (λ) of 229 nm for AG, 343 nm for PP, 279 nm for P-I, 264 nm for P-II, 254 nm for AC, and 280 nm for both 6S and 6G by HPLC–PDA detector.ResultsThe marker compounds in JKC formulations were observed in different retention times (Rt) i.e. AG at 3.060 ± 0.01 min, PP at 5.460 ± 0.03 min, P-I at 2.789 ± 0.02 min, P-II at 2.553 ± 0.03 min, AC at 10.951 ± 0.02 min, 6S at 6.302 ± 0.03 min, and 6G at 4.111 ± 0.02 min respectively. The proposed method was validated with acceptable linearity (r2 0.9995–0.9999), precision, robustness, ruggedness, and accuracy (RSD < 2%) under optimum conditions. The limit of detection and quantification of bioactive markers were as: AG (1.386; 4.200 ppm), PP (2.033; 6.161 ppm), P-I (2.822; 8.553 ppm), P-II (2.538; 7.691 ppm), AC (0.269; 0.815 ppm), 6G (0.158; 0.480 ppm), 6S (0.188; 0.569 ppm). The amount (mg/g) of bioactive markers detected and estimated in plants and formulation were as: AG (41.282 ± 0.48; 10.06 ± 0.18), PP (53.81 ± 0.25, 13.82 ± 0.37 in PN, PL; 4.27 ± 0.07), P-I (15.97 ± 0.01; 0.48 ± 0.003), P-II (63.24 ± 0.35; 2.31 ± 0.006), AC (0.42 ± 0.01; 0.36 ± 0.006), 6G (0.71 ± 0.03; 0.16 ± 0.001), and 6S (2.64 ± 0.09; 0.12 ± 0.004) respectively. Method was found to be rugged and robust. The results found for all the validation parameters were within the limits according to ICH guidelines.ConclusionThe proposed method is fast, precise, economic, and specific and used for the simultaneously quantifiable analysis of seven major bioactive markers in the ingredients (herbs) and the JKC formulations.

Evaluation of Hepatoprotective activity of a Formulation containing Andrographolide (AGP) and Choline Chloride on Poultry Chickens

Evaluation of Hepatoprotective activity of a Formulation containing Andrographolide (AGP) and Choline Chloride on Poultry Chickens

Anti-inflammatory and anti-oxidative activities of andrographolide determined using atherosclerosis induced mice in Malaysia

Atherosclerosis and relative cardiovascular complications remain the main reasons for death worldwide. This study stimulated atherosclerosis in C57BL/6J mice using P-407 via intraperitoneal injection, and treatment with Andrographolide (AGP) (15, 30 and 45 mg/kg BW) was carried out for six weeks. The heart and aorta were harvested after six weeks and assessed using Enzyme-Linked Immunosorbent Assay (ELISA) and histological studies. The results demonstrated that the treatment with AGP reversed the effects of P-407 induced atherosclerosis. The doses of AGP correlated with the reduction of atherosclerosis biomarkers, and a high dose (45 mg/kg BW) was the most significant dose. The Low-Density Lipoprotein (LDL), Triglycerides (TG), and Atherogenic Index (AI) were significantly reduced by the AGP treatment. The histological results showed a reduction in inflammation, fibrosis and hypertrophy in the heart tissues of the groups treated with AGP compared to the disease control. In addition, AGP treatment significantly decreased Reactive Oxygen Species (ROS) and the inflammation marker (NF-kB). Furthermore, the AGP-treated groups showed typical morphological characteristics of the aorta, while the disease control cells were highly affected. The results demonstrated that AGP is highly recommended as a natural treatment to reduce the symptoms of atherosclerosis by reducing oxidative stress and inflammation.

Bletilla striata polysaccharide-coated andrographolide nanomicelles for targeted drug delivery to enhance anti-colon cancer efficacy.

Vitamin E, which is also known as tocopherol, is a compound with a polyphenol structure. Its esterified derivative, Vitamin E succinate (VES), exhibits unique anticancer and healthcare functions as well as immunomodulatory effects. Natural polysaccharides are proved to be a promising material for nano-drug delivery systems, which show excellent biodegradability and biocompatibility. In this study, we employed a novel bletilla striata polysaccharide-vitamin E succinate polymer (BSP-VES) micelles to enhance the tumor targeting and anti-colon cancer effect of andrographolide (AG). BSP-VES polymer was synthesized through esterification and its structure was confirmed using 1H NMR. AG@BSP-VES was prepared via the dialysis method and the drug loading, entrapment efficiency, stability, and safety were assessed. Furthermore, the tumor targeting ability of AG@BSP-VES was evaluated through targeted cell uptake and in vivo imaging. The antitumor activity of AG@BSP-VES was measured in vitro using MTT assay, Live&Dead cell staining, and cell scratch test. In this study, we successfully loaded AG into BSP-VES micelles (AG@BSP-VES), which exhibited good stability, biosafety and sustained release effect. In addition, AG@BSP-VES also showed excellent internalization capability into CT26 cells compared with NCM460 cells in vitro. Meanwhile, the specific delivery of AG@BSP-VES micelles into subcutaneous and in-situ colon tumors was observed compared with normal colon tissues in vivo during the whole experiment process (1-24 h). What's more, AG@BSP-VES micelles exhibited significant antitumor activities than BSP-VES micelles and free AG. The study provides a meaningful new idea and method for application in drug delivery system and targeted treatment of colon cancer based on natural polysaccharides.

Andrographolide influences IDD cell autophagy and oxidative stress under mechanical pressure via miR-9/FoxO3/PINK1/Parkin molecular axis.

Intervertebral disc degeneration (IDD) is characterized by the decreased function and number of nucleus pulposus cells (NPCs) caused by excessive intervertebral disc (IVD) pressure. This research aims to provide novel insights into IDD prevention and treatment by clarifying the effect of andrographolide (ANDR) on IDD cell autophagy and oxidative stress under mechanical stress. Human primary NPCs were extracted from the nucleus pulposus tissue of non-IDD trauma patients. An IDD cell model was established by posing mechanical traction on NPCs. Through the construction of an IDD rat model, the influence of ANDR on IDD pathological changes was explored in vivo. The proliferation and autophagy of NPCs were decreased while the apoptosis rate and oxidative stress reaction were increased by mechanical traction. ANDR intervention obviously alleviated this situation. MiR-9 showed upregulated expression in IDD cell model, while FoxO3 and PINK1/Parkin were downregulated. Decreased proliferation and autophagy as well as enhanced apoptosis and oxidative stress response of NPCs were observed following miR-9 mimics and H89 intervention, while the opposite trend was observed after FoxO3 overexpression. FoxO3 is a direct target downstream miR-9. The in vivo experiments revealed that after ANDR intervention, the number of apoptotic cells in rat IVD tissue decreased and the autophagy increased. In conclusion, ANDR improves NPC proliferation, and autophagy, inhibits apoptosis and oxidative stress, and alleviates the pathological changes of IDD via the miR-9/FoxO3/PINK1/Parkin axis, which may be a new and effective treatment for IDD in the future.

Polymer Matrix and Manufacturing Methods in Solid Dispersion System for Enhancing Andrographolide Solubility and Absorption: A Systematic Review.

Background: Andrographolide (ADG) has poor aqueous solubility and low bioavailability. This study systematically reviews the use of solid dispersion (SD) techniques to enhance the solubility and absorption of ADG, with a focus on the methods and polymers utilized. Methodology: We searched electronic databases including PubMed, Web of Science, Scopus®, Embase and ScienceDirect Elsevier® up to November 2023 for studies on the solubility or absorption of ADG in SD formulations. Two reviewers independently reviewed the retrieved articles and extracted data using a standardized form and synthesized the data qualitatively. Results: SD significantly improved ADG solubility with up to a 4.7-fold increase and resulted in a decrease in 50% release time (T1/2) to less than 5 min. SD could also improve ADG absorption, as evidenced by higher Cmax and AUC and reduced Tmax. Notably, Soluplus-based SDs showed marked solubility and absorption enhancements. Among the five SD techniques (rotary evaporation, spray drying, hot-melt extrusion, freeze drying and vacuum drying) examined, spray drying emerged as the most effective, enabling a one-step process without the need for post-milling. Conclusions: SD techniques, particularly using Soluplus and spray drying, effectively enhance the solubility and absorption of ADG. This insight is vital for the future development of ADG-SD matrices.

Andrographolide induced cytotoxicity and cell cycle arrest in Giardia trophozoites

Giardiasis is a prevalent parasitic diarrheal disease caused by Giardia lamblia, affecting people worldwide. Recently, the availability of several drugs for its treatment has highlighted issues such as multidrug resistance, limited effectiveness and undesirable side effects. Therefore, it is necessary to develop alternative new drugs and treatment strategies that can enhance therapeutic outcomes and effectively treat giardiasis. Natural compounds show promise in the search for more potent anti-giardial agents. Our investigation focused on the effect of Andrographolide (ADG), an active compound of the Andrographis paniculata plant, on Giardia lamblia, assessing trophozoite growth, morphological changes, cell cycle arrest, DNA damage and inhibition of gene expression associated with pathogenic factors. ADG demonstrated anti-Giardia activity almost equivalent to the reference drug metronidazole, with an IC50 value of 4.99 μM after 24 h of incubation. In cytotoxicity assessments and morphological examinations, it showed significant alterations in trophozoite shape and size and effectively hindered the adhesion of trophozoites. It also caused excessive ROS generation, DNA damage, cell cycle arrest and inhibited the gene expression related to pathogenesis. Our findings have revealed the anti-giardial efficacy of ADG, suggesting its potential as an agent against Giardia infections. This could offer a natural and low-risk treatment option for giardiasis, reducing the risk of side effects and drug resistance.

The Potential Anti-psoriatic Effects of Andrographolide: A Comparative Study to Topical Corticosteroids.

Andrographolide (AP), a bioactive anti-inflammatory compound of Sambiloto, inhibits NF-κB, TNF-α, and interleukin IL-6. Nowadays, molecular docking simulation between AP and dexamethasone against NF-κB receptor presented the energy AP higher than dexamethasone. This becomes a potential treatment for psoriasis. This manuscript reported the effectiveness of AP from Sambiloto in treating psoriasis compared to topical steroids. This study conducted TLC analysis of AP content and its metabolite impurities, emulgel formulation, molecular docking, in-silico skin toxicity study, and in-vivo anti-psoriatic activity. This was a combination study of an in-silico study and an in-vivo study. This in-silico study was analyzed through multivariate statistical analysis (PCA) to elucidate the data constellation relationship of andrographolide derivatives with several target proteins. The intervention was performed in seven days. The PASI score, molecular parameters (IL-6, IL-17, VEGF, and TNF-a levels), and histopathological findings were assessed. Molecular docking results revealed andrographolide to exhibit a relatively high binding affinity towards IL-6, NF-kB, and TNF-α which is comparable to the corticosteroids, andrographolide also shares similar residue interaction profile with each of the respective protein's native ligand. In the in-vivo study, we found several parameters statistically significantly different regarding the intervention, including final PASI score (p = 0.017), redness (p = 0.017), scale (p = 0.040), thickness (p = 0.023), total histopathology of psoriasis score (p = 0.037), keratin layer score (p = 0.018). Emulgel AP 0.1% could lower the anti-inflammatory agent, which is vital to psoriasis progression.

Andrographolide Attenuates NLRP3 Inflammasome Activation and Airway Inflammation in Exacerbation of Chronic Obstructive Pulmonary Disease.

The aim of this study is to uncover the anti-inflammatory propertity of andrographolide (AGP) in acute exacerbation of chronic obstructive pulmonary disease (AECOPD) and the underlying mechanisms related to the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome pathway. An in vivo experiment was conducted on murine model of AECOPD through endotracheal atomization of elastase and lipopolysaccharide (LPS). Intraperitoneal AGP was administered four times. NLRP3 inflammasome pathway molecules were examined using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. By using enzyme-linked immunosorbent assay (ELISA), we tested interleukin (IL)-1β levels in bronchoalveolar lavage fluid. An in vitro study was conducted to determine how AGP impacts the NLRP3 inflammasome in THP-1 derived macrophages. The levels of molecules involved in the pathway were measured. Furthermore, molecular docking analyses were carried out to investigate the interactions between AGP and pathway targets. In the in vivo study, NLRP3 inflammasome activation was observed in mice experiencing AECOPD. The administration of high-dose AGP demonstrated a mitigating effect on inflammatory cells infiltration in the lungs. Moreover, AGP administration effectively suppressed the expression of NLRP3, apoptosis associated speck-like protein that contains a CARD (PYCARD), cysteinyl aspartate-specific protease-1 (Caspase-1), IL-1β, and IL-18 at both the genetic and protein levels. In the in vitro experiment, IL-1β levels were significantly elevated in THP-1 derived macrophages with activated inflammasome compared to the control group. Furthermore, the downregulation of NLRP3, CASP1, and IL1B genes was observed upon the inhibition of NLRP3 expression through small interfering RNA (siRNA). AGP demonstrated inhibitory effects on the gene expression and protein levels of NLRP3, Caspase-1, and IL-1β. Additionally, molecular docking analysis confirmed that AGP exhibited a favorable binding affinity with all five targets of the pathway. AGP effectively inhibited NLRP3 inflammasome activation and mitigated the inflammatory reaction of AECOPD both in animal models and in vitro experiments, highlighting the potential of AGP as a treatment for AECOPD with anti-inflammatory properties.

HPTLC Stability Indicating Analytical Method of Andrographolide and 5-fluorouracil with Network Pharmacology Analysis against Cancer.

Herbal drugs when used in combination with chemotherapeutic drugs can reduce the side effects and increase the efficacy by acting on multiple targets. Andrographolide (AG), a diterpene lactone isolated from Andrographis paniculata Nees, is a bioactive compound with anticancer potential, and 5-fluorouracil (FU), a pyrimidine analogue, is used in the treatment of cancer. Both drugs are used to formulate combination nanoformulation to increase absorption, thereby increasing their oral bioavailability. The study aimed to develop and validate stability indicating simultaneous HPTLC method for quantification of FU and AG in combination nanoformulation along with in silico docking and network pharmacology analysis to understand the interaction between the drugs and cancer targets. Chromatographic separation was performed using mobile phase chloroform: methanol: formic acid (9: 0.5: 0.5, v/v/v) on HPTLC silica plates 60 F254 as a stationary phase using UV-Vis detector and HPTLC scanner at 254 nm. Further, in silico docking analysis was performed to predict the binding affinity of AG and FU with different proteins and network pharmacology to find out the exact biomolecular relationship of AG and FU in alleviating cancer. The data from the calibration curve showed a good linear regression relationship with r² = 0.9981 (FU) and r² = 0.9977 (AG) in the concentration range of 0.1-2.0 μg/mL. The developed method was validated according to the ICH guidelines. Stability studies showed changes in peak patterns and areas. Bioinformatic and network pharmacology analyses of AG and FU with target proteins and genes associated with cancer play a multimechanistic role in alleviating cancer. The developed method has been concluded to be robust, simple, precise, reproducible, accurate, and stability indicating for simultaneous quantification of AG and FU, and the molecular interaction studies have further indicated that the combination nanoformulation of AG and FU could be effective against cancer.

Abstract 5757: Graphene quantum dots-covered AuAg alloy nanoparticles as drug delivery platforms for anticancer applications

Abstract Cancer continues to be a global health challenge, with millions suffering from its various forms. Prostate cancer, in particular, emerges as a significant health risk for men. Originating in the prostate gland—a small organ nestled in the male pelvis—this type of cancer the second most common cause of cancer-related deaths among men in the U.S. Despite numerous medical breakthroughs over the years, finding an effective treatment for prostate cancer remains a challenge. Our research is venturing into a new frontier in the fight against prostate cancer, focusing on an innovative method, it explores the potential of nanoparticles for targeted drug delivery. This targeted approach promises to not only intensify the therapeutic effect, but also minimize adverse side effects on healthy cells, ensuring a more balanced and effective treatment regimen. A key in this study is the drug Andrographolide (ADG), which has been previously recognized for its robust anti-cancer properties. However, one of its limitations is its hydrophobicity, which makes it challenging to administer due to solubility concerns. To navigate this challenge, we are pioneering the synthesis of AuAg (gold and silver) graphene quantum dots (GQDs) alloy nanoparticles. AuAg particles, in have been the subject of numerous studies due to their profound impact on cellular processes. By integrating a protective layer of GQDs, we aim to boost the nanoparticles' stability, all while preventing any potential release of harmful metallic ions. The primary objective of this advanced approach is to augment the solubility of andrographolide, paving the way for its targeted and efficient delivery to PC-3 cells. An integral part of our research involves determining the IC50 value, which is pivotal in indicating the concentration at which the drug can inhibit the growth of 50% of prostate cancer cells. Once the IC50 value for the AuAgGQDs is firmly established, our focus will shift to evaluating its effects on the morphology of Red Blood Cells (RBCs). By encapsulating andrographolide within the designed nanoparticles, such as AuAgGQDs and their derivatives, we aim to observe changes in morphology. If there is a change in morphology, it indicates successful drug encapsulation. Through this research, we are not just seeking a viable treatment method, but also aiming to expand the horizons of drug delivery systems. The insights gained from this study could very well revolutionize our understanding of therapeutic potential and underscore the importance of nanoparticles in the realm of biomedical applications. Citation Format: Alondra Del Mar Veloz Bonilla, Brad Weiner, Gerardo Morell, Nataniel Medina-Berríos. Graphene quantum dots-covered AuAg alloy nanoparticles as drug delivery platforms for anticancer applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5757.

Andrographolide attenuates sepsis-induced acute kidney injury by inhibiting ferroptosis through the Nrf2/FSP1 pathway

Sepsis is a systemic inflammatory response syndrome caused by infection, which causes renal dysfunction known as sepsis-associated acute kidney injury (S-AKI). Ferroptosis is a form of lipid peroxidation dependent on iron and reactive oxygen species that differs from other forms of programmed cell death at the morphological and biochemical levels. Andrographolide (AG), a natural diterpenoid lactone compound extracted from Andrographis paniculata, has been shown to have therapeutic effects in kidney disease. In this study, we investigated the novel mechanism by which AG attenuates septic acute kidney injury by inhibiting ferroptosis in renal tubular epithelial cells (HK-2) through the Nrf2/FSP1 pathway. Cecum ligation and puncture (CLP)-induced septic rats and lipopolysaccharide (LPS)-induced HK-2 cells were used for in vivo and in vitro experiments. Firstly, in septic rats and HK-2 cells, AG effectively decreased the levels of kidney injury indicators, including blood creatinine, urea nitrogen, and markers of kidney injury such as neutrophil gelatinase-associated lipid transport protein and kidney injury molecule-1 (KIM-1). In addition, AG prevented ferroptotosis, by avoiding the accumulation of iron and lipid peroxidation, and an increase in SLC7A11 and GPX4 in AG-treated HK-2 cells. Furthermore, AG attenuated mitochondrial damage, including mitochondrial swelling, outer membrane rupture, and a reduction in mitochondrial cristae in LPS-treated HK-2 cells. Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, significantly inhibited LPS-induced ferroptosis in HK-2 cells. Importantly, our results confirm that Nrf2/FSP1 is an important pathway for ferroptosis resistance. Nrf2 siRNA hindered the effect of AG in attenuating acute kidney injury and inhibiting ferroptosis. These findings demonstrate that Nrf2/FSP1-mediated HK-2 ferroptosis is associated with AG, alleviates septic acute kidney injury, and indicates a novel avenue for therapeutic interventions in the treatment of acute kidney injury in sepsis.

ApCPS2 contributes to medicinal diterpenoid biosynthesis and defense against insect herbivore in Andrographis paniculata

Kalmegh (Andrographis paniculata) spatiotemporally produces medicinally-important ent-labdane-related diterpenoids (ent-LRDs); andrographolide (AD), 14-deoxy-11,12-didehydroandrographolide (DDAD), neoandrographolide (NAD). ApCPS1 and ApCPS2, the ent-copalyl pyrophosphate (ent-CPP)-producing class II diterpene synthases (diTPSs) were identified, but their contributions to ent-CPP precursor supply for ent-LRD biosynthesis were not well understood. Here, we characterized ApCPS4, an additional ent-CPP-forming diTPS. Further, we elucidated in planta function of the ent-CPP-producing diTPSs (ApCPS1,2,4) by integrating transcript-metabolite co-profiles, biochemical analysis and gene functional characterization. ApCPS1,2,4 localized to the plastids, where diterpenoid biosynthesis occurs in plants, but ApCPS1,2,4 transcript expression patterns and ent-LRD contents revealed a strong correlation of ApCPS2 expression and ent-LRD accumulation in kalmegh. ApCPS1,2,4 upstream sequences differentially activated β-glucuronidase (GUS) in Arabidopsis and transiently-transformed kalmegh. Similar to higher expression of ApCPS1 in kalmegh stem, ApCPS1 upstream sequence activated GUS in stem/hypocotyl of Arabidopsis and kalmegh. However, ApCPS2,4 upstream sequences weakly activated GUS expression in Arabidopsis, which was not well correlated with ApCPS2,4 transcript expression in kalmegh tissues. Whereas, ApCPS2,4 upstream sequences could activate GUS expression at a considerable level in kalmegh leaf and roots/calyx, respectively, suggesting the involvement of transcriptional regulator(s) of ApCPS2,4 that might participate in kalmegh-specific diterpenoid pathway. Interestingly, ApCPS2-silenced kalmegh showed a drastic reduction in AD, DDAD and NAD contents and compromised defense against insect herbivore Spodoptera litura. However, ent-LRD contents and herbivore defense in ApCPS1 or ApCPS4-silenced plants remained largely unaltered. Overall, these results suggested an important role of ApCPS2 in producing ent-CPP for medicinal ent-LRD biosynthesis and defense against insect herbivore.