Emerging Roles of Gossypol in Therapy: Innovations in Prodrug Design and Nanoformulation Frontiers.

Stimuli activatable systems have the potential to deliver drugs to targeted areas by releasing therapeutic agents in response to diseased specific microenvironments such as the acidic environment commonly found in diseased tissues. This review article focuses on gossypol, a bioactive compound with inherent toxicity due to its formyl groups. It highlights the potential of imine-linked gossypol-based prodrugs and nanoparticle formulations for targeted delivery and controlled release. The unique presence of polyphenolic cores on gossypol can be utilized to prepare nanoparticles. This review offers valuable insights into designing safer and more effective drug delivery systems by elucidating the masking effect and stimuli-responsive release mechanisms. Numerous examples demonstrate the conversion of formyl groups to imines, creating prodrugs that mask reactive functionalities and offer pH-responsive release. This insight can guide the design of combination therapeutics, where a second drug with an amine terminal group can form imine-linked prodrugs. Additionally, the second part discusses the use of polyphenolic moieties to create stable nanoparticles from infinite polymeric networks. Through a comprehensive examination of gossypol's properties and applications, this review emphasizes the broader implications of such a masking strategy for optimizing the therapeutic benefits of many similar bioactive compounds while minimizing adverse effects.

Fish gut-derived probiotic Pediococcus pentosaceus alleviates gossypol-induced intestinal inflammation by inhibiting NLRC3/NF-κB/IL-1ß signal pathway in Nile tilapia.

Cottonseed meal (CSM) and cottonseed protein concentrate (CPC) serve as protein alternatives to fish meal and soybean meal in the feed industry. However, the presence of gossypol residue in CSM and CPC can potentially trigger severe intestinal inflammation, thereby restricting the widespread utilization of these two protein sources. Probiotics are widely used to prevent or alleviate intestinal inflammation, but their efficacy in protecting fish against gossypol-induced enteritis remains uncertain. Here, the protective effect of Pediococcus pentosaceus, a strain isolated from the gut of Nile tilapia (Oreochromis niloticus), was evaluated. Three diets, control diet (CON), gossypol diet (GOS) and GOS supplemented with P. pentosaceus YC diet (GP), were used to feed Nile tilapia for 10 weeks. After the feeding trial, P. pentosaceus YC reduced the activity of myeloperoxidase (MPO) in the proximal intestine (PI) and distal intestine (DI). Following a 7-day exposure to Aeromonas hydrophila, the addition of P. pentosaceus YC was found to increase the survival rate of the fish. P. pentosaceus YC significantly inhibited the oxidative stress caused by gossypol, which was evidenced by lower reactive oxygen species (ROS) and malondialdehyde (MDA), as well as higher activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in PI and DI. Addition of P. pentosaceus YC significantly inhibited enteritis, with the lower expression of pro-inflammatory cytokines (il-1ß, il-6, il-8) and higher expression of anti-inflammatory cytokines tgf-ß. RNA-seq analysis indicated that P. pentosaceus YC supplementation significantly inhibited nlrc3 and promoted nf-κb expression in PI and DI, and the siRNA interference experiment in vivo demonstrated that intestinal inflammation was mediated by NLRC3/NF-κB/IL-1ß signaling pathway. Fecal bacteria transplantation experiment demonstrated that gut microbiota mediated the protective effect of P. pentosaceus YC. These findings offer valuable insights into the application of P. pentosaceus YC for alleviating gossypol-induced intestinal inflammation in fish.

[The anti-reproductive active compound gossypol: Progress in research].

Gossypol is a natural product extracted from cotton seeds, roots and stems, once used as a male contraceptive and later found with an anti-tumor effect. Recent studies show that it has an antiviral effect after structurally modified. This review focuses on the status quo of present studies on the effects of gossypol and its derivatives in anti-reproduction and anti-PCa, with an introduction of the application of the new compounds obtained from structural modification of gossypol in the treatment of PCa.

Rumen Microbiota Transplantation Alleviates Gossypol Diet-Induced Reproductive, Liver, and Intestinal Damage in Male Mice.

Ruminants exhibit stronger tolerance to gossypol, an anti-nutritional factor, compared to monogastric animals. We transplanted Hu sheep rumen microbiota into male mice to investigate the role of rumen microbiota in animal gossypol tolerance. Thirty specific-pathogen-free (SPF) male C57BL/6 mice were randomly divided into three groups: normal diet (CK group), gossypol diet (FG group), and rumen microbiota transplantation (FMT group, gossypol diet). The pathological changes in the liver and small intestine of the mice, the organ coefficient, and sperm parameters were analyzed. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the blood and lactate dihydrogen-X (LDH-X) levels in the testicular tissue were also measured. The results showed that body weight, feed intake, sperm concentration, sperm motility, and LDH-X levels in the FMT group increased (p < 0.05) compared with the FG group, while the enzyme activities of ALT, AST, and AST/ALT decreased (p < 0.05). In the FMT group, the injury to liver cells was alleviated, the structure of the small intestine was intact, and the villus height and the ratio of villus height to crypt depth (V/C) were higher than those in the FG group (p < 0.05). And there were no differences in various organ coefficients and sperm deformity rates among the three groups (p > 0.05), but compared with the FG group, mice in the FMT group showed tendencies closer to those in the CK group. Rumen microbiota transplantation relieved the reproductive toxicity and liver damage induced by gossypol in male mice and improved the tolerance of recipient animals to gossypol. Additionally, rumen microbes improved the intestinal structural integrity of recipients.

Blockade of CaV3 calcium channels and induction of G0/G1 cell cycle arrest in colon cancer cells by gossypol.

BACKGROUND AND PURPOSE: Gastrointestinal tumours overexpress voltage-gated calcium (CaV3) channels (CaV3.1, 3.2 and 3.3). CaV3 channels regulate cell growth and apoptosis colorectal cancer. Gossypol, a polyphenolic aldehyde found in the cotton plant, has anti-tumour properties and inhibits CaV3 currents. A systematic study was performed on gossypol blocking mechanism on CaV3 channels and its potential anticancer effects in colon cancer cells, which express CaV3 isoforms. EXPERIMENTAL APPROACH: Transcripts for CaV3 proteins were analysed in gastrointestinal cancers using public repositories and in human colorectal cancer cell lines HCT116, SW480 and SW620. The gossypol blocking mechanism on CaV3 channels was investigated by combining heterologous expression systems and patch-clamp experiments. The anti-tumoural properties of gossypol were estimated by cell proliferation, viability and cell cycle assays. Ca2+ dynamics were evaluated with cytosolic and endoplasmic reticulum (ER) Ca2+ indicators. KEY RESULTS: High levels of CaV3 transcripts correlate with poor prognosis in gastrointestinal cancers. Gossypol blockade of CaV3 isoforms is concentration- and use-dependent interacting with the closed, activated and inactivated conformations of CaV3 channels. Gossypol and CaV3 channels down-regulation inhibit colorectal cancer cell proliferation by arresting cell cycles at the G0/G1 and G2/M phases, respectively. CaV3 channels underlie the vectorial Ca2+ uptake by endoplasmic reticulum in colorectal cancer cells. CONCLUSION AND IMPLICATIONS: Gossypol differentially blocked CaV3 channel and its anticancer activity was correlated with high levels of CaV3.1 and CaV3.2 in colorectal cancer cells. The CaV3 regulates cell proliferation and Ca2+ dynamics in colorectal cancer cells. Understanding this blocking mechanism maybe improve cancer therapies.

Silica Gel Chromatographic Methods for Identification, Isolation and Purification of Gossypol Acetic Acid.

Several cottonseed varieties are cultivated in different countries. Each variety produces a different amount of gossypol as a natural toxic compound. The rising interest in cottonseed products (oil and feed) increases the demand for establishing simple methods for gossypol detection. Silica gel-based methods are ideal for its isolation, purification, and characterization. Silica gel-based methods are variants and can be used as simple methods for tracking plants' compounds. In this study, gossypol was isolated, characterized, and purified as gossypol acetic acid in the form of yellow crystals. Methods used for its characterization were TLC, preparative TLC, silica gel column, UV/IR spectrophotometer, and HPLC (robust spherical silica gel). A comparative study between its amount in both the Egyptian and Chinese varieties was performed. Under the experimental conditions, the Egyptian's cottonseed contains 8.705 gm/kg, while the Chinese's cottonseed contains 5.395 gm/kg. The TLC used in this study proved to be fast, accurate, and inexpensive. It can be used for gossypol acetic acid evaluation and quantification. Additionally, using TLC as a pre-purification step will give a pre-judgment for the sample's purity and quality. This step will protect the expensive HPLC silica gel-based column from any unexpected impurities. During each step, the silica gel itself could be simply removed by paper filtration. Collectively, the different silica gel-based methods as well as the other used methods are recommended for better Gossypol acetic acid isolation, purification, and characterization, as well as for maintaining HPLC columns.

Genetic Analysis of an F2 Population Derived from the Cotton Landrace Hopi Identified Novel Loci for Boll Glanding.

Landraces are an important reservoir of genetic variation that can expand the narrow genetic base of cultivated cotton. In this study, quantitative trait loci (QTL) analysis was conducted using an F2 population developed from crosses between the landrace Hopi and inbred TM-1. A high-density genetic map spanning 2253.11 and 1932.21 cM for the A and D sub-genomes, respectively, with an average marker interval of 1.14 cM, was generated using the CottonSNP63K array. The linkage map showed a strong co-linearity with the physical map of cotton. A total of 21 QTLs were identified, controlling plant height (1), bract type (1), boll number (1), stem color (2), boll pitting (2), fuzz fiber development (2), boll shape (3), boll point (4), and boll glanding (5). In silico analysis of the novel QTLs for boll glanding identified a total of 13 candidate genes. Analysis of tissue-specific expression of the candidate genes suggests roles for the transcription factors bHLH1, MYB2, and ZF1 in gland formation. Comparative sequencing of open reading frames identified early stop codons in all three transcription factors in Hopi. Functional validation of these genes offers avenues to reduce glanding and, consequently, lower gossypol levels in cottonseeds without compromising the defense mechanisms of the plant against biotic stresses.

Computational analysis of ligands from natural products on the cellular targets of combined hepatocellular carcinoma and cholangiocarcinoma.

Therapeutic effects of the bioactive compounds obtained from three common plants against the human combined hepatocellular carcinoma and cholangiocarcinoma (cHCC-CC) was explored in silico. These phytoconstituents viz. berberine, gossypol, and parthenolide were subjected for their drug likeliness, ADMET properties and molecular interactions to the cell surface receptors viz. FGFR1-4, VEGFR1-3, and PDGFR -A & -B. Interestingly, all these phytoconstituents had drug likeliness and ADMET properties similar to the anti-cancer drug, irinotecan. Gossypol exhibited binding energies -14.14 , -11.09, -13.49, -15.27, -14.51, -8.42, -14.72, and -9.39 kcal/mol on the cell receptors of human cHCC-CC viz. FGFR1, FGFR2, FGFR3, VEGFR1, VEGFR2, VEGFR3, PDGFRA, and PDGFRB, respectively. Whereas, berberine had binding energies -12.71 and -8.88 kcal/mol and -9.51 kcal/mol on the receptors viz. FGFR3, VEGFR3, and PDGFRB, respectively. The order of gossypol, berberine and parthenolide was determined as effective, whereas, the order of berberine, parthenolide and gossypol was found safer for human use.

MiR-214_L-1R+4 regulate gossypol-induced immune response through MyD88-dependent signaling pathway in Cyprinus carpio.

MicroRNAs (miRNAs) have been demonstrated to act as crucial modulators with considerable impacts on the immune system. Cottonseed meal is often used as a protein source in aqua feed, cottonseed meal contains gossypol, which is harmful to animals. However, there is a lack of research on the role of miRNAs in fish exposed to gossypol stress. To determine the regulatory effects of miRNAs on gossypol toxicity, Cyprinus carpio were given to oral administration of 20 mg/kg gossypol for 7 days, and the gossypol concentration in the tissues was tested. Then, we detected spleen index, histology, immune enzyme activities of fish induced by gossypol. The results of miRNA sequencing revealed 8 differentially expressed miRNAs in gossypol group, and miR-214_L-1R+4 was found involved in immune response induced by gossypol. The potential targets of miR-214_L-1R+4 were predicted, and found a putative miR-214_L-1R+4 binding site in the 3'UTR of MyD88a. Furthermore, dual-luciferase reporter assays displayed miR-214_L-1R+4 decreased MyD88a expression through binding to the 3'UTR of MyD88a. Moreover, miR-214_L-1R+4 antagomir were intraperitoneally administered to C. carpio, down-regulated miR-214_L-1R+4 could increase MyD88a expression, as well as inflammatory cytokines and anti-inflammatory cytokines expression. These findings revealed that miR-214_L-1R+4 via the MyD88-dependent signaling pathway modulate the immune response to gossypol in C. carpio spleen.

Effects of gossypol acetate on growth, serum biochemical parameters, and intestinal health of goslings.

Free gossypol (FG), the primary antinutritional component in cottonseed meal, can adversely affect the growth and health of poultry. Although younger geese are particularly sensitive to FG, the precise effects of FG on geese remain elusive. This study aimed to investigate the effects of gossypol acetate (GA), a form of FG, on the growth, serum biochemical parameters, and intestinal health of goslings. Seventy-two healthy male goslings, aged 7-day-old with similar body weight (BW), were randomly divided into 3 groups: a control group and 2 GA-treated groups (GA25 and GA50), which were orally administered GA (25 and 50 mg/kg BW) daily for 14 d. The results showed that oral administration of GA significantly suppressed BW, altered serum parameters, and impaired intestinal health in a dose- and time-dependent manner. Specifically, GA adversely affected intestinal morphology, induced oxidative stress, and inflammation, diminished immune function, and increased intestinal permeability and apoptosis of intestinal cells, consequently impairing nutrient absorption and utilization of goslings. Overall, these data indicate that GA adversely affects the growth, serum parameters, and intestinal health of goslings, providing valuable information further to understand the toxic effects of gossypol on goslings.

A Simple and Rapid Quantitative Assay for Gossypol via Reactive Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

The development of simple and rapid analytical tools for gossypol (GSP) is important to the food industry and medical field. Here, we report a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method for the detection of GSP by using a reactive matrix 4-hydrazinoquinazoline (4-HQ). The two aldehyde groups of GSP react with the 4-HQ and therefore improve the detection sensitivity and selectivity of GSP. Moreover, GSP forms homogeneous crystals with the 4-HQ matrix, allowing the quantification of the GSP by the proposed method. With the optimized experimental conditions, GSP could be detected at concentrations as low as 0.1 µM and quantified in a wide linear range (1-500 µM). After a brief extraction with an organic solvent, the GSP contents in cottonseeds and cottonseed kernels from different provinces of China were determined successfully. The spiked recovery of GSP in cottonseed/cottonseed kernel samples was obtained as 97.88-105.80%, showing the reliability of the assay for GSP determination in real samples.

Loss of Gcn2 exacerbates gossypol induced oxidative stress, apoptosis and inflammation in zebrafish.

Gossypol, a naturally occurring compound found in cottonseed meal, shows promising therapeutic potential for human diseases. However, within the aquaculture industry, it is considered an antinutritional factor. The incorporation of cottonseed meal into fish feed introduces gossypol, which induces intracellular stresses and hinders overall health of farmed fish. The aim of this study is to determine the role of General control nonderepressible 2 (gcn2), a sensor for intracellular stresses in gossypol-induced stress responses in fish. In the present study, we established two gcn2 knockout zebrafish lines. A feeding trial was conducted to assess the growth-inhibitory effect of gossypol in both wild type and gcn2 knockout zebrafish. The results showed that in the absence of gcn2, zebrafish exhibited increased oxidative stress and apoptosis when exposed to gossypol, resulting in higher mortality rates. In feeding trial, dietary gossypol intensified liver inflammation in gcn2-/- zebrafish, diminishing their growth and feed conversion. Remarkably, administering the antioxidant N-acetylcysteine (NAC) was effective in reversing the gossypol induced oxidative stress and apoptosis, thereby increasing the gossypol tolerance of gcn2-/- zebrafish. Exposure to gossypol induces more severe mitochondrial stress in gcn2-/- zebrafish, thereby inducing metabolic disorders. These results reveal that gcn2 plays a protective role in reducing gossypol-induced oxidative stress and apoptosis, attenuating inflammation responses, and enhancing the survivability of zebrafish in gossypol-challenged conditions. Therefore, maintaining appropriate activation of Gcn2 may be beneficial for fish fed diets containing gossypol.

Structural identification and comprehension of human ALDH1L1-Gossypol complex.

The folate metabolism enzyme ALDH1L1 catalyzed 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Non-small cell lung cancer cells (NSCLC) strongly express ALDH1L1. Gossypol binds to an allosteric site and disrupts the folate metabolism by preventing NADP+ binding. The Cryo-EM structures of tetrameric C-terminal aldehyde dehydrogenase human ALDH1L1 complex with gossypol were examined. Gossypol-bound ALDH1L1 interfered with NADP+ by shifting the allosteric site of the structural conformation, producing a closed-form NADP+ binding site. In addition, the inhibition activity of ALDH1L1 was targeted with gossypol in NSCLC. The gossypol treatment had anti-cancer effects on NSCLC by blocking NADPH and ATP production. These findings emphasize the structure characterizing ALDH1L1 with gossypol.

Targeting leucine-rich PPR motif-containing protein/LRPPRC by 5,7,4'-trimethoxyflavone suppresses esophageal squamous cell carcinoma progression.

JAK2/STAT3/MYC axis is dysregulated in nearly 70 % of human cancers, but targeting this pathway therapeutically remains a big challenge in cancer therapy. In this study, genes associated with JAK2, STAT3, and MYC were analyzed, and potential target genes were selected. Leucine-rich PPR motif-containing protein (LRPPRC) whose function and regulation are not fully understood, emerged as one of top 3 genes in terms of RNA epigenetic modification. Here, we demonstrate LRPPRC may be an independent prognostic indicator besides JAK2, STAT3, and MYC. Mechanistically, LRPPRC impairs N6-methyladenosine (m6A) modification of JAK2, STAT3, and MYC to facilitate nuclear mRNA export and expression. Meanwhile, excess LRPPRC act as a scaffold protein binding to JAK2 and STAT3 to enhance stability of JAK2-STAT3 complex, thereby facilitating JAK2/STAT3/MYC axis activation to promote esophageal squamous cell carcinoma (ESCC) progression. Furthermore, 5,7,4'-trimethoxyflavone was verified to bind to LRPPRC, STAT3, and CDK1, dissociating LRPPRC-JAK2-STAT3 and JAK2-STAT3-CDK1 interaction, leading to impaired tumorigenesis in 4-Nitroquinoline N-oxide induced ESCC mouse models and suppressed tumor growth in ESCC patient derived xenograft mouse models. In summary, this study suggests regulation of m6A modification by LRPPRC, and identifies a novel triplex target compound, suggesting that targeting LRPPRC-mediated JAK2/STAT3/MYC axis may overcome JAK2/STAT3/MYC dependent tumor therapeutic dilemma.

Gossypol acetic acid regulates leukemia stem cells by degrading LRPPRC via inhibiting IL-6/JAK1/STAT3 signaling or resulting mitochondrial dysfunction.

BACKGROUND: Leukemia stem cells (LSCs) are found to be one of the main factors contributing to poor therapeutic effects in acute myeloid leukemia (AML), as they are protected by the bone marrow microenvironment (BMM) against conventional therapies. Gossypol acetic acid (GAA), which is extracted from the seeds of cotton plants, exerts anti-tumor roles in several types of cancer and has been reported to induce apoptosis of LSCs by inhibiting Bcl2. AIM: To investigate the exact roles of GAA in regulating LSCs under different microenvironments and the exact mechanism. METHODS: In this study, LSCs were magnetically sorted from AML cell lines and the CD34+CD38- population was obtained. The expression of leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) and forkhead box M1 (FOXM1) was evaluated in LSCs, and the effects of GAA on malignancies and mitochondrial function were measured. RESULTS: LRPPRC was found to be upregulated, and GAA inhibited cell proliferation by degrading LRPPRC. GAA induced LRPPRC degradation and inhibited the activation of interleukin 6 (IL-6)/janus kinase (JAK) 1/signal transducer and activator of transcription (STAT) 3 signaling, enhancing chemosensitivity in LSCs against conventional chemotherapies, including L-Asparaginase, Dexamethasone, and cytarabine. GAA was also found to downregulate FOXM1 indirectly by regulating LRPPRC. Furthermore, GAA induced reactive oxygen species accumulation, disturbed mitochondrial homeostasis, and caused mitochondrial dysfunction. By inhibiting IL-6/JAK1/STAT3 signaling via degrading LRPPRC, GAA resulted in the elimination of LSCs. Meanwhile, GAA induced oxidative stress and subsequent cell damage by causing mitochondrial damage. CONCLUSION: Taken together, the results indicate that GAA might overcome the BMM protective effect and be considered as a novel and effective combination therapy for AML.

An Enzymatic and Proteomic Analysis of Panus lecomtei during Biodegradation of Gossypol in Cottonseed.

Cotton is an important plant-based protein. Cottonseed cake, a byproduct of the biodiesel industry, offers potential in animal supplementation, although the presence of the antinutritional sesquiterpenoid gossypol limits utilization. The macrofungus Panus lecomtei offers potential in detoxification of antinutritional factors. Through an enzymatic and proteomic analysis of P. lecomtei strain BRM044603, grown on crushed whole cottonseed contrasting in the presence of free gossypol (FG), this study investigated FG biodegradation over a 15-day cultivation period. Fungal growth reduced FG to levels at 100 µg/g, with a complex adaptive response observed, involving primary metabolism and activation of oxidative enzymes for metabolism of xenobiotics. Increasing activity of secreted laccases correlated with a reduction in FG, with enzyme fractions degrading synthetic gossypol to trace levels. A total of 143 and 49 differentially abundant proteins were observed across the two contrasting growth conditions after 6 and 12 days of cultivation, respectively, revealing a dynamic protein profile during FG degradation, initially related to constitutive metabolism, then later associated with responses to oxidative stress. The findings advance our understanding of the mechanisms involved in gossypol degradation and highlight the potential of P. lecomtei BRM044603 in cotton waste biotreatment, relevant for animal supplementation, sustainable resource utilization, and bioremediation.

Keap1 Negatively Regulates Transcription of Three Counter-Defense Genes and Susceptibility to Plant Toxin Gossypol in Helicoverpa armigera.

Expressions of a wide range of cytoprotective counter-defense genes are mainly regulated by the Keap1-Nrf2-ARE signaling pathway in response to oxidative stress from xenobiotics. Gossypol is the major antiherbivore secondary metabolite of cotton, but how the polyphagous pest Helicoverpa armigera copes with this phytochemical to utilize its favorite host plant cotton remains largely elusive. In this study, we first suppressed the Keap1 gene in newly hatched larvae of cotton bollworm by feeding them the siRNA diet for 4 days. All of the larvae were subsequently fed the artificial diet supplied with gossypol or the control diet for 5 days. We identified that the knockdown of the Keap1 gene significantly decreased larval mortality and significantly increased the percentages of larval survival, reaching the fourth instar, compared with ncsiRNA when exposed to a diet containing gossypol. Three counter-defense genes CYP9A17, CYP4L11 and UGT41B3, which were related to the induction or metabolism of gossypol according to the report before, were all significantly up-regulated after the knockdown of the Keap1 gene. The Antioxidant Response Elements (AREs) were also detected in the promoter regions of the three counter-defense genes above. These data indicate that the suppression of the Keap1 gene activates the Keap1-Nrf2-ARE signaling pathway, up-regulates the expressions of counter-defense genes involved in the resistance of oxidative stress and finally contributes to reducing the susceptibility of gossypol. Our results provide more knowledge about the transcriptional regulation mechanisms of counter-defense genes that enable the cotton bollworm to adapt to the diversity of host plants including cotton.

A comprehensive tool in recycling plant-waste of Gossypium barbadense L agricultural and industrial waste extracts containing gossypin and gossypol: hepatoprotective, anti-inflammatory and antioxidant effects.

Improper management of agricultural and industrial cotton wastes causes environmental pollution and worsens the climate change challenge. Green recycling of cotton could contribute to a circular economy. One of the economic values of cotton wastes lies in their bioactive components. Two types of cotton wastes-agricultural and industrial-of the species Gossypium barbadense L. Giza 95 were targeted in the current study, aiming to maximize their medicinal value and investigate the anti-inflammatory, hepatoprotective, and antioxidant activities of their phytochemical extracts. Phytochemical extraction was performed using different solvents extraction. An anti-inflammatory effect was tested in carrageenan-induced acute edema in a rat paw model. A carbon tetrachloride chronic model of liver injury was used for the assessment of hepatoprotective potential. Liver enzymes (AST and ALT), oxidative stress markers (MDA and GSH), inflammatory biomarkers (C-reactive protein), and histopathological features were investigated. As a result, ethyl acetate proved to be the solvent of best choice to extract the gossypin polyphenolics, where the extracted amount reached 14,826.2 µg/g, followed by butanol (8751.4 µg/g extract). The chloroform (CHCL3) fraction showed the highest amounts of gossypol (190.7 µg/g extract), followed by petroleum ether. Cotton waste's composition analysis showed a wide range of components, including 33 metabolites such as gossypetin, polyphenolics, and other metabolites that possess therapeutic effects. Both chloroform extract and industrial waste extracts showed superior anti-inflammatory and hepatoprotective effects in comparison to other extracts. All tested extracts (ethyl acetate, chloroform, and industrial waste) showed proper antioxidant activities.