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Betulinic acid (BA) is a lupinane-type pentacyclic triterpenoid natural product derived from lupeol that has favorable anti-inflammatory and anti-tumor activities. Currently, BA is mainly produced via botanical extraction, which significantly limits its widespread use. In this study, we investigated the de novo synthesis of BA in Saccharomyces cerevisiae, and to facilitate the synthesis and storage of hydrophobic BA, we adopted a dual-engineering strategy involving peroxisomes and lipid droplets to construct the BA biosynthetic pathway. By expressing Betula platyphylla-derived lupeol C-28 oxidase (BPLO) and Arabidopsis-derived ATR1, we succeeded in developing a BA-producing strain and following multiple expression optimizations of the linker between BPLO and ATR1, the BA titer reached 77.53 mg/L in shake flasks and subsequently reached 205.74 mg/L via fed-batch fermentation in a 5-L bioreactor. In this study, we developed a feasible approach for the de novo synthesis of BA and its direct precursor lupeol in engineered S. cerevisiae., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

During in vitro maturation, excess levels of reactive oxygen species (ROS) are a major cause of developmental defects in embryos. Betulinic acid (BA) is a naturally produced antioxidant in white birch bark. Recent studies have shown that BA exhibits antioxidant properties in various cells through the activation of antioxidant genes. Therefore, we investigated the effect of BA treatment on porcine oocytes and its underlying mechanism during oocyte maturation. Treatment with 0.1 μM BA significantly increased the proportion of MII oocytes compared with controls, and BA-treated oocytes had significantly higher development rates, trophectoderm cell numbers, and cell survival rates than controls. These results demonstrate that BA treatment improved the developmental competence of oocytes. Following BA treatment, oocytes exhibited reduced ROS levels and elevated glutathione (GSH) levels, accompanied by the enhanced expression of antioxidant genes, compared with control oocytes. To evaluate the antioxidant effects of BA, oocytes were exposed to H2O2, a potent ROS activator. Impaired nuclear maturation, ROS levels, and GSH levels induced in oocytes by H2O2 exposure was restored by BA treatment. As these antioxidant genes are regulated by the Nrf2/Keap1 signaling pathway, which is involved in antioxidant responses, we applied the Nrf2 inhibitor brusatol to investigate the effects of BA on this pathway. The negative effects of brusatol on meiotic maturation and oocyte quality, including levels of ROS, GSH, and antioxidant-related gene expression, were mitigated by BA treatment. Our results suggested that BA plays an effective role as an antioxidant in porcine oocyte maturation through adjusting the Nrf2/Keap1 signaling pathway. This finding provides valuable insights into the mechanisms governing oocyte maturation and embryonic development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Leea indica (Burm. f.) Merr. (Vitaceae) is used for the treatment of wounds in traditional medicine practiced in Sri Lanka. The current study is carried out to investigate its wound healing potential in terms of in vitro cell migration and proangiogenic activity. The scratch wound assay (SWA) guided fractionation of dichloromethane extract of L. indica led to the isolation of a rare phytosterol, stigmast-5-en-3 β ,7 α ,22 α -triol ( 1 ), betulin ( 2 ), lupeol ( 3 ), and β -sitosterol ( 4 ) all of which showed enhanced cell migration in SWA and significant proangiogenic response in chorioallantoic membrane (CAM) assay. The identities of compounds 1 - 4 were established by the analysis of NMR spectroscopic data and comparison with those reported. This is the first report of the occurrence of compounds 1 and 2 in L. indica .

Betulinic acid (BA) is a natural triterpenoid extracted from Bacopa monnieri. BA has been reported to be used as a neuroprotective agent, but their molecular mechanisms are still unknown. Therefore, in this study, we attempted to investigate the precise mechanism of BA for its protective effect against Titanium dioxide nanoparticles (TiO2NP) induced neurotoxicity in zebrafish. Hence, our study observation showed that 10 µg/ml dose of TiO2NP caused a rigorous behavioral deficit in zebrafish. Further, biochemical analysis revealed TiO 2 NP significantly decreased GSH, and SOD, and increased MDA, AChE, TNF-α, IL-1β, and IL-6 levels, suggesting it triggers oxidative stress and neuroinflammation. However, BA at doses of 2.5,5,10 mg/kg improved behavioral as well as biochemical changes in zebrafish brain. Moreover, BA also significantly raised the levels of DA, NE, 5-HT, and GABA and decreased glutamate levels in TiO 2 NP-treated zebrafish brain. Our histopathological analysis proved that TiO 2 NP causes morphological changes in the brain. These changes were expressed by increasing pyknotic neurons, which were dose-dependently reduced by Betulinic acid. Likewise, BA upregulated the levels of NRF-2 and HO-1, which can reduce oxidative stress and neuroinflammation. Thus, our study provides evidence for the molecular mechanism behind the neuroprotective effect of Betulinic acid. Rendering to the findings, we can consider BA as a suitable applicant for the treatment of AD-like symptoms., (Copyright © 2024. Published by Elsevier B.V.)

Background: Betulinic acid (BA), which is a pentacyclic triterpenoid found in the bark of plane, birch, and eucalyptus trees, has emerged as a compound of significant interest in scientific research due to its potential therapeutic applications. BA has a range of well-documented pharmacological and biological effects, including antibacterial, immunomodulatory, diuretic, antiviral, antiparasitic, antidiabetic, and anticancer activities. Although numerous research studies have explored the potential anticancer effects of BA, there is a noticeable gap in the literature, highlighting the need for a more up-to-date and comprehensive evaluation of BA's anticancer potential., Purpose: The aim of this work is to critically assess the reported cellular and molecular mechanisms underlying the cancer preventive and therapeutic effects of BA., Methods: Relevant research on the inhibitory effects of BA against cancerous cells was searched using Science Direct, Scopus, Web of Science, and PubMed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines., Results: The anticancer properties of BA are mediated by the activation of cell death and cell cycle arrest, production of reactive oxygen species, increased mitochondrial permeability, modulation of nuclear factor-κB and Bcl-2 family signaling. Emerging evidence also underscores the combined anticancer effects of BA with other natural bioactive compounds or approved drugs. Notably, several novel BA nanoformulations have been found to exhibit encouraging antineoplastic activities., Conclusion: BA, whether used alone or in combination, or as a form of nanoformulation, shows significant potential for cancer prevention and treatment. Nevertheless, further detailed studies are necessary to confirm the therapeutic effectiveness of this natural compound., Competing Interests: Declaration of competing interest Given their role as Editor/Associate Editor/Editorial board members, Anupam Bishayee had no involvement in the peer-review of this article and has no access to information regarding its peer-review., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Purpose of Review: This review evaluates the therapeutic potential of Ziziphus jujuba and its main components in managing complications of metabolic syndrome, including diabetes, dyslipidemia, obesity, and hypertension., Recent Findings: The reviewed studies provide evidence supporting the use of Z. jujuba and its main components (lupeol and betulinic acid) as natural treatments for complications of metabolic syndrome. These substances enhance glucose uptake through the activation of signaling pathways such as phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), reduce hepatic glucose synthesis, and increase glucose uptake by adipocytes and skeletal muscle cells. They also improve insulin sensitivity by modulating AMP-activated protein kinase (AMPK) activity and regulating insulin signaling proteins and glucose transporters. In the field of dyslipidemia, they inhibit triglyceride synthesis, lipid accumulation, and adipogenic enzymes, while influencing key signaling pathways involved in adipogenesis. Z. jujuba and its constituents demonstrate anti-adipogenic effects, inhibiting lipid accumulation and modulating adipogenic enzymes and transcription factors. They also exhibit positive effects on endothelial function and vascular health by enhancing endothelial nitric oxide synthase (eNOS) expression, NO production, and antioxidant enzyme activity. Z. jujuba, lupeol, and betulinic acid hold promise as natural treatments for complications of metabolic syndrome. They improve glucose metabolism, insulin sensitivity, and lipid profiles while exerting anti-adipogenic effects and enhancing endothelial function. However, further research is needed to elucidate the mechanisms and confirm their efficacy in clinical trials. These natural compounds offer potential as alternative therapies for metabolic disorders and contribute to the growing body of evidence supporting the use of natural medicines in their management., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Betulinic acid (BA) has been well investigated for its antiproliferative and mitochondrial pathway-mediated apoptosis-inducing effects on various cancers. However, its poor solubility and off-target activity have limited its utility in clinical trials. Additionally, the immune modulatory role of betulinic acid analogue in the tumor microenvironment (TME) is largely unknown. Here, we designed a potential nanotherapy for colorectal cancer (CRC) with a lead betulinic acid analogue, named as 2c, carrying a 1,2,3-triazole-moiety attached to BA through a linker, found more effective than BA for inhibiting CRC cell lines, and was chosen here for this investigation. Epithelial cell adhesion molecule (EpCAM) is highly overexpressed on the CRC cell membrane. A single-stranded short oligonucleotide sequence, aptamer (Apt), that folds into a 3D-defined architecture can be used as a targeting ligand for its specific binding to a target protein. EpCAM targeting aptamer was designed for site-specific homing of aptamer-conjugated-2c-loaded nanoparticles (Apt-2cNP) at the CRC tumor site to enhance therapeutic potential and reduce off-target toxicity in normal cells. We investigated the in vitro and in vivo therapeutic efficacy and anti-tumorigenic immune response of aptamer conjugated nanotherapy in CRC-TME., Methods: After the characterization of nanoengineered aptamer conjugated betulinic acid nanotherapy, we evaluated therapeutic efficacy, tumor targeting efficiency, and anti-tumorigenic immune response using cell-based assays and mouse and rat models., Results: We found that Apt-2cNP improved drug bioavailability, enhanced its biological half-life, improved antiproliferative activity, and minimized off-target cytotoxicity. Importantly, in an in vivo TME, Apt-2cNP showed promising signs of anti-tumorigenic immune response (increased mDC/pDC ratio, enhanced M1 macrophage population, and CD8 T-cells). Furthermore, in vivo upregulation of pro-apoptotic while downregulation of anti-apoptotic genes and significant healing efficacy on cancer tissue histopathology suggest that Apt-2cNP had predominantly greater therapeutic potential than the non-aptamer-conjugated nanoparticles and free drug. Moreover, we observed greater tumor accumulation of the radiolabeled Apt-2cNP by live imaging in the CRC rat model., Conclusions: Enhanced therapeutic efficacy and robust anti-tumorigenic immune response of Apt-2cNP in the CRC-TME are promising indicators of its potential as a prospective therapeutic agent for managing CRC. However, further studies are warranted., (© 2024. The Author(s).)

Cyclophosphamide (CYP) is a chemotherapeutic drug used to treat various cancers. However, its clinical use is limited due to severe organ damage, particularly to the kidneys. While several phytochemicals have been identified as potential therapeutic targets for CYP nephrotoxicity, the nephroprotective effects of boswellic acid (BOSW) and betulinic acid (BET) have not yet been investigated. Our study used 42 rats divided into six equal groups. The study included six groups: control, CYP (200 mg/kg), CYP+BOSW20 (20 mg/kg), CYP+BOSW40 (40 mg/kg), CYP+BET20 (20 mg/kg), and CYP+BET40 (40 mg/kg). The pre-treatments with BOSW and BET lasted for 14 days, while the application of cyclophosphamide was performed intraperitoneally only on the 4th day of the study. After the experimental protocol, the animals were sacrificed, and their kidney tissues were isolated. Renal function parameters, histological examination, oxidative stress, and inflammation parameters were assessed both biochemically and at the molecular level in kidney tissue. The results showed that oxidative stress and inflammatory response were increased in the kidney tissue of rats treated with CYP, leading to impaired renal histology and function parameters (p < 0.05). Oral administration of both doses of BET and especially high doses of BOSW improved biochemical, oxidative, and inflammatory parameters significantly (p < 0.05). Histological studies also showed the restoration of normal kidney tissue architecture. BOSW and BET have promising biological activity against CYP-induced nephrotoxicity by attenuating inflammation and oxidative stress and enhancing antioxidant status., (© 2024. Pleiades Publishing, Ltd.)

Context: Obesity is related to insulin resistance, and adipose tissue-secreted TNF- α may play a role in inducing obesity. TNF- α activates inflammatory protein kinase and impairs insulin signalling., Objectives: We investigated the effect of betulinic acid on insulin resistance caused by TNF- α treatment in 3T3-L1 adipocytes., Material and Methods: 3T3-L1 was exposed to TNF- α in the presence and absence of betulinic acid. Various parameters such as glucose uptake assay, cell viability, expression of proteins involved in insulin resistance were studied., Results: Betulinic acid increased glucose uptake in TNF- α pre-treated cells and inhibited the activation of PTP1B and JNK and reduced IκB α degradation. Tyrosine phosphorylation was increased, and serine phosphorylation was decreased in IRS-1., Discussion: Betulinic acid restored TNF- α impaired insulin signalling and increased PI3K activation and phosphorylation of Akt and increased plasma membrane expression of GLUT 4, which stimulated glucose uptake concentration-dependently., Conclusion: These results suggest that betulinic acid is effective at improving TNF- α -induced insulin resistance in adipocytes via inhibiting the activation of negative regulator of insulin signalling and inflammation-activated protein kinase and may potentially improve insulin resistance.

Betulinic acid is a lupane-type pentacyclic triterpene mostly found in birch bark and thoroughly explored for its wide range of pharmacological activities. Despite its impressive biological potential, its low bioavailability has challenged many researchers to develop different formulations for achieving better in vitro and in vivo effects. We previously reported the synthesis of fatty acid esters of betulinic acid using butyric, stearic, and palmitic acids (But-BA, St-BA, and Pal-BA) and included them in surfaced-modified liposomes (But-BA-Lip, St-BA-Lip, Pal-BA-Lip). In the current study, we evaluated the cytotoxic effects of both fatty acid esters and their respective liposomal formulations against MCF-7, HT-29, and NCI-H460 cell line. The cytotoxic assessment of BA derivatives revealed that both the fatty esters and their liposomal formulations acted as cytotoxic agents in a dose- and time-dependent manner. But-BA-Lip exerted stronger cytotoxic effects than the parent compound, BA and its liposomal formulation, and even stronger effects than 5-FU against HT-29 cells (IC 50 of 30.57 μM) and NCI-H460 cells (IC 50 of 30.74 μM). BA's fatty esters and their respective liposomal formulations facilitated apoptosis in cancer cells by inducing nuclear morphological changes and increasing caspase-3/-7 activity. The HET-CAM assay proved that none of the tested compounds induced any irritative effect, suggesting that they can be used safely for local applications.

Betulinic acid (BA) is a lupane-type triterpenoid with potent anticancer and anti-HIV activities. Its great potential in clinical applications necessitates the development of an efficient strategy for BA synthesis. This study attempted to achieve efficient BA biosynthesis in Saccharomyces cerevisiae using systematic metabolic engineering strategies. First, a de novo BA biosynthesis pathway in S. cerevisiae was constructed, which yielded a titer of 14.01 ± 0.21 mg/L. Then, by enhancing the BA synthesis pathway and dynamic inhibition of the competitive pathway, a greater proportion of the metabolic flow was directed toward BA synthesis, achieving a titer of 88.07 ± 5.83 mg/L. Next, acetyl-CoA and NADPH supply was enhanced, which increased the BA titer to 166.43 ± 1.83 mg/L. Finally, another BA synthesis pathway in the peroxisome was constructed. Dual regulation of the peroxisome and cytoplasmic metabolism increased the BA titer to 210.88 ± 4.76 mg/L. Following fed-batch fermentation process modification, the BA titer reached 682.29 ± 8.16 mg/L. Overall, this work offers a guide for building microbial cell factories that are capable of producing terpenoids with efficiency.

The demand for antibiofilm molecules has increased over several years due to their potential to fight biofilm-associated infections, such as those including the interkingdom Staphylococcus aureus-Candida albicans occurring in clinical settings worldwide. Recently, we identified a pentacyclic triterpenoid compound, betulinic acid, from invasive macrophytes, with interesting antibiofilm properties. The aim of the present study was to provide insights into the mechanism of action of betulinic acid against the clinically relevant bi-species S. aureus-C. albicans biofilms. Microscopy examinations, flow cytometry and crystal violet assays confirmed that betulinic acid was effective at damaging mature S. aureus-C. albicans biofilms or inhibiting their formation, reducing biofilm biomass by 70% on average and without microbicidal activity. The results suggested an action of betulinic acid on cell membranes, inducing changes in properties such as composition, hydrophobicity and fluidity as observed in C. albicans, which may hinder the early adhesion step, biofilm growth and the physical interactions of both microbial species. Further results of real-time polymerase chain reaction argued in favour of a reduction in S. aureus-C. albicans physical interaction due to betulinic acid by the modulation of biofilm-related gene expression, as observed in early stages of biofilm formation. This study revealed the potential of betulinic acid as a candidate agent for the prevention and treatment of S. aureus-C. albicans biofilm-related infections., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Betulinic acid (BA), a naturally occurring lupane-type triterpenoid, possesses a wide range of potential activities against different types of cancer. However, the molecular mechanisms involved in anti-cervical cancer about BA were rarely investigated. Herein, the role of BA in cervical cancer suppression by ROS-mediated endoplasmic reticulum stress (ERS) and autophagy was deeply discussed. The findings revealed that BA activated Keap1/Nrf2 pathway and triggered mitochondria-dependent apoptosis due to ROS production. Furthermore, BA increased the intracellular Ca 2+ levels, inhibited the expression of Beclin1 and promoted the expression of GRP78, LC3-II, and p62 associated with ERS and autophagy. Besides, BA initiated the formation of autophagosomes and inhibited autophagic flux by the co-administration of BA with 3-methyladenine (3-MA) and chloroquine (CQ), respectively. The in vivo experiment manifested that hydroxychloroquine (HCQ) enhanced the apoptosis induced by BA. For the first time, we demonstrated that BA could initiate early autophagy, inhibit autophagy flux, and induce protective autophagy in HeLa cells. Thus, BA could be a potential chemotherapy drug for cervical cancer, and inhibition of autophagy could enhance the anti-tumor effect of BA. However, the interactions of signaling factors between ERS-mediated and autophagy-mediated apoptosis deserve further attention., (© 2024. The Author(s) under exclusive licence to The Japanese Society of Pharmacognosy.)

Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of lung cancer patients with mutated EGFR. However, the efficacy of EGFR-TKIs in wild-type EGFR tumors has been shown to be marginal. Methods that can sensitize EGFR-TKIs to EGFR wild-type NSCLC remain rare. Hence, we determined whether combination treatment can maximize the therapeutic efficacy of EGFR-TKIs., Methods: We established a focused drug screening system to investigate candidates for overcoming the intrinsic resistance of wild-type EGFR NSCLC to EGFR-TKIs. Molecular docking assays and western blotting were used to identify the binding mode and blocking effect of the candidate compounds. Proliferation assays, analyses of drug interactions, colony formation assays, flow cytometry and nude mice xenograft models were used to determine the effects and investigate the molecular mechanism of the combination treatment., Results: Betulinic acid (BA) is effective at targeting EGFR and synergizes with EGFR-TKIs (gefitinib and osimertinib) preferentially against wild-type EGFR. BA showed inhibitory activity due to its interaction with the ATP-binding pocket of EGFR and dramatically enhanced the suppressive effects of EGFR-TKIs by blocking EGFR and modulating the EGFR-ATK-mTOR axis. Mechanistic studies revealed that the combination strategy activated EGFR-induced autophagic cell death and that the EGFR-AKT-mTOR signaling pathway was essential for completing autophagy and cell cycle arrest. Activation of the mTOR pathway or blockade of autophagy by specific chemical agents markedly attenuated the effect of cell cycle arrest. In vivo administration of the combination treatment caused marked tumor regression in the A549 xenografts., Conclusions: BA is a potential wild-type EGFR inhibitor that plays a critical role in sensitizing EGFR-TKI activity. BA combined with an EGFR-TKI effectively suppressed the proliferation and survival of intrinsically resistant lung cancer cells via the inhibition of EGFR as well as the induction of autophagy-related cell death, indicating that BA combined with an EGFR-TKI may be a potential therapeutic strategy for overcoming the primary resistance of wild-type EGFR-positive lung cancers., (© 2024. The Author(s).)

Background: Gray horses are predisposed to equine malignant melanoma (EMM) with advancing age. Depending on the tumor's location and size, they can cause severe problems (e.g., defaecation, urination, feeding). A feasible therapy for EMM has not yet been established and surgical excision can be difficult depending on the location of the melanoma. Thus, an effective and safe therapy is needed. Naturally occurring betulinic acid (BA), a pentacyclic triterpene and its synthetic derivate, NVX-207 (3-acetyl-betulinic acid-2-amino-3-hydroxy-2-hydroxymethyl-propanoate) are known for their cytotoxic properties against melanomas and other tumors and have already shown good safety and tolerability in vivo. In this study, BA and NVX-207 were tested for their permeation potential into equine skin in vitro in Franz-type diffusion cell (FDC) experiments after incubation of 5 min, 30 min and 24 h, aiming to use these formulations for prospective in vivo studies as a treatment for early melanoma stages. Potent permeation was defined as reaching or exceeding the half maximal inhibitory concentrations (IC 50 ) of BA or NVX-207 for equine melanoma cells in equine skin samples. The active ingredients were either dissolved in a microemulsion (ME) or in a microemulsion gel (MEG). All of the formulations were transdermally applied but the oil-in-water microemulsion was administered with a novel oxygen flow-assisted (OFA) applicator (DERMADROP TDA)., Results: All tested formulations exceeded the IC 50 values for equine melanoma cells for BA and NVX-207 in equine skin samples, independently of the incubation time NVX-207 applied with the OFA applicator showed a significant time-dependent accumulation and depot-effect in the skin after 30 min and 24 h (P < 0.05)., Conclusions: All tested substances showed promising results. Additionally, OFA administration showed a significant accumulation of NVX-207 after 30 min and 24 h of incubation. Further in vivo trials with OFA application are recommended., (© 2024. The Author(s).)

The abnormal activation of the mammalian target of rapamycin(mTOR) signaling pathway in non-small cell lung cancer(NSCLC) is closely associated with distant metastasis, drug resistance, tumor immune escape, and low overall survival. The present study reported that betulinic acid(BA), a potent inhibitor of mTOR signaling pathway, exhibited an inhibitory activity against NSCLC in vitro and in vivo. CCK-8 and colony formation results demonstrated that BA significantly inhibited the viability and clonogenic ability of H1299, A549, and LLC cells. Additionally, the treatment with BA induced mitochondrion-mediated apoptosis of H1299 and LLC cells. Furthermore, BA inhibited the mobility and invasion of H1299 and LLC cells by down-regulating the expression level of matrix metalloproteinase 2(MMP2) and impairing epithelial-mesenchymal transition. The results demonstrated that the inhibition of mTOR signaling pathway by BA decreased the proportion of M2 phenotype(CD206 positive) cells in total macrophages. Furthermore, a mouse model of subcutaneous tumor was established with LLC cells to evaluate the anti-tumor efficiency of BA in vivo. The results revealed that the administration of BA dramatically retarded the tumor growth and inhibited the proliferation of tumor cells. More importantly, BA increased the ratio of M1/M2 macrophages in the tumor tissue, which implied the enhancement of anti-tumor immunity. In conclusion, BA demonstrated the inhibitory effect on NSCLC by repolarizing tumor-associated macrophages via the mTOR signaling pathway.

Phytochemicals are now increasingly exploited as remedial agents for the management of diabetes due to side effects attributable to commercial antidiabetic agents. This study investigated the structural and molecular mechanisms by which betulinic acid exhibits its antidiabetic effect via in vitro and computational techniques. In vitro antidiabetic potential was analysed via on α -amylase, α -glucosidase, pancreatic lipase and α -chymotrypsin inhibitory assays. Its structural and molecular inhibitory mechanisms were investigated using Density Functional Theory (DFT) analysis, molecular docking and molecular dynamics (MD) simulation. Betulinic acid significantly ( p  < 0.05) inhibited α -amylase, α -glucosidase, pancreatic lipase and α -chymotrypsin enzymes with IC 50 of 70.02 μg/mL, 0.27 μg/mL, 1.70 μg/mL and 8.44 μg/mL, respectively. According to DFT studies, betulinic acid possesses similar reaction in gaseous phase and water due to close values observed for highest occupied molecular orbital (HOMO) and lowest occupied molecular orbital (LUMO) and the chemical descriptors. The dipole moment indicates that betulinic acid has high polarity. Molecular electrostatic potential surface revealed the electrophilic and nucleophilic attack-prone atoms of the molecule. Molecular dynamic studies revealed a stable complex between betulinic acid and α-amylase, α-glucosidase, pancreatic lipase and α-chymotrypsin. The study elucidated the potent antidiabetic properties of betulinic acid by revealing its conformational inhibitory mode of action on enzymes involved in the onset of diabetes.

Mast cells play important role in acquired and natural immunity making these favorable therapeutic targets in various inflammatory diseases. Here we observed that, pentacyclic tri terpenoid betulinic acid (BA) treatment resulted in a significantly high number (9%) of cells positive for Hoechst and negative for annexin-V indicating that BA could interfere with plasma membrane integrity. The degranulation of both activated and non-activated mast cells was enhanced upon treatment with BA. The pre-treatment of BA had remarkable effect on calcium response in activated mast cells which showed increased calcium influx relative compared to untreated cells. The results also showed potentially less migration of BA treated mast cells signifying the possible effect of BA on cell membrane. BA treatment resulted in a significant increase in mRNA levels of IL-13 while as mRNA levels of other target cytokines, IL-6 and TNF-α seem to be not affected. Moreover, there was global Increase in phosphorylation of signaling proteins and no significant change in phosphorylation of FcεRI receptors indicating that the effect of BA was independent of signaling cascade or FcεRI receptor mediated mast cell aggregation. Overall, these results portray BA potentiates mast cell effector functions by compromising the membrane integrity and independent of FcεRI involvement.

This investigation delves into the inhibitory capabilities of a specific set of triterpenoic acids on diverse isoforms of human carbonic anhydrase (hCA). Oleanolic acid (1), maslinic acid (2), betulinic acid (3), platanic acid (4), and asiatic acid (5) were chosen as representative triterpenoids for evaluation. The synthesis involved acetylation of parent triterpenoic acids 1-5, followed by sequential reactions with oxalyl chloride and benzylamine, de-acetylation of the amides, and subsequent treatment with sodium hydride and sulfamoyl chloride, leading to the formation of final compounds 21-25. Inhibition assays against hCAs I, II, VA, and IX demonstrated noteworthy outcomes. A derivative of betulinic acid, compound 23, exhibited a K i value of 88.1 nM for hCA VA, and a derivative of asiatic acid, compound 25, displayed an even lower K i value of 36.2 nM for the same isoform. Notably, the latter compound displayed enhanced inhibitory activity against hCA VA when compared to the benchmark compound acetazolamide (AAZ), which had a K i value of 63.0 nM. Thus, this compound surpasses the inhibitory potency and isoform selectivity of the standard compound acetazolamide (AAZ). In conclusion, the research offers insights into the inhibitory potential of selected triterpenoic acids across diverse hCA isoforms, emphasizing the pivotal role of structural attributes in determining isoform-specific inhibitory activity. The identification of compound 25 as a robust and selective hCA VA inhibitor prompts further exploration of its therapeutic applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer. However, the effect of current treatment strategies by inducing tumor cell apoptosis alone is not satisfactory. The growth, metastasis and treatment sensitivity of tumors can be strongly influenced by cancer-associated fibroblasts (CAFs) in the microenvironment. Effective cancer therapies may need to target not only the tumor cells directly but also the CAFs that protect them., Methods: Celastrol and small-sized micelles containing betulinic acid were co-encapsulated into liposomes using the thin-film hydration method (CL@BM). Folic acid was further introduced to modify liposomes as the targeting moiety (F/CL@BM). We established a novel NIH3T3+4T1 co-culture model to mimic the tumor microenvironment and assessed the nanocarrier's inhibitory effects on CAFs-induced drug resistance and migration in the co-culture model. The in vivo biological distribution, fluorescence imaging, biological safety evaluation, and combined therapeutic effect evaluation of the nanocarrier were carried out based on a triple-negative breast cancer model., Results: In the present study, a novel multifunctional nano-formulation was designed by combining the advantages of sequential release, co-loading of tretinoin and betulinic acid, and folic acid-mediated active targeting. As expected, the nano-formulation exhibited enhanced cytotoxicity in different cellular models and effectively increased drug accumulation at the tumor site by disrupting the cellular barrier composed of CAFs by tretinoin. Notably, the co-loaded nano-formulations proved to be more potent in inhibiting tumor growth in mice and also showed better anti-metastatic effects in lung metastasis models compared to the formulations with either drug alone. This novel drug delivery system has the potential to be used to develop more effective cancer therapies., Conclusion: Targeting CAFs with celastrol sensitizes tumor cells to chemotherapy, increasing the efficacy of betulinic acid. The combination of drugs targeting tumor cells and CAFs may lead to more effective therapies against various cancers., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Li et al.)

Endoplasmic reticulum stress (ERS) plays a vital role in the pathogenesis of the alcoholic liver disease (ALD). Betulinic acid (BA) has been reported to be effective in the attenuation of ALD; however, its role in ERS and associated stress-signaling pathways remains elusive. Here, we found that the BA pretreatment significantly reduced the alcohol-induced liver injury by decreasing the activities of serum alanine aminotransferase and aspartate aminotransferase, alleviating fat deposition and rupturing the ER in hepatocytes. Moreover, the protective effect of BA on ALD was associated with the inhibition of reactive oxygen species accumulation and ERS, accompanied by the downregulation of glucose-regulated protein 78 (Grp78), Grp94, phosphorylation-inositol-requiring enzyme 1α (p-IRE1α), and phosphorylation-protein kinase R-like endoplasmic reticulum kinase (p-PERK), activating the transcription factor 6 (ATF6) and C/EBP homologous protein (CHOP). Moreover, the alcohol-induced hepatocyte apoptosis was reduced, along with the downregulation of the mitogen-activated protein kinase pathway, caspase-12, caspase-3, and caspase-7, following BA administration. Additionally, the BA-mediated mitigation of alcohol-induced liver injury and deactivation of the ER pathways were the same with 4-PBA, an inhibitor of ERS, indicating that the protective effect of BA on ALD may be regulated by ERS-associated pathways. Collectively, BA is a potentially desirable agent for the ALD, which may reduce hepatocyte apoptosis by suppressing excessive ERS in the liver.

Myocardial ischemia-reperfusion injury (MIRI) leads to cardiac remodeling and heart failure associated with acute myocardial infarction, which is one of the leading causes of death worldwide. Betulinic acid (BA), a widely distributed lupane-type triterpenoid, has been reported to possess antioxidative activity and inhibit apoptosis in MIRI. Due to the low bioavailability and water insolubility of BA, a previous study found a series of BA-derivative compounds by microbial transformation. In this study, we observe whether there are anti-MIRI effects of BTA07, a BA derivative, on cardiac injuries induced by hypoxia/reoxygenation (H/R) in adult rat cardiomyocytes in vitro and in Langendorff-perfused hearts ex vivo, and further explore its mechanism of cardioprotection to find more efficient BA derivatives. The hemodynamic parameters of isolated hearts were monitored and recorded by a Lab Chart system. The markers of oxidative stress and apoptosis in isolated hearts and adult rat cardiomyocytes (ARCMs) were evaluated. The expression levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), protein kinase B (Akt) and phospho-Akt (pAkt, Ser473) induced by H/R were detected via Western blot. The Langendorff experiments showed that BTA07 improves hemodynamic parameters, reduces myocardium damage and infarct size, inhibits levels of myocardial tissue enzymes lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary outflow and reduces oxidative stress and the activation of caspase-3 in the myocardium. In vitro, BTA07 reduced cell death and caspase-3 activation and inhibited reactive oxygen species (ROS) generation. Furthermore, the protective effects of BTA07 were attenuated by inhibition of the PI3K/Akt signaling pathway with LY294002 in ARCMs. BTA07 protects ARCMs and isolated hearts from hypoxia-reperfusion partly by inhibiting oxidative stress and cardiomyocyte apoptosis.

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin, which mainly contaminates grains and has estrogen-like effects on the reproductive system. Betulinic acid (BA), a natural lupane-type pentacyclic triterpene, has anti-oxidative and anti-inflammatory properties. This study aimed to investigate whether BA alleviates ZEA-induced testicular damage and explore the possible mechanism. Here, BA ameliorated testicular damage by mitigating the disordered arrangement of seminiferous tubules, the exfoliation of lumen cells, and the increase of cell apoptosis caused by ZEA. Meanwhile, BA alleviated ZEA-triggered testicular damage by restoring hormone levels and sperm motility, and reconstructing the blood-testis-barrier. Moreover, BA alleviated ZEA-exposed testicular oxidative stress by activating Nrf2 pathway. Furthermore, BA moderated ZEA-evoked testicular inflammation by inhibiting p38/ERK MAPK pathway. Overall, our results revealed that BA has a therapeutic protective effect on ZEA-induced testicular injury and oxidative stress via p38/ERK MAPK inhibition and Nrf2-mediated antioxidant defense activation, which provides a viable alternative to alleviate ZEA-induced male reproductive toxicology., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The effectiveness of betulinic acid (B) and PLGA loaded nanoparticles of B (Bnp) against hepatocellular carcinoma (HCC) was established and reported earlier. In continuation of our previous report, the present study described the molecular mechanisms of their antineoplastic responses. In this context, the antineoplastic properties of both B and Bnp were evaluated on DEN-induced HCC rat model. The quantitative real-time polymerase chain reaction and western blot analyses revealed that HCC was developed through lower expressions of e-NOS, BAX, BAD, Cyt C and higher expressions of i-NOS, Bcl-xl, Bcl-2. B and Bnp normalised the expressions of these apoptogenic markers. Particularly, both activated i-NOS and e-NOS mediated Bcl-2 family proteins→CytC→Caspase 3 and 9 signalling cascades. The 1 H-NMR-based metabolomics study also demonstrated that the perturbed metabolites in DEN-induced rat serum restored to the normal level following both treatments. Moreover, the antineoplastic potential of Bnp was found to be comparable with the marketed product, 5-flurouracil.

Inhibiting the intestinal α-glucosidase can effectively control postprandial hyperglycemia for type 2 diabetes mellitus (T2DM) treatment. In the present study, we reported the binding interaction of betulinic acid (BA), a pentacyclic triterpene widely distributed in nature, on α-glucosidase and its alleviation on postprandial hyperglycemia. BA was verified to exhibit a strong inhibitory effect against α-glucosidase with an IC 50 value of 16.83 ± 1.16 μM. More importantly, it showed a synergistically inhibitory effect with acarbose. The underlying inhibitory mechanism was investigated by kinetics analysis, surface plasmon resonance (SPR) detection, molecular docking, molecular dynamics (MD) simulation and binding free energy calculation. BA showed a non-competitive inhibition on α-glucosidase. SPR revealed that it had a strong and fast affinity to α-glucosidase with an equilibrium dissociation constant ( K D ) value of 5.529 × 10 -5 M and a slow dissociation. Molecular docking and MD simulation revealed that BA bound to the active site of α-glucosidase mainly due to the van der Waals force and hydrogen bond, and then changed the micro-environment and secondary structure of α-glucosidase. Free energy decomposition indicated amino acid residues such as PHE155, PHE175, HIE277, PHE298, GLU302, TRY311 and ASP347 of α-glucosidase at the binding pocket had strong interactions with BA, while LYS153, ARG210, ARG310, ARG354 and ARG437 showed a negative contribution to binding affinity between BA and α-glucosidase. Significantly, oral administration of BA alleviated the postprandial blood glucose fluctuations in mice. This work may provide new insights into the utilization of BA as a functional food and natural medicine for the control of postprandial hyperglycemia.

Betulinic acid (BA) and its derivatives exhibit a variety of biological activities, especially their anti-HIV-1 activity, but generally have only modest inhibitory potency against influenza virus. The entry of influenza virus into host cells can be competitively inhibited by multivalent derivatives targeting hemagglutinin. In this study, a series of hexa-, hepta- and octavalent BA derivatives based on α-, β- and γ-cyclodextrin scaffolds, respectively, with varying lengths of flexible oligo(ethylene glycol) linkers was designed and synthesized using a microwave-assisted copper-catalyzed 1,3-dipolar cycloaddition reaction. The generated BA-cyclodextrin conjugates were tested for their in vitro activity against influenza A/WSN/33 (H1N1) virus and cytotoxicity. Among the tested compounds, 58 , 80 and 82 showed slight cytotoxicity to Madin-Darby canine kidney cells with viabilities ranging from 64 to 68% at a high concentration of 100 μM. Four conjugates 51 and 69 - 71 showed significant inhibitory effects on influenza infection with half maximal inhibitory concentration values of 5.20, 9.82, 7.48 and 7.59 μM, respectively. The structure-activity relationships of multivalent BA-cyclodextrin conjugates were discussed, highlighting that multivalent BA derivatives may be potential antiviral agents against influenza infection.

Background: Betulinic acid (BA), a pentacyclic triterpene glycoside extract from guava (Psidium guajava Linn.) leaves, displays a variety of biological activities which exhibit cancer therapeutic properties associated with cancer growth inhibition in various kinds of human cancer cells including brain, breast, colorectal, cervical, lung and prostate gland. However, the effects on human cholangiocarcinoma cells have not previously been reported. Current study, we evaluated the activity of BA against human cholangiocarcinoma (HuCCA) cells., Methods: The cytotoxicity analysis was measured by using MTT assay on HuCCA and BHK-21 cells. Analysis of apoptosis was evaluated by using staining with Hoechst 33342 and quantitative real-time PCR., Results: The BA (50-800 µg/mL) significantly reduced the viability of HuCCA cells in a dose-dependent action with 50% inhibitory concentration (IC50) of 92.45 µg/mL at 24 h. It also induced apoptosis signaling pathway, such as nuclear chromatin condensation and fragmentation. Quantitative real-time PCR analysis demonstrated that BA increased p53, Bax and caspase-3 expression whilst it decreased Bcl-2 expression in the HuCCA cells in a dose dependent manner., Conclusion: BA can inhibit the HuCCA cell viability and induce apoptosis of neoplastic cells. This study indicates that BA has effective treatment for cholangiocarcinoma in vitro. Consequently, BA may be used as a novel therapeutic agent for the treatment of cholangiocarcinoma in the future.

Background: Glioblastoma (GBM) is the most common and fatal primary tumor in the central nervous system (CNS). Due to the existence of blood-brain barrier (BBB), most therapeutics cannot efficiently reach tumors in the brain, and as a result, they are unable to be used for effective GBM treatment. Accumulating evidence shows that delivery of therapeutics in form of nanoparticles (NPs) may allow crossing the BBB for effective GBM treatment., Methods: Betulinic acid NPs (BA NPs) were synthesized by the standard emulsion approach and characterized by electron microscopy and dynamic light scattering analysis. The resulting NPs were characterized for their anti-tumor effects by cell viability assay, EdU-DNA synthesis assay, cell cycle assay, mitochondrial membrane potential, and PI-FITC apoptosis assay. Further mechanistic studies were carried out through Western Blot and immunostaining analyses. Finally, we evaluated BA NPs in vivo for their pharmacokinetics and antitumor effects in intracranial xenograft GBM mouse models., Results: BA NPs were successfully prepared and formed into rod shape. BA NPs could significantly suppress glioma cell proliferation, induce apoptosis, and arrest the cell cycle in the G0/G1 phase in vitro. Furthermore, BA NPs downregulated the Akt/NFκB-p65 signaling pathway in a concentration dependent manner. We found that the observed anti-tumor effect of BA NPs was dependent on the function of CB1/CB2 receptors. Moreover, in the intracranial GBM xenograft mouse models, BA NPs could effectively cross the BBB and greatly prolong the survival time of the mice., Conclusions: We successfully synthesized BA NPs, which could cross the BBB and demonstrated a strong anti-tumor effect. Therefore, BA NPs may potentially be used for effective treatment of GBM., (© 2022. The Author(s).)

Alcoholic abuse is one of the most serious causes of liver diseases worldwide. Although detailed molecular pathogenesis of alcohol-induced liver damages remains elusive with intensive debates, it has been widely recognized that hepatic damage caused by free radicals generated from alcohol metabolism is one of the most critical factors for alcohol-induced liver diseases. Betulinic acid is a potent antioxidant with additional known pharmacological safety characteristics and minimal toxicity. However, poor solubility limited its usage. In this study, we assessed the efficacy of BAN, a betulinic acid and nucleoside hybrid with good water solubility, in reversing acute liver damages using an established alcohol overdose animal model. The results indicated that BAN is an extremely promising therapeutic agent against acute alcohol-induced liver damage. BAN effectively protects liver from alcohol damage by reducing serum ALT level by up to 47%, as well as liver oxidative stress indicated by significantly increased SOD, CAT, and GSH-Px levels. Moreover, hepatic FXR activation and a corresponding downstream anti-oxidative stress transcriptional cascade including Nrf2, HO-1, and NOQ1 induce the protective role of BAN. On the other hand, BAN administration also leads to increase cellular autophagy response, as indicated by the key ATG protein activation. We concluded that BAN, comparing with Betulinic acid, prevents acute alcohol-induced liver damages more effectively, with the dual mechanisms of neutralizing oxidative stress and promoting autophagy., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

In this work, we designed three new ligands by conjugating cholesterol metabolites 3-hydroxy-5-cholestenoic acid (3-HC) and 3-oxo-4-cholestenoic acid (3-OC) and the natural tri-terpenoid betulinic acid with the tumor-targeting peptide YHWYGYTPQNVI. Molecular interactions with the unconjugated peptide and the conjugates were examined with three receptors that are commonly overexpressed in pancreatic adenocarcinoma cells using ligand docking and molecular dynamics. This study demonstrated the utility of the designed conjugates as a valuable scaffold for potentially targeting EGFR and LDLR receptors. Our results indicate that the conjugates showed strong binding affinities and formation of stable complexes with EGFR, while the unconjugated peptide, BT-peptide conjugate, an 3-HC-peptide conjugate showed the formation of fairly stable complexes with LDLR receptor. For EGFR, two receptor kinase domains were explored. Interactions with the N-terminal domain of CCKA-R were relatively weaker. For LDLR, binding occurred in the beta-propeller region. For the N-terminal fragment of CCKA-R, the conjugates induced significant conformational changes in the receptor. The molecular dynamic simulations for 100 ns demonstrate that BT-peptide conjugates and the unconjugated peptide had the highest binding and formed the most stable complexes with EGFR. RMSD and trajectory analyses indicate that these molecules transit to a dynamically stable configuration in most cases within 60 ns. NMA analysis indicated that amongst the conjugates that showed relatively higher interactions with the respective receptors, the highest potential for deformability was seen for the N-terminal-47 amino acid region of the CCKA-R receptor with and the lowest for the LDLR-receptor. Thus, the newly designed compounds may be evaluated in the future toward developing drug delivery materials for targeting tumor cells overexpressing LDLR or EGFR., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Phytochemicals have been suggested as an effective strategy for cancer prevention. Within this context, triterpene betulinic acid (BA) exhibits several biological properties but its chemopreventive effect has not been fully demonstrated. The present study investigated the antigenotoxic potential of BA against doxorubicin (DXR)-induced genotoxicity using the mouse peripheral blood micronucleus assay, as well as its anticarcinogenic activity against 1,2dimethylhydrazine (DMH)-induced colorectal lesions in rats. Micronuclei (MN) assay and aberrant crypt foci assay were used to assess the antigenotoxic and the anticarcinogenic potential, respectively. The molecular mechanisms underlying the anticarcinogenic activity of BA were evaluated by assessing anti-inflammatory (COX-2) and antiproliferative (PCNA) pathways. The results demonstrated that BA at the dose of 0.5 mg/kg bodyweight exerted antigenotoxic effects against DXR, with a reduction of 70.2% in the frequencies of chromosomal damage. Animals treated with BA showed a 64% reduction in the number of preneoplastic lesions when compared to those treated with the carcinogen alone. The levels of COX-2 and PCNA expression in the colon were significantly lower in animals treated with BA and DMH compared to those treated with the carcinogen alone. The chemopreventive effect of BA is related, at least in part, to its antiproliferative and anti-inflammatory activity, indicating a promising potential of this triterpene in anticancer therapies, especially for colorectal cancer., (© 2021 Wiley Periodicals LLC.)

Betulinic acid (3β-Hydroxy-20(29)-lupaene-28-oic acid, BA) has excellent anti-cancer activity but poor solubility and low bioavailability. To improve the antitumor activity of BA, a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol (PVCL-PVA-PEG) graft copolymer (Soluplus) encapsulated BA micelle (Soluplus-BA) was fabricated. The Soluplus-BA micelles presented a mean size of 54.77 ± 1.26 nm and a polydispersity index (PDI) of 0.083. The MTT assay results showed that Soluplus-BA micelles increased the inhibitory effect of BA on MDA-MB-231 cells, mainly due to the enhanced accumulation of reactive oxygen species (ROS) and the destruction of mitochondrial membrane potential (MMP). Soluplus-BA micelles induced the DNA double-strand breaks (DSBs) as the γH2AX foci increased. Moreover, Soluplus-BA also inhibited the tube formation and migration of human umbilical vein endothelial cells (HUVECs), and inhibited the neovascularization of the chicken chorioallantoic membrane (CAM). This angiogenesis inhibitory effect may be accomplished by regulating the HIF-1/VEGF-FAK signaling pathway. The in vivo study confirmed the improved anti-tumor effect of Soluplus-BA and its inhibitory effect on angiogenesis, demonstrating the possibility of Soluplus-BA as an effective anti-breast cancer drug delivery system.

Betulinic acid (BA) is a pentacyclic triterpenoid compound with potential antioxidant and anti-inflammatory effects. In this study, T-2 toxin was injected intraperitoneally in mice to establish kidney damage model and to evaluate the protective effects of BA and further reveal the molecular mechanism. BA pretreatment inhibited the T-2 toxin-stimulated increase in serum Crea, but showed no significant effect on serum Urea. BA pretreatment alleviated excessive glomerular hemorrhage and inflammatory cell infiltration in kidneys caused by T-2 toxin. Moreover, pretreatment with BA mitigated T-2 toxin-induced renal oxidative damage by up-regulating the activities of SOD and CAT, and the content of GSH, while down-regulating the accumulation of ROS and MDA. Meanwhile, BA pretreatment markedly attenuated T-2 toxin-induced renal inflammatory response by decreasing the mRNA expression of IL-1β, TNF-α and IL-10, and increasing IL-6 mRNA expression. Furthermore, mechanism research found that pretreatment with BA could activate Nrf2 signaling pathway. It was suggested that BA ameliorated the oxidative stress and inflammatory response of T-2 toxin-triggered renal damage by activating the Nrf2 signaling pathway., (Copyright © 2021. Published by Elsevier B.V.)

A set of new substituted dienes were synthesized from betulinic acid by its oxidation to 30-oxobetulinic acid followed by the Wittig reaction. Cytotoxicity of all compounds was tested in vitro in eight cancer cell lines and two noncancer fibroblasts. Almost all dienes were more cytotoxic than betulinic acid. Compounds 4.22, 4.30, 4.33, 4.39 had IC 50 below 5 μmol/L; 4.22 and 4.39 were selected for studies of the mechanism of action. Cell cycle analysis revealed an increase in the number of apoptotic cells at 5 × IC 50 concentration, where activation of irreversible changes leading to cell death can be expected. Both 4.22 and 4.39 led to the accumulation of cells in the G0/G1 phase with partial inhibition of DNA/RNA synthesis at 1 × IC 50 and almost complete inhibition at 5 × IC 50 . Interestingly, compound 4.39 at 5 × IC 50 caused the accumulation of cells in the S phase. Higher concentrations of tested drugs probably inhibit more off-targets than lower concentrations. Mechanisms disrupting cellular metabolism can induce the accumulation of cells in the S phase. Both compounds 4.22 and 4.39 trigger selective apoptosis in cancer cells via intrinsic pathway, which we have demonstrated by changes in the expression of the crucial apoptosis-related protein. Pharmacological parameters of derivative 4.22 were superior to 4.39, therefore 4.22 was the finally selected candidate for the development of anticancer drug., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

The race against ovarian cancer continue to motivate the research worldwide. It is known that many antitumor drugs have limited penetration into solid tumor tissues due to its microenvironment, thus contributing to their low efficacy. Therapeutic modalities have been exploited to elicit antitumor effects based on microenvironment of tumor, including Photodynamic therapy (PDT). Prospection of natural small molecules and nanotechnology are important tools in the development of new ways of obtaining photoactive compounds that are biocompatible. The Betulinic acid (BA) has shown potential biological effect as bioactive drug, but it has low water solubility. Thus, in the present study, owing to the poor solubility of the BA, its free form (BAF) was compared to a spray dried microparticle betulinic acid/HP-β-CD formulation (BAC) aiming to assess the BAF and BAC efficacy as a photosensitizer in PDT for application in ovarian cancer. BAF and BAC were submitted to assays in the presence of LED (λ = 420 nm) under different conditions (2.75 J/cm 2 , 5.5 J/cm 2 , and 11 J/cm 2 ) and in absence of irradiation, after 5 min or 4 h of contact with ovarian carcinoma cells (A2780) or fibroblast murine cells (3T3). Furthermore, HPLC-MS/MS and MALDI-MSI methods were developed and validated in plasma and tumor of mice proving suitable for in vivo studies. The results found a greater photoinduced cytotoxic effect for the BAC at low concentration for A2780 when irradiated with LED with similar results for fluorescence microscopy. The results motivate us to continue the studies with the BA as a potential antitumor bioactive compound., (Copyright © 2021 Elsevier B.V. All rights reserved.)

T-2 toxin is highly cytotoxic to animals, which causes damage to animal health and great economic losses to agriculture and livestock production. Betulinic acid (BA), a naturally occurring pentacyclic lupane-type triterpenoid, has various biological and medicinal activities in vivo and in vitro. The objective of the present study was to investigate the toxic effects of T-2 toxin and the reversal effect of BA on porcine kidney (PK-15) cells. We evaluated T-2 toxin-induced apoptotic responses via oxidative stress and endoplasmic reticulum stress pathways by assessing the repair effect of BA in PK-15 cells. The results proved that T-2 toxin (1 μM, treated for 24 h) is highly toxic to PK-15 cells. After pre-treatment with BA (0.25, 0.5, and 1 μM) for 24 h, the cell viabilities were significantly increased, and the lactate dehydrogenase (LDH) in the culture media was dramatically decreased compared to that in the T-2 toxin treatment group. BA also enhanced the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and catalase (CAT) and reduced the production of reactive oxygen species (ROS) and malondialdehyde (MDA) in cells. BA also dose-dependently increased the expression of glucose regulated protein (GRP78), reduced expression of activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), the phosphorylation of protein kinase R-like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor 2α (eIF2α), and intracellular Ca 2+ concentration in a dose-dependent manner. In addition, BA significantly decreased the expression of cleaved-caspase-3 and caspase-12, consequently reducing T-2 toxin-induced PK-15 cell apoptosis in a dose-dependent manner. Collectively, we suggest that BA has a protective effect on T-2 toxin-induced cytotoxicity by ameliorating oxidative stress and endoplasmic reticulum stress in PK-15 cells., (Copyright © 2021 Elsevier Inc. All rights reserved.)

N-nitrosamines have been linked with cancer in humans due to their presence in drinking water and diets. This study evaluated the role of betulinic acid (BA) in abating oxidative stress, inflammation, and hyperlipidemia in rats treated with N-nitrosodimethylamine (NDMA). Twenty-four male rats were assigned into four equal groups. Group I served as the control, Group II received BA (25 mg/kg), Group III received NDMA (5 mg/kg) and, Group IV received BA (25 mg/kg) and NDMA (5 mg/kg). Results showed that the administration of NDMA significantly (p < 0.05) elevated malondialdehyde in the liver and kidney relative to controls. Activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione-S-transferase, and the level of glutathione were significantly (p < 0.05) decreased by NDMA, while treatment with BA elevated the activities of these enzymes in the liver and kidney. The BA lowered serum interleukin-6 and tumor necrosis factor-alpha levels against the NDMA effect. Furthermore, NDMA increased hepatic and renal triglyceride while phospholipids levels were decreased. NDMA significantly modulated the activities of drug-metabolizing enzymes (aniline hydroxylase, aminopyrine-N-demethylase, and uridyldiphosphoglucuronyltransferase), while BA was able to restore these enzymes to values close to controls. Histology revealed the presence of infiltration and fibroplasia in the liver, while cortical degeneration was noticed in the kidney in NDMA-administered rats. These lesions were reduced in the NDMA rats treated with BA. The findings suggest that BA improves NDMA-induced damage in the liver and kidney of rats through reactions that can be linked with antioxidant, anti-inflammatory, and lipid-lowering pathways., (© 2021 Wiley Periodicals LLC.)

Betulinic acid (BA) is a pentacyclic triterpene usually isolated from botanical sources. Numerous studies have reported the inhibitory effect of BA against human colorectal cancer cells (CRC). However, its effect on the expression of the molecular chaperone HSPA is unclear. The aim of this research is to investigate the anti-cancer activities of BA purified from Piper retrofractum and study its effect on the expression of HSPA in colorectal cancer HCT116 and SW480 cells. The viability of both cancer cells was reduced after they were treated with an increasing dosage of BA. Flow cytometry assay revealed that levels of cell apoptosis significantly increased after incubation with BA in both cancer cells. Pro-apoptotic markers including Bax, cleaved-caspase-3 and cleaved-caspase-9 were increased while anti-apoptotic marker Bcl-2 was decreased after BA treatment. Western blot also showed that the expression of HSPA fluctuated upon BA treatment, whereby HSPA was increased at lower BA concentrations while at higher BA concentrations HSPA expression was decreased. Preliminary molecular docking assay showed that BA can bind to the nucleotide binding domain of the HSP70 at its ADP-bound state of the HSP70. Although further research is needed to comprehend the BA-HSPA interaction, our findings indicate that BA can be considered as potential candidate for the development of new treatment for colorectal cancer.

It is known that Senna obtusifolia has been used in medicine since ancient times due to the content of many valuable compounds with a pro-health effect. One of them is betulinic acid, which is a pentacyclic triterpene with antimalarial, antiviral, anti-inflammatory and anticancer properties. In this work, a continuation of our previous research, an attempt was made to increase the level of betulinic acid accumulation by the cultivation of transgenic hairy roots that overexpress the squalene synthase gene in a 10 L sprinkle bioreactor with methyl jasmonate elicitation. We present that the applied strategy allowed us to increase the content of betulinic acid in hairy root cultures to the level of 48 mg/g dry weight. The obtained plant extracts showed a stronger cytotoxic effect on the U87MG glioblastoma cell line than the roots grown without elicitors. Additionally, the induction of apoptosis, reduction of mitochondrial membrane potential, chromosomal DNA fragmentation and activation of caspase cascades are demonstrated. Moreover, the tested extract showed inhibition of topoisomerase I activity.

Periodontitis is one of the most common chronic inflammations of the oral cavity, which eventually leads to tooth loss. Betulinic acid (BetA) is an organic acid that has anti-inflammatory effects and is derived from fruits and plants, but its effect on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) is still unclear. This study aimed to explore the effect of BetA on the osteogenic differentiation of hPDLSCs and its mechanism. Our results revealed that BetA not only promoted the viability of hPDLSCs but also induced their osteogenic differentiation in a dose-dependent manner. In addition, RNA sequencing was used to screen the differentially expressed genes (DEGs) after hPDLSCs were treated with BetA, and 127 upregulated and 138 downregulated genes were identified. Gene Ontology enrichment analysis showed that DEGs were mainly involved in the response to lithium ions and the positive regulation of macrophage-derived foam cell differentiation. The Kyoto Encyclopedia of Genes and Genomes analysis results revealed that DEGs were enriched in the nuclear factor-κB and interleukin-17 signaling pathways. More importantly, we confirmed that early growth response gene 1 (EGR1), one of the three DEGs involved in bone formation, significantly promoted the expression of osteogenic markers and the mineralization of hPDLSCs. Knockdown of EGR1 obviously limited the effect of BetA on the osteogenic differentiation of hPDLSCs. In conclusion, BetA promoted the osteogenic differentiation of hPDLSCs through upregulating EGR1, and BetA might be a promising candidate in the clinical application of periodontal tissue regeneration., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Betulinic acid (BA) is a pentacyclic triterpene compound that can be obtained by separation, chemical synthesis and biotransformation from birch. BA has antitumour activity, and its mechanisms of action mainly include the induction of mitochondrial oxidative stress; the regulation of specificity protein transcription factors, and the inhibition of signal transducer and activator of transcription 3 and nuclear factor-κB signalling pathways. In addition, BA can increase the sensitivity of cancer cells to other chemotherapy drugs. Recent studies have shown that BA plays an anticancer role in several kinds of tumour diseases. In this article, the anticancer mechanism of BA and its application in the treatment of tumour diseases are reviewed., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Cardiac hypertrophy can lead to congestive heart failure and is a leading cause of morbidity and mortality worldwide. In recent years, it has been essential to find the treatment and prevention of cardiac hypertrophy. Betulinic acid (BA), the main active ingredient in many natural products, is known to have various physiological effects. However, as the potential effect of BA on cardiac hypertrophy and consequent renal dysfunction is unknown, we investigated the effect of BA on isoprenaline (ISO)-induced cardiac hypertrophy and related signaling. ISO was known to induce left ventricular hypertrophy by stimulating the β2-adrenergic receptor (β 2 AR). ISO was injected into Sprague Dawley rats (SD rats) by intraperitoneal injection once a day for 28 days to induce cardiac hypertrophy. From the 14th day onwards, the BA (10 or 30 mg/kg/day) and propranolol (10 mg/kg/day) were administered orally. The study was conducted in a total of 5 groups, as follows: C, control; Is, ISO (10 mg/kg/day); Pr, positive-control, ISO + propranolol (10 mg/kg/day); Bl, ISO + BA (10 mg/kg/day); Bh, ISO + BA (30 mg/kg/day). As a result, the total cardiac tissue and left ventricular tissue weights of the ISO group increased compared to the control group and were significantly reduced by BA treatment. In addition, as a result of echocardiography, the effect of BA on improving cardiac function, deteriorated by ISO, was confirmed. Cardiac hypertrophy biomarkers such as β-MHC, ANP, BNP, LDH, and CK-MB, which were increased by ISO, were significantly decreased by BA treatment. Also, the cardiac function improvement effect of BA was confirmed to improve cardiac function by inhibiting calcineurin/NFATc3 signaling. Renal dysfunction is a typical complication caused by cardiac hypertrophy. Therefore, the study of renal function indicators, creatinine clearance (Ccr) and osmolality (BUN) was aggravated by ISO treatment but was significantly restored by BA treatment. Therefore, it is thought that BA in cardiac hypertrophy can be used as valuable data to develop as a functional material effective in improving cardiac-renal dysfunction.

Chemodynamic therapy (CDT) based on intracellular Fenton reactions is attracting increasing interest in cancer treatment. A simple and novel method to regulate the tumor microenvironment for improved CDT with satisfactory effectiveness is urgently needed. Therefore, glutathione (GSH)/ROS (reactive oxygen species) dual-responsive supramolecular nanoparticles (GOx@BNPs) for chemo-chemodynamic combination therapy were constructed via host-guest complexation between water-soluble pillar[6]arene and the ferrocene-modified natural anticancer product betulinic acid (BA) prodrug, followed by encapsulation of glucose oxidase (GOx) in the nanoparticles. The novel supramolecular nanoparticles could be activated by the overexpressed GSH and ROS in the tumor microenvironment (TME), not only accelerating the dissociation of nanoparticles-and, thus, improving the BA recovery and release capability in tumors-but also showing the high-efficiency conversion of glucose into hydroxyl radicals (·OH) in succession through intracellular Fenton reactions. Investigation of antitumor activity and mechanisms revealed that the dramatic suppression of cancer cell growth induced by GOx@BNPs was derived from the elevation of ROS, decrease in ATP and mitochondrial transmembrane potential (MTP) and, finally, cell apoptosis. This work presents a novel method for the regulation of the tumor microenvironment for improved CDT, and the preparation of novel GSH/ROS dual-responsive supramolecular nanoparticles, which could exert significant cytotoxicity against cancer cells through the synergistic interaction of chemodynamic therapy, starvation therapy, and chemotherapy (CDT/ST/CT).

A series of pyrazole-fused betulinic acid (BA) derivatives were designed and synthesized by replacing the carboxyl group at C-17 with aliphatic amine, amide, and urea groups. The suppressive effects of the compounds on osteoclast (OC) formation and inflammatory cytokine production were evaluated on murine macrophages, RAW264.7 cells, conditioned with receptor activator for nuclear factor-κB ligand (RANKL)/macrophage colony stimulating factor (M-CSF) or lipopolysaccharide (LPS), respectively. Results showed that, compared with betulinic acid, most of these compounds exhibited significant improvements in inhibitory potency. Compound 25 exhibited distinguished activities on inhibiting OC differentiation with an IC 50 value of 1.86 μM. Meanwhile, compound 25 , displaying the most promising suppression on IL-1β secretion from RAW264.7 cells, was further found to possess therapeutic effects in the sodium monoiodoacetate (MIA)-induced osteoarthritis rat model. Dose-dependent benefits were observed in MIA-elicited rats with ameliorated joint pain as well as decreased cartilage damage and bone changes after compound 25 treatment.

Ischemia/reperfusion (I/R) events are involved in the pathophysiology of numerous ocular diseases. The purpose of this study was to test the hypothesis that betulinic acid protects from I/R injury in the mouse retina. Ocular ischemia was induced in mice by increasing intraocular pressure (IOP) to 110 mm Hg for 45 min, while the fellow eye served as a control. One group of mice received betulinic acid (50 mg/kg/day p.o. once daily) and the other group received the vehicle solution only. Eight days after the I/R event, the animals were killed and the retinal wholemounts and optic nerve cross-sections were prepared and stained with cresyl blue or toluidine blue, respectively, to count cells in the ganglion cell layer (GCL) of the retina and axons in the optic nerve. Retinal arteriole responses were measured in isolated retinas by video microscopy. The levels of reactive oxygen species (ROS) were assessed in retinal cryosections and redox gene expression was determined in isolated retinas by quantitative PCR. I/R markedly reduced cell number in the GCL and axon number in the optic nerve of the vehicle-treated mice. In contrast, only a negligible reduction in cell and axon number was observed following I/R in the betulinic acid-treated mice. Endothelial function was markedly reduced and ROS levels were increased in retinal arterioles of vehicle-exposed eyes following I/R, whereas betulinic acid partially prevented vascular endothelial dysfunction and ROS formation. Moreover, betulinic acid boosted mRNA expression for the antioxidant enzymes SOD3 and HO-1 following I/R. Our data provide evidence that betulinic acid protects from I/R injury in the mouse retina. Improvement of vascular endothelial function and the reduction in ROS levels appear to contribute to the neuroprotective effect.

Betulinic acid (BA), a pentacyclic triterpenoid isolated from tree bark, exhibits antitumor effects against solid malignancies and triggers autophagy and/or apoptosis in human cancer cells. Nonetheless, the relationship between autophagy and apoptosis and the potential modulatory actions of BA on autophagy-dependent bladder cancer cell death remain unclear. The present study showed that BA exposure significantly suppressed viability, proliferation, and migration of EJ and T24 human bladder cancer cells. These effects reflected caspase 3-mediated apoptosis and could be attenuated or abolished by inhibiting ROS production with N-acetyl-L-cysteine, inhibiting autophagy with chloroquine, or silencing ATG7 with targeted siRNA. BA-induced autophagy was evidenced by epifluorescence imaging of lentivirus-induced expression of mCherry-GFP-LC3B and increased expression of two autophagy-related proteins, LC3B-II and TEM. Moreover, enhanced AMPK phosphorylation and decreased mTOR and ULK-1 phosphorylation suggested BA activates autophagy via the AMPK/mTOR/ULK1 pathway. Accordingly, exposure to dorsomorphin (Compound C), an AMPK inhibitor, and AICAR, an AMPK activator, respectively inhibited and stimulated BA-induced autophagy in EJ and T24 cells. The effects of Bmi-1 overexpression in vitro and decreased Bmi-1 expression in BA-treated T24 cell xenografts in nude mice suggested that downregulation of Bmi-1 is the underlying mechanism in BA-mediated, autophagy-dependent apoptosis.

Objective: To investigate the effects of betulinic acid on apoptosis of human gastric cancer SGC-7901 cells. Methods: The human gastric cancer SGC-7901cells were divided in to 4 groups, and each group was set with 3 replicates. The SGC-7901cells in control group were not treated with betulinic acid; the other 3 experimental groups were treated with betulinic acid at the concentrations of 10, 20 and 30 mg/L, respectively; each group was incubated in a 5% carbon dioxide incubator for 48 h. Laser confocal microscope was used to observe morphological changes of SGC-7901 cells; Flow cytometry was applied to determine apoptosis rate and mitochondrial membrane potential. The mRNA and protein levels of Bcl-2 , Bax and Caspase-3 were also detected by qRT-PCR and western blot respectively. Results: Compared with the control group, SGC-7901 cells in the treated group at final concentrations of 10, 20 and 30 mg/L shrinked, appeared apoptosis body along with nuclear splitting. The percentage of cells in early and advanced period of apoptosis were markedly increased ( P ＜0.05 or P ＜0.01), mitochondrial membrane potential was obviously reduced ( P ＜0.05 or P ＜0.01). qRT-PCR and western blot analysis showed that the mRNA and protein expressions of Bax and Caspase-3 were increased significantly ( P ＜0.01), while the expressions of Bcl-2 were decreased significantly ( P ＜0.01). Conclusion: Within a certain range of concentrations, betulinic acid induces cell apoptosis by regulating the expression of Bcl-2 , Bax and Caspase-3 in human gastric cancer.

Takeda G protein-coupled receptor 5 (TGR5) is a promising target for treating metabolic syndrome and inflammatory diseases. Herein, we identified a new series of betulinic acid derivatives as potent TGR5 agonists, which show remarkable activity on human (h) and canine (c) TGR5 but exhibit unpromising activity on murine (m) TGR5. Species difference was also observed with many other reported TGR5 agonists. Therefore, we screened 29 amino acids which were conserved in hTGR5 and cTGR5 but different in mTGR5 and found a key amino acid, H88 in mTGR5 (Y89 in hTGR5), which contributed to the species difference. With the CRISPR/Cas9 system, the mTGR5 H88Y mutation was introduced into mice, and the optimized compound 11d-Na displayed a significant glucose-lowering effect and stimulated GLP-1 and insulin secretion in TGR5 H88Y mice but not in wild-type animals. Taken together, our study provides a useful tool to bridge the gap of species difference and discovers a potent TGR5 agonist for further investigation.

Hypoxia-regulated protein carbonic anhydrase IX (CA IX) is up-regulated in different tumor entities and correlated with poor prognosis in breast cancer patients. Due to the radio- and chemotherapy resistance of solid hypoxic tumors, derivatives of betulinic acid (BA), a natural compound with anticancer properties, seem to be promising to benefit these cancer patients. We synthesized new betulin sulfonamides and determined their cytotoxicity in different breast cancer cell lines. Additionally, we investigated their effects on clonogenic survival, cell death, extracellular pH, HIF-1α, CA IX and CA XII protein levels and radiosensitivity. Our study revealed that cytotoxicity increased after treatment with the betulin sulfonamides compared to BA or their precursors, especially in triple-negative breast cancer (TNBC) cells. CA IX activity as well as CA IX and CA XII protein levels were reduced by the betulin sulfonamides. We observed elevated inhibitory efficiency against protumorigenic processes such as proliferation and clonogenic survival and the promotion of cell death and radiosensitivity compared to the precursor derivatives. In particular, TNBC cells showed benefit from the addition of sulfonamides onto BA and revealed that betulin sulfonamides are promising compounds to treat more aggressive breast cancers, or are at the same level against less aggressive breast cancer cells.

Betulinic acid, which is found in transgenic roots of Senna obtusifolia (L.) H.S.Irwin & Barneby, is a pentacyclic triterpene with distinctive pharmacological activities. In this study, we report the differences in the content of betulinic acid and selected anthraquinones in transgenic S. obtusifolia hairy roots with overexpression of the PgSS1 gene (SOPSS2 line) and in transformed hairy roots without this genetic construct (SOA41 line). Both hairy root lines grew in 10 L sprinkle bioreactor. Additionally, the extracts obtained from this plant material were used for biological tests. Our results demonstrated that the SOPSS2 hairy root cultures from the bioreactor showed an increase in the content of betulinic acid (38.125 mg/g DW), compared to the SOA41 hairy root line (4.213 mg/g DW). Biological studies have shown a cytotoxic and antiproliferative effect on U-87MG glioblastoma cells, and altering the level of apoptotic proteins (Bax, p53, Puma and Noxa). Antimicrobial properties were demonstrated for both tested extracts, with a stronger effect of SOPSS2 extract. Moreover, both extracts showed moderate antiviral properties on norovirus surrogates., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.)