Cellular and molecular mechanisms underlying the potential of betulinic acid in cancer prevention and treatment.

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.

Neuroprotective potential of Betulinic acid against TIO2NP induced neurotoxicity in zebrafish.

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 TiO2NP significantly decreased GSH, and SOD, and increased MDA, AChE, TNF-α, IL-1ß, 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 TiO2NP-treated zebrafish brain. Our histopathological analysis proved that TiO2NP 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.

Cytotoxic Potential of Betulinic Acid Fatty Esters and Their Liposomal Formulations: Targeting Breast, Colon, and Lung Cancer Cell Lines.

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 (IC50 of 30.57 µM) and NCI-H460 cells (IC50 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.

An investigation of quantum dot theranostic probes for prostate and leukemia cancer cells using a CdZnSeS QD-based nanoformulation.

Anticancer theranostic nanocarriers have the potential to enhance the efficacy of pharmaceutical evaluation of drugs. Semiconductor nanocrystals, also known as quantum dots (QDs), are particularly promising components of drug carrier systems due to their small sizes and robust photoluminescence properties. Herein, bright CdZnSeS quantum dots were synthesized in a single step via the hot injection method. The particles have a quasi-core/shell structure as evident from the high quantum yield (85 %), which decreased to 41 % after water solubilization. These water solubilized QDs were encapsulated into gallic acid / alginate (GA-Alg) matrices to fabricate imaging QDs@mod-PAA/GA-Alg particles with enhanced stability in aqueous media. Cell viability assessments demonstrated that these nanocarriers exhibited viability ranging from 63 % to 83 % across all tested cell lines. Furthermore, the QDs@mod-PAA/GA-Alg particles were loaded with betulinic acid (BA) and ceranib-2 (C2) for in vitro drug release studies against HL-60 leukemia and PC-3 prostate cancer cells. The BA loaded QDs@mod-PAA/GA-Alg had a half-maximal inhibitory concentration (IC50) of 8.76 µg/mL against HL-60 leukemia cells, which is 3-fold lower than that of free BA (IC50 = 26.55 µg/mL). Similar enhancements were observed with nanocarriers loaded with C2 and simultaneously with both BA and C2. Additionally, BA:C2 loaded QDs@mod-PAA/GA-Alg nanocarriers displayed a similar enhancement (IC50 = 3.37 µg/mL compared against IC50 = 11.68 µg/mL for free BA:C2). The C2 loaded QDs@mod-PAA/GA-Alg nanocarriers had an IC50 = 2.24 µg/mL against HL-60 cells. C2 and BA loaded QDs@mod-PAA/GA-Alg NCr had IC50 values of 7.37 µg/mL and 24.55 µg/mL against PC-3 cells, respectively.

Analgesic and anti-inflammatory activities of NPK 500 capsules, a Cassia sieberiana DC. - Based herbal analgesic medicine used to treat dysmenorrhea and peptic ulcer, is mediated through the inhibition of PGE2 and iNOS.

ETHNOPHARMACOLOGICAL RELEVANCE: Pain and inflammation are the most frequent reasons for which people seek medical care. Currently available analgesics against these conditions produce fatal adverse effects. NPK 500 capsules is an alternative herbal analgesic employed to treat dysmenorrhea, peptic ulcer and pain. NPK 500 is produced from Cassia sieberiana. A plant used in traditional medicine to treat pain and inflammation. AIM OF THE STUDY: This study reports the analysis, phytochemical characterization and mechanism of analgesic and anti-inflammatory activities of two NPK 500 capsules, called old and new NPK500 capsules (ONPK500 and NNPK500) respectively. MATERIALS AND METHODS: Physicochemical, organoleptic, GC-MS and LC-MS methods were employed to analyze the NPK 500 capsules. Analgesic activity was evaluated using tail immersion, Randall-Selitto and acetic acid induced writing tests. Anti-inflammatory activity was evaluated using carrageenan-induced rat paw inflammation. Additionally, pro-inflammatory mediators such as prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase 1 and 2 (COX-2 and COX-1) were quantified in the sera of the rats using Enzyme Linked Immunosorbent Assay (ELISA) kits. RESULTS: Thirteen major compounds were characterized in the NNPK 500 capsules via the GC-MS and LC-MS spectroscopies. Kaempferol was the major compound characterized in addition to physcion, ß-sitosterol 3-O-ß-D-glucopyranoside, betulinic acid and nine others. Physicochemical and organoleptic indices of the capsules were also derived for its authentication and quality control. Furthermore, NNPK 500 0.5-1.5 mg/kg p.o. produce significant (P < 0.5) analgesic activity (160-197%) higher than that of ONPK500 (109.8%) and Morphine (101%) in the tail immersion test. The analgesic activity of NNPK 500 0.5-1.5 mg/kg p.o. (171.0-258.3%) and ONPK 500 (179.5%) were also significant (P < 0.01) and higher than that of Aspirin (103.00%) in the Randall-Selitto test. Both capsules also demonstrated significant (P < 0.5) analgesic and anti-inflammatory activities in the acetic acid-induced writhing and carrageenan-indued paw edema tests respectively. The two NPK500 capsules also, significantly (P < 0.5) inhibited PGE2 and iNOS but not COX-2 and COX-1 in the carrageenan-indued paw edema test. CONCLUSION: These results show that NNPK 500 and ONPK 500 capsules possessed potent analgesic and anti-inflammatory activities via inhibition of PGE2 and iNOS as a result of their chemical constituents. NPK500 capsules thus, relief acute pain and inflammation without causing gastrointestinal, renal or hepatic injuries, since they did not inhibit COX-1.

Boswellic Acid and Betulinic Acid Pre-treatments Can Prevent the Nephrotoxicity Caused by Cyclophosphamide Induction.

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.

Potent drug delivery enhancement of betulinic acid and NVX-207 into equine skin in vitro - a comparison between a novel oxygen flow-assisted transdermal application device and microemulsion gels.

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 (IC50) 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 IC50 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.

[Betulinic acid inhibits non-small cell lung cancer by repolarizing tumor-associated macrophages via mTOR signaling pathway].

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.

Phytochemical evaluation of Ziziphus mucronata and Xysmalobium undulutum towards the discovery and development of anti-malarial drugs.

BACKGROUND: The development of resistance by Plasmodium falciparum is a burdening hazard that continues to undermine the strides made to alleviate malaria. As such, there is an increasing need to find new alternative strategies. This study evaluated and validated 2 medicinal plants used in traditional medicine to treat malaria. METHODS: Inspired by their ethnobotanical reputation of being effective against malaria, Ziziphus mucronata and Xysmalobium undulutum were collected and sequentially extracted using hexane (HEX), ethyl acetate (ETA), Dichloromethane (DCM) and methanol (MTL). The resulting crude extracts were screened for their anti-malarial and cytotoxic potential using the parasite lactate dehydrogenase (pLDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. This was followed by isolating the active compounds from the DCM extract of Z. mucronata using silica gel chromatography and structural elucidation using spectroscopic techniques (NMR: 1H, 12C, and DEPT). The active compounds were then targeted against P. falciparum heat shock protein 70-1 (PfHsp70-1) using Autodock Vina, followed by in vitro validation assays using ultraviolet-visible (UV-VIS) spectroscopy and the malate dehydrogenase (MDH) chaperone activity assay. RESULTS: The extracts except those of methanol displayed anti-malarial potential with varying IC50 values, Z. mucronata HEX (11.69 ± 3.84 µg/mL), ETA (7.25 ± 1.41 µg/mL), DCM (5.49 ± 0.03 µg/mL), and X. undulutum HEX (4.9 ± 0.037 µg/mL), ETA (17.46 ± 0.024 µg/mL) and DCM (19.27 ± 0.492 µg/mL). The extracts exhibited minimal cytotoxicity except for the ETA and DCM of Z. mucronata with CC50 values of 10.96 and 10.01 µg/mL, respectively. Isolation and structural characterization of the active compounds from the DCM extracts revealed that betulinic acid (19.95 ± 1.53 µg/mL) and lupeol (7.56 ± 2.03 µg/mL) were responsible for the anti-malarial activity and had no considerable cytotoxicity (CC50 > µg/mL). Molecular docking suggested strong binding between PfHsp70-1, betulinic acid (- 6.8 kcal/mol), and lupeol (- 6.9 kcal/mol). Meanwhile, the in vitro validation assays revealed the disruption of the protein structural elements and chaperone function. CONCLUSION: This study proves that X undulutum and Z. mucronata have anti-malarial potential and that betulinic acid and lupeol are responsible for the activity seen on Z. mucronata. They also make a case for guided purification of new phytochemicals in the other extracts and support the notion of considering medicinal plants to discover new anti-malarials.

Muscle relaxant and antipyretic effects of pentacyclic triterpenes isolated from the roots of Diospyros lotus L.

The present article describes the muscle relaxant and antipyretic effects of pentacyclic triterpenes, oleanolic acid (OA), ursolic acid (UA) and betulinic acid (BA) isolated from roots of Diospyros lotus in animal models. The muscle relaxant effects of isolated pentacyclic triterpenes were determined by chimney and inclined plane tests. In the chimney test, pretreatment of pentacyclic triterpenes evoked significant dose dependent influence on muscle coordination. When administered intraperitoneally (i.p.) to mice at 10 mg/kg for 90 min, OA, UA, and BA exhibited muscle relaxant effects of 66.72 %, 60.21 %, and 50.77 %, respectively. Similarly, OA, UA, and BA (at 10 mg/kg) illustrated 65.74 %, 59.84 % and 51.40 % muscle relaxant effects in the inclined plane test. In the antipyretic test, significant amelioration was caused by pretreatment of all compounds in dose dependent manner. OA, UA, and BA (at 5 mg/kg) showed 39.32 %, 34.32 % and 29.99 % anti-hyperthermic effects, respectively 4 h post-treatment, while at 10 mg/kg, OA, UA, and BA exhibited 71.59 %, 60.99 % and 52.44 % impact, respectively. The muscle relaxant effect of benzodiazepines is well known for enhancement of GABA receptors. There may exist a similar mechanism for muscle relaxant effect of pentacyclic triterpenes. The in-silico predicted binding pattern of all the compounds reflects good affinity of compounds with GABAA receptor and COX-2. These results indicate that the muscle relaxant and antipyretic activities of these molecules can be further improved by structural optimization.

Synthesis of Rhodamine-Conjugated Lupane Type Triterpenes of Enhanced Cytotoxicity.

Various conjugates with rhodamines were prepared by starting with betulinic acid (BA) and platanic acid (PA). The molecules homopiperazine and piperazine, which were identified in earlier research, served as linkers between the rhodamine and the triterpene. The pentacyclic triterpene's ring A was modified with two acetyloxy groups in order to possibly boost its cytotoxic activity. The SRB assays' cytotoxicity data showed that conjugates 13-22, derived from betulinic acid, had a significantly higher cytotoxicity. Of these hybrids, derivatives 19 (containing rhodamine B) and 22 (containing rhodamine 101) showed the best values with EC50 = 0.016 and 0.019 µM for A2780 ovarian carcinoma cells. Additionally, based on the ratio of EC50 values, these two compounds demonstrated the strongest selectivity between malignant A2780 cells and non-malignant NIH 3T3 fibroblasts. A375 melanoma cells were used in cell cycle investigations, which showed that the cells were halted in the G1/G0 phase. Annexin V/FITC/PI staining demonstrated that the tumor cells were affected by both necrosis and apoptosis.

Exploring the inhibitory action of betulinic acid on key digestive enzymes linked to diabetes via in vitro and computational models: approaches to anti-diabetic mechanisms.

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 IC50 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.

Combinatorial Metabolic Engineering for Improving Betulinic Acid Biosynthesis in Saccharomyces cerevisiae.

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.

Combination of betulinic acid and EGFR-TKIs exerts synergistic anti-tumor effects against wild-type EGFR NSCLC by inducing autophagy-related cell death via EGFR signaling pathway.

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.

Insights into betulinic acid as a promising molecule to fight the interkingdom biofilm Staphylococcus aureus-Candida albicans.

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.

Comparative Phytoprofiling of Achillea millefolium Morphotypes: Assessing Antioxidant Activity, Phenolic and Triterpenic Compounds Variation across Different Plant Parts.

Achillea millefolium L., commonly known as yarrow, is a versatile and widely distributed plant species with a rich history of ethnopharmacological significance. This study aimed to evaluate the comparative differences of A. millefolium inflorescence morphotypes. The phytochemical profile of white and pink inflorescence morphotypes was characterised by a complex of thirty-four phenolic and triterpene compounds. The species has distinct morphotypes of white and pink inflorescence. Phenolic and triterpenic profiles were determined, and individual compounds were quantified in inflorescence, leaf, and stem samples of two morphotypes tested. The antioxidant activity of plant extracts was evaluated by free radical scavenging (ABTS) and ferric-reducing antioxidant power (FRAP) assays. Caffeoylquinic acids predominated in all parts of the plant tested. Chlorogenic acid and 3,5-dicaffeoylquinic acid were the principal compounds in the phenolic profile. Betulin, betulinic acid, and α-amyrin were the prevailing triterpenic components in the triterpenic profiles of Achillea millefolium morphotypes. The predominant flavonoids in inflorescences were flavones, while in leaves, flavonols were the organ-specific compounds. The quantitative differences were observed between plant parts of morphotypes. Leaves consistently displayed the highest amounts of identified compounds and have been testified as the main source of antioxidant activity. Overall, white inflorescences accumulated a higher total amount of compounds compared to pink ones. The observed differences between morphotypes derived from the same population reflect the differences in specialised metabolites and their chemotypes. This study addresses gaps in knowledge, particularly in phenolic and triterpenic profiling of coloured inflorescence morphotypes, enhancing our understanding of chemotypes and morphotypes within the species.

Tuning of the Anti-Breast Cancer Activity of Betulinic Acid via Its Conversion to Ionic Liquids.

Betulinic acid (BA) is a natural pentacyclic triterpene with diverse biological activities. However, its low water solubility limits its pharmaceutical application. The conversion of pharmaceutically active molecules into ionic liquids (ILs) is a promising strategy to improve their physicochemical properties, stability, and/or potency. Here, we report the synthesis and characterization of 15 novel ILs containing a cation ethyl ester of a polar, non-polar, or charged amino acid [AAOEt] and an anion BA. Except for [ValOEt][BA], we observed preserved or up to 2-fold enhanced cytotoxicity toward hormone-dependent breast cancer cells MCF-7. The estimated IC50 (72 h) values within the series varied between 4.8 and 25.7 µM. We found that the most cytotoxic IL, [LysOEt][BA]2, reduced clonogenic efficiency to 20% compared to that of BA. In addition, we evaluated the effect of a 72 h treatment with BA or [LysOEt][BA]2, the most cytotoxic compound, on the thermodynamic behavior of MCF-7 cells. Based on our data, we suggest that the charged amino acid lysine included in the novel ILs provokes cytotoxicity by a mechanism involving alteration in membrane lipid organization, which could be accompanied by modulation of the visco-elastic properties of the cytoplasm.

Betulinic Acid Potentiates Mast Cell Degranulation by Compromising Cell Membrane Integrity and Without Involving Fcεri Receptors.

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.

NF-kB affects migration of vascular smooth muscle cells after treatment with heparin and ibrutinib.

The migration of vascular smooth muscle cells (VSMCs) is one of the most important events in the remodeling of atherosclerosis plaque. The aim of study was to investigate the role of Heparin in the VSMC migration and its association with the NF-kB, collagen 1 and collagen 3 expression levels. Moreover, the incorporation of Heparin was studied in the VSMC cultures including Betulinic acid and Ibrutinib. Twelve cell groups were cultured and treated with the Heparin, Betulinic acid and Ibrutinib based on the viability and toxicity in 24-h and 48-h periods. The gene and protein expression levels were measured by RT-qPCR and western blotting techniques. The VSMC migration was determined by scratch test. In contrast with Ibrutinib (2 µM), Heparin (30 IU) increased significantly (P < 0.05) the NF-kB gene and protein expression levels and the VSMC migration during the exposure periods. Heparin (15 IU and 30 IU) also increased the collagen 1 gene expression level in the 48-h period while Heparin (5 IU and 15 IU) increased the collagen 3 gene expression levels in both periods. Incorporating Heparin into the cultures including Betulinic acid and Ibrutinib affected the collagen 1 and collagen 3 expression levels. The data suggested that the cell migration relates to NF-kB in the VSMCs treated with Heparin and Ibrutinib. Furthermore, the Heparin doses (5 IU and 15 IU) were safe for VSMCs based on the NF-kB, and collagen 3 expression levels.

Combination of Betulinic Acid Fragments and Carbonic Anhydrase Inhibitors-A New Drug Targeting Approach.

Human carbonic anhydrase IX (hCA IX) is a zinc(II)-dependent metalloenzyme that plays a critical role in the conversion of carbon dioxide and water to protons and bicarbonate. It is a membrane-bound protein with an extracellular catalytic center that is predominantly overexpressed in solid hypoxic tumors. Sulfamates and sulfonamides, for example acetazolamide (AZA), have been used to inhibit hCA IX in order to improve the response to solid hypoxic tumors. In the present study, we propose a new drug targeting approach by attaching the natural cytotoxic substances betulin and betulinic acid (BA) via a linker to sulfonamides. The conjugate was designed with different spacer lengths to accumulate at the target site of hCA IX. Computational and cell biological studies suggest that the length of the linker may influence hCA IX inhibition. Cytotoxicity tests of the newly synthesized bifunctional conjugates 3, 5, and 9 show effective cytotoxicity in the range of 6.4 and 30.1 µM in 2D and 3D tumor models. The hCA IX inhibition constants of this conjugates, measured using an in vitro enzyme assay with p-nitrophenyl acetate, were determined in a low µM-range, and all compounds reveal a significant inhibition of hypoxia-induced CA activity in a cell-based assay using the Wilbur-Anderson method. In addition, the cells respond with G1 increase and apoptosis induction. Overall, the dual strategy to produce cytotoxic tumor therapeutics that inhibit tumor-associated hCA IX was successfully implemented.