Citrus seeds are full of phenolic compounds, such as flavonoids. The aims of this study were to identify the types of flavonoids in Citrus seed extracts, the cytotoxic effect, mode of cell death, and signaling pathway in human hepatic cancer HepG2 cells. The flavonoids contain anticancer, free radical scavenging, and antioxidant activities. Neohesperidin, hesperidin, and naringin, active flavanone glycosides, were identified in Citrus seed extract. The cytotoxic effect of three compounds was in a dose-dependent manner, and IC₅₀ levels were determined. The sensitivity of human HepG2 cells was as follows: hesperidin > naringin > neohesperidin > naringenin. Hesperidin induced HepG2 cells to undergo apoptosis in a dose-dependent manner as evidenced by the externalization of phosphatidylserine and determined by annexin V-fluorescein isothiocyanate and propidium iodide staining using flow cytometry. Hesperidin did not induce the generation of reactive oxygen species, which was determined by using 2′,7′-dichlorohydrofluorescein diacetate and flow cytometry method. The number of hesperidin-treated HepG2 cells with the loss of mitochondrial transmembrane potential increased concentration dependently, using 3,3′-dihexyloxacarbocyanine iodide employing flow cytometry. Caspase-9, -8, and -3 activities were activated and increased in hesperidin-treated HepG2 cells. Bcl-xL protein was downregulated whereas Bax, Bak, and tBid protein levels were upregulated after treatment with hesperidin in a dose-dependent manner. In conclusion, the bioflavanone from Citrus seeds, hesperidin, induced human HepG2 cell apoptosis via mitochondrial pathway and death receptor pathway. Citrus seed flavonoids are beneficial and can be developed as anticancer drug or food supplement, which still needs further in vivo investigation in animals and human beings.

Dihydrochalcone derivatives are active compounds that have been purified from the Thai medicinal plant Cyathostemma argenteum. The objectives of this study were to investigate the effects of two dihydrochalcone derivatives on human breast cancer MDA-MB-231 and MCF-7 cell proliferation and to study the relevant mechanisms involved. The two dihydrochalcone derivatives are 4′,6′-dihydroxy-2′,4-dimethoxy-5′-(2″-hydroxybenzyl)dihydrochalcone (compound 1) and calomelanone (2′,6′-dihydroxy-4,4′-dimethoxydihydrochalcone, compound 2), both of which induced cytotoxicity toward both cell lines in a dose-dependent manner by using MTT assay. Treatment with both derivatives induced apoptosis as determined by annexin V-FITC/propidium iodide employing flow cytometry. The reduction of mitochondrial transmembrane potential (staining with 3,3′-dihexyloxacarbocyanine iodide, DiOC₆, employing a flow cytometer) was established in the compound 1-treated cells. Compound 1 induced caspase-3, caspase-8, and caspase-9 activities in both cell lines, as has been determined by specific colorimetric substrates and a spectrophotometric microplate reader which indicated the involvement of both the extrinsic and intrinsic pathways. Calcium ion levels in mitochondrial and cytosolic compartments increased in compound 1-treated cells as detected by Rhod-2AM and Fluo-3AM intensity, respectively, indicating the involvement of the endoplasmic reticulum (ER) stress pathway. Compound 1 induced cell cycle arrest via enhanced atm and atr expressions and by upregulating proapoptotic proteins, namely, Bim, Bad, and tBid. Moreover, compound 1 significantly inhibited the EGFR/MAPK signaling pathway. In conclusion, compound 1 induced MDA-MB-231 and MCF-7 cell apoptosis via intrinsic, extrinsic, and ER stress pathways, whereas it ameliorated the EGFR/MAPK pathway in the MCF-7 cell line. Consequently, it is believed that compound 1 could be effectively developed for cancer treatments.

Calomelanone, 2 ^′,6 ^′‐dihydroxy‐4,4 ^′‐dimethoxydihydrochalcone, possesses anticancer activities. This study was conducted to investigate the cytotoxic effect of calomelanone, a dihydrochalcone analogue, on human cancer cells and its associated mechanisms. The cytotoxic effect of calomelanone was measured by MTT assay. Annexin V‐FITC/propidium iodide and DiOC₆ staining that employed flow cytometry were used to determine the mode of cell death and reduction of mitochondrial transmembrane potential (MTP), respectively. Caspase activities were measured using specific substrates and colorimetric analysis. The expression levels of Bcl‐2 family proteins were determined by immunoblotting. Reactive oxygen species were also measured using 2 ^′,7 ^′‐dihydrodichlorofluorescein diacetate and dihydroethidium (fluorescence dyes). Calomelanone was found to be toxic towards various human cancer cells, including acute promyelocytic HL‐60 and monocytic leukemic U937 cells, in a dose‐dependent manner at 24 h and human hepatocellular HepG2 cells at 48 h. However, the proliferation of HepG2 cells increased at 24 h. Calomelanone was found to induce apoptosis in HL‐60 and U937 at 24 h and HepG2 apoptosis at 48 h via the intrinsic pathway by inducing MTP disruption. This compound also induced caspase‐3, caspase‐8, and caspase‐9 activities. Calomelanone upregulated proapoptotic Bax and Bak and downregulated antiapoptotic Bcl‐xL proteins in HepG2 cells. Moreover, signaling was also associated with oxidative stress in HepG2 cells. Calomelanone induced autophagy at 24 h of treatment, which was evidenced by staining with monodansylcadaverine (MDC) to represent autophagic flux. This was associated with a decrease of Akt (survival pathway) and an upregulation of Atg5 (the marker of autophagy). Thus, calomelanone induced apoptosis/regulated cell death in HL‐60, U937, and HepG2 cells. However, it also induced autophagy in HepG2 depending on duration, dose, and type of cells. Thus, calomelanone could be used as a potential anticancer agent for cancer treatment. Nevertheless, acute and chronic toxicity should be further investigated in animals before conducting investigations in human patients.

Encapsulation serves as a critical strategy for the preservation of sensitive bioactive compounds, ensuring their stability and functionality within complex food matrices. Cellulose nanocrystals (CNCs) upcycled from by-products are being favoured as wall materials because they offer a sustainable yet powerful solution for maintaining compound stability. This study evaluated the encapsulation of fish oil (FO) within a nanocomposite matrix of garlic skin-derived cellulose nanocrystals (GCNCs) and soy protein isolate (SPI). The synergistic effects of FO loading and GCNC:SPI ratios on the microcapsules’ structural, physicochemical, and digestive properties were investigated. Higher FO loading significantly reduced the moisture content of the resulting microcapsule powders while increasing bulk and tapped densities by minimising internal porosity. Microstructural analysis showed irregularly shaped agglomerates. Higher FO loading also increased surface oil retention and inter-particle adhesion of the microcapsule powders; however, elevated SPI levels effectively counteracted these effects. Colour analysis further revealed that higher FO loading reduced powder lightness (L*) and increased yellowness (b*), while greater GCNC content positively influenced redness (a*). The formulation containing 10% FO, 3% GCNCs, and 7% SPI was identified as the optimal treatment. This ratio achieved the highest encapsulation efficiency (65.77% ± 1.10) and demonstrated superior flowability, characterised by the lowest Carr’s Index (20.65% ± 0.29) and Hausner Ratio (1.23 ± 0.05). Additionally, it maintained oxidative stability, with TBARS values (2.42 ± 0.08 mg MDA/kg oil) remaining consistently below the established 3 mg MDA/kg threshold. Fourier Transform Infrared Spectroscopy confirmed the successful entrapment of FO within the GCNC–SPI matrix. According to the in vitro digestion assays, the wall material provided a durable barrier in acidic media because the gastric release (28.04–55.28%) was significantly lower than the intestinal release (64.38–77.62%). The predominant fatty acids identified in both encapsulated and unencapsulated products were myristic acid (saturated fatty acid), elaidic acid (monounsaturated fatty acid), and docosadienoic acid (polyunsaturated fatty acid). Superior nutritional quality index (NQI) values in the encapsulated samples underscore the effectiveness of the wall material in providing a critical defence against fatty acid degradation and preserving overall oil quality. These findings suggest that the GCNC/SPI binary system is a highly effective delivery vehicle for protecting sensitive polyunsaturated fatty acids in functional food applications.