Introduction

Growing health awareness has intensified the search for safer and more effective therapeutic and preventive alternatives. Natural compounds derived from plants, particularly those from the genus Citrus, have demonstrated significant potential in diverse health applications. Citrus fruits are rich in bioactive compounds, such as ascorbic acid, flavonoids, and phenolic acids, which are known for their antioxidant, antimicrobial, and anticancer properties1,2,3,4. However, the limited bioavailability and stability of these compounds often pose a challenge to their use as therapeutic agents. To address this limitation, current research focuses on nanotechnology-based approaches, particularly the development of nanoparticles synthesized from citrus extracts5,6. These nanoparticles are expected to enhance the effectiveness of bioactive compounds through improved formulation and increased absorption7.

Nanoparticles derived from natural sources, such as citrus fruits, present numerous advantages, including safety and sustainability. Research has revealed that biogenic nanoparticles have significant potential as carriers for bioactive compounds8increasing their delivery to target cells9,10. The antioxidant and anticancer activities of CMNs are expected to provide valuable insights for the development of more effective alternative therapies. Current studies investigating the antioxidant activity of biogenic nanoparticles have shown variable results, with some demonstrating significant antioxidant properties and others showing limited efficacy11,12,13. These findings suggest that CMNs have the potential to reduce oxidative stress, which often contributes to the development of degenerative diseases and cancer. Furthermore, incorporating CMNs into therapeutic applications could increase the bioavailability of essential nutrients and phytochemicals found in citrus fruits. By improving the efficacy of these compounds, CMNs may play a crucial role in preventive healthcare strategies. Continued research in this area could lead to innovative treatments that utilize the natural properties of CMNs for improved health outcomes.

Furthermore, the anticancer activity of CMNs is strong and significant particularly in reducing cancer cell viability14,15,16. These findings are particularly promising, especially considering the challenges associated with conventional cancer treatments, which often involve adverse side effects. Recent findings indicate that ascorbic acid and flavonoids from citrus can induce apoptosis and inhibit the proliferation of cancer cells1,17. In this context, the development of nanoparticles based on citrus extracts may increase the absorption of these compounds into cancer cells, thereby increasing therapeutic efficacy and minimizing side effects.

A meta-analysis of data on the antioxidant and anticancer activities of citrus plays a crucial role in systematically evaluating and quantifying the factors that affect its effectiveness18,19. These factors include the citrus species, the plant part used, the type and size of the nanoparticle formulation, the coating materials, and the specific cancer cell line or type. By synthesizing data from multiple studies, a meta-analysis provides more reliable and generalizable conclusions than those drawn from individual studies alone. Comparative analyses with positive controls such as Trolox demonstrated that nanoparticle type, size, and the chemical composition of citrus extracts are critical determinants of therapeutic efficacy20,21. These findings create opportunities for further research on optimizing CMNs, which could enhance the therapeutic effects of the bioactive compounds contained within. This study hypothesizes that nanoformulations derived from citrus phytochemicals, demonstrate stronger antioxidant and anticancer activities than both positive controls (butylated hydroxytoluene and doxorubicin) and negative controls (non-nano citrus extract and untreated cancer cells). Through systematic evaluation of multiple cancer cell lines and nanoparticle formulations, we investigated the dual efficacy of these nanoformulations in reducing oxidative stress and inhibiting cancer cell proliferation.

The primary objective of this study was to evaluate the effectiveness and mechanisms of action of CMNs in the context of health, as well as to provide data supporting the development of CMN-based therapies as safe and effective alternatives for cancer treatment. Therefore, this research aims to identify the potential of nanoparticles in combating degenerative diseases and cancer through antioxidant and anticancer mechanisms. This study has the potential to make significant contributions to public health and the development of improved natural health products.

Materials and methods

Topic selection, literature search, and literature evaluation

The topic was selected on the basis of the framework of previous meta-analyses, utilizing the Population, Intervention, Comparison, and Outcome (PICO) system and following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines22,23. The topic description based on PICO was as follows: P = in vitro experiments on the measurement of antioxidant and anticancer activities, I = the level or dosage of CMN affecting the measurements, C = negative control (citrus extract or others) and positive control (standard compounds used as antioxidants and anticancer agents), and O = the resultant values of antioxidant and anticancer activity. The PICO framework was then used to generate search keywords for cloud databases, including Scopus, PubMed, WoS, and JSTOR. The detailed search results are presented in Table 1. The search yielded 110 articles related to antioxidants and 321 articles related to anticancer agents. Duplicate data were removed via Mendeley Desktop version 1.19.8, resulting in 97 articles related to antioxidants and 136 articles related to anticancer agents. Further selection was conducted using the following criteria: (a) original research articles published in English and indexed with a digital object identifier (DOI); (b) inclusion of treatment versus control groups; (c) use of nanoparticles derived from citrus fruits (with unspecified varieties); and (d) reported results on antioxidant and/or anticancer activities. The final selection included 21 articles on antioxidants, 6 articles on anticancer agents, and 4 articles covering both parameters. The article selection process is illustrated in Fig. 1.

Table 1 Original research investigations from cloud databases.
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Fig. 1
figure 1

Process of discovering, assessing, and selecting original articles on the antioxidant and anticancer activities of nanoparticles from citrus phytochemicals.

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Estimation of risk of bias (ROB) and data extraction

The determination of the risk of bias (ROB), including individual scores per article and overall summaries, was based on a detailed review of each article. The assessment considered various sources of bias: randomization in the sample (D1), the intended intervention (D2), missing data (D3), the measurement process of variables (D4), and the selection of reported outcome results (D5). The results of this risk of bias assessment for articles A1–A25 (studies on antioxidants) and B1–B10 (studies on anticancer agents) are illustrated in Fig. 2. Articles with overall high bias were excluded, and data extraction was not performed.

Fig. 2
figure 2

Risk of bias assessment: The risk of bias at the individual level (A) and summary of risk of bias (B). D1 is bias from randomization, D2 is bias from deviations from the intended intervention, D3 is bias due to missing data, D4 is bias from measurement outcomes, and D5 is bias from selecting reported results.

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The selected articles served as the data source from which secondary data on antioxidants and anticancer activities were extracted. These data were then systematically organized into a spreadsheet, including the number of samples (sample size or n), average values (X̄), and standard deviations (SD) for both the control groups (C) and treatment (E) groups. The total number of studies addressing antioxidant and anticancer research or experiments was denoted as k.

Research from 24 articles on CMN antioxidants covered various aspects: citrus species, plant parts (leaf, juice, peel, pomace, and seed), nanoparticle types, nanoparticle sizes (10 to 1026 nm), synthesis methods [green synthesis (GSy), nanocomposite (NPC), nanoemulsion (NPE), and nanoparticle (NP)], antioxidant activity (measured the radical inhibition (%) and IC50 (µg/mL)), control (negative and positive), antioxidant assays, and dosages (in µg/mL; Table 2). The negative controls included silver nitrate (AgNO₃), citrus extract, sodium alginate, and pectin. The positive controls used were common antioxidant standards such as ascorbic acid (AsA), butylated hydroxytoluene (BHT), catechol, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Antioxidant assays were used to measure the 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), ferric reducing antioxidant power (FRAP), and hydroxyl radical (OH) contents. The CMN dose ranged from 15 to 50,000 µg/mL.

In Table 3, a summary of 10 studies on CMNs and their anticancer effects highlights various details. It included information on citrus species, plant parts (juice and peel), nanoparticle types and sizes (approximately 17.5 to 150 nm), synthesis methods (GSy), and anticancer activity (e.g., cancer cell growth inhibition (%) and IC50 (µg/mL)). It also included controls (both negative and positive), cancer cell lines (A-549, C6-Neural, DU-145, HeLa, HepG2, MCF-7, MDA-MB-468, melanoma, and SH-SY5Y), and dosages (in µg/mL). The cancer cell lines used in the study included A-549 (human lung cells, lung cancer), C6-Neural (rat brain cells, glioma or neural cancer), DU-145 (human prostate cells, prostate cancer), HeLa (human cervical cells, cervical cancer), HepG2 (human liver cells, liver cancer/hepatoblastoma), MCF-7 (human breast cells, hormone receptor-positive breast cancer), MDA-MB-468 (human breast cells, triple-negative breast cancer), Melanoma (human skin cells, melanoma/skin cancer), and SH-SY5Y (human neuroblastoma cells, sympathetic nervous system cancer) cells. The negative controls included untreated cancer cells and Citrus limetta carbon dots (CDOT), whereas doxorubicin (DOX) served as the positive control. The dosages ranged from 0.03 to 800 µg/mL.

The CMNs identified in the literature include silver nanoparticles with chitosan (Ag chitosan-NP), silver nanoparticles (Ag-NP), gold–iron oxide nanoparticle composite (Au@Fe₂O₃-NPC), gold nanoparticle (Au-NP), hydroxyapatite nanoparticle (Ca₁₀(PO₄)₆ (OH)₂-NP), cadmium oxide nanoparticle (CdO-NP), cerium dioxide nanoparticle (CeO₂-NP), cobalt nanoparticle (Co-NP), iron oxide nanoparticle (FeO-NP), hydrogel-based nanocomposite (Hydrogel-NPC), lime-derived nanoemulsion (Lime-NPE), lipid-based nanoemulsion (Lipid-NPE), luminescent nanoparticle (Luminescent-NP), nickel oxide nanoparticle (NiO-NP), pectin-based nanoemulsion (Pectin-NPE), tellurium nanoparticle (Te-NP), titanium dioxide nanoparticle (TiO₂-NP), vanadium nanoparticle (V-NP), whey-derived nanoemulsion (Whey-NPE), and zinc nanoparticle (Zn-NP).

Table 2 Recent studies on the use of citrus phytochemicals in the synthesis of nanocompounds with antioxidant activity.
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Table 3 Studies on the engineering of nanoparticles based on citrus phytochemicals for anticancer applications.
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Analysis and validation of a meta-analysis

The meta-analysis conducted was based on the methodology introduced by Marín-Martínez and Sánchez-Meca (2010), utilizing Hedges’ effect size (d) calculations18. Hedges’ method quantifies the difference between treatment and control groups by dividing the mean difference of both averages by the pooled standard deviation of both groups (Eq. 1). Equation 2 provides the formula for computing the pooled standard deviation (SD pooled), with adjustments made for small sample sizes (5–20 units per treatment) via Eq. 3 or Hedges’ correction factor (J). The combined effect size (d++) was determined via Eq. 4, where a random effects model was used to estimate the variance of heterogeneity and assess its impact on dependent variables such as antioxidant and anticancer activities. The value of d++indicates a larger effect size (> 1) and a small effect size (< 0.8)47. The variation in Hedges’ (Vd++) is calculated via Eq. 5. Sources of heterogeneity include study types (measured by IC50 in µg/mL, radical inhibition (%), and cell viability in %), citrus species, plant parts, nanoparticle characteristics [type and size (1–50, 51–100, 101–500, or > 500 nm)], coating materials, control groups (negative and positive), cancer cell lines, and types of cancer. Heterogeneity levels are evaluated via the Q statistic and I2 statistic (Eq. 6); significant heterogeneity is indicated by P < 0.05 or I2 > 50%48. High heterogeneity necessitates d++calculations for each source or subgroup analysis in the meta-analysis. The validation metrics for meta-analysis models involve the use of the fail-safe N (FsN) calculation, as defined by Eq. 749. The model is deemed robust (Rb.) if the computed FsN ≥ FsN model (5k + 10), where k denotes the number of studies in the meta-analysis. Conversely, if the computed FsN is less than the FsN model, the meta-analysis model is considered not robust (NRb.).

$$\:d = \:\frac{{\overline{{\text{X}}} E - \overline{{\text{X}}} C}}{{{\text{SD}}\:_{{pooled}} }}$$
(1)
$$\:{\text{S}\text{D}\:}_{pooled}=\sqrt{\frac{\left(nE-1\right){\left(\text{S}\text{D}\text{E}\right)}^{2}+\left(nC-1\right){\left(\text{S}\text{D}\text{C}\right)}^{2}}{\text{n}\text{E}+nC-2}\times\:J}$$
(2)
$$\:J=1-\:\frac{3}{4\left(nE+\:nC\right)-9}$$
(3)
$$\:{d}_{++}=\frac{{\sum\:}_{i=1}^{n}{W}_{i}{d}_{1}}{{\sum\:}_{i=1}^{n}{W}_{i}}$$
(4)
$$\:{Vd}_{++}=\frac{nC+nE}{nC.nE}+\frac{{d}^{2}}{2\left(nC+nE\right)}$$
(5)
$$\:{I}^{2}=\frac{Q-df}{Q}\times\:100\%$$
(6)
$$\:Fail-safe\:N=\left(\frac{{\left(\frac{\varSigma\:Z}{1.64}\right)}^{2}-k}{{1.64}^{2}-k}\right)$$
(7)

The symbols used for Eqs. 1–6 are as follows: X̄E denotes the average of the treatment group, whereas X̄C represents the mean of the control group. The pooled SDs represent the combined standard deviation of both the treatment and control groups. Thus, nE and nC refer to the respective sample sizes of the treatment and control groups. Additionally, SDE and SDC indicate the standard deviations of the treatment and control groups, respectively. Furthermore, J served as the correction factor for small sample sizes, Wi represented the weight assigned to each study within the meta-analysis, Q measured the Q statistic for assessing heterogeneity, and df represented the degrees of freedom associated with the Q statistic. Z represents the total z score from each kcalculated within Hedges’. The calculation of Hedges’ and FsN was conducted via OpenMEE, version 20165.

Meta-network analysis is used to rank CMNs based on citrus species and types of nanoparticles for antibacterial and anticancer activities. The results for antioxidant and anticancer activities, which show significant findings for the species and nanoparticle subgroups, were selected for ranking via network meta-analysis. Additionally, a trend analysis (using a meta-regression approach) of the effects of the dosage of CMN on the antioxidant and anticancer activities of citrus species and types of nanoparticles was conducted. The meta-network analysis and meta-regression analysis were conducted via R software version 4.3.2 with the primary packages “lme4” version 1.1–35 and “multinma” version 0.2.151.

Results

Antioxidant activity

Table 4 presents the results of the meta-analysis of CMNs. Overall, CMNs did not exhibit statistically significant antioxidant activity, as reflected by the small effect sizes observed for general antioxidant capacity (d++ = 0.2; P = 0.309) and radical scavenging activity (d++ = 0.1; P = 0.602). In contrast, the IC50-based assay demonstrated a significant and robust effect (d++ = 3.49; P < 0.05), indicating strong antioxidant potential under this specific measurement.

Table 4 Antioxidant and anticancer activities of citrus-mediated nanoformulations.
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Table 5 presents the results of the subgroup meta-analysis of IC50 antioxidant values. The analysis revealed that citrus peel (plant part subgroup) and polyvinyl alcohol (coating material subgroup) had significant and substantial effect sizes (d++ >1; P < 0.05). In comparison, the other subgroups, which included species, nanoparticle type, particle size, and control type, did not have statistically significant effects.

Table 5 Antioxidant IC50 (µg/mL) values of citrus-mediated nanoformulations by citrus species, plant parts, nanoparticle types, sizes, coatings, and control types.
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Table 6 presents the subgroup analysis for radical inhibition, while Fig. 4 illustrates the percentage of radical inhibition across various citrus species. Species such as C. limetta, C. limon, C. maxima, C. paradisi, C. reticulata, and C. sinensis presented strong effect sizes (|d++| > 0.8; P < 0.05). Notably, C. limetta, C. maxima, and C. sinensis presented negative effect sizes, indicating that the treatment groups presented lower values than did their respective controls. The models involving C. limetta, C. limon, C. paradisi, and C. sinensis remained statistically robust. Within the plant-based subgroup, the leaf, pomace, and seed samples derived from citrus sources presented significant and robust effect sizes (|d++| > 0.8; P < 0.05). Among these, pomace had a negative effect size, implying reduced radical inhibition relative to that in the control groups. For the nanoparticle subgroup, all categories showed statistically significant results, each with a strong effect size (|d++| > 0.8; P < 0.05). The nanoparticles that yielded positive effect sizes included Ag-NPs, Ag chitosan-NPs, CeO₂-NPs, hydrogel-NPC, pectin-NPE, TiO₂-NPs, and whey-NPE. In contrast, Au-NPs, Au@Fe₂O₃-NPC, CdO-NPs, Co-NPs, Fe₃O₄-NPs, NiO-NPs, and V-NPs presented negative values. However, the model involving Fe₃O₄-NPs lacked robustness. The nanoparticle size also influenced the efficacy. Particles within the 101–500 nm range produced the greatest effect size (d++ = 3.44), surpassing those in the 1–50 nm (d++ =0.77), 51–100 nm (d++ = −1.56), and > 500 nm (d++ = −13.7) ranges (P < 0.01). Subgroup analysis further indicated that coating materials such as citrus extract, hydroxypropyl methylcellulose, pectin, and whey significantly enhanced radical inhibition (P < 0.01). All models were robust, except for citrus extract, which, although statistically strong, exhibited an exceptionally high positive effect size. Finally, the meta-analysis based on the control types used in the radical inhibition assays revealed a clear contrast. The positive controls (including AsA, BHT, catechol, and Trolox) had strong negative effects (|d++| >1; P < 0.001), reflecting greater antioxidant capacity than other treatments did. Conversely, negative controls (such as AgNO₃, C. clementina, C. limon, C. reticulata, C. unshiu, hydrogel films, pectin, and sodium alginate) yielded positive effect sizes (P < 0.05), indicating lower radical inhibition compared to treatments.

Table 6 Radical Inhibition (%) by citrus species, plant parts, nanoparticle types, sizes, coatings, and control types.
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Anticancer activity

The anticancer activity of CMNs had strong and significant effects on overall outcomes, including IC50 values and a reduction in cancer cell viability (|d++| >1; P < 0.05; Table 4). Notably, only the IC50 value for cancer inhibition positively influenced both the reduction in cancer cell viability and the overall effect size. This likely reflects the fundamental nature of the cell viability data, which typically indicates decreased viability in cancer cells treated with CMNs compared with untreated control groups (usually cancer cells without any intervention). Accordingly, the administration of therapeutic agents such as CMNs is expected to reduce cancer cell viability.

In the evaluation of 50% inhibition of cancer cells, several effect sizes were observed within the subgroup meta-analysis (Table 7). The species group, specifically C. macroptera, exhibited a robust and positive effect size (d++ = 2.25; P < 0.001). Additionally, plant parts such as juice presented a similarly robust effect size of 2.26 (P < 0.001). Subgroups involving nanoparticles, namely, Au-NPs and Te-NPs, also yielded significant results (|d++| >1; P < 0.001; Rb.). Furthermore, nanoparticles with sizes between 101 and 500 nm demonstrated an effect size of 2.26 (P < 0.001; Rb.). Citrus extract, employed as the sole coating, had a significant effect size (|d++| >1; P < 0.05). The positive control groups treated with DOX presented a notable effect size (d++ = 1.4; P < 0.001), whereas the negative control groups treated with CDOT and UCC presented divergent results (|d++| >1, P < 0.05; Rb.). An examination of various cancer cell lines and types revealed that cervical cancer (HeLa), lung cancer (A-549), and skin cancer (melanoma) all had effect sizes > 1 and were statistically significant (P < 0.01). Notably, only HeLa cells exhibited a negative effect size with a non-robust model.

Table 7 Anticancer IC50 (µg/mL) values of citrus-mediated nanoformulations by citrus species, plant parts, nanoparticle types, sizes, coating, control types, cancer cell lines, and cancer types.
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Table 8 summarizes the subgroup meta-analysis results regarding the effects of citrus phytochemicals in nanoparticle form on cancer cell inhibition. Subgroups categorized by citrus species, such as C. aurantiifolia, C. clementina, C. limetta, C. limon, C. macroptera, and C. sinensis, presented significant negative effect sizes (|d++| >1; P < 0.001; Rb.). Similar findings were observed across subgroups based on plant part and type of nanoparticle. Specifically, both citrus juice and peel had notable negative effects (|d++| >1; P < 0.001; Rb.). Moreover, the Ag-NPs, Au-NPs, CdO-NPs, CeO2-NPs, Ca10(PO4)6(OH)2-NPs, and Te-NPs generally had negative effect sizes, except V-NPs, which had a positive effect size (|d++| >1; P < 0.05; Rb.). In terms of the CMNs’ size, those ranging from 1 to 50 nm and 101–500 nm presented negative effects, whereas those ranging from 51 to 100 nm presented positive effects (|d++| >1; P < 0.001; Rb.). Additionally, the citrus extract used as a coating yielded an effect size of −3.26 (P < 0.001; Rb.). The positive control (DOX) had an effect size of 5.54, whereas the negative controls CDOT and UCC had effect sizes of −3.4 and − 3.61, respectively (P < 0.001; Rb.). Finally, various cancer cell types, such as A-549 (lung cancer), C6-neural and SH-SY5Y (nerve cancer), DU-145 (prostate cancer), HeLa (cervix cancer), HepG2 (lung cancer), MDA-MB-468 (breast cancer), and melanoma (skin cancer), manifested negative effect sizes (|d++| >1; P < 0.05). Notably, only the SH-SY5Y cell line exhibited a less robust meta-analysis model.

Table 8 Inhibition of cancer cells (%) by citrus-mediated nanoformulations across citrus species, plant parts, types, sizes, coatings, controls, cancer cell lines, and cancer types.
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Dosages of the antioxidant and anticancer activities of CMNs

AsA and BHT inhibited free radicals by nearly 100% at concentrations below 200 µg/mL (Fig. 3). However, the data for AsA and was less consistent than those for BHT, with R2 values less than 0.75. Previous studies have confirmed that BHT had a stronger antioxidant activity than both AsA did52. Further, the IC₅₀ values (µM) in the DPPH assay were 16.1 for BHT, 23.8 for Trolox, and 39.4 for AsA52. Theoretical calculations also ranked the bond dissociation energy (BDE) in ascending order as BHT < Trolox < AsA52. BDE reflects an antioxidant’s ability to donate hydrogen atoms to neutralise free radicals52,53. A lower BDE corresponds to greater antioxidant potential54,55.

Fig. 3
figure 3

Effects of radical inhibition (%) from various control groups on antioxidant activity.

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Regression analysis indicated that CMNs generally presented a lower antioxidant capacity than the positive controls did (Fig. 3), with most effective antiradical doses exceeding 1,000 µg/mL (Fig. 4). However, CMNs derived from C. clementina and nanoparticles such as Au-NPs, CdO-NPs, and NiO-NPs showed relatively high radical inhibition at much lower doses (< 100 µg/mL), resulting in inhibition rates between 80% and 100%. In green synthesis, phytochemicals from C. clementina facilitated the formation of Ag-NPs with an average size of 17.5 nm. Moreover, the Au-NPs, CdO-NPs, and NiO-NPs were synthesised via phytochemicals extracted from C. limetta, C. limetta, and C. aurantium, respectively (Table 2).

Fig. 4
figure 4

Radical inhibition (%) by different types of citrus-mediated nanoformulations.

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Figure 5 shows the percentage inhibition of cancer cells by various control groups in relation to anticancer activity. The inhibitory effects of CMNs on cancer cells have been consistently demonstrated across multiple citrus species, including C. aurantiifolia, C. clementina, C. limetta, C. limon, C. macroptera, and C. sinensis (Fig. 6). These CMNs exhibit large effect sizes (d++ > 0.8), whether derived from juice or peel extracts. Compared to others, cerium oxide nanoparticles (CeO₂-NPs) most significantly reduce cancer cell viability. Specifically, CeO₂-NPs synthesised from C. aurantiifolia and averaging 22.5 nm in size, reduced the viability of HeLa (cervical cancer) cells by 7.5%, 23.5%, 33.1%, 46.6%, 57.8%, and 67.1% at concentrations of 10, 25, 50, 75, 100, and 125 µg/mL, respectively34. The reduction in cancer cell populations induced by CMNs surpasses that induced by carbon dots synthesised from C. limetta (CDOT), although doxorubicin continues to demonstrate superior efficacy13,40,42,44,46. Notably, the most pronounced anticancer response was observed in CMNs derived from C. sinensis peel extract, which achieved an 82.4% reduction in DU-145 (prostate cancer) cell viability45.

Fig. 5
figure 5

Cancer cell inhibition (%) by different control groups in terms of anticancer activity.

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Fig. 6
figure 6

Cancer cell inhibition (%) by various citrus fruits.

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Meta-network analysis of the effects of CMNs on variables related to antioxidant and anticancer activity

The antioxidant activity extensively studied in the experiment involves comparisons such as Trolox (used as a positive control) versus CMNs derived from C. limetta and BHT versus CMN extracts from C. aurantium, C. reticulata, and C. limon. Additionally, minor discussions touch upon comparisons of AsA versus C. sinensis (Fig. 7a). With respect to nanoparticle types, comparisons include Trolox versus Au-NPs and the relationship between BHT and several CMN types, such as NiO-NPs, Co-NPs, and Ag-NPs (Fig. 7b). According to the rankings obtained from Fig. 8a, among the species categories, C. sinensis ranks highest compared with the other citrus varieties and positive controls (AsA, BHT, catechol, and Trolox). Following C. sinensis are C. reticulata, C. limon, C. limetta, C. clementina, and C. aurantium. The sequences of nanoparticle types were Co-NPs, NiO-NPs, pectin-NPEs, Ag-NPs, CdO-NPs, and Au-NPs (Fig. 8b).

Fig. 7
figure 7

Meta-network analysis of the antioxidant and anticancer activities of citrus-mediated nanoformulations.

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Fig. 8
figure 8

Meta-network analysis ranking of variables related to citrus-mediated nanoformulations in terms of antioxidant and anticancer activities.

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Concerning the anticancer activities shown in Fig. 7c and d, the most frequent studies discuss the use of CDOT versus C. limetta as anticancer agents. Additionally, several studies have also examined UCC versus various citrus variants, such as C. macroptera, C. limon, C. aurantium, and C. aurantiifolia. Moreover, the most frequently studied nanoparticle types involve CDOTs versus Ag-NPs and UCCs versus various CMN variants, including Te-NPs, Au-NPs, and Ag-NPs. The anticancer effectiveness of CMNs based on species and nanoparticle type is discussed in Fig. 8c and d. Specifically, C. aurantiifolia showed the greatest effectiveness, followed by C. limon, C. macroptera, C. aurantium, and C. limetta. For the various nanoparticle types, the ranking order was Ag-NPs, Ca10(PO4)6(OH)2-NPs, Te-NPs, and Au-NPs.

Discussion

Antioxidant activity of CMNs

A lower IC50 value reflects greater potency in scavenging DPPH radicals, indicating greater antioxidant activity56. The meta-analysis results revealed a significant difference in the IC50 values between CMNs and the control group. The higher IC50 observed in the CMN treatment group suggested that the control had greater antioxidant activity. However, several studies have reported that CMNs exhibit lower IC50 values than citrus extracts do, indicating superior radical-scavenging capacity57,58. In contrast, other findings suggest that CMNs have higher IC50 values than citrus extracts do59. The antioxidant efficacy of CMNs largely depends on the redox potential of the capping agents, such as quercetin and gallic acid, present on the surface of Ag-Se nanoparticles60,61. These compounds function as potent reducing agents, hydrogen ion donors, and singlet oxygen1O2) scavengers, thereby enhancing the antioxidant performance of CMNs62. Additionally, phytochemicals in Citrus sinensis contribute to the neutralisation of reactive nitrogen species (RNS) and reactive oxygen species (ROS)63. In advanced applications, juice from C. limon has been used to form nanovesicles that protect cells from oxidative stress. This protection occurs through the activation of the aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (Nrf2) signalling pathways64.

The antioxidant results, based on the IC50 values, revealed a significant effect, particularly in CMNs derived from citrus peel extracts. Subgroup analysis confirmed that only peel-derived CMNs demonstrated statistically significant differences. This notable potential may stem from the high flavonoid content in the peel65. Flavonoids are predominantly located in the flavedo and albedo layers of citrus peels, whereas carboxylic acids are mainly found in the fruit segments66. Citrus peels are rich in bioactive compounds, making them a sustainable and renewable source of phenolics65. Compared with the pulp and seeds, the peel contains higher concentrations of phenolic compounds and exhibits greater antioxidant activity67. In addition to the type of citrus species and the plant part used, the geographical origin of the citrus is crucial. Different citrus species, depending on their location, present varying levels of antioxidants, flavonoids, and phenols68,69. Extraction techniques influenced the concentration of antioxidant compounds, as indicated by a meta-analysis that reported a weak correlation for citrus extracts (R² = 0.206; Fig. 3e). In contrast, standard controls commonly used in antioxidant evaluations showed stronger regression values, including AsA, BHT, and catechol (Fig. 3b–d). Incorporating citrus extracts into CMN-based compounds improved their bioavailability as radical-scavenging agents, particularly those derived from C. aurantium, C. limon, C. clementina, C. reticulata, and C. sinensis, all of which showed R² > 0.75. An exception occurred in C. limetta, which exhibited a decreasing trend with R² = 0.92 (Fig. 4). Previous findings supported this observation, where green synthesis modification of C. aurantium peel extract produced a DPPH IC₅₀ value of 19 µg/mL, which surpassed BHT37. Although BHT showed much stronger oxidative power than AsA, BHA, and Trolox52, the findings remained promising. The abundant availability of citrus peel waste in nature indicated antioxidant potential comparable to BHT, as previously reported.

The radical inhibition of the CMN was slightly greater than that of the control, with the strongest to weakest order being Ag chitosan-NP > Whey-NPE > Pectin-NPE > Hydrogel-NPC > CeO2-NP > TiO2-NP > Ag-NP > Fe3O4-NP > Au-NP > NiO-NP > Co-N > CdO-NP > Au@Fe2O3-NPC > V-NP. The ratios of Fe3O4-NPs to V-NPs were lower than those of the control. It would be helpful to explain the pros and cons related to nanoparticles based on the previous statement, such as whether Ag chitosan-NPs truly have a greater antioxidant capacity. This discovery confirms that nanoparticles derived from citrus phytochemicals can be used as antioxidants in various applications. However, to fully understand the nature, physicochemical properties, and mechanisms of action of nano-antioxidant composites, it is crucial to study their catalytic and biological activities62. Phytochemical substances are crucial reducing agents and stabilizers in nanoparticle synthesis22,59,70. Each substance has a distinct function. Alkaloids serve as reducing agents, flavonoids and terpenoids act as stabilizers and capping agents, and proteins and carbohydrates function as both stabilizers and reducing agents in the transformation of metallic salts into metallic nanoparticles71. Finally, nanoparticles with biologically active phytochemical components exhibit bacteriostatic properties due to various functional groups, such as OH, NH2, COOH, and NO272,73,74.

The therapeutic efficacy of CMNs strongly depends on the physicochemical characteristics of their constituent metals. Studies have shown that nanoparticles synthesised from Clerodendrum inerme (CI) leaf extracts using gold (Au) and silver (Ag) exhibit different levels of antioxidant activity75. Specifically, CI-Ag-NPs demonstrate a higher DPPH scavenging rate than CI-Au-NPs do, with values of 78.8% and 75.9%, respectively75. Further evidence suggests that Ag-NPs prepared with 2.5 mM gallic acid possess a greater absolute zeta potential than do Au-NPs, with values of 40 mV and 38 mV, respectively76. A higher absolute zeta potential enhances the stability of nano-antioxidant systems77,78,79. Researchers have successfully incorporated natural antioxidants into nanospheres by forming complex covalent bonds between metal ions and phytochemicals, thereby improving their antioxidant functionality80. This bonding also contributes to nanoparticle stability59,81. Moreover, phytochemical extracts can neutralise the intrinsic oxidative properties of nanoparticles, enabling their antioxidant activity82. Numerous studies support these observations. For example, one investigation reported that CMNs exhibit substantial antioxidant capacity, with ABTS and DPPH assay results comparable to those of the standard antioxidant Trolox58. In contrast, another study revealed no significant differences in antioxidant activity between various lemon essential oils and their corresponding nanoemulsions, as measured by both ABTS and DPPH assays5. Furthermore, compared with those produced via conventional synthesis, zinc oxide nanoparticles synthesised via green methods with C. limetta peel extract demonstrated superior antioxidant capacity35. Another finding concerns the green synthesis of silver nanoparticles using extracts from Punica granatum and Plectranthus rugosus, which demonstrated free radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl at 70% and 68%, respectively83. In addition, extracts from Azadirachta indica have also been reported to possess antidiabetic properties84.

Anticancer activity of CMNs

Differences in cell viability responses are likely due to the ability of CMNs to reduce cancer cell viability. In a subgroup meta-analysis, several citrus species and specific plant parts, such as juice, had significant positive effects on cancer inhibition, with C. macroptera and nanoparticles of certain sizes demonstrating strong results. The synthesis of nanoparticles throughout the study revealed that the green synthesis used to obtain CMNs has biological properties as an anticancer agent40. Previous findings indicate that nanoparticles from C. macroptera incorporated with gold (Au) at a dose of 300 ng/mL significantly reduce the populations of A-549 (lung cancer), MDA-MB-468 (breast cancer), and HepG2 (liver cancer) cells16. The size of C. macroptera nanoparticles is estimated to be approximately 31 nm via DLS tools16. Moreover, C. macroptera has an IC50 value of 70.2 ng/mL against HepG2 cells, while the values for A549 and MDA-MB-468 cells are 143 ng/mL and 157.9 ng/mL, respectively16.

Other findings also indicate that CMNs from C. aurantium, C. aurantiifolia, and C. limon combined with Te-NPs (101, 150, and 150 nm, respectively) have IC50 values of 204, 39, and 135 µg/mL against melanoma15. A size range of 101–500 nm was confirmed to be the most effective anticancer agent. Additionally, the incorporation of C. aurantium extract with V-NPs 80 nm in size has an IC50 of 15.9 µg/mL against MCF-7 (breast cancer) cells13. Furthermore, Cd-NPs from C. limetta (peel extract) and Ag-NPs from C. clementina (peel extract) had IC50 values of 152.2 (against A-549, lung cancer) and 60 µg/mL (C6-Neural, nerve cancer), respectively. Although the meta-analysis model confirmed that only the inhibition of A-549 cells and melanoma by CMNs was robust, data supporting other cancer cell models remain scarce.

The cytotoxicity to cancer cells caused by CMNs is attributed to two primary factors: particle aggregation and ROS formation. Particle aggregation triggers lipid beta-oxidation in the cell membrane, leading to increased cell viability (Fig. 9)16. ROS can induce apoptosis, which has been observed in DU-145 cells treated with Ag-NPs (C. sinensis)45. Additionally, the antioxidant properties of V-NP from C. aurantium are believed to involve alkaloids, ascorbic acid, flavonoids, saponins, and tannins that are correlated with DPPH. V-NP also scavenges free radicals, which can trigger mutagenesis and proliferation of cancer cells13. The results of previous experiments clearly demonstrated that nanoparticles derived from Olea paniculata and Bauhinia variegata Linn extracts exhibit increased antioxidant activity, which strongly correlates with the concentration of the extracts85,86. Other possible mechanisms resulting from the use of nanoparticles as cancer cell inhibitors include DNA damage, mitochondrial disruption, disturbance of cellular signalling pathways, lysosomal dysfunction, increased free metal ions, and autophagy (Fig. 9).

Au-NPs targeted at the cancer cell nucleus can cause significant DNA damage, leading to apoptosis (Fig. 9). Au-NPs disrupt cytokinesis, resulting in binucleated cells and increased double-strand DNA damage87. Similarly, iron oxide nanoparticles can also induce apoptosis in cancer cells through oxidative stress, which includes increased ROS and lipid peroxidation, as well as reduced antioxidant activity. Oxidative stress causes DNA damage, which can be detected through comet assays and chromatin condensation. The activation of caspase-3 indicates the apoptotic pathway, resulting in programmed cell death due to DNA damage. Thus, iron oxide nanoparticles induce apoptosis in cancer cells through oxidative stress mechanisms that cause DNA damage88. Furthermore, ROS from metal ion nanoparticles can also induce mitochondrial dysfunction in cancer cells. Such damage includes decreased mitochondrial membrane potential, which is essential for ATP synthesis and cellular homeostasis. This process induces the expression of proapoptotic genes such as p53 and bcl-2-associated X protein activation (BAX). Mitochondrial damage also reduces the number of healthy mitochondria, alters mitochondrial morphology, and causes broader dysfunction in cancer cells89,90. Additionally, when nanoparticles accumulate in lysosomes, their pH can be altered, leading to lysosomal damage and increased ROS production91. This generates oxidative stress that can damage cellular components and trigger apoptosis91,92. Moreover, the increase in free metal ions released from nanoparticles can exacerbate oxidative stress and influence signalling pathways that regulate cancer cell growth93. Nanoparticles can also induce or disrupt autophagy, which is crucial for maintaining cellular homeostasis94. Therefore, while nanoparticles can provide significant therapeutic effects, they may also contribute to cancer therapy resistance, depending on their physical and chemical characteristics94,95.

Nanoparticles can affect cellular signalling pathways through various mechanisms (Fig. 9). First, they can bind to receptors on the cell surface, triggering cell proliferation or death. Second, they increase ROS production, which can activate signalling pathways such as the MAPK and STAT pathways, which play roles in cell growth, proliferation, and death96,97. Additionally, nanoparticle exposure can lead to cell death through apoptosis pathways, depending on the concentration and duration of exposure. Nanoparticles also affect chaperone proteins and the p53 gene, which are involved in DNA repair and cell cycle regulation. Finally, magnetic nanoparticles can activate signalling pathways by applying physical forces to cells. Overall, interactions between nanoparticles and cells can lead to significant changes in cellular function and health, necessitating further research to understand their long-term effects96,98.

Fig. 9
figure 9

Molecular pathways of cytotoxicity and immunomodulation induced by citrus-mediated metal nanoparticles in cancer therapy99,100,101,102.

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Potential CMNs as anticancer agents also raise concerns regarding their cytotoxicity toward healthy cells. Reports suggest that they can induce oxidative stress by increasing ROS production and damaging DNA, lipids, and proteins103,104. Furthermore, nanoparticles may disrupt cell membrane integrity, potentially leading to cell death63,105,106. Among CMNs, Au-NPs, Te-NPs, and larger particles strongly inhibit cancer, while certain sizes of CMNs have significant effects. Citrus extracts are also effective as coatings, enhancing nanoparticle efficacy. Positive controls such as doxorubicin show notable results, whereas some negative controls show the opposite results, underscoring the importance of composition. These findings support the therapeutic potential of CMNs in cancer treatment, although further research is necessary to refine their properties and applications.

Conclusion

This study reveals that various types of citrus-mediated nanoformulations (CMNs) display distinct antioxidant and anticancer activities. Among them, C. reticulata and C. sinensis exhibit the highest free radical scavenging potential. In contrast, CMNs synthesised from C. aurantiifolia and C. macroptera show superior anticancer effects, particularly against specific cancer cell lines. Particle size also plays a crucial role, with CMNs ranging from 101 to 500 nm being more effective at neutralising free radicals, while those ranging from 51 to 100 nm better inhibit cancer cell proliferation. In terms of nanoparticle type, gold-iron oxide nanocomposites (Au@Fe₂O₃-NPC), cadmium oxide nanoparticles (CdO-NPs), hydrogel-based nanocomposites (hydrogel-NPC), and vanadium nanoparticles (V-NPs) demonstrate strong antioxidant capacity. Moreover, silver nanoparticles (Ag-NPs), cerium dioxide nanoparticles (CeO₂-NPs), and V-NPs have notable cytotoxic effects against cancer cells. Nevertheless, the bioactivity of these CMNs remains lower than that of standard controls such as butylated hydroxytoluene (BHT) and doxorubicin. Despite these limitations, CMNs demonstrate considerable promise in suppressing the proliferation of various cancer cell lines, including human lung carcinoma (A549), rat glioma (C6), human prostate carcinoma (DU-145), human cervical cancer (HeLa), human hepatocellular carcinoma (HepG2), human triple-negative breast cancer (MDA-MB-468), and melanoma cells.

This meta-analysis has several limitations. The primary constraint lies in the efficacy effects on living organisms, which still require clinical validation. This limitation restricts the generalisability of the findings. In addition, despite the focus on a single species and the use of citrus fruit extracts, substantial heterogeneity remains across the included studies. A broader exploration of bioactive compound evaluations is necessary to identify consistent metabolomic patterns across different citrus extracts, which may vary depending on extraction techniques, sample types, and environmental conditions.

Therefore, further research is essential to address the existing gaps concerning the toxicity of CMNs, particularly to support their practical application in medical fields. Fundamental molecular assays and detailed investigations into the mechanisms of action of CMNs are also needed to better understand their efficacy and potential side effects. Furthermore, the standardisation of CMN processing protocols is crucial, especially considering their prospective use in pharmaceuticals and even functional foods, to ensure both effectiveness and product safety.