Cytotoxic Activities of Some Cynipid (Hymenoptera: Cynipidae) Gall Extracts

ABSTRACT

Natural products derived from plants are valuable sources of bioactive compounds with potential anticancer properties. This study primarily investigated the cytotoxic activities of gall extracts of Andricus caputmedusae (ACA), Andricus curtisii (ACU), and Andricus quercustozae (AQU) in human cancer cell lines (LNCaP, Caco‐2, HeLa) and a non‐cancerous cell line (HEK293). The phenolic compositions of the extracts were analyzed by LC‐MS/MS to help interpret their biological effects. The extracts contained notable levels of shikimic acid, gallic acid, protocatechuic acid, and catechin, with AQU exhibiting the richest phenolic profile. All extracts showed marked cytotoxicity, particularly against Caco‐2 cells (EC₅₀: 17.64–19.67 µg/mL), followed by HeLa (37.06–81.31 µg/mL) and LNCaP (59.33–160.19 µg/mL), while higher EC₅₀ values in HEK293 indicated selective anticancer potential. Apoptosis assays demonstrated that these cytotoxic effects were associated with apoptosis induction, especially in LNCaP and HeLa cells. Overall, the findings suggest that cynipid gall extracts possess promising anticancer potential linked to their phenolic constituents, warranting further in vitro and in vivo studies.

Phenolic‐rich cynipid gall extracts from Andricus caputmedusae, A. curtisii, and A. quercustozae exhibit selective cytotoxicity against Caco‐2, HeLa, and LNCaP cancer cells, with strongest effects in Caco‐2 cells. LC‐MS/MS profiling reveals shikimic acid, gallic acid, protocatechuic acid, and catechin, while apoptosis assays link cytotoxicity to programmed cell death induction.

1. Introduction

Galls are abnormal growths or vegetative tumors that plants form to trap and provide nutrients to a foreign organism in order to protect their conduction bundles [1]. The galls provide a microenvironment in which insects can feed and be sheltered, facilitating their development and protection [2, 3]. The majority of members of the family Cynipidae (Hymenoptera: Cynipoidea) are known as gall wasps because they form galls on host plants [4, 5]. Species of the tribe Cynipini (Cynipidae) are commonly known as oak gall wasps because they induce gall on oaks and other genera related to oaks (Fagaceae) [6, 7, 8]. Andricus species form galls on various plant parts (flowers, leaves, roots, etc.) of their host plant, oaks (Quercus spp.) [9]. While approximately 375 species have been described worldwide [6], 84 of these species are distributed in Türkiye [3, 10, 11, 12].

Each cynipid species induces a morphologically distinct gall structure unique to itself on its host plant, and these structures vary considerably among species [4]. However, in studies investigating the bioactive compounds and biological properties of cynipid galls, the specific cynipid species responsible for gall formation is rarely identified. Even when galls from different wasp species are used, the literature often employs generic and overlapping terminology. Commonly used names include “Quercus infectoria galls (QIG),” “oak galls,” “Turkish galls,” “gallnuts,” “nutgalls,” “Mecca galls,” “Aleppo galls,” and “Galla Turcica.” This lack of taxonomic specificity leads to the conflation of galls produced by different cynipid species, making it difficult to accurately distinguish among them [13, 14, 15, 16, 17, 18]. In light of all this information, in studies on gall extracts, identifying the cynipid wasp responsible for gall formation and from which the gall takes its name, and specifying the gall's name, accordingly, is of great importance for future research.

Pioneering studies highlighting the presence of bioactive compounds in cynipid galls [19, 20] have increasingly attracted the attention of researchers in recent years. Contemporary investigations have revealed that these galls, particularly due to their high phenolic content, exhibit a broad spectrum of biological activities, including antiviral, antibacterial, antifungal, astringent, antiparkinsonian, antidiabetic, antitumor, local anesthetic, anti‐inflammatory, cytotoxic, and antioxidant effects [13, 16, 21, 22, 23, 24, 25, 26, 27, 28]. These properties have historically underpinned the extensive use of cynipid galls in traditional medicine, such as Traditional Turkic or Uyghur Medicine, without reports of significant toxicity [29]. However, extracts derived from galls formed on Quercus brantii Lindl. have been shown to induce mild tissue damage when administered at high doses (≥ 500 mg/kg/day) [30]. Recent studies have further demonstrated that galls from various cynipid species possess strong antioxidant, antimicrobial, and cytotoxic activities, and have highlighted their specific therapeutic potential, such as in the treatment of ulcerative colitis and in promoting wound healing through modulation of the inflammatory response [31, 32]. Despite the well‐documented chemical composition and antioxidant properties of cynipid gall extracts, research on their cytotoxic and anticancer effects remains notably limited [33, 34]. Therefore, this study aims to investigate the cytotoxic and apoptosis‐inducing potential of gall extracts obtained from galls of Andricus caputmedusae (ACA), Andricus curtisii (ACU), and Andricus quercustozae (AQU) in various human cancer cell lines using in vitro models. Additionally, the study seeks to characterize the phenolic composition of these extracts to better understand the bioactive components responsible for their anticancer activities.

2. Results

2.1. Extraction Yield of Cynipid Gall Extracts

The extraction yields of ACA, ACU, and AQU were calculated as 7.5%, 6.2%, and 5.6%, respectively. Although ACA showed the highest extraction yield, the yield values did not directly parallel the cytotoxic and apoptosis‐inducing effects of the extracts. Therefore, the biological effects should be interpreted together with the phytochemical composition and cellular response data.

2.2. LC‐MS/MS Analysis

Analysis of phenolic compounds in extracts of ACA, ACU, and AQU galls was performed by LC‐MS/MS. Targeted screening of phenolic compounds was performed using the multiple reaction monitoring (MRM) mode. The MRM chromatogram of the standard compounds is shown in Figure S1, while the representative MRM chromatograms of the compounds detected in ACA, ACU, and AQU extracts are presented in Figures S2, S3, and S4, respectively. The quantitative results were expressed as milligrams of compound per 100 g of gall extract (mg/100 g) and are presented in Table 1. According to the analysis, shikimic acid (886.213), gallic acid (146.556), protocatechuic acid (28.021), catechin (28.722), vanillic acid (15.294), and syringic acid (11.433) in AQU; shikimic acid (1025.474), gallic acid (99.830), and protocatechuic acid (15.443) in ACU; and salicylic acid (5.879) in ACA were found at relatively high levels. While AQU showed the richest phenolic profile among all gall extracts, ACA showed the lowest detected phenolic content.

TABLE 1.

Compounds and their amounts found in cynipid gall extracts by using LC‐MS/MS.

	Compounds	ACA (mg/100 g)	ACU (mg/100 g)	AQU (mg/100 g)	RT	Transition
1	Shikimic acid	nd	1025.474	886.213	1.398	173.0 → 93.1
2	Gallic acid	nd	99.830	146.556	3.226	169.0 → 125.1
3	Protocatechuic acid	0.925	15.443	28.021	5.409	153.0 → 109.0
4	Catechin	nd	nd	28.722	6.863	288.9 → 245.1
5	Chlorogenic acid	nd	nd	nd	7.330	353.0 → 191.0
6	4‐Hydroxybenzaldehyde	nd	nd	nd	7.679	121.0 → 92.0
7	Vanillic acid	nd	nd	15.294	7.743	167.0 → 151.8
8	Caffeic acid	nd	0.190	1.849	7.798	178.9 → 135.1
9	Syringic acid	nd	nd	11.433	8.401	197.1 → 181.8
10	Caffeine	nd	nd	nd	8.431	195.0 → 137.9
11	Vanillin	nd	0.583	1.030	8.649	153.0 → 125.0
12	p‐coumaric acid	0.344	0.334	1.918	9.441	163.0 → 119.1
13	Salicylic acid	5.879	nd	4.603	9.539	137.0 → 93.1
14	Resveratrol	nd	nd	nd	9.791	229.0 → 107.0
15	trans‐ferulic acid	nd	nd	3.597	10.132	193.1 → 133.9
16	Sinapic acid	2.658	nd	nd	10.398	223.1 → 208.0
17	Scutellarin	nd	nd	nd	11.151	462.8 → 286.8
18	o‐coumaric acid	nd	nd	nd	11.502	163.0 → 119.0
19	Protocatehuic ethyl ester	nd	0.198	0.485	11.622	181.0 → 107.9
20	Rutin	nd	nd	nd	11.818	611.0 → 302.8
21	Isoquercitrin	nd	nd	4.378	11.851	464.9 → 302.8
22	Hesperidin	nd	5.130	4.020	12.301	611.0 → 302.9
23	Quercetin‐3‐D‐xyloside	nd	nd	nd	12.433	432.7 → 299.5
24	Kaempferol‐3‐glucoside	nd	0.107	1.705	13.087	448.8 → 286.9
25	Fisetin	nd	nd	nd	13.327	287.0 → 137.0
26	Baicalin	nd	nd	nd	13.653	446.8 → 270.9
27	trans‐cinnamic acid	nd	nd	nd	14.272	149.0 → 131.1
28	Quercetin	nd	nd	1.502	14.821	300.8 → 151.0
29	Naringenin	nd	nd	0.974	14.999	270.9 → 119.1
30	Hesperetin	nd	nd	nd	15.815	300.9 → 164.0
31	Kaempferol	nd	nd	nd	16.431	284.9 → 116.9
32	Baicalein	nd	nd	nd	17.084	271.0 → 123.0
33	Luteolin	nd	nd	nd	17.909	285.0 → 133.1
34	Biochanin A	nd	nd	nd	17.910	284.9 → 151.9
35	Chrysin	nd	nd	nd	17.963	254.9 → 153.0

Abbreviation: nd, not detected.

2.3. Cytotoxic Effects of Cynipid Gall Extracts

We performed the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay to investigate the cytotoxic effects of ACA, ACU, and AQU on LNCaP, Caco‐2, HeLa, and HEK293 cell lines (Figure 1). The cells were treated with different concentrations (6.25, 12.5, 25, 50, and 100 µg/mL) of extracts for 24 h. According to the results, ACU, AQU, and ACA were demonstrated to be effective on the cancer cell lines, with EC₅₀ values of 89.55, 160.19, and 59.33 µg/mL for LNCaP, 17.64, 19.65, and 19.67 µg/mL for Caco‐2, and 81.31, 76.04, and 37.06 µg/mL for HeLa, respectively. Additionally, the effects of ACU, AQU, and ACA on the HEK293 cell line were observed to be 55.61, 121.90, and 90.31 µg/mL, respectively. Hence, gall extracts exhibit cytotoxicity toward LNCaP, Caco‐2, and HeLa cell lines in a dose‐dependent manner.

FIGURE 1.

Cell viability of LNCaP, Caco‐2, HeLa, and HEK293 cells after 24 h treatment with cynipid gall extracts. Data are presented as mean ± SD of triplicate experiments. *p < 0.05 compared with the untreated control group.

2.4. Apoptosis Analysis

For the apoptotic effect of ACA, ACU, and AQU on human prostate adenocarcinoma (LNCaP) after 24 h of treatment, cisplatin used as positive control was detected by using the Annexin V‐FITC/7‐AAD Apoptosis Assay Kit. According to the results, percentages of apoptotic cells in negative control and positive controls were 3.69%, and 18.88% respectively. However, the EC₅₀ values of ACA, ACU, and AQU on the LNCaP cell line showed significant (13.03%, 40.33%, and 43.78% respectively) apoptotic activity (Figure 2).

FIGURE 2.

Apoptosis analysis of ACA, ACU, and AQU extracts in the LNCaP cell line. After 24 h of treatment, cells were stained with Annexin V/PI and were analyzed by flow cytometry. Cisplatin (25 µM) was used as a positive control. *p < 0.05 compared with the untreated control group.

The Caco‐2 cell line was treated with the EC₅₀ concentrations of ACU, AQU, and ACA for 24 h, after analysis by CytoFLEX Flow Cytometer, the results showed that negative and positive controls were 4.22% and 22.59%, and ACA, ACU, and AQU were 23.97%, 19.45%, and 15.55%, respectively, apoptosis effect described (Figure 3).

FIGURE 3.

Apoptosis analysis of ACA, ACU, and AQU extracts in the Caco‐2 cell line. After 24 h of treatment, cells were stained with Annexin V/PI and were analyzed by flow cytometry. Cisplatin (25 µM) was used as a positive control. *p < 0.05 compared with the untreated control group.

The apoptotic effects of ACA, ACU, and AQU extracts on the HeLa cell line were evaluated after 24 h of treatment. Cells were harvested, and the percentage of apoptotic cells was analyzed using the CytoFLEX Flow Cytometer according to the Annexin V‐FITC/PI Apoptosis Assay Kit. Apoptotic cell percentages were 4.1% in the negative control and 14.89% in the positive control, whereas treatment with ACA, ACU, and AQU induced apoptosis at rates of 20.09%, 18.67%, and 22.9%, respectively (Figure 4).

FIGURE 4.

Apoptosis analysis of ACA, ACU, and AQU extracts in the HeLa cell line. After 24 h of treatment, cells were stained with Annexin V/PI and were analyzed by flow cytometry. Cisplatin (25 µM) was used as a positive control. *p < 0.05 compared with the untreated control group.

3. Discussion

Plant‐derived natural products, owing to their bioactive compounds, have attracted increasing attention in cancer research in recent years [13]. In particular, secondary metabolites such as tannins, flavonoids, and phenolic acids are considered promising candidates for the prevention and treatment of various cancer types due to their antimicrobial, antiviral, antioxidant, anti‐inflammatory, and antiproliferative properties [15, 25, 35, 36].

Galls formed by species belonging to the Cynipidae family are remarkable for their richness in phenolic acids and flavonoids [28, 37]. While these compounds are involved in plant defense mechanisms, they also exhibit important biological effects that may be relevant to human health [28, 38]. In this context, the phenolic profiles of the galls of ACA, ACU, and AQU were determined by LC‐MS/MS, and their cytotoxic and apoptotic effects were evaluated in LNCaP, Caco‐2, and HeLa cancer cell lines. Although the present study does not establish a direct causal relationship between individual phenolic compounds and cytotoxic activity, the phytochemical findings provide a useful basis for discussing the possible contribution of phenolic constituents to the observed biological effects.

LC‐MS/MS analyses revealed that cynipid gall extracts possess a rich phenolic profile (Table 1). In particular, shikimic acid and gallic acid, detected at relatively high levels in the AQU extract, have previously been reported to exhibit anticancer potential [39, 40]. Shikimic acid, in addition to being a precursor metabolite in aromatic amino acid biosynthesis, has been reported to reduce oxidative stress and regulate cellular metabolism [41]. Similarly, gallic acid has been shown to exert antitumor effects in various cancer cell lines by inducing DNA damage, increasing ROS production, and activating caspase‐mediated apoptotic pathways [42, 43]. Protocatechuic acid and catechin, which were also detected in ACU and AQU extracts, have been associated with suppression of cell proliferation and induction of apoptosis in previous studies [44, 45, 46]. By contrast, ACA extract contained lower phenolic content, which may partially explain its weaker biological activity in some experimental settings. However, since correlation analysis, bioactivity‐guided fractionation, and testing of isolated compounds were not performed in the present study, the specific contribution of each phenolic constituent to the observed cytotoxic and apoptotic effects remains to be clarified.

Cell viability assay results demonstrated that ACA, ACU, and AQU extracts reduced the viability of LNCaP, Caco‐2, and HeLa cells following 24 h exposure (Figure 1). Among the tested cancer cell lines, Caco‐2 cells appeared to be more sensitive to the gall extracts, as reflected by the lower EC₅₀ values compared with LNCaP and HeLa cells. This cell line‐dependent sensitivity may be related to differences in cellular metabolism, uptake capacity, oxidative stress response, or intrinsic susceptibility to phytochemical‐induced damage. Moreover, ACU and AQU extracts, which exhibited richer phenolic profiles, generally showed stronger cytotoxic effects in several experimental conditions. Nevertheless, this relationship should be interpreted cautiously, as the present study did not include a direct statistical correlation between phenolic content and cytotoxic activity. The higher EC₅₀ values observed in HEK293 cells indicate lower cytotoxicity in this non‐cancerous, transformed kidney‐derived reference cell line under the present experimental conditions. However, since HEK293 cells are transformed and do not fully represent normal primary cells, these findings should not be interpreted as definitive evidence of cancer‐selective cytotoxicity.

Previous studies have shown that phenolic compounds, especially gallic acid and catechin, can affect lung and colon cancer cells through mechanisms involving cell cycle arrest and induction of apoptosis [43, 45, 47]. In addition Q. infectoria gall extract has been reported to significantly inhibit the proliferation of colon (HT29), lung (A549), gastric (BGC823), and esophageal (KYSE‐30) cancer cell lines [26, 48]. Yosuf and colleagues further demonstrated cytotoxicity of Q. infectoria against HeLa (EC₅₀ = 6.33 ± 0.33 µg/mL) and MCF‐7 (EC₅₀ = 25 µg/mL) cancer cells [25, 49]. Similarly, A. sternlichti gall extract exhibited antiproliferative activity against MIA PaCa‐2 cells [34]. Moreover, Q. brantii gall extract showed stronger cytotoxicity in human melanoma A375 cells compared to normal human fibroblasts AGO‐1522, suggesting that gall‐derived extracts may exert selective effects against cancer cells [50]. In line with these reports, the present findings support the potential anticancer relevance of cynipid gall extracts, while also indicating that their activity may vary depending on gall species, phenolic composition, and cancer cell type.

Apoptosis analyses showed that the cytotoxic effects of cynipid gall extracts were accompanied by increased apoptotic cell death (Figures 2, 3, 4). ACU and AQU induced higher apoptotic rates in LNCaP cells, whereas ACA was more effective in Caco‐2 cells, suggesting that gall extracts may act through cell line‐dependent mechanisms. Previous studies have reported that phenolic compounds such as gallic acid, protocatechuic acid, and catechin can trigger apoptosis by disrupting mitochondrial membrane potential, increasing ROS levels, and activating the caspase cascade [51, 52]. Therefore, the apoptotic effects observed in the present study may be partly associated with the phenolic constituents detected in the extracts. However, this interpretation remains indirect, since individual compounds were not isolated or tested separately. In HeLa cells, the marked apoptotic response induced by ACU suggests that this extract may deserve further investigation in cervical cancer‐related models. Supporting this, Q. infectoria gall extract has been shown to induce apoptosis in colon, cervical, lung, and gastric cancer cells by regulating caspase‐3, caspase‐9, and TP53, BAX, and BCL2 gene expression, while also inhibiting migration and causing cell cycle arrest [26, 48, 53]. Similarly, A. sternlichti gall extract was reported to modulate the expression of Bax, Bcl‐2, FAS, BID, FADD, TRADD, caspase‐3, caspase‐8, caspase‐9, and caspase‐10 genes, thereby inducing apoptosis in MIA PaCa‐2 cells [34].

Although the present findings provide important evidence that cynipid gall extracts exert cytotoxic and apoptosis‐inducing effects in cancer cell lines, some points should be considered when interpreting the results. The study was performed using in vitro cell culture models, which are useful for initial biological evaluation but do not fully reflect the complexity of tumor biology in vivo, including bioavailability, metabolism, tumor microenvironment, and systemic responses. In addition, LC‐MS/MS analysis revealed the presence of several phenolic compounds in the extracts; however, correlation analysis, bioactivity‐guided fractionation, and testing of isolated compounds were beyond the scope of the present study. Therefore, the possible contribution of individual phenolic constituents to the observed biological effects requires further investigation. Moreover, HEK293 cells were used as a non‐cancerous, transformed kidney‐derived reference cell line; therefore, the selectivity‐related findings should be interpreted cautiously and validated using additional non‐transformed normal cell models. Future studies involving isolated bioactive constituents, detailed molecular mechanism analyses, time‐course experiments, additional normal cell models, and in vivo validation would further clarify the anticancer relevance of cynipid gall extracts.

4. Conclusions

The results of this study indicate that cynipid gall extracts contain diverse phenolic constituents, including shikimic acid, gallic acid, protocatechuic acid, and catechin, as identified by LC‐MS/MS analysis. The tested extracts exhibited cytotoxic and apoptosis‐inducing effects in Caco‐2, HeLa, and LNCaP cancer cell lines, with lower cytotoxicity observed in HEK293 cells under the present experimental conditions. These findings suggest that cynipid gall extracts may have potential biological relevance in cancer‐related in vitro models. However, a direct causal relationship between individual phenolic compounds and the observed anticancer effects cannot be established based on the current data. Therefore, further studies involving bioactivity‐guided fractionation, isolated compounds, detailed molecular mechanism analyses, and in vivo models are required to better clarify their anticancer potential.

5. Experimental Section

5.1. Gall Material

In the field studies conducted in 2021, the asexual generation galls of a total of three cynipid species (A. caputmedusae (Hartig, 1843), A. curtisii (Müller, 1870), and AQU (Bosc, 1792)) were individually collected from host plants (Q. infectoria G. Olivier) by Prof. Dr. Yusuf Katılmış and Dr. Musa Tataroğlu, who also identified the specimens. Gall specimens from the first two species (A. caputmedusae, A. curtisii) were obtained from Denizli province (Türkiye), while gall specimens from the last species (AQU) were obtained from Burdur province (Türkiye) to prepare a sufficient amount of extract for analysis. The emergence of adult wasps was awaited from gall specimens collected for the sole purpose of extracting gall tissue. Moreover, gall specimens in which the emergence of adults was not completed were dissected, and insect larvae were separated from them.

5.2. Gall Extraction

The asexual generation galls of the three cynipid species were collected at the appropriate phenological stage and identified. The gall samples were shade‐dried separately and then chopped into small pieces using an electric blender. For extraction, 15 g of each powdered gall sample was transferred into an Erlenmeyer flask, and 150 mL of ethanol was added, corresponding to a mass‐to‐solvent ratio of 1:10 (w/v). The extraction was carried out overnight in a shaking water bath at 55°C. After extraction, the samples were filtered into amber bottles using Whatman No. 1 filter paper. The same extraction procedure was repeated three times under identical conditions. The combined filtrates were concentrated under reduced pressure using a rotary evaporator to remove the ethanol and obtain crude extracts. The extraction yield was calculated using the following formula: extraction yield (%) = (weight of dried extract/weight of dried sample) × 100. The abbreviations of the gall extracts are presented as follows: ACA, A. caputmedusae gall extract; ACU, A. curtisii gall extract; and AQU, AQU gall extract.

5.3. Analysis of Phytochemicals

A liquid chromatography system coupled with triple quadrupole mass spectrometry (LC–MS/MS; Agilent Technologies, CA, USA) was used for the qualitative and quantitative analysis of the phytochemical compounds shown in Figure 1 in cynipid gall extracts. Chromatographic separation was achieved using a Poroshell 120 EC‐C18 analytical column (100 × 4.6 mm i.d., 2.7 µm). The column temperature was maintained at 40°C, the injection volume was 5.12 µL, and the flow rate was set to 0.4 mL/min. The mobile phase consisted of eluent A (0.1% formic acid and 5 mM ammonium format in water) and eluent B (0.1% formic acid in methanol). A gradient elution program was applied as follows: 25% B for 3–12 min, 50% B for 12–16 min, 90% B for 16–21 min, followed by re‐equilibration to 2.5% B. Mass spectrometric conditions were set as follows: capillary voltage, +3.5 kV; nebulizer gas pressure, 35 psi; drying gas flow rate, 8 L/min at 300°C. Data acquisition was performed in both positive and negative ionization modes. Compound‐specific optimization studies were conducted, and sample analyses were carried out using scheduled MRM mode.

To evaluate the analytical performance of the method, accuracy, precision, selectivity/specificity, and limits of detection (LOD) and quantification (LOQ) were assessed. Calibration curves for phenolic compounds were constructed by plotting the peak areas against the corresponding concentrations of standards prepared at a minimum of five different concentration levels. For the validation study, blank samples, standard solutions, and plant extract samples were prepared and analyzed in triplicate. In plant extract samples spiked with the standard mixture, the retention times of the analytes were confirmed, and chromatographic selectivity was evaluated in terms of potential interferences. For the evaluation of accuracy and precision, the standard mixture within the linear range was analyzed in triplicate. Accuracy was expressed as percentage bias (%bias), while precision was expressed as coefficient of variation (%CV). Accuracy was evaluated as percentage bias (%bias) by comparing the measured concentrations with the nominal concentrations, while precision was expressed as coefficient of variation (%CV) calculated from replicate analyses. LOD and LOQ values were determined using the signal‐to‐noise (S/N) approach. The LOD and LOQ values corresponded to signal‐to‐noise ratios of approximately 3 and 10, respectively. Thus, validation parameters, MRM transitions, and other analytical conditions are provided in the Supporting Information (Table S1).

$$\begin{array}{ccc}& & \mathrm{Bias}\left(\%\right)=[(\mathrm{Mean}\mathrm{Measured}\mathrm{Concentration}\hfill \\ & & -\mathrm{Nominal}\mathrm{Concentration})/\mathrm{Nominal}\mathrm{Concentration}]\times 100\hfill \end{array}$$

$$\begin{array}{ccc}\hfill \mathrm{CV}\left(\%\right)& =& \left(\mathrm{Standard}\mathrm{Deviation}/\mathrm{Mean}\mathrm{Measured}\mathrm{Concentration}\right)\hfill \\ & & \times 100\hfill \end{array}$$

For sample preparation and injected into the LC‐MS system, 50 mg of dried gall extract was completely dissolved in methanol. Liquid–liquid extraction was performed by adding 1 mL of hexane, followed by centrifugation at 9000 rpm for 10 min. After phase separation, the lower phase was diluted with a water–methanol (v/v) solution containing 0.1% formic acid and 5 mM ammonium format. The final solution was filtered and analyzed by LC–MS/MS [54].

5.4. Cell Culture

Human prostate adenocarcinoma LNCaP cell line (RRID:CVCL_0395), human colorectal adenocarcinoma Caco‐2 cell line (RRID:CVCL_0025), human cervical adenocarcinoma HeLa cell line (RRID:CVCL_0030), and human embryonic kidney HEK293 cell line (RRID:CVCL_0045), were obtained from the European Collection of Cell Cultures (ECACC). LNCaP cell line maintained in RPMI (Gibco) medium. Caco‐2, HeLa and HEK293 cell lines maintained in Dulbecco's modified eagle's medium (DMEM)(Gibco). all mediums contained 10% fetal bovine serum (FBS, Sigma), 1% streptomycin‐penicillin (Gibco). The cell lines incubated at 37°C in a 5% CO₂ humidified atmosphere as described previously [55].

5.5. Cytotoxicity Assay

The MTT assay was used to evaluate the cytotoxic effects of the cynipid gall extracts on the human prostate carcinoma LNCaP cell line, human colorectal adenocarcinoma Caco‐2 cell line, human cervical adenocarcinoma HeLa cell line, and the transformed human embryonic kidney HEK293 cell line was used as a non‐cancerous reference control cell line. The cells were cultured in RPMI /DMEM mediums with 10% FBS. After cell density reached 80%–90%, the cells were harvested with 0.025% trypsin‐EDTA (Sigma), seeded in 96‐well plates at a concentration of 2 × 10³ cells per well, incubated at 37°C in a 5% CO₂ humidified atmosphere overnight, and treated with various concentrations of gall extracts (6.25, 12.5, 25, 50, and 100 µg/mL) for 24 h. Following incubation, each plate received 5 mg/mL of MTT reagent and was incubated for 2‐4 h, then formazan crystals were dissolved in 50 µL of dimethyl sulfoxide (DMSO). Absorbance was measured at 590 nm using a microplate reader (Epoch spectrophotometer (BioTek, USA). Cell viability was calculated relative to untreated controls, as described previously [56].

5.6. Apoptosis Assay

The apoptotic effects of gall extract (ACA, ACU, and AQU) were determined by using the Annexin V‐FITC/7‐AAD Apoptosis Assay Kit (Biovision, CA, USA) according to the manufacturer's protocol, with minor modifications as described previously [57]. Briefly, after the density reached 80%–90% of the cancer cell lines (LNCaP, Caco‐2, and HeLa), cells were harvested with 0.025% trypsin‐EDTA (Sigma), seeded into 6‐well cell culture plates at a concentration of 3 × 10⁴, and incubated overnight under standard conditions of cell culture. Following incubation, the cells were treated with EC₅₀ concentrations of the gall extracts for 24 h. After 24 h of treatment, the cells were trypsinized and washed twice with 1X PBS, and the cell pellets were resuspended in 100 µL of 1x Annexin binding buffer including 2.5 µL of Annexin V‐FITC and 2.5 µL 7AAD, and incubated for 20 min in dark at room temperature as described previously [58]. Finally, 400 µL of 1x Annexin binding buffer was added and 3 × 104 cells were analyzed by CytoFLEX Flow Cytometer (Beckman Coulter, USA).

6. Statistical Analysis

All experiments were performed in triplicate, and the results are presented as mean ± standard deviation (SD). Statistical analyses were carried out using SPSS v23.0 and GraphPad Prism v9.0, while all graphs and data visualizations were generated with GraphPad Prism. Differences between the control and treatment groups were analyzed using one‐way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. EC₅₀ values were calculated by nonlinear regression analysis using GraphPad Prism v9.0. A p value of <0.05 was considered statistically significant.

Author Contributions

All the authors are responsible for the following: study conception and design, data collection, analysis, interpretation of results, and manuscript preparation. All the authors reviewed the results and approved the final version of the manuscript.

Ethics Statement

This study did not involve human participants or experimental animals. All experimental procedures were performed using commercially available cell lines; therefore, ethical approval was not required.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71604‐sup‐0001‐SuppMat.docx

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.