Abstract Hyperhomocysteinemia is considered to be one of the risk factors for inflammatory bowel disease (IBD), it is a chronic, relapsing, and remittent inflammatory disease of the gastrointestinal tract. Citrus sinensis L. has been used traditionally to treat bowel disorders. The present study aims to quantify the phenolic and flavonoid contents of the Citrus sinensis L. fruit (ORF) extract and to evaluate in vitro the antioxidant activity and the anti-inflammatory effect of ORF in vivo on intestinal inflammation induced by hyperhomocysteinemia. The total phenolic and flavonoid contents of the extract were determinated using the spectrophotometric method and the evaluation of antioxidant activity was performed by four methods: DPPH, ABTS, CUPRAC, and reducing power. The inflammatory marker (plasma homocysteine), the reduced glutathione (GSH) content in the liver tissue were measured and the histological sections of the intestines of the mice used were examined to assess the anti-inflammatory activity of ORF. The results of this study showed that the ethanolic extract of ORF possessed high phenolic content and exhibited good antioxidant activity. The use of ORF powder in the in vivo study showed an increase in GSH levels, a decrease in plasma homocysteine levels, and a restoration of the integrity of the intestinal epithelium.

Keywords: Hyperhomocysteinemia, Intestinal Inflammation, Citrus sinensis L. fruit, Antioxidant activity, Anti-inflammatory   

Citation: Khelfi, S., Zerizer, S., Foughalia, A., Tebibel, S., Bensouici, C., and Kabouche, Z. 2023. The antioxidant activity and the anti-inflammatory effect of Citrus sinensis L. fruit on intestinal inflammation induced by hyperhomocysteinemia in mice. Nat. Life Sci. Commun. 22(1): e2023009.

INTRODUCTION

Homocysteine (Hcy) is a non-protein sulfur amino acid derivative of demethylated methionine, the accumulation of which may be caused by genetic defects or vitamin B deficiency. A serum level above 15 μmol/L is defined as hyperhomocysteinemia (HHcy) (Ledda et al., 2020; Moretti et al., 2021). HHcy, activate NFκB, a transcription factor that regulates the transcription of various genes involved in inflammatory and immune responses inducing the increase of pro-inflammatory cytokines and down-regulation of anti-inflammatory cytokines. Moreover, high levels of Hcy have been detected in various inflammatory diseases such as inflammatory bowel disease (Elsherbiny et al., 2020; Al Mutairi, 2020).

Inflammatory Bowel Disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a group of disorders involving alterations in gastrointestinal physiology and chronic inflammation of the mucous membranes (Gampierakis et al., 2021; Lohning et al., 2021)

Previous studies have confirmed that HHcy is a risk factor associated with cardiovascular disease and possibly an important independent risk factor for inflammatory bowel disease (Vezzoli et al., 2020; Chen et al., 2021).

Inflammation and oxidative stress are closely related to pathophysiological events (Pepe et al., 2018). Oxidative stress results from an imbalance between an excess production of reactive oxygen species and antioxidant defenses, which has been linked to numerous pathologies (Liguori et al., 2018; Hayes et al., 2020).

Citrus sinensis L., known as orange or sweet orange belongs to the Rutaceae family, which is the most cultivated and most traded species in the world (Sathiyabama et al., 2018; Juibary et al., 2021). C. sinensis is an excellent source of secondary metabolites which have been identified in the fruits, peel, leaves, juice, and roots that contribute to the pharmacological activities attributed to this plant (Favela-Hernández et al., 2016). Eminently, Citrus sinensis L. fruit (ORF) is a rich source of vitamin C known to have beneficial effects on health (Liu et al., 2012; Oikeh, 2020).

C. sinensis identified metabolites include: flavonoids, steroids, hydroxyamides, alkanes and fatty acids, coumarins , peptides, carbohydrates, carbamates, alkylamines, carotenoids, volatile compounds, and nutritional elements such as potassium, magnesium, calcium and sodium (Grosso et al., 2013; Favela-Hernández et al., 2016). Additional nutrients are shown in Table 1 (Etebu and Nwausoma, 2014).

Table 1. Nutrient composition of Citrus sinensis.

The nutritional benefits of citrus fruit consumption on human health are well demonstrated (Campone et al., 2020). C. sinensis has been used in traditional medicine to treat medical conditions such as constipation, cramps, colic, diarrhea, bronchitis, tuberculosis, cough, cold, menstrual disorder, angina, hypertension, anxiety, depression and stress (Favela-Hernández et al., 2016; Bentahar et al., 2020). In addition, these fruits are used also to treat bowel disorders, respiratory disorders, cardiovascular disease, and stress (Mannucci et al., 2018). The genus Citrus has been recognized for its antibacterial, antioxidant, and anti-inflammatory properties (Huang et al., 2010; Nawrin et al., 2021). In addition, it has demonstrated a protective role against oxidative stress and gastric ulcer (Selmi et al., 2017; Nawrin et al., 2021).

Several studies have asserted that Citrus fruits and their derivatives can be effective in the prevention or treatment of inflammatory diseases such as IBD (Ferlazzo et al., 2016; Musumeci et al., 2020).

Studies have demonstrated the beneficial effects of Citrus and other extracts on different experimental models of colitis (Impellizzeri et al., 2015; Abe et al., 2018), whereas others have questioned their real applicability (Farzaei et al., 2015), emphasizing the need for further investigation.

In this perspective, considering the content of ORF in biologically active constituents, the present study aims to provide new research on the possible mechanism of action of the protective effect of C. sinensis fruit against experimental intestinal inflammation caused by L-methionine-induced hyperhomocysteinemia in a mouse model to confirm its pharmacological use as a remedy for IBD.

MATERIALS AND METHODS

Reagents and solvent

Folin-Ciocalteu reagent, sodium carbonate, aluminium nitrate, potassium acetate, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline6sulfonicacid) diammonium salt (ABTS), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol, ethanol, methanol. Ethylenediaminetetraacetic acid (EDTA), 5,50-dithiobis (2- nitrobenzoic) acid (DTNB), 5-sulfosalicylic acid, potassium persulfate, copper (II) chloride, neocuproine, ammonium acetate, trichloroacetic acid, ferric chloride, potassium ferricyanide, 2-Amino-2-hydroxymethyl-propane-1, L- methionine. All chemicals were purchased from Sigma Aldrich, Merck and Fluka Chemika.

Extraction and preparation of samples

The ripe of Citrus sinensis L. fruits were obtained from the local market in Constantine (North-East Algeria) in November 2019. The fruits were fresh, of taste quality, and free from damage. Two methods of ORF sample preparation were used. The fruits were washed, peeled with a knife, then divided into small pieces, then ground before being macerated with the mixture of ethanol (80%) for 48 hours at room temperature. The solvent was removed under reduced pressure using a rotor evaporator (Buchi R-215) at 40°C. The extract obtained was stored in an airtight bottle for use in vitro experiments. A portion of crushed ORF was frozen for two days and dried using a laboratory freeze-dryer (ALPHA 1-4 LD plus, Martin Christ) then made into a fine powder and kept in an airtight flask for use in the in vivo experiment.

The determination of total phenolic and flavonoid contents and evaluation of the antioxidant activity

Determination of total phenolic content (TPC)

The determination of polyphenols in the extract using the Folin-Ciocalteu reagent (Singleton and Rossi, 1965) according to a microplate assay method described by Muller et al. (2010) with some modifications. A volume of 20 μL of ORF extract (1 mg/mL) was mixed with 100 μL of Folin-Ciocalteu reagent (2 N) and 75 μL of sodium carbonate (7% w/v) in each well of the microplate. The mixture was incubated in the dark at room temperature for 2 h. The absorbance was measured at 740 nm in the microplate reader (Perkin Elmer Enspire, Singapore). Gallic acid was used as a standard for the calibration curve and construction of a linear regression line. Results are expressed as μg of gallic acid equivalent (GAE)/mg extract.

Determination of total flavonoid content (TFC)

TFC of the extract was determined using the modified method described by (Topçu et al., 2007). Briefly, 50 μL of ORF extract (1 mg/mL) was added to 10 μL of 10% aluminum nitrate, 10 μL of 1 M potassium acetate, and 130 μL of methanol. The mixture was then allowed to stand for 40 min at room temperature. The absorbance was measured at 415 nm. TFC is expressed as quercetin equivalent (QE)/mg extract.

DPPH free radical scavenging assay

The scavenging activity of the extract against DPPH ^• radical was measured according to the modified method of Blois (1958). 160 µL of the DPPH solution (6 mg DPPH dissolved in 100 mL of methanol) was added to 40 µL of ORF extract at different concentrations in all wells. The mixture was incubated in dark for 30 min at room temperature before taking the absorbance readings at 517. DPPH solution in methanol was used as a control. α-tocopherol, BHT, and BHA are used as antioxidant standards.

The results are expressed as IC₅₀ (µg/mL). IC₅₀ value corresponding to the concentration of the extract, which inhibits 50% of the radical DPPH, and the % inhibition was calculated by applying the following formula:

Inhibition (%) = [(A Control − A Sample) ∕ A Control] × 100

A Control: absorbance of the control; A sample: absorbance of the extract or standard.

ABTS scavenging activity assay

The ABTS scavenging activity was determined according to the modified method of Re et al. (1999). In all wells, a mixture of 160 μL of diluted ABTS^•+ solution (7 mM ABTS in water and 2.45 mM potassium persulfate, stored in the dark at room temperature for 12 h) was added to 40 μL of ORF extract in ethanol at different concentrations. The absorbance was measured at 734 nm after 10 min. ABTS^•+ solution in ethanol was used as a control, BHT and BHA are used as antioxidant standards.

The results were given as IC₅₀ value (mg/mL) corresponding the concentration of 50% inhibition using the following equation:

Inhibition (%) = [(A Control − A Sample) ∕ A Control] × 100

A Control: absorbance of the control; A sample: absorbance of the extract or standard.

Cupric reducing antioxidant capacity (CUPRAC)

The cupric reducing antioxidant capacity was measured according to Apak et al. (2004). A volume of 50 μL of copper (II) chloride solution (10 mM), 50 μL neocuproine (7.5 mM), and 60 μL ammonium acetate buffer (1 M, pH 7.0) solutions were added to each well. Then, 40 μL of ORF extract at different concentrations was added to the mixture. The absorbance was measured at 450 nm after 1h. The results were calculated as A_0.5 (µg/mL) corresponding to the concentration indicating 0.50 absorbance. BHT and BHA are used as antioxidant standards.

Reducing power assay

The reducing power activity was determined according to the modified method by Oyaizu (1986). A mixture of 10 μL of ORF extract at various concentrations, 40 μL of 0.2 M phosphate buffer and 50 μL of potassium ferricyanide (1%) were added to each well of the plate and incubated for 20 min at 50°C in the water bath. After 50 μL of 10% trichloroaceticacid (TCA) and 10 μL of ferric chloride (0.1%) were added to the mixture and completed with 40 μL of distilled water. The absorbance was read at 700 nm. Ascorbic acid and α-tocopherol were used as standards. A_0.5 values were calculated from the absorbance curves.

Evaluation of anti-inflammatory effect of Citrus sinensis L. fruit in vivo

Anti-inflammatory activity was tested in vivo, evaluating the effect of
C. sinensis fruits on homocysteine levels during L-methionine-induced intestinal inflammation in 28 adult male Mus musculus weighing (20–35 g). The mice were obtained from the Faculty of Pharmacy, University Constantine, Algeria. They were maintained under standard laboratory conditions with free access to water and food. The animal study was conducted according to the procedure of the research project code number (F00920140076) obtained from our Ministry of Scientific Research, Algeria, and the ethical principles and guidelines provided by the committee for the purpose of control and supervision of the experiments on animals (CPCSEA). Each treatment dose was prepared in distilled water and administered by oral gavage for 21 consecutive days at a dose of (200 mg/kg/day). The animals were divided into four experimental groups: the control group (C) received only distilled water; the second group (M) received (200 mg/kg) of L-methionine; the third group (OM) received (200 mg/kg) of L-methionine and was treated with (200 mg/kg) of ORF powder; the last group (O) received (200 mg/kg) of ORF powder only. The blood samples were taken after 21 days of treatment, it was immediately centrifuged for 15 minutes at 3,000 /rpm. Plasma homocysteine values are measured by immunoassay using the analyzer (IMMULITE 2000 XPi) and the homocysteine values were expressed in (µmol/L).

Reduced Glutathione assay (GSH)

Preparation of the homogenate

The animals were sacrificed and 0.5 g of liver from each mouse was homogenized in a volume of 2 mL of TBS (50 mM Tris, 150 mM NaCl, pH 7.4). The obtained homogenates were centrifuged for 15 min at 4°C at 9,000 g and the supernatant was used for the measurement of glutathione reduced (GSH).

GSH measurement

Reduced glutathione (GSH) concentration was performed according to Sakhri et al. (2021) method. First 800 μL of the liver homogenate was added to 200 μL of the sulfosalicylic acid (0.25%) solution, and then the mixture was incubated in an ice bath for 15 min. Next, centrifugation at 1000 tours/min for 5 min was realized. After that, 500 μL of the supernatant and 1 ml of the buffer Tris-EDTA (pH 9.6) were added to 25 μL ml of DTNB of (0.01 M) and after 5 min the absorbance was measured at 412. The GSH concentration was expressed as nmol of GSH nmol/mg protein.

Histological examination

Intestine fragments were removed after sacrifice the mice and were fixed in 10% formalin, sections were stained with hematoxylin-eosin and examined under a digital photographic microscope (OPTECH MICROSCOPE).

Statistical analyses

All results are given as mean ± standard errors of the mean (S.E.M). The in vitro experiments were performed in triplicate. The data from in vivo study was analyzed by SPSS 20.0 statistics software with one-way ANOVA followed by Turkey’s post hoc test for multiple comparisons and P < 0.05 was considered as statistically significant.

RESULTS

The determination of total phenolic and flavonoid contents and the antioxidant activity of Citrus sinensis L. fruit extract

The contents of total phenol and flavonoid in the extract were quantified. The results showed that the ethanolic extract of ORF exhibited the highest total phenolic content (173.41 ± 3.06µg GAE/mg extract), the extract gave the value of 17.01 ± 0.96µg QE/mg for the total flavonoidcontent. The results are shown in Table 2.

Table 2. Antioxidant activity of Citrus sinensis L. fruit extract.

Note: Values are expressed as means ± SEM of three parallel measurements.

The evaluation of the antioxidant activity of the ORF extract showed the strongest antioxidant activity in the ABTS and in the reducing power assays with (IC₅₀ = 76.27 ± 0.07 µg/mL and A_0.5 = 80.16 ± 0.89 µg/mL) respectively. According to the Table 3, the extract showed good activity in DPPH (153.48 ± 0.98 µg/mL) and in the CUPRAC (161.81 ± 1.50 µg/mL).

Table 3. Total phenolic and flavonoid contents of Citrus sinensis L. fruit extract.

Note: Values were expressed as means ± (n=3).

The anti-inflammatory effect of Citrus sinensis L. fruit powder

The results of this study showed that there is a significant difference in the concentration of homocysteine between groups (C, M, OM and O) at P = 0.01. In addition, we have detected that the level of homocysteine has decreased significantly in goup (O) when it is compared with group (M) administered by L- methionine, and the concentration of the homocysteine in the group (M) is increased significantly when it is compared to the control group (P <0.05). On the other hand, we have obtained that the level of homocysteine is decreased in group (OM) but not significantly (Figure 1).

Figure 1. Effect of Citrus sinensis L. fruit powder on homocysteine values

Results are shown as mean ± S.E.M (n = 4) and significant between groups is shown as *P = 0.01. The letters a and b indicate a significant difference between (C, M) and (M, O) respectively.

(C): control group; (M): group administered with L-methionine (200 mg/kg); (OM): group administered with L- methionine (200 mg/kg) and treated with ORF (200 mg/kg); (O): group treated with ORF (200 mg/kg).

Effect of Citrus sinensis L. fruit powder on glutathione reducing values

Our results showed that there are very highly significantly in the GSH values between groups at P =0.001 (Figure 2). The Tukey test showed that the GSH values was decreased highly and significantly in group (M) when it is compared to the groups (C and OM) (P < 0.01) and significantly when it is compared to the group (O) (P < 0.05).

Figure 2. Effect of Citrus sinensis L. fruit powder on glutathione reducing values.

Results are shown as mean ± S.E.M (n = 4) and significant between groups is shown as *P = 0.001. The letters a and b indicate a significant difference between the groups.

(C): control group; (M): group administered with L-methionine (200 mg/kg); (OM): group administered with L- methionine (200 mg/kg) and treated with RCF (200 mg/kg); (O): group treated with RCF (200 mg/kg).

Histological estimation

The results obtained from the histological study of the intestines of the experimental groups are represented in figure 3. The groups (C) and (O) showed normal architecture of the intestinal membrane (Figures 3 A and D). Whereas in the group (M) which have administered with L-methionine (200 mg/kg), showed a granuloma with significant lymphocytic infiltration and degeneration in the enterocytes membrane cells (Figures 3 B). However, the group animal (OM) showed a normal structure with distinct villi, markedly reduced lymphocyte infiltration, and restoration of enterocyte membrane cell integrity (Figure 3 C).

Figure 3. Histological sections of intestine of (A) received distilled water, (B) administered with L-methionine (200 mg/kg), (C) administered with L- methionine (200 mg/kg) and treated with ORF powder (200 mg/kg), (D) received ORF powder (200 mg/kg). Hematoxylin - eosin staining (A, B, C and D × 100). Mucosa (M), Muscularis Mucosa (MM), Submucosa (SM), Muscularis (ML), Granuloma (G), Degeneration (D), Lymphocytic Infiltration (LI), Villi (V).

DISCUSSION

The ORF extract revealed a high content of total phenolic with a moderate quantity of total flavonoids. Our results are in agreement with Campone et al. (2020) and AbdGhafar et al. (2021).

Antioxidant capacity tests can be broadly classified as tests based on electron transfer (ET) and hydrogen atom transfer (HAT). ET based tests measure an antioxidant's ability to reduce an oxidant, which changes color when reduced (Apak et al., 2007). In vitro antioxidant activities were measured in ethanol extract obtained using ABTS, DPPH, CUPRAC, and reducing power assays.

DPPH^• is a free radical that accepts electrons or hydrogen radicals from donor compounds. On the other hand, ABTS^• assay based on the inhibition of the formation of ABTS^• by one-electron oxidants (Sridhar and Charles, 2018). The results showed that the ethanolic extract of C. sinensis fruits exhibited the highest activity in the ABTS assay and good activity against the DPPH assay. Our results are in agreement with the study of (Campone et al., 2020).

The reducing power is based on the capacity of substances, which have reduction potential, react with potassium ferricyanide to form potassium ferrocyanide, which then reacts with ferric chloride to form a ferric complex (Singhal et al., 2014). The extract showed the strongest reducing power, this agrees with the study of Bentahar et al. (2020) who showed that the ethanolic extract of C. sinensis fruits exhibited a strong reducing power. CUPRAC method is one of the most widely used antioxidant methods. It is based on the reduction of the copper (II) -neocuproine to copper (I) -neocuproine chelate complex (Akar and Burnaz, 2019). Our data showed that the extract exhibited a good copper reducing antioxidant power.

The above results may be due to the richness of ORF extract in total phenolic and the presence of flavonoids which have the principal contribution to the antioxidant capacity of extract. Previous studies showed that a good correlation significant was found between antioxidant activity and polyphenols and flavonoids contents (Canan et al., 2016; Bentahar et al., 2020).

Glutathione reductase (GR) is an important antioxidant enzyme essential for maintaining the GSH / GSSG ratio by catalyzing the recovery of reduced glutathione (GSH) from oxidized glutathione (GSSG) (Güller et al., 2021; Robbins et al., 2021).

In the present study, treatment with C. sinensis fruit and L-methionine showed a significant increase in the level of hepatic GSH, however, it is suggested that C. sinensis fruits increased glutathione reductase enzymatic activity and GSH levels and enhanced ROS scavenging capacity by inhibiting ROS over expression and oxidative damage.

Abdelghffar et al. (2021) worked on the extract of orange fruit peel (Citrus sinensis) and reported that the extract revealed increased GSH levels in tissue homogenates and confirmed its protective efficacy against chemotherapy-induced toxicity in male rats.

Our research confirmed that the treatment with ORF powder significantly reduced plasma homocysteine levels in animals administered with high dose of L-methionine. Numerous studies have confirmed that Citrus fruits have an anti-inflammatory potential (Leguizamón et al., 2019; Denaro et al., 2021) and inhibited NF-κB (Impellizzeri et al., 2015).

Citrus fruits are a good source of vitamin C, folate, vitamins B6 and, flavonoids (Rauf et al., 2014; Rao et al., 2021). Through this, their consumption has beneficial effects on human health due to the antioxidant and anti-inflammatory properties (Ma et al., 2020; Rao et al., 2021) and their gross use reduces the risk of gastric and colorectal diseases (Roussos et al., 2011; Rauf et al., 2014).

Previous work links increased levels of homocysteine to IBD, indicating a major role for vitamin B deficiency in intestinal damage and inflammation (Lurz et al., 2020). We suggest that ascorbic acid, flanovoids and phenolic contents prevent the oxidation process thereby protecting the intestine from damage caused by ROS and vitamin B supplementation provided by C sinensis corrected the deficiency of B vitamins resulting in lower levels of homocysteine.

Activation of the intestinal immune system and recruitment of inflammatory cells in the intestine help maintain inflammation and damage in the intestines (Wera et al., 2016). Histological examination revealed that the treatment with ORF powder restored the integrity of the intestine and the histopathological changes caused by the elevated homocysteine levels. Khan et al. (2016) reported that there is a marked reduction in histopathological damage and a protective role against inflammation in intestinal tissue in rat colitis treated with Citrus sinensis L.

The study of Gholap et al. (2012) also reported that C. sinensis fruit peel extract is effective in the treatment of UC in mice who showed less ulceration in histopathological observation. On the basis of these results, we can confirm that C. sinensis fruits exhibit protective role against intestinal inflammation.

A study carried out by Fusco et al. demonstrated that Orange juice decreased oxidative stress and inflammatory response in a murine model of IBD (Fusco et al., 2017).

Gholap et al. (2012) reported that the combination of Moringa oleifera root and a peel extract of Citrus sinensis fruit is effective in mice with acetic acid (AA)-induced UC, by decreasing malondialdehyde (MDA) content and myeloperoxidase (MPO) activity.

Khan and collaborators tested the effect of Citrus sinensis L., Citrus paradisi L. and their combinations in rats subjected to experimental trinitrobenzene sulfonic acid (TNBS)-induced colitis. By histological and biochemical analyses, they have found that these fruit juices exerted antioxidant and anti-inflammatory activities (Khan et al., 2016).

On the other hand, it was reported that the use of Citrus aurantium L. and its flavonoids have proved to exhibit anti-inflammatory activity, reduce weight loss and diarrhea as well as suppress isolated jejunal contraction in TNBS induced IBD rats (He et al., 2018).

Previous studies have confirmed that HHcy is a risk factor for several pathological disorders (Cordaro et al., 2021; Ji et al., 2022). On the grounds of this fact, we have planned to evaluate the impact of hyperhomocysteinemia induced by L- methionine to provoke intestinal inflammation and at the same time to estimate the protective ability of ORF, this work is an original never done before. Based on results, induced hyperhomocysteinemiahas showed significant intestinal alterations where the inflammatory process was well manifested. Besides, ORF has exhibited an important anti-hyperhomocysteinemic and anti-inflammatory effect.

CONCLUSION

The results of this study confirmed that hyperhomocysteinemia induced intestinal inflammation and the ethanolic extract of ORF had a high content of phenols, contained flavonoids, and exhibited good antioxidant activity. The use of C. sinensis fruit powder in the in vivo study showed increased GSH levels, decreased plasma homocysteine levels, and restored the integrity of the intestinal epithelium. The C. sinensis fruit is therefore recommended for the prevention of inflammatory bowel diseases due to its antioxidant and anti-inflammatory power.

ACKNOWLEDGEMENTS

The authors are grateful to the MESRS (Ministry of Scientific Research, Algeria).

AUTHOR CONTRIBUTIONS

Sara Khelfi performed the experiments, the statistical analysis and data visualization and wrote the manuscript. Sakina Zerizer was the supervisor of this work and contributed to its technical and academic realization and followed the revision of the manuscript. Soraya TEBIBEL was the second supervisor, followed the revision of the manuscript. Amina FOUGHALIA help ın pratıcal work. Chawki BENCOUICI and Zahia KABOUCHE provıded materials of the part of extraction and in vitro antioxidant activities for the experimental study.

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