Antioxidant and Antitumor Activity Against Colorectal Cancer Cells of Lycium chinense Mill. Cultivated in Ukraine ^†

Abstract

The polyphenolic extract from Lycium chinense fruits cultivated in Ukraine was surveyed for antioxidant and anticancer activity against HT-29 colorectal cancer cells. The total phenolic compounds and total flavonoid content were analyzed through the Folin–Ciocalteu and aluminum chloride procedure, respectively, while the antioxidant activity in vitro was assessed using DPPH radical scavenging and β-carotene bleaching assays. Anticancer effects were established using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The TPC was 11.3 mg GAE/g dry weight, while the TFC was 7.3 mg rutin/g dry weight. The extract demonstrated an antioxidant capacity (DPPH IC₅₀ = 125.9 ± 12.6 µg/mL), lipid peroxidation inhibition (23.5 ± 2.1% in the β-carotene assay) and the inhibition of HT-29 colorectal cancer cells (GI₅₀ at 72 h = 250 µg/mL). These findings provide the first evidence of the anticancer potential of Lycium chinense against human colorectal cancer cells, supporting its application as a source of bioactive compounds for the development of functional foods with antitumor properties.

1. Introduction

Colon cancer, one of the most prevalent human malignancies, represents a major public health concern in developed countries and ranks as the third leading cause of cancer-related mortality worldwide. A significant limitation of current chemotherapeutic regimens is their high cytotoxicity to normal cells. Consequently, the discovery of novel anticancer agents with selective activity and minimal side effects remains a critical priority [1]. Moreover, up to 90% of colorectal cancer cases could be prevented through dietary modifications. In this context, edible berries have garnered increasing attention as potential sources of bioactive compounds with chemopreventive properties [2].

The Lycium genus, which comprises 97 recognized species, is well known for its dual use in food and traditional medicine; of these, 31 species have documented pharmacological applications [3]. Despite the broad interest in their bioactive properties, only two species—Lycium barbarum and Lycium ruthenicum—have been studied for their anticancer effects against human colon cancer. Carotenoid extracts and nanoemulsions from Lycium barbarum were effective at inhibiting the growth of HT-29 colon cancer cells, with an IC₅₀ of 4.5 and 4.9 μg/mL, respectively [4]; the LoVo cells, a typical human colorectal carcinoma cell line, were significantly inhibited by the mixture of the polysaccharides and anthocyanins from Lycium ruthenicum, without imposing any cytotoxicity on normal immune cells [5].

In Ukraine, Lycium chinense Mill. (L. chinense) (пoвiй китaйcький in Ukrainian) was introduced in 1998 into the dendrological park “Olexandria,” located in the northeastern sector of the Right-Bank properties region. The park is situated at an elevation of 80–106 m and experiences a temperate continental (Dfb) climate, with a mean annual temperature of 6.93 °C, ranging between 5.8 °C and 8.5 °C across different years. Data concerning the biological activity of Lycium fruits cultivated in temperate continental climates—particularly in Eastern Europe—remain exceedingly scarce.

The present study was therefore designed to obtain a polyphenolic extract from L. chinense, to evaluate its cytotoxicity against human colon cancer HT-29 cells and to characterize its antioxidant capacity.

2. Materials and Methods

2.1. Reagents and Chemicals

Unless otherwise indicated, all chemicals and solvents were purchased from Merck (Madrid, Spain). Aluminum chloride and sodium carbonate were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). Water was purified using a Milli-Q system (Millipore, Burlington, MA, USA). All the chemicals used, including the solvents, were of analytical grade.

2.2. Plant Material

Fruits were manually harvested at maturity from three plants in the dendrological park “Olexandria”. The voucher number of the collected species is 09113/2024. Washed fruits were dried at 40 °C for 24 h and stored at 4 °C for further use.

2.3. Extraction and Determination of Total Phenolic and Flavonoid Contents

The berries were defatted with hexane, and then we extracted methanol/water (60:40, v/v) using a Soxhlet apparatus. After experimenting with different solvent ratios, the ratio between both solvents was selected because it yields the highest amount of phenolics. After centrifuging at 3000× g for 15 min [6], the supernatants were collected and combined, and the solvent was evaporated under vacuum at 60 °C to dryness. The residue was used for the determination of total phenolic content (TPC) and total flavonoid content. The determination of the TPC was conducted following the Folin–Ciocalteu procedure [7]. Absorbance values at 725 nm were then converted to Gallic Acid Equivalents (GAEs) using gallic acid as the reference compound. The total flavonoid content was determined using the aluminum chloride method, with the absorbance of the samples being measured at 420 nm. The results were expressed as rutin equivalents in milligrams per gram dry weight of plant material [8].

2.4. Antioxidant Activity

2.4.1. DPPH Method

The DPPH assay was performed according to the method described [9]. Ascorbic acid was used as positive antioxidant control.

2.4.2. β-Carotene Bleaching Method

Antioxidant activity was determined by measuring inhibition of β-carotene bleaching as described [10] with modifications. The reagent consisted of 1 mL β-carotene (2 mg/mL in chloroform), 20 mg of linoleic acid and 100 mg of Tween 80, which were vigorously mixed by vortexing and flushed with nitrogen to remove chloroform before addition of oxygen-sparged distilled water (100 mL). Samples (100 µg/mL; 200 µL) were added to 800 µL of the β-carotene reagent and incubated in the dark at 54 °C for 60 min before determination of absorbance at 450 nm. Butylated hydroxytoluene (BHT) was used as reference antioxidant.

2.5. Cell Assays on Cancer and Normal Cell Lines

The HT-29: Human Colorectal Adenocarcinoma Cell Line (ATCC HTB-38American Type Culture Collection) and the CCD-18Co colonic human myofibroblasts cells line (CRL-1459 ™) were used to check antiproliferative activities. HT-29 cell line is a well-established model used to study the biology of human colon cancers but also in studies focused on food bioavailability and digestion due to epithelial morphology [2]. Cultures were supplied by the Technical Instrumentation Service of University of Granada (Granada, Spain). Cells were grown at 37 °C and 5% CO₂ humidified atmosphere in medium RPMI-1640 supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.125 mg/mL amphotericin and 100 mg/mL penicillin–streptomycin.

All cultures were plated in 25 cm² plastic tissue culture flasks (Sarstedt, Newton, NC, USA). All culture media and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture and cell assays, that is, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, were accomplished as previously described [11]. Values presented are mean ± standard error of the mean. The selectivity index (SI) was calculated as GI₅₀ of the extract against the CCD-18 normal cell line/GI₅₀ of the same extract against the HT-29 cancer cell line [12].

2.6. Statistical Analysis

All analyses were performed in triplicate. All data were expressed as the average ± SD. The statistical significance was calculated using Student’s t-tests and a one-way analysis of variance (ANOVA) and expressed as the average SD. Differences among mean values were tested by Duncan’s test at p < 0.05 and for antiproliferative activity at p < 0.05 and p < 0.01. Statistical analyses were performed using Statgraphics Centurion XVIII version. 18.1.12 (Warrenton, VA, USA).

3. Results and Discussion

3.1. Total Phenolics and Flavonoids Content

The hydroalcoholic extraction of goji berries, carried out under the previously described conditions, resulted in a yield of 16.9% dry weight. The TPC was 11.3 mg GAE/g dry weight, while the total flavonoid content was 7.3 mg rutin/g dry weight. These values are comparable to those reported for commercial samples obtained from the Brazil Municipal Market [13]. The TPC was higher than the TPC in the small cranberry Vaccinium oxycoccos (3.72 GAE/g DW) and was comparable with the TPC in other well-known edible berries, such as Vaccinium myrtillus (2.20–37.15 GAE/g DW) and Hippophae rhamnoides (9.3 GAE/g DW) [14]. The extract exhibited an antioxidant activity, with a DPPH radical scavenging IC₅₀ value of 125.9 ± 12.6 µg/mL and a lipid peroxidation inhibition of 23.5 ± 2.1% in the β-carotene bleaching assay. However, its antioxidant capacity was considerably lower when compared to the positive controls: the ascorbic acid (IC₅₀ = 4.2 ± 1.0 µg/mL) in the DPPH assay and BHT (78.6 ± 0.8% inhibition) in the β-carotene assay.

3.2. Anticancer Activity Against HT-29 Colon Cancer Cells

The L. chinense extract did not inhibit the growth of HT-29 after the 48 h incubation. After 72 h of treatment, the MTT assay demonstrated a concentration- and time-dependent inhibitory effect on HT-29 cells (Figure 1).

The GI₅₀ values of the extract, doxorubicin (control) and selected phenolic compounds are shown in Figure 2.

It is important to consider that polysaccharide-rich fractions from Lycium species have been previously reported as highly bioactive [15]. Given that the water/methanol extracts used in this study may contain certain amounts of polysaccharides, a synergistic interaction between phenolic compounds and seed-derived polysaccharides may contribute to the observed anticancer effects—particularly considering that HT-29 cells are known to exhibit a limited sensitivity to phenolic compounds alone [10]. A similar synergistic effect has previously been reported in L. ruthenicum, where a combination of polysaccharides and anthocyanins demonstrated an inhibitory activity against human colorectal carcinoma LoVo cells [5]. Additionally, the SI was calculated to compare the extract’s cytotoxicity toward HT-29 cancer cells and normal cells CCD-18. An SI value greater than two indicates a high selectivity toward cancer cells, whereas values below two suggest a general cytotoxicity to both normal and cancer cells. Such a value for any research on herbal drugs and/or isolated compounds is critical for determining whether further research can be continued [16]. The SI at 72 h was 10, from which it can be concluded that the Lycium extract has a high selectivity against HT-29 human colorectal cancer cells. This means that the extract evaluated here constitutes a promising candidate for the possible obtainment of active compounds against colorectal cancer.

4. Conclusions

This study provides the first evidence of the anticancer potential of L. chinense fruits cultivated in Ukraine. The phenolic extract exhibited a selective cytotoxicity against HT-29 colorectal cancer cells, highlighting its promise as a source of bioactive compounds for nutraceutical or pharmaceutical applications. Future research should prioritize elucidating the molecular mechanisms underlying its antiproliferative effects, conducting a detailed characterization of phenolic constituents, developing regionally adapted cultivars with optimized bioactivity and promoting the sustainable valorization of processing by-products. Given the influence of environmental factors on chemical compositions, long-term studies are essential to ensure consistency and efficacy in the context of human health.