Introduction

Cancer is the leading cause of death in the world. Empirical evidence compiled by the National Center for Health Statistics reveals that the trend of the incidence and mortality rates is cancer. In the United States, an estimated 1,958,310 new cancer cases and 609,820 cancer-related fatalities are expected in 2023 [1].  In Thailand, the annual number of cancer related deaths is increasing every year. Among the various types of cancer prevalent in Thailand, liver and bile duct cancer, lung cancer, and colon and rectal cancer emergence are the most commonly diagnosed forms [2]. Various cancer treatments are available, including alternative medicine used in conjunction with modern medicine to boost patient immunity. Diet plays a crucial role in cancer prevention, with foods rich in antioxidants. Thai medicinal plants are a major source of those compounds such as phenolic, flavonoid, vitamin, and catechin which involve in preventing and reducing the risk of cancer [3].

In the Thai traditional medicine framework, cancer is the results of human body deterioration and an imbalance in the tri-elemental system consisting of Pitta, Wata, and Semha. This imbalance disrupts the body's equilibrium. Thai folk healers, with extensive cancer treatment knowledge, often rely on local herbal medicines. Among these remedies, one commonly used herb is from the Euphorbiaceae family, known for its laxative property and its ability to facilitate detoxification of the body. In the northern region of Thailand, folk healers in Umphang District, Tak Province often use Euphorbia heterophylla L. as a laxative, which can be beneficial for cancer patients who have constipation symptom in this regard. The plant has been investigated for its cytotoxicity against A549 (human lung cancer cells) and HepG2 (human liver cancer cells) [4]. Its extract demonstrated an impact on intestinal motility in mice similar to acetylcholine, histamine, and potassium chloride [5]. Euphorbia heterophylla L., a member of the Euphorbiaceae family, shows promise as a potential laxative and exhibits potential anti-cancer properties, particularly within the gastrointestinal system. However, there is no report on the antioxidant and cytotoxic activities against human colon adenocarcinoma cell lines (SW480 ATCC CCL-228) and human gastric carcinoma cell lines (Kato III) of this plant. Therefore, the objective of this study was to investigate the antioxidant activity and cytotoxic effects of Euphorbia heterophylla L. on these cancer cell lines.

Materials and Methods

Plant materials

Plant parts were collected in The Umphang District, Tak Province. The collected plant materials were identified by authentication procedures, ensuring its accuracy and reliability. A voucher specimen were keep in Department of Thai Traditional and Alternative Medicine in Thailand annotated as TTM-C No.1000701-4.

Preparation of crude extracts

Maceration Mehod. The dried plant materials (300 g) were macerated with 95% ethanol or hexane for 3 days. It was then filtered and dried using an evaporator. Subsequently, the hexane residue, was extracted with 95% ethanol using the same procedure.

Decoction method. The dried plant materials (100 g) were boiled with water (decoction) for 15 minutes, then filtered   and evaporated down to one-third of the volume, then freeze dried using a freeze dryer (Lyophilizer). All extracts of each plant part were calculated percentage of yield and kept in freezer (-20^oC) until used.

DPPH Radical Scavenging Assay

The antioxidant activity was determined using DPPH assay, according to the modified method [6]. Briefly, testing procedure began with preparation of a stock solution of extracts and standard substances to a concentration equal to 1 mg/mL, then adjust the concentration to different concentrations. The final concentrations were as follow: 1, 10, 50, 100 mg/ mL which were tested for antioxidant activity in 96 well plates. A 100 ml of DPPH was added to 100 ml of test substance, held for 30 minutes, then absorbance was measured at 520 nm using a spectrophotometer. The scavenging activity of the samples were calculated as a percent inhibition in the formula below:

Inhibition (%) = [(ODcontrol – ODsample)/ODcontrol] x 100

BHT (Butylated hydroxytoluene) was used as standard control. The obtained values were calculated for radical scavenging activity (EC₅₀). The EC₅₀ value below 20 mg/mL indicates as good antioxidant activity.

Cell culture

Cancer cell lines used in this study were the human colon adenocarcinoma cell lines (SW480 ATCC CCL-228) and Human gastric carcinoma cell lines (Kato III) were purchased from RIKEN BRC, Japan. They were cultured in RPMI 1640 medium (BIOCHROMAG) supplemented with 10% heated fetal bovine serum, 1% of 2mM 1-glutamine, 50 IU/mL penicillin and 50 mg/mL streptomycin.

Sulforhodamine B (SRB) Assay

The cytotoxicity activity assay, SRB assay, was performed essentially according to the previous method [7]. This assay is used for cell density determination, which is performed to assess growth inhibition by a colorimetric assay by staining total cellular protein with the dye SRB. Cell cultures were fixed by 100 ml of ice-cold 40% Trichloroacetic acid (TCA) per well, incubated at 4 °C for 1 hour in the refrigerator and washed off non-viable cells with distilled water, add 50 ml of SRB solution (0.4% w/v in 1% acetic acid, Sigma) per well, held for 30 minutes and wash with 1% acetic acid. The plate was dried then 100 ml of 10 mM Tris, pH 10.5 was added. The absorbance (OD) of each well was measured at 492 nm. The IC50 values were calculated from the Prism program obtained by plotting the percentage of survival versus the concentrations, interpolated by cubic spine. According to National Cancer Institute guidelines extracts with IC50 values < 20 mg/mL were considered active. The IC50 values were calculated from the Prism program.

Inhibition (%) = [(ODcontrol – ODsample)/ODcontrol] x 100

Statistical analysis

All experiments were carried out in triplicate and presented as mean ± SEM (standard error of the mean).

Results & Discussion

Plant materials

The percentage yield was calculated after the extraction process. The 95% ethanol extract of the hexane residue (AHE) of aerial part exhibited the highest yield of 11.43%. Similarly, the underground part was extracted using n-hexane (RH) and hexanes residue with 95% ethanol (RHE), yielded 13.43% and 13.32%, respectively (Table 1).

Table 1. Percentage yield of all Euphorbia heterophylla L. extracts.

DPPH Radical Scavenging Assay

The results revealed that Euphorbia heterophylla L. exhibited little antioxidant activity, as assessed through the DPPH assay. The EC₅₀ values for both aerial and underground parts in all extracts were found to more than 100 µg/mL. In comparison, the standard substance BHT demonstrated superior antioxidant activity, with an EC₅₀ value of 15.49 ± 1.27 µg/mL. However, the underground part of the water extract (RW) demonstrated the highest percentage inhibition at a concentration of 100 µg/mL, with a value of 49.11 ± 0.89% (Table 2). These results suggested that this plant showed little antioxidant effect. This study agreed with previous research, which reported the maximum concentration of 500 µg/mL for the aqueous extract of Euphorbia heterophylla L. leaves which showed EC₅₀ value as 141.11 ± 4.23 µg/mL [8].

Sulforhodamine B (SRB) Assay

According to the National Cancer Institute (NCI) of the US, plant extracts are classified as high cytotoxic activity if their IC₅₀ is less than 20 mg/mL, moderate cytotoxic activity if the IC₅₀ ranges between 21-200 mg/mL, weak cytotoxic activity if the IC₅₀ ranges between 201-500 mg/mL, and no cytotoxic activity if the IC₅₀ exceeds 500 μg/mL [9]. This study revealed the remarkable cytotoxic potential of Euphorbia heterophylla L. against gastric cancer and adenocarcinoma cells using diverse extraction methods. The aerial parts, including AE and AH, exhibited highly potent cytotoxic activity against gastric cancer cell lines (Kato III), with IC₅₀ values of 5.67 ± 0.62 μg/mL and 6.64 ± 0.17 μg/mL, respectively. Conversely, the underground parts, RE and RHE, demonstrated moderate cytotoxic activity, with IC₅₀ values of 38.52 ± 0.79 μg/mL and 37.84 ± 0.75 μg/mL, respectively.

Furthermore, the aerial parts in the AE, AHE, and AH extracts exhibited highly potent cytotoxic activity against human colon adenocarcinoma cell lines (SW480), with IC₅₀ values of 5.01 ± 0.31 μg/mL, 6.17 ± 0.33 μg/mL, and 17.87 ± 1.28 μg/mL, respectively. Similarly, the underground parts, including RHE, RH, and RE, also displayed highly potent cytotoxic activity against these cell lines, with IC₅₀ values of 7.76 ± 0.34 μg/mL, 7.84 ± 0.23 μg/mL, and 12.56 ± 1.78 μg/mL, respectively. Notably, these extracts exhibited high selectivity in their cytotoxic effects against all types of cancerous cell lines. However, all water extracts showed no effect on any of the cancer cell lines tested (IC₅₀ > 100 μg/mL).

Figure 1 Cytotoxicity of the Euphorbia heterophylla L. extracts against Gastric cancer cell-lines (Kato III), human colon adenocarcinoma cell lines (SW 480) using SRB assay (n=3).

Conclusion

Euphorbia heterophylla L. has been traditionally used by folk healers as a laxative for cancer patients. The present study provides substantial support for the potential utilization of Euphorbia heterophylla L. in the treatment of cancer, especially gastric cancer and adenocarcinoma cells. Despite the fact that all its extracts showed little antioxidant activity, but they showed high cytotoxic effects. This current study also showed that the extraction method by 95% ethanol demonstrated cytotoxic activity, suggesting its potential as a selective approach for extracting the active components. Nevertheless, the cytotoxic compound should be investigated for the development of its product for gastrointestinal cancer treatment.

Acknowledgement

This research was financially supported by the Thailand Science Research and Innovation Fundamental Fund through Grant No. TUFF 28/2565. The authors are grateful to the Center of Excellence in Applied Thai Traditional Medicine Research and the Faculty of Medicine, Thammasat University, for providing laboratory facilities.

References

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2. National Cancer Institute: Additional Links and Resources United States of Thailand 2022 [cited 2022 May 12].

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4. Lingaraju, K.; Naika, H. R.; Nagaraju, G.; Nagabhushana, H. Biocompatible synthesis of reduced graphene oxide from Euphorbia heterophylla L. and their in-vitro cytotoxicity against human cancer cell lines. Biotechnology Reports, 2019, 24, e00376.

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6. Yamazaki, K.; Nakajima, T.; Gemmell, E.; Polak, B.; Seymour, G. J.; Hara, K. IL‐4‐and IL‐6‐producing cells in human periodontal disease tissue. Journal of oral pathology & medicine, 1994, 23(8), 347-353.

7. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Boyd, M. R. New colorimetric cytotoxicity assay for anticancer-drug screening. JNCI: Journal of the National Cancer Institute, 1990, 82(13), 1107-1112.

8. Keerthana, K.; Deepa, A.; Shobana, G.; Jothi, G.; Sridharan, G. Preliminary phytochemical screening and in vitro antioxidant potential of Euphorbia heterophylla L. International Journal of Pharmacy and Pharmaceutical Sciences, 2014, 6(8), 550-553.

9. Boik, J. Natural compounds in cancer therapy. Oregon Medical Press, 2001.

 

The above documents were reviewed and edited by editorial board of NATPRO8 Conference.

 

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