A novel anticancer effect of Licuala longecalyculata Furtado extracts on lung cancer cell line

Purpose: This study was designed to investigate anticancer effect of ethanol and aqueous extracts of Lygodium microphyllum (Cav.) R.Br., Lygodium salicifolium Presl, and Licuala longecalyculata Furtado on human lung adenocarcinoma A549 cell line, and to determine total phenolic and flavonoid contents of those medicinal plants. Methods: The three medicinal plants were extracted with ethanol and water using a rotary evaporator and freeze dryer, respectively. Cytotoxicity of the crude extracts at concentrations ranging from 3.125 to 200 µg/ml on A549 cells was determined using MTT assay at various times. Aluminium chloride colorimetric assay and the Folin-Ciocalteu colorimetric method were used to analyze total flavonoid and phenolic content, respectively. Results: L. longecalyculata Furtado extracted with ethanol strongly inhibited A549 cell proliferation in a dose- and time-dependent manner with an IC 50 value of 12.00±0.41 µg/ml, whereas L. microphyllum (Cav.) R.Br. and L. salicifolium Presl did not. In addition to the morphological changes, an anticancer effect was observed in L. longecalyculata Furtado-treated A549 cells. L. longecalyculata Furtado showed the presence of phenolic and flavonoid compounds. Conclusion: These results indicate that in accordance with local wisdom, L. longecalyculata Furtado crude extract can empirically inhibit lung cancer cell line, but L. microphyllum (Cav.) R.Br. and L. salicifolium Presl cannot. However, further study of L. longecalyculata Furtado is needed to identify phytochemical compounds and their mechanism of anticancer action, in order to develop them as a new therapeutic agent for lung cancer management.


INTRODUCTION
Cancer currently ranks as a "leading cause of premature death in 134 of 183 countries in the world", including Thailand [1]. According to GLOBOCAN 2020 estimates, Asia accounts for 49.3% of global cancer incidence and 58.3% of cancer mortality. The predicted global burden of cancer in 2040 is 28.4 million cases, a 47% increase from 2020. Worldwide, lung cancer is a leading cause of cancer mortality-ranking highest for both incidence and mortality in men, but third and second, respectively, in women [2]. Meanwhile, lung cancer ranks second in terms of both cancer incidence and mortality in Thailand [3]. Also, bronchial and lung cancer were reported as one of ten ranking causes of death in Narathiwat province [4].
In our increasingly globalized world, natural products are popular and widely used as complementary and alternative medicine (CAM). Reasons given for use of CAM include a belief in its effectiveness, dissatisfaction with conventional medicine, and perceived safety [5]. In the United States, the National Center for Complementary and Integrative Health (NCCIH) has classified herbal products as the most popular biologically-based therapies [6,7]. As with previous reports, natural-derived compounds, including phytochemicals, from medicinal plants play a significant role in cancer prevention and treatment. Several are undergoing clinical trials and clinical use, including curcumin (Zingiberaceae), camptothecin (Nyssaceae), vincristine sulfate (Apocynaceae), and paclitaxel (Taxaceae) [8][9][10]. Narathiwat is located in southern Thailand and has a unique natural resource in Sirindhorn peat swamp forest. It is Thailand's largest peat swamp forest and has high biodiversity. According to local wisdom in the Sirindhorn peat swamp forest area, Lygodium microphyllum (Cav.) R.Br., Lygodium salicifolium Presl, and Licuala longecalyculata Furtado are used as medicinal plants for cancer treatment [11]. In a traditional medicine practice [11], stems of L. microphyllum (Cav.) R.Br. or L. salicifolium Presl were boiled and drunk thrice a day after meals by early-stage cancer patients. Young shoots of L. longecalyculata Furtado, raw or cooked, were eaten at least once a day with meals to treat cancer patients. However, anticancer activities and medical properties of the three medicinal plants have never been scientifically reported. L. microphyllum (Cav.) R.Br. has only been reported to affect the hepatoprotective mechanism, mitigating carbon tetrachloridemediated oxidative stress by maintaining antioxidant enzyme activity [1 2] . In the same family as L. longecalyculata Furtado, Licuala spinosa has been reported to be active against Mycobacterium tuberculosis H37Rv [13], Candida albicans, and malaria parasite Plasmodium falciparum [14]. L. microphyllum (Cav.) R.Br. and L. salicifolium Presl are in family Lygodiaceae, which are native to Asia, Africa, Australia, and United States [15,16]. L. longecalyculata Furtado, in family Arecaceae, is distributed throughout the peat swamp forest of Narathiwat, and its shoots are eaten both raw and cooked as part of the local diet [17].
The objectives of this study were to investigate anticancer effect of ethanol and aqueous extracts of L. microphyllum (Cav.) R.Br., L. salicifolium Presl, and L. longecalyculata Furtado on lung cancer cell line, and to determinate total phenolic and flavonoid contents of those medicinal plants. These exploratory findings may lead to the development of a new therapeutic drug for lung cancer management.

EXPERIMENTAL Preparation and extraction of medicinal plants
The stems and leaves of L. microphyllum (Cav.) R.Br. and L. salicifolium Presl, as well as the young shoots of L. longecalyculata Furtado were obtained from Sirindhorn Peat Swamp Forest Nature Research and Study Centre, Narathiwat, Thailand. These were identified by Mr. Jarearnsak Sae Wai and the specimens were deposited at Prince of Songkla University (PSU) Herbarium.
The aforementioned parts of those medicinal plants were cleaned and cut into small pieces and then dried in a hot air oven (Memmert) at 45 ºC for 3 days. Maceration ratio of each dried plant to absolute ethanol (Merck KGaA; Darmstadt, Germany) was 1:4 (g: ml) for 72 h at room temperature while decoction ratio of each dried plant to distilled water was 1:20 (g: ml) for 15 minutes [18]. After maceration and decoction, the supernatants were filtered using Whatman filter paper (Grade 1). Subsequently, ethanol crude extract was concentrated in a vacuum below 45 ºC using a rotary evaporator (Heidolph/Hei-VAP Precision) and aqueous crude extract was lyophilized using a freeze dryer (Labconco/ FreeZone Plus-84C). The crude extracts obtained were kept in humidity controllers (Weifo/DRY-70) at 40% RH and 25 ºC until further analysis. Percentage yield of extraction was determined as follow: Percentage yield =

Cytotoxic assay
Two cell lines were used in this assay, namely A549 cells (human lung carcinoma) with L929 cells (Mouse fibroblast) as non-cancerous control cells. A549 cells were maintained in RP10 (RPMI-1640 medium containing L-glutamine [Gibco; New York, USA] supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate). L929 cells were maintained in DM10 (DMEM [Gibco; New York, USA] with the same supplements as RP10).
Cytotoxicity of ethanol and aqueous crude extracts in A549 and L929 cells was assessed by MTT assay (Invitrogen; Oregon, USA). This assay measures metabolic activity as a proxy for cell viability. Briefly, the cells were seeded at a density of 1x10 4 cells/well into 96-well flatbottomed plates in the absence or presence of various concentrations of each crude extract, in triplicate, and incubated in a humidified incubator with 5% CO2 and 37 ºC for 24, 48 and 72 h. Each concentration of crude extract was used in two-fold serial dilutions from 3.125 to 200 µg/ml. The cell control wells without crude extract were used as control cells. In conditions without cells, a control blank was set without each crude extract, and the treated blank was set with each concentration of crude extracts. Both conditions were used as negative controls to determine background absorbance. Doxorubicin and curcumin served as positive controls. Subsequently, the supernatant in each well was gently discarded. Next, 100 µl of MTT reagent (5 mg/ml) was added to each well, and the plates were incubated at 37 ºC for 2 h. After incubation, the supernatant was gently discarded, and then insoluble purple formazan sediment was dissolved completely with 100 µl DMSO (Sigma-Aldrich; Missouri, USA) for 30 minutes. Finally, OD of absorbance was measured at 570 nm for test wavelength and 630 nm for reference wavelength. Results were recorded as mean ± SD of triplicate cultures of three separate experiments. Percentage of cell viability determined as follow: Cell viability = The percentage of cell viability values and concentration of crude extracts were plotted in order to determine the median inhibition concentration (IC50) values of each extract. Based on IC50 values, criteria of cytotoxic activity for the crude extract according to National Cancer Institute guideline and Nguyen et al [19] are as follows: ≤20 µg/ml considered highly active, 21-200 µg/ml considered moderately active, 201-500 µg/ml considered weakly active, and >501 µg/ml considered inactive.

Determination of total phenolic content
Total phenolic content was analyzed using Folin-Ciocalteu colorimetric method with modification according to Yildirim et al [20]. Briefly, 20 µl of crude extract at a 1 mg/ml concentration was added to a 96-well flat-bottomed plate, and 100 µl of tenfold diluted 2M Folin-Ciocalteu's reagent was added into each well. Then 80 µl of 7.5%Na2CO3 was added to the mixture, and the plate was incubated for 30 minutes in the dark at room temperature. After incubation, absorbance was detected with a microplate reader (Bio-Rad; California, USA) at 765 nm. The total phenolic content was compared with gallic acid standards curve. Data were calculated as mean ± SD (n=3) and showed as milligrams of gallic acid equivalents (GE) per gram of crude extract.

Determination of total flavonoid content
Total flavonoid content was determined by aluminium chloride colorimetric assay with modification according to Yildirim et al [20]. Briefly, 500 µl of 1 mg/ml extract were mixed with 75 µl of 5% NaNO2 for 6 minutes. 150 µl of 10% AlCl3 were added to the mixture, and then 500 µl of 1M NaOH and 275 µl of distilled water were added. The mixture was incubated at room temperature for 15 minutes. After incubation, absorbance was detected with a microplate reader (Bio-Rad; California, USA) at 510 nm. The total flavonoid content was compared with quercetin standards curve. Data were calculated as mean ± SD (n=3) and showed as milligrams of quercetin equivalents (QE) per gram of crude extract.

Statistical analysis
Results were recorded as mean ± SD. The data were analyzed using Microsoft Excel and GraphPad Prism Software 9 (GraphPad Software; California, USA). An unpaired t-test was performed for cytotoxicity to determine differences between treated A549 cells and treated L929 cells. P-value of less than 0.01 was considered statistically significant.

Cytotoxicity of crude extracts
To investigate cytotoxicity of crude extracts, A549 cells were treated with various concentrations of each crude extract for 72 h and its cytotoxicity was assessed by MTT assay. The According to the National Cancer Institute guideline for a natural product extract, criteria of highly cytotoxic activity for the crude extract is an IC50≤20 µg/ml [19]. N/A is not available cytotoxic effect of each crude extract is shown in Table 1. Regarding L. longecalyculata Furtado, the ethanol extract strongly inhibited A549 cell proliferation with an IC50 value of 12.00±0.41 µg/ml, whereas the aqueous extract was moderately active (181.40±5.19 µg/ml) against cancer cells. The ethanol and aqueous extracts of L. microphyllum (Cav.) R.Br. and L. salicifolium Presl at concentrations ranging from 3.125 to 200 µg/ml showed no inhibitory effect on the proliferation of lung cancer cells.
The ethanol extract of L. longecalyculata Furtado was highly cytotoxic activity against A549 cell for 72 h. To investigate the anticancer effect of this extract on lung cancer cells (A549), MTT assays were performed at intervals. L929 cells were taken as non-cancerous control cells.
As Table 2 Table 2).  Our study revealed that L. longecalyculata Furtado extracted with ethanol showed high efficacy against lung cancer cell line and was composed of phenolic and flavonoid compounds.
In previous reports, natural phenolic compounds like flavonoid (green tea), curcumin (turmeric), cucurbitacin B (Cucurbitaceae family), benzyl isothiocyanate (papaya seeds), and isoflavone (soy) showed potential to inhibit lung cancer. They can affect multiple biochemical pathways involved in lung cancer, for example, inducing apoptosis through downregulation of Bcl-2 and NK-κB pathway or modulation of PI3K/AKT/mTOR signaling pathway, and epigenetic alterations in cells via modulation of DNA methylation and chromatin modeling [21][22][23]. Consequently, we hypothesize that L. longecalyculata Furtado crude extract may have a similar mechanism of anticancer actions. Identification of these inhibitory mechanisms and phytochemical compounds will remain part of our plans for future study.
For normal cells treated with L. longecalyculata Furtado, an increase in survival adaptations was observed over time. Interestingly, nanotechnology-based delivery of phytochemicals as chemotherapeutic drugs against cancer has potential to overcome the limiting side-effects on normal cells, due to target-specific manner of delivery [24,25]. Recent advancements in phytochemical-based nanomedicine approved by Food and Drug Administration (USA) for clinical trials and clinical use in the treatment of various cancers are commercially available, including paclitaxel (Taxaceae), Abraxane ® ; vincristine (Apocyna-ceae), Marqibo ® ; and camptothecin (Nyssaceae), L9-NC. Also, paclitaxel and camptothecin are currently under clinical trial as effective treatments for non-small cell lung cancer [10,26]. Our pioneering research suggests that L. longecalyculata Furtado has the potential to enable the development of a new therapeutic drug for lung cancer management.

CONCLUSION
This study has not only revealed that L. longecalyculata Furtado extracted with ethanol can inhibit lung cancer cells with morphological changes in a dose-and time-dependent manner, but also showed the presence of phenolic and flavonoid compounds. The non-detection of cytotoxic effects in the other plants may be attributed to the extraction procedure adopted in this work. However, further studies are needed not only to identify the phytochemical compounds present in L. longecalyculata Furtado and their mechanism of anticancer action but on the possible anticancer properties of the other plants.
In addition, more studies will be conducted on L. longecalyculata Furtado to explore its possible development as a new therapeutic agent for cancer prevention and treatment.

Acknowledgements
We would like to thank Assoc. Prof. Dr. Pritsana Raugrut for providing human lung adenocarcinoma A549 cell line, and Sirindhorn Peat Swamp Forest Nature Research and Study Center for providing Lygodium microphyllum (Cav.) R.Br., Lygodium salicifolium Presl, and Licuala longecalyculata Furtado.

Funding
This study was supported by Faculty of Medicine, Princess of Naradhiwas University.

Ethical approval
None provided.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.