Investigating the role of Cinnamomum verum in zebrafish swim bladder development and anti-cancer activity in human lung cancer cell lines

2.1 Plant material and extraction

C. verum bark was obtained from Sorrah Saudi Arabia (https://www.sorrah.sa). Sixty grams of the powder was used for each solvent extraction. The extracts were prepared by the Soxhlet extraction method described previously (Farooq Khan et al., 2021).

2.2 Zebrafish

The wild-type zebrafish were obtained from the zebrafish international resource centre in Oregon, USA, and are kept in the animal facility bioproducts research chair, college of Science, Department of Zoology Saudi Arabia. The animals are fed twice daily with the Zeigler zebrafish diet (Zeigler Bros, Inc. Gardners, PA 17324 USA). The zebrafish is maintained by following the local and international regulations regarding the use and care of laboratory animals.

2.3 Zebrafish embryos treatment

2.4 In vitro anti-cancer activity

Three types of human lung cancer cell lines were used to evaluate the anti-cancer potential of C. verum extracts on lung cancer. The cell lines are obtained from American type cell collection Human lung cancer cell line A549 (ATCC cat # CCL-185), H-1650 (ATCC cat # CRL-5883), H-1975 (ATCC cat # CRL-5908) and one type of primary cell line HUVEC (ATCC cat # PCS-100–013). Cells were grown in DMEM (Dulbecco's Modified Eagle Medium) in a humified incubator at 37 ℃ with 5% CO2. In vitro cytotoxicity assays were carried out as described previously (Alqahtani et al., 2022). Briefly Cells were seeded at 2 × 10⁴ per well of 6 well cell culture plates, and incubated with serial dilution (0, 0.5, 1.0, 3.0, 9.0, 27.0, 100.0 μg/mL) of extracts for 24 h. Control cells were treated with solvent (methanol 0.5% v/v). Three replicates were done for all experiments. DMEM with 10% FBS and 1% penicillin–streptomycin was used to grow the cells. The anticancer activity of C. verum extracts was assessed in vitro by MTT assay. To each well, 10 μL of 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) solution (made in PBS) was added and incubated for an additional 4 h. Finally, acidified isopropanol (0.01 N HCl) was added to dissolved MTT (formazan product). The dose–response curve was used to compute IC 50 values using Probit analysis. Cell viability was measured by using following formula. $$\text{Cell}\text{viability}\left(\%\right)=\text{Mean}/\text{Control}\text{OD}\times 100$$

2.5 Statistical analysis

All the experiments repeated for at least three times and data are represented as the mean of triplicates ± standard deviation (SD). IC₅₀ for cell lines and LD₅₀ for zebrafish embryo were calculated using Probit analysis (Mekapogu 2017). Using a 2-tailed Student's t-test (GraphPad Prism v6), statistical significance was determined. The following P-values were considered significant: **P < 0.01; ***P < 0.001; ****P < 0.0001.

3.1 Percent yield of different solvent extract

The cinnamon bark weighing 28.4 g was grinded to powder. Around 7 g of powder was used to prepare four types of extracts in solvents of methanol, chloroform, ethyl acetate and hexane. 500 ml of solvent was used for each extract. The percent yield for each solvent fraction is shown in Table 1. The maximum yield was from the methanol fraction (35%) while the least yield was from the chloroform fraction (6%).

3.2 In vivo toxicity of C. verum extracts in zebrafish embryos

Zebrafish embryos were treated with serial dilution of each extract. The response of the embryos varied depending on the extract’s nature and the dose used. As shown in Fig. 1 and Table 2, the methanol extract (LD 50 ≥ 1 mg/ml) was safest for the embryos as compared with chloroform, hexane, and ethyl acetate extracts. The chloroform extract was the most toxic, with LD ₅₀ values of just 2.42 µg/ml. In this study, the chloroform and hexane extract of C. verum showed more toxicity as compared to methanol, and ethyl acetate extracts. The hexane and chloroform extracts induced lethality in zebrafish treated embryos at concentrations of more than at 150 µg/ml. All the extracts were safe and did not induce any noticeable phenotype when used ≤ 50 µg/ml.

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Fig. 1

The dose–response of zebrafish embryos towards different extracts of C. verum. The data presented is the mean of three replicates, and the error bars represent the standard deviation.

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3.3 Hexane, ethyl acetate, and chloroform extracts of C. Verum induced craniofacial cartilage and notochord abnormities in zebrafish embryos

To investigate whether C. verum extracts induced any developmental toxicity (teratogenicity) in zebrafish embryos, the embryos were treated with sublethal a dose of extracts by which the treated embryos survived up to 3 days of treatment. To see the comparative teratogenic effects of different extracts, the zebrafish embryos were treated with an equal amount (100 µg/ml) of extracts. Each of the extracts was dissolved in methanol to prepare the final working solution, and hence the methanol (0.5% V/V) was used as mock control. As shown in Fig. 2A, no teratogenic effect was observed in mock treated embryos. The notochord in control embryos was straight and connected (NC pointed by black arrow). Similarly, no deformities were noted in craniofacial cartilage in mock-treated embryos (small black arrows under the mouth). The embryos which were treated with methanol extract of C. verum did not show any observable embryonic abnormalities (Fig. 2C). The highest level of embryonic abnormalities were observed in zebrafish embryos treated with C. verum chloroform extract. As shown in Fig. 2B, comparing to control embryos, these embryos are developmentally retarded and also cardiac edema is quite prominent in these embryos (Fig. 2B white arrow). The posterior trunk did not develop in 100% (n = 150) of chloroform treated embryos resulting in shortened bodies. The hexane and ethyl acetate extracts showed similar types of embryonic abnormalities as observed by chloroform extracts. Fig. 2D and E show the zebrafish embryos treated with hexane and ethyl acetate extract respectively. The craniofacial cartilage did not form in treated embryos and they showed an undulated notochord (black arrow NC).

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Fig. 2

Craniofacial cartilage and notochord defects induced by C. verum extracts in zebrafish larvae. The photomicrograph is the representative image of the live zebrafish larvae snapped at four dpf. A) Control. Small arrows under the mouth indicate the normal development of craniofacial cartilage and the black arrows indicate the notochord (NC). B) Zebrafish embryos treated with 1 µg/ml of chloroform extract. The treated embryos showed severe developmental abrnomalities, smaller as compared to control larvae at the same stage.The posterior trunk did not develop as well. The embryos had severe cardiac edema (red arrow). C) zebrafish embryos treated with 500 µg/ml of methanol extract. The methanol extract did not induce any obvious defects, and the larvae's development and size were the same as control larvae at 4dpf. D) Zebrafish embryos treated with hexane extract. Moreover, craniofacial cartilage was also absent in these embryos. The embryos were smaller size as compared to the control. E). A clear undulation of notochord can easily be visualized in zebrafish embryos treated with ethyl acetate extract. Moreover, the craniofacial cartilage did not form in these larvae. F). All images are taken by keeping the larvae anterior to the left under the same magnification. The scale bar is shown at the right lower side of each image.

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To evaluate whether the toxic effects were due to the extracts or solvents, the zebrafish embryos were treated with relevant solvents (as mock control for each solvent) which were used for the extraction. The concentration of the solvent was kept equal to the final highest working concentrations of the solvent (50 µL in 5 ml). As shown in Fig. 3, we have not detected any lethality and embryonic abnormalities in solvent alone treated embryos, which means that the teratogenic effects which were observed in zebrafish embryos were specific to the C. verum.

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Fig. 3

Solvent toxicity profiling in zebrafish embryos. Representative live images of zebrafish larvae at 3 days post fertilization. Control (no treatment), chloroform, methanol, hexane and ethyl acetate treated with 1% (V/V) solvents alone. The embryos were treated at shield stage (6 h post fertilization) and images were recorded at 3 days post fertilization.

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C. verum has been extensively studied to investigate its various level of biological activities in many in-vitro and in-vivo systems. However, its safety profile on embryonic development is largely not known. Many studies have reported that C. verum did not induce toxicity when tested in adult experimental animals (mostly rodents). One study has reported normal body weight in rats after ingestion of C. verum by a 13-week repeat-dose oral toxicity assay. The same study has shown that C. verum extract was not mutagenic or clastogenic by in-vitro mammalian cell, and in vivo, bone marrow micronucleus assays (Yun et al., 2018). The aqueous extract of C. verum was used to treat female Sprague Dawley rats, and the authors suggested that the C. verum extract is safe when used below 0.5 g/kg dose (Abdeen et al., 2019, Hussain et al., 2019). Similarly, the ameliorative activity of C. verum essential oil has been demonstrated in rats against carbon tetrachloride-induced hepatoxicity (Bellassoued et al., 2019).

The chloroform extract of hexane in this study induced severe teratogenic phenotype at sublethal dose (100 µg/ml) in zebrafish embryos. Developmental delay was one of the most noticeable effects of chloroform extracts of C. verum at this concentration.

3.4 C. verum extracts perturbed the formation of the swim bladder in treated zebrafish embryos

Zebrafish embryos were treated with an equal concentration (100 µg/ml) of different extracts. The extracts were added at the shield stage (6 hpf), and the experiment lasted for four days, and swim bladder development was evaluated at 4dpf. The Mock (0.05% methanol V/V) treated embryos served as control. The black arrow in Fig. 4A, shows a fully developed and inflated swim bladder (SB) in control larvae. Similarly, methanol extract of C. verum did not cause toxicity and swim bladder formation in the zebrafish larvae. As the methanol extract did not cause swim bladder formation defects at 100 µg/ml, the concentration of methanol extract was increased up to 1000 µg/ml to see whether it could affect swim bladder development at higher concentration but it was well tolerated by zebrafish larvae and did not cause any obvious developmental defects in 10 times more concentration than other solvents extract. Zebrafish larvae treated with C. verum ethyl acetate extract (100 µg/ml) exhibited an inflated but small swim bladder (back arrow Fig. 4C). As shown in Fig. 4D, the swim bladder did not form in zebrafish embryos treated with C. verum hexane extract (100 µg/ml) at 4dpf, (absence of swim bladder is indicated by a white asterisk.) Similarly, the swim bladder did not form in zebrafish embryos treated with chloroform C. verum extract (100 (Fig. 4E).

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Fig. 4

C. verum extracts blocked the formation and growth of swim bladder in zebrafish embryos. Representative images of live zebrafish larvae at 4dpf. Zebrafish embryos were treated with sub-lethal doses of C. verum extracts starting from the shield stage (6 hpf), and swim bladder development in control and treated larvae were recorded at 4dpf. A) Mock (0.5% methanol) treated control larvae with fully developed and inflated swim bladder (black arrow) B) Zebrafish larvae treated with methanol extract of C. verum (500 µg/ml). The methanol extract also did not affect the formation and growth of the swim bladder. C) Zebrafish larvae treated with C. verum ethyl acetate extract (10 µg/ml) show un-inflated and small-size swim bladder (back arrow) D). Hexane extract (5 µg/ml) treated larvae show the absence of swim bladder at 4dpf (white asterisk). E) Chloroform extract (2 µg/ml) treated larvae shows absence of swim bladder (white asterisk). All images were taken under the same magnification. The scale bar is shown at the bottom right corner.

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Teleost (bony) fish do not have lungs, but they have a swim bladder, an organ that functions like the lungs. The swim bladders of fish develop similarly to the lungs of higher vertebrates. The anatomical structure, morphological development, and transcriptional patterns of the zebrafish swim bladder, which functions as a variable buoyancy device, are similar to those of the lung (Zheng et al., 2011, Cass et al., 2013). Both arise as outgrowths from the esophagus, with the glottis occupying the same position (M. 2023). We have not come across any report in which the effect of C. verum on embryonic lung development was studied in any animal model. Hence this is the first report demonstrating the role of C. verum on lung formation using zebrafish.

3.5 Ani-inflammatory activity of C. Verum extracts in CuSO4 induced oxidative stress in zebrafish embryos

The ameliorative function of C. verum against CuSO4 induced toxicity in zebrafish larvae was used as in-vivo model to measure the anti-inflammatory activity of extracts. Zebrafish larvae (3dpf) were exposed to 10 µM of CuSO4 for one hour and then co-treated with C. verum extracts with LD₅₀ concentration (Table 1) for 12 to 15 h. Untreated larvae were negative control, and CuSO4 alone treated larvae as a positive control. The protective effect of C. verum extracts is shown in Fig. 5. Exposure to CuSO4 at a concentration of 10 µM induced 96% mortality in zebrafish larvae in 24 h. In contrast, no mortality was found in the negative control. The methanol extract (500 µg/ml) showed the weakest level of anti-inflammatory activity against CuSO4-induced oxidative stress and only 5% of zebrafish larvae survived by co-administration of methanol extract. Twenty one percent (21%) of zebrafish larvae survived, which were treated with 10 µM of CuSO4 and 110 µg/ml chloroform extract. Ethyl acetate (150 µg/ml) and hexane extract (110 µg/ml) showed strong protective activity against CuSO4-induced toxicity with 61 and 80% larvae survival. The protective action of ethyl acetate and hexane against CuSO4-induced toxicity was statistically significant, with a p-value of 0.05 compared with CuSO4 alone treated larvae.

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Fig. 5

Organic extract of C. verum exhibited the highest anti-inflammatory activity in zebrafish larvae. Zebrafish larvae were exposed to 10 µM of CuSO4 for one hour and then co-exposed to C. verum extracts equal concentration (1 µg/ml) for 24 h. As indicated by bar graph, only 4% of larvae survived after 24 h treatment with CuSO4 alone. 100% larval survival was observed in negative control larvae (not exposed to CuSO4 or C. verum extracts). Methanol extract of C. verum showed the weakest anti-inflammatory activity, and only 2% of larvae survived, which were treated with CuSO4 and methanol extract. Hexane extract of C. verum showed strong ameliorated activity against CuSO4-induced toxicity with an 80% survival rate. The ethyl acetate showed moderate anti-inflammatory activity against CuSO4-induced toxicity. The data presented are the average of three independent experiments. *: indicates degree of statistical significance (*** p-value 0.0001, ** p value 0.001, * p value 0,01) between the CuSO4 alone and the larvae treated with CuSO4 and C. verum extracts. Error bars represent the ± standard deviation between three replicates.

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Many in vitro and in vivo studies have documented C verum extracts or their components, especially cinnamaldehyde, as an anti-inflammatory agent (Abeysekera et al., 2022, Chen et al., 2022). Gunawardena et al. also reported that the highest level of anti-inflammatory activity was present in the organic fractions than the methanol and water extract of C. zeylanicum and C. cassia (Gunawardena et al., 2015). However, there is no report in which the anti-inflammatory activity of C. verum has been checked in live animals. Zebrafish possess an excellent in vivo model of inflammation as it shares several inflammatory genetic signatures with mammals (Zanandrea et al., 2020). Copper adversely affects organisms' development, physiology, reproduction, and survival and has immunosuppressive effects on certain fish species (Schink et al., 2018, Hong et al., 2020). Ethanol extract of leaves of C. cyrtophyllum showed protective activity against CuSO4 toxicity in 3dpf zebrafish larvae, and researchers proved that the protective activity was due to the high content of flavonoids in ethanol extract of leaves of C. cyrtophyllum which resulted in a high level of anti-oxidant (IC₅₀ of 16.45 µg/mL) and anti-inflammatory. The co-administration of ethanol extract of leaves of C. cyrtophyllum and CuSO4 downregulated inflammatory response genes in zebrafish larvae (Nguyen et al., 2020). Similarly, the protective effect of acacetin against CuSO4-induced toxicity was also attributed to the anti-inflammatory activity of acacetin. The co-administration of acacetin resulted in upregulation of anti-oxidant genes and suppressing pro-inflammatory response genes in CuSO4-exposed zebrafish larvae (Singh et al., 2022).

3.6 The anticancer activity of C. Verum against human lung cancer cell lines

As presented in the previous section, most of the C. verum extracts affected the swim bladder formation in zebrafish embryos. We next tested whether these extracts could induce cytotoxicity in human lung carcinoma cell lines? Three types of human lung carcinoma cell lines, namely A549, H-1650, and, H-1975, and a primary cell (non-cancer) human umbilical vein endothelial cell (HUVEC), were selected for such investigation.

The 50% inhibitory concentration (IC₅₀) of the extract against lung cancer cell and primary cell lines are presented in Table 3. The hexane extract of C. verum was most active against A549, H-1650, and H-1975 with IC₅₀ values of 44.30 ± 0.58, 42.12 ± 057, 30.33 ± 0.57 µg/ml respectively. The methanol extract was not active in all three types of lung cancer cell line and HUVEC. The cytotoxicity C. verum extract in human lung cancer cell lines indicates that the co– related with the in vivo toxicity profile of these extracts in zebrafish embryos. The hexane extract showed maximum level of cytotoxicity with minim concentration as compared to chloroform, ethyl acetate and methanol. Its LD₅₀ values in zebrafish embryos and IC₅₀ concentration in HUVEC cell were more than almost double as compared its IC50 values in cancer cell lines, which shows that hexane extract showed higher toxicity towards cancer cell the normal cell.

The anti-cancer activity of cinnamon against human cancer cell lines and xenograft cancer models has been reported previously. Cinnamomum cassia essential oil (CEO) infused chitosan nanoparticles suppressed the growth of 4T1 breast cancer cells in a mice xenograft model via inhibiting the expression of the Ki-67 protein (Xu et al., 2023). Cinnamaldehyde, the main ingredient has been suggested for the treatment of breast Cancer in women (Liu et al., 2020). The aqueous extract of cinnamon has been shown to inhibit the proliferation of bladder cancer 5637 cells by inhibiting glycolysis and induction of apoptosis (Aminzadeh et al., 2022). Proteasome inhibition by aromatic monophenols (procyanidin-B2), from cinnamon induced the death of prostate cancer cells by autophagy-dependent apoptosis (Gopalakrishnan and Ismail 2021). The anti-proliferative function of the cinnamon extract was also reported in three hematological cancer cell lines (Schoene et al., 2005).

3.7 Chemical composition of C. verum in different solvent fractions

The biological activity of any crude extract is due to the presence of one or many phytochemicals in the crude extract. So, it is important to know what kind of phytochemicals are present in the crude extract. Gas chromatography coupled with mass spectrophotometry (GC–MS) is any easy technique to identify the different active compounds especially the volatile compounds in crude extract. The four solvent fractions (methanol, chloroform, ethyl acetate and hexane) of C. verum were analyzed by GC–MS and results are presented in Tables 4–7. We detected only two peaks in the methanol fraction and the identified compounds are Phenol, 3,5-bis(1,1-dimethylethyl), and 9-octadecenamide. Both of these compounds have been reported previously from the C. verum bark. The Phenol, 3,5-bis(1,1-dimethylethyl) from the bark of specie Cinnamomum Zeylanicum (Hameed et al., 2016), and 9-octadecenamide was identified in small quantity from the bark of C. cambodianum and C. caryophyllus (Son et al., 2014).

Table 4 Methanol fraction.

No	Retention Time	Compound in %	Compound Name	Formula	Structure
1	15.82	9.74	Phenol, 3,5-bis(1,1-dimethylethyl)	C14H22O
2	29.697	90.26	9-octadecenamide	C18H35NO

Table 5 Chloroform fraction.

No	Retention Time	Compound in %	Compound Name	Formula	Structure
1	10.767	9.10	Cinnamaldehyde	C9H8O
2	13.535	69.31	Cinnamaldehyde dimethyl acetal	C11H14O2
3	14.290	4.33	2H-1-Benzopyran-2-one	C10H9NO2
4	14.4870	1.55	2-Thiapropane	C3H8S
5	16.618	3.50	1,3-Bis(cinnamoyloxymethyl)adamantane	C30H32O
6	18.082	12.2	2-METHYL-6-NITRO-2H-INDAZOLE	C8H7N3O2

Table 6 Hexane fraction.

No	Retention Time	Compound in %	Compound Name	Formula	Structure
1	10.743	11.6	Cinnamaldehyde	C9H8O
2	13.059	8.50	Copaene	C15H24
3	13.535	64.50	Cinnamaldehyde dimethyl acetal	C11H14O2
4	14.833	2.20	2-Propenoic acid	C3H4O2
5	15.144	3.23	1,2,3,4,4a,5,6,8a-octahydro-7-methyl-4-methylene-1-(1-methylethyl)-, (1.alpha.,4a.beta.,8a.alpha.)-	C15H24
6	15.619	3.01	(+)-Cyclosativene	C15H24
7	16.06	6.6	1 S-CIS-CALAMENENE	C15H22
8	18.094	4.73	1H-Benzimidazole	C7H6N2

Table 7 Ethyl acetate fraction.

No	Retention Time	Compound in %	Compound Name	Formula	Structure
1	10.767	4.819	Cinnamaldehyde	C9H8O
2	13.528	78.734	Cinnamaldehyde dimethyl acetal	C11H14O2
3	16.174	1.755	ORTHO METHOXY CINNAMIC ALDEHYDE	C10H10O2
4	18.094	14.692	2-METHYL-6-NITRO-2H-INDAZOLE	C8H7N3O2

The Cinnamaldehyde was found in abundance in chloroform, hexane and ethyl acetate fractions. These fractions have Cinnamaldehyde mostly in cinnamaldehyde dimethyl acetal form. Cinnamaldehyde converted to Cinnamaldehyde dimethyl while using methanol for GC–MS which is a common phenomenon also has been reported previously (He et al., 2021). That mean that cinnamaldehyde dimethyl acetal is actually Cinnamaldehyde itself and is present from 60 to 80% in these fractions.

Several studies have reported cinnamaldehyde as the major ingredient (present as 65.00 to 80.00% in bark) and most of the biological properties of C. verum are attributed towards cinnamaldehyde (Singh et al., 2007, Rao and Gan 2014, Lu et al., 2022). We have observed the craniofacial cartilage and notochord deformities in zebrafish embryos exposed to 100 µg/ml of hexane, ethyl acetate and chloroform crude extract of C. verum. Surprisingly such type of deformities in zebrafish larvae were observed when zebrafish embryos were treated with pure cinnamaldehyde ((Bhattacharya et al., 2021)., which means that these toxicities are mainly due to cinnamaldehyde, and thus care should be taken by pregnant mothers, while using C. verum as whole or pure cinnamaldehyde. The neurotoxicity in zebrafish embryos upon exposure to cinnamaldehyde is also reported. The treated embryos had damaged ventricular structures, abnormal eyes, shortened body length, undulated trunk, and pericardial edema in zebrafish (Chang et al., 2022). We also have observed similar kind of neurotoxicity in treated embryos by hexane extracts. We have performed an online program (http://www.swisstargetprediction.ch/) to identify the protein targets in humans using the chemical structure of Cinnamaldehyde. The results have been shown as Supplementary Table S1. The Swiss protein target prediction shows the high probability of “Transient receptor potential cation channel subfamily A member 1 (TRPA1)” being a specific target of cinnamaldehyde. TRPA1 channel plays a vital role in regulating brain development and the physiological function of astrocytes (Shigetomi et al., 2011). Hence, it could be postulated that cinnamaldehyde present in C. verum could have blocked the activity of TRPA1 which resulted the neurotoxicity in treated zebrafish larvae. One study also reported the inhibition of angiogenesis blood vessels in transgenic zebrafish line (flk1:GFP) embryos by high doses of C. verum extract (Bansode et al., 2013). We have not observed any defects in angiogenesis blood vessels by exposure of C. verum extracts to TG (fli; EGFP) zebrafish embryos (data not shown). This could be due to the nature of the solvent used, as we have not used the DMSO, or the angiogenesis defects could be due to secondary affect due to the high dose of C. verum. The neuro and cardiovascular toxicity with high dose of C. verum in zebrafish embryos has been reported by other studies, but we have not found any study which reported the effect of C. verum on lung toxicity in embryos. This study is unique in the sense that the effect of C verum on zebrafish lung (swim bladder) development was being reported for the first time an also its anticancer potential against lung cancer.