Cedrus atlantica extract exerts antiproliferative effect on colorectal cancer through the induction of cell cycle arrest and apoptosis

Abstract Cedrus atlantica is a tree species found in Morocco with many clinical benefits in genitourinary, musculoskeletal, and skin systems. Previous studies have reported that extracts of Cedrus atlantica have antioxidant, antimicrobial, and anticancer properties. However, its role in colorectal cancer (CRC) remains unclear. The present study investigated the effects and underlying mechanisms of Cedrus atlantica extract (CAt) using HT‐29 (human colorectal adenocarcinoma) and CT‐26 CRC cell lines. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay was performed to measure cell viability. Flow cytometry analysis and terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) assay were used to study the cell cycle and cell apoptosis, respectively. The expression of cell cycle and apoptosis‐associated proteins was detected by western blotting or immunohistochemical (IHC) staining. CAt showed significant inhibitory effects on the proliferation of HT‐29 and CT‐26 cells, and combined with the clinical drug, 5‐fluorouracil (5‐FU), exhibited synergistic effects. CAt induced cell cycle arrest at the G0/G1 phase through the upregulation of p53/p21 and the downregulation of cyclin‐dependent kinases (CDKs)/cyclins. In addition, CAt‐treated cells exhibited chromatin condensation, DNA fragmentation, and apoptotic bodies, which are typical characteristics of apoptosis activated via both the extrinsic (Fas ligand (FasL)/Fas/caspase‐8) and intrinsic (Bax/caspase‐9) pathways. Importantly, CAt suppressed tumor progression and prolonged the life span of mice within a well‐tolerated dose. Therefore, our findings provide novel insights into the use of CAt for the treatment of CRC.


| INTRODUC TI ON
Colorectal cancer (CRC) is one of the most common cancers worldwide: global cancer statistics show that the prevalence of CRC ranks third in the world in 2018, with approximately 1.8 million people diagnosed with CRC each year (Bray et al., 2018). It is estimated that by 2030, 2.2 million new cases of CRC will be diagnosed and 1.1 million will die from CRC (Arnold et al., 2017). Most CRC cases are sporadic, and 18%-35% of cases are from family inheritance, indicating that the environment and genetic background are relevant to the occurrence of CRC (Lynch & de la Chapelle, 2003;Rawla et al., 2019). Among the environmental risk factors associated with CRC, the most important are high-calorie diets of rich animal fat, smoking, increased alcohol consumption, and insufficient intake of vegetables, fruits, and fibers. Despite advances in CRC screening, approximately 35% of colorectal cancer patients present with stage IV metastasis at the time of diagnosis, while 20%-50% of patients with stage II or III metastasis will develop into stage IV as the disease progresses (Zacharakis et al., 2010). Although CRC therapy has improved, the 5-year survival rate of patients with distant metastasis is still only 10%-15%.
Methods commonly used in the treatment of CRC include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy, which are based on tumor size, location, cancer stage, and patient health status. 5-Fluorouracil (5-FU), irinotecan (Camptosar), oxaliplatin (Eloxatin), capecitabine (Xeloda), and trifluridine/tipiracil (Lonsurf) are commonly used to treat CRC, and they can be used alone or in combination to increase response rates and reduce the development of drug resistance (Kim, 2015). However, the long-term use of these drugs can cause serious side effects and reduce the quality of life. Recent studies have indicated that many natural products are targeted agents that can induce tumor cell apoptosis, inhibit proliferation, initiate cell cycle arrest, and have great anticancer potential .
For centuries, extensive research has been conducted on drug discovery and development from plant extracts and natural products, which contain numerous molecules with proven cytotoxicity inducing apoptosis via different signaling pathways against cancers (Benarba & Pandiella, 2018). For example, anticancer compounds such as Vinca alkaloids isolated from Catharanthus roseus (Shams et al., 2009), podophyllotoxin isolated from Podophyllum peltatum (Ardalani et al., 2017), camptothecin isolated from Camptotheca acuminata (Ran et al., 2017), taxol isolated from Taxus brevifolia (Kuriakose et al., 2020), and their derivatives are widely used as firstline and second-line cancer therapies.
Cedrus species (Pinaceae) classified by their morphological diversities include C. atlantica in Morocco and Algeria, C. libani in Lebanon, Syria, and Turkey, C. brevifolia in Cyprus, and C. deodara in the Himalaya Mountains (Panetsos et al., 1992). Essential oils extracted from different species of Cedus have traditionally been used in aromatherapy for many clinical benefits of the genitourinary, musculoskeletal, and skin systems (Lovell, 1998;Gabriel Mojay, 2002;G. Mojay, 2004). Cedrus atlantica is the largest remaining population and the main forest species in Morocco used for timber production, and sawdust is usually refined by hydrodistillation to provide essential oils. It exerts antimicrobial (Dakir et al., 2005;Shin, 2003) and anticancer (Chang et al., 2021;Huang et al., 2020;Saab et al., 2012) activities and alleviates pain behavior via inhalation (Martins et al., 2015). However, there is currently a lack of information regarding the potential anticancer properties of C. atlantica extract (CAt) against CRC. The present study assessed the anticancer effects of CAt in CRC cells and investigated the underlying molecular mechanisms in vitro and in vivo.
The preparation of CAt was commissioned to Phoenix (New Jersey, USA) according to the following conditions: the bark of Cedrus atlantica was extracted through steam distillation at a flow rate of approximately 7.2 ml/min at 100-105°C for 90 min (Chang et al., 2021). CAt and 5-FU (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (DMSO) and diluted in fresh medium.
The final concentration of DMSO for cell treatment was <1%. and apoptotic bodies, which are typical characteristics of apoptosis activated via both the extrinsic (Fas ligand (FasL)/Fas/caspase-8) and intrinsic (Bax/caspase-9) pathways. Importantly, CAt suppressed tumor progression and prolonged the life span of mice within a well-tolerated dose. Therefore, our findings provide novel insights into the use of CAt for the treatment of CRC.  at 37°C, and incubated in a humidified 5% CO 2 atmosphere. The status of TP53 exon8 in HT-29 cells was mutant type (R273H) using automated nucleic acid extraction (AccuBioMed Co., Ltd., Taipei, Taiwan) and sequencing using Femtopath Human TP53 Primer Sets (HongJing Biotech, Taipei, Taiwan).
Then, 100 μl of MTT solution in medium (400 μg/ml) (Sigma-Aldrich) was added to each well and the cells were incubated for 4 h. A microplate reader (Spec384; Molecular Devices) was used to measure the absorbance at 550 nm. Cell viability was calculated as the OD percentage relative to the control (100%).

| Detection of the cell apoptosis
Apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay using the Situ Cell Death Detection Kit (Roche, Mannheim, Germany). Cells were treated with 35 μg/ml CAt for 48 h, collected, and washed with phosphate-buffered saline (PBS). After fixation with 10% formaldehyde, the cells were smeared and dried on silane-coated glass slides.
Then, cells or deparaffinized sections were rehydrated with PBS, inactivated endogenous peroxidase using 3% H 2 O 2 in methanol, and permeabilized using 0.1% Triton X-100 in 0.1% sodium citrate on ice.
Samples were incubated with TUNEL solution for 2 h at 37°C, counterstained with PI, and observed under a fluorescence microscope (Axioskop 2; Zeiss) at 400× magnification.

| Western blot analysis
HT-29 cells were seeded at a density of 2 × 10 6 cells in a 100-mm dish. The next day, the cells were treated with 35 μg/ml CAt for 0, 6, 12, 24, and 48 h. Cell lysates were prepared by adding radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor (Bio Basic Inc., Canada) and phosphatase inhibitor (Bionovas, Toronto, Canada), and incubated on ice for 30 min. The extracted proteins were estimated according to the protocol of bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Approximately 20 μg of the proteins was separated by 8%-12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.22μm polyvinylidene difluoride (PVDF) membranes (Pall Corporation, USA).
The PVDF membranes were blocked with 5% nonfat dry milk for 30 min, followed by probing the membranes with blocking bufferdiluted specific primary antibodies (1:1,000 dilution) at 4°C overnight with continuous shaking. The membranes were washed three times with 0.5% Tween-20 in Tris-buffered saline, incubated with biotin-conjugated secondary antibodies (Santa Cruz, CA, USA) for 2 h, followed by interaction with peroxidase-conjugated streptavidin (Jackson ImmunoResearch Inc., USA) for 1 h. After washing and treatment with an enhanced chemiluminescence reagent (ECL) (T-Pro Biotechnology, Taiwan), the blots were scanned and analyzed using a chemiluminescence imaging analyzer (GE LAS-4000; GE Healthcare Life. Sciences, NJ, USA) and ImageJ software 1.47t (National Institutes of Health, Bethesda, MD, USA). The density ratio of sample to control was calculated as follows: density ratio = (normalized sample/normalized control).   (1 × 10 6 ) were subcutaneously injected into the right flank of the mice. The vehicle group (n = 4) and CAt group (n = 6) received solvent (100 μl of mineral oil) and 200 mg/kg of CAt, respectively (every 2 days for 20 times, subcutaneous injection). 5-FU (n = 4) was intraperitoneally injected 3 times a week at the dose of 25 mg/ kg for 21 days as positive control (Cho et al., 2020). Tumor size (L × H × W × π/6 mm 3 ) and body weight were recorded every 2 days.

| Animal study
Mice were sacrificed using carbon dioxide when the tumor volume was greater than 1,500 mm 3 . The tumors and organs were fixed with 4% formaldehyde, embedded in paraffin, and sliced for hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining.

| H&E and immunohistochemistry stain
In H&E staining, the deparaffinized sections (7 μm) were stained with hematoxylin and eosin (Muto Pure Chemicals, Tokyo, Japan), after which the tissue morphology was observed and photographed under a bright field microscope. For IHC staining, the sections (4 μm) were heated at 65°C for 30 min, deparaffinized, and hydrated through a series of xylene and alcohol baths. The slides were microwaved in an antigen retrieval solution (Thermo Fisher Scientific Inc., MA, USA) for 5 min. The sections were immersed in 3% H 2 O 2 for 10 min to inactivate endogenous peroxidase activity and then blocked with 10% bovine serum albumin (BSA) for 30 min. Thereafter, immunohistochemical staining was performed using rabbit anti-PCNA antibody and mouse anti-caspase-3 antibody and incubated at 4°C overnight.
After washing with 0.1% Tween-20 in PBS for 3 times, the antibodybinding proteins were detected and visualized using Super Sensitive Polymer-HRP IHC Detection System (BioGenex, CA, USA) and 3,3'-diaminobenzidine (DAB) substrate (BioGenex, CA, USA). Finally, the sections were photographed using a bright field microscope and scored using the Quickscore method performed according to a previously published protocol (Chang et al., 2021).

| Statistical analysis
The results are expressed as the mean ± SD (in vitro) or mean ± SEM (in vivo). Statistical analysis was performed using an unpaired Student's t-test or one-way analysis of variance (ANOVA) to analyze the differences between each group, and the Kaplan-Meier method was used for the survival rate analysis. Statistical significance was set at p < .05. Experiments were repeated at least three times in duplicate or triplicate.

| Effects of CAt on the viability of CRC cells
In our previous study, the major components of CAt included α-cedrene (37.98%), cedrol (23.03%), thujopsene (19.45%), γmuurolene (6.68%), and cuparene (2.14%), identified using a gas chromatography-mass spectrometry (GC-MS) spectrometer (Chang et al., 2021). To evaluate the anticancer activity of CAt against CRC cells, we performed an MTT cell proliferation assay F I G U R E 1 Cedrus atlantica extract (Cat) inhibited the cell proliferation of colorectal cancer (CRC) cells. Human colorectal adenocarcinoma (HT-29) (a), mouse colorectal carcinoma (CT-26) (b), mouse vascular endothelial (SVCE) (c), and canine kidney epithelial (MDCK) (d) cells were treated with CAt (0, 3.125, 6.25, 12.5, 25. 50, and 100 μg/ml) for 24, 48, and 72 h. The 3-(4,5-dimethylthiazol-2-yl )-2,5-diphenyltetrazolium bromide (MTT) assay was used to monitor cell viability. Data were shown as the mean ± SD of three independent experiments on two CRC cell lines, HT-29 and CT-26. First, different concentrations of CAt (0-100 μg/ml) were used to treat CRC and normal cells (SVEC and MDCK). Cell viability was measured at different time points (24, 48, and 72 h), and a cell growth inhibition curve was constructed ( Figure 1). The results showed that CAt inhibited the proliferation of CRC cells in a concentration-and time-dependent manner, but had less effect on normal cells. Furthermore, the halfmaximal inhibitory concentration (IC 50 ) of all cells (HT-29, CT-26, SVEC, and MDCK) was determined at 48 h, and the values were 31.21 ± 1.36 μg/ml, 19.77 ± 0.7 μg/ml, 45.62 ± 0.88 μg/ml, and 69.71 ± 2.1 μg/ml, respectively ( Table 1). Comparison with normal cells indicated that CAt had more drug selectivity to CRC cells, but this effect was not observed with 5-FU. Based on the data, it can be concluded that CAt showed effective inhibitory effects in CRC cells but not in normal cells.

| CAt combined with 5-FU revealed synergistic effects in CRC cells
The first-line chemotherapy drug for CRC is 5-FU; however, it has a short half-life, which limits its effectiveness. Next, we evaluated whether combination treatment could enhance the growth inhibition of CRC cells. HT-29 cells were treated with CAt (0-80 μg/ml) with or without 5-FU or 5-FU (0-8 μg/ml) with or without CAt, and cell viability was detected by the MTT assay. The results showed that the combination treatment of CAt and 5-FU significantly decreased the viability of CRC cells compared with CAt or 5-FU alone (Figure 2a,b). To investigate the drug interaction of CAt and 5-FU, a combination index (CI) was calculated using Compusyn software, which quantitatively determined synergism (CI < 1), additive effect (CI = 1), and antagonism (CI > 1), respectively. As shown in Figure 2c,d, most of the combined drug doses showed a synergistic effect (CI > 1). Collectively, these data suggest that CAt in combination with 5-FU synergistically inhibits the growth of CRC cells.

cells
To determine whether the antiproliferation effect is attributable to the inhibitory effect of CAt on the cell cycle, cell cycle analysis was performed. HT-29 cells were treated with IC 50 concentration of CAt (35 μg/ml) for 0-48 h; CAt (0, 25, 35, and 45 μg/ml) for 24 h, stained with PI, and cell cycle distribution was analyzed by flow cytometry.
The results showed that in comparison to 0 h, the cells treated with CAt for 48 h increased the percentage in G0/G1 phase from 56.77% to 69.82%, whereas the percentage of cells significantly decreased in S and G2/M phases (Figure 3a). Compared to treatment with various doses for 24 h, cell cycle arrest at the G0/G1 phase was induced by treatment with 25 and 35 μg/ml CAt (Figure 3b).
Furthermore, the expression level of cell cycle-related proteins was determined by western blotting after the HT-29 cells were treated with CAt (35 μg/ml) for 0-48 h. This indicated that the protein expression levels of p-53/p-p53 and p21 were increased, while the protein expression levels of Rb/p-Rb and PCNA were decreased after CAt treatment (Figure 3c). In addition, the expression of cell cycle regulators, such as CKD4/cyclin D1, CKD2/cyclin A, and cyclin B1, was reduced in CAt-treated cells in a time-dependent manner.
These findings suggest that CAt arrested the cell cycle at the G0/ G1 phase in CRC cells by regulating the expression of p53/p21 and CDK4/cyclin D1 proteins. To further understand the specific mechanism of CAt leading to the apoptosis of HT-29 cells, western blotting was used to detect the expression of apoptosis-related proteins. Compared with the control, the protein levels of FasL/Fas/caspase-8, Bax/caspase-9, and cleaved caspase-3 were increased (Figure 4d). These results showed that CAt effectively promoted apoptosis in CRC cells through extrinsic and intrinsic caspase-dependent pathways.   (Figure 5a). The survival time of tumor-bearing mice was prolonged from 25 to 49 days after CAt treatment (p < .05; Figure 5b). This indicated that the tumor growth of implanted tumors was strongly suppressed after 38 days of CAt intervention (once every 2 days). In addition, the clinical drug 5-FU had obvious inhibitory effects in the early stage of treatment, however, the tumor grew rapidly after the second treatment cycle.

| CAt inhibited tumor growth of CRC in vivo
There was no significant difference in tumor volume between the CAt group and the 5-FU group before day 21, but the CAt group showed more suppressive effects than the 5-FU group after day 23.
In this animal study, no obvious loss of body weight was observed in vehicle, CAt, or 5-FU groups (Figure 5c). F I G U R E 2 Cedrus atlantica extract (Cat) combined with 5-fluorouracil (5-FU) synergistically inhibited the growth of human colorectal adenocarcinoma (HT-29) cells. HT-29 cells were incubated with a combination of (a) CAt (0, 10, 20, 40, and 80 μg/ml) and/or 1.5 μg/ ml 5-FU; (b) 5-FU (0, 1, 2, 4, and 8 μg/ ml) and/or 30 μg/ml CAt for 48 h, and the cell viability measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. The data are expressed as the mean ± SD. *p < .05 versus single drug group. Combination index (CI) plot (c) and normalized isobologram (d) were calculated and analyzed using CompuSyn software The negative control animals received the same volume of solvent (mineral oil). No obvious morphological changes in organs in the heart, liver, spleen, kidney, intestine, and stomach were observed between the CAt and vehicle groups ( Figure 6). The no-observedadverse-effect level for CAt in the 38-day repeated injection study in mice was greater than 200 mg/kg body weight/2 days. These results suggest that the dose of CAt was well tolerated and effective for CRC treatment.

| DISCUSS ION
Herbal medicines have been used to treat cancer for millennia and are currently used alone or in combination with conventional therapies to treat various diseases (Sultana et al., 2014). It is known that plant-based bioactive components exhibit anticancer activities in various ways, including changes in carcinogen metabolism, the activation of the immune system, the stimulation of DNA damage, the inhibition of cell cycle progression, and the induction of cell apoptosis.
Compared with traditional anticancer agents, inducing cell growth arrest and apoptosis are the safest strategies for cancer treatment, as they are less toxic and have a lower risk of causing inflammation and side effects as a result of damaged and necrotic cells (Pfeffer & Singh, 2018;Samadi et al., 2015). In addition, natural products with potential anticancer activities are inexpensive compared to conventional anticancer agents (Seca & Pinto, 2018). Our previous studies indicated that a plant extract of C. atlantica (CAt) showed high potential for bioactivity against CRC screened from 24 plants through a drug screening platform based on the growth inhibition of CRC and normal cells. In the present study, we found that CAt inhibited the Significant advances in CRC therapy have improved the overall survival rate of patients through the use of various promising drugs, such as oxaliplatin and 5-FU, as well as antibodies, such as bevacizumab and cetuximab, inducing programmed cell death (Schwartz et al., 2004). In recent decades, 5-FU has been used as the first-line treatment for CRC (Vodenkova et al., 2020). However, the adverse effects and emergence of drug resistance remain a critical limitation to the clinical application of conventional chemotherapy (Hu et al., 2016). Recent progress has shown that combination treatment has many advantages over conventional treatment, including enhancing chemosensitivity and reducing the necessary drug dosage (Hu et al., 2016). Based on these results, in this study, we investigated the anticancer effect of the combination of CAt and 5-FU.
The data revealed that CAt improved the inhibitory effects of 5-FU and showed that the combination of CAt and 5-FU achieved better treatment effects than a single drug. Previous studies on glioblastoma showed that CAt enhanced the antiproliferative effects of temozolomide on DBTRG-05MG and RG2 cells (Chang et al., 2021).
Therefore, CAt may be a potentially effective antiproliferative drug or anticancer adjuvant agent combined with clinical drugs.
Several studies have shown that cell cycle progression controls cell proliferation (Dickson & Schwartz, 2009), and its dysfunction is a crucial stage in cancer development (Williams & Stoeber, 2012).
Therefore, controlling cell cycle progression by inducing cell cycle arrest may be an appropriate strategy for cancer treatment (Carnero, 2002). In this study, flow cytometry analysis showed a significant accumulation of cells in the G0/G1 phase, along with a decrease in the percentage of cells in the S and G2/M phases after CAt treatment. The results implied that CAt induced cell cycle arrest at the G0/G1 phase in a time-dependent manner, resulting in the discontinuing proliferation of damaged cells. It has been reported that p21, activated by the transcriptional factor p53, acts as an inhibitor of the CDK4/Cyclin D complex, which plays a vital role in the progression of the cell from the G1 phase to the S phase (Satyanarayana & Kaldis, 2009). Our data showed that CAt increased the protein expression levels of p53 and p21, and decreased the p-Rb. In conjunction with these changes, the vital proteins CDK4/cyclin D1 in the G0/G1 phase were significantly inhibited. These results suggest that CAt may induce cell cycle arrest at the G0/G1 phase, resulting in the growth inhibition of CRC cells via the regulation of p53/p21 and CDK4/Cyclin D1.
Apoptosis (programmed cell death) plays a crucial role in the control of carcinogenesis and cancer treatment. Studies have indicated that treatment strategies, such as chemotherapy, radiotherapy, and F I G U R E 5 Cedrus atlantica extract (Cat)-mediated inhibition of tumor growth in vivo. The tumor volume (a), survival rate (b), and body weight (c) of tumor-bearing mice were recorded after administration of mineral oil (vehicle), CAt at 200 mg/ kg, and 5-fluorouracil (5-FU) at 25 mg/ kg. (d) The expression of proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 in tumor tissues was detected by immunohistochemical (IHC) assays (×400), quantified, and presented as percentages or IHC scores, respectively. Scale bar = 100 µm. All data are shown as the mean ± SEM. *p < .05 versus vehicle surgery, usually involve inducing the apoptosis signaling pathway in most cancer cells (Ghobrial et al., 2005;Lee et al., 2014). The caspase protein family plays an important role in apoptosis and is produced as an inactive precursor (procaspase) in cells. Then, a series of caspases are activated, and cleaved caspases are produced after triggering the apoptotic pathway. Active initiator caspases, including caspase-8 (extrinsic pathway) and caspase-9 (intrinsic pathway), can activate other downstream caspases called executioner caspases (caspase-3, −6, and −7). Caspase-3 is the central enzyme responsible for plasma membrane reversion, nuclear and cytoplasmic protein degradation, and DNA fragmentation, which ultimately leads to cell death (Chinnaiyan, 1999;Degterev et al., 2003). In the present study, we found that CAt-treated cells showed TUNELpositive results and apoptotic morphology, including chromatin condensation, DNA fragmentation, and apoptotic bodies. In addition, CAt induced apoptosis in HT-29 cells through the activation of initiator and effector caspases (caspase-8, −9, and −3). These findings indicate that CAt may induce HT-29 cell apoptosis via extrinsic and intrinsic pathways.
In conclusion, the results of the present study showed that CAt inhibited the proliferation of HT-29 cells by changing the cell cycle distribution, leading to cell cycle arrest at the G0/G1 phase via the regulation of p53/p21 and CDK4/cyclin D1. Moreover, CAt induced apoptosis through the activation of the extrinsic (FasL/Fas/ caspase-8) and intrinsic (Bax/caspase-9) apoptotic pathways. In an animal study, subcutaneous treatment with CAt suppressed tumor growth in mice, which was well tolerated. In view of the above safe and effective anticancer effects, CAt may be exploited for the development of novel anticancer agents or dietary supplements against CRC.

ACK N OWLED G M ENTS
The research was supported by grants from the Ditmanson Medical University, which is supported by the National Science Council, the Ministry of Education, and Chung Shan Medical University.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
This study was approved by Institutional Animal Care and Use Committee (IACUC) of Chung Shan Medical University (Approval No. CSMU-IACUC-1543).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author by reasonable request.

F I G U R E 6
Cedrus atlantica extract (Cat) showed good tolerance with no significant toxicity after treatment. The mice were treated with 200 mg/kg CAt for 38 days once every 2 days, collected organs, including heart, liver, spleen, kidney, intestine, and stomach, and analyzed histological change using hematoxylin and eosin (H&E) staining. Scale bar = 50 µm