Abstract: Background and Objective: Medicinal plant species represent a large source of new compounds that help for the preparation of new drugs. In this research was evaluated the cytotoxic activity of leaves and fruits extract of Juniperus procera, commonly used in folk medicine in Saudi Arabia against Carcinoma colon (Caco2) cell lines. Materials and Methods: Caco2 cell lines were exposed to different concentrations of leaves and fruits extract of J. procera with silver nanoparticles (AgNPs). The MTT assay was used to determine the cytotoxic effect of all treatments. Morphology of Caco2 cell lines was monitored using an inverted microscope. Nuclei of Caco2 cell lines was counted using hemocytometer chamber. The DNA fragmentation of Caco2 cell lines was separated electrophoretically on a 2% agarose gel containing 1 μg mL–1 ethidium bromide and visualized under ultraviolet transillumination. Results: Toxicity percentage of leaves and fruits extracts of J. procera against Caco2 cells was dose dependent, with the concentrations. Fruits extract was more effective (IC50 8.80 μg mL–1) than leaves (IC50 11.44 μg mL–1). Mixing IC50 of AgNPs (47.32 ppm) with IC50 of leaves or IC50 of fruits showed strong positive anti-cancer activity where the toxicity reached to 65.39 and 74.44%, respectively. Number of nuclei in treated cell decreasing with increasing extract concentration and not detected at high concentration of leaves (500 μg mL–1) and fruits extract (250 and 500 μg mL–1). The DNA of treated cells with IC50 of fruit and leaves extract remained intact as in the controls and DNA smearing was not detected but DNA fragmentation was clear with IC50 of AgNPs treatment. Conclusion: Present investigation concluded that the obtained IC50 fruits or leaves J. procera extract against Caco2 cells meaning that the extracts have potential anti-cancer properties.
INTRODUCTION
Cancer is a pathological condition characterized by excessive cell growth and deriving from loss of control over the cell cycle and/or decreased apoptosis1. Colon cancer is one of the most common types of cancers worldwide. While chemotherapy is one of the most widely used therapeutic strategies against colon cancer, it also has some limitations, such as normal cell toxicity and gradually increasing resistance in cancer cells. The discovery of new drugs for use in alternative strategies in cancer treatment is therefore highly desirable. Plants are regarded as very promising from this perspective, since they represent substantial sources of substances with various therapeutic uses. Most anti-cancer drugs are today produced from plants2,3.
All developing countries and Gulf countries, most of the population still depends on folk medicine to treat serious diseases including cancers and various types of inflammations. Nowadays, use of medicines from plant source increases significantly with conventional therapies. Hence, the plants are gaining more attention by the researchers to find out new and effective agents for different diseases4. Over 50% of drugs used in clinical trials for anti-cancer activity have been isolated from natural sources or are related to them. Hence, the search for natural products to be used in cancer therapy represents an area of great interest in which the plant kingdom has been the most important source, providing many anti-tumor agents with novel structures and unique mechanisms of action5. About 61% of new drugs developed between 1981 and 2002 were based on natural products and they have been very successful, especially in the areas of infectious disease and cancer6. The herbal medicines have a vital role in the prevention and treatment of cancer and they are also commonly accessible7. Natural extracts and biologically active compounds isolated from plant species used in traditional medicine could be resources for new drugs8-11.
Junipers are long lived trees which sometimes live up to 2000 years. It is belong to the Cupressaceae family. Economic importance of genus Juniperus was attributed to its various phytochemical constituents as coumarins and flavonoids12, phenylpropanoid13 and essential oils14. Junipers species are known for their potential as a source for two important chemical products, the anti-cancer drug synthetic precursor, podophyllotoxin and essential oils15. The medicinal uses of Juniperus spp. are widespread in many countries such as Saudi Arabia, Bosnia, Lebanon and Turkey and according to folk medicine was used for treating skin and respiratory tract diseases16, urinary problems, rheumatism and gall bladder stones17. Juniperus procera is used in the southern part of Saudi Arabia as a traditional remedy for tuberculosis and jaundice18. Three alpha-hinokiol (3) and 3 alpha-hydroxymannol (9) isolated from Juniperus przewalskii, exhibited effective anti-tumor activities to cervical carcinoma (HeLa) and human ovarian carcinoma (HO-8910) cell lines19. Juniperus chinensis, commonly known as Chinese juniper, is a native and widely used ornamental plant in East Asian countries. The J. chinensis and plants of the same genus exhibit many bio-activities, such as anti-microbial, anti-fungal, anti-viral, anti-insect, anti-fertility, vasorelaxing and anti-tumor activities20. Several anti-parasitic, nematicidal, anti-microbial, anti-mycobacterial and hepatoprotective compounds were isolated from bark, leaves and berries of J. procera including abieta-7, 13-diene, ferruginol, isocupressic acid, (+)-Z-communic acid, (+)-totarol, 4-epi-abietol and sugiol21.
Several studies indicated that deoxypodophyllotoxin isolated from Juniperus communis and from bark of J. procera22 significantly induced cell apoptosis of breast cancer cells15 and non-small cell lung cancer cells23. Previous studies reported anti-cancer activity of J. phoenicea24,25. Native Americans used J. communis berries as an appetite suppressant and in the treatment of diabetes26, antioxidant27, antimicrobial28 activity. Juniperus excelsa berries extract and its fractions showed good and moderate levels of tumor inhibition29. All scientific reports reflect the unique properties silver nanoparticles (AgNPs) possess that find myriad applications such as antibacterial, antifungal and anti-cancer drugs, very good antioxidants, treatment of diabetes-related complications and wound healing activities30-34. The anti-tumor properties of AgNPs may be a cost-effective alternative in the treatment of cancer35. The purpose of this study was therefore to determine the cyto and genotoxic effect of J. procera leaves and fruits extract against colon cancer cell lines, one of the most common forms of cancer worldwide, with assessment the cytotoxicity of AgNPs combined with J. procera extracts.
MATERIALS AND METHODS
Location and total time duration of research work: Location of research work in King Abdulaziz University, Jeddah, Saudi Arabia. Work planed begin at November, 2018 and continued to March, 2019.
Collection and preparation of plant extracts: The J. procera aerial parts (leaves and fruits) were collected in November, 2018 from Fifa mountains, Jizan Region, southwest Saudi Arabia. The plant was identified according to Migahid36 and Chaudhary37. The fresh leaves and fruits (100 g for each it) of J. procera were air dried at room temperature under shade and ground into powder using an electric grinder. About 10 g from dried powder of leaves and fruits were incubated with 50 mL ethanol with shaking overnight, the extract was filtrated using filter paper and then drayed and the dry weight measured as 1.25 and 0.82 g for leaves and fruits, respectively.
Silver nanoparticles (AgNPs) used: The AgNPs (chemically synthesized <100 nm) were obtained from Sigma-Aldrich.
Cell line: The Caco2 cells supplied by the America Type Culture Collection (ATCC, USA) were used (Organism, homo sapiens human, tissue, colon, cell type, epithelial, culture properties, adherent, disease, colorectal adenocarcinoma, ATCC, ATB-37).
Cytotoxicity of plant extract (leaves and fruits) and AgNPs: Viable cells were measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay according to MTT kit (R and D Systems) manufacturer’s instructions. Caco2 cells were inoculated in 96 well tissue culture plates at 1×105 cells mL–1 (100 μL well–1) and incubated at 37°C for 24 h to develop a complete monolayer sheet. Growth medium was decanted from 96 well micro titer plates after confluent sheet of cells were formed, cell monolayer was washed twice with wash media. Two-fold dilutions of tested sample were made in RPMI medium with 2% serum (maintenance medium). Each dilution (0.1 mL) was tested in different wells leaving 3 wells as control, receiving only maintenance medium. Plate was incubated at 37°C and examined. Cells were checked for any physical signs of toxicity, e.g., partial or complete loss of the monolayer, rounding, shrinkage or cell granulation. The MTT solution was prepared (5 mg mL–1 in PBS) (BIO BASIC CANADA INC.,) then 20 μL MTT solution were added to each well. Place on a shaking table, 150 rpm for 5 min , to thoroughly mix the MTT into the media. Incubate (37°C, 5% CO2) for 1-5 h to allow the MTT to be metabolized. Resuspend formazan (MTT metabolic product) in 200 μL DMSO. Place on a shaking table, 150 rpm for 5 min, to thoroughly mix the formazan into the solvent. Read optical density at 560 nm and subtract background at 620 nm. Optical density should be directly correlated with cell quantity. The OD of formazan formed in control cells was taken as 100% of viability and the positively stained cells with MTT are expressed as the percentage (%) compared to control. Log concentrations versus (%) cell viabilities were plotted with a logarithmic graph, which was then used to determine the IC50 values38.
Morphological analysis: The morphology of cell was monitored using an inverted microscope. The Caco2 cells were checked for morphologic changes after 48 h exposure to range concentrations of plant extract or as compared to control and photographs were taken39.
Combination assay of fruits and leaves extracts of J. procera with AgNPs: Combined assay of IC50 of fruits and leaves of J. procera with AgNPs (47.32 ppm) was evaluated to determine its toxicity on Caco2 cells using MTT assay as described previously40.
Nuclei counting of treated Caco2 cells: The treated cells incubated in a mixture of citric acid and crystal violet that causes cells to lyse and the released nuclei to stain purple. Allow micro carriers from a culture sample (1 mL) to settle to the bottom of a centrifuge tube. Then the clear supernatant was removed by aspiration, 1 mL of crystal violet reagent was added and incubate at 37 at least 1 h. Introduce a sample into the hemocytometer chamber to count the purple-stained nuclei for whole cells as following: Volume of cell solution (mL)×Dilution factor in PBS blue (1:10)×Mean number of stained cells×104 (Conversion of 0.1 mm3 to mL)40.
DNA fragmentation: The culture medium of treated cells by IC50 of fruits (8.80 μg mL–1) and leaves (11.44 μg Ml–1) of J. procera extract and AgNPs (47.32 ppm) was removed and centrifuged at 3000 rpm for 5 min to collect detached cells. Adherent cells were lysed with a hypotonic lysis buffer (10 mM Tris-HCl, pH 8.0) containing EDTA (10 mM) and Triton X-100 (0.5%) and then pooled with pellets made of detached cells. RNA was digested using RNase (0.1 mg mL–1 at 37°C for 1 h ) followed by proteinase K treatment for 2 h at 50°C. The DNA was extracted with a mixture of phenol, chloroform and isoamyl alcohol (25:24:1). The DNA was precipitated by adding an equal volume of isopropyl alcohol, stored overnight at 20°C and centrifuged at 12,000 rpm for 15 min at 4°C. The pellet was air-dried, resuspended in 20 μL Tris acetate EDTA buffer supplemented with 2 μL of sample buffer (0.25% bromphenol blue, 30% glyceric acid) and electrophoretically separated on a 2% agarose gel containing 1 μg mL–1 ethidium bromide and visualized under ultraviolet transillumination41.
Statistical analysis: The results are reported as mean±standard error S.E. of three independent replicates. Statistical analysis of data were carried out by computer using SPSS ver. 22.0 software.
RESULTS
Cytotoxicity of J. procera extract and AgNPs: Leaves and fruits extracts of J. procera were tested against Caco2 colorectal carcinoma cells to determine their ability to inhibit cancer cell growth (Table 1). Toxicity (%) of leaves and fruits extracts of J. procera against Caco2 cells were dose dependent, with the concentrations. Toxicity (%) of leaves and fruits extracts of J. procera was similar up to 125 μg mL–1. However, fruits extract was more effective because its IC50 (8.80 μg mL–1) was less than leaves (11.44 μg mL–1) extracts. The IC50 of leaves and fruits extracts of J. procera indicated the strongest active against Caco2. Further evaluation of AgNPs outlined that lowest level of cytotoxic activity against Caco2 cells at concentration less than 62.5 μg mL–1 with the IC50 values of 47.32 μg mL–1 but more than 62.5 μg mL–1 concentration, it showed highest toxicity (Table 1).
Combined effect of IC50 of AgNPs with J. procera extract on Caco2 cell line: The synergistic potential of AgNPs IC50 together with the IC50 of leaves and fruits extracts were tested against Caco2 cells (Table 2). Mixing AgNPs IC50 (47.32 ppm) with leaves IC50 (11.44 μg mL–1) or fruits IC50 (8.80 μg mL–1) showed strong positive anti-cancer activity where the toxicity reached to 65.397 and 74.44%, respectively. But unfortunately 1/10 IC50 of leaves or fruits antagonize the activity of AgNPs against Caco2 cells where the toxicity was 47.46 and 40.64%, respectively. Nuclei not detected at high concentration of leaves (500 μg mL–1) and fruits (250 and 500 μg mL–1) extract. At high concentration (250 and 500 ppm) of AgNPs, nuclei was detected (Table 3).
Effect of J. procera extract and AgNPs on morphological features of Caco2 cell line: Morphological features of Caco2 cells treated with different concentration of leaves extract of J. procera were reported (Fig. 1) and compared with the untreated cells. At 250 and 500 μg mL–1 concentrations of leaves extracts of J. procera the treated cells showed remarkable difference with the control. Also, morphological changes of Caco2 cells treated with fruits extract of J. procera were reported (Fig. 2), where destructuration of cells were clear at high concentration up to IC50 (8.80 μg mL–1) of fruits extract. From the current results, the cells treated with fruits extract was more affected than treated with leaves extract, where the cells appeared less uniform with the loss of membrane integrity, rounding, shrinkage, however still intact at 31.25 and 62.50 μg mL–1.
On the other hand, Identifiable morphological features of apoptosis were observed in the treated cells with AgNPs (Fig. 3), morphological changes were clear in a concentration-dependent manner in case AgNPs treatment.
| Table 1: | Cytotoxicity of J. procera extract (leaves and fruits) and AgNPs against Caco2 cells |
| Table 2: | Cytotoxicity of combined IC50 of J. procera extract (leaves and fruits) with IC50 AgNPs against Caco2 cells |
| Fig. 1: | Morphological features of Caco2 cells treated with different concentration of leaves extract of J. procera |
| Table 3: | Nuclei counting of Caco2 cells lines treated with leaves, fruits extracts of J. procera and AgNPs |
Effect of J. procera extract and AgNPs on DNA fragmentation of Caco2 cell line: The DNA fragmentation assay was performed on Caco2 cells to elucidate the mechanism of cells death. As shown in Fig. 4, treatment with IC50 of fruit and leaves extract of J. procera (8.80 and 11.44 μg mL–1) remained intact as in the controls and DNA smearing was not detected. In cells treated with IC50 of AgNPs light smearing was observed.
DISCUSSION
The leaves and fruits extracts of J. procera displayed patented the inhibitory activity against Caco2 cells. Generally, Juniperus species are a good bet in the development of new drugs with natural compounds, it was reported that phytochemical analysis J. procera leaves indicated the presence of diterpenes, alkaloids and flavonoids in extract.
| Fig. 2: | Morphological features of Caco2 cells treated with different concentration of fruits extract of J. procera |
These constituents play an important role as anti-tumor activity. The IC50 obtained in current study on Caco2 cell using J. procera extract of fruits or leaves meaning that the extracts have potential anti-cancer properties42. Topcu et al.43 showed weakly active (IC50 = 17.7 μg mL–1) of J. excels leaves extract against A2780 (human ovarian cancer cell line). In recent study, the anti-proliferative activity of J. communis berry extracts against Caco2 carcinoma cell line was noted, with IC50 value44 500 μg mL–1. In previous studies it was investigated that the anti-cancer activities of J. procera leaves extract against hepatocellular carcinoma (Hep G-2) cells lines with IC50 value 131.9 μg mL–1. The differences in IC50 may be due to species of plant, target cells, types of extracted solvent and ingredient of plant but all these factors don’t eliminate effectiveness of J. procera against cancer cells42. Similar findings were reported by Nabi et al.29, where the anti-tumor activities of J. excelsa extract and its fractions showed good levels of tumor inhibition 86.6% inhibition. AgNPs also showed anti-cancer activity against Caco2 cells (Table 1). Several studies reported that, good anti-cancer activity of AgNPs against HCT-116 colon cancer cells45 and LoVo cells line46. Synergistic potential of AgNPs IC50 with leaves extract IC50 or of fruits extract IC50 were reported in the current study against cancer cells suggested that a better interaction of the mixture with tested cells. Generally, synergistic abilities of leaves and fruits extract of J. procera with AgNPs may reduce the use of AgNPs and therefore, reduce the side effects of metal.
Number of nuclei in treated cell decreasing with increasing extracts concentration. The toxic effect of treatments and its concentration reflected by the number of nuclei and suggestive for the apoptotic activities of treatments. Actually, the cytotoxic effect observed in current study is probably due to the presence of Hinokiol (3,12-dihydroxy-abieta-8,11,13-trien) whose presence in the J. procera has been reported by Wang et al.19.
| Fig. 3: | Morphological features of Caco2 cells treated with different concentration of AgNPs |
| Fig. 4: | Effect of leaves (Lane 1) and fruit (Lane 2) extract of J. procera and AgNPs (Lane 3) on DNA fragmentation in Caco2 cells. DNA standard 100 bp (Lane St.) |
A very recent study detected that sugiol (2-hydroxy-abieta-8,11,13-triene-7-one) as a constituent in J. procera reduced the cell viability of human pancreatic cancer cells (Mia-PaCa2)47. Therefore, the results indicated that apoptosis may be induced by the use of these two plant extracts but further studies should be performed to confirm this conclusion. Compared to control cells, morphology of treated Caco2 cells were changed particularly at high concentrations strongly suggesting that cell death is occurring in the wells treated with the plant extract and AgNPs. George et al.48 detected morphological alterations in the Caco2 cells exposed to Rubusfairholmianus root extract. Therefore, the results obtained from this study confirm that AgNPs induced cell death via apoptosis. The data obtained by Krishnaraj et al.49 exhibited cytotoxic effects of AgNPs on MDA-MB-231, human breast cancer cells and the apoptotic features were confirmed through DNA fragmentation assays.
CONCLUSION
These findings support and extend previous studies examining the anti-cancer effects of J. procera extracts against cancer cell. The J. procera fruits extract was more anti-cancer effective than leaves extract. The IC50 of J. procera extracts of leaves or fruits induced the cytotoxic effects of AgNPs, therefore J. procera extract may minimize the concentration of AgNPs used in cancer treatment. The DNA fragmentation assay confirmed that AgNPs induced Caco2 cells death via apoptosis. The results indicated that J. procera was a promising anti-cancer agent for Caco2 cells. The performed experiments add scientific evidence to conduct further studies.
SIGNIFICANCE STATEMENT
Public pressure to increase the use of natural therapeutic agent in treatment of diseases has increased in the recent years. Therefore; this study discovered the J. procera extract (leaves and fruits) for cancer treatment in vitro. This study will help the researchers to uncover the natural compounds from medicinal traditional plants that many researchers were not able to explore and helpful information in cancer therapy.
ACKNOWLEDGMENT
Thankful to King Abdulaziz University, Jeddah, Saudi Arabia.