|Year : 2017 | Volume
| Issue : 1 | Page : 31-36
Lack of in vitro anticancer and antimicrobial activities from Karanda (Carissa carandas) fruit extracts
Faculty of Science and Technology, Suan Sunandha Rajabhat University, Bangkok, Thailand
|Date of Web Publication||21-Apr-2017|
Faculty of Science and Technology, Suan Sunandha Rajabhat University, Bangkok
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Carissa carandas L. (Apocynaceae), commonly known as Karanda, is a widely used medicinal plant. In Thailand, Karanda fruits are favorite fruits especially in central region due to attractive shape and color with health promoting activities. Aims: To evaluate its antimicrobial and anticancer activities for claim promoting activities. Cytotoxicity of extracts was also evaluated with normal cells to claim for health safety. Materials and Methods: Anticancer activities of Karanda fruits extracted with dichloromethane (KD) and methanol (KM) were performed by resazurin microplate assay and tested with five cell lines, including KB-oral cavity cancer, MCF7-breast cancer, NCI-H187-small lung cancer, HepG2-hepatocarcinoma, and Caco2-colon adenocarcinoma cell lines. Cytotoxicity of KD and KM were performed as above and used HDFn-neonatal dermal fibroblast as normal cells. Antimicrobial activities of KD and KM against herpes simplex virus type I (HSV-1) and Mycobacterium tuberculosis were tested according by green fluorescent protein-based assay; Candida albicans were tested according to resazurin microplate assay, and Plasmodium falciparum K1 strain was tested according to microculture radioisotope techniques. Results: Both extracts were not possessed anticancer activity to any cancer cell lines at maximum concentration = 100 µg/mL. In the same way to anticancer activity assays, both extracts were not inhibited HSV-1, P. falciparum K1 strain, and M. tuberculosis (maximum concentration = 50 µg/mL). Also, both extracts were nontoxic to normal cells. Conclusion: KD and KM extracts of ripped Karanda fruits had no significant anticancer and antimicrobial activities with noncytotoxicity.
Keywords: Anticancer, antimicrobial, Carissa carandas, cytotoxicity, Karanda fruit
|How to cite this article:|
Sudjaroen Y. Lack of in vitro anticancer and antimicrobial activities from Karanda (Carissa carandas) fruit extracts. J Pharm Negative Results 2017;8:31-6
|How to cite this URL:|
Sudjaroen Y. Lack of in vitro anticancer and antimicrobial activities from Karanda (Carissa carandas) fruit extracts. J Pharm Negative Results [serial online] 2017 [cited 2018 Jul 19];8:31-6. Available from: http://www.pnrjournal.com/text.asp?2017/8/1/31/204914
| Introduction|| |
Carissa carandas L. (Apocynaceae), commonly known as Karanda, is a widely used medicinal plant. C. carandas is large dichotomously branched evergreen shrub with short stem and strong thorn in pairs. This species is a rank-growing, straggly, woody, climbing shrub, usually growing to 10 or 15 ft. (3–5 m) high, sometimes ascending to the tops of tall trees. The fruits, leaves, barks, and roots of C. carandas have been used for ethnomedicine in the treatment of human diseases, such as diarrhea, stomachic, anorexia, intermittent fever, mouth ulcer, and sore throat, syphilitic pain, burning sensation, scabies, and epilepsy. The prominent biological activities reported, include antidiabetic, antimicrobial, cytotoxicity, anticonvulsant, hepatoprotective, antihyperlipidemic, cardiac depressant, analgesic, anti-inflammatory, antipyretic, and antiviral properties.,,,,, Traditionally, whole plant and its parts were used in the treatment of various ailments. The roots were employed as a bitter stomachic, vermifuge, and as an ingredient in the remedy for itches. The roots were reported to contain salicylic acid and cardiac glycosides. It also contains carissone; D-glycoside of ß-sitosterol; glucosides of odoroside H; carindone; a terpenoid lupeol; ursolic acid and its methyl ester; also carinol, a phenolic lignan. Fruits contain good amount of vitamin C. The fruits, leaves, and bark are rich in tannins., It is useful in treatment of diarrhea, anorexia, and intermittent fevers. Fruits have also been studied for its analgesic, anti-inflammatory, and lipase 1 activities. It is used by tribal healers of Western Ghat region of Karnataka as hepatoprotective and antihyperglycemic, however, no scientific data are vailable to validate the folklore claim., Antimicrobial activities of Karanda fruits (5 mg/mL of 50% ethanol extract) against Staphylococcus aureus (ATCC 2593) and Escherichia More Details coli (ATCC 8739) were reported. Ethanol extract. Antioxidant activities of Karanda fruit extracts were relatively high when compared to other tropical fruits. In Thailand, Karanda fruits are favorite fruits especially in central region due to attractive shape and color with health promoting activities. Many local products are made from Karanda fruits, such as juice, jam, and desserts. However, concern for health promoting information and few anticancer activities of Karanda fruits are reported; this present study has been designed to evaluate its antimicrobial and anticancer activities. Furthermore, antimicrobial activities were few more in vitro antimicrobial activities that need to be tested, e.g., antituberculosis, antimalarial (Plasmodium falciparum), antiviral (herpes simplex type I), antifungal (Candida albicans), and in vitro screening for anticancer activity test against cell lines also interesting. Cytotoxicity of Karanda fruit extracts was also evaluated with normal cells to claim for health safety. Biological activities of Karanda fruits on ripping state as edible Karanda fruits were confirmed for health benefits and were discussing with previous studies.
| Materials and Methods|| |
Cells and chemicals
Cells: The anticancer activity and cytotoxicity tests were done in National Center for Genetic Engineering and Biotechnology, Thailand as the Laboratory service and cell lines and normal cells, including five cell lines, epidermoid carcinoma of oral cavity (KB) ATCC CCL-17, breast adenocarcinoma (MCF-7) ATCC HTB-22, small cell lung carcinoma (NCI-H187) ATCC CRL-5804, human hepatocarcinoma (HepG2) ATCC HB-8065, and human colon adenocarcinoma (Caco2) ATCC HTB-37; and two normal cells, Vero cells (African green monkey) and human dermal fibroblasts, neonatal (HDFn) C-004-5C. Other assayed pathogens were also provided and done in National Center for Genetic Engineering and Biotechnology, Thailand.
Chemicals: Dichloromethane, methanol, dimethyl sulfoxide (DMSO), and Folin–Ciocalteu reagent were purchased from Fluka (Singapore); gallic acid, resazurin, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) were purchased from Sigma-Aldrich (Germany); ellipticine, doxorubicin, tamoxifen, rifampicin, ofloxacin, isoniazid, ethambutol, dihydroartemisinin, acyclovir, and amphotericin B were purchased from Roche (Germany).
In this study, Amphawa Agricultural Office, Samut Songkhram, Thailand provided help to collect the sample Karanda fruits. Fresh ripped fruits (511 g) were selected, cut in small pieces, and then air dried. The air-dried Karanda fruit (48 g) was grinded in powder form and kept for screening by biological tests. Bring Karanda fruit powder for continuous extraction using soxhlet apparatus was extracted with hexane, then, extracted with dichloromethane and methanol, respectively. Finally, get the solvent evaporated through rotary evaporation apparatus under vacuum. The extracts could also be dissolved in DMSO and be test to the anticancer and antimicrobial activities onward.
Total phenolic content
In this step, 0.1 mL of 1 mg sample extract was input into the test tube, mixing with 4.6 mL distilled water and 1 mL of Folin–Ciocalteu reagent. After that, the extract was left inside the room in room temperature for 3 min. Next, 3 mL of 2% Na2CO3 (w/v) was filled into the tube and shaken with the speed of 150 rpm for 2 h. Then, the extract was measured to find out the light absorbance at 760 nm by comparing with the gallic acid at the intensity of 1, 0.875, 0.75, 0.625, 0.5, 0.375, 0.25, and 0.125 mg/mL. The total phenolic content (TPC) was calculated into mg of gallic acid per g of the extract.
Anticancer activity and cytotoxicity tests
A total of five cell lines, including epidermoid carcinoma of oral cavity (KB) ATCC CCL-17, breast adenocarcinoma (MCF-7) ATCC HTB-22, small cell lung carcinoma (NCI-H187) ATCC CRL-5804, human hepatocarcinoma (HepG2) ATCC HB-8065, and human colon adenocarcinoma (Caco2) ATCC HTB-37, were used in this study. The resazurin microtiter assay (REMA) developed by O'Brien et al. was performed for anticancer test. In brief, the cells were cultured in proper condition and diluted by culture medium at 2.2-3.3 x 104 cells/mL. The next step was to add the 50 μL of 5% DMSO into cell suspension and 45 μL in the 384-well plates. Then, the extract was incubated at 37°C in the incubator which contained 5% of CO2. After incubation (3–5 days), 12.5 μL of resazurin (62.5 μg/mL) was added. The incubation was continued for 4 h, then measured fluorescence signal by SpectraMax M5 multidetection microplate reader (Molecular Devices, USA) at excitation and emission wavelength of 530 and 590 nm, respectively. Dose–response curve could be done in the 6th test. Threefold serial intensity dilution and the intensity of the cell-restraint extract 50% (IC50) could be calculated by SoftMax Pro software (Molecular Devices, USA). Ellipticine, doxorubicin, and tamoxifen were used as positive control. A total of 0.5% DMSO and water were used as negative control. For cytotoxicity test, Vero cells (African green monkey) and human dermal fibroblasts, neonatal (HDFn) C-004-5C were used for evaluating cytotoxicity of Karanda fruit by same method.
Antimicrobial activity test
Antiherpes simplex virus type I (HSV-1) test
Before the test of antivirus activity, there should be the cytotoxicity test conducted first to make sure that the extract is noncytotoxic. The antivirus test was conducted by green fluorescent protein (GFP)-based assay. The extracts diluted by 10% DMSO at 10 μL/well were added into 96-well plates. Next, added green fluorescent protein-expressing Vero cell suspension 1 * 105 cells/mL mixed with HSV-1 (ATCC VR260) 5 * 105 PFU/mL for 190 μL/well. Then, the sample was incubated at 37°C by incubator which has 5% of CO2 for 4 days. After that, fluorescence signal was measured by SpectraMax M5 multidetection microplate reader (Molecular Devices, USA) at excitation and emission wavelength 485 and 535 nm, respectively (bottom-reading mode). Fluorescence signal from the 4th day of incubation will be deducted on the 1st day (day = 0) of incubation. The IC50 was calculated by SoftMax Pro Software (Molecular Devices, USA) from testing six levels of twofold serial dilution extracts. Acyclovir was used as positive control and 0.5% DMSO was used as negative control.
Anticandida albicans test
The test was performed by taking C. albicans yeast (ATCC 90028) to culture on potato dextrose agar plate at 30°C for 3 days. After that, 3–5 colony of yeast was taken to culture in shaking flask that had RPMI-1640 medium until the density was 5 * 105 colony forming unit(CFU) /mL. Next, the yeast cell suspension was brought to be tested in antiyeast activity by REMA. A total of 45 μ of cell suspension and 5 μL extracts from each density diluted by 0.5% DMSO were added into 384-well plates. The plate was cultured at 37°C for 4 days, then 10 μL/well of 62.5 μg/mL resazurin solvent was added and incubated for another 30 min. After that, fluorescence signal was measured by SpectraMax M5 multidetection microplate reader (Molecular Devices, USA) at excitation and emission wavelength 530 and 590 nm, respectively. The IC50 was calculated by SoftMax Pro Software (Molecular Devices, USA) from testing six levels of twofold serial dilution extracts. Amphotericin B was used as positive control and 0.5% DMSO was used as negative control.
Antimalarial activity test
P. falciparum (K1, multidrug-resistant strain) was cultured in the test tube (in vitro), developed by Trager and Jensen method. It was cultured by RPMI 1640 medium which had 20 mM of HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 32 mM of NaHCO3, and 10% of heat inactivated human serum with 3% of erythrocyte mixed together. Then, it was incubated at 37°C by 3% CO2 in CO2 incubator. The culture medium and erythrocyte were changed every day during the test. The evaluation of in vitro antimalarial test was performed by microculture radioisotope techniques. And 200 µL mixture which has 1.5% of erythrocyte infected by 1% of malaria (1% parasitemia). In early ring stage, it was mixed with 25 µL of medium that mixed with sample extract in each density distilled by 1% DMSO (the total was 0.1% DMSO). After that, the sample was incubated for 24 h. Then, 25 µL of [3H] hypoxanthine (Amersham, USA) would be added into medium (0.5 µCi) in each plate and incubated again for another 24 h. Radioactive labeled on hypoxanthine indicates the growth of cell. TopCount microplate scintillation counter (Packard, USA) was used to find out the radioactive volume. And 50% inhibition concentration (IC50) could tell that the cell development was reduced to 50%. This experiment used 1 and 10 g/mL extract to prevent P. falciparum to calculate the IC50. Dihydroartemisinin and 0.1% DMSO was used as positive control and negative control, respectively.
GFP expressing Mycobacterium tuberculosis H37Ra strain [H37Ra gfp] culture was developed by Changsen et al. and Collins et al. H37Ra gfp was cultured on plate 7H10 agar consisting of kanamycin 30 μg/mL. The incubation at 37°C was 4 week long. After that, the single colony of the cell was taken to culture on 7H9 broth which had 0.2% of glycerol v/v, 0.1% of casitone w/v, 0.05% of Tween 80 v/v, 10% of Middlebrook OADC enrichment solution (BD Biosciences, India) v/v, and 30 μg/mL of kanamycin. All substances were incubated at 37°C in 200 rpm shaker incubator until the 550 nm optical density was around 0.5–1.For batch cultivation, 1/10 of the ingredient above were taken to incubate at the same condition. Then, the cells were cleansed and suspended by phosphate buffered saline buffer and then were sonicated eight times (15 s per time). The cultures were divided into tubes and kept at -80°C for 2–3 months before experiment session. During test session, the cells were tested their density in 384-well plates at around 1 x 105 CFU/mL/well. The tests were took four times (quadruplicate), or within four wells/each test. Each testing plate contained 5 μL of 0.5% DMSO (diluted by serial dilution) and 45 μL cell suspension. The plate was incubated at 37°C for 10 days. To find out the fluorescence signal, I used SpectraMax M5 multidetection microplate reader (Molecular Devices, USA) with excitation and emission wavelength 485 and 535 nm, respectively (bottom-reading mode). The fluorescence signal on 10th incubation day were deducted on = 0 of incubation. It could be calculated in MIC by rifampicin, ofloxacin, isoniazid, and ethambutol as positive control and 0.5% DMSO as negative control.
| Results|| |
It was found that Karanda fruits extracted with dichloromethane (KD) contained crude extract lower than Karanda fruits extracted with methanol (KM) by concentration = 0.054 ± 0.001 and 0.098 ± 0.001 mg of gallic acid equivalent/g of extract, respectively which was related with the TPC. KD and KM extracts were not possessed anticancer activity to KB-oral cavity cancer, MCF7-breast cancer, NCI-H187-small lung cancer, HepG2-hepatocarcinoma, and Caco2-colon adenocarcinoma cell lines at maximum concentration = 100 µg/mL [Table 1]. In the same way to anticancer activities, both extracts were not significantly inhibited HSV-1, C. albicans, P. falciparum, K1 strain, and M. tuberculosis H37Ra strain at maximum concentration of test (50 μg/mL) [Table 2]. In addition, no any cytotoxic effect of KD and KM extracts to Vero cells and HDFn-neonatal dermal fibroblast [Table 1], which implied that both extract were not harm human normal cells and also human cancer cell lines.
|Table 1: Cytotoxic effect of KD and KM against KB, MCF7, NCI-H187, HepG2, and Caco2 cell linesh|
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|Table 2: Antimicrobial of KD and KM against HSV-1, M. tuberculosis, C. albicans, and P. falciparume|
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| Discussion|| |
The phytochemical screening of KD and KM revealed the presence of phenolic compounds. This finding correspond to previous study by Mamun et al. that report high amount of TPC contained in Karanda fruit extract (ethanol extract) and antioxidant activity was also relatively high (4.882 μmol Trolox equivalent/g) when compared with other tropical fruits and concluded that this fruit contained potent phenolic compounds. This present study revealed lack of anticancer activities of KD and KM (maximum concentration = 100 μg/mL) against KB, MCF7, NCI-H187, HepG2, and Caco2 cell lines, and no cytotoxicity to normal cell (Vero and HDFn cells). However, rare anticancer activity of Karanda fruits was reported in literature review to compare and discuss with the results. Thus, there may conclude that KD and KM had no cytotoxic effect with cancer cell lines and normal cells. In case of antimicrobial activities, this study showed negative results of KD and KM along with various pathogens, including HSV-1, C. albicans, P. falciparum, K1 strain, and M. tuberculosis H37Ra strain at preferable concentration (50 μg/mL). Previous study reported antibacterial activities of Karanda fruit extracted by ethanol, which revealed antimicrobial activities against S. aureus (ATCC 2593) and E. coli (ATCC 8739) at 5 mg/mL. When compared, the concentration of Karanda extract between previous (5 mg/mL) and present studies (50 μg/mL) were evaluated for antimicrobial assays, which were too different about 100 times. At this concentration (mg/mL), this study was difficult to dissolve in all in vitro assays (in presented of DMSO) and it means that KD and KM did not possess anticancer and antimicrobial activities at preferable concentration (maximum conc. = 100 and 50 μg/mL, respectively). The concentration unit of plant extract (50–100 μg/mL) was the common maximum concentration used to “cut off” for the significant of biological activity, which was referred by the National Center for Genetic Engineering and Biotechnology, Thailand.
The previous studies revealed cytotoxicity of unripe fruits, fully-ripe fruits, and leaves of Karanda extracts (40% of ethanol) inhibited HepG2 cells  and cytotoxicity of leaf extract inhibit the proliferation of human cervical cancer cells (HeLa), prostate cancer cells (PC-3), and normal mouse fibroblasts (3T3). Hence, there concluded that cytotoxicity of leaf extract was highest at 100 μg/mL; however, fruits (unripe and ripe) lack cytotoxicity to inhibit HepG2 cells at same concentration and increase the dose of assay up to 200 μg/mL. They may imply that KD and KM had no cytotoxic effect with normal cells same concentration as reported previously (100 μg/mL); however, they may be cytotoxic when increase the concentration of extract. Thus, further studies will be conducted to determine the response of normal cells, as well as cell lines and pathogens, with higher concentrations of the extract should be considered. The different results may due to solubility of extract and type of assay. Variation of plant harvest is also important to concern that Karanda fruits were in unripe or ripe stages. The result interpretation needs to consider, such as method of biological assay, solubility of extract, and assay solution, units of extract and cut-off of method. Lack of in vitro anticancer and antimicrobial activities from Karanda fruit in this study represented that there was insufficient effects for treatment; however, they might prevent or relieve effects. The present study will be helpful to avoid any study repeated in this direction in the future.
| Conclusion|| |
Dichloromethane and ethanol extracts of ripping Karanda fruits had no significant anticancer activities against to KB, MCF7, NCI-H187, HepG2, and Caco2 cell lines, and also no cytotoxicity to normal cell (Vero and HDFn cells), which tested resazurin by microplate assay. Both extracts had no significant antimicrobial activities against to HSV-1 and M. tuberculosis were tested according to GFP-based assay; C. albicans (ATCC 90028) were tested according by resazurin microplate assay; and P. falciparum, K1 strain was tested according to microculture radioisotope techniques.
The author would like to express their gratitude to Research and Development Institute of Suan Sunandha Rajabhat University, Bangkok, Thailand for the funding support. And he is also grateful to Faculty of Science and Technology, Saun Sunandha Rajabhat University, National Center for Genetic Engineering and Biotechnology for research facility service support.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]