|Year : 2018 | Volume
| Issue : 1 | Page : 33-38
Inappropriate of In vitro antimicrobial and anticancer activities from cashew (Anacardium occidentale L.) nut shell extracts
Yuttana Sudjaroen1, Kanittada Thongkao1, Kowit Suwannahong2
1 Department of Applied Science, Faculty of Science and Technology, Suan Sunandha Rajabhat University, Bangkok, Thailand
2 Department of Occupational Health, Safety and Environment, Faculty of Public Health, Western University, Kanchanaburi, Thailand
|Date of Web Publication||21-Aug-2018|
Faculty of Science and Technology, Suan Sunandha Rajabhat University, 1 U-Thong-Nok Rd, Dusit, Bangkok 10300
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: Cashew nut is one of the major agriculture products in Southern of Thailand, and the volume of cashew nut shell is become increase as a by-product. Thus, cashew nut shell is attractive source for evaluation of antimicrobial activities as inexpensive source. This study was aimed to evaluates in vitro antimicrobial properties of the water extract (CW) and ethanol extract (CE) from cashew nut shell and to evaluate anticancer activity and cytotoxicity of extracts by in vitro screening test against cell lines and normal mammalian cells, respectively. Materials and Methods: CW and CE extracts were determined total phenolic compound by Folin-Ciocalteu reagent. Antibacterial activity of CW and CE against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii were evaluated by resazurin microplate assay (REMA). Antimicrobial activities of both extracts against herpes simplex virus Type I and Mycobacterium tuberculosis were tested according by green fluorescent protein (GFP)-based assay; Candida albicans was tested according by REMA; and Plasmodium falciparum was tested according by microculture radioisotope techniques. Anticancer activities of CW and CE were tested with MCF7, NCI-H187, HepG2-, and Caco2 cell lines; and cytotoxicity test was used Vero and human dermal fibroblasts, neonatal (HDFn) cells. Results: CW and CE were lack of in vitro antibacterial (maximum concentration = 100 μg/ml), antimicrobial, and anticancer activities (maximum concentration = 50 μg/ml). CW and CE (0-50 μg/ml) were lack of cytotoxicity against Vero cells and HDFn-neonatal dermal fibroblast. Conclusions: CW and CE extracts of cashew nut shell had no significant in vitro anticancer and antimicrobial activities with noncytotoxicity.
Keywords: Anacardium occidentale L, anticancer, antimicrobial, cashew nut shell, cytotoxicity
|How to cite this article:|
Sudjaroen Y, Thongkao K, Suwannahong K. Inappropriate of In vitro antimicrobial and anticancer activities from cashew (Anacardium occidentale L.) nut shell extracts. J Pharm Negative Results 2018;9:33-8
|How to cite this URL:|
Sudjaroen Y, Thongkao K, Suwannahong K. Inappropriate of In vitro antimicrobial and anticancer activities from cashew (Anacardium occidentale L.) nut shell extracts. J Pharm Negative Results [serial online] 2018 [cited 2019 Feb 17];9:33-8. Available from: http://www.pnrjournal.com/text.asp?2018/9/1/33/239511
| Introduction|| |
Resistance to antibiotics in numerous pathogens has become a real public health problem; it concerns specific pathogens causing diseases such as tuberculosis but also opportunistic pathogens involved in nosocomial infections.,,, Recently, the WHO expressed urgent need for the development of new antibacterial compounds. However, the number of new drugs in development is low, raising the question for alternative research. As plants have evolved since hundreds of millions of years in the presence of bacteria and develop rarely bacterial diseases, this provides a rational basis for the search for vegetal antibacterial compounds. In addition, the long-standing empirical use of herbal medicine against infectious ailments is consistent with the previous observation. At this point, natural products have garnered substantial interest as lead sources for identification of new pharmaceutical agents.
Cashew (Anacardium occidentale L.) is well-known species which belongs to the Anacardiaceae family. Currently, Brazil, India, Vietnam, Tanzania, and also Thailand are the main producers of cashew nuts. Southern Thailand is located in tropical zone and characterized by high humidity and rain throughout the year, which is the major region of cashew nut production., Different parts of the cashew tree have traditionally been used across the world to treat various diseases. Leaves or nut shell extracts from cashew, commonly known as the cashew tree, have long been used to treat inflammation and other conditions including asthma, ulcers, and cancer., Although the efficacy of these compounds for treating such disorders has not been established in controlled trials, the major component of cashew nut shell extract, anacardic acid, has been shown to exert a variety of effects on both prokaryotic and eukaryotic cells., Anacardic acid is a blanket term applied to a family of closely related compounds consisting of salicylic acid with a 15-carbon alkyl chain, which exist either in a fully saturated form or as a monoene, diene, or triene. Anacardic acid has been shown to exhibit direct antimicrobial activity against a number of bacterial species including Propionibacterium acnes, Staphylococcus aureus, and Helicobacter pylori.,,
Cashew nut is one of the major agriculture products in Southern of Thailand, especially in Ranong province, and the volume of cashew nut shell is become increase as by-product. Cashew nut shell liquid (CNSL), the reddish brown viscous liquid extracted from the pericarp of the cashew nut, is the major by-product of the cashew nut industry and has numerous medicinal and industrial applications. Its major constituents include anacardic acid, cardol, cardanol, and methyl cardol  and the ratio of these components varies based on the method of extraction. Thus, cashew nut shell is attractive source for evaluation of antimicrobial activities as inexpensive source and simple extraction for local use were focused. Hence, this study was undertaken (1) To study on in vitro antimicrobial properties of the water and ethanol extracts from cashew nut shell against Gram-negative bacteria (including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii), yeast (Candida albicans), Mycobacterium tuberculosis, malaria (Plasmodium falciparum), and herpes simplex virus Type I (HSV-1); (2) to evaluate anticancer activity of cashew nut shell by in vitro screening test against cell lines and cytotoxicity was also evaluated with normal mammalian cells to claim for health safety.
| Materials and Methods|| |
Cells and chemicals
The anticancer activity and cytotoxicity tests were done from National Center for Genetic Engineering and Biotechnology, Thailand as the Laboratory service and cell lines and normal cells including four cell lines, 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 (Kidney epithelial cells from African green monkey), and human dermal fibroblasts, neonatal (HDFn) C-004-5C. Other assayed pathogens were also provided and done from National Center for Genetic Engineering and Biotechnology, Thailand.
dichloromethane, methanol, dimethyl sulfoxide (DMSO), and Folin-Ciocalteu reagent were purchased from Fluka (Singapore); gallic acid, resazurin, and N-2-hydroxyeth ylpiperazine-N'-2-ethanesulfonic acid (HEPES) were purchased from Sigma-Aldrich (Germany); ellipticine, doxorubicin, tamoxifen, rifampicin, ofloxacin, isoniazid, ethambutol, dihydroartemisinine (DHA), acyclovir, and amphotericin B were purchased from Roche (Germany).
In this study, expert local agriculturalist who living Suan Sunandha Rajabhat University, Ranong campus, Thailand, was provided cashew nut shell. Fresh cashew nut shell (1 kg) was cleaned, cut in small pieces, and then air dried. The air-dried nut shell (780 g) was grinded in powder form. Shell powder was macerated with water and with 95% ethanol by 1:10 (W/V) during 3 days. Each extract solution was evaporated through rotary evaporation apparatus under vacuum in constant weight and kept for a biological test screening. The extracts were dissolved in DMSO for biological assays 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% Na2 CO3 (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 was calculated into mg of gallic acid per grams of the extract.
Antimicrobial activity tests
Antibacterial activity test
Four Gram-negative bacteria, E. coli(ATCC 25922), K. pneumoniae (ATCC 70063), P. aeruginosa strain PAO1, and Acinetobacter baumannii (ATCC 19606) were used as tested organisms. They were cultured by streaking on to medium called Tryptic Soy Agar. The culturing lasted 1-day incubation at 37°C. Next, the single colony was cultured in tryptic soy broth (TSB) for 5 mL and incubated at 37°C in 200 rpm shaker incubator for 30 min (Optical density at 600 nm, OD600~ 0.1). Activated cultures were diluted for 200 times. The 20 mL TSB medium was incubated at 37°C in 200 rpm shaker incubator for another 3 h (OD600~ 0.4–0.5). After that, the test to find out antimicrobial activity continued adding 7.5 μL extract, 25 μL of resazurin 0.25 mM, and bacterial cell suspension until the final volume. Furthermore, 75 μL (~15,000 cells) in Mueller-Hinton broth was put onto 384-well plate. The sample was taken to incubate at 37°C for 2 h, following by measuring signal by SpectraMax M5 multi-detection microplate reader (Molecular Devices, USA) at excitation and emission wavelength of 530 and 590 nm. Amikacin and ofloxacin were used as positive control and 0.5% DMSO used as negative control.
Anti-herpes simplex virus Type I 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 plate. Next, added GFP-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 inhibition concentration (IC50) was calculated by SOFTMax ® Pro software (Molecular Devices, California, USA) from testing six levels of 2-fold serial dilution extracts. Acyclovir was used as positive control and 0.5% DMSO was used as negative control.
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 CFU/ml. Next, the yeast cell suspension was brought to be tested in anti-yeast activity by resazurin microplate assay (REMA). 45 μ of cell suspension and 5 μl extracts from each density diluted by 0.5% DMSO were added into 384-well plate. The plate was cultured at 37°C for 4 days, then 10 μl/well of 62.5 μg/ml resazurin solvent were 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 6 levels of 2-fold 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, 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. 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. After that, 25 μl of (3 H) 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. Top Count microplate scintillation counter (Packard, USA) was used to find out the radioactive volume. IC50 could tell that the cell development was reduced to 50%. This experiment used 1 and 10 μg/ml extract to prevent P. falciparum to calculate the IC50. DHA and 0.1% DMSO were used as positive control and negative control, respectively.
Green Fluorescent Protein (GFP) expressing M. tuberculosis H37 Ra strain (H37 Ra gfp) culture was developed by Changsen et al. and Collins et al. H37 Ra gfp was cultured on plate 7H10 agar consisting of kanamycin 30 μg/ml. The incubation at 37°C was 4 weeks 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 Middle brook OADC enrichment solution (BD Biosciences) 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 was taken to incubate at the same condition. Then, the cells were cleansed and suspended by PBS buffer, and then were sonicated eight times (15 s/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 plate at around 1 × 105 CFU/ml/well. The tests took four times (quadruplicate) or within 4 wells/test. Each testing plate was 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, we were 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 was deducted by starting signal of incubation period. It could be calculated in minimal inhibitory concentration (MIC) by rifampicin, ofloxacin, isoniazid, and ethambutol as positive control and 0.5% DMSO as negative control.
Anticancer activity and cytotoxicity tests
Four cell lines including 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 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 × 104 cells/ml. The next step was to add the 5% DMSO 50 μl into cell suspension 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. Three-fold 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. 0.5% DMSO and water were used as negative control. For cytotoxicity test, Vero cells and HDFn C-004-5C were used for evaluated cytotoxicity of cashew shell extracts by the same method.
| Results|| |
The yield of water and ethanol extractions and total phenolic content from cashew nut shell were 39.1 ± 1.1 and 24.6 ± 1.8 g/100 g of dry weight and 180.15 ± 9.47 and 125.6 ± 8.4 mg of gallic acid equivalent/gram, respectively. Maximum concentration (100 μg/ml) of water (CW) and ethanol (CE) extracts were lack of in vitro antibacterial activity against four Gram-negative bacteria including E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii and unable to calculate 90% of MIC [Table 1]. Moreover, CW and CE (maximum concentration = 50 μg/ml) were lack of in vitro antimicrobial activity against to HSV-1 virus, M. tuberculosis, C. albicans, and P. falciparum [Table 2] and also were lack of anticancer activity for MCF7 breast cancer, NCI-H187-small lung cancer, HepG2-hepatocarcinoma, and Caco2-colon adenocarcinoma cell lines. In addition, there were lacks of cytotoxicity in present of to Vero and HDFn cells [Table 3].
|Table 1: The antibacterial activity of water and ethanol extracts of cashew nut shell against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumanniia,b|
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|Table 2: Antimicrobial activity of water and ethanol extracts of cashew nut shell against herpes simplex virus Type I, Mycobacterium tuberculosis, Candida albicans, and Plasmodium falciparuma,b|
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|Table 3: Cytotoxicity (%) of water and ethanol extracts of cashew nut shell against KMCF7, NCI-H187, and Caco2 cell lines; and HDFn and Vero mammalian cellsh|
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| Discussion|| |
The use of an abundant and cheap source of natural waste products was according to the concept of green chemistry and environmental friendly safe and also in case of antibiotics and folk medicinal uses. Leaves or nut shell extracts from cashew have long been folk medicinal used to treat inflammation and other conditions including asthma, ulcers, and cancer. The enduring research and emerging evidence suggests that anacardic acid could be a potent target molecule with bactericide, fungicide, insecticide, antitermite, and molluscicide properties and as a therapeutic agent in the treatment of the most serious pathophysiological disorders such as cancer, oxidative damage, inflammation, and obesity. Furthermore, anacardic acid was found to be a common inhibitor of several clinically targeted enzymes such as NFκB kinase, histone acetyltransferase (HATs), lipoxygenase-1, xanthine oxidase, tyrosinase, and ureases.
The high content of phenolic compounds in cashew nut shell was corresponding to various reports for its antioxidant properties from previous studies.,,,,, The amount of phenolic content and antioxidant activity are depended on agricultural and/or industrial processes. Anacardic acid is most of active compound, which exhibit bacterial species, including P. acnes, S. aureus, H. pylori, and Bacillus subtilis.,,, However, in our study, the CW and CE were inappropriate antibacterial activity for four Gram-negative bacteria. We were note that the phenolic compounds from cashew nut shell are commonly effective to Gram-positive bacteria rather than Gram-negative.,,, Lack of in vitro antimicrobial activity of CW and CE against to HSV-1 virus, M. tuberculosis, C. albicans, and P. falciparum were reported in our study. In contrast, inhibit of hepatitis C virus from anacardic acid had been reported due to HATs inhibition with dose specifically on anacardic acid (>5 μM) and prolonged administration (12 h). Lack of in vitro anticancer activity for MCF7-breast cancer, NCI-H187-small lung cancer, HepG2-hepatocarcinoma, and Caco2-colon adenocarcinoma cell lines were also found in our results. However, antitumor effects of anacardic acid had been reported by modulating the nuclear factor-κB and it is exhibit cytotoxic effect to lung, liver, and gastric tumor cells through epigenetic mechanisms by HATs. Moreover, anacardic acid can be potentiating apoptosis induced by tumor necrotic factor. In previous study, toxicity testing of anacardic acids in the CNSL had measured the acute, subacute, and mutagenic effects of anacardic acid administration in BALB/c mice and doses <300 mg/kg did not produce biochemical and hematological alterations in BALB/c mice. In addition, CNSL had been reported sustainable and environmental safe plant-based larvicide on larvae of Aedes aegypti.,,
Due to lack of antimicrobial activity and mammalian cell toxicity from our results and previous studies, cashew nut shell waste may apply for food additives used, as well as, to reduce or diminish agricultural waste production. Maximum concentration of CW and CE was partial dissolving in all in vitro assay environments and it was implied that both extracts were not possessed anticancer and antimicrobial activities at preferable concentration. The common maximum concentration in our study was used to “cutoff” for the significant of biological activity, which was referred by the National Centre for Genetic Engineering and Biotechnology, Thailand., Hence, the target evaluation, appropriate dose, and duration of administration on bioassays are need to awareness. In addition, extraction protocols and cutoff point are also need to consider., Lack of in vitro antimicrobial and anticancer activities from CW and CE on this study was represented that there was insufficient effects for treatment; however, there might prevent or relieve effects. The present study will be helpful to avoid any study repeated in this direction in the future.
| Conclusions|| |
Lack of in vitro antibacterial activity of CW and CE (up to 100 μg/ml) against four Gram-negative bacteria (including E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii) were reported. Lack of antimicrobial and anticancer activities of CW and CE (up to 50 μg/ml) against HSV-1 virus, M. tuberculosis, C. albicans, and P. falciparum and for MCF7, NCI-H187, HepG2, and Caco2-cell lines were also reported with no toxicity to mammalian cells (Vero and HDFn cells).
The researcher would like to express their gratitude to Research and Development Institute of Suan Sunandha Rajabhat University, Bangkok, Thailand, for the funding support. I am 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], [Table 3]