Sidika Genc1, Yesim Yeni1, Ahmet Hacımüftüoğlu1 and Ali Taghizadehghalehjoughi1,2,*
1Department of Medical Pharmacology, Faculty of Medicine,Ataturk University, Turkey 2Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Ataturk University, Turkey
*Corresponding Author: Ali Taghizadehghalehjoughi, Department of Medical Pharmacology, Faculty of Medicine,Ataturk University, Turkey.
Received:March 01, 2021; Published: : April 14, 2021
Coronavirus disease 2019 (COVID-19) caused by acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a major threat to global health. The rapid spread of the epidemic around the world has caused it to become a global pandemic.Disinfection is one of the best practice measures used to eradicate viral/bacterial pathogens in homes and community settings and reduces the spread of infection. However, it has been shown in studies that the long-term use of degenerate agents with different pH values triggers the development of itching, rash, urticaria and even allergic rhinitis. Graviola (Annona muricata) and its fruits, leaves, stems and roots are known to be rich in flavonoids and isoquinoline alkaloids. It has been used for thousands of years to treat many diseases from arthritis to liver problems. It is also used to treat a wide variety of human diseases such as inflammation, rheumatism, diabetes, hypertension, insomnia, parasitic infections and cancer. The aim of this study is to prevent toxicity that develops against different pH changes with the use of Graviola plant. For this reason, Human Fibroblast cell line was grown under the conditions specified by the manufacturer and inoculated into 96 well plates and culture medium with different pH (5 - 6 - 7 - 8) was prepared and different doses of Graviola (20-40-80-160 µgr/ml) were prepared. It is planned to eliminate the toxicity caused by using it for 24 hours. At the end of the study, cell viability was determined by performing MTT analysis and LDH and GR. According to the results of our study, cell viability increased from %71 to 132 compared to positive control at pH 5. The vitality that decreased to %79 at 6 pH increased to 132 as a result of the application of the highest concentration of Graviola (160 µgr/ml). While it increased from % 71 to %74 at pH 7, the damage received by fibroblast cells at pH 8 was determined to be high (viability rate %50) and by removing this toxicity, it was observed that the vitality was increased up to %75. Considering the results obtained, Graviola is recommended to be used at doses of 80 and 160 µgr/ml, as it has a protective effect. The LDH and GR results show correlation with MTT.
Keywords: Graviola; Covid 19; pH; MTT; LDH
SARS-CoV-2: Coronavirus-2; COVID-19: Coronavirus Disease 2019; MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; LDH: Lactate Dehydrogenase; GR: Glutathione Reductase; WHO: World Health Organization; GST: Glutatyon-transferaz; CAT: Chloramphenicol Acetyltransferase; SOD: Superoxide Dismutase
The coronavirus disease 2019 outbreak (COVID-19), which emerged in Wuhan, China in December 2019, was declared by the World Health Organization (WHO 2020b) as an emergency case of international importance and threatening public health. WHO has announced the official name of the virus as "COVID-19 virus" previously known as "2019-nCoV" or "Wuhan Coronavirus" . In the first three months of the epidemic, this epidemic spread rapidly across the country and the world and became a global epidemic. The COVID-19 pandemic has shaken the entire scientific community, and researchers around the world have turned to work to devise various intervention strategies to treat and prevent the disease. There are two main routes of transmission of SARS-CoV-2: infected respiratory droplets and transmission by contact . Current evidence suggests that droplets containing SARS-CoV-2 can survive for hours on surfaces made of various materials  in temperature ranges from 4°C to -70°C (4°C to -70°C) . For this reason, the use of disinfectants has gained importance and the use of degenerates with different chemical formulations has become widespread.
Disinfection is one of the best practice measures used to eradicate viral/bacterial pathogens in homes and community settings and reduces the spread of infection. Therefore, as COVID-19 spread around the world, the use of disinfectants has increased at the same rate. However, its use is thought to cause global secondary disasters in human health and ecosystems. These chemicals, when used regularly, increase the risk of chronic obstructive pulmonary disease (COPD), asthma and eye irritation in healthcare workers and individuals [5-7]. Chemical residues left on a surface can be airborne and inhaled, and often cause reactions in asthmatic, allergic or sensitive individuals. These residues have been shown to cause cancer, reproductive disorders, respiratory disorders (including occupational asthma), skin reactions with symptoms such as eye and skin itching, rash, urticaria, central nervous system (CNS) deterioration, and oxidative damage .
Herbal treatments are traditionally used in many parts of the world. Annona muricata (also known as Graviola) is a tropical fruiting tree of the Annonaceaefamily found in the rainforests of Africa, South America and Southeast Asia [9,10]. Its leaves, pericarp, fruits, seeds and roots are widely used in traditional medicine. Different parts of A. muricata have been used in the treatment of diabetes [11,12], cough, cancer [13,14], arthritis , hypertension , skin diseasesand malaria . In addition, there are studies showing that it is used as antimicrobial , antidiabetic , anti-inflammatory , antiprotozoan, antioxidant , insecticide, larvicideand anticancer .
The aim of this study is to prevent the toxicity of the fibroblast cell line with the use of Graviola against different pH changes caused by increased disinfectant use with Covid 19.
It was obtained from GraviolaSolgar (U.K). All chemicals derived from Dulbecco Modified Eagles Medium (DMEM), Fetal bovine serum (FBS), phosphate buffer solution (PBS), antibiotic antimitotic solution (Penicillin/Streptomycin/Amphotericin B) (100 ×), L glutamine and trypsin-EDTA has been. HCl and NaOH were obtained from Sigma Aldrich (St. Louis, MO, USA).Cell culture
For the study, a fibroblast cell line, Ataturk University (Erzurum, Turkey) were obtained from medical pharmacology department. Briefly, the cell suspension was centrifuged at 1200 rpm for 5 minutes. Cells were resuspended in fresh medium DMEM, %10 FBS and %1 antibiotic (penicillin, streptomycin and amphotericin B) and cells were collected in a 25cm2 flask. (Corning, USA) and stored in incubator (%5 CO2; 37°C). When %80 of the flask was covered with the cell, it was removed with Trypsin-Ethylene di amine tetra acetic acid (EDTA) (%0.25 trypsin-%0.02 EDTA) and planted in 96 well plates to be centrifuged.PH toxicity
DMEM medium was prepared as full medium and added to the cells by adjusting various pH values (5, 6, 7 and 8) with the use of HCl and NaOH by pH meter.Graviolaapplication
After the cells reached %85 confluence, graviola of different concentration (20, 40, 80 and 160 µg/ml) was added to the corresponding wells and the plates were left in the incubator for 24 hours.MTT test
At the end of the experiment (after 24 hours of treatment) 10 μL of MTT solution was added to each well plate (1 mM final concentration). Plates were then incubated for 4 hours at 37°C in a CO2 incubator. After 4 hours, 100 µL of DMSO solution was added to each well to dissolve the formazan crystals. The density of the formazan crystals was read by the Multiskan™ GO Microplate Spectrophotometer reader at a wavelength of 570 nm [25-27].LDH (Lactate Dehydrogenase) assay
LDH assay test was performed using a commercially available test kit from Cayman Chemical Co. Ltd, (Ann Arbor, MI, USA). Briefly, the cell culture medium was centrifuged at 400g for 5 minutes at the room temperature 100 µl of the supernatant was added to 100 µl of the reaction solution (LDH Assay Buffer, LDH Substrate Mix) and incubated with gentle shaking on an orbital shaker for 30 min at room temperature. Finally, the absorbance was read at 490 nm wavelength [28,29].GR (Glutathione Reductase) assay
In the activity measurement of the GR enzyme, the principle that the reacted NADPH gives maximum absorbance at 450 nm was used. The GR enzyme causes a decrease in NADPH in the reaction it catalyzes. Enzyme activity was determined by following this reduction spectrophotometrically at 450 nm .Statistical analysis
Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey's HSD for posthoc comparisons using SPSS 22.0 software. P<0.05 was accepted as the statistical threshold for each analysis.
MTT analysis results are shown in figure 1. Viability percentages of all groups were calculated by comparing with the positive control (pH application only) values and the control values were set as %100 viability. The lowest effect was seen in the Graviola (20 µg/ml) group among the treatment groups compared to the PH control groups (P>0.05). In addition, it was observed that the survival rate in the Graviola (20-40-80 and 160 µg/ml) groups increased depending on the concentration (Figure 1). According to the results of our study, cell viability increased from %71 to 131 compared to positive control at pH 5. The vitality that decreased to %79 at 6 pH increased to 132 as a result of the application of the highest concentration of Graviola (160 µg/ml). While it increased from %71 to %74 at pH 7, the damage received by fibroblast cells at pH 8 was determined to be high (viability rate %50) and it was observed that this toxicity was removed and the vitality was increased up to %83 (P<0.05).
Figure 1:Different PH concentrations and Graviola treatments effects on fibroblast cells viability ratio *P<0.05, **P<0.01.
Damage to the cell membranes is reflected as elevated LDH levels in the cell medium after the cells were exposed to Graviola (20- 40- 80 and 160 µg/ml) for 24 h. The LDH activity of the control group was defined as %100, and the other groups were rated accordingly. Our results show that pH 8 was most toxic as indicated by the greatest amount of LDH activity in the media from the fibroblast cells in comparison to the other pH group. Figure 2 shows that combinations of Graviola treatment group at all pH parameters reduce cytotoxicity in fibroblast cells in a time- and dose-dependent manner.Also, the high concentration Graviola groups (80 and 160 µg/ml) differed statistically from the PH control group (P<0.05). A linear correlation was observed between LDH activity and cell viability.
Figure 2:LDH activity (AU) was measured and the result proportioned to control group. *P < 0.05.
Glutathione reductase levels in the cell medium after the cells were exposed to Graviola (20-40-80 and 160 µg/ml) was evaluated for 24h. The GR activity of the control group was defined as %100, and the other groups were rated accordingly. Figure 3 shows that combinations of Graviola treatment group at all pH parameters increased GR level in fibroblast cells.Also, the high concentration Graviola groups (80 and 160 µg/ml) differed statistically from the PH control group (P<0.05 and P<0.001).
Various studies have shown that A. muricata has chemical components of different alkaloids and essential oils [31,32]. However, species belonging to the Annonaceae family, including A. muricata, have various acetogenin compounds that act as the main bioactive compounds .Studies have shown that Annona species have wound healing capacity. An in vivo study on the wound-healing activity of Graviola trunk bark revealed a significant reduction in the wound area after topical treatment with ethanol extract of the root bark . In another study, Annona squamosa leaves showed a promising wound-healing effect against streptozotocin-induced diabetic rats . Given the wound healing activity of graviola extract, the wound healing process is thought to be accompanied by skin ischemia, which promotes the formation of reactive oxygen species by activated leukocytes in the tissue region.
Prolonged exposure to disinfectant agents may cause skin damage or hypersensitivity. Such exposures can cause immediate or delayed skin reactions. Highly concentrated solutions have caused severe chemical skin burns. The long-term use of these agents is thought to result in greater release of oxygen-derived free radicals and increased tissue oxidative damage .
Under normal conditions, body homeostasis balances the level of free radicals by utilizing the human body's endogenous antioxidant capacity. However, when this level exceeds the normal balancing capacity of antioxidants, highly activated radicals will cause different structural changes and cell damage[37,38]. The cells' antioxidant defense systems contain a variety of enzymatic and non-enzymatic scavengers. Enzymatic antioxidants of cells such as GR, glutathione-transferase (GST), chloramphenicol acetyltransferase (CAT), Superoxide Dismutase (SOD) play a critical role in weakening oxidative stress caused by reactive oxygen species. The first defense mechanism against reactive oxygen species is provided by the GR, which reduces oxidative stress through the reduction of NADPH. The antioxidant activity of Graviola leaves has also been reported in previous in vitro and in vivo studies [39,40].
Among the different Annona species, Graviola leaves were found to have the highest antioxidant activity assessed by FRAP (Ferric reducing antioxidant property), DRSA (DPPH radical scavenging activity) and HRSA (hydroxyl scavenging activity) techniques .An in vivo study also showed that ethyl acetate extract of Graviola leaves caused an increase in CAT, glutathione and SOD activity on stomach cells.In our in vitro study, it was observed that Graviola concentrations increased fibroblast proliferation in a dose-dependent manner, and also increased antioxidant levels by eliminating oxidative damage. After treatment with Graviola for 24 hours against toxicity applied at different PH, it was observed that the high concentration of Graviola extract (160 µg/ml) at pH 5 and 6 had a statistically significant positive effect on enhancing cellular viability and alleviating oxidative stress.
Today, the rapidly spreading coronavirus is a great danger threatening humanity. Despite the rapid development of vaccine and drug studies, disinfectants continue to be a part of our lives. If the use of disinfectants destroys viral agents, it also damages our skin cells. According to our results, skin cells suffer great damage during PH change. According to our study, Graviola has a strong protective effect on PH between 6 – 8 damage induction. Therefore, we recommend the use of Graviola after hand disinfections.
Citation: : Ali Taghizadehghalehjoughi., et al. “Investigation of Protective Effects of Graviola (Annona muricata) Plant against Disinfectants Used in Different PHs in Covid-19 Process". Acta Scientific Pharmaceutical Science 5.5 (2021): 45-50 .
Copyright: © 2021 :Ali Taghizadehghalehjoughi., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.