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Antioxidant, Anti-Inflammatory and Anticancer Activities of Methanolic bioRxiv preprint doi: https://doi.org/10.1101/2020.03.29.014324; this version posted March 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Antioxidant, Anti-inflammatory and Anticancer activities of Methanolic 2 Extract of Thottea siliquosa: An In Vitro study 3 Amrutha Koottasseri1, Amal Babu1, Anna Augustin1, Joice Tom Job2 and 4 Arunaksharan Narayanankutty3* 5 1 PG & Research Department of Zoology, St. Joseph’s College (Autonomous), Devagiri, 6 Calicut, Kerala, India 7 2 Assistant Professor, PG & Research Department of Zoology, St. Joseph’s College 8 (Autonomous), Devagiri, Calicut, Kerala 9 3 Assistant Professor (Ad-hoc), PG & Research Department of Zoology, Malabar Christian 10 College, Nadakkavu, Calicut, Kerala 11 12 13 * Corresponding Author, 14 Dr. Arunaksharan Narayanankutty 15 Assistant Professor (Ad-hoc), 16 PG and Research Department of Zoology, 17 Malabar Christian College, Nadakkave, 18 Calicut- 673 001, Kerala, India 19 Email: [email protected] 20 [email protected] 21 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.29.014324; this version posted March 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 22 Highlights 23 Thottea siliquosa is a traditional Ayurvedic medicine 24 T. siliquosa exhibits significant anti-inflammatory and anticancer properties 25 Polyphenolic compounds may be responsible for the bioactivities of T. siliquosa 26 T. siliquosa may be a source for novel bioactive components 27 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.29.014324; this version posted March 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 28 Abstract 29 Background and Aim: Oxidative stress and inflammation are the predominant cause of 30 chronic diseases including cancers, thereby making it as an essential target for preventing 31 these disorders. Various natural products and plants have been shown to prevent the process 32 of free radical induced damages. The present study evaluated the biological properties of 33 Thottea siliquosa, belonging to the family Aristolochiaceae, which is a traditionally used 34 Ayurvedic plant. 35 Experimental Procedure: Antioxidant assays carried out were DPPH, FRAP, hydrogen 36 peroxide scavenging and hemolysis inhibition assay; nitric oxide and lipoxygenase inhibition 37 assays were used for anti-inflammatory studies. Anticancer activity was done using human 38 endometrial and breast cancer cells by MTT assay. 39 Results and Conclusion: The results of the present study showed antioxidant properties for 40 the methanolic crude extract of T. siliquosa as DPPH radical scavenging (110.40± 41 4.5µg/mL), FRAP capacity (41.1± 6.2), peroxide scavenging (233.4± 14.2 µg/mL) etc. In 42 addition, anti-inflammatory properties are also evident in terms of nitric oxide radical 43 scavenging (28.76± 3.9 µg/mL) and lipoxygenase inhibition (39.2± 3.2 µg/mL) assays. 44 Further, the anticancer activity of these extract has been identified against human endometrial 45 and breast cancer cells. Further studies together with a bioassay guided fractionation may 46 identify possible bioactive compounds. 47 Keywords: Thottea siliquosa; antioxidant activity; anti-inflammatory activity; anticancer 48 activity; polyphenols 49 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.29.014324; this version posted March 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 50 1. Introduction 51 Inflammation is the primary protective response of the body against exogenous molecules/ 52 organisms. This involves a cascade of events which involve the recruitment of inflammatory 53 cells to the site of action, release of inflammatory mediators from the granules of cells and 54 subsequent tissue damage and edema. Acute inflammation is often described as protective 55 response, as it doesn’t damage tissues for a long time. On the other hand, chronic 56 inflammation, which lasts for a longer period of time are reported to be deleterious to the 57 body. 58 Numerous studies have described the association of chronic inflammation with the onset and 59 progression of various diseases such as cancers 1, cardiovascular diseases 2, fatty liver 3, and 60 Alzheimer’s disease 4. Several studies have indicated that use of non-steroidal anti- 61 inflammatory drugs can reduce the incidence of various cancers 5, cardiovascular diseases 6, 62 acute respiratory problems 7, and Alzheimer’s disease 8. Thus, inflammatory molecules have 63 become the primary target for the prevention of various diseases. 64 Natural products has always been the source of drugs, likewise several different anti- 65 inflammatory drugs have been isolated from the Mother Nature 9,10. In addition, the search for 66 new anti-inflammatory drugs is also been intensified due to its multi-targeted nature and lack 67 of side effects. Aristolochiaceae is an important family which is well known for its 68 antioxidant and anticancer compound Aristolochicacids (AAs) 11. The pharmacological 69 properties of the family include anti-diarrhoeal 12, anticancer 13, anti-inflammatory and 70 antipruritic activity 14. Several novel alkaloid and terpenoids have also been isolated from the 71 different plants belonging to this family 15. The plants belonging to this family are also used 72 traditionally in Chinese and Ayurvedic medicines as well as different folklore medicinal 73 systems 16. Thottea siliquosa is an unexplored medicinal plant which is traditionally used for 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.29.014324; this version posted March 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 74 digestive tract disorders and inflammatory conditions. Very limited literature is available on 75 the biological and pharmacological properties of the plant. The only available published 76 research indicates that the leaf and root extracts of T. siliquosa exhibits antibacterial activity 77 against Streptococcus, E. coli and Pseudomonas strains 17. Apart from these there are no 78 available reports on the antioxidant and anti-inflammatory activities of the plant. Thus, the 79 present study aimed to evaluate the antioxidant, anti-inflammatory and anticancer activities of 80 the plant and further to elucidate the mechanism of action of the same. 81 2. Materials and Methods 82 2.1 Collection of plant material and extraction 83 The leaves of Thottea siliquosa plant was collected from Western Ghats regions of Kerala. 84 The whole plant was cleaned properly, shade dried, and coarsely powdered. The powdered 85 materials (10 g) were extracted with methanol (100 mL) by Soxhlet apparatus and the 86 extracts obtained were concentrated under reduced pressure at 40°C using rotary evaporator. 87 A portion of the residue was dissolved at 10 mg/mL stock solution in Dimethyl sulfoxide 88 (DMSO) for the study and remaining portion was stored at 4°C for further use. 89 2.2 Cell lines and maintenance 90 Cells (HeLa and MCF7) were procured from National Centre for Cell Science, Pune and 91 maintained at 5% carbon dioxide at 95% humidity. The cells were cultured in RPMI-1640 or 92 DMEM media supplemented with 10% foetal bovine serum. The cells was passaged every 93 third day or before reaching 80% confluency. 94 2.3 Total phenolic and total flavonoids content 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.29.014324; this version posted March 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 95 Total phenolic content was quantified using a modified Folin-Ciocalteu method 18. The 96 results was compared to the standard curves and expressed as mg Gallic acid equivalent per 97 gram dry powder for the samples. 98 Total flavonoid contents was quantified using the method explained by Khodaie, Bamdad, 99 Delazar, Nazemiyeh 19 and the results were expressed as mg quercetin equivalent per gram 100 dry powder for the samples. 101 2.4 In vitro antioxidant activities 102 In vitro antioxidant capacity of the extract was determined in terms of DPPH radical 103 scavenging 20, Ferric reducing antioxidant power (FRAP) 21, and hydrogen peroxide 104 scavenging potentials. 105 2.5 In vitro anti-inflammatory activity 106 Lipoxygenase is an important mediator of inflammation, thereby making it a target for anti- 107 inflammatory activity. Inhibition of 5-Lipoxygenase was assessed according to the previously 108 described methods 22. Likewise nitric oxide scavenging activity had also been carried out as 109 an indicator of anti-inflammatory potential. Percentage inhibition was calculated and IC50 110 was determined as per the methods described by Kakatum, Jaiarree, Makchucit, Itharat 23. 111 2.6 Anti-hemolytic potential 112 The anti-hemolytic activity induced by a peroxyl radical initiator, AAPH was measured 113 according to the method established by Salini, Divya, Chubicka, Meera, Fulzele, 114 Ragavamenon, Babu 24. Two hundred microliters of blood were mixed with 10 μL of plant 115 extract at various concentrations (0-100 μg/mL) and then 600 μL of AAPH (10%) was added. 116 The mixture was incubated at 37o C for 30 minutes and the absorbance of the mixture was 6 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.29.014324; this version posted March 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 117 measured at 450 nm.
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