ReseaRch PaPeR Journal of Agricultural and Marine Sciences 2021, 26(1): 47–52 DOI: 10.24200/jams.vol26iss1pp47-52 Reveived 21 April 2020 Accepted 22 Nov 2020 Protective effect of Oxalis corniculate and Pteropyrum scoparium Leaf Extracts against Azoxymethane-induced oxidative stress and colon carcinogenesis Mostafa I. Waly*, Mohammed Al-Khusaibi, Nejib Guizani Mostafa I. Waly*( ) mostafa@squ.edu.om, Department of Food Science and Nutrition, College of Agricultural and Marine Sciences, Sultan Qaboos University, Muscat, Oman Introduction Oman has around 1200 species of wild plants, many of which are reported to be used in tradi-tional medicine (Divakar et al., 2016). However, less than 10% of these plants have been screened for their medicinal use (Waly et al., 2014). Among the important wild plants of Oman are two edible plants (Oxalis cor- niculate, commonly known as Hamd, and Pteropyrum scoparium, commonly known as Sidaf) which have ther- apeutic properties based on their bioactive constituents (Al Attabi et al., 2015). Oxalis corniculate is short-lived perennial plant growing only 5 – 10 cm tall but spread- ing at the roots to form a mat of growth 30 cm or wider, and it is harvested from the wild for food, medicine and various other commodities (Divakar et al., 2016). Ptero- pyrum scoparium has been used for generations as an additive to Omani traditional dishes; it is considered as التأثري الوقائي ملستخلص أوراق النبااتت الربية العمانية ) السيداف و احلميضة( ضد اإلجهاد التأكسدي الناجم عن تسرطن القولون يف حيواانت التجارب مصطفى وايل*, حممد اخلصييب, جنيب قيزاين Abstract. Oxalis corniculate and Pteropyrum scoparium are two edible wild Omani plants with known preventive effects on various human diseases, yet their therapeutic role in colon cancer has not been studied yet. Azoxymethane (AOM) is a common oxidizing agent that induces colon cancer in experimental animal models. In the current study, the protective effect of Oxalis corniculate and Pteropyrum scoparium leaves extracts against AOM-induced cancer and oxidative stress in rat colon was examined. Sixty Sprague-Dawley rats were randomly divided into 6 groups (10 rats/group). Control group was fed a standard diet; the AOM-treated group was fed a standard diet and received an intraperitoneal injection of AOM at a dose of 30 mg/kg body weight for each rat. The other four groups received an intra-gastric intubation of Pteropyrum scoparium or Oxalis corniculate leaves extracts (0.1 mg extract/1 mL water/ day) in the absence or presence of AOM injection. After 8 weeks, all rats were sacrificed and the colon tissues were dissected for Aberrant Crypt Foci (ACF) enumeration of cancer lesions development, and for measurements of gluta- thione (GSH), total antioxidant capacity (TAC), and DNA oxidative damage. The results in this study showed that the AOM-injected rats showed significant increased level of DNA oxidative damage, lower levels of GSH and TAC, and higher ACF as compared to the control group. Oxalis corniculate and Pteropyrum scoparium leaf extracts significantly suppressed the oxidative damage associated with AOM injection and mitigated its carcinogenic effect in rat colon. Both Oxalis corniculate and Pteropyrum scoparium leaf extracts acted as potent antioxidants and combat the AOM-associ- ated oxidative stress and colon carcinogenesis. The data from this study suggest that dietary supplementation of these two wild plants might be applied as a therapeutic agent for colon cancer treatment. Keywords: Antioxidants, Colon cancer, Omani Wild Plants, Oxidative stress املســتخلص:النبااتت العمانيــة )الســيداف Pteropyrum scoparium( و )احلميضــة Oxalis corniculate( هــي نبــااتت تنمــو يف الربيــة و صاحلــة لــأكل و أيضــاً هلمــا آاثر وقائيــة معروفــة ضــد العديــد مــن األمــراض املزمنــة ، ومــع ذلــك مل يتــم دراســة دورمهــا العالجــي يف ســرطان القولــون حــى اآلن. فمــن املعــروف أن العوامــل املؤكســدة تســبب مــرض ســرطان القولــون لذلــك يف الدراســة احلاليــة ، مت فحــص التأثــر الوقائــي ملســتخلصات أوراق النبــااتت العمانيــة ضــد الســرطان الناجــم عــن اإلجهــاد التأكســدي يف قولــون الفئــران كنمــوذج حليــواانت التجــارب. ولقــد اشــتملت التجربــة علــى ســتون فــأراً مــن ســاللة ســرباغ داويل ومقســمة إىل 6 جمموعــات )10 فئــران / جمموعــة( كاأليت: اجملموعــة الضابطــة و اجملموعــة املعاجلــة بســرطان القولــون )الــي تلقــت حقنــة مــن مركــب AOM جبرعــة 30 جمــم / كجــم مــن وزن اجلســم لــكل فــأر( بينمــا تلقــت اجملموعــات األربــع األخــرى مســتخلصات أوراق نبــات الســيداف أو احلميضــة يف غيــاب أو وجــود مركــب AOM . اســتمرت التجربــة ملــدة 8 أســابيع ، و بعــد هــذه املــدة مت التضحيــة جبميــع الفئــران ومت تشــريح أنســجة القولــون مــن أجــل رصــد تطــور األورام الســرطانية يف القولــون ، ومت ايضــاً قيــاس مركــب اجللواتثيــون )GSH( ، والقــدرة اإلمجاليــة ملضــادات األكســدة )TAC( ، وكذلــك أضــرار أكســدة احلمــض النــووي يف مجيــع أنســجة القولــون جلميــع الفئــران. ولقــد أظهــرت نتائــج هــذه الدراســة أبن الفئران احملقونة بـ AOM أظهرت زايدة ملحوظة يف مســتوى ضرر أكســدة احلمض النووي ، واخنفاض مســتوايت GSH و TAC ، وارتفاع نســبة األورام الســرطانية ابلقولــون مقارنــة ابجملموعــة الضابطــة. بينمــا أظهــرت النبــااتت العمانيــة املســتخدمة أتثــر دفاعــي ضــد الضــرر التأكســدي املرتبــط حبقــن AOM وخففــت أيضــاً مــن التأثــرات املســرطنة يف القولــون. ختامــاً فــإن كاًل مــن أوراق نبــات الســيداف و احلميضــة تعمــل كمضــادات أكســدة قويــة وتكافــح اإلجهــاد التأكســدي املرتبــط بـــسرطان القولــون. و تشــر البيــاانت الــواردة مــن هــذه الدراســة إىل أن هــذه النبــااتت الربيــة العمانيــة ميكــن اســتخدامها كمكمــل غذائــي عالجــي ملكافحــة مــرض ســرطان القولــون. الكلمات املفتاحية: مضادات األكسدة ، سرطان القولون ، النبااتت الربية العمانية ، اإلجهاد التأكسدي 48 SQU Journal of Agricultural and Marine Sciences, 2021, Volume 26, Issue 1 Protective effect of Oxalis corniculate and Pteropyrum scoparium Leaf Extracts against Azoxymethane-induced oxidative stress and colon carcinogenesis an important herbal drug for treating various diseases, such as diabetes, fever, skin diseases and inflammation (Divakar et al., 2016). Colon cancer is the third most common type of can- cer in terms of incidence and the fourth in cause of death world-wide (WHO, 2020). Clinical studies continue to support the notion that oxidative stress is involved in the etiology of colon cancer (Afshari et al., 2019). Oxidative stress is a condition in which oxidants level is counter- balancing the antioxidant-dependent cellular defense mechanisms and contributes among other factors to the pathogenesis of cancer (Monteiro et al., 2020). It has been suggested that higher intake of antioxidants is associated with a lower risk of oxidative stress-me- diated colonic carcinogenesis (Waly et al., 2014). Azoxymethane (AOM) induces cancer in the co- lon and rectum of animal models by causing oxida- tive damage to DNA and its associated gene muta- tion and adductions (Waly et al., 2015). Glutathione (GSH) is the major intracellular antioxidant which scavenged oxidants, and accordingly an increase in its level is crucial to combat oxidative stress-mediat- ed colon carcinogenesis (Padmanabhan et al., 2019). Total antioxidant capacity (TAC) reflects the cellu- lar redox status (enzymatic and non-enzymatic sys- tems), and TAC impairment activity was reported in animals’ models for colon cancer (Waly et al., 2016). There are negligible published reports that identi- fy the in vivo antioxidant potential properties of Ox- alis corniculate and Pteropyrum scoparium in relation to colon cancer pathogenesis. Therefore, this study was undertaken to evaluate these two Omani plants for their phytochemical contents and antioxidant properties in an experimental model of colon cancer. Methods Chemicals AOM (catalogue # A5486) and sodium chloride phys- iological solution (catalogue# 52455) were purchased from Sigma-Aldrich Co. (St. Louis, Missouri, USA). Plant Collection and Identification Three kg of each plant (Oxalis corniculate and Pteropy- rum scoparium) were collected from their natural habi- tat and immediately washed of dust and impurities using tap water. The plants were kept at room temperature un- der shade until they are completely dried, and the leaves of each plant were ground to fine powder by use of elec- tric grinder (Moulinex AR1043-UK0). The powdered samples were macerated in aqueous methanol, and the filtrates were then evaporated using rotary evaporator. The resulting crude extract (50 g dry solids) was stored at - 40 ºC till use for subsequent experiments. Phytochemical contents of plants leaves extracts Folin-Ciocalteu assay was used to determine the total phenol content (Suresh et al., 2016). The total pheno- lic content of samples was expressed as mg Gallic Acid Equivalents (mg GAE/100 g sample). The total flavo- noids content was determined using Catechin as stan- dard, and the results were expressed as mg Catechin (mg of CAE/100 g sample). (Suresh et al., 2016). Evaluation of the free radical scavenging capacity of plant leave extracts The capacity of each one of the two plants leaves extract to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical was performed by a spectrophotometric meth- odology. Briefly, each plant leave extract at different concentrations (10 - 100 µg/mL), were mixed with stable DPPH radical in methanolic solution (0.3 mL of DPPH radical solution 0.5 mM in 3 mL ethanol). When DPPH reacts with an antioxidant compound, which can donate hydrogen, it is reduced. The changes in color (from deep violet to light yellow) were read [Absorbance (Abs)] at 517 nm after 30 min of reaction using a UVVIS spec- trophotometer (DU 800; Beckman Coulter, Fullerton, CA, USA). Controls contained all the reaction reagents except plant leaves extracts or 2,6-di-tert-butyl-4-hy- droxytoluene (BHT), the positive control. The free radical scavenging capacity of different samples were expressed as %DPPH inhibition, a higher %free radical scavenging activity value indicates a higher antioxidant activity and it was calculated as follows: % DPPH inhibition = [(Absorbance of control–Absor- bance of sample)/ Absorbance of control] X 100 Animal and Experimental Design The protocol used in this study was conducted in ac- cordance with international laws and policies and ap- proved by the Animal Ethics Committee at the Sultan Qaboos University (SQU/AEC/2019-2020/8). Sixty male Sprague Dawley rats weighing 150 ± 5 gm were obtained from the animal breed at the animal house facility, Sul- tan Qaboos University, Muscat, Oman. The rats were housed in individual cages at standard conditions, and they were fed a standard diet and given water ad-libitum. The rats were randomly divided into 6 groups (n=10 rats/group). Control group was fed a standard diet and also received a single intraperitoneal injection of 0.9% physiological saline in week one; AOM-injected group was fed a standard diet and a single intra-peritoneal dose of AOM (30 mg/kg body weight) dissolved in 0.9% phys- iological saline in week one. The other four groups were fed a standard diet and received intra-gastric intubation of 1 mL of either Oxalis corniculate or Pteropyrum sco- parium leaves extracts (100 µg extract/mL sterile dis- tilled water/day) in the presence or absence of AOM in- jection. The therapeutic dose used effective doses for the 49Research Paper Waly, Al-Khusaibi, Guizani two plants leaf extracts were determined based on the results of DPPH assay. The plant leaf extracts were given on daily basis at the same timing early morning, through- out the 8 weeks study period. Body weight was record- ed weekly for the entire duration of the experiment. Animal Sacrifice After 8 weeks, the rats were fasted overnight, anesthe- tized with a lethal dose of a cocktail containing ketamine (1 mg), xylazine (5 mg), and acepromazine (0.2 mg) and then sacrificed. The colon tissue of each rat was excised for histo-pathological examination of any cancer lesions development, and for biochemical measurements of ox- idative stress indices. Colon Preparation The colons were carefully removed from rats and were kept on a glass plate in ice jackets. The colons were then opened longitudinally, rinsed with ice-cold physiologi- cal saline, and sectioned longitudinally into two equal halves. The first half was fixed flat in 10% buffered for- malin (Fisher Scientific, Fair Lawn, NJ) between two fil- ter papers for one week before Aberrant Cypt Foci (ACF) enumeration. Meanwhile, the other half was rinsed with ice-cold physiological saline and was immediately ho- mogenized in 10 mL of 100 mM potassium phosphate buffer (pH 7.2) by a glass-Teflon homogenizer with an ice-cold jacket and centrifuged at 6,000 g at 4°C for 60 minutes. The resulting supernatant was used for deter- mination of protein, GSH and TAC measurements. Aberrant Cypt Foci (ACF) Enumeration Fixed colons were stained with 0.2% methylene blue in Kreb’s ringer bicarbonate buffer for 20 minutes in a Petri dish and rinsed with physiological saline. After staining, the colons were placed with the mucosal surfaces up on a slide, to be examined with a light microscope under 40X magnification and scored for ACF. In brief, the ACF were distinguished from normal crypts by their darker stain, enlarged and slightly elongated size, thick epithe- lial lining, slightly elongated cryptal opening and often slit shaped. The total number of ACF was recorded for all examined colons (Waly et al., 2014). Protein Content Analysis The Protein content of colon tissues was as- sayed by the method of Lowry et al. (1951) us- ing bovine serum albumin as standard and pro- tein content was expressed as mg/ml of sample. GSH Measurements Aliquots of supernatant (100 µL) were transferred to fresh Eppendorf tubes and 2 µL of monochlorobimane (25 mmol/L) and 2 µL of glutathione-S-transferase re- agent were added, as provided by a commercial kit (Bio- vision, Mountain View, CA, USA, Catalog # K251). After 30 minutes of incubation at 37oC, the samples and stan- dards were read in a fluorescence plate reader at 380/460 nm. GSH content was determined by comparison with values from a standard curve using freshly prepared GSH and normalized to the protein content of the as- sayed colon tissue homogenates. Total Antioxidant Capacity (TAC) Measurements A colorimetric method (Randox Assay Kit, Randox Laboratories Ltd, UK) was used to measure the TAC. The assay is based on the incubation of samples with 2, 2’-azino-di-[3-ethylbenzthiazoline sulphonate (6)] diammonium salt (ABTS) with a peroxidase (metmyo- globin) and hydrogen peroxide to produce the radical cation ABTS+ which has a relatively stable blue-green color that is measured at 600 nm. Antioxidants present in the assayed colonic mucosal tissue homogenate sam- ples inhibit the oxidation of ABTS to ABTS+ (cause sup- pression of the color production) to a degree that is pro- portional to their concentration. The assay results were normalized to the protein content of the assayed colon tissue homogenates. DNA Oxidative Damage Assay The DNA was isolated from the colon tissues homog- enates and the DNA oxidative damage was measured using 8-oxo-7,8-dihydro-20-deoxyguanosine (8-oxod- Guo) assay as described earlier (Suresh et al., 2017). The 8-oxodGuo and 2dG in the DNA were detected using an ESA Coulochem II electrochemical detector in line with a UV detector. Statistical analysis The results are expressed as means ± standard deviation (SD). The statistical analysis was performed using one- way analysis of variance (ANOVA) followed by Tukey’s test and a P value of less than 0.05 was considered signif- icant (GraphPad Prism version 5.03; GraphPad Software Inc. San Diego, CA). Results Polyphenols and flavonoids Oxalis corniculate leaf extract had as higher total poly- phenol contents as compared to that of Pteropyrum sco- parium leaf extracts (320±13 and 252±10 mg GAE/100 g sample, respectively, P < 0.05). A similar significant dif- ference in the flavonoid contents was observed for both Oxalis corniculate and Pteropyrum scoparium leaves extracts (245±9 and 117±8 mg of CAE/100 g sample, re- spectively, P < 0.05). DPPH As presented in Figure 1, the Oxalis corniculate and Pteropyrum scoparium leaves extracts inhibited DPPH 50 SQU Journal of Agricultural and Marine Sciences, 2021, Volume 26, Issue 1 Protective effect of Oxalis corniculate and Pteropyrum scoparium Leaf Extracts against Azoxymethane-induced oxidative stress and colon carcinogenesis formation in a dose-dependent manner (10-100 µg/mL), and it was observed that Oxalis corniculate leaves ex- tract exhibited a higher inhibition rate as compared to the Pteropyrum scoparium leaf extracts, P < 0.05. The plateau of the effective doses for both extracts was 50- 100 µg/ml. Therefore, the effective dose, 100 µg/mL, was used for subsequent animal in vivo studies. Body Weight Gain of Animals No mortality occurred in any group, and the body weight for each rat was recorded weekly for the whole duration of the experiment and as demonstrated in Fig- ure 2. The body weight increased gradually throughout the experimental period for all the groups. However, all rats in the AOM-injected group showed a consistent de- crease in body weight throughout week 2 to week 8 as compared to control groups, P < 0.05. It was notable that the supplementation of AOM-injected group with ei- ther Oxalis corniculate or Pteropyrum scoparium leaves extracts compensated for the weight loss due to AOM injection, P < 0.05. ACF Enumeration All the AOM-injected rats developed the ACF; mean- while the concomitant supplementation of AOM-inject- ed groups with Oxalis corniculate or Pteropyrum scopar- ium leaves extracts has abrogated the ACF production, P < 0.05. The histopathological examination of colonic tissues of rats supplemented with Oxalis corniculate, or Pteropyrum scoparium leaves extracts displayed the normal architecture of colon tissue as compared to the control group and did not show any ACF development (Figure 3). AOM-Associated Oxidative Stress It has been observed that the Oxalis corniculate, or Pteropyrum scoparium leaves extracts supplementation to the AOM-injected group resulted in combating the observed AOM-induced oxidative stress by restoring the level of depleted GSH to a level that is comparable to the control group, P > 0.05 (Figure 4). The same trend was observed for the protective effects of the plants ex- tracts supplementation on abrogating the AOM-medi- ated effect on TAC, (Figures 5). As illustrated in Figure Figure 1. Scavenging effect of Effects of Oxalis cornic- ulate and Pteropyrum scoparium leaves extracts and 2,6-di-tert-butyl-4-hydroxytoluene (BHT) against 1,1-di- phenyl-2-picrylhydrazyl (DPPH) free radical formation. *Significantly higher as compared to Pteropyrum scoparium leaves extracts, P< 0.05. Figure 2. Changes in body weight of rats supplement- ed with Oxalis corniculate and Pteropyrum scoparium leaves extracts in the presence or absence of azoxy- methane (AOM) carcinogen. Animals in the six groups were examined for the changes in their body weight ev- ery week for 8 weeks. *Significantly lower as compared to control group, P< 0.05. Values without superscript are not significantly different as compared to control group. Figure 3. Effects of Oxalis corniculate and Pteropyrum scoparium leaves extracts on aberrant crypt foci (ACF) development in control and azoxymethane (AOM)-in- jected groups. *Significantly higher than the control group, P<0.05. **Significantly lower than AOM-injected group, P<0.05. Values without superscript are not signifi- cantly different as compared to control group, P>0.05. 51Research Paper Waly, Al-Khusaibi, Guizani 6, AOM caused oxidative damage to the DNA in the colon tissues of rat injected with AOM and the differ- ence was significantly higher than the control group, P<0.05, meanwhile, the plants extract supplementation showed a significant reduction in the DNA damage in the AOM-injected group, P < 0.05. Discussion AOM injection acted as a colon cancer inducing agent in a mechanism that involves oxidative stress as evidenced by the significant reduction of the intracellular GSH lev- el, impairment of TAC, and increase in the level of DNA oxidative damage as compared to control non-treated group. The present study elucidated the role of Oxalis corniculate and Pteropyrum scoparium leaves extracts in alleviating AOM-induced oxidative stress and its associ- ated colon carcinogenesis. We reported that rats injected with AOM developed oxidative stress in the colon tissues as evidenced by GSH depletion, reduction of TAC and increased DNA oxida- tive damage, as well as histo-pathological changes which manifested carcinogenic effect in the examined colonic tissues. This is consistent with previous reports from our research group which indicated that AOM injection in rats resulted in a reduction in the colonic redox cellular status (Waly et al., 2014). However, Oxalis cornicu- late and Pteropyrum scoparium supplementation has suppressed the oxidative damage associated with AOM injection and mitigated its carcinogenic effect. These findings suggested that these two Omani wild plants can have a colon-protective effect against AOM-induced oxidative stress and its associated colon carcinogenesis. The findings in this work are consistent with the well documented role of natural plants in the treatment and prevention of chronic diseases, including cancer (Wang et al., 2020). In particular, Oxalis corniculate and Ptero- pyrum scoparium have wide medicinal applications, as previous studies have shown that Oxalis corniculate and Pteropyrum scoparium are rich in phytonutrients and Figure 4. Glutathione (GSH) measurements in colon- ic tissue homogenates of rats supplemented with Oxalis corniculate and Pteropyrum scoparium leaves extracts in the presence or absence of azoxymethane (AOM) car- cinogen.*Significantly lower as compared to control group, P<0.05. **Significantly higher than AOM injected group, P<0.05. Values without superscript are not significantly dif- ferent as compared to control group, P>0.05. Figure 5. Total antioxidant capacity (TAC) measurements in colonic tissue homogenates of rats supplemented with Oxalis corniculate and Pteropyrum scoparium leaves ex- tracts in the presence or absence of azoxymethane (AOM) carcinogen. *Significantly lower as compared to control group, P<0.05. **Significantly higher than AOM injected group, P<0.05. Values without superscript are not significant- ly different as compared to control group, P>0.05. Figure 6. DNA oxidative damage (8-hydroxydeoxygua- nosine, 8-OHdG) in colonic tissue homogenates of rats supplemented with Oxalis corniculate and Pteropyrum scoparium leaves extracts in the presence or absence of azoxymethane (AOM) carcinogen. *Significantly lower as compared to control group, P<0.05. **Significantly higher than AOM injected group, P<0.05. Values without super- script are not significantly different as compared to con- trol group, P>0.05. 52 SQU Journal of Agricultural and Marine Sciences, 2021, Volume 26, Issue 1 Protective effect of Oxalis corniculate and Pteropyrum scoparium Leaf Extracts against Azoxymethane-induced oxidative stress and colon carcinogenesis prevent generation of cellular oxidative stress and in- flammation, hence offers a novel therapeutic approach to prevent oxidative stress-induced pathogenesis (Al-Atta- bi et al., 2015). Our study documented that Oxalis cornic- ulate and Pteropyrum scoparium scavenged the oxida- tive stress-induced colon carcinogenic effect in the used experimental model, these results address the primary prevention of oxidative stress in relation to colon cancer. Conclusion AOM administration resulted in oxidative stress and increased ACF formation in the examined colon tissues in rats. Oxalis corniculate and Pteropyrum scoparium are rich in total phenolic and flavonoid compounds, and their supplementation have significantly abrogated the AOM-mediated oxidative stress, and combated the AOM-induced AFC formation. Our study suggests that dietary supplementation of these two wild plants might be applied as a therapeutic agent for prevention against colo rectal cancer rather than its treatment as abberant crypt foci are more of premalignant lesions and dyspla- sia, however it is essential to conduct further studies to evaluate the colon-protective effect of these two Omani wild plants in human-based clinical trials. Acknowledgment The authors declared no conflicts of interest. This re- search was supported by a grant obtained from the Col- lege of Agricultural and Marine Sciences, Sultan Qaboos University, Muscat, Oman (IG/AGR /FOOD /20/01). An oral presentation of this manuscript was presented at the 21st ISANH Middle East World Congress in Muscat, Sultan Qaboos University on March 4-5, 2019. The pre- sentation entitled: Medicinal plants from chemopreven- tion to identification of potential biomarkers for colon cancer. References Afshari K, Haddadi NS, Haj-Mirzaian A, Farzaei MH, Rohani MM, Akramian F, Naseri R, Sureda A, Gha- naatian N, Abdolghaffari AH. (2019). Natural fla- vonoids for the prevention of colon cancer: A com- prehensive review of preclinical and clinical studies. Journal of Cell Physiology 234(12): 21519-21546. Al-Attabi Z, AlMamri R, Al AbdAslam K. (2015). Antiox- idant potential properties of three Wild Omani plants against hydrogen peroxide-induced oxidative stress. Canadian Journal of Clinical Nutrition (2): 16-22. Divakar MC, Al-Siyabi A, Varghese SS, Rubaie MA. (2016). The Practice of Ethnomedicine in the North- ern and Southern Provinces of Oman. Oman Medical Journal (4):245-52. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry. 193(1): 265-75. Monteiro HP, Rodrigues EG, Amorim Reis AKC, Longo LS Jr, Ogata FT, Moretti AIS, da Costa PE, Teodoro ACS, Toledo MS, Stern A. (2019). Nitric oxide and interactions with reactive oxygen species in the de- velopment of melanoma, breast, and colon cancer: A redox signaling perspective. Nitric Oxide 89: 1-13. Omara T, Kiprop AK, Ramkat RC, Cherutoi J, Kagoya S, Moraa Nyangena D, Azeze Tebo T, Nteziyaremye P, Nyambura Karanja L, Jepchirchir A, Maiyo A, Jema- tia Kiptui B, Mbabazi I, Kiwanuka Nakiguli C, Nak- abuye BV, Chepkemoi Koske M. (2020). Medicinal Plants Used in Traditional Management of Cancer in Uganda: A Review of Ethnobotanical Surveys, Phy- tochemistry, and Anticancer Studies. Evidence Based Complementary Alternative Medicine 2020: 1-26 (Article ID 3529081). Padmanabhan S, Waly MI, Taranikanti V, Guizani N, Ali A, Rahman MS, Al-Attabi Z, Al-Malky RN, Al-Mas- kari SNM, Al-Ruqaishi BRS, Dong J, Deth RC. (2019). Folate/Vitamin B12 Supplementation Combats Oxi- dative Stress-Associated Carcinogenesis in a Rat Mod- el of Colon Cancer. Nutrition Cancer 71(1):100-110. Suresh S, Waly MI, Rahman MS, Guizani N, Al-Kindi MAB, Al-Issaei HKA, Al-Maskari SNM, Al-Ruqa- ishi BRS, Al-Salami A. Broccoli (Brassica oleracea). (2017). Reduces Oxidative Damage to Pancreatic Tis- sue and Combats Hyperglycaemia in Diabetic Rats. Preventive Nutrition and Food Science 22(4): 277-284. Waly MI, Al-Ghafri BR, Guizani N, Rahman MS. (2015). Phytonutrient Effects of Date Pit Extract against Azoxymethane--Induced Oxidative Stress in the Rat Colon. Asian Pacific Journal of Cancer Prevention (8): 3473-3477. Waly MI, Al Alawi AA, Al Marhoobi IM, Rahman MSh. Red Seaweed (Hypnea bryodies and Melanothamnus somalensis) Extracts Counteracting Azoxymeth- ane-Induced Hepatotoxicity in Rats. Asian Pacific Journal of Cancer Prevention 2016 Dec 1; 17(12): 5071-5074. Waly MI, Al-Rawahi AS, Al Riyami M, Al-Kindi MA, Al-Issaei HK, Farooq SA, Al-Alawi A, Rahman MS. (2014). Amelioration of azoxymethane induced-car- cinogenesis by reducing oxidative stress in rat colon by natural extracts. BMC Complementary Alterna- tive Medicine 14: 1-10. Wang J, Seyler BC, Ticktin T, Zeng Y, Ayu K. (2020). An ethnobotanical survey of wild edible plants used by the Yi people of Liangshan Prefecture, Sichuan Prov- ince, China. Journal of Ethnobiology and Ethnomed- icine 16: 1-27. WHO. 2020. World Health Organization report on cancer: setting priorities, investing wisely and pro- viding care for all. ISBN 978-92-4-000129-9 (elec- tronic version). ISBN 978-92-4-000130-5 p. 28-30.