Khảo sát hiệu quả kháng oxy hóa và bảo vệ gan trên mô hình chuột của một số cây thuộc họ cà phê (rubiaceae) TT TIENG ANH

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Major: Biotechnology Code: 62420201 PHAN KIM DINH STUDY ON ANTIOXIDANT AND HEPATOPROTECTIVE ACTIVITIES IN MICE MODEL OF SOME PLANTS OF THE RUBIACEAE FAMILY Can Tho, 2021 THE THESIS IS COMPLETED AT CAN THO UNIVERSITY Supervisor: 1, Assos Prof Dr Dai Thi Xuan Trang 2, Assos Prof Dr Nguyen Trong Tuan The thesis can be found at: - Learning Resource Center, Can Tho University - National Library Chapter 1: INTRODUCTION The liver is one of the largest organs in the human body, playing an important role in metabolism and excretion (Ahsan et al., 2009) In the process of performing the function, the liver is the first and continuously attacked organ by toxic substances, resulting in liver damage and dysfunction (Bodakhe and Ram, 2007) There are many causes of liver damage Most of these agents cause oxidative stress, producing many free radicals in the liver that attack biological macromolecules that damage liver cells The chemicals are hepatotoxic mainly by inducing lipid peroxidation in hepatocytes and other oxidative damage (Abou Sief, 2016) The common progression of liver disease usually ranges from fatty liver to hepatitis and eventually fibrosis and cirrhosis If untreated, fibrosis can progress to cirrhosis, eventually leading to liver failure and death Liver diseases are one of the most serious health problems in the world today Despite great advances in modern medicine, the prevention and treatment of liver diseases is still limited Using antioxidants to reduce peroxidation and oxidative stress is one of the effective therapies to prevent and treat liver damage A natural antioxidant found in many different plants (Prakash et al., 2007) and has also been shown to be effective and safe for the liver (Ranawat et al., 2010) Currently, drugs to treat liver diseases are mainly made from plants, but not much The objective of the study was to investigate the antioxidant and hepatoprotective effects of some plants of the Rubiaceae family, in order to select the plants or parts of the plants with the best antioxidant and liver protective effects , contributing to providing information on new sources of medicinal herbs for the prevention and treatment of liver disease The study was conducted with the following specific contents: (1) Investigation of medicinal plants used to treat liver disease; (2) Sampling and extracting some plants of the Rubiaceae family; (3) Screening for antioxidant activity of extracts; (4) Investigation of the hepatoprotective effect of the extracts; (5) Toxicity test of the extracts; (6) Extraction of fractions and bioactivity testing of fractions; (7) Isolation of compounds from bioactive fractions The meaning of the thesis The results of the thesis are the scientific basis for the antioxidant, anti-inflammatory and hepatoprotective abilities of methanol extracts of plants in the Rubiaceae family, including: Ixora duffii (leaves, flowers), Paederia lanuginosa Wall (leaves), Paederia scandens L (leaves), Neolamarckia cadamba (Roxb.) Bosser (leaves, stem bark, roots), Nauclea orientalis L (leaves, stem bark, roots), Hedyotis corymbosa L (whole plant) and Hedyotis diffusa Willd (whole plant) The thesis has determined that Nauclea orientalis L root has the best antioxidant and hepatoprotective effects, especially proving that the ethyl acetate fraction of Nauclea orientalis L root has hepatoprotective activity, resistance to lipid peroxidation and increase antioxidant content in the liver From there, contributing to creating a premise for the research and production of new products to prevent and treat liver disease New point of the thesis The study was carried out systematically to investigate the biological activities of plants in the Rubiaceae family from in vitro screening for antioxidant, anti-inflammatory to in vivo animal experiments The methanol extracts extracted from the studied plants were determined to have the ability to protect the liver based on a combination of biochemical tests investigating the ability to reduce oxidative stress in vivo and histopathological observations The plant extract with good antioxidant and hepatoprotective activity was fractionated, retested, selected for efficient fractionation for compounds isolation and chemical structure determination Research results have provided a scientific basis for the use of plants in the Rubiaceae family as medicine to treat liver disease Chapter 2: OVERVIEW Briefly describe free radicals, their harmful effects, antioxidants and the role of antioxidants Several methods of assessing antioxidant and hepatoprotective capacity An overview of liver structure and function, causes of liver damage, and types of liver damage Some hepatoprotective drugs and a brief overview of the Rubiaceae family plant species in this study Chapter 3: RESEARCH METHODS 3.1 Facilities for research 3.1.1 Time and place of study (As presented in the thesis) 3.1.2 Materials The plants of the Rubiaceae family studied, including: Ixora duffii, Paederia lanuginosa Wall., Paederia scandens L., Neolamarckia cadamba (Roxb.) Bosser, Nauclea orientalis L., Hedyotis corymbosa L and Hedyotis diffusa Willd were collected in Vinh Long and Hau Giang provinces The study used Mus musculus mice provided by Pasteur Institute, Ho Chi Minh City (167 Pasteur, Ward 8, District 3, Ho Chi Minh City) 3.1.3 Equipment and chemicals (As presented in the thesis) 3.2 Research Methods 3.2.1 Research diagram (As presented in the thesis) 3.2.2 Investigation of medicinal plants used to treat liver disease The study used the method of interviewing according to the form of a survey sample of medicinal plants in the community (Institute of Medicinal Materials, Ministry of Health) The locations selected for the survey as presented in the thesis 3.2.3 Collection and extraction of some plants of the Rubiaceae family Parts of some plants include Ixora duffii (leaves, flowers), Paederia lanuginosa Wall (leaves), Paederia scandens L (leaves), Neolamarckia cadamba (Roxb.) Bosser (leaves, stem bark, roots), Nauclea orientalis L (leaves, stem bark, roots), Hedyotis corymbosa L (whole plant) and Hedyotis diffusa Willd (whole plant) were sampled and processed into material powder The methanol extract was extracted by the method of soaking the material powder in methanol solvent, filtering the extract and evaporating the solvent 3.2.4 Screening for in vitro antioxidant activity of extracts 3.2.4.1 Determination of total polyphenol, flavonoid and alkaloid content of extracts Determination of chemical composition of the extracts The extracts were preliminarily qualitative for the presence of chemical components such as alkaloids, flavonoids, steroids, glycosides, saponins and tannins as described by Nguyen Kim Phi Phung (2007) Quantitative method of total polyphenol content The total polyphenol content was determined by the FolinCiocalteu colorimetric method (Singleton et al., 1999) The polyphenol content was determined to be equivalent to milligrams of gallic acid per gram of extract (mg GAE/g extract) Quantitative method of total flavonoid content The total flavonoid content was determined according to the procedure described by Sultana et al (2009) corrected as presented in the thesis The flavonoid content was determined to be equivalent to milligrams of quercetin per gram of extract (mg QE/g extract) Quantitative method for total alkaloid tent The alkaloid content was determined by complexation with bromocresol green (BCG), forming a yellow colored product (Shamsa et al., 2008) The alkaloid content (mg AE/g extract) was determined based on the atropine standard curve equation 3.2.4.2 Study in vitro antioxidant activity of extracts Determination of DPPH radical scavenging activity The DPPH test was carried out according to the method of Shekhar and Anju (2014) with correction as presented in the thesis EC50 was calculated based on the linear regression of DPPH free radical scavenging efficiency Determination of reducing power The reducing power of the extracts and BHA was performed according to the method of Oyaizu (1986) with correction as presented in the thesis The reducing power of the extracts was determined based on the antioxidant content equivalent to BHA and EC50 Investigation of total antioxidant capacity The total antioxidant capacity (TAC) of the extracts was evaluated by the phosphomolybdenum method of Prieto et al (1999) The total antioxidant capacity of the extracts was expressed by antioxidant content equivalent to µg/mL trolox and EC50 3.2.4.3 Investigation of in vitro anti-inflammatory activities of extracts The anti-inflammatory activity of the extract was investigated through inhibition of BSA protein denaturation by the method of Shah et al (2017) with corrections as presented in the thesis 3.2.5 Study the hepatoprotective effect of the extract in mice 3.2.5.1 Investigate the ability to reduce AST and ALT enzymes The extracts were investigated for their hepatoprotective effects in a mouse model of CCl4 toxicity as described by Kang and Koppula (2014) with correction as presented in the thesis The experimental groups of mice are presented in Table 3.1 Table 3.1 Hepatoprotective test design Group 5,6,7 8,9,10 Survey content Normal control Olive oil, DMSO 1% CCl4 2,5 mL/kg (20% of CCl4 in olive oil) CCl4, silymarin 16 mg/kg CCl4, methanolic leaf extract of Paederia lanuginosa at 100, 200, and 400 mg/kg, respectively CCl4, methanolic leaf extract of Paederia scandens at 100, 200, and 400 mg/kg, respectively 11,12,13 CCl4, methanolic leaf extract of Ixora duffii at 100, 200, and 400 mg/kg, respectively 14,15,16 CCl4, methanolic leaf extract of Neolamarckia cadamba at 100, 200, and 400 mg/kg, respectively 17,18,19 CCl4, methanolic roots extract of Neolamarckia cadamba at 100, 200, and 400 mg/kg, respectively CCl4, methanolic sterm bark extract of Neolamarckia cadamba at 100, 200, and 400 mg/kg, respectively 20,21,22 23,24,25 CCl4, methanolic leaf extract of Nauclea orientalis at 100, 200, and 400 mg/kg, respectively 26,27,28 CCl4, methanolic roots extract of Nauclea orientalis at 100, 200, and 400 mg/kg, respectively 29,30,31 CCl4, methanolic sterm bark extract of Nauclea orientalis at 100, 200, and 400 mg/kg, respectively After weeks, the groups of mice were weighted, anesthetized, and blood was taken from the heart to be tested for AST and ALT enzymes Animal livers were separated for observation and photography The liver was then divided into two sections, one to analyze the ability to inhibit lipid peroxidation (MDA test) and to regulate antioxidant activity (GSH test) in liver, the other part was immobilized in formol to perform histological specimen 3.2.5.2 Investigation of in vivo antioxidant capacity of extracts Quantification of malonyldialdehyde and glutathione content Quantification of malonyldialdehyde (MDA) and glutathione (GSH) was carried out according to the method of Ohkawa et al (1979) and Moron et al (1979) was adjusted according to Nguyen Bao Tran et al (2011) as presented in the thesis 3.2.5.3 Investigate the ability to protect liver tissue of extracts Perform histopathology specimen of mice liver Histology specimen was performed according to the procedure described by Saalu et al (2012) adjusted as presented in the thesis 3.2.6 Toxicity test of extracts 3.2.6.1 Acute toxicity test Single dose of 2000 or 5000 mg/kg over a 14-day period 3.2.6.2 Sub-chronic toxicity test Dose of 400 mg/kg/time/day for 90 days 3.2.7 Fractional extraction and bioactivity test of fractions The study selected an extract (methanol extract) with good antioxidant and hepatoprotective activity to extract fractions 3.2.7.1 Separation of fractional extracts The total methanol was extracted with the solvents of increasing polarity, n-hexane and ethyl acetate, respectively, by liquid-liquid extraction to obtain the corresponding fractional extracts, n-hexane, ethyl acetate and high water 3.2.7.2 Quanlitative and quantification of chemical composition in total methanol extract and fractions The fractionated extracts were preliminarily identified with some chemical components and quantified the total polyphenols, flavonoids and alkaloids according to the methods described in section 3.2.4.1 3.2.7.3 Investigation of antioxidant, anti-inflammatory and hepatoprotective capacity of fractionated extracts The in vitro antoxidant and anti-inflammatory activity of the studied extracts were similar to those presented in sections 3.2.4.2, 3.2.4.3 The hepatoprotective activity in mice of the studied fractions was similar to that described in section 3.2.5 at the extracts dose of 200 mg/kg BW 3.2.8 Isolation, purification and structural determination of compounds from the extract with the most effective hepatoprotective activity Using conventional methods in classical column chromatography, including rapid dry column chromatography, normal phase column chromatography, combined with thin layer chromatography and recrystallization were isolated and purificated of compounds The structures of the compounds were determined by modern spectroscopy methods 11 Table 4.5: EC50 of extracts in DPPH test Extract PLLE Linear regression equation y=0.7583x–1.0814 (R²=0.996) EC50 (g/mL) 67.3±1.35c PSLE y=0.4621x+0.1701 (R²=0.941) 107.8±1.22a IDLE y=0.8109x+2.0818 (R²=0.988) 59.3±1.26d IDFE y=0.8067x+4.3979 (R²=0.994) 56.5±1.65de NOLE y=0.6052x+5.7882 (R²=0.998) 73.0±0.99b NOSBE y=0.6309x+15.628 (R²=0.993) 54.4±0.2ef NORE y=0.6459x+2.525 (R²=0.997) 73.4±1.25b NCLE y=0.7637x+10.6 (R²=0.996) 51.5±1.61g NCSBE y=0.7356x+11.427 (R²=0.984) 52.4±1.93fg NCRE y=0.703x+10.553 (R²=0.961) 56.1±3.93e HCE - - HDE - - y =2.3915x+6.1567 (R²=0.977) 18.3±0.33h Vitamin C Note: The values shown are mean±SEM, n=3; Values followed by the same letter in the same column are not significantly different at the 5% level I duffii leaf extract (IDLE), I duffii flower extract (IDFE), P lanuginosa leaf extract (PLLE), P scandens leaf extract (PSLE), N cadamba leaf extract (NCLE), N cadamba stem bark extract (NCSBE), N cadamba root extract (NCRE), N orientalis leaf extract (NOLE), N orientalis stem bark extract (NOSBE), N orientalis root extract (NORE) H.s corymbosa extract (HCE) and H diffusa extract (HDE) 4.3.2.2 Reducing power of extracts The reducing power of the extracts was determined based on the antioxidant content equivalent of the BHA standard and EC50 Based on EC50 are presented in Table 4.7, it can be concluded that the reducing power of the extracts decreased in order of high order of N cadamba leaf, N cadamba stem bark, I duffii flower, N orientalis stem bark, I duffii leaf, N cadamba root, N orientalis leaf, P lanuginosa leaf, N orientalis root, P scandens leaf, H diffusa and H corymbosa 12 Table 4.7: EC50 of extracts in reducing power test Linear regression equation EC50 (g/mL) PLLE y=0.0007x+0.2331 (R²=0.986) 381.2±3.29b PSLE y=0.0005x+0.2089 (R²=0.984) 582.2±7.68a IDLE y=0.0015x+0.1717 (R²=0.967) 218.9±1.46e IDFE y=0.0015x+0.2678 (R²=0.978) 162.0±3.26fg NOLE y=0.0009x+0.2185 (R²=0.968) 325.3±11.51c NOSBE y=0.0014x+0.2434 (R²=0.992) 183.9±1.13f NORE y=0.001x+0.1374 (R²=0.995) 375.9±25.39b NCLE y=0.0089x+0.3514 (R²=0.996) 16.6±0.29i NCSBE y=0.0019x+0.2285 (R²=0.990) 140.0±8.02g NCRE y=0.0004x+0.4044 (R²=0.980) 282.8±36.56d - - - - y=0.0118x+0.1448 (R =0.993) 30.1±0.13h Extract HCE HDE BHA Note: The values shown are mean±SEM, n=3; Values followed by the same letter in the same column are not significantly different at the 5% level I duffii leaf extract (IDLE), I duffii flower extract (IDFE), P lanuginosa leaf extract (PLLE), P scandens leaf extract (PSLE), N cadamba leaf extract (NCLE), N cadamba stem bark extract (NCSBE), N cadamba root extract (NCRE), N orientalis leaf extract (NOLE), N orientalis stem bark extract (NOSBE), N orientalis root extract (NORE) H.s corymbosa extract (HCE) and H diffusa extract (HDE) 4.3.2.3 Total antioxidant capacity of the extracts The total antioxidant capacity (phosphomolybdenum method) of the extracts was determined based on the antioxidant equivalent of trolox standard and EC50 Based on EC50 in Table 4.9, there were 10 extracts with total antioxidant capacity except for H corymbosa and H diffusa 13 Table 4.9: EC50 of extract in phosphomolybdenum method Linear regression equation EC50 (g/mL) PLLE y=0.0041x+0.2093 (R²=0.997) 71.5±1.23b PSLE y=0.0041x+0.1667 (R²=0.996) 81.2±0.55a IDLE y=0.0062x+0.1717 (R²=0.994) 52.9±0.3e IDFE y=0.007x+0.1563 (R²=0.995) 48.8±0.27f NOLE y=0.0061x+0.1707 (R²=0.997) 54.5±0.36d NOSBE y=0.0073x+0.2261 (R²=0.988) 37.1±0.05i NORE y=0.0042x+0.2448 (R²=0.991) 61.2±0.27c NCLE y=0.0084x+0.2336 (R²=0.997) 31.5±0.17j NCSBE y=0.0076x+0.1864 (R²=0.995) 40.9±0.22h NCRE y=0.0076x+0.1379 (R²=0.993) 47.4±0.59g HCE - - HDE - - y=0.145x+0.163 (R²=0.989) 2.32±0.08k Extract Trolox Note: The values shown are mean±SEM, n=3; Values followed by the same letter in the same column are not significantly different at the 5% level I duffii leaf extract (IDLE), I duffii flower extract (IDFE), P lanuginosa leaf extract (PLLE), P scandens leaf extract (PSLE), N cadamba leaf extract (NCLE), N cadamba stem bark extract (NCSBE), N cadamba root extract (NCRE), N orientalis leaf extract (NOLE), N orientalis stem bark extract (NOSBE), N orientalis root extract (NORE) H.s corymbosa extract (HCE) and H diffusa extract (HDE) Through surveying the antioxidant capacity of 12 extracts, there were 10 extracts for antioxidant capacity, including N cadamba leaf, N orientalis stem bark, N cadamba stem bark, N cadamba root, I duffii flower, I duffii leaf, N orientalis leaf, N orientalis root, P lanuginosa leaf, P scandens leaf However, the collection of I duffii flower samples was limited, so I.duffii flower was not selected for further study 14 4.3.3 In vitro anti-inflammatory effects of extracts The anti-inflammatory effect of the extract was determined based on its inhibition of BSA protein denaturation and EC50 EC50 of the extracts presented in Table 4.11 showed that, all studied extracts had antiinflammatory activity, EC505000 mg/kg Results of the subchronic toxicity test that the extract of N orientalis root extract did not cause toxicity in mice The methanolic extract of N orientalis root was extracted fractions and quantified total polyphenols, flavonoids, alkaloids contents, tested for antioxidant, anti-inflammatory and hepatoprotective activities The ethyl acetate fraction with the highest activities was selected for compound isolation The study isolated and determined the structure of compounds from the ethyl acetate fraction, including naucleficine and 3-O-rhamnoside quinovic acid compounds 5.2 Recommendations Continue to study the isolation of substances to determine the remaining substances in the ethyl acetate fraction of N orientalis root Study some biological activities such as antioxidant, antiinflammatory and hepatoprotective activities of isolated compounds Investigation of antioxidant and hepatoprotective activities of a mixture of some effective extracts in this study LIST OF WORKS HAS BEEN PUBLISHED Phan Kim Dinh, Dai Thi Xuan Trang, 2017 Study on the antioxidant and anti-cancer activities in HEPG2 cells of Ixora duffii Science and Technology Development Journal, 1(6) 2017 13-21 Phan Kim Dinh, Nguyen Trong Tuan, Dai Thi Xuan Trang, 2018 Studies on in vitro antioxidant and antiinflammatory activity of the methanol leaf extract of Mo leo (Paederia scandens L.) Journal of Anatytical Sciences, 3, 2018 152-158 Phan Kim Dinh, Nguyen Thi Thanh Lan, Nguyen Trong Tuan, Dai Thi Xuan Trang, 2018 Study on hepatoprotective activity of the methanol leaf extract of skunkvine (Paederia scandens L.) on carbon tetrachloride-induced hepatic damage in mice Can Tho UniversityJournal of Sciences, 54(7), 2018 94-100 Phan Kim Dinh, Nguyen Thi Thanh Lan, Dai Thi Xuan Trang, 2019 Antioxidant and hepatoprotective activities of the methanol leaf extract of Paederia lanuginose W Journal of Biotechnology, 17(1) 2019 157-166 Phan Kim Dinh, Nguyen Trong Tuan, Dai Thi Xuan Trang, 2019 Antioxidant, anti-inflammatory and hepatoprotective activities on carbon tetrachloride-induced hepatic damage in mice of Ixora duffii leaf extract Journal of Biology, 41(1) 2019 117-128 Phan Kim Dinh, Nguyen Trong Tuan, Dai Thi Xuan Trang, 2019 Antioxidant and hepatoprotective activities of the methanol leaf extract of Neolamarckia cadamba (Roxb.) Bosser Can Tho UniversityJournal of Sciences, 55(5), 2019 24-31 ... largest organs in the human body, playing an important role in metabolism and excretion (Ahsan et al., 2009) In the process of performing the function, the liver is the first and continuously attacked... 2009) In the process of performing the function, the liver is the first and continuously attacked organ by toxic substances, resulting in liver damage and dysfunction (Bodakhe and Ram, 2007) There... damage Most of these agents cause oxidative stress, producing many free radicals in the liver that attack biological macromolecules that damage liver cells The chemicals are hepatotoxic mainly by