Summary of doctoral thesis: Chemical investigation and immunomodulatory potential of Euphorbia hirta L. in the Mekong Delta on striped catfish

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The objectives of this study are to investigate the main chemical compositions of Euphorbia hirta L. to search for chemical constituents in relation to biological activities, contributing to further studies to create products for application in the aquarculture industry in particular and for life in general.

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Nội dung Text: Summary of doctoral thesis: Chemical investigation and immunomodulatory potential of Euphorbia hirta L. in the Mekong Delta on striped catfish

MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ------------------- LE THI BACH CHEMICAL INVESTIGATION AND IMMUNO-MODULATORY POTENTIAL OF EUPHORBIA HIRTA L. IN THE MEKONG DELTA ON STRIPED CATFISH Major: Organic Chemistry Code: 9 44 01 14 SUMMARY OF DOCTORAL THESIS Can Tho – 2020
INTRODUCTION 1. The urgency of the thesis Vietnam is one of Asian leaders in aquaculture production and has experienced extraordinary growth rates in recent years. However, aquaculture in Vietnam is coping with many problems, of which disease outbreaks from parasites, fungi and bacteria are among the most important concerns. Chemicals or antibiotics have been applied quite extensively to control pathogens, sometimes under improper management. The application of these chemicals brings a lot of disadvantages such as their bioconcentration in cultivated organisms, human carryover, pollution, development of antibiotic resistant bacteria, destabilization of helpful bacterial populations in environment and immunosuppression in fish. For that reason, nowadays, more environmental-friendly prophylactic and preventive solutions are required and natural bio-active products are searched for to enhance the immune system and health status of cultivated animals. From this perspective, bio-products resulting from natural plants have attracted considerable attention as a source of eco- friendly prophylactic compounds for using in the aquaculture industry. Vietnam is one of the tropical countries with diverve ecosystems and numerous plant compositions. According to botanists, Vietnam has possessed about 12.000 species of higher plants, 800 species of mosses and 600 species of mushrooms. Of which, more than 5000 species of medicinal plants, from the past, have been used as traditional medicines for disease treatments and 1
for health improvement. Especially, more than 90% of them are natural, wild medicinal plants. This is an extremely valuable source of natural compounds that need to be studied their bioactivities and chemical properties in order to find out active compounds that can be developed into products for agriculture, aquaculture and human life. Euphorbia hirta L. has the effect of purifying, detoxifying, clearing, antipruritic, and has been used by folk medicine for the treatment of coughing, colds, runny nose, pneumonia and asthma, thanks to bronchodilator effect. According to our initial screening results on the effect of strengthening the immune system of Pangasianodon hypophthalmus (Sauvage), this species showed good activity. However, study on the chemical constituents and biological activities about Euphorbia hirta L. is rather rare. Therefore, we carried out this thesis: "Chemical investigation and immuno- modulatory potential of Euphorbia hirta L. in the Mekong Delta on striped catfish", with the aims at contributing to the knowledge of the chemical composition as well as bioactivities of this species in Vietnam. 2. The objectives of the thesis The objectives of this study are to investigate the main chemical compositions of Euphorbia hirta L. to search for chemical constituents in relation to biological activities, contributing to further studies to create products for application in the aquarculture industry in particular and for life in general. 3. The main contents of the thesis 2
1. Screening of medicinal herbs for ability of strengthening the immune system of Pangasianodon hypophthalmus; 2. Isolation of compounds from the whole plant of Euphorbia hirta L. collected in Can Tho city; 3. Determination of chemical structures of isolated compounds from Euphorbia hirta by physical and chemical methods; 4. Evaluation of immunomodulatory effects on the striped catfish; antibacterial activity; antioxidant resistance and the protection efficiency on the MIN6 cell from the ER stress of fractioned extracts and some isolated pure compounds. CONTENT OF THE THESIS CHAPTER 1. OVERVIEW The overview on national and international studies about issues: + General plant characteristics, chemical compositions and biological activities of genus Euphorbia. + Plant characteristics, folk use, chemical compositions and biological activities of Euphorbia hirta L. + Overview of immunity. CHAPTER 2. EXPERIMENTS 2.1 Samples, chemicals and equipments 3
2.1.1 Samples Based on bibliography review data and on a survey in fish farms of the Mekong Delta, 20 plants possessing potential immunostimulatory activities were selected for in vitro bioactivity evaluation. The whole plants of E. hirta were collected at Can Tho city - Vietnam, in February 2016. Identification and authentication of the plant were carried out by Dr. Dang Minh Quan- a taxonomist at Can Tho University and a voucher specimen was deposited. 2.1.2 Chemicals and equipments  Chemicals Solvents including n-hexane, chloroform, ethyl acetate, n- butanol, methanol (purity ≥ 99.0%), and ethanol 96% were purchased from Chemsol company (Vietnam). Silica gel Merck 60 F254; RP-18 and Sephadex LH-20 (GE Healthcare).  Equipments NMR spectra were recorded on a Bruker AM500 FTNMR spectrometer (Bruker, Karlsruhe, Germany) using TMS as an internal standard, at the Institute of Chemistry - Vietnam Academy of Science and Technology, and also on a Bruker AVANCE (600 MHz) in Kyoto Institute of Technology, Japan. HR-ESI-MS was also performed on SCIEX X500 QTOF at Institute of Chemistry - Vietnam Academy of Science and Technology, and also on 4
MicroTOF, Bruker Daltonics Inc., Billerica, MA, USA, at Kyoto Institute of Technology, Japan. Melting point was measured on SMP3. UV-Vis spectra were recorded on Jasco V-730. The specific rotation was measured on ADP 440. All these experiments were conducted at the Department of Chemistry, College of Natural Sciences, Can Tho University. 2.2 Methods 2.2.1 Preparation of extracts From 20 plant samples collected from the Mekong Delta in Vietnam, the powdered plant material of each type (100 g) was cold- extracted using 96% ethanol for one day with occasional shaking. This was repeated 5 times to ensure complete extraction of the chemical constituents of the plant. The extracts were combined, filtered and the filtrate was evaporated to dryness using a rotary evaporator at 40ºC to obtain 20 types of crude ethanol extracts. 2.2.2 Isolation of compounds from E. hirta Using thin layer chromatography (TLC) and column chromatography (CC). 2.2.3 Chemical characterization of isolated compounds Compounds were isolated and identified from the extracts of E. hirta L. by analysis UV-Vis, ESI-, HR-ESI-MS, NMR spectra, and comparison with literature data. 2.2.4 Biological activities 5
2.2.4.1 Immuno-modulatory potential of different plant extracts using striped catfish (Pangasianodon hypophthalmus) leukocyte-based in vitro tests Experimental fish: Farm-raised striped catfish juveniles (body weight = 50 ± 5 g) were obtained from a local fish farm in Vinh Long province, Vietnam, and transported to the laboratory in plastic bags filled with oxygenated water. The fish were acclimatized to laboratory conditions for 15 days then maintained into composite tanks (250 L) under a flow-through freshwater supply system, and fed twice a day with the formulated diets at a rate of 2% of their body weight/day. Few randomly sampled animals were examined for the presence of any abnormal lesions or parasites on body surfaces and internal organs. In vitro stimulation of cells and detection of immune parameters: After isolation of striped catfish peripheral blood mononuclear cells (PBMCs) and head kidney leukocytes (HKLs), five hundred μL of cell suspension (5 × 106 cells/mL) in L-15 medium supplemented with 5% FBS, 1% Hepes and 1% of a T-cell- specific mitogen agent, phytohemagglutinin A were added to each well of 48-well plate (Greiner Bio-One, Vilvoorde, Belgium). Afterward, leukocytes stimulation was carried out with 20 ethanol plant extracts to reach final concentrations at 10 and 100 μg/mL. Cells cultivated in the same medium containing 1% DMSO served as control. Each experiment was realized in triplicates. The humoral immune response was assessed for 24 hrs at 28ºC in a humidified atmosphere of 5% CO2. Collected leukocyte membranes were disrupted by 50 μL lysis buffer (50 mM tris HCl, 150 mM NaCl, 6
0.1% Triton X 100, PMSF 0.1 μg/mL). Samlpes were centrifuged at 2000 rpm for 10 min to remove debris. Supernatants were collected for immune assays (Lysozyme assay, Complement assay and Total immunoglobulin assay). 2.2.4.2 Antibacterial activity Antibacterial activity of the extract, fractions and some isolated compounds were evaluated using disc diffusion and microplate dilution methods. Cefixim and Vancomycin HCl were used as the positive control and the extracting solvents were used as negative controls. The zone of inhibition of the negative controls was subtracted from the zone of the plant extracts so as to find the true zone of inhibition of the extract. This experiment was done in triplicates. The test organisms used in this study consisted of reference strains obtained from Research Institute for Aquaculture No. 2, Ho Chi Minh city, namely Aeromonas hydrophila-N, Aeromonas dhakensis-D, Aeromonas hydrophila-D, Aeromonas dhakensis-W and Vibrio parahaemolyticus. 2.2.4.3 Antioxidant activity Antioxidant activity of the crude extract, fractions and some isolated compounds were investigated using two antioxidant assays namely DPPH, ABTS•+ radical scavenging effect. Total phenol, flavonoid contents were also estimated. In addition, this is the first time the antioxidant capacities of medicinal plant Euphorbia hirta L. had been conducted on pancreatic -cells MIN6 exposed to hydrogen peroxide-oxidative stress conditions which was more 7
biological than the popular chemistry antioxidant activity measures as DPPH, ABTS•+. 2.2.4.4 The protective effect on pancreatic β-cells MIN6 Thapsigargin (TG) 2 µM was used as a model to induce ER stress and β-cell death. The effects of extracts or isolated compounds from E. hirta that protect MIN6 cells from the death were assessed by comparing the survival rate of the cell group treated with TG and then treated with the samples compared with the group treated only with TG. Cell survival was assessed by a colorimetric method based on the activity of dehydrogenase enzyme. CHAPTER 3. RESULTS AND DISCUSSION 3.1. Screening of immuno-modulatory potential of different plant extracts using striped catfish (Pangasianodon hypophthalmus) leukocyte-based in vitro tests Based on bibliography review data and on a survey in fish farms of Mekong Delta, 20 plants possessing potential immunostimulatory activities were selected. Table 3.1 The list of 20 crude ethanol extracts No Specimens Scientific name Place of Part Yield collection studied (%) 1 Dây vác Cayratia trifolia (L.) Sóc Trăng Aerial 7.51 Domin part 2 Rau sam Portulaca oleracea Vĩnh Long Whole 5.73 L. plant 3 Tỏi Allium sativum L. Cần Thơ Bulbs 6.87 4 Húng quế Ocimum basilicum Cần Thơ Aerial 5.15 L. part 8
5 Bình bát Annona reticulata L. Sóc Trăng Leaves 13.71 nước 6 Cỏ sữa lá Euphorbia hirta L. Cần Thơ Whole 5.38 lớn plant 7 Gừng Zingiber officinale Cần Thơ Bulbs 5.65 Rosc. 8 Rau má Centella asiatica Hậu Giang Whole 5.41 (L.) Urb plant 9 Sài đất Wedelia chinensis Cần Thơ Whole 5.76 (Osbeck) Merr. plant 10 Diệp hạ Phyllanthus amarus Sóc Trăng Aerial 6.29 châu L. part 11 Sầu đâu Azadirachta indica Cần Thơ Leaves 8.84 A. Juss 12 Ổi Psidium guajava L. Vĩnh Long Leaves 10.41 13 Trầu Piper betle L. Hậu Giang Leaves 5.47 không 14 Mướp Momordica Vĩnh Long Aerial 5.98 đắng charantia L. part 15 Giấp cá Houttuynia cordata Vĩnh Long Aerial 8.93 Thunb. part 16 Mắc cỡ Mimosa pudica L. Cần Thơ Aerial 6.28 part 17 Tía tô Perilla frutescen (L.) Cần Thơ Aerial 5.42 Britt part 18 Cỏ cứt lợn Ageratum Hậu Giang Aerial 8.40 conyzoides L. part 19 Rau dệu Alternanthera Cần Thơ Aerial 6.10 sessilis (L.) A. DC part 20 Cỏ mực Eclipta prostrata Cần Thơ Aerial 5.07 (L.) L. part 9
Our results suggest a positive contribution of several herbal extracts to increase immune responses in a dose dependent manner in striped catfish PBMCs and HKLs after 24 hrs. Based on the achieved results, Euphorbia hirta L. has selected on chemical composition, and also investigated on antibacterial, antioxidant and cell-protective abilities on extracts as well as on some pure compounds isolated from this species. 3.1 Extraction and isolation of compounds from E. hirta L. 3.1.1 Preparation of various extracts 3.1.2 Isolation of compounds From the various extracts, with the appropriate solvent systems, 22 compounds were isolated on the chromatographic column. 3.1.3 Physical constants and spectral data of compounds isolated from E. hirta This section details the physical characteristics, spectral data of 22 compounds isolated from E. hirta. 3.2 Results about immune parameters in PBMCs and HKLs of 20 plant extracts Most examined extracts, which were selected by their previously known for influence on the immune response, stimulated the release of lysozyme activity after 24 hrs in PBMCs and/or HKLs (both 10 and 100 µg/mL). In addition, the complement activity levels increased in cells treated with several extracts compared with control treatment at 24 hrs. In both PBMCs and HKLs, total Ig activity was 10
noticed to be statistically higher in most plant extracts treated groups compared with control group. A compilation of the humoral immune results obtained in PBMCs and HKLs after stimulating with 20 plant extracts has been done in order to select the good extracts including Euphorbia hirta L. Although Euphorbia hirta L. has been studied in several countries around the world; in Vietnam, this medicinal plant has not been used much in the pharmaceutical industry. Although E. hirta has many medicinal effects in folk, many valuable biological activities but a few research works in Vietnam. For the above reasons, this thesis has selected Euphorbia hirta L. to conduct chemical composition research, and additionally evaluated some biological activities of the fractions as well as some isolated compounds from this medicinal plant. 3.3 The structural elucidation of the compounds 22 compounds were isolated from the various solvent extracts of Euphorbia hirta L., various chromatographic techniques were employed for the fractionation and purification of the isolates. Spectroscopic methods NMR, MS, HR-MS were employed for the structural elucidation of the isolated compounds. 11
Table 4.24 Sumarize of isolated compounds from Euphorbia hirta L. New compound Flavonoid compounds 12
13
Phenolic compounds Diterpenoid compounds 14
Triterpenoid Steroid compound 15
Other compound 2 7 1' HO 1 3 OH O O OH 4 6 6' 5 O 6" 1" H3 C O HO HO OH Eup18: 1'-O-benzyl-rutinoside 3.4 Results about immune parameters in HKLs Based on the achieved results, Euphorbia hirta L. has selected to investigate on increase immune responses in a dose dependent manner in striped catfish HKLs after 24 hrs at 10 and 100 μg/mL for extracts and 10 and 50 μg/mL for pure compounds. Most examined extracts and isolated compounds stimulated the release of lysozyme activity after 24 hrs in HKLs. At the dose of 100 μg/mL of crude, MeOH and n-hexane extracts significantly enhanced the lysozyme levels compared with control (p < 0.01) in HKLs. The strongest effect was observed in HKLs treated with 100 μg/mL of crude extract (p < 0.01). In addition, at the low dose of 10 μg/mL of quercitrin stimulate the lysozyme levels compared with control (p < 0.1), while taraxerol và rutin showed no statistical influence on lysozyme activity. The complement levels increased in cells treated with EtOAc, BuOH extracts as well as rutin at 10 µg/mL compared with control treatment at 24 hrs (p
Total Ig activity was noticed to be statistically higher in crude, MeOH and BuOH extracts treated groups at 24 hrs compared with control group (p
significant antibacterial activity against Aeromonas hydrophila-N (MIC = 1.5 µg/mL). Interestingly, the disc diffusion method result showed rutin showed antibacterial activities for A. hydrophila-D with zone of inhibition ranging from 13.60 to 29.33 mm at the concentration of 64 µg/mL; while vancomycin HCl presented zone of inhibition ranging from 9.33 to 19.00 mm at the same concentration. All examined samples exhibited varying antibacterial activity against the bacterial strains Aeromonas hydrophila-N. n-butanol extract showed the highest antibacterial activity Aeromonas dhakensis-W with MIC = 18 µg/mL. Vancomycin HCl showed good antibacterial activities for A. dhakensis-W with zone of inhibition ranging from 14.33±0.58 mm at 4 µg/mL to 20.67±2.52 mm at the concentration of 64 µg/mL with the value of MIC = 2.0 µg/mL. While cefixim showed no activity against this bacterial at the concentration of 4.0 µg/mL. MIC values of ethyl acetate fraction, taraxerol and Vancomycin HCl against Vibrio parahaemolyticus were 18.0; 2.0 and 3.0 µg/mL, respectively. The present results further confirm the activity of the extracts and constituents isolated from E. hirta against tested bacteria and justify the potential use of this medicinal plant in folk medicine, as well as expand our knowledge on the bacterial activity of this species. Some of the compounds isolated are candidates for further work to evaluate their therapeutic potential, especially in aquaculture. 18
3.6 Antioxidant activity 3.6.1 DPPH and ABTS•+ assays All the crude extract and fractions showed IC50 greater than 4.39 µg/mL against the DPPH radical. The IC50 values of the BuOH and MeOH extracts were 11.43 and 12.69 µg/mL. Furthermore, the IC50 values of quercitrin và rutin were 5.32 and 6.43 µg/mL; it was apparent that the pure compounds markedly influences the antioxidant activity of plant extract. The results obtained by ABTS•+ method has similar pattern in extract activity with those of the DPPH method. The ABTS•+ scavenging activity of crude extract and fractions expressed in the term of IC50 with the strongest antioxidant potency for the n-butanol extract (IC50 = 11.59 µg/mL); howerver this value was considerably higher than those obtained from the positive control trolox (IC50 = 2.90 µg/mL). Quercitrin và rutin were most active with IC50 values 6.92 and 7.57 µg/mL. Both the ABTS•+ and DPPH assays measure the total antioxidant activity of E. hirta. The results of both the assays are in agreement in that plant displayed the good antioxidant activitiy. The crude extract and fractions as well as some major compounds isolated from E. hirta were strong radical-scavengers, indicating that active compounds of different polarity are present in this species. The high antioxidant activities of this plant might be due to their flavonoid and phenolic contents. 19