Chemical investigation and immuno - Modulatory potential of euphorbia hirta l. in the mekong delta on striped catfish

ruhe, 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 5 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 6 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, 7 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 8 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 collection Part studied Yield (%) 1 Dây vác Cayratia trifolia (L.) Domin Sóc Trăng Aerial part 7.51 2 Rau sam Portulaca oleracea L. Vĩnh Long Whole plant 5.73 3 Tỏi Allium sativum L. Cần Thơ Bulbs 6.87 4 Húng quế Ocimum basilicum L. Cần Thơ Aerial part 5.15 9 5 Bình bát nước Annona reticulata L. Sóc Trăng Leaves 13.71 6 Cỏ sữa lá lớn Euphorbia hirta L. Cần Thơ Whole plant 5.38 7 Gừng Zingiber officinale Rosc. Cần Thơ Bulbs 5.65 8 Rau má Centella asiatica (L.) Urb Hậu Giang Whole plant 5.41 9 Sài đất Wedelia chinensis (Osbeck) Merr. Cần Thơ Whole plant 5.76 10 Diệp hạ châu Phyllanthus amarus L. Sóc Trăng Aerial part 6.29 11 Sầu đâu Azadirachta indica A. Juss Cần Thơ Leaves 8.84 12 Ổi Psidium guajava L. Vĩnh Long Leaves 10.41 13 Trầu không Piper betle L. Hậu Giang Leaves 5.47 14 Mướp đắng Momordica charantia L. Vĩnh Long Aerial part 5.98 15 Giấp cá Houttuynia cordata Thunb. Vĩnh Long Aerial part 8.93 16 Mắc cỡ Mimosa pudica L. Cần Thơ Aerial part 6.28 17 Tía tô Perilla frutescen (L.) Britt Cần Thơ Aerial part 5.42 18 Cỏ cứt lợn Ageratum conyzoides L. Hậu Giang Aerial part 8.40 19 Rau dệu Alternanthera sessilis (L.) A. DC Cần Thơ Aerial part 6.10 20 Cỏ mực Eclipta prostrata (L.) L. Cần Thơ Aerial part 5.07 10 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 11 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. 12 Table 4.24 Sumarize of isolated compounds from Euphorbia hirta L. New compound Flavonoid compounds 13 14 Phenolic compounds Diterpenoid compounds 15 Triterpenoid Steroid compound 16 Other compound 6' O OH OH HO O O OH HO HO H3C 1" 6" O 1 2 3 4 5 6 7 Eup18: 1'-O-benzyl-rutinoside 1' 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<0.01). 17 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<0.01). The level of total Ig was found to be the highest in BuOH extract (at 100 μg/mL), increased two times than those of the control. However, no significant changes were observed in HKLs stimulated with examined purified compounds. In the present study, the immunomodulatory effects of E. hirta on the lysozyme and complement activities as well as on the total immunoglobulin in the striped catfish HKLs were analyzed. These results indicated that the humoral immune responses was activated in striped catfish by E. hirta. 3.5 Antibacterial activity The antibacterial activities of the extract and fractions as well as pure compounds isolated from E. hirta were carried out with the organisms, namely Aeromonas hydrophila-N, Aeromonas dhakensis- D, Aeromonas hydrophila-D, Aeromonas dhakensis-W and Vibrio parahaemolyticus which are major pathogens for the aqualculture industry. The plant showed significant antibacterial activity against almost all the organisms except n-hexane extract. It is apparent that the crude extract and fractions of E. hirta posses varying degree of different antibacterial activity against tested bacteria. The ethyl acetate fraction was the most active fraction across selected bacterial while the n-hexane fraction showed the least activity among the selected bacteria. The ethyl acetate extract displayed good antibacterial activities against Aeromonas dhakensis-D. and V. parahaemolyticus (MIC = 18 µg/mL). Rutin and taraxerol showed 18 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. 19 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. 20 3.6.2 Antioxidant activity against H2O2-induced cytotoxicity 3.6.2.1 Cytotoxicity assay This result shows that DMSO 0.5% has no toxicity to MIN6 cell lines. In addition, in the culture media with different concentrations of tested samples, the cell survival was not significantly different for the control group (only DMEM medium). This result shows that the examined samples at the concentrations studied (0.01 - 10.00 mg/mL) have no toxicity to MIN6 cells. 3.6.2.2 Antioxidant activity against H2O2-induced cytotoxicity + Effects of the concentration of H2O2 on cell viability The toxic effects of hydrogen peroxide to MIN6 cells were investigated with different concentrations of H2O2. This result demonstrated that the treatment with 0.1 to 0.5 mM H2O2 alone for two hours induced significant cell death compared with the blank control experiment and the effect was dose-dependant. On the basis of these results and some previous reports, 0.4 mM H2O2 (49% survival) was chosen for the following experiments. + Effects of crude extract and different fractioned extracts on cell viability Crude extract and some fractioned extracts including n- hexane, ethyl acetate, n-butanol and methanol fractions were tested for protective effect on H2O2-induced MIN6 cells. The addition of extracts improved cell viabilities compared with the H2O2 treated cultures, with the exception of the n-hexane extract at 0.01 mg/mL. The ethyl acetate extract showed the strongest protective activities 21 and yielded the maximum cell viability of 81% at the dose of 0.1 mg/mL comparable to vitamin E (84% at 0.2 mg/mL). The crude extract gave the highest cell viability of 78% at the dose of 1.0 mg/mL, while the methanol extract exhibited relatively good antioxidant activity and the apolar extract (n-hexane extract) was less effective. We also observed that the highest concentrations (10 mg/mL) have less positive effects, which may perhaps be explained by some cytotoxicity of the extracts. + Effects of the isolated compounds on cell viability The six compounds protected MIN6 cells from H2O2 toxicity from 1.0 to 1000 µg/mL. Quercetin and luteolin at the dose of 100 µg/mL had the strongest protective effect, comparable with Vitamin E (0.2 mg/mL, 84%). Quercetin and luteolin have the best effect at 100 µg/ml (82 and 78% cell viability, respectively), while other compounds were also active but less effective. In addition, quercitrin (10 µg/mL) and rutin (100 µg/mL) gave the cell protection effectively (65 and 70% cell viability, respectively). Taraxerol showed no cell protection at the concentration of 100 µg/mL and only 61% cell protection at 1000 µg/mL. It is noteworthy that individual compounds possessed significant antioxidant activities while ethyl acetate extract could protect pancreatic β-cells against hydrogen peroxide-induced toxicity remarkably. Flavonoids, known as sensitive to oxidative stress, were also found as the one of most commonly phytochemicals from E hirta L. and the effects of which could come from their polyphenol structures. 22 The results of this study revealed that Euphorbia hirta L. collected in Can Tho city showed high potency of anti-oxidative property. It is a naturally antioxidative plant and worth testing for further pharmacological investigations in the treatment of oxidative stress as for example those related to neurological diseases. 3.7 The protective effect of some extracts and isolated compounds from E. hirta on pancreatic β-cells MIN6 The cells were pretreated with different samples for 24 hours and then 2 µM TG (a concentration inducing about 60%cell death) was added for 24 additional hours. CCK-8 assay was performed to determine the cell viability. TG decreased cell viability compared to control, but pre-treatment with plant extracts (0.01–10 mg/mL), the percentage of cell viabilities was improved. The MeOH extract (0.1 mg/mL) showed the strongest protective effect giving a maximum cell viability of 69%. The crude extract with the concentration 1.0 mg/mL gave the lower cell viability of 68%. The values of % cell viability of ethyl acetate, BuOH and n-hexane extracts treated groups ranged (59-64)%, (58- 65)%, and (54-58)%, respectively at concentration of (0.01-10) mg/mL. The six selected compounds protected MIN6 cells from TG toxicity from 1.0 to 1000 µg/mL. All compounds promoted cell survival against TG-induced cytotoxicity of MIN6 cells. The highest protective effect against TG was observed for quercitrin (78%, 10 µg/mL). Depending on the concentration (1.0-1000 µg/mL), the % cell viability of luteolin and quercetin treated groups ranged from 23 66% to 69%, from 62% to 70%, respectively. As a result, quercetin and luteolin have proved to enhance the protective effect against toxicity in MIN6 cells and showed their maximum efficacies at the concentration of 10 µg/mL, and caffeic acid also showed the highest cell-protective activity (68%) at this concentration. The above results indicated that the crude extract and all the fractioned extracts as well as some examined constitutents have enhanced the protective effect against toxicity in MIN6 cells. Our results support the potential antidiabetic effect of E. hirta from the Mekong Delta of Vietnam. CONCLUSION AND FUTURE WORKS 1. Conclusion  The chemical investigation of the whole plant Euphorbia hirta L. collected in Can Tho City led to the isolation of 22 compounds. The structures of the isolated compounds were elucidated using the NMR, MS techniques and the compounds were identified as sodium β-D-glucopyranosyl 12-hydroxyjasmonate (Eup16, a new compound) and 21 known compounds including 11 flavonoid: quercitrin (Eup01), rutin (Eup02), avicularin (Eup04), astragalin (Eup05), luteolin (Eup08), quercetin (Eup09), (+)- catechin (Eup11), afzelin (Eup14), myricitrin (Eup15), hymenoxin (Eup19) and cynaroside (Eup21); three phenolic compounds: caffeic acid (Eup10), protocatechuic acid (Eup12) and gallic acid (Eup13); five triterpenoids: taraxerol (Eup06), taraxerone (Eup07), 3β,7β,25- trihydroxycucurbita-5,(23E)-dien-19-al (Eup03), cycloart-23-ene- 3β,25-dioltrifolin (Eup17) and campesterol (Eup20); one 24 diterpenoid: 2β,16α,19-trihydroxy-ent-kaurane (Eup23) as well as another compound: 1-O-benzyl-rutinoside (Eup18). In which, there are seven compounds that were isolated for the first time from this species: avicularin (Eup04), (+)-catechin (Eup11), hymenoxin (Eup19), cynaroside (Eup21), 3β,7β,25-trihydroxycucurbita- 5,(23E)-dien-19-al (Eup03), cycloart-23-ene-3β,25-dioltrifolin (Eup17) and 1-O-benzyl-rutinoside (Eup18).  The results of bioactivities have shown:  The immunomodulatory effects on the lysozyme and complement activities as well as on the total immunoglobulin in the striped catfish blood mononuclear cells (PBMCs) and head kidney leukocytes (HKLs) indicated that the humoral immune responses was activated by E. hirta.  Compound Eup02 (rutin) displayed significant antibacterial activity against A. hydrophila-D with zone of inhibition as 29.33 ±0.35 at the concentration of 64 µg/mL. Similarly, compound Eup06 (taraxerol) gave zone of inhibition 24.50±0.44 mm against A. hydrophila-N at the same concentration of 64 µg/mL.  Compound Eup01 (quercitrin) and Eup02 (rutin) showed IC50 values as 5.32; 6.43 and 6.92; 7.57 µg/mL, similar to than those obtained from the positive control: vitamin C (3.66 µg/mL) and trolox (IC50 = 2.90 µg/mL) for the ABTS•+ and DPPH assays, respectively. In addition, the antioxidant capacities of fractioned extracts and isolated compounds from Vietnamese E hirta L. had been conducted on pancreatic -cells MIN6 exposed to hydrogen peroxide-oxidative stress conditions and compared with positive 25 control vitamin E (0.2 mg/mL). The results showed that EtOAc extract (0.1 mg/mL) and compound Eup09 (quercetin) at 100 µg/mL gave the cell protection effectively (81 and 82% cell viability, respectively), comparable with vitamin E (0.2 mg/mL, 84%).  E. hirta also enhanced the protective effect against toxicity in MIN6 cells. The MeOH extract (0.1 mg/mL) and compound Eup01 (quercitrin) showed the strongest protective effect giving a maximum cell viability of 69% and 78%. 2. Future works From above results on the chemical composition and biological activity of Euphorbia hirta L., it is clear that there are many flavonoid compounds (11/22 substances) isolated as well as diverse biological applications of this species. However, at present, Euphorbia hirta L. has not been applied and exploited much in Vietnam. Therefore, more biological and pharmacological studies are needed to confirm the scientific value of this plant. Research on E. hirta should be encouraged for the development and production of new pharmaceuticals or herbal extracts of therapeutic value. 3. New findings of the thesis For the first time