The present study was carried out to investigate the antioxidant and antibacterialactivities of ethyl acetate, n-butanol and ethanol extracts of Breynia vitis-idaeaBurm.f.. Ethyl acetate
Isolation, refinement and structure determination of compound
Find the suitable development solvent system by thin layer chromatography
The name of Determine structure by spectrometry d compoun
Diagram 2.5 General process of purification and determination of structure of compound
After choosing the suitable process of extraction, the amount of material was increased up to 200 g Then | liter of ethanol 96% was added, standing for 24 hours at room temperature and the first extract was collected The ground was extracted twice more Each time was with | liter of ethanol 96% in 24 hours at the same condition above The extracts were combined and concentrated to constant weight in a rotary shaker evaporator to get ethanol residue The ethanol residue (80 g) was removed impurities by shaking with ten 80-ml proportions of n-hexane and the layer of n- hexane was removed The rest after being shaken with n-hexane was extracted with fifteen 80-ml proportions of ethyl acetate The extracts were collected and concentrated to get 14.2 g of ethyl acetate residue (see diagram 2.6).
>| Shake with 80 ml of n-hexane —+ Layer of n-hexane /
Ad ằ Shake with 80 ml of ethyl acetate y
15 times j The residue / / Layer of ethyl acetate /
Diagram 2.6 Process of handling filtrate 2.2.4.2 Survey development solvent systems by thin layer chromatography
+ Systems of solvents for development:
S3: chloroform: ethyl acetate: formic acid = 7.5: 2: 0.5
S5: chloroform: ethyl acetate: formic acid = 5: 4: 1
S6: chloroform: ethyl acetate: formic acid = 6.5: 3: 0.5
+ Detect spots by observing under UV 254 nm, 365 nm ultraviolet lamp or make spots appear by using 1% FeCl, solution.
2.2.4.3 Separation on silica gel column
Preparation of chromatography column: a column of glass which had 4-cm diameter and 60-cm length was assembled vertically on a support; below the column had a valve to adjust speed of solvent This valve was closed and a small amount of solvent was added into the column The bottom of the column was lined by a piece of cotton so that silica gel after being cramed into couldn’t obstruct the column.
Filling the column: 200 g of silica gel and 500 ml of n-hexane were mixed carefully and poured in the column slowly The valve was opened in order for solvent to run slowly and silica gel to deposit (a glass stirring rod covered by rubber should be used to knock slightly on the column in order to impulse process of settlement) During this time, solvent was added so that the column was not dry Finally, the distance between upper side of filler and upper side of solvent was kept about 10 cm, closed the valve The column was stabilized over night.
Loading sample: 5 g of ethyl acetate extract was dissolved by 50 ml of methanol. Then 12.5 g of silica gel was added, mixed carefully and let the solvent evaporate completely to get a mixture of scattered powder This powder was dissolved by 50 ml of n-hexane and transferred lightly to the column, avoiding disordering the filler in the column Before adding developing sovent, it had been necessary to rinse around the inner side of the column by that solvent, using a pipette The systems of developing solvents were showed in table 2.1.
Table 2.1 Ingredient and volume of systems of developing solvent in column chromatography
Ratio of solvents (v/v) Volume Number of System n-hexane | Ethyl acetate | Methanol (ml) erlens
Speed of flow was adjusted at 10 ml/minute Solution was caught by 100-ml erlens All of the solution in erlens was checked by thin layer chromatography The samples which had the same result of TLC were combined and waited for evaporating solvent and crystalizing.
2.2.4.4 Structure determination of the isolated compounds by spectrometry
High-resolution mass spectrometry (HRMS), one-direct nuclear magnetic resonance (H-NMR, C-NMR, and DEPT) and two-direct nuclear magnetic resonance (COSY, HSQC and HMBC) were used to find specific structural characteristics of the isolated compounds These experiments were conducted inCentral Laboratory for Analysis, Ho Chi Minh City University of Science.
RESULTS Q.7 SG HH ghe 35 3.1 Preliminary experiMent 5G <0 0g re 35 3.1.1 Identification of herbal DẽATIE - ô<1 1 ng ng, 35 3.1.2 Moisture content and total ash content of mater1aè -ôô s sôô+ 35 3.1.3 Experiments of preliminary phytochemical composIfIon
Bioactivities of fractionated €XÍTACẨS - G SH ng ke 38 3.3 Enzyme-assIsted €XITACẨIOIN - - G00 gọn re 39 3.3.1 Effect of enzyme conC€rIfFAfIOTIS - - - - <5 ôsgk, 39 3 Effect of treatment ẨITT© - - - <5 - + 111111 9.1 ng vn 43 3.4 Results of purification and structure determination of compounds
Choosing solvent must depend on properties of material, active elements and impurities that are available in material The requirement for solvent is its ability to extract the most quantity of active elements and the least quantity of impurities To meet this demand, it is necessary to combine several solvents It is noticeable that the least volume of solvent should be used for extracting in order not to last time for concentrating later Ethanol is an organic solvent which has strong polarity and can dissolve many compounds, so it is usually used to get total extract From the total extract, a lot of fractions are subdivided In this study, two smaller partitions obtained were ethyl acetate (EtOAc) and n-butanol (n-BuOH) Two these extracts together with ethanol extract (EtOH) were tested with antimicrobial and antioxidant activities The results were presented in Table 3.4, 3.5 and 3.6.
Antimicrobial activity of three extracts was relatively low EtOH extract and EtOAc extract could only inhibit Gram-positive bacteria (E faecalis, S aureus and MRSA) but could not resist Gram-negative bacteria (E coli, Sal typhi and P. aeruginosa) whereas n-BuOH extract didn’t have any activity in all tested bacteria. MIC values of EtOAc extract in Gram-positive bacteria were two folds lower than that of EtOH extract; this demonstrated that activity of EtOAc extract was the highest.
Table 3.4 Inhibition zone diameter of the extracts on tested bacteria (mm)Extract E coli E faecalis Sal typhi P aeruginosa S qureus MRSAEtOH 11 16 14 12 18 16EtOAc 9 13 9 15 15 16 n-BuOH 12 9 9 11Gentamycin 27 25 26 33 34 35
Table 3.5 MIC value of the extracts on tested bacteria (mg/ml) Extract E coli E faecalis Sal typhi P aeruginosa S qureus MRSA EtOH - 3 - - 2 1.5 EtOAc - 1.5 - - 1 1 n-BuOH - - - -
Table 3.6 Antioxidant activities of the fractionated extracts from B vitis-idaea
DPPH ABTS EtOH 109 + 1.66 115.5 +0.88 EtOAc 99,55 + 1.26 94.66 + 0.69 n-BuOH 370.3 + 2.48 181.1 + 0.68 Vitamin C 16.36 + 1.46 16.21 +2.48
All of the three extracts of B vitis-idaea showed significant antioxidant activity in two different methods of antioxidant assays tested (Table 3.6) The highest DPPH scavenging activity was observed in EtOAc (ICs59 99.55 + 1.26 ug/ml) followed by EtOH (109 + 1.66 ug/ml) and n-BuOH (370.3 + 2.48 ug/ml) Similar trend was observed in scavenging activity of ABTS (EtOAc > EtOH > n-BuOH) The activities of these extracts were concentration-dependent.
From the above data, it could be inferred that EtOAc extract had the highest bioactivity because it contained the most bioactives (the fraction rich in glycosides, see table 3.3) This fraction was considered as a standard to evaluate efficiency of enzyme- assisted extraction and used in isolation and purification experiments to get pure compound.
The experiments were conducted as in section 2.2.3 with enzyme concentrations of 1,2,3,4,5 and 6% (v/w) and the control (sample without enzyme, marked 0) The obtained results of efficiency of extraction process and content of ethyl acetate extract were recorded in Table 3.7 and illustrated in Figure 3.2 and 3.3.
Table 3.7 Efficiency of extraction and content of ethyl acetate extract at different concentrations of enzyme
X: content of total compound in sample; m: weight of compounds extracted; M: weight of initial material; ha: moisture content of initial material; Y: content of ethyl acetate extract; n: weight of ethyl acetate residue per 1g of total extract.
There was a direct ratio between enzyme concentration and efficiency of extraction The increase was rapid when enzyme concentration was from | to 5%, but later this was slower (Fig 3.2) Similarly, enzyme concentration was also in direct proportion to content of ethyl acetate extract However, it was noticeable that the content in all samples was lower than the sample without enzyme At concentrations from | to 3% of enzyme, content of ethyl acetate extract rose significantly (from 4.06 to 7.39%) After that, it remained stable around 7.40% (ranged from 7.39 to 7.50%) when raising enzyme concentration (Fig 3.3).
Content of ethyl acetate extract 10
Figure 3.2 Efficiency of extraction at different concentrations of enzyme
Figure 3.3 Content of ethyl acetate extract at different concentrations of enzyme
Table 3.8 and 3.9 showed antimicrobial activity of the extracts on six tested bacteria via two methods: diffusion and dilution (see 2.2.2.2 section and appendix 2).
Table 3.8 Inhibition zone diameter of the extracts at different concentrations of enzyme on tested bacteria (mm)
Enzyme E coli E faecalis Sal typhi P aeruginosa S qureus MRSA
All extracts had antimicrobial activity on Gram-positive bacteria and could not inhibit Gram-negative bacteria The higher enzyme concentration was, the weaker this activity was At enzyme concentration of | — 3%, all of the extracts resisted three Gram-positive bacteria From enzyme concentration of 4% then on, the number of bacteria which was repressed decreased little by little For details, sample of 4% inhibited E feacalis and MRSA, sample of 5% inhibited only S aureus and sample of 6% inhibited only MRSA The MIC values of enzyme-contained samples were much higher than that of the control without enzyme.
Table 3.9 MIC value of the extracts at different concentrations of enzyme on tested bacteria (mg/ml) Enzyme E coli E faecalis Sal typhi P aeruginosa S qureus MRSA
All extracts were tested antioxidant activities based on DPPH and ABTS radical scavenging capacities (see section 2.2.2.1) The data were presented in Table 3.10,Figure 3.4 and 3.5 Antioxidant activities increased when enzyme concentration was up to 3%, but later these activities decreased Therefore, 3% was the best concentration in both DPPH and ABTS radical scavenging assays because it gave the lowest ICso value among the others However, this ICs) value was higher than that of the positive control (the sample marked Q).
Table 3.10 Antioxidant activities of the extracts at different concentrations of enzyme
Figure 3.4 DPPH radical scavenging activity of the extracts at different concentrations of enzyme
Figure 3.5 ABTS radical scavenging activity of the extracts at different concentrations of enzyme 3.3.2 Effect of treatment time
Enzyme concentration was 3% and periods of treatment time were 1, 2, 3 and 4 hours The obtained results of efficiency of extraction process and content of ethyl acetate extract were recorded in Table 3.11 The general trends of the results were illustrated in Figure 3.6 and 3.7.
Table 3.11 Efficiency of extraction and content of ethyl acetate extract at different periods of treatment time Time (hour) m (g) M (g) n(g) ha (%) X (%) Y (%)
X: content of total compound in sample; m: weight of compounds extracted; M: weight of initial material; ha: moisture content of initial material; Y: content of ethyl acetate extract; n: weight of ethyl acetate residue per 1g of total extract.
When treatment time was prolonged, yield of extract was increased (Fig 3.6).The rise was quick at the two first periods of treatment time (by 3.69%) but later it was slow (by 1.91%) For content of ethyl acetate extract (Fig 3.7), the pattern was similar,too After material was treated by Viscozyme L in | hour, content of ethyl acetate extract was 7.39% This content increased to 7.97% after 2 hours treating with enzyme and leveled off at around 8% when extending treatment time However, the figures were lower than that of the sample without enzyme (8.26%).
Efficiency of extraction Content of ethyl acetate extract
0 1 2 3 4 0 1 2 3 4 Treatment time (hour) Treatment time (hour)
Figure 3.6 Efficiency of extraction at different Figure 3.7 Content of ethyl acetate extract at periods of treatment time different periods of treatment time
Inhibitory activity of five extracts against test microorganisms was reported in Table 3.12 and 3.13.
Table 3.12 Inhibition zone diameter of the extracts at different periods of treatment time on tested bacteria (mm) Time (hour) E coli E faecalis Sal typhi P aeruginosa S qureus MRSA
Table 3.13 MIC value of the extracts at different periods of treatment time on tested bacteria (mg/ml) Time (hour) E coli E faecalis Sal typhi P aeruginosa S qureus MRSA
In general, all extracts were only inhibitory to Gram-positive bacteria, but Gram- negative bacteria were found resistant MIC value of the extract after 1 hour treating was too high in comparison with that of the control sample while there was an insignificant difference in MIC values of the others It was noticeable that MIC values of two extracts which were treated by Viscozyme L within 3 and 4 hours were the same and lower than that of the extract which was treated in 2 hours This meant extending treatment time helped the extracts increase their antibacterial activity. However, in comparison with the control sample, the activity of enzyme-assisted extracts was weaker.
DPPH and ABTS radical scavenging activities of five extracts were recorded in Table 3.14 and Figure 3.8 and 3.9.
Table 3.14 Antioxidant activities of the extracts at different periods of treatment time
DPPH radical scavenging activity ABTS radical scavenging activity
0 - 0 vitC O0 1 2 3 4 vitC O0 1 2 3 4 Treatment time (hour) Treatment time (hour)
Figure 3.8 DPPH radical scavenging activity of the extracts at different periods of treatment time
Figure 3.9 ABTS radical scavenging activity of the extracts at different periods of treatment time
All extracts of B vitis-idaea showed significant antioxidant activity in two assays.
In DPPH and ABTS assay, ICs) value decreased from 120.4 + 1.59 g/ml and 121.3 + 0.68 g/ml (after |-hour treating) to 116 + 1.57 wg/ml and 118.5+0.65 g/ml (after 2- hour treating), respectively However, the figure increased sharply to 177.9 + 1.10 pg/ml and 132.6 + 0.71 wg/ml after 4-hour treating In sumary, the suitable period of treatment time was 2 hours; however, at this time, antioxidant activities of the extract weaker than that of the control extract without enzyme.
In conclusion, using Viscozyme L did not bring positive effect on extraction of glycosides because there was a downward trend in content of ethyl acetate extract and their bioactivities of enzyme-assisted samples in comparison with the control sample without enzyme.
3.4 Results of purification and structure determination of compounds
3.4.1 Results of thin layer chromatography analysis