Extraction and bioactivity evaluation of the extracts from cleistocalyx operculatus l leaves

Vietnam Journal of Science and Technology 58 (6A) (2020) 52-62 doi:10.15625/2525-2518/58/6A/15450 EXTRACTION AND BIOACTIVITY EVALUATION OF THE EXTRACTS FROM CLEISTOCALYX OPERCULATUS L LEAVES Nguyen Thi Ngoc Tuyet1, Nguyen Minh Anh1, Le Thi Kim Phung1, 2, * Refinery and Petrochemicals Technology Research Center (RPTC), Ho Chi Minh City University of Technology (HCMUT), 268 Ly Thuong Kiet Street, District 10, Ho Chi Minh City, Viet Nam Vietnam National University Ho Chi Minh City, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, Viet Nam * Emails: phungle@hcmut.edu.vn Received: September 2020; Accepted for publication: 25 December 2020 Abstract Cleistocalyx Operculatus L was known as a natural resource of bioactivity compounds with high potential of anti-inflammatory, anti-bacterial, and antioxidant In this study, two conventional extraction methods (maceration and liquid-solid extraction) at different extraction conditions (solvent concentration, extraction temperature and time, liquid-to-solid ratio and number of extractions) were conducted The extracts were analyzed for total phenolic content, anti-inflammatory activity, anti-bacterial, and antioxidant activity Three parameters (liquid-solid ratio, extraction temperature, and time) were the important factors that affected on the extraction yield and antioxidant activity The results showed that the highest values of the extraction yield and antioxidant activity were obtained at liquid-solid ratio of 5:1 (v/w – mL/g), extraction temperature and time of 50 °C and 50 min, respectively The antioxidant activity of the extract was confirmed by IC50 values (29.94 ± 2.34 μg/mL) Moreover, Cleistocalyx Operculatus L extract showed an average anti-inflammatory activity of 381.14 ± 5.48 μg/mL Keywords: Cleistocalyx Operculatus L., extraction, anti-inflammatory, anti-bacterial, antioxidant Classification numbers: 1.2.1, 1.3.1, 1.4.7 INTRODUCTION Cleistocalyx Operculatus L is a medium to large timber tree, which can attain the height up to 20 m It contains many branches, with grey or brownish grey bark C Operculatus L flowering time is from February to March, and leaves can be harvested throughout the year [1] C Operculatus L is a well-known perennial tree, widely distributed throughout Asia countries such as China, Viet Nam, Malaysia, Myanmar, Thailand, Sri Lanka, India, Nepal, and several other tropical countries [2] In Viet Nam, this plant is cultivated or grows native, especially in Northern regions Fresh C Operculatus L leaves are washed off, all the resin, sliced and incubated, their color turns black thoroughly On that occasion, they are taken out, cleaned, and dried and added to tea, which is exceedingly common in Vietnam countryside In essence, Extraction and bioactivity evaluation of the extracts from Cleistocalyx Operculatus L leaves bioactivity compounds had been isolated from C Operculatus L., for example, pentacyclic triterpenoids and essential oil from leaves [3], flavonoids and ursolic acid from flower buds [4] Previous reports revealed that the C Operculatus had various biological activities in vitro and in vivo such as radical scavenging effect [5], anti-inflammatory [6] and antimicrobial [7] However, the study of the effect of extraction conditions on these bioactivity compounds has not been conducted in C Operculatus L leaf in Viet Nam According to previous research, the extraction conditions can be achieved by varying of many factors, namely, solvent concentration, extraction temperature and time, liquid-to-solid ratio, and number of extractions, etc [8], [9] Therefore, the essential purposes of this work were to determinate the extraction yield and antioxidant activity of the extracts from C Operculatus L leaves by using solid-liquid extraction method under different conditions MATERIALS AND METHODS 2.1 Materials preparation Fresh C Operculatus L were collected in Song Ray, Dong Nai province in 2018 They were naturally dried in the shadow until the moisture content was lower than 12 % They were crushed into powder and then stored in zipper bags with the desiccant pack inside until used for the extraction step 2.2 Extraction The solvent was an essential criterion to dissolve chemical compounds This factor was prioritized since the dissolution of bioactive compounds significantly relies on the physical properties of the solvent (selectivity, solubility, toxicity, cost, etc.) Otherwise, the using the solvent must be safe and eco-friendly [10] According to previous research, using ethanol was better than consuming acetone, water, or methanol for extraction of phenolic compounds from the plant [11] The phenolic compounds and antioxidant properties of the extract were significantly dependent on the concentration of ethanol-water mixture [12] Therefore, ethanol– water mixtures were also recommended for the preparation of plant extracts due to their acceptability for human consumption [13] Therefore, C Operculatus L leaf powder was extracted with ethanol by using solid-liquid extraction method: 30 g of dry powder was added into the beaker with ethanol and liquid-to-solid ratio of 5:1 mL/g, at a temperature of 50 °C, stirring speed of 395 rpm during 45 Additionally, the mixture was vacuum filtered, the liquor collected was evaporated using a rotavapor Ethanol crude extract was obtained and preserved in a sealed bottles placed in the freezer Ethanol crude extraction yield was calculated as the following equation: where H%, m1 and m2 were extraction yield (%w/w), weight of dry material (g) and weight of crude extract (g), respectively There were many factors affecting on the extraction process of bioactive compounds In this study, the effect of extraction conditions (solvent concentration, extraction temperature and time, liquid-to-solid ratio, and number of extractions) on the extraction yield and antioxidant activity of C Operculatus L leaf powder extracts were investigated These criteria were evidenced to significantly affect the polyphenol content, flavonoid content, and the radical53 Nguyen Thi Ngoc Tuyet, Nguyen Minh Anh, Le Thi Kim Phung scavenging activity of the extract [14] Extraction process: 30 g of leaf powder were extracted at various conditions (Table 1), then mixture of the solid and solvent was filtrated by vacuum filter to collect filtrate The filtrate was concentrated to dryness by a vacuum rotary evaporator at 55 °C The extract was kept in dark at °C until analysis the extraction yield and antioxidant activity Table Various extraction conditions Ethanol concentration (%) Extraction temperature (°C) Extraction time (min) Liquid-to-solid ratio (mL/g) Number of extractions 0, 20, 40, 60, 80, 100 50 45 5:1 two-times - 40, 50, 60, 70, 80 45 5:1 two-times - - 15, 30, 45, 60, 90 5:1 two-times - - - 4:1, 5:1, 6:1, 7:1, 8:1 two-times - - - - one-time, two-times, three-times 2.3 Analytical methods 2.3.1 Total phenolic content Total phenolic content was determined by Folin-Ciocalteu method Polyphenols in the extracts, react with specific redox reagents (Folin–Ciocalteu reagent), will form a blue complex (molydenium) that could be quantified by visible-light spectrophotometry By measuring the absorbance at the wavelength 760 nm and combining the result with the gallic acid calibration curve, the total phenolic content was estimated and calculated [15] as the following equation: where GAE (mg/mL) is Gallic Acid Equivalent (estimated phenolic content derived from Gallic acid calibration curve), TPC (mg GAE/g dry extract) is Total Phenolic Content in g extract, V (mL) is volume of extract and m (g) is weight of dry extract 2.3.2 Antioxidant activity In this study, antioxidant activity was determined by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay [8, 9] The DPPH assay measured the scavenging capacity of the radical dissolved in different solvent mixtures (i.e., methanol, methanol/water) DPPH shows strong absorption at 517 nm, but upon reduction by an antioxidant, the absorption disappears The purple color in the initial solution turned to yellow when the amount of the free radical was blocked by the antioxidants The antioxidants scavenge of the extract and the reduction of DPPH can be monitored by the decrease of the absorbance at 517 nm; and vitamin C was used as 54 Extraction and bioactivity evaluation of the extracts from Cleistocalyx Operculatus L leaves positive control The ability of the sample to neutralize the radicals was expressed as IC50, which was the sample concentration required to exhibit the radical’s neutralization by 50 % Extract solution: the extract was weighed and calculated for the dry weight extract based on extract humidity, then 0.02 g of dry extract was dissolved into 20 mL methanol 80% to obtain extract solution of 1000 μg/mL, this solution was diluted to different concentration (5, 15, 30, 45, 60, 75, 90 μg/mL) Test sample: 4.2 mL of DPPH (50 μg/mL) was added into 2.8 mL of these above extract solutions, then these mixtures were shaken thoroughly, left in darkness at room temperature for 30 and measured of the absorbance at wavelength of 517 nm Blank sample: similar to test sample, yet extract solution was replaced by 2.8 mL methanol 80 % Color sample: 4.2 mL of methanol 80 % was added into 2.8 mL of extract solution Positive control: similar to extract sample, yet extract solution was replaced by vitamin C which has been prepared in methanol 80 % at 5, 6, 7, 8, 9, 10, 11 μg/mL The percentage of DPPH radical inhibition is calculated by the formula: where Ab, At and Ac are absorbances of blank, test sample and color sample, respectively Considering the relationship between concentrations possessing inhibition percentage around 50 % (one lower and one higher) as linear A linear line equation presenting the change of inhibition percentage over extract concentration can be written: %I = A + BC (where C (μg/mL): extract concentration) Substituting %I with 50 value, extraction concentration that inhibit the antioxidant activity by 50 %, which is IC50, was obtained RESULTS AND DISCUSSION 3.1 Evaluation of ethanol crude extract from C Operculatus L leaves 3.1.1 Extraction yield Bioactive compounds with different polarity were usually extracted from methanol or ethanol to get their crude extract The phenolic compounds and antioxidant properties of the extract were dependent on the concentration of ethanol-water mixture [12] In this study, the ethanol crude extract from C Operculatus L leaves collected after vacuum evaporation were sticky, dark green color and light fragrance The highest amount of extract was collected in the first-time extraction since most of the bioactive compounds have dissolved in ethanol [12] The result from Figure 1a indicated that the first-time extraction had the highest yield (22.95 ± 1.29 %) The extract amount experienced a dramatic fall for the following extraction The second-time extraction exhibited that the extraction yield was about half of the first-time extraction (10.97 ± 2.56 %) and the fourth-time extraction gave the lowest extraction yield (1.03 ± 0.14 %) It can be referred from Figure 1a that the extraction experiment can be conducted for the maximum of three times with the aim of “economical” solvent consumption 3.1.2 Total phenolic content Polyphenolic compounds from plants have been extensively studied for their antioxidant activity, which represents a biological function, important in keeping the oxidative stress levels below a critical point in the human body [16] It was considered that the antioxidant activity of phenolic compounds was due to their high redox potentials, which allow them to act as reducing 55 Nguyen Thi Ngoc Tuyet, Nguyen Minh Anh, Le Thi Kim Phung agents, hydrogen donors, and singlet oxygen quenchers [14] Therefore, quantification of total phenolic content was useful to predict the strength of their bioactivities Ethanol crude extracts in three time extraction were mixed together to determine total phenolic content (TPC) TPC of C Operculatus L leaf ethanol crude extract was 50.29 ± 1.10 mgGAE/g dry material The total phenolic content of methanolic extracts from various kinds of teas (e.g., green tea, oolong tea, black tea, pu-erh tea) was also determined by Gawron-Gzelle et al [17] TPC of C Operculatus L leaf ethanol crude extract was about 1.83 times lower than that of green tea, but equal to that of Oolong tea and higher than that of black tea and Pu-erh tea (3.25 and 8.90 times, respectively) TPC of C Operculatus L leaf ethanol crude extract was 4.28 times higher than that of ethanolic extract from guava [18] Total phenolic content of C Operculatus L leaf ethanol crude extract is relatively high, and therefore, C Operculatus L is believed to possess many outstanding bioactivities 3.1.3 Antioxidant activity evaluation In fact, free radicals oxidative damage all biological molecules To be against the oxidative process, antioxidants are consumed for neutralizing deleterious free radicals or their actions and offer protection at varying levels [19] The IC50 value of C Operculatus L leaf ethanol crude extract was 29.73 ± 1.42 μg/mL, which was higher than this value of the vitamin C (7.75 ± 0.19 μg/mL) According to the research in antioxidant activity from some Indian medicinal plants, IC50 values of the methanolic extract were higher than vitamin C and decreased in the following order: C spinosa (36.33 ± 0.33 μg/mL), C asiatica (17.33 ± 0.17 μg/mL), D falcata (7.93 ± 0.03 μg/mL), S japonica (6.25 ± 0.25 μg/mL) and vitamin C (2.12 ± 0.01 μg/mL) [20] It indicated that antioxidant activity of C Operculatus L ethanol crude extract was at medium level Otherwise, it was also reported by Dung et al [5] that the IC50 value of ethanol extract from the buds of C Operculatus L was 39.27 ± 2.40 μg/mL, representing the antioxidant activity of the bud extract was lower than that of the leaf extract (29.73 ± 1.42 μg/mL) This was no doubt that C Operculatus L ethanol crude extract possessed high antioxidant activity comparing to others 3.2 The effect of different extraction conditions on the extraction yield and antioxidant activity of C Operculatus L leaf extract 3.2.1 Effect of solvent concentration on the extraction yield and antioxidant activity Figure 1b illustrated the extraction yield was stable with an increase of ethanol concentration from % to 60 % and slightly decreased with an increase of ethanol concentration from 60 % to 100 % (see Table for extraction conditions) Radical-scavenging ability reached the highest value at extracting with ethanol 60 % (IC50 of 24.96 ± 1.87 μg/mL) The antioxidant activity was lowest at the extract with water (IC50 of 37.38 ± 2.34 μg/mL) The variation of antioxidant activity was caused by the change in solvent polarity, affected the dissolving rate of bioactive compounds in the solvent [21] The extraction yield and antioxidant activity were high with an extraction of ethanol concentration lower than 60 % With ethanol concentrations higher than 60 %, the extract had low extraction yields as well as low antioxidant activity since bioactive compounds exhibiting strong radical-scavenging ability such as flavonoids and polyphenols were not soluble in high-concentrated ethanol These compounds were not soluble in water so that extracts with ethanol 40 % and below had given high yield but low antioxidant activity [12] The high extraction yield was obtained because of the dissolution of some residues 56 Extraction and bioactivity evaluation of the extracts from Cleistocalyx Operculatus L leaves Therefore, ethanol 60 % was chosen for studying the next factors because of both high bioactivity and extraction yield 3.2.2 Effect of extraction temperature on the extraction yield and antioxidant activity Temperature was considered to represent a critical parameter in the extraction process The increase of temperature led to a larger diffusion coefficient, which also means a rise in the amount of material diffusing in the solvent Moreover, the more elevated the temperature was, the lower the solvent viscosity was This will create the favorable condition for the extraction process and increase number of bioactive compounds However, the bioactive compounds were heat-sensitive and easy to denaturation at high temperature The antioxidant compounds significantly exhibited decomposition at temperatures above 60 °C [22] Figure Extraction yield and antioxidant activity of C Operculatus L leaf powder extract at different extraction conditions: (a) extraction times of ethanol crude extract, (b) ethanol concentration, (c) extraction temperature, (d) extraction time, (e) liquid-to-solid ratio and (f) and number of extraction (see Table for extraction conditions) Additionally, the presence of polyphenol oxidases may decrease the number of antioxidant compounds present to extract At the extraction temperature above 60 °C, the polyphenol 57 Nguyen Thi Ngoc Tuyet, Nguyen Minh Anh, Le Thi Kim Phung oxidase was activated to instigate the degradation Metabolites of polyphenols possessed considerable antioxidant properties, but at elevated temperature, they can be degraded into other compounds with insignificant antioxidant potential [23] Therefore, in this study, a range of extraction temperature from 40 °C to 80 °C was chosen to evaluate the effect of extraction temperature on the extraction yield and antioxidant activity The antioxidant activity rose with an increase in the extraction temperature from 40 °C to 60 °C (see Table for extraction conditions) and gained the most considerable value at 60 °C (IC50 of 23.09 ± 3.87 μg/mL, see Figure 1c) This could be explained by the fact that under the effect of extraction temperature, the flexibility of molecules increased, they would chaotically move due to an increase in mass transfer driving force and made the diffusion process better [23] However, when the temperature exceeded 70 °C, the bioactive compounds became decomposed and led to a decrease in antioxidant activity Besides, at elevated temperature, residues (resin, starch) in C Operculatus L leaf powder was swollen and gelatinized This phenomenon increased the solvent viscosity and inhibits the extraction process Too high temperature led to the loss of the solvent due to vaporization Despite losing bioactivity, these compounds presented in the extract, thus the extraction yield did not vary considerably at all extraction temperature conditions, the average value was 28.81 ± 0.38 % 3.2.3 Effect of extraction time on the extraction yield and antioxidant activity If the extraction time was too short, bioactive compounds did not have enough time to dissolve completely into solvent Otherwise, carrying the extraction too long can lead to the appearance of residues, making it difficult for the purifying step The increase in extraction time potentially increased the loss of solvent by evaporation It was therefore suggested that an extraction time was of no longer than h [24] It was understandable from Figure 1d that, at the initial, the antioxidant activity rose with an increase in extraction time (from 15 to 45 min) and reached the highest values at 45 (IC50 of 22.96 ± 2.07 μg/mL) This could be explained by Fick’s second law of diffusion/high extraction time that led to a considerable number of bioactive compounds in the material diffusing out, which generated the radical-scavenging ability to be more effective However, long extraction time led to the appearance of more residues and the denaturation of bioactive compounds, which can reduce the extraction process quality [25] When the extraction time increased from 15 to 45 min, the extraction yield increased slightly owing to the fact that it took time for the solvent to completely contact with the material particles and for the temperature to be raised to the desired point (60 °C) The mass transfer process needed a certain time to take place, effectively, the bioactive compounds in the material could, as follows, diffuse to the extract solution However, the extraction time did not show a significant effect on the extraction yield (Figure 1d) For this reason, after 45 min, the extraction yield flattened off at the average value of 28.43 ± 0.16 % As a consequence, the extraction time of 45 was appropriate for the following experiments 3.2.4 Effect of liquid-to-solid ratio on the extraction yield and antioxidant activity Liquid-solid extraction process was the concentration gradient of extracted components between the solvent and the materials [26] The concentration gradient was changed when a different solvent-to-solid ratio was used, resulting in a difference in the diffusion rate that allows more significant solid extraction by the solvent [27] In addition, bioactive compounds have a chance to contact with the extraction solvent when the solvent amount increased, leading to a higher extraction rate [28] However, the extraction yield could not increase once equilibrium 58 Extraction and bioactivity evaluation of the extracts from Cleistocalyx Operculatus L leaves was reached [29] Figure 1e demonstrated that liquid-to-solid ratio did not significantly affect the antioxidant activity as well as the extraction yield The antioxidant activity attained the most significant value at the ratio of 5:1 mL/g (IC50 of 23.22 ± 2.19 μg/mL) Considerably, an increase in the liquid-to-solid ratio caused a gradual decrease in antioxidant activity This can be explained by the fact that when the material ratio achieved a certain level, the bioactive compounds were well dissolved in the solution [25] However, the residues could still dissolve and reduce the extract quality The extraction yields slightly went up with the increase of the ratio from 4:1 to 5:1 due to two reasons Firstly, less contact area between the solvent and material led to low extraction efficiency Secondly, during the agitating process, a considerable amount of material was splashed onto the wall of the container and was not extracted In addition, a high liquid-to-solid ratio means high concentration gradient and better diffusivity [30] Because of not observed higher extraction yield and antioxidant activity with an increase in solvent amount, liquid-to-solid ratio 5:1 mL/g was chosen for further surveyed experiments 3.2.5 Effect of extraction number on the extraction yield and antioxidant activity According to the previous study, when the number of extraction times increased, the extraction yield could increase [31] At a low number of extractions, the bioactive compounds in the material were not extracted completely, otherwise, excessively repeating the experiment means high consumption of solvent volume and time That was a reason to take into account the number of extractions It was clear from Figure 1f that the extraction yield increased considerably with an increase in the number of extraction times, while the antioxidant activity was stable IC50 value was most limited at 22.62 ± 2.26 μg/mL by three-times extraction and it was observed for the highest extraction yield of 30.90 ± 0.43 % This was due to the fact that after two-times extraction, most of the bioactive compounds were extracted, the extraction yield between two-times and three-times extraction was thus not noticeable The experiment with twotimes extraction would cover the economic efficiency and antioxidant activity CONCLUSIONS The compounds with high potential of total phenolic content and antioxidant from C Operculatus L leaf were extracted and their bioactivities were evaluated The results showed that the highest values of the extraction yield and antioxidant activity were obtained at liquidsolid ratio of 5:1 with ethanol 60 % and two-time extraction temperature and time of 60 °C and 45 min, respectively TPC and antioxidant activity (IC50 value) of the extract were 271.50 ± 3.87 mg GAE/g dry extract and 22.98 ± 1.54 μg/mL, respectively However, the liquid-solid extract possessed an average anti-inflammatory activity In vivo bioactivity assessment should be applied in further research for more extraordinary accuracy in results Drying of extract solution to obtain C Operculatus L leaf bioactive powder can be proceeded Acknowledgements We acknowledge the support of time and facilities from Ho Chi Minh City University of Technology (HCMUT), VNU-HCM for this study CRediT authorship contribution statement NTNT: Methodology, Paper writing NMA: Formal analysis and Experiments LTKP: Formal analysis, Supervision Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper 59 Nguyen Thi Ngoc Tuyet, Nguyen Minh Anh, Le Thi Kim Phung REFERENCES Subhadrabandhu S - Under-Utilized Tropical Fruits of Thailand, Thailand, 2001 Nguyen P T M., Schultze N., Boger C., Alresley Z., Bolhuis A., and Lindequist U Anticaries and antimicrobial 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