Effect of different polarity solvents on the anti-inflammatory activity of Symplocos cochinchinensis leaves and correlation with total polyphenol content

Cite this paper: Vietnam J. Chem., 2021, 59(1), 106-114 Article DOI: 10.1002/vjch.202000136 106 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Effect of different polarity solvents on the anti-inflammatory activity of Symplocos cochinchinensis leaves and correlation with total polyphenol content Ly Hai Trieu1*, Le Vu Khanh Trang2, Nguyen Thai Minh3, Phan Thi Anh Dao4 1Research Center of Ginseng and Medicinal Materials, National Institute of Medicinal Materials, No. 41, Dinh Tien Hoang Str., Ben Nghe Ward, Dist. 1, Ho Chi Minh City 70000, Viet Nam 2The University of Danang - University of Science and Education, No. 459, Ton Duc Thang Str., Hoa Khanh Nam Ward, Lien Chieu Dist., Da Nang City 50000, Viet Nam 3Hong Bang International University, No. 215, Dien Bien Phu Str., Ward 15, Binh Thanh Dist., Ho Chi Minh City 70000, Viet Nam 4Faculty of Chemical and Food Technology, Ho Chi Minh City University of Technology and Education, No. 1, Vo Van Ngan Str., Linh Chieu Ward, Thu Duc Dist., Ho Chi Minh City 70000, Viet Nam Submitted August 17, 2020; Accepted September 16, 2020 Abstract Symplocos cochinchinensis leaves known as Vietnamese traditional herbal medicines which have been utilized anciently to support the medication of infirmities. The study aimed to explore the correlation of total polyphenol content (TPC) with anti-inflammatory activity of S. cochinchinensis leaves. S. cochinchinensis leaf extracts (ethanol extract, chloroform, ethyl acetate, n-butanol, aqueous fractions) was determined the TPC by Folin-Ciocalteu reagent, anti- inflammatory proficiency by in vitro protein denaturation and membrane lysis assays. The correlation between TPC and anti-inflammatory activities was analyzed in silico by Pearson’s method. Results illustrated that the TPC was arranged in sort of descending order as ethyl acetate fraction > n-butanol fraction > ethanol fraction > aqueous fraction > chloroform fraction. S. cochinchinensis leaf extracts exhibited anti-inflammatory effect via their protein denaturation inhibition capacity as well as hemolysis by heat and hypotonicity, which was better than the standard drug diclofenac sodium. There was a significantly negative correlation between TPC in S. cochinchinensis leaf extracts with their IC50 inhibition of protein denaturation, hemolysis by heat and hypotonicity with Pearson’s correlation coefficient (r) of -0.6302, -0.7241, and -0.9101, respectively. It was suggested that S. cochinchinensis leaves have great potential for anti- inflammatory activity during the high total polyphenol content. Hence, polyphenol compounds of S. cochinchinensis leaves should be considered since they can have good effects when in pure form. Keywords. Symplocos cochinchinensis leaves, polyphenols, anti-inflammatory activity, correlation. 1. INTRODUCTION Inflammation known as a biological response of the immune system involves a complex array of mediators and pathways. These may trigger acute as well as chronic inflammatory responses in many organs, potentially resulting in tissue damage or disease.[1] Inflammation is involved with several phenomena such as pain, membrane changes, increasing protein denaturation, and vascular permeability.[2] To resolve inflammation, many anti- inflammatory drugs are applied; in which, non- steroidal anti-inflammatory drugs (NSAIDs) are ubiquitously applied for inflammation control. Unfortunately, there are still the existence of side effects, particularly for stomachs such as stomach ulcers and stomach aches.[3] In recent decades, the medicinal plant materials sources used in folk have increasingly been concerned not only by local but also by foreign researchers for scientific pieces of evidence in their pharmacological effects. Plants are considered to be one of the ideal alternative remedies because of their safety, no or few side effects. Besides, these medicinal plants are also known as rich sources of polyphenols in their extracts with excellent biological activities, for example, anti-inflammatory, antioxidant, antidiabetes, anticancer, and anti-aging properties.[4] Polyphenols are known as secondary metabolites, which help plants reduce the effects of Vietnam Journal of Chemistry Ly Hai Trieu et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH Verlag GmbH www.vjc.wiley-vch.de 107 ultraviolet rays as well as eliminate the attack of pathogens.[5] Recently, the researchers on over the world have been identified more than 8000 phenolic compounds in various plant species.[6] Strong pieces of evidence have confirmed that the anti- inflammatory activities of polyphenolic substances were high evaluated such as rutin, flavanols, hesperetin, quercetin reported in acute and chronic inflammations.[7,8] On the other hand, some kinds of polyphenols such as genistein, apigenin, luteolin can disturb directly the signaling and enzymatic systems in inflammatory processes. Particularly, they disrupt B lymphocytes activation and proliferation of T cells as well as inhibit the secreting of lysozyme of neutrophils and arachidonic acid from cell membranes.[9,10] Symplocos cochinchinensis (Lour.) Moore ssp. Laurina (Retz.) Nooteb belongs to the Symploceaceae family and widely distributed in many tropical as well as subtropical areas in the world, including Vietnam. The plant is commonly used as folk medicines for effective treatments of various pathologicals like inflammation, cancer, diarrhea, menorrhagia, uterine problems, and some other diseases.[11] The Symplocos species have been used in treating various diseases based on their biological activities including anti-diabetic, anti- HIV, antimicrobial, anti-inflammatory, antioxidant, and antitumor applications.[12] However, the knowledge about potential biological activities of this indigenous Vietnamese plant has been still limited on scientific publishing. Therefore, the present study would conduct comprehensively on both chemical composition and biological activities of the indigenous S. cochinchinensis which collected from Cham Island, Quang Nam Province, Vietnam. The objectives of this research is to evaluate systematically in vitro anti-inflammatory activities as well as total polyphenols concentration of S. cochinchinensis leaves extract for potential medical applications. Thereby the correlation between anti- inflammatory ability and the total concentration of polyphenols in that indigenous species would be displayed in the study. 2. MATERIALS AND METHODS 2.1. Plant material and sample preparation The plant sample was collected in August 2019 in Cham Island, Quang Nam Province, which was identified and authenticated by The Greenviet Biodiversity Conservation Centre and Southern Institute of Ecology, Vietnam Academy of Science and Technology and a voucher specimen was deposited for Symplocos cochinchinensis (Lour.) Moore ssp. Laurina (Retz.) Nooteb. Before grinding into the fine powder, the leaves were cleaned with tap water and distilled water, then air dried to the standard of losing weight due to drying (LOD) following the Vietnam Pharmacopoeia 5th Edition (LOD = 9.73±0.41 %). The samples were kept in an airtight sealed bag at the Research Center Ginseng and Medicinal Materials in Ho Chi Minh City (Sample code: TTS-SC-0819). 2.2. Chemicals and reagents Ethanol 96 % (OPC Pharmaceutical Company, Vietnam), n-hexane, chloroform, ethyl acetate, n- butanol (VN-Chemsol, Co. Ltd, Vietnam), egg abumin (HIMEDIA), methanol, Folin-Ciocalteu’s phenol reagent, gallic acid (HPLC  98 %), sodium carbonate, and diclofenac sodium were purchased from Sigma-Aldrich® Co. Ltd (USA). 2.3. Extraction and fractionation Ethanol extract was obtained by using ethanol 45 % to extract dried powdered material with material- solvent ratio of 1/15 (w/v) for 24 hours at room temperature using a device called percolator apparatus, then the liquid extract was collected at a rate of 2 mL/min and a rotary evaporator was used to concentrate at 60 °C under reduced pressure to gain 45 % ethanol extract (total extract). To get fractionated extracts, the 45 % ethanol extract (LOD = 15.50±0.34 %) was solubilized in distilled water and sequentially extracted with solvents of increasing polarity (n-hexane, chloroform, ethyl acetate, and n-butanol). Eventually, five dried extracts including TE (45 % ethanol extract), chloroform (F1), ethyl acetate (F2), n-butanol (F3), and water (F4) extracts were obtained as fractional extracts which were dissolved in a suitable solvent to yield a stock solution. The extracts were preserved in sterilized vials and stored at 4 °C. 2.4. Determination of total polyphenols content The total polyphenol content (TPC) was estimated by Folin-Ciocalteu’s method using gallic acid as a standard.[13] Briefly, the reaction including 200 μL test sample, 6 mL of double distilled water and 500 μL of Folin-Ciocalteu’s reagent was held for 5 min, then 1.5 mL of 20 % w/v sodium carbonate (Na2CO3) solution was added into this mixture and the volume was adjusted up to 10 mL by distilled water. The reaction was kept at room temperature in the dark. After 2 h, the absorbance was recorded at Vietnam Journal of Chemistry Effect of different polarity solvents © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH Verlag GmbH www.vjc.wiley-vch.de 108 758 nm. The TPC was calculated based on the standard curve of gallic acid and expressed as mg of gallic acid equivalent (GAE)/g of dry mass. 2.5. In vitro anti-inflammatory activity 2.5.1. Inhibition of protein denaturation Anti-inflammatory activity of the extracts was carried out by protein denaturation method according to the study of Alhakmani[14] with slightly adjustments. To briefly illustrate, 2 mL of different concentrations of extract was mixed with 2.9 mL of phosphate buffered saline (pH 6.4), then 100 μL of egg albumin was added and incubated for another 15 min at 37±1 °C. The reaction mixture was kept at 70 °C in a water bath for 10 min for protein denaturation to be formed. The mixture after cooling, its absorbance is measured at 660 nm, distilled water was used as a blank sample. A powerful non-steroidal anti-inflammatory drug, diclofenac sodium was used as a positive control. The tests were carried out in triplicate and the average was calculated. The proportion of protein denaturation inhibition was determined by the expression: In which, At and Ac are absorbance of test sample and control sample, respectively. Simultaneously, the IC50 values (the concentration of sample that results in 50 % inhibition of maximal activity) were determined. 2.5.2. Membrane lysis assay Preparation of red blood cells (RBCs) suspension The blood was taken from the tails of healthy Swiss albino mice (20-25 g) and transferred to the centrifuge tubes containing anticoagulant. After which, these tubes were centrifuged at 3000 rpm for 10 min and washed with equal volume of normal saline for three times. The blood was suspended in normal saline to reach a concentration of 10 % (v/v).[15] Heat-induced hemolysis The resulting reaction mixture consists of 1 mL different concentrations of test extract or diclofenac sodium (positive control) and 1 mL of 10 % RBCs suspension were put into centrifuge tube. For the control sample, the extract was replaced with saline. The centrifuge tubes were incubated at 56 ºC for 30 min and then were cooled by tap water. Subsequently, these tubes were centrifuged at 2500 rpm for 5 min and the optical density of the supernatants was recorded at 560 nm.[15] All tests were carried out in triplicates and the rate of inhibition of hemolysis was determined as follows: In which, At and Ac are absorbance of test sample and control sample, respectively. Simultaneously, the IC50 value of test samples were also determined in this assay. Hypotonicity-induced hemolysis The reaction mixture consists of 0.5 mL test extract at various concentrations or diclofenac sodium (positive control), 1 mL of phosphate buffer, and 2 mL of hyposaline were mixed with 0.5 mL of RBCs suspension. Subsequently, all the reaction mixtures were incubated at 37 oC for 30 min and then centrifuged at 3000 rpm. The supernatant liquid was decanted and optical density was measured at 560 nm using a spectrophotometer to determine hemoglobin content.[16] The percentage inhibition of hemolysis was estimated using the expression: In which, At and Ac are absorbance of test sample and control sample, respectively. Simultaneously, the IC50 value of test samples was also determined in this assay. 2.6. Statistical analysis All experiments were repeated triplicate. The obtained results were expressed in terms of mean ± SD (Standard deviation). The IC50 value was determined based on the equation illustrating the correlation between the test substance concentration and the percentage of anti-inflammatory activity using Graphpad Prism software (version 8.0.1, Inc., La Jolla, CA, USA). Data were analyzed by Graphpad Prism software using t-test and One-way ANOVA. The correlation statistic was based on the Pearson’s correlation coefficient (r) and coefficient of determination (R2) for total polyphenols content versus IC50 values of anti-inflammatory activity (p < 0.05 is statistically significant). 3. RESULTS AND DISCUSSION 3.1. Total polyphenols content of extracts Vietnam Journal of Chemistry Ly Hai Trieu et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH Verlag GmbH www.vjc.wiley-vch.de 109 b, c a, c c d23.04 12.64 25.46 23.97 17.17 0 10 20 30 40 50 0 200 400 600 800 1000 TE F1 F2 F3 F4 E x tr ac ti o n y ie ld ( % ) T P C ( m g G A E /g d . w .) Extracts TPC (mg GAE/g d.w) Extraction yield (%) The concentration of total polyphenol contents (TPC), which expressed as mg of gallic acid equivalent (GAE)/g of dry mass, was determined by equation y = 0.0024x - 0.2135 from the standard gallic acid graph (figure 1). As shown in figure 2, the highest concentration of TPC is ethyl acetate fraction (852.74 mg GAE/g d. w), followed by n- butanol and 45 % ethanol extract corresponding to 757.51 and 738.47 mg GAE/g d. w., respectively and are nearly double when compared to chloroform fraction (397.08 mg GAE/g d. w.). Meanwhile, the TPC of aqueous fraction is 511.36 mg GAE/g d. w. Concentration (g/mL) Figure 1: Calibration line of gallic acid standard for total polyphenol content Figure 2: Extraction yield and total polyphenol content of S. cochinchinensis leaf extracts. All values were expressed as mean ± SD (n = 3). ap < 0.01 compared with F3 fraction, bp < 0.001 compared with F2 fraction, cp < 0.0001 compared with F1 and F4 fractions, dp < 0.0001 compared with F1 fraction. TE, F1, F2, F3, and F4: 45 % ethanol extract, chloroform, ethyl acetate, n-butanol, and aqueous fractions, respectively 3.2. In vitro anti-inflammatory activity 3.2.1. Inhibition of protein denaturation The results which were given in figure 3 showed the anti-inflammatory capabilities of five studied extracts and diclofenac sodium (standard drug). Increasing level of the samples resulted in higher inhibition percentage of protein denaturation. The Vietnam Journal of Chemistry Effect of different polarity solvents © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH Verlag GmbH www.vjc.wiley-vch.de 110 figure for IC50 values of all extracts revealed significantly higher than diclofenac sodium and were arranged in order of F3 (n-butanol fraction) < F2 (ethyl acetate fraction) < F1 (chloroform fraction) < TE (45 % ethanol extract) < F4 (aqueous fraction) < Diclofenac sodium. The n-butanol and ethyl acetate fractions were the two lowest IC50 values; consequently, the inhibition of protein denaturation from two fractions would be higher than other extracts. Figure 3: In vitro inhibition of protein denaturation of S. cochinchinensis leaf extracts. DS, TE, F1, F2, F3, and F4: Diclofenac sodium, 45 % ethanol extract, chloroform, ethyl acetate, n-butanol, and aqueous fractions, respectively. (A) Percentage inhibition of protein denaturation activity, (B) The IC50 values. Values were expressed as Mean±SD (n = 3). ap < 0.01 compared with F2 fraction, bp < 0.001 compared with TE extract and F1 fraction, cp < 0.0001 compared with F3 fraction, dp < 0.0001 compared with F4 fraction, ep < 0.0001 compared with all extracts 3.2.2. Membrane lysis assay Activity of the studied extracts and DS which known as the standard drug on in vitro membrane stabilization was presented in figure 4. The inhibition percentage of both heat-induced hemolysis (figure 4A) and hypotonicity-induced hemolysis (figure 4C) by 45 % ethanol extract and different fractions were increased depending dose- increase. In terms of IC50 values, almost extracts were found to be lower than that of DS (figure 4B and figure 4D). It can be concluded that the inhibitory capacity for heat-induced hemolysis as well as hypotonicity-induced hemolysis from all extracts of S. cochinchinensis leaves was higher than the standard diclofenac sodium. 3.3. Correlation between total polyphenols content and anti-inflammatory activity Figure 5 showed the correlation between total polyphenols content and anti-inflammatory activities through the assessment on protein denaturation, heat-induced hemolysis, and hypotonicity-induced hemolysis. Following these graphs, the higher was the TPC, the lower was the IC50. Therefore, the higher were the TPC, the higher were anti- inflammatory activity of the extracts. The coefficient of Pearson correlation was significantly negative if - 0.61  r  -0.97 and significantly positive if 0.61  r  0.97.[17] The correlation coefficients of protein denaturation inhibition were -0.6302 while they were -0.7241 and -0.9101 for heat-induced hemolysis inhibition and hypotonicity-induced hemolysis inhibition, respectively (table 5). This indicated that TPC in S. cochinchinensis leaf extracts had a significantly negative correlation with their IC50 of extracts in investigated assays. These correlations indicated that anti-inflammatory properties may be related to the presence of bioactive antioxidants such as polyphenols. Anciently, the leaves and the bark of the S. cochinchinensis have been used predominantly more than other parts of this plant as folk medicines for treatment of ulcers, arthritis, bronchitis, leprosy, asthma, diarrhea, dysentery, and skin diseases.[18] In this study, the anti-inflammatory ability of S. cochinchinensis leaves extracts was assessed on the inhibition of protein denaturation as well as membrane lysis assay. a, d b, d b c c e Vietnam Journal of Chemistry Ly Hai Trieu et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH Verlag GmbH www.vjc.wiley-vch.de 111 a b c, d e f, g a, b c d d Figure 4: In vitro membrane stabilization activity of S. cochinchinensis leaf extracts. DS, TE, F1, F2, F3, and F4: Diclofenac sodium, 45 % ethanol extract, chloroform, ethyl acetate, n-butanol, and aqueous fractions, respectively. (A) Percentage inhibition of heat-induced hemolysis, (B) The IC50 values of heat- induced hemolysis: ap < 0.05 compared with TE extract and DS standard