Secondary metabolites and in vitro biological activities of fractions from Stemona collinsae Craib root extract

BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM - HỘI NGHỊ KHOA HỌC QUỐC GIA LẦN THỨ 4 DOI: 10.15625/vap.2020.000109 SECONDARY METABOLITES AND IN VITRO BIOLOGICAL ACTIVITIES OF FRACTIONS FROM Stemona collinsae Craib ROOT EXTRACT Van Hoang Lan, Le Thi Phuong Hoa* Abstract: Stemona collinsae Craib is a medicinal plant, which has been reported previously with various biological properties such as insecticidal, anticancer, antioxidant and antifungal activities. In this study, further characterization of S. collinsae roots was performed with four fractions, namely n-hexane, ethyl acetate, butanol and water fractions from methanolic root extract. Thin-layer chromatography (TLC) analysis revealed the presence of various compounds especially alkaloids and phenolics in four fractions. Their total phenolic content ranged from 7.60 to 154.64 mg GAE (gallic acid equivalent)/g dry weight. The total flavonoids of four fractions were at low level varying from 4.045 to 11.662 mg QE (quercetin equivalent)/g dry weight. Antioxidant activity of four fractions was evaluated through 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay with IC50 values from 0.676 to 4.884 μg/mL. Ethyl acetate fraction showed the highest phenolic (154.64 mg GAE/g) and flavonoid content (11.662 mg QE/g) and moderate antioxidant activity with IC50 of 0.676 μg/mL. Besides, the fractions of S. collinsae root extract showed weak ferric ion reducing capacity and no significant inhibition on tested microorganisms and trypsin in this study. The results suggest further characterization of antioxidants from ethyl acetate fraction of S. collinsae methanolic root extract for its future application. Keywords: Antioxidant activity, Stemona collinsae root, thin layer chromatography, total flavonoids, total phenolics, trypsin inhibition. 1. INTRODUCTION Stemona collinsae Craib, known as “Bach Bo hoa tim” in Vietnam, is a subshrub with tuberous, fusiform roots and mainly distributes in Laos, Thailand (Inthachub et al., 2010). The roots have been traditionally used in Thailand for liver cancer protection, skin infection, anti-parasitic and insecticidal agent (Inthachub et al., 2010, Rutnakornpituk et al., 2018). Up to date, there were some alkaloids (H. Greger, 2019), stilbenoids (Pacher et al., 2002) and phenolics (Rutnakornpituk et al., 2018) isolated from the roots or rhizomes of S. collinsae. The presence of alkaloids related mainly to reported biological activities of S. collinsae. Two alkaloids, 16,17-didehydro-16(E)-stemofoline and 16,17-didehydro- 4(E)-16(E)-stemofoline, from S. collinsae root extract exhibited strong insecticidal activity against diamondback moth (Plutella xylostella) larvae with LD50 of 0.63 and 5.5 μg/mL, respectively (Jiwajinda et al., 2001). Bioactivity - guided purification of S. collinsae root dichloromethane extract revealed another alkaloid, hydroxystemofoline, as a potential Hanoi National University of Education *Email: lephhoa@yahoo.com PHẦN II. NGHIÊN CỨU ỨNG DỤNG SINH HỌC PHỤC VỤ ĐỜI SỐNG VÀ PHÁT TRIỂN XÃ HỘI 879 insecticide against P. xylostella larvae (Phattharaphan et al., 2010). Moreover, acetylcholinesterase inhibition, which is one of insect toxicity mechanisms in many plants, was found in S. collinsae root extracts with their major alkaloids, didehydrostemofoline and stemofoline (Kongkiatpaiboon et al., 2013). Besides, most of stilbenoids isolated from S. collinsae showed antifungal capacity against 5 microfungal strains with EC50 ranging mainly from 0.8 - 34 μg/mL (Pacher et al., 2002). The crude extract from S. collinsae root had antiproliferative effect on medullary thyroid carcinoma cells (Rinner et al., 2004). Also, the extracts of S. collinsae roots showed moderate activity against herpes simplex virus type 1 and type 2 with IC50 of 105 and 107.3 µg/mL, respectively, when using a plaque reduction assay. All the extracts exerted moderate to low antiproliferative activity against malignant cell lines KB and MCF-7, with EC50 ranging from 85 to 289 µg/mL (Akanitapichat et al., 2005). However, there has not been any research about biological activities of S. collinsae in Vietnam. Therefore, this study is aimed to evaluate some secondary metabolic composition mainly phenolics of four fractions from methanolic extract of S. collinsae roots and their in vitro biological activities like antioxidant and antimicrobial activity. 2. MATERIALS AND METHODS Stemona collinsae roots were collected in Savannakhet, Laos. Microbial strains including Staphylococcus aureus ATCC 13709, Bacillus subtilis ATCC 6633, Lactobacillus fermentum N4, Escherichia coli ATCC 25922, Pseudomonas aeruginosa 15422, Salmonella enterica ATCC 13076 and Candida albicans ATCC 10231 were obtained from the Laboratory of Applied biochemistry, Institute of Chemistry, Vietnam Academy of Science and Technology. Chemicals and reagents were of analytical grade and purchased from Sigma Chemicals (MO, USA) and Merck Chemicals (Darmstadt, Germany). Sample extraction and fractionation Stemona collinsae roots were dried, ground and soaked in methanol for one week at room temperature and further extracted in an ultrasonic bath for 30 minutes in three replicates. The extract was filtered and concentrated in a rotary evaporator. The crude extract was fractionated sequentially with n-hexane, butanol, ethyl acetate and water. The fractions were vacuum concentrated and lyophilyzed. Thin layer chromatography The fractions were mixed with ethanol at the concentration of 10 mg/mL. Various solvent systems were used including n-hexane/ethyl acetate 5 : 1 for n-Hexane fraction; chloroform/methanol/H2O 25 : 2.5 : 0.1 for butanol fraction; chloroform/ethyl acetate 4 : 1 for ethyl acetate fraction; chloroform/methanol/H2O 5 : 2 : 0.1 for water fraction. The plates were dried and observed under ultraviolet radiation (302 nm, 365 nm). They were then sprayed with 5% H2SO4 and observed under visible light. 880 BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM Determination of total phenolic content and total flavonoid content The total phenolic content was determined according to Waterhouse (2002) using Folin - Ciocalteau reagent and gallic acid as the standard. The absorbance was measured at 765 nm. The total phenolic content was expressed as mg GAE per gram of fraction dry weight, based on the calculation using gallic acid standard curve. The total flavonoid content was measured following the method of Sapkota et al., (2010) with the standard as quercetin. The total flavonoid content was determined as mg QE per gram of sample dry weight. Free radical scavenging capacity The DPPH radical scavenging ability of samples was determined at 517 nm according the method described by Blois (1958) with ascorbic acid as a positive control. The negative control was prepared with ethanol instead of the sample. DPPH scavenging activity was calculated using the following formula: DPPH scavenging activity (%) = [(Acontrol-Asample)/ (Acontrol)]  100 Where: Acontrol represents the absorbance of the negative control, Asample represents the absorbance of the test sample. The IC50 value is deduced from the logarithm curve of scavenging activity with sample concentration. Reducing power assay Reducing power was evaluated according to the method of Sapkota et al., (2010) with quercetin and ascorbic acid as positive controls. Samples were prepared at the concentration range of 0.025; 0.05; 0.1; 0.2; 0.4 mg/mL. The higher the absorbance the stronger the reducing power is. Antimicrobial activity assay Antimicrobial activity assay was performed based on the method described by Hadacek et al., (2000). Each fraction was dissolved in dimethyl sulfoxide and water. Dimethyl sulfoxide served as a negative control and ampicillin, streptomycin and amphotericin B were used as positive controls. The IC50 value was determined through a graph of the correlation between the percentage inhibiting the growth of microorganisms and the test concentration. Trypsin inhibitory activity Trypsin inhibition was determined according to Hoang Thu Ha and Pham Thi Tran Chau (2006) with 0.1% casein as the substrate. Each sample was prepared at the concentration of 20 mg/mL and mix with 0.05 mg/mL enzyme at the ratio of 1:1. The control consisted of only enzyme and the blank contained only buffer. The agar plates were stained with 0.1% amidoblack. Statistical analysis The experimental results were performed in triplicate. The data were recorded as mean ± standard deviation and analyzed by SPSS and Microsoft Excel software. A level of p value less than 0.05 was regarded as significant. PHẦN II. NGHIÊN CỨU ỨNG DỤNG SINH HỌC PHỤC VỤ ĐỜI SỐNG VÀ PHÁT TRIỂN XÃ HỘI 881 3. RESULTS AND DISCUSSION Thin layer chromatography Secondary metabolite composition of four fractions from S. collinsae methanolic root extract has been investigated preliminarily by thin layer chromatography. n-Hexane Butanol Ethyl acetate Water 302 365 Vis 302 365 Vis 302 365 Vis 302 365 Vis Figure 1. Thin layer chromatogram of S. collinsae root fractions under UV radiation (302 nm, 365 nm) and visible light (Vis) Under visible light (Figure 1), all fractions show violet and dark grey color, indicating they are rich in terpene and alkaloid compounds, respectively. Yellow-orange bands in ethyl acetate and n-hexane fractions reveal the presence of flavonoids. Additionally, under 302 nm radiation, butanol, ethyl acetate and n-hexane fractions have blue fluorescent bands, suggesting these fractions contain phenolic acids. The fluorescent bands under 365 nm radiation indicate the presence of alkaloids in those fractions. The results of TLC showed that the n-hexane and ethyl acetate fractions have the most chemical compounds, especially alkaloids and phenolics. In previous researches, alkaloids were predominant compounds in S. collinsae roots, which contributed to its biological properties (Greger, 2019). Recently, Rutnakornpituk et al., (2018) reported the presence of phenolics in the crude extract of S. collinsae roots. The TLC results of fractions from S. collinsae root extract especially ethyl acetate fraction confirmed their constituents including phenolics, flavonoids, which need further characterizing. Total phenolic and flavonoid content Natural phenolics widely distribute in plant for various bioactivities such as antioxidant, anticancer and anti-inflammatory effect. (Dai J & Mumper R. J, 2010). There is significant difference in phenolic content among S. collinsae fractions (Table 1). Particularly, the phenolic content of ethyl acetate fraction was about 5 times higher than that of butanol fraction and about 9 times than that of n-hexane fraction. However, the flavonoid content of all four fractions from roots extracts of S. collinsae were rather low. Ethyl acetate fraction also contained significantly highest flavonoid content. 882 BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM Table 1. Total phenolic and flavonoid content of S. collinsae root fractions Fractions Total phenolic content (mg GAE/g fraction) Total flavonoid content (mg QE/g fraction) n-Hexane 16.81 ± 2.65c 5.10 ± 0.35 a Butanol 32.19 ± 7.81a 5.12 ± 0.41 a Ethyl acetate 154.64 ± 4.29b 11.66 ± 2.12 b Water 7.60 ± 1.30c 4.04 ± 0.37 a a,b,c: Significant difference among fractions at p < 0.05. The low level of flavonoids was also reported for all crude extracts from roots, stems and leaves of S. collinsae (Rutnakornpituk et al., 2018). However, the amount of phenolic compounds in ethyl acetate, butanol and n-hexane fractions in this study was much higher than the figure of root crude extracts of S. collinsae from Thailand, only 15.58 ± 0.67 mg GAE/g (Rutnakornpituk et al., 2018). More phenolics were concentrated in the fractions especially ethyl acetate fraction. Additionally, S. collinsae fractions have moderate to low phenolic content as those of S. cochinchinensis in previous report (Ha Thi Minh Tam & Le Thi Phuong Hoa, 2015). Higher phenolic content in the ethyl acetate fraction might be expected to exhibit various bioactivities such as antioxidant activity. Antioxidant activity DPPH radical scavenging activity Based on the correlation between DPPH scavenging activity (%) and concentration of S. collinsae fractions and ascorbic acid, IC50 values was calculated. Table 2. DPPH radical scavenging activity of S. collinsae root fractions compared with ascorbic acid Sample IC50 (μg/mL) n-Hexane fraction 3.258c Butanol fraction 4.884b Ethyl acetate fraction 0.676a Ascorbic acid 0.034d a,b,c,d: significant difference among fractions and ascorbic acid at p<0.05. As the lower IC50 value indicates the higher antioxidant activity, ethyl acetate fraction had a much higher ability to scavenge free radical as compared to n-hexane and butanol fractions (Table 2). Scavenging activity of water fractions reached only 2.88% at the concentration of 6.4 mg/mL (data not shown). Furthermore, the ethyl acetate fraction of S. collinsae root extract showed stronger radical scavenging capacity than that of S. cochinchinensis in previous study, 0.681 ± 0.063 µg/mL vs. 0.849 ± 0.167 µg/mL, respectively (Ha Thi Minh Tam and Le Thi Phuong Hoa, 2015). High antioxidant activity displayed by ethyl acetate fraction has a positive correlation with its phenolic content. This signified that ethyl acetate fraction of S. collinsae root extract contained phenolic compounds that can donate hydrogen easily. PHẦN II. NGHIÊN CỨU ỨNG DỤNG SINH HỌC PHỤC VỤ ĐỜI SỐNG VÀ PHÁT TRIỂN XÃ HỘI 883 Reducing power In order to examine further antioxidant mechanism of S. collinsae root fractions, we used the reducing power assay. This method is based on the principle of increase in the absorbance of the reaction mixtures in coincidence with the increase of the ferric ion reducing activity. Figure 2. Reducing power of S. collinsae root fractions Figure 2 illustrates the correlation between concentration and absorbance at 700 nm of S. collinsae root fractions. All S. collinsae root fractions performed low reducing power when comparing with quercetin and ascorbic acid. In particular, at the concentration of 0.4 mg/mL, the absorbance value of ethyl acetate, n-Hexane, butanol and water fraction only reached 0.58, 0.53, 0.43 and 0.23, respectively. Compounds in S. collinsae fractions are suggested to have low electron-donating activity through ferric ion reduction. Their antioxidant activity might be signifed by hydrogen donation. This is the first report on reducing power of S. collinsae. Antimicrobial activity assay S. tuberosa was reported to have antibacterial activity in China and former Soviet Union (Do Tat Loi, 2004). However, our result showed that S. collinsae root fractions had weak inhibition on the growth of three Gram negative, three Gram positive bacterial strains and one microfungal strain (IC50 > 128 μg/mL). In previous research by Akanitapichat et al., 2005, dichloromethane–methanol (1:1), 95 % ethanol and aqueous extracts of S. collinsae roots also exhibited no activity against S. aureus, E. coli, S. typhimurium, P. aeruginosa, C. albicans and A. niger by a disc diffusion assay. It might be assumed that antibacterial activity is not a property of S. collinsae roots. 0 0.5 1 1.5 2 2.5 0 0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 A b so rb an ce a t 7 0 0 n m Concentration (mg/mL) Ascorbic acid Quercetin Butanol Ethyl acetate n-Hexane Water 884 BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM Trypsin inhibitory activity Trypsin is one of the best characterized serine proteinases. Proteinases play essential roles not only in many physiological processes (food digestion, blood coagulation, fibrinolysis, and control of blood pressure) but also in a wide range of important pathological processes (atherosclerosis, inflammation, and cancer) (DeClerck et al., 2004). Trypsin expression is increased in human cancer cells of the ovary, prostate, lung, stomach, colon, and others. Therefore, trypsin inhibitors might have anticancer activity. In this experiment, we conducted trypsin inhibitory activity assay on four fractions of S. collinsae using casein agar plate. Figure 3. Trypsin inhibitory activity of S. collinsae root fractions (1_Blank; 2_Butanol; 3_Ethyl acetate; 4_n-Hexane; 5_Water; 6_Control) However, all the S. collinsae root fractions showed low activity in inhibiting trypsin at the concentration of 20 mg/mL (Figure 3). Diameter of hydrolytic ring ranging from 29.67 ± 0.58 mm for ethyl acetate fraction to 31.33 ± 1.15 mm for water fraction as compared to 31.67 ± 1.15 mm for the control. CONCLUSIONS n-Hexane, ethyl acetate, butanol, and water fractions from methanolic root extract of Stemona collinsae contain various secondary metabolites especially alkaloids and phenolics. The ethyl acetate fraction had the highest total phenolic content (154.64 ± 4.29 mg GAE/g) and total flavonoid content (11.662 ± 2.125 mg QE/g). S. collinsae root fractions showed moderate antioxidant activity through DPPH free radical scavenging activity. 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