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African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1964-1970, 15 July, 2012 Available online at http://www.academicjournals.org/AJPP DOI: 10.5897/AJPP12.484 ISSN 1996-0816 © 2012 <strong>Academic</strong> <strong>Journals</strong> Full Length Research Paper Preparation and biological activity of saponin from Ophiopogon japonicus Shuang-Li Xiong*, Da-Bin Hou, Ni Huang and Anlin Li College of Biological Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, Sichuan, China. Accepted 11 June, 2012 Saponin from Ophiopogon japonicus was isolated and preliminary identified by physical and chemical methods. The antioxidant and macrophages-modulating activities of the saponin were also investigated employing various established systems in vitro. The results of physical-chemical identification indicated that saponin was steroidal saponin. It was observed that the scavenging activity of saponin on 2, 2-diphenyl-1-picry-hydrazyl (DPPH) significantly increased in a concentration-dependent manner. The scavenging rate of it on DPPH radical was 99.64% when the concentration of the saponin was up to 5 mg/ml. For scavenging hydroxyl radical, it first increased and then decreased gradually with the increase of concentration, and reached the strongest scavenging rate at the concentration of 2 mg/ml. Saponin also exhibited prominent macrophages-modulating activity by the promotion of phagocytic capacity, macrophage viability, nitric oxide (NO) production and interleukin-1 release. It follows that saponin from O. japonicus can be explored as a novel and potential natural antioxidant and immunostimulants. Key words: Ophiopogon japonicas, saponin, antioxidant activity, macrophages. INTRODUCTION Saponins are a diverse group of natural compounds occurring in a wide variety of plants, food, and a few marine animals. They are classified into two categories (steroidal saponins and triterpenoid saponins) according to the nature of their aglycone skeleton (Sparg et al., 2004). Although, saponins are extremely toxic to coldblooded animals, their oral toxicity to mammals is low (Dini et al., 2001). Many plant drugs and folk medicines contain saponins that are found to have several kinds of bioactivities, such as antiviral, anti-inflammatory, antiparasitic, immune-enhancing, anti-cancer, and antimicrobial activities (Navarroa et al., 2001; Estrada et al., 2000). Thus, plants containing saponins are becoming research focus all over the world. The tuber of Ophiopogon japonicus (Thunb.) Ker-Gawl, *Corresponding author. E-mail: lxberry225@yahoo.com.cn. Tel: 86 816 6089531. the liliaceous plant, is an important traditional Chinese herbal medicine and has many health benefits in treating a wide range of disorders, mainly thrombosis, myocardial ischemia, arrhythmias, respiratory disease and hyperglycemia (Li et al., 2010) based on a wide variety of bioactive components, such as homoisoflavonoid, saponins, and polysaccharide (Anh et al., 2003; Chen et al., 2011). It has always been the food material which can be used as medicine and food in China. In recent years, some studies have shown that saponins from tuber of O. japonicus have the pharmacological activity of antimyocardial ischemia (He and Dai, 2005). However, few studies have so far been carried out regarding the antioxidant and immunoregulatory activity. In the context of the present work, we have isolated and evaluated antioxidant (2, 2-diphenyl-1-picry-hydrazyl (DPPH) and hydroxyl radical scavenging assay) and macrophagesmodulating activities (phagocytic activity, macrophage viability, NO release and interleukin-1 production) of saponins from tuber of O. japonicus.
EXPERIMENTAL PROCEDURE Materials and reagents Fresh tuber of O. japonicus was collected in Mianyang known as Sichuan O. japonicus. Butylated hydroxyanisole (BHA), DPPH radical, concanavalin A (ConA) and lipopolysaccharide (LPS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Roswell Park Memorial Institute (RPMI) Medium 1640 and fetal bovine serum (FBS) were obtained from GIBCO (Grand Island, NY, USA). 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl tetrazolium bromide (MTT) were purchased from Amresco. All the reagents were of analytical grade. Extraction and isolation of saponin Crushed tuber of O. japonicus was firstly extracted with 70% alcohol at 60°C in ultrasonic washing machine (200 W) for 1.5 h. The extracts were evaporated of alcohol at 45°C in vacuum on rotary evaporation instrument (SHANG HAI HU XI ANAYSIS INSTRUMENT FACTORY CO., LTD). The resulting solution was extracted with diethyl ether, and the treatment was repeated twice. The obtained water layer was again extracted with normal butyl alcohol saturated with water, and the treatment was repeated four times. The obtained normal butanol layer was re-extracted with 0.4 M NaOH (twice). The normal butanol layer was evaporated just to dryness with a vacuum evaporator at 45°C to obtain saponin which was again purified by non-polar macroporous adsorption resin D 101 (particle size of 0.3 to 1.25 mm, pore size of 6.0 to 10.0 nm, TIANJIN HAIGUANG CHEMICAL CO., Ltd., China). A portion of it was dissolved in deionized water and loaded onto a chromatographic column (2.0 × 20 cm). It was firstly eluted with 0.1% of NaOH to colourless eluent, then eluted by deionized water for removing carbohydrate. It was again eluted by 70% of ethanol at a flow rate of 2 BV/h for collecting saponin after eluent exhibited Mollish negative reaction. The purified saponin was evaporated just to dryness with a vacuum evaporator at 45°C and was stored at 20°C for further evaluation of antioxidant and macrophagesmodulating activities. Physico-chemical properties of saponin Saponin was preliminary identified by chemical methods including foam experiment and Liebermann reaction, and thin layer chromatography (Chen, 2004). Briefly, sample containing methanol solution was spotted on silica gel G thin-layer plates, with chloroform-ether (1:9) as developing solvent. Vanillin-sulfuric acid reaction system was used for identification of saponin. DPPH radical scavenging activity of saponin The DPPH radical scavenging activity of saponin was measured using the method reported by Song et al. (2010) with slight modification. Briefly, 1 ml sample with different concentrations (1 to 5 mg/ml) was added to screw-capped tube containing 1.5 ml of DPPH (40 mg/L, m/v) anhydrous alcohol solution. The mixture was vortexed and set for 30 min, then the absorbance was measured at 517 nm against a blank (water instead of test sample solution) using UNICO 2102 spectrophotometer (UNICO Instruments Co., Ltd., Shanghai, China). Ascorbic acid and BHA were used for positive comparison. DPPH radical scavenging rate (SC) is calculated as follows: DPPH radical scavenging rate (%) = 100 × (A0 - A1)/A0. where A0 is absorbance of blank control and A1 is absorbance value Shuang-Li et al. 1965 of the sample. The IC50 value was calculated by nonlinear regression algorithm SC via sample concentration. This is defined as the amount of antioxidant necessary to reduce the concentration of DPPH radical by 50%. Hydroxyl radical scavenging activity of saponin Hydroxyl radical (·OH) scavenging activity of saponin was assessed by the method of Zhu et al. (2010). Briefly, 2 ml deionized water or tested sample was added to screw-capped tubes which contained 0.35 ml of 8.8 mM H2O2 and 0.35 ml of 6 mM FeSO4. The mixture was thoroughly shaken and set for 10 min before salicylic acid was added, and shaken vigorously, which was set for 10 min and absorbance at 510 nm was recorded. The ·OH scavenging activity was calculated according to the following equation: ·OH scavenging rate (%) = 100 × (A0 - A1)/A0 where A0 is absorbance value of blank and A1 is absorbance value of the sample. IC50 value was the same as that of DPPH radical. Isolation and culture of peritoneal macrophage Male Kunming strain mouse (7 to 8 weeks old, 19 ± 0.5 g body weight) was purchased from the Experimental Animal Institute of Sichuan Academy of Medical Science. Main members in research teams have obtained the permission to work with experimental animals. Mouse was intraperitoneally injected with 5% soluble starch three days in advance before experiments. Macrophages were prepared from mouse as described earlier with some modifications (Li et al., 2011; Yang et al., 2008). Briefly, peritoneal cell suspensions were harvested by peritoneal cavity lavage with 5 ml ice-cold sterile phosphate buffer solution (PBS). The recovered peritoneal fluid was centrifuged at 2380 g for 10 min, the cell pellets were suspended with RPMI-1640 cell culture media supplemented with 10% (v/v) heat-inactivated FBS, and was cultivated for 3 h at 37°C in a humidified 5% CO2 incubator. Non-adherent cells were removed by gentle washing twice with sterile PBS. 1 ml fresh RPMI- 1640 was added to the adherent macrophages and it was adjusted to the concentration of 5 × 10 5 cells/ml. Assay of phagocytic activity The effects of saponin on the phagocytic activity of macrophage were determined using neutral red assay as reported earlier with some modifications (Yi et al., 2010; Xiong et al., 2011). In brief, 100 μl of macrophages (5 × 10 5 cells/ml) were pre-incubated for 3 h in a 96-well plate before removal of non-adherent with RPMI- 1640. Various tested samples and RPMI-1640 supplemented with 10% FBS were added to each well, and were incubated for 48 h. 100 μl of 1% neutral red physiologic saline solution was then added. After an additional incubation for 3 h, the mixture was removed of supernatant and was washed 3 times with PBS prior to the addition of cytolysate (glacial acetic acid:alcohol, 1:1, 100 μl). The absorbance at 540 nm was recorded on Multiskan Spectrum Microplate Spectrophotometer (Thermo Scientific, America) after cytolysis. Higher absorbance indicates stronger phagocytic activity. Assessment of macrophage viability The effect of saponin on the macrophage viability was detected by the MTT assay as previously described with some modifications (Davicino et al., 2006). Briefly, 20 μl of MTT was added to macrophage (100 μl, 5 × 10 5 cells/ml) which was pre-incubated for
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African Journal of Pharmacy and Pha
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Editors Sharmilah Pamela Seetulsing
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Electronic submission of manuscript
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Fees and Charges: Authors are requi
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Table of Contents: Volume 6 Number
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Minimum inhibitory concentration (M
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Kim et al. 1887 Table 4. Antimicrob
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Klevens RM, Morrison M A, Nadle J,
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Figure 1. Chemical structure of ten
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ng/ml by 4.9 ml blank plasma with 5
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Figure 5. Chromatogram of plasma sp
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Figure 7. Chromatogram of a healthy
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Figure 11. Chromatograms of standar
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Shatoor 1903 Figure 1. The effect o
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Heart rate (Beats/min.) 345 330 315
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Table 2. Percent of changes in hear
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heart rate on coronary flow and car
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Urinary tract infections are the mo
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