The immunomodulatory activity of extract from
The ascidians are commonly found in waters all over the world, along the coasts and deep to the bottoms, which is the most important marine source for active agents except sponge. In the early 1970s, it had been found that the extract from ascidians had a variety of bioactivities such as cytotoxicity, antitumor
By strengthening organisms’ defense and host immune system against tumor cells, cell immunity can improve its overall antitumor ability to inhibit tumor and thus play an important role in immune antitumor treatment. Alcohol from
The ascidian has become the hot topic for research and development both at home and abroad for its numerous active agents with high activity.
This paper is a study on
Kunming mice of clean grade were purchased from Shandong Animal Experiment Center, with weight being 18–22 g for each.
ConA, LPS, MTT, peptone, 1,1-DPPH,
Tissue of
After activity screening, normal phase silica gel column chromatography (CC) and ODS reversed phase silica gel column chromatography were adopted, respectively, for the most active ET, while petroleum ether and acetone, chloroform and carbinol were used for silica gel column chromatography gradient elution. Seven components (E1–E7) were isolated, among which components E2, E3, E4, and E5 for their large quantity were further purified by Sephadex LH-20, PTLC, Amberlite XAD-2, and recrystallization and 5 compounds were obtained and their structures were identified by IR (Nicolet impact 400), MS (HP 5988A GC/MS), and 1D NMR and 2D NMR (Bruker-500 MHz-FT-NMR).
Ten days after vaccinated S180, Kunming mice of clean grade were killed by cervical dislocation and spleens were taken out under asepsis for the preparation of lymphocyte suspension which was incubated in the 96-well plates (Coastar, USA) at the concentration of
2% (w/v) peptone of 2 mL was injected into the mice’s peritoneal cavity every day for three days. After cervical dislocation, precooled Hank’s of 5 mL was injected into the peritoneal cavity; then abdominal fluid was sucked and the supernatant was removed by centrifugation at 2000 rpm; the precipitate cells were washed twice.
Macrophages were prepared with the same method as in Section
1.5 mL DPPH of 0.2 mmol in anhydrous ethanol was added to all the samples of 1.5 mL, after mixing vigorously, and kept at room temperature in dark for 30 min. The absorbance of the resulting solution was measured at 517 nm with water as the control and the ability to scavenge the DPPH radical was expressed by clearance rate
The half clean concentration (EC50) is calculated by regression equation of E and sample concentration.
All data were expressed by mean
As organism’s first line defending tumor, immune system plays an important role in the recognition and removal of malignant cells. The immune status evaluation of the body bearing neoplasm is important for mechanism study of antitumor drugs.
As shown in Figure
Effects of extracts on the proliferation of the mouse splenocytes.
Figure
Effects of extracts on the proliferation of the mouse peritoneal macrophages.
ET and CE are close on the effect of proliferation. ET reaches the lowest point at 25
Macrophages are the important components in the body immune system, functioning as immunization effect regulating the immune system, and NO is one of the active agents produced by macrophages, after they are activated [
Effects of extracts on NO production.
The DPPH free radicals scavenging method is a colorimetric assay and can be used to evaluate the radical scavenging activity in a short time. DPPH is an organic free radical with maximal absorption at 517 nm. When with free radical scavenger, DPPH’s lone electron will be paired and its absorbance will decrease, which can be used to evaluate its antioxidant activity. As shown in Table
DPPH scavenging activity of different components isolated from
Component | DPPH scavenging rate (%) | |||
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100 ( |
150 ( |
200 ( |
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CE |
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PE |
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ET |
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BU |
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AR |
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E1–E7 subcomponents are isolated from ET with silica gel column chromatography, whose DPPH scavenging activity is shown in Table
DPPH scavenging activity of subcomponents E1–E7 isolated from ET of
Subcomponent | DPPH scavenging rate (%) | |||
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50 ( |
100 ( |
150 ( |
200 ( |
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ET |
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E1 |
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E2 |
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E3 |
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E4 |
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E5 |
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E6 |
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E7 |
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Antioxidant activity of different components isolated from
Component | Antioxidant activity (%) | |||
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10 ( |
50 ( |
100 ( |
200 ( |
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CE |
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PE |
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ET |
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BU |
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AR |
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AscA |
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The antioxidant activity of subcomponents E1–E7 isolated from ET in the
Antioxidant activity of subcomponents E1–E7 isolated from ET in the
Subcomponent | Antioxidant activity (%) | |||
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10 ( |
50 ( |
100 ( |
200 ( |
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ET |
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E1 |
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E2 |
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E3 |
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E4 |
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E5 |
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E6 |
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E7 |
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AscA |
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GA |
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Table
Reducing power of extract and subcomponents E1–E7 isolated from ET.
Component | AscAE (mg/g AscA) |
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CE |
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PE |
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ET |
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BU |
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WT |
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E1 |
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E2 |
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E3 |
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E4 |
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E5 |
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E6 |
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E7 |
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Compound 1, colorless needle crystal, is identified as C27H46O with EI-MS and NMR.
Compound 2, colorless needle crystal, mp 244
Compound 3 is a white needle crystal, mp 148
Compound 4 is white powder, mp 68
Compound 5, molecular formula is C36H71NO3 according to EI-MS and NMR.
The structures of these compounds are shown in Figure
Structures of compounds.
The immunomodulatory and antioxidant activity of crude and fractionated extracts of the ascidian
The authors declare that there is no conflict of interests regarding the publication of this paper.
Ju Bao and Chen Bin contributed equally to this project.
This study was supported by the National Natural Science Foundation of China (no. 31070368) and the Science and Technology Department of Shandong Province (BS2010HZ022 and ZR2011DL008).