Sephacryl S-200 Column Chromatography Research Articles

Production of laccase enzyme from Curvularia lunata MY3: purification and characterization

Laccase-producing fungus (MY3) was successfully isolated from soil samples collected from Mansoura Governorate, Egypt. This fungal isolate has shown a high laccase production level over other isolated fungi. The identity of this isolate was determined by the molecular technique 18SrRNA as Curvularia lunata MY3. The enzyme purification was performed using ammonium sulfate precipitation followed by Sephacryl S-200 and DEAE-Sepharose column chromatography. The denatured enzyme using SDS-PAGE had a molar mass of 65 kDa. The purified laccase had an optimum temperature at 40 °C for enzyme activity with 57.3 kJ/mol activation energy for 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) oxidation. The enzyme had an optimum pH of 5.0, and it has shown a high stability at the acidic range (4.5 to 5.5). Mn2+ and Mg2+ ions enhanced the enzyme activity, while most of the enzyme activity was inhibited by Hg2+. Some compounds such as 2-mercaptoethanol, L-cysteine, and sodium azide at a concentration of 10 mmol/L had shown a high suppression effect on the enzyme activity. The enzyme strongly oxidized ABTS and syringaldazine and moderately oxidized DMP and guaiacol. The antimicrobial activity of the purified enzyme towards three pathogenic strains (Escherichia coli ATCC-25922, Staphylococcus aureus NRRLB-767, and Candida albicans ATCC-10231) was evaluated for the potential use as an antimicrobial therapeutic enzyme.

Identification and Characterization of Mosquitocidal Toxins From <i>Bacillus cereus</i> VCRC-641

Bacillus cereus VCRC 641 was found to be an alternate larvicidal bacterial agent which was newly isolated from fresh water fish, Clarias batrachus (walking cat fish). The protein responsible for mosquito toxic effect was separated and purified by Sephacryl S-200 column chromatography and NATIVE PAGE. It was observed that a unique protein with molecular weight of 70kDa was the responsible factor for mosquito killing effect. Further analysis of the protein by liquid chromatography-mass spectrometry (LC-MS) it was identified as “Uncharacterized Protein”. The length of the uncharacterized protein was around 158bp. The uncharacterized protein was expected to have a molecular mass of 70 kDa, 51 amino acids, confidence score of 78.45 and mass of 206772.57. Therefore, in the present study we report for the first time that uncharacterized protein (70kDa) was the responsible factor for the mosquito killing effect.

A new polysaccharide isolated from Dendrobium offcinale, stimulates aquaporin-3 expression in human keratinocytes

Polysaccharides are the major components of the water extract from Dendrobium officinale, which have been thoroughly studied and found to have favorable moisturizing properties on mammalian skin. However, the structures of polysaccharides on skin moisturizing properties remains unknown. The aim of this study was to extract and characterize D. officinale polysaccharides, which have anti-oxidant activity and the role of promoting effects of aquaporin-3 (AQP3). Two crude polysaccharide fractions were extracted using water and biological fermentation, respectively. The crude polysaccharides were further purified by graded alcohol precipitation, and finally purified polysaccharides DOP-S and DOP-F were obtained by Sephacryl S-300 column chromatography. The average molecular weights of DOP-S and DOP-F were 3.604 × 105 and 1.027 × 105 Da, respectively. Monosaccharide units and structures of DOP-S and DOP-F were further elucidated by gas chromatography-mass spectrometry (GC-MS) and Fourier Transform infrared spectroscopy (FTIR). At the same time, DOP-S and DOP-F induced aquaporin-3 (AQP3) expression was evaluated by immuno-fluorescence analysis. The antioxidant activities of the polysaccharides were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging capacities, and ferric reducing antioxidant power (FRAP). The results showed that biological fermentation effectively achieved the hydrolysis of the polysaccharides and stimulated antioxidant activity and AQP3 expression.

Structural characterization and antioxidant activity of alkali-extracted polysaccharides from quinoa

A polysaccharide component (SQAP-2) was isolated from quinoa by alkaline extraction and purified with DEAE-Sepharose Fast Flow and Sephacryl S-300 column chromatography. The chemical structure of the polysaccharide was characterized, and the impact on antioxidant activity was evaluated. Polysaccharide SQAP-2 (2.02 × 103 Da) is a type of glucose whose glycosidic bond Glc-(1→, →4)-Glcp-(1→, →4, 6)-Glcp-(1→ linkages were identified through gas chromatography-mass spectrometry. Nuclear magnetic resonance confirmed the monosaccharide composition and glycosidic linkage content, and a suggestion of the structural formula was provided. Their radical scavenging capacity was evaluated, and scavenging activity against DPPH, ABTS, hydroxyl, superoxide radicals, and ferric reducing ability exhibited excellent radical scavenging activities of approximately 21.71 ± 1.25%, 49.46 ± 1.59%, 70.10 ± 3.39%, 70.49 ± 2.09%, 51.25 ± 1.16%, respectively, at 3 mg/mL. The isolated quinoa polysaccharides could represent an antioxidant agent once the structural features and antioxidant activities are definitively characterized.

A simple and efficient purification method of native immunoreactive antigen for diagnosis of camel hydatidosis.

Background:Cystic echinococcosis (CE), a zoonotic disease that affects animal and human health, is of increasing economic importance due to high morbidity rates and high economic losses in the livestock industry.Aim:The present study was conducted to purify the antigen from hydatid cyst fluid (HCF) with high diagnostic efficacy of camel hydatidosis using indirect enzyme-linked immunosorbent assay (ELISA).Materials and Methods:The HCF antigen was purified using Sephacryl S-300 column chromatography. Characterization of fractions was performed using reducing and non-reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Further, antibodies against Echinococcus granulosus cysts in camel serum were detected using indirect ELISA.Results:The purification process resulted in three fractions of antigens: FI, FII, and FIII. Indirect ELISA showed that higher diagnostic efficacy was observed in FI than in FII and FIII. Indirect ELISA, in which FI was utilized, showed 88% sensitivity and 91.7% specificity. Non-reducing SDS-PAGE showed that FI had two bands of molecular weights 120 and 60 kDa. Western blot analysis of FI demonstrated that 60, 38, and 22 kDa were antigenic bands when reacted with naturally infected camel sera with E. granulosus cysts. Using indirect ELISA, F1 recorded an infection percentage of 81.7% in randomly collected camel serum samples.Conclusion:FI is a promising antigen for accurate diagnosis of camel CE using indirect ELISA.

Structural characterization of a galacturonic acid-rich polysaccharide from Ziziphus Jujuba cv. Muzao

Jujube (Zizyphus jujuba Mill.) have been widely used as a health food and medicinal herb in oriental medicine. In the present study, a novel galacturonic acid-rich polysaccharide (PZMP3-2) was isolated from Zizyphus Jujuba cv. Muzao through hot water extraction, ethanol precipitation, and purification with DEAE-Sepharose Fast Flow and Sephacryl S-300 column chromatography. The chemical structures of PZMP3-2 were elucidated by methylation analysis, along with HPGPC, FT-IR spectroscopy, GC-MS, 1D NMR spectroscopy, and 2D NMR spectroscopy. Its morphological properties were further characterized by SEM, AFM, and XRD. Monosaccharide compositional analysis of PZMP3-2 revealed the presence of rhamnose, arabinose, galactose, and galacturonic acid at a molar ratio of 1.74:2.00: 1.00:18.69, and the HPGPC data demonstrated an average molecular weight of 58.21 kDa. Structural and linkage analysis by GC-MS and NMR spectroscopy suggested that PZMP3-2 was a homogalacturonan (HG)-like pectic polysaccharide branched at C-2 with some Araf and Rhap residues. Such unique structure of PZMP3-2 may indicate distinct bioactivities and further application in food and even clinic.

An alkali-extracted polysaccharide from Zizyphus jujuba cv. Muzao: Structural characterizations and antioxidant activities

An acidic polysaccharide (SAZMP3) was isolated from jujube fruit residues by alkaline extraction and purified with DEAE-Sepharose Fast Flow and Sephacryl S-300 column chromatography. The structural characterizations of SAZMP3 were determined by high-performance gelpermeation chromatography, gas chromatography, Fourier transform infrared spectroscopy, methylation analysis, NMR spectroscopy, scanning electron microscopy, and atomic force microscopy. The results determined SAZMP3 was an acidic polysaccharide mainly contained rhamnose, galactose, and galacturonic acid with a molecular weight of 9.37 kDa and its backbone might mainly be composed of 1,4-α-D-GalAp with side chains of 1,3-β-D-Galp, 1,3,5-linked Araf, 1,2,4-α-L-Rhap and terminals of 1-linked Araf, 1-linked Rhap, 1-linked Galp by methylation and NMR analysis. Besides, SAZMP3 has shown potential antioxidant activity in scavenging free radicals suggesting that it is an antioxidant agent, which might contribute to the functional food qualities of jujube fruit.

An acidic polysaccharide from Ziziphus Jujuba cv. Muzao: Purification and structural characterization

A novel acidic polysaccharide fraction (PZMP2-2) was isolated from Ziziphus Jujuba cv. Muzao fruit via hot water extraction and purified by DEAE-Sepharose FF and Sephacryl S-300 column chromatography. PZMP2-2 consisted of rhamnose, arabinose, xylose, galactose, and galacturonic acid in a ratio of 1.18: 5.23: 0.22: 2.68: 2.20, with a molecular weight of 62.73 kDa. Structural features of PZMP2-2 were investigated using FT-IR, SEM, AFM, GC–MS analysis and NMR spectroscopy. Methylation and GC–MS analysis revealed that the main glycosidic bonds in PZMP2-2 comprised 1-linked Galp, 1,3-linked Araf, 1,2,4-linked Rhap, 1,3-linked Galp, 1,4-linked GalpA and 1,3,5-linked Araf. NMR analysis indicate that PZMP 2–2 has a linear backbone of (1 → 4)-linked GlcpA and (1 → 2,4)-linked Rhap residues, with branches at the O-4 position, consisting of Araf and Galp residues.

Structural characterization of a polysaccharide from Sargassum henslowianum, and its immunomodulatory effect on gastric cancer rat

A complicated sulfated fucoidan, SHPPB2, was purified from Sargassum henslowianum by DEAE-cellulose 52 and Sephacryl S-300 column chromatography. Via chemical and spectral method, SHPPB2 was found to contain mannose, glucuronic acid, galactose, xylose, and fucose, in a ratio of 17.4: 13.5: 10.5: 16.8: 41.8, as well as 21.4% of sulfate. The methylation analysis demonstrated terminal, 3-, 4-, 2, 3-, and 3, 4- linked fucose, 2-, 2, 3-, 2, 4-, and 2, 4, 6- linked mannose, terminal, 4-, 6-, 2, 4-, 3, 4-, and 3, 6- linked galactose, terminal and 4- linked xylose, and 4- linked glucuronic acid. In addition, the sulfate groups were substituted on the C-2, C-3 or C-4 of 3- and 4- linked fucose, on the C-4 or C-6 of 2- linked mannose, and on C-2 or C-3 of 4- and 6- linked galactose. With the treatment of SHPPB2 in the N-methyl-N’-nitro-nitrosoguanidine (MNNG) induced gastric cancer rats, it was observed with an increased body weight, and improved immune organ indices. Furthermore, SHPPB2 could significantly promote splenocyte proliferation induced by ConA or LPS in gastric cancer rats, and improve anti-inflammatory cytokines (IL-2, IL-4, and IL-10) secretion, but reduce pro-inflammatory cytokines (IL-6 and TNF-α). Taken together, it suggested that SHPPB2 could improve immune function in gastric cancer rats. Thus, it could be explored as a potential immuno-therapy for gastric cancer treatment.

Purification, structural characterization, and hypolipidemic effects of a neutral polysaccharide from Ziziphus Jujuba cv. Muzao

A neutral polysaccharide (PZMP1) with a molecular weight of 16.97 kDa was isolated from Ziziphus Jujuba cv. Muzao with water extraction, ethanol precipitation, and purification with DEAE-Sepharose Fast Flow and Sephacryl S-300 column chromatography. PZMP1 was structurally characterized with high-performance gel-permeation chromatography, gas chromatography, Fourier transform infrared spectroscopy, methylation analysis, NMR spectroscopy, X-ray diffraction, scanning electron microscopy, and atomic force microscopy. The results show that PZMP1 is a homogeneous heteropolysaccharide composed of arabinose, galactose, glucose, mannose, and xylose in a molar percentage of 17.36: 3.29:2.68:1.05:1.00. The majorly backbone of PZMP1 is composed of 1,3,5-linked Araf, 1,3-linked Araf, 1,5-linked Araf, 1,4-linked Glcp, and 1-linked Araf, 1-linked Glcp. According to an NMR analysis, the repeating unit of PZMP1 is a linear backbone with branches at positions O-3, and O-5. PZMP1 also displays effective dose-dependent hypolipidemic activity, suggesting that it is a natural hypolipidemic agent, which could contribute to the functional food qualities of jujube fruit.

A novel function of vitellogenin subdomain, vWF type D, as a toxin-binding protein in the pufferfish Takifugu pardalis ovary

Marine pufferfish of the Tetraodontidae family contain high levels of tetrodotoxin (TTX) in the liver and ovary. TTX is suggested to transfer from the liver to the ovary in female pufferfish during maturation. TTX in pufferfish eggs may act as a repellent against predators and as a sexual pheromone to attract male pufferfish. The toxification mechanism of the pufferfish ovary is poorly understood. Here we evaluated the chemical form of TTX and its related substances in the ovary of the panther pufferfish Takifugu pardalis by LC-ESI/MS. TTX and its analogs 4-epi-TTX, 4, 9-anhydroTTX, deoxyTTX, dideoxyTTX, and trideoxyTTX were detected in a low molecular weight fraction by Sephacryl S-400 column chromatography. The finding of an unknown TTX-related substance in a high molecular weight fraction from the Sephacryl S-400 column suggested the occurrence of toxin-binding protein in the ovary. The toxin-binding protein in the ovary was purified by ion-exchange HPLC, gel filtration HPLC, and SDS-PAGE. Amino acid sequencing and cDNA cloning revealed that the toxin-binding protein, TPOBP-10 (Takifugu pardalis ovary toxin-binding protein with a molecular mass of 10 kDa) was homologous with the predicted vitellogenin-1-like protein [Takifugu rubripes] subdomain, a von Willebrand factor type D domain. TPOBP-10 mRNA was highly expressed in the ovary and liver and less in other organs of female individuals based on RT-PCR. These findings reveal a novel function of the vitellogenin subdomain as binding with TTX-related substances, and its involvement in the toxification of the pufferfish ovary.

Biochemical properties of a serine protease from Aspergillus flavus and application in dehairing

The proteases are enzymes produced by several filamentous fungi with important biotechnological applications. In this work, a protease from Aspergillus flavus was characterized. The culture filtrate of A. flavus was purified to homogeneity by Sephacryl S-200 column chromatography followed by CM–cellulose. The molecular weight of the purified enzyme was estimated to be approximately 32 kDa by SDS–PAGE. The enzyme hydrolysed BTpNA (N-α-benzoyl-dl-tyrosyl-p-nitroanilide), azo-casein and casein as substrates. Optimal temperature and pH were 55 °C and 6.5, respectively. The enzyme was stimulated by Mg2+, Ca2+, Zn2+ and inhibited by Hg2+ and Ag2+ and Cu2+. The protease showed increased activity with detergents, such as Tween 80 and Triton X, and was stable to the reducing agents, such as β-mercaptoethanol. The protease activity was strongly inhibited in the presence of phenylmethylsulfonyl fluoride, indicating it is a serine protease. The enzyme entrapped in calcium alginate beads retained its activity for longer time and could be reused up to 10 times. The thermostability was increased after the immobilization and the enzyme retained 100% of activity at 45 °C after 60 min of incubation, and 90% of residual activity at 50 °C after 30 min. In contrast, the free enzyme only retained 10% of its residual activity after 60 min at 50 °C. The enzymatic preparation was demonstrated to be efficient in the capability of dehairing without destruction of the hide. The remarkable properties such as temperature, pH and immobilization stability found with this enzyme assure that it could be a potential candidate for industrial applications.

Structure and antitumor activity of the extracellular polysaccharides from Aspergillus aculeatus via apoptosis and cell cycle arrest.

Two extracellular polysaccharides, designated as WPA and WPB, were isolated from the fungus Aspergillus aculeatus using Q-Sepharose fast flow and Sephacryl S-300 column chromatography. WPA composed of mannose and galactose in a molar ratio of 3.9:1.0, and WPB mainly contained mannose. The molecular weight of WPA and WPB was about 28.1kDa and 21.0kDa, respectively. On the basis of methylation and NMR analysis, the possible main chain of WPA was [→5)-β-D-Galf-(1→2,6)-α-D-Manp(1→], and WPB was mainly [→2,6)-α-D-Manp(1→], both with [α-D-Manp(1→2)-α-D-Manp(1→2)-α-D-Manp(1→] substituted at C-2 of [→2,6)-α-D-Manp(1→]. Meanwhile, WPA displayed a stronger anti-proliferative effect than WPB on HeLa, MCF-7 and MGC-803 cells in vitro. WPA and WPB could arrest HeLa cells in G2/M phase and induce HeLa cells apoptosis. Thus, our study provides evidence that WPA and WPB may be taken as potential candidates for treating cervical carcinoma.

Structural and physical properties of sanxan polysaccharide from Sphingomonas sanxanigenens

Sphingomonas sanxanigenens, a new species of the genus Sphingomonas, synthesizes extracellular biopolymer termed sanxan. Sanxan polysaccharide was purified from the fermentation broth by Sephacryl S-400 column chromatography. The molecular weight of sanxan polysaccharide was 408kDa by the method of size-exclusion chromatography combined with laser light scattering. Based on FT-IR, periodate oxidation, Smith degradation, composition analysis and nuclear magnetic resonance experiments, the structure of sanxan polysaccharide was elucidated as follows: [Display omitted] The solution of sanxan polysaccharide showed properties of high viscosity and shear-thinning. By cooling hot solutions, sanxan polysaccharide could form elastic thermoreversible gel.

Separation, characterization and anticancer activities of a sulfated polysaccharide from Undaria pinnatifida

The purpose of this paper was to investigate separation, characterization and anticancer activities of a sulfated polysaccharide (SPUP) from Undaria pinnatifida. Firstly, polysaccharide from U. pinnatifida was separated by DEAE-52 cellulose and Sephacryl S-400 column chromatography. As results, SPUP was obtained with the yield of 19.42%. Then, SPUP was characterized using chemical analysis, gas chromatography, size-exclusion HPLC chromatography, UV–vis spectra and FT-IR spectrum. The content of total sugar, uronic acid, protein and sulfate radical were 80.48%, 3.21%, 7.12% and 29.14%, respectively. SPUP was a heteropolysaccharide composed of fucose, glucose and galactose in a molar percentage of 27.15:19.34:53.51 with molecular weight of 97.9kDa. Finally, the strongly against breast cancer activity of SPUP was confirmed by DMBA-induced breast cancer rats model. AS results, SPUP can significantly restrain breast abnormal enlargement, prolong tumor latency and reduced tumor incidence. Immunomodulatory activity and regulating abnormal sex hormones level might contribute to its anticancer activities.

Isolation and structural elucidation of a novel homogenous polysaccharide from Tricholoma matsutake

A crude polysaccharide possessing antitumour, radiation-resistant and anti-ageing attributes was extracted from Tricholoma matsutake by water extraction and alcohol precipitation. From this crude polysaccharide, a homogeneous polysaccharide, TMP-5II, was successfully purified by Sephacryl S-300 column chromatography. The average molecular weight (Mw) of TMP-5II was 15.76 kDa. Monosaccharide analysis indicated that the homogeneous polysaccharide contained four different residues: d-glucose, d-galactose, d-mannose and d-fucose. Attenuated total reflectance infrared spectroscopy revealed characteristics typical of carbohydrate polymers and a peak typical of a β-type glycosidic bond. TMP-5II was selected for structural characterisation by nuclear magnetic resonance (NMR) analysis. According to 1H NMR, 13C NMR and two-dimensional-NMR analysis, TMP-5II contains two kinds of linkages, β and α, at a ratio of 4:1. Preliminary results indicated that the polysaccharide had (1-4)-beta-pyran glucose as the main chain, and a branched chain in the O-6 location with fucose (1-2) mannose (1-3)-alpha-pyran galactose.

Purification, Physicochemical Characterization, and Bioactivities of Polysaccharides from Puerh Tea

Two fractions of polysaccharides, named PTPS-1 and PTPS-2, was extracted and purified from puerh tea by Sephacryl S-300 column chromatography. The physicochemical properties of these two polysaccharides were investigated by high-performance liquid chromatography (HPLC), Fourier Transform IR spectra, scanning electron microscope (SEM) and atomic force microscope (AFM). Analysis of chemical compositions (protein, neutral sugars, uronic acid and monosaccharide composition) suggested that they were both kinds of acid heteropolysaccharides bound with protein, and contained seven monosaccharides with different molar ratio. Meanwhile, evaluation of antioxidant activities by in vitro assays of DPPH, ABTS and FRAP showed that PTPS-1 demonstrated stronger antioxidant ability than PTPS-2. Similarly, PTPS-1 exhibited remarkable inhibitory potential on α-glycosidase in vitro, significantly stronger than that of PTPS-2 and acarbose. Moreover, the results from animal test indicated PTPS-1 possessed significant inhibition on postprandial hyperglycemia in diabetic mice compared with the model group.

Purification, antioxidant activity and antiglycation of polysaccharides from Polygonum multiflorum Thunb

Polysaccharides, one of the most important constituents in Polygonum multiflorum Thunb, a famous Chinese medicinal herb, were isolated by DEAE-52, Sepharose 4B and Sephacryl S-300 column chromatography. Two polysaccharides (PMP-1 and PMP-2) were identified as homogeneous in molecular weight with HPLC. The molecular weights were 4.8×102 and 6.1×102kDa, respectively. Antioxidant activity tests were performed with two polysaccharides at concentrations of 0.1–1.5mg/mL. The results indicated that the inhibitory activity on oxidation and glycation exhibited a dose-dependent response. PMP-2 exhibited a much stronger antioxidant capacity against free radical, lipid oxidation and protein glycation. The IC50 values of PMP-2 were 0.47, 0.6 and 0.93mg/mL for superoxide anion scavenging, hydroxyl radical scavenging, and hydroxyl peroxide scavenging, respectively. The inhibitory ability of PMP-2 on lipid oxidation was most markedly in rat liver, followed by heart and kidney. Meanwhile, PMP-2 also showed satisfactory suppression of AGEs formation. This suggested that the polysaccharides present in PM can contribute to the biological effects.

Characterization and antioxidant activity of a polysaccharide extracted from Sarcandra glabra

Response surface methodology (RSM) was employed to optimize the parameters for polysaccharide extracted from the aerial parts of Sarcandra glabra (SGP). The optimum conditions were predicted as follows, ratio of water to raw material at 30, extracting temperature at 85°C, extracting duration at 3h, and yield was estimated at 4.55%. The experimental yield of SGP under the optimum conditions was 4.49±0.09%. A homogenous polysaccharide (SGP-1) was obtained by purification using DEAE-cellulose-52 and Sephacryl S-400 column chromatography. SGP-1 showed a single symmetrical peak in high performance size-exclusion chromatography (HPSEC) and the average molecular weight (Mw) was estimated to be 1.06×104Da. It was composed of glucose, galactose, and mannose in a ratio of 8.38:3.13:1 determined by gas chromatography (GC). The in vitro antioxidant tests showed that SGP-1 has significant inhibition effects on hydroxyl, superoxide anion, DPPH, ABTS radicals in a dose-dependent manner. This study indicated that SGP-1 could be used as a potential natural antioxidant.

In vivo macrophage activation and physicochemical property of the different polysaccharide fractions purified from Angelica sinensis

The water-soluble crude polysaccharide (CAP) was extracted from Angelica sinensis and was further fractionated by Sephacryl S-400 column chromatography, giving three polysaccharide fractions termed APF1, APF2 and APF3. The obtained results showed that the uronic acid linkages in the polysaccharides were more stable and resistant to thermal hydrolysis than neutral sugar linkages. APF2 exhibited higher thermal stability, followed by APF1, APF3 and CAP in decreasing order. APF1 contained the highest carbohydrate content (80.5%) and the lowest uronic acid content (28.4%) in comparison with APF2 (77.7% and 39.2%) and APF3 (70.1% and 34.6%). Further bioactive investigation showed that intraperitoneal administration of the polysaccharide fractions significantly induced peritoneal macrophage to release nitric oxide (NO), reactive oxygen species (ROS) and enhanced cellular lysosomal enzyme activity ( p < .05) and the highest activity was achieved by APF2. Taken together, APF2 had the high thermal stability and immunostimulatory activity and so should be explored as a novel potential immunostimulants.