Alpha-Momorcharin, a type I ribosome inactivating protein, induced apoptosis of hepatocellular carcinoma SK-HEP-1 cells through mitochondrial pathway.

Alpha-Momorcharin (α-MMC), as one of the most important type I RIPs, has been reported to exert inhibitory effects against various tumour cells through its N-glycosidase activity. The present study was designed to propose an efficient purification strategy and explored its mechanism of apoptosis signalling pathway against human liver cancer cells SK-Hep-1. α-MMC can be successfully obtained by our purification strategy combining ion-exchange and gel-filtration chromatography. The functional studies revealed that α-MMC obviously increased the level of ROS and apoptosis rate, induced cell cycle arrest in the G1 phase, and depolarised MMP of SK-Hep-1 cells. To further confirm whether α-MMC could induce mitochondria involved apoptosis, we found that PARP-1, Caspase-3, Caspase-9, and BCL-2 were downregulated upon α-MMC. Taken together, these results suggested that this natural purified α-MMC can induce apoptosis involved mitochondria and may serve as a potential novel therapeutic drug in the treatment of human liver cancer in the future.

Ribosome-inactivating protein MAP30 isolated from Momordica charantia L. induces apoptosis in hepatocellular carcinoma cells.

BACKGROUND: Ribosome-inactivating proteins (RIPs) have been reported to exert anti-tumor and anti-virus activities. A recent patent CN202011568116.7 has developed a new method to prepare Momordica anti-HIV protein of 30 kDa (MAP30). MAP30 is a type I RIP, which kills various tumor cells through the N-glycosidase activity and irreversibly inhibits protein synthesis. OBJECTIVE: To assess the potential role of MAP30 in inducing apoptosis of human hepatocellular carcinoma HCC-LM3 cells and elucidate the molecular mechanism of MAP30. METHODS: CC-8 assay was used to assess the proliferation of HCC-LM3 cells. Flow cytometry was used to measure the cycle, the level of ROS and apoptosis in HCC-LM3 cells. Western blots were used to measure protein levels Results: Treatment with MAP30 reduced survival and proliferation of human liver cancer HCC-LM3 cells in a dose-dependent manner. PI staining showed cell cycle arrest in G0/G1 phase. Furthermore, MAP30 increased the level of ROS in HCC-LM3 cells in 24 h treatment. To further confirm the role of MAP30 in inducing cell apoptosis, immunoblotting was carried out to detect the change of apoptosis-related proteins including PARP poly (ADP-ribose) polymerase (PARP-1), Casepase3 and Cleaved-Caspase9. We found that PARP-1 and Caspase-3 were downregulated, whereas Cleaved-Caspase9 were up-regulated in HCC-LM3 cells treated with MAP30. CONCLUSION: This study indicated that MAP30 has the potential to be a novel therapeutic agent for human hepatocellular carcinoma.

Chemosynthesis and characterization of site-specific N-terminally PEGylated Alpha-momorcharin as apotential agent.

Alpha-momorcharin (α-MC), a type I ribosome-inactivating protein (RIP) isolated from Momordica charantia seeds, has been extensively studied for its antitumor, antiviral and antifungal activities. However, as an exogenous protein, problems associated with short half-life and strong immunogenicity have limited its clinical application. Poly (ethylene glycol) (PEG), as a polyether compound, is a well established and efficient modifier to develop it as a potential agent. Nevertheless, conventional PEGylation is not site-controlled and the conjugates are often not homogenous due to the generation of multi-PEGylated derivatives. To obtain a homogenous mono-PEGylated α-MC, the PEGylation was carried out by coupling a 20 kDa mPEG-butyraldehyde (mPEG-ALD) with α-MC. The product was separated and purified by MacroCap SP chromatography. Results from SDS-PAGE and MALDI-TOF MS revealed that the PEGylated α-MC consisted of one molecule mPEG and α-MC. Edman degradation confirmed that the N-terminal residue of α-MC was successfully coupled with mPEG-ALD. The mono-PEGylated α-MC possessed an extremely similar secondary structure to native α-MC through spectral analyses. In addition, it also showed low immunogenicity by double immunodiffusion and preserved moderate antitumor activity to three kinds of tumor cell lines in vitro. Finally, trypsin resistance was also considerably improved.

Mono-PEGylation of Alpha-MMC and MAP30 from Momordica charantia L.: Production, Identification and Anti-Tumor Activity.

PEGylation is a well-established and effective strategy to decrease immunogenicity, which can increase the stability and in vivo half-life time. However, the generation of multi-site modified products is inevitable due to the lysine chemistry, which will bring difficulties in subsequent research, such as purification and quantification. Site-specific modification by mPEG-succinimidyl carbonate (mPEG-SC) is a widely used method for N-terminal conjugation. In this study, we used it for site-directed modification on two ribosome-inactivating proteins (RIPs), alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30), from Momordica charantia L. According to the optimization of previous modification conditions, we compared Macro-Cap SP with SP-Sepharose FF chromatography for separating the final mPEGylated RIPs. Two kinds of methods both can obtain homogenous mPEGylated RIPs which were identified by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing electrophoresis (IEF), and matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI-TOF/TOF) analysis. We also used iodine staining method to detect the amount of unmodified PEG. Furthermore, the inhibition activity of both mPEGylated and non-PEGylated RIPs against human lung adenocarcinoma epithelial A549 cells was detected. All of the results suggested that the mPEGylated α-MMC/MAP30 might be potentially developed as new anti-tumor drugs.

PEGylation alleviates the non-specific toxicities of Alpha-Momorcharin and preserves its antitumor efficacy in vivo.

Alpha-Momorcharin (α-MMC) is a ribosome inactivating protein from Momordica charantia with anti-tumor activity. Previously, we had observed that modification of α-MMC with polyethylene glycol (PEG) could reduce toxicity, but it also reduces its anti-tumor activity in vitro. This study aims to investigate whether the metabolism-extended properties of α-MMC resulting from PEGylation could preserve its anti-tumor efficacy in vivo through pharmacokinetics and antitumor experiments. The pharmacokinetics experiments were conducted in rats using the TCA (Trichloroacetic Acid) method. Antitumor activity in vivo was investigated in murine mammary carcinoma (EMT-6) and human mammary carcinoma (MDA-MB-231) transplanted tumor mouse models. The results showed that PEGylation increased the plasma half-life of α-MMC in rats from 6.2-7.5 h to 52-87 h. When administered at 1 mg/kg, α-MMC-PEG and α-MMC showed similar anti-tumor activities in vivo, with a T/C% of 38.56% for α-MMC versus 35.43% for α-MMC-PEG in the EMT-6 tumor model and 36.30% for α-MMC versus 39.88% for α-MMC-PEG in the MDA-MB-231 tumor model (p > 0.05). Importantly, at the dose of 3 mg/kg, all the animals treated with α-MMC died while the animals treated with α-MMC-PEG exhibited only moderate toxic reactions, and α-MMC-PEG exhibited improved anti-tumor efficacy with a T/C% (relative tumor growth rate) of 25.18% and 21.07% in the EMT-6 and MDA-MB-231 tumor models, respectively. The present study demonstrates that PEGylation extends the half-life of α-MMC and alleviates non-specific toxicity, thereby preserving its antitumor efficacy in vivo, and a higher lever of dosage can be used to achieve better therapeutic efficacy.

Α-MMC and MAP30, two ribosome-inactivating proteins extracted from Momordica charantia, induce cell cycle arrest and apoptosis in A549 human lung carcinoma cells.

α­Momorcharin (α­MMC) and momordica anti­human immunodeficiency virus protein (MAP30), produced by Momordica charantia, are ribosome­inactivating proteins, which have been reported to exert inhibitory effects on cultured tumor cells. In order to further elucidate the functions of these agents, the present study aimed to investigate the effects of α­MMC and MAP30 on cell viability, the induction of apoptosis, cell cycle arrest, DNA integrity and superoxide dismutase (SOD) activity. α­MMC and MAP30 were purified from bitter melon seeds using ammonium sulfate precipitation in combination with sulfopropyl (SP)­sepharose fast flow, sephacryl S­100 and macro­Cap­SP chromatography. MTT, flow cytometric and DNA fragmentation analyses were then used to determine the effects of α­MMC and MAP30 on human lung adenocarcinoma epithelial A549 cells. The results revealed that A549 cells were sensitive to α­MMC and MAP30 cytotoxicity assays in vitro. Cell proliferation was significantly suppressed following α­MMC and MAP30 treatment in a dose­ and time­dependent manner; in addition, the results indicated that MAP30 had a more potent anti­tumor activity compared with that of α­MMC. Cell cycle arrest in S phase and a significantly increased apoptotic rate were observed following treatment with α­MMC and MAP30. Furthermore, DNA integrity analysis revealed that the DNA of A549 cells was degraded following treatment with α­MMC and MAP30 for 48 h. The pyrogallol autoxidation method and nitrotetrazolium blue chloride staining were used to determine SOD activity, the results of which indicated that α­MMC and MAP30 did not possess SOD activity. In conclusion, the results of the present study indicated that α­MMC and MAP30 may have potential as novel therapeutic agents for the prophylaxis and treatment of cancer.

Immobilization of Aspergillus terreus lipase in self-assembled hollow nanospheres for enantioselective hydrolysis of ketoprofen vinyl ester.

The aim of this study was to improve the ability of Aspergillus terreus lipase to separate the racemic ketoprofen vinyl ester into individual enantiomers using hollow self-assembly alginate-graft-poly(ethylene glycol)/α-cyclodextrins (Alg-g-PEG/α-CD) spheres as enzyme immobilization carriers. The morphology and size of the Alg-g-PEG/α-CD particles were investigated by transmission electron microscopy (TEM) and were found to be nanoscale. To facilitate recycling, calcium alginate (CA) beads were developed to encapsulate Alg-g-PEG/α-CD particles, thereby producing Alg-g-PEG/α-CD/CA composite beads. The influence of buffer pH and enzyme concentration during immobilization was studied along with the biocatalyst's kinetic parameters. When the immobilized enzyme was under optimal conditions in the resolution reaction, maximal conversion (approximately 45.9%) and enantioselectivity (approximately 128.8) were obtained. The immobilized A. terreus lipase maintained excellent performance even after 20 reuses and retained nearly 100% of its original activity after 24 weeks of storage at 4°C.

A novel method for simultaneous production of two ribosome-inactivating proteins, α-MMC and MAP30, from Momordica charantia L.

Alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) from Momordica charantia L. have been confirmed to possess anti-tumor and anti-virus activities. Traditional purification methods of these two ribosome-inactivating proteins (RIPs) were separate which was time consuming and cost effective as well as low efficient. In order to obtain sufficient samples for researches, a strategy combining ion-exchange and gel filtration chromatography was developed and optimized in this study. Using this novel purification method, averagely 1162 mg of α-MMC and 535 mg of MAP30 were obtained from 400 g of Momordica charantia L seeds. The homogeneities of them were assessed by electrophoresis analysis. Determination of molecular weights of α-MMC and MAP30 were 28.585 kDa and 29.094 kDa by MALDI-TOF/TOF and pI were 9.02 and 9.12, respectively. The single glycoproteins were identified by Periodate-Schiff's base (PAS) and the saccharide content was tested to be 1.25% and 1.1% by anthrone-sulfuric acid method. Biological activities were evidenced by their ability to inhibit proliferation of lung adenocarcinoma A549 cell and to convert supercoiled plasmid pUC18 into relaxed forms. Finally, we also found that both two RIPs exhibited no superoxide dismutase (SOD) activity.

Purification and characterization of a hydrolysis-resistant lipase from Aspergillus terreus.

Lipase from Aspergillus terreus was purified to homogeneity using ammonium sulfate precipitation and chromatographies with Q-Sepharose and Sephacryl S-200. It showed a single band on SDS-PAGE and IEF-PAGE with a relative molecular mass of 37.2 kDa and pI of 3.2. Its glycoprotein nature was confirmed with the percentage of saccharides of 5.02% and 3.88% determined by the phenol/sulfuric acid and anthrone/ sulfuric acid methods, respectively. The lipase hydrolyzed both plant oils and animal oils, with the K(m) value for substrate p-NPP of 16.42 mM at pH 6.0, 50 °C. The enzyme was tolerant in a wide range of pH (pH 3-12) with optimum activity at pH 4.0. It remained stable under the highest temperature of 65 °C, with maximal activity at 50 °C. Ca²âº, Co²âº, Mn²âº, and Ni²âº stimulated enzyme activity, but Hg²âº caused inhibition. Detected detergents had no obvious effect on enzyme activity, except SDS, which stimulated the activity at lower concentrations but inhibited the activity at higher concentrations. The inhibitory effect on enzyme activity of phenylmethanesulfonyl fluoride revealed that the Ser was involved in catalysis. Saccharides had no obvious effect on enzyme activity but could enhance its thermostability. Furthermore, the enzyme was resistant to trypsin digestion.

[Effect of PEGylation of alpha-Momorcharin against its hepatotoxicity in rats].

OBJECTIVE: To explore the effect of PEGylation of alpha-Momorcharin (alpha-MMC), one of ribosome-inactivating proteins from bitter melon seed, against its hepatotoxicity in rats. METHODS: SD rats were randomized into NS group, alpha-MMC treated groups, and alpha-MMC-PEG treated groups. The doses of alpha-MMC and alpha-MMC-PEG were high, middle, and low dose (6.25, 2.08, 0.70 mg/kg). The rats were given different dose of alpha-MMC, or alpha-MMC-PEG via caudal vein every other day for consecutive 28 days and then left for 14 days recovery. The general condition of animals was observed, blood and liver samples were collected for liver function study and pathological examination on day 28 after initiation of administration and on day 14 after withdrawal. RESULTS: On day 28 after initiation of administration, the liver function damages were found in high-dose and middle-dose of alpha-MMC treated groups, such as the decreasing of ALB, increasing of GLB, A/G ratio decreasing and the dose-dependant increasing of AST, BIL and CHO. The pathological changes of hepatotoxicity were also observed in these two groups, including the massive hepatocyte, swelling degeneration, inflammatory cell infiltration, congestion and diffusive necrosis. However, the liver function and pathological changes in alpha-MMC-PEG treated groups were better than those in alpha-MMC treated groups. CONCLUSION: PEGylation could reduce the hepatotoxicity of alpha-MMC to rats.

[The preparation and identification of diagnostic recombinant glycerol kinase].

In order to establish an efficient and low-cost production procedure of recombinant glycerol kinase (r-GK), we expressed the r-GK gene at high level in E. coli by induction with lactose on a large-scale fermentation of 300L. The results showed that the biomass concentration reached OD600 of 42 and the expression of r-GK in E. coli accounted for about 30% of total soluble protein. The cell-free extract was processed by selective thermo-denaturation and then purified with Ni sepharose FF column chromatography. Finally, highly purified r-GK was obtained and its purity reached 97% by using analysis on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), polyacrylamide gel electrophoresis (PAGE) and gradient polyacrylamide gel electrophoresis (Gradient PAGE). Further identification study showed that the molecular weight of r-GK was 120kDa with two subunit of 58kDa. Contaminants of NADH oxidase and catalase were not detected in the sample pool of r-GK. The purified r-GK was able to retain about 85% of its initial activity at 4 degrees C for 30 days. After lyophilized, it can retain 93% of its initial activity at 4 degrees C for one year.

Alpha-momorcharin, a RIP produced by bitter melon, enhances defense response in tobacco plants against diverse plant viruses and shows antifungal activity in vitro.

Alpha-momorcharin (α-MMC) is type-1 ribosome inactivating proteins (RIPs) with molecular weight of 29 kDa and has lots of biological activity. Our recent study indicated that the α-MMC purified from seeds of Momordica charantia exhibited distinct antiviral and antifungal activity. Tobacco plants pre-treated with 0.5 mg/mL α-MMC 3 days before inoculation with various viruses showed less-severe symptom and less reactive oxygen species (ROS) accumulation compared to that inoculated with viruses only. Quantitative real-time PCR analysis revealed that the replication levels of viruses were lower in the plants treated with the α-MMC than control plants at 15 days post inoculation. Moreover, the coat protein expression of viruses was almost completely inhibited in plants which were treated with the α-MMC compared with control plants. Furthermore, the SA-responsive defense-related genes including non-expressor of pathogenesis-related genes 1 (NPR1), PR1, PR2 were up-regulated and activities of some antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) were increased after the α-MMC treatment. In addition, the α-MMC (500 µg/mL) revealed remarkable antifungal effect against phytopathogenic fungi, in the growth inhibition range 50.35-67.21 %, along with their MIC values ranging from 100 to 500 µg/mL. The α-MMC had also a strong detrimental effect on spore germination of all the tested plant pathogens along with concentration as well as time-dependent kinetic inhibition of Sclerotinia sclerotiorum. The α-MMC showed a remarkable antiviral and antifungal effect and hence could possibly be exploited in crop protection for controlling certain important plant diseases.

Preparation of an antitumor and antivirus agent: chemical modification of α-MMC and MAP30 from Momordica Charantia L. with covalent conjugation of polyethyelene glycol.

BACKGROUND: Alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) derived from Momordica charantia L. have been confirmed to possess antitumor and antivirus activities due to their RNA-N-glycosidase activity. However, strong immunogenicity and short plasma half-life limit their clinical application. To solve this problem, the two proteins were modified with (mPEG)(2)-Lys-NHS (20 kDa). METHODOLOGY/PRINCIPAL FINDINGS: In this article, a novel purification strategy for the two main type I ribosome-inactivating proteins (RIPs), α-MMC and MAP30, was successfully developed for laboratory-scale preparation. Using this dramatic method, 200 mg of α-MMC and about 120 mg of MAP30 was obtained in only one purification process from 200 g of Momordica charantia seeds. The homogeneity and some other properties of the two proteins were assessed by gradient SDS-PAGE, electrospray ionization quadruple mass spectrometry, and N-terminal sequence analysis as well as Western blot. Two polyethylene glycol (PEG)ylated proteins were synthesized and purified. Homogeneous mono-, di-, or tri-PEGylated proteins were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The analysis of antitumor and antivirus activities indicated that the serial PEGylated RIPs preserved moderate activities on JAR choriocarcinoma cells and herpes simplex virus-1. Furthermore, both PEGylated proteins showed about 60%-70% antitumor and antivirus activities, and at the same time decreased 50%-70% immunogenicity when compared with their unmodified counterparts. CONCLUSION/SIGNIFICANCE: α-MMC and MAP30 obtained from this novel purification strategy can meet the requirement of a large amount of samples for research. Their chemical modification can solve the problem of strong immunogenicity and meanwhile preserve moderate activities. All these findings suggest the potential application of PEGylated α-MMC and PEGylated MAP30 as antitumor and antivirus agents. According to these results, PEGylated RIPs can be constructed with nanomaterials to be a targeting drug that can further decrease immunogenicity and side effects. Through nanotechnology we can make them low-release drugs, which can further prolong their half-life period in the human body.

PEGylation is effective in reducing immunogenicity, immunotoxicity, and hepatotoxicity of α-momorcharin in vivo.

BACKGROUND AND AIM: α-momorcharin (α-MMC), a type I ribosome-inactivating protein (RIP) from Momordica charantia, is well known for its antitumor and antivirus activities. However, the immunotoxicity and hepatotoxicity hampers its potential therapeutic usage. In order to reduce its toxicity, we had modified the α-MMC with polyethylene glycol (PEG), and detected the toxicity of the PEGylated α-MMC conjugates (α-MMC-PEG) in vivo. MATERIALS AND METHODS: After α-MMC purified from bitter melon seeds, α-MMC-PEG was constructed with a branched 20 kDa (mPEG) 2-Lys-NHS, the tests of immunogenicity, immunotoxicity, and general toxicity of α-MMC-PEG were conducted in guinea pig and rat. RESULTS: The titer of specific IgG in rats, immunized by α-MMC-PEG, were approximately one-third of those that by α-MMC, all the guinea pigs treated with α-MMC died of anaphylaxis shock within 5 min, while no animals treated with α-MMC-PEG died in the active systemic anaphylaxis (ASA) test. The passive cutaneous anaphylaxis (PCA) reaction of α-MMC-PEG challenge in rats was significantly smaller than that of the α-MMC. The liver damage was greatly released, such as the change of globulin (GLB), aspartate aminotransferase (AST), total bilirubin (TBIL) cholesterol (CHOL), albumin (ALB), and the degree of hepatocyte necrosis in repeated toxicity study. CONCLUSIONS: PEGylation is effective in reducing the immunogenicity, immunotoxicity, and hepatotoxicity of α-MMC in vivo.

Immunoaffinity purification of α-momorcharin from bitter melon seeds (Momordica charantia).

α-Momorcharin (α-MMC), a type I ribosome-inactivating protein (RIP), has shown therapeutic potential such as anti-tumor and anti-viral agent. Traditional process of α-MMC purification from bitter melon seeds was time consuming and low efficient. To take this challenge, we made an affinity matrix by coupling the monoclonal antibody (McAb) with Sepharose 4B. Using this attractive strategy, 196 mg of α-MMC was obtained from 100 g of bitter melon seeds as the starting material. The yield of the protein was 2.7%. The homogeneity and properties of the protein were assessed by SDS-PAGE, acidic PAGE, RP-HPLC and N-terminal sequence as well as Western blot. Purified α-MMC showed remarkable inhibition to the melanoma cell line JAR and EMT-62058. In addition, it also displayed obvious inhibition on hepatitis B virus (HBV). This work provided a simple, rapid and efficient approach for α-MMC purification from Momordica charantia.

[Preparation and identification of monoclonal antibody against alpha-momorcharins].

Three BALB/c mice were immunized four times with alpha-momorcharins (alpha-MMC). Using polyethylene glycol (PEG) method, the immunized splenocytes were fused with SP2/0 cells. One strain of hybridoma cells was obtained which secrete antibodies against alpha-MMC. To get ascites, the hybridoma cells were injected into the abdominal cavity of mice. The antibodies were purified from ascites. Indirect enzyme linked immunosorbent assay (ELISA) and Western blot assay were applied to determine the specifity of the monoclonal antibody (McAb). The results showed that the McAb was specific to alpha-MMC without detectable cross-activity with MAP30. The McAb provided detecting method for further research of the structure and function of alpha-MMC.

PEGylation of alpha-momorcharin: synthesis and characterization of novel anti-tumor conjugates with therapeutic potential.

Alpha-momorcharin (alpha-MMC) is a ribosome-inactivating protein (RIP) with excellent cytotoxicity to tumor cells. However, its strong immunogenicity and short plasma half-life limit its clinical applications. To overcome this, we have to PEGylated alpha-MMC using a branched 20 kDa (mPEG) (2)-Lys-NHS. Homogeneous mono-, di- and tri-PEGylated alpha-MMCs were synthesized, purified and characterized. In vitro and in vivo analysis indicated that the serial PEG-conjugates preserved moderate anti-tumor activity with 36% acute toxicity and at most 66% immunogenicity decrease. These results suggested the potential application of alpha-MMC-PEG conjugates as an anti-tumor agent.

Anti-tumor activity and immunological modification of ribosome-inactivating protein (RIP) from Momordica charantia by covalent attachment of polyethylene glycol.

Ribosome-inactivating proteins (RIPs) are a family of enzymes that depurinate rRNA and inhibit protein biosynthesis. Here we report the purification, apoptosis-inducing activity, and polyethylene glycol (PEG) modification of RIP from the bitter melon seeds. The protein has a homogenous N-terminal sequence of NAsp- Val-Ser-Phe-Arg. Moreover, the RIP displayed strong apoptosis-inducing activity and suppressed cancer cell growth. This might be attributed to the activation of caspases-3. To make it available for in vivo application, the immunogenicity of RIP was reduced by chemical modification with 20 kDa (mPEG)(2)-Lys-NHS. The inhibition activity of both PEGylated and non-PEGylated RIP against cancer cells was much stronger than against normal cells, and the antigenicity of PEGylated RIP was reduced significantly. Our results suggested that the PEGylated RIP might be potentially developed as anti-cancer drug.

[Study on the immunotoxicity of ribosome inhibiting protein of Momordica charantia].

OBJECTIVE: To separate and purify ribosome inhibiting protein (RIP) from Momordica charantia (bitter melon) seeds and to evaluate its acute toxicity and immunotoxicity in animal. METHODS: Ion exchange chromatography and gel filtration chromatography were applied in the separating and purifying of RIP from Momordica charantia seeds. Then the acute toxicity testing of RIP in mice was conducted to obtain its half lethal dose (LD50). Active systemic anaphylaxis(ASA)test in guinea pig and passive cutaneous anaphylaxis test (PCA) in rat were performed to evaluate its immunotoxicity. RESULTS: The LD50 (iv) in mice of RIP was 25.2 mg/kg in ASA, guinea pigs of the higher and lower RIP group all appeared stong allergic responses and most of them died quickly. In PCA, obvious blue dye in skin were observed in SD rats of the RIP group. CONCLUSION: RIP getting from Momordica charantia seeds had a relatively strong immunotoxicity in animals.

[Preparation of 5-fluorouracil-peanut agglutinin conjugate and determination of the ratio of drug to conjugate].

5-fluorouracil was combined with peanut agglutinin by a water-soluble carbodiimide to prepare the tumor target conjugate of 5-fluorouracil-peanut agglutinin and the ratio of drug to conjugate was determined by the modified trinitrobenzenesulfonic acid method (TNBS). The ratio of drug to conjugate was 76.33%. The result showed that 5-fluorouracil could link to the peanut agglutinin by EDC/NHS crosslinking with high drug ratio.