相思子化学成分研究

本研究从相思子种子的乙醇提取物中首次分离得到8个化合物,通过理化性质及光谱分析鉴定其结构为N-9-甲基-β-咔啉(1)、异喹啉酮(2)、吲哚-3-羧酸(3)、2,3-二甲氧基-5,7-二羟基-二氢黄酮(4)、3-羟甲基呋喃醛(5)、3-羟基-2-甲基-4-吡喃酮(6)、豆甾醇-4,22-二烯-3-酮(7)和(6E,6’E)-2-hydroxypropane-1,3-diylbis(octadec-6-enoate)(8)。同时还得到13个其他成分。     

Immunomodulatory role of native and heat denatured agglutinin from Abrus precatorius

Lectins are known as polyclonal activators of lymphocytes and work through the induction of battery of cytokines, which vary from lectin to lectin. Most widely used biological response modifier Mistletoe lectin (ML-1) in therapy stimulates lymphocytes, macrophages, and natural killer cells and induces both TH1 and TH2 type cytokines. Abrus agglutinin, similar to ML-1 with respect to carbohydrate specificity [gal (beta1-->3) gal/Nac], was studied both in native (NA) and heat denatured (HDA) condition for murine splenocyte proliferation, cytokine secretion, NK-cell activation, and thymocyte proliferation in vitro with a view to assess its potential as an immunomodulator. Both NA and HDA activate splenocytes and induce production of cytokines like IL-2, IFN-gamma and TNF-alphabeta indicating a TH1 type of immune response. Native agglutinin and HDA induced conditioned media of adherent splenocytes could stimulate non-adherent splenocytes and vice versa. Heat denatured agglutinin was able to induce NK-cell activation at much lower concentration than that of NA, but the extent of NK-cell activation was higher for NA. Proliferation of thymocytes by NA and HDA was also observed. This study indicates that Abrus agglutinin could be a potential immunomodulator both in native as well as in heat denatured form.     

Immunopotentiating activity of abrin,a lectin from Abrus precatorius Linn

A non-toxic dose of abrin, (1.25 microg/kg body wt) consecutively for five days in normal mice stimulated specific humoral responses. A noticeable increase was observed in total leucocyte count, lymphocytosis, weights of spleen and thymus, circulating antibody titre, antibody forming cells, bone marrow cellularity and alpha-esterase positive bone marrow cells. The results suggest that abrin can potentiate the humoral immune response of the host.     

Physical studies on three lectins from the seeds of Abrus precatorius

The physical properties of three lectins from the seeds of the Abrus precatorius plant, abrin C, abrin A and the Abrus agglutinin, were studied. All three exhibited similar circular dichroic (CD) spectra in the near-ultraviolet having negative maxima at 286 and 293 nm. In addition, d-galactose induced similar conformational alterations in the three proteins as observed through changes in the near-ultraviolet CD from 280 to 295 nm. The near-ultraviolet CD spectrum of the toxic subunit of abrin C was very different from that of the parent molecule. The fluorescence emission spectra of the three proteins were also studied. All exhibited fluorescence near 335 nm which is quenched 9% by galactose. Iodide quenching of fluorescence using the Stern-Volmer analysis indicated different tryptophan accessibilities in the presence and absence of d-galactose for the Abrus agglutinin. The result suggest that there is a saccharide-induced conformational change which buries several partially exposed tryptophan residues. A comparable analysis of the closely related Ricinus agglutinin revealed that its tryptophan residues are more buried than those of the Abrus agglutinin and, unlike the Abrus agglutinin, there was no saccharide-induced change in tryptophan accessibility.     

A characterization of abrin a from the seeds of the Abrus precatorius plant

Abrin A was purified from the seeds of the Abrus precatorius plant and its physical and biological properties were studied. The biological properties of abrin A were found to be similar to the better studied Abrus protein, abrin C, in that it is toxic to cell-free protein synthesis and binds d-galactose.Abrin A contains carbohydrate moieties including both neutral and amine sugars but no metals, similar to the other two Abrus proteins (abrin C and the Abrus agglutinin). Amino acid compositions of the subunits of abrin A indicated that it consists of two different subunits of comparable size. Furthermore, one of the subunits showed microheterogeneity suggesting that abrin A is a mixture of isolectins. A comparative study of abrin A and abrin C based on compositions and tryptic maps reveals them to be closely related. The evidence suggests that the two abrins may have the same mechanisms of toxic action.Far-ultraviolet circular dichroic studies of abrin A show it to contain 47% β-pleated sheet and 10% α-helix, again similar to the other two Abrus proteins.     

Extraction,purification, and biochemical characterization of serine protease from leaves of Abrus precatorius

A protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ～?28?kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co²⁺, Ni²⁺, Hg²⁺, and Zn²⁺ while its activity has increased in the presence of Ca²⁺ and Mg²⁺. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its V_max and K_m determined using casein as a substrate were 94.34?U/mL and 349.07?µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.     

Anti-inflammatory activity of compounds isolated from the aerial parts of Abrus precatorius (Fabaceae).

Two triterpenoid saponins 1 and 2 isolated from the aerial parts of Abrus precatorius and their acetates derivatives, 3 and 4 have been tested for anti-inflammatory activity using the croton oil ear model. All the compounds exhibited anti-inflammatory activity but the acetates showed greater inhibition than the parent compounds.     

Inhibition, of hormone-induced ethylene synthesis by the indole plant-growth inhibitor from Abrus precatorius seeds

The plant growth inhibitory fraction from Abrus precatoriusseeds inhibited protein and RNA synthesis and IAA- and kinetin-stimulatedethylene production by lettuce seedlings as well as IAA-stimulatedgrowth and ethylene production by wheat coleoptile sections. (Received August 28, 1974; )     

Ultraviolet difference spectroscopic studies on the saccharide-binding properties of Abrus precatorius agglutinin

The nature of the binding of specific saccharides to Abrus precatorius agglutinin (APA) was studied by ultraviolet difference spectroscopy. Upon binding of saccharides, APA displayed difference spectra with maxima at 291-292 nm and 284-285 nm. Such spectra suggest that the state of the tryptophan residue closely associated with the saccharide-binding activity of APA is perturbed by the binding of a saccharide. The difference spectra value (delta epsilon) increased with increasing saccharide concentration. From the increase in delta epsilon at 291-292 nm, the association constant (Ka) was obtained for the binding of individual saccharides to APA. Lactose bound to APA with the highest affinity among the saccharides examined and its Ka value (8.3 X 10(3) M-1 at pH 7.0 and 25 degrees C) was approximately four times as large as that of galactose (2.2 X 10(3) M-1). Raffinose and methyl beta-galactopyranoside showed larger association constants than galactose. Galactosamine, N-acetylgalactosamine and 2-deoxy galactose were found to bind with APA with fairly low affinity. The shape of the lactose-induced difference spectrum changed with pH and the spectrum in the acidic region showed characteristic broadening of the difference maximum peaks. The affinity of lactose to APA was nearly equal in the range of pH 6-8, but decreased outside this pH region and with increasing temperature.     

Studies on the structure and properties of the lectins from Abrus precatorius and Ricinus communis.

The amino acid composition of the isolated A- and B-chains of the toxic lectins abrin and ricin was determined and compared. Even though the two toxins originate from widely different plants, statistical analysis of the amino acid content indicates extensive homologies in the amino acid sequence of the 4 chains. The intact lectins contain no free SH-groups whereas the isolated A- and B-chains contain close to one free SH-group each. The results indicate that in both toxins the A- and B-chains are connected by a single S-S bond. The B-chains of abrin and ricin contain similar amounts of mannose and glucosamine. The A-chain of ricin also contains some carbohydrate, whereas the A-chain of abrin appears not to be a glycoprotein. The non-toxic abrus and ricinus agglutinins contain more carbohydrate than abrin and ricin. The isoelectric points of the different lectin preparations were measured by isoelectrofocusing. The intact lectins are much more resistant to heat, freezing and chemical treatments than the isolated A- and B-chains. The intact lectins are also very resistant to treatment with proteolytic enzymes, whereas the isolated chains are easily digested. Evidence indicating that the toxins and their chains undergo extensive conformational changes upon reduction of the S-S bond is discussed.     

The interaction of Abrus precatorius agglutinin with saccharides as analyzed by fluorescence spectroscopy

The binding of saccharides to Abrus precatorius agglutinin (APA) was analyzed by fluorescence spectroscopy. Upon binding of specific saccharides, the fluorescence emission maximum of APA (338 nm) shifted to shorter wavelength by 5 nm, owing to the change in the environment of tryptophan. By analyzing the change in the fluorescence intensity at 338 nm as a function of concentration of saccharides, the association constants for binding of saccharides to APA were determined. The results suggest that in the saccharide binding site on each B-chain of APA, there may be a site which interacts with the saccharide residue linked to galactopyranoside at the non-reducing end, in addition to the site which recognizes the galactopyranosyl residue. Fluorescence quenching data indicate that 8 out of 24 tryptophans in APA are located at or near the surface of the protein molecule and are available for quenching with both KI and acrylamide, and 10 tryptophans are involved in the environment to which acrylamide has access but KI does not. Binding of lactose to APA reduced by 4 the number of tryptophan residues accessible to quenchers. Based on the results, it is suggested that the tryptophan residues at the saccharide binding site on each B-chain of APA are present on the surface of the APA molecule, and they are shielded from quenching by KI and acrylamide upon binding with specific saccharides.     

Isolation, purification, and some properties of a lectin and abrin from Abrus precatorius linn.

An alternative and simple procedure has been described for the simultaneous separation of the lectin and abrin from the seeds of Abrus precatorius and their purification free from each other. Both the lectin and abrin have been crystallized, the latter as salt-free crystals. One variety of abrin which was nonhemagglutinating and did not bind d-galactose was obtained. The lectin found homogeneous by immunodiffusion, immunoelectrophoresis and sedimentation had a molecular weight of 132,000 which underwent pH-dependent reversible association-dissociation at pH 7 and 2, dissociating into non-covalently bound subunits of approximately 64,000 molecular weight. The protein was stable in the pH range 2–10. The abrin molecule did not undergo any change at low pH values. The C- and NH₂-terminal groups of the lectin were found to be Ala-Leu (or Leu-Ala) and valine, respectively. Crystalline lectin showed the presence of three isolectins in isoelectric focusing.     

Effect of steroidal fraction of seeds of Abrus precatorius Linn. on rat testis

Dose-dependent degenerative changes in the testicular weights, sperm count, later stages of spermatogenesis and Leydig cells are observed in testis of rats treated with steroidal fraction of seeds of A. precatorius. These are correlated with the dose-dependent decrease in the enzyme activity of 3 alpha, 3 beta, 17 beta-hydroxysteroid dehydrogenases, glucose-6-phosphate dehydrogenase, sorbitol dehydrogenase and leucine aminopeptidase. The steroidal fraction may also exert its influence indirectly at the pituitary level by a feedback mechanism, leading to decrease in production and release of testosterone which results in significant alterations in the testis.     

Structure-function analysis and insights into the reduced toxicity of Abrus precatorius agglutinin I in relation to abrin

Abrin and agglutinin-I from the seeds of Abrus precatorius are type II ribosome-inactivating proteins that inhibit protein synthesis in eukaryotic cells. The two toxins share a high degree of sequence similarity; however, agglutinin-I is weaker in its activity. We compared the kinetics of protein synthesis inhibition by abrin and agglutinin-I in two different cell lines and found that approximately 200-2000-fold higher concentration of agglutinin-I is needed for the same degree of inhibition. Like abrin, agglutinin-I also induced apoptosis in the cells by triggering the intrinsic mitochondrial pathway, although at higher concentrations as compared with abrin. The reason for the decreased toxicity of agglutinin-I became apparent on the analysis of the crystal structure of agglutinin-I obtained by us in comparison with that of the reported structure of abrin. The overall protein folding of agglutinin-I is similar to that of abrin-a with a single disulfide bond holding the toxic A subunit and the lectin-like B-subunit together, constituting a heterodimer. However, there are significant differences in the secondary structural elements, mostly in the A chain. The substitution of Asn-200 in abrin-a with Pro-199 in agglutinin-I seems to be a major cause for the decreased toxicity of agglutinin-I. This perhaps is not a consequence of any kink formation by a proline residue in the helical segment, as reported by others earlier, but due to fewer interactions that proline can possibly have with the bound substrate.