Spheroplast induction inAnabaena variabilis Kütz andA. azollae stras

Summary Spheroplasts were obtained by lysozyme treatment of 48 hour (4– 8cells) akinete germlings of the cultured cyanobacteriaAnabaena variabilis andA. azollae originally isolated from the leaf cavity of the fernAzolla pinnata. The osmotic stabilizer was 0.5 M sucrose. At least 50% of the cells in a short filament became spheroplasts after 1–4 hours in lysozyme (1 mg/ml) in incubation medium at 34 °C, with greater than 75% viability after 2 hours. The spheroplasts were osmotically fragile and showed intense chlorophyll autofluorescence in UV light. In phase microscopy, treated cells appeared larger, became spherical and lost some of their optical refraction. Transmission electron microscopy confirmed the loss of the peptidoglycan layer and the partial remains of the outer membrane after lysozyme exposure. We previously obtained protoplasts ofAzolla fern leaf cells so that we now can study the recognition sites in both members of theAzolla/Anabaena nitrogen fixing symbiosis during cell wall degradation and regeneration.     

Electron Microscopy and Viability of Lysostaphin-induced Staphylococcal Spheroplasts, Protoplast-like Bodies, and Protoplasts

The cell walls of a selected isolate of Staphylococcus aureus FDA 209P were observed undergoing progressive disintegration when exposed to lysostaphin (1 unit/ml) in 24% NaCl solution. Electron micrographs of ultrathin sections of test cells after exposure to lysostaphin for 2 min showed only superficial evidence of lytic damage. However, an average of 89% of these cells were osmotically fragile, and 21% were damaged beyond their capacity to regenerate cell walls and to grow as normal staphylococci. The 68% (average) of the osmotically fragile cells which retained the capacity to revert to normal staphylococci were designated spheroplasts. Neither perforations of the cell walls nor separation of the cell walls from the plasma membranes were observed in the micrographs of these 2-min spheroplasts. Thus, it appears that the osmotic fragility of these and possibly all lysostaphin-induced staphylococcal spheroplasts results from the hydrolysis of a critical number of the pentapeptide cross-linkages of the murein of the cell wall. Electron micrographs of cells exposed to lysostaphin for 5 to 10 min showed perforations and more extensive damage, including the separation of walls from the plasma membranes and the disintegration of large sections of the walls. Smaller numbers of spheroplasts (21 and 8%) were recovered from these 5- and 10-min preparations; those recovered probably represent cells which were attacked more slowly than the majority by the lytic enzyme. The nonrevertible, osmotically fragile cells that retained segments of cell wall were designated protoplast-like bodies. After 20-min exposure to lysostaphin, all of the cell wall was digested away from most of the cells, and true staphylococcal protoplasts were produced. These lysostaphin-induced, osmotically fragile forms appear to have different osmotic properties from the staphylococcal "protoplasts" reported by other investigators and should serve as the basis for a variety of fundamental investigations.     

Osmotic Fragility and Viability of Lysostaphin-induced Staphylococcal Spheroplasts

When Staphylococcus aureus FDA 209P cells were treated with lysostaphin (1 unit/ml) in hypertonic sodium chloride or sucrose environments, viable, osmotically fragile spheroplasts were produced. Turbidimetric studies indicated that 64% (w/v) sucrose or 20 to 28% (w/v) sodium chloride gives maximal protection against lysis of the lysostaphin-treated cells. The NaCl appeared to give greater protection than the sucrose and proved to be much more suitable for viability and related studies. Viability of both shocked and nonshocked treated cells was determined by S. aureus colony counts on agar plates overlayered with the test dilution of the cells suspended in 4 ml of semisolid agar containing 72% sucrose. The difference in the counts represented the number of revertible spheroplasts. Under these conditions, 30 to 50% of the test cells were recovered as osmotically fragile, but revertible, spheroplasts after 5 to 10 min of exposure to lysostaphin in 24% NaCl. This rewere obtained after 5 to 10 min of exposure to lysostaphin in 24% NaCl. This recovery rate fell off rapidly with prolonged exposure. In view of residual turbidity of 30- and even 60-min exposure preparations, it appeared probable that most of the osmotically fragile cells were eventually converted to protoplasts by the prolonged lysostaphin treatment. Osmotically fragile cells were converted to osmotic stability by fixation with 4% (v/v) Formalin.     

Characterization of Escherichia coli nucleoids released by osmotic shock

Nucleoids were isolated by osmotic shock from Escherichia coli spheroplasts at relatively low salt concentrations and in the absence of detergents. Sucrose-protected cells, made osmotically sensitive by growth in the presence of ampicillin or by digestion with low lysozyme concentrations (50-5 μg/ml), were shocked by 100-fold dilution of the sucrose buffer. Liberated nucleoids stained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI), the dimeric cyanine dye TOTO-1, or fluorescent DNA-binding protein appeared as cloud-like structures, in the absence of phase contrast. Because UV-irradiation disrupted the DAPI-stained nucleoids within 5-10 s, they were imaged by time-lapse microscopy with exposure times less than 2 s. The volume of nucleoids isolated from ampicillin- or low-lysozyme spheroplasts and minimally exposed to UV (<2 s) was on average ～42 μm(3). Lysozyme at concentrations above 1 μg/ml in the lysate compacted the nucleoids. Treatment with protease E or K (20-200 μg/ml) and sodium dodecyl sulfate (SDS; 0.001-0.01%) caused a twofold volume increase and showed a granular nucleoid at the earliest UV-exposure; the expansion could be reversed with 50 μM ethidium bromide, but not with chloroquine. While DNase (1 μg/ml) caused a rapid disruption of the nucleoids, RNase (0.1-400 μg/ml) had no effect. DAPI-stained nucleoids treated with protease, SDS or DNase consisted of granular substructures at the earliest exposure similar to UV-disrupted nucleoids obtained after prolonged (>4 s) UV irradiation. We interpret the measured volume in terms of a physical model of the nucleoid viewed as a branched DNA supercoil crosslinked by adhering proteins into a homogeneous network.     

Preparation and characteristics of protoplasts of Streptomyces kanamyceticus

To prepare actively regenerating protoplasts of S. kanamyceticus, the influence of the conditions of the mycelium cultivation, the culture age, lytic conditions, composition of the regeneration medium, the procedure of the culture inoculation to the regeneration medium and other parameters were studied. The study resulted in development of optimal conditions for preparation of S. kanamyceticus protoplasts in a number of 1.10(9) protoplasts per ml. The cultivation on the ST medium with 10 to 15% sucrose and addition of glycine up to 1% for 30 hours (the stationary growth phase) followed by treatment of the culture with lysozyme in an amount of 2 mg/ml for 1 hour at 32 degrees C provided preparation of up to 100% of actively regenerating protoplasts free of mycelium fragments. The size of the protoplasts increased up to 1.5 micron against the usually observed size of 0.7 to 1.0 micron with using modified lyzing buffer with 20% of sucrose according to the method recommended for S. erythreus. However, 50 to 70% of the protoplasts had point of linear regions in the cell walls, which suggested that spheroplasts were mainly forming and the phenomenon was associated with the characteristic properties of the strain cell wall structure.     

Transformation in Quasi Spheroplasts of Bacillus subtilis

RECENTLY DEVELOPED DIFFERENTIAL PLATING MEDIA PERMIT THE DISTINCTION OF FOUR CELL TYPES IN INCOMPLETELY PROTOPLASTED POPULATIONS: intact, osmotically insensitive bacilli; osmotically sensitive rods; spheres with adherent wall residues, called quasi spheroplasts; and protoplasts. Such population mixtures were washed free of lysozyme, and then transforming deoxyribonucleic acid (DNA) was added. Transformation was nil in the protoplasts, very low in the residual osmotically insensitive bacilli, and markedly enhanced in both osmotically sensitive rods and quasi spheroplasts. Transformation in the latter two population fractions was reduced, respectively, by about 60% and about 80% by deoxyribonuclease treatment. DNA adhering to the quasi spheroplasts transforms these cells only if they are permitted to resume wall synthesis; when the same cells are plated on a medium where they shed the residual wall and form L colonies, no transformant L colonies are recovered. It is inferred that far-reaching or complete protoplasting blocks all entry of transforming DNA into the cell interior. This may be owing to eversion of mesosomes. Evidence that intact mesosomes may be required for DNA entry is provided by the finding that the recovery of transformants in the intact cell system is sharply reduced on plating media containing 25% gelatin. On such media, cells expel their mesosomes and 75% of them do not re-form any. Our own data and a survey of published results suggest the generalization that partial depolymerization of the cell wall by lysozyme may enhance competence, whereas its complete removal abolishes it.     

Electrotransformation of intact and osmotically sensitive cells of Corynebacterium glutamicum

Summary Intact and osmotically sensitive cells of Corynebacterium glutamicum can be efficiently transformed by electroporation. This was shown by using the plasmid vector pUL-330 (5.2 kb), containing the kanamycin resistance gene of transposon Tn5. The following electric parameters yielded efficient transformation. For intact cells: one exponentially decaying field pulse with time constants and with initial field intensities of E ₀=35–40 kV cm^-1; prepulse temperature 20°C. Cell regeneration (survival) was 100%–80%. Transformation efficiency can be increased by an additional freeze and thaw cycle of the cells, prior to electroporation. Lysozyme treated cells (osmotically sensitive) were transformed with three successive pulses of E ₀=25–30 kV cm^-1. Cell regeneration under these conditions was found to be 20–30%. The optimum yield of transformants/g plasmid-DNA was 3×10³ for intact cells, 2×10⁴ for intact cells which were frozen and thawed twice and 7×10⁴ for osmotically sensitive cells if the cell suspension was pulsed at a cell density of 1–3×10⁸/ml and at a DNA concentration of 0.2 g/ml up to 2 g/ml. The data obtained for osmotically sensitive cells suggest that the temperature increase accompanying the electric field pulse enhances colony formation and transformation efficiency if the initial prepulse temperature is 20°C, although regeneration of electroporated C. glutamicum cells starts to decrease at temperatures20°C.     

Active accumulation of tetracycline by Escherichia coli

1. At low concentrations of tetracycline (10mug/ml) net accumulation of the drug by Escherichia coli cells ceased after 7-10min. 2. At higher concentrations of tetracycline (>30mug/ml) the period of net accumulation of the drug was significantly extended. 3. The efflux of tetracycline from E. coli cells transferred from medium containing 10mug of tetracycline/ml to drug-free medium was a rapid temperature-dependent process and was accelerated by 2,4-dinitrophenol. 4. As the concentration of tetracycline in the preloading phase was increased, the rate of subsequent efflux of the drug progressively declined. The efflux of drug from cells preloaded in medium containing 200mug of tetracycline/ml was negligible, although efflux was readily provoked by 2,4-dinitrophenol, by N-ethylmaleimide or by omission of glucose from the medium. 5. The initial rate of uptake of tetracycline by E. coli cells was linearly proportional to the concentration of tetracycline in the medium up to the maximum concentration of drug obtainable under the experimental conditions used (400mug/ml, 0.83mm). 6. Although N-ethylmaleimide strongly inhibited the accumulation of tetracycline by E. coli, no evidence was obtained for the direct involvement of thiol groups in the transport process. It was concluded that N-ethylmaleimide inhibited accumulation by interruption of the energy supply of the cells. 7. Osmotic shock of E. coli cells did not significantly affect the influx of tetracycline, but promoted both efflux of tetracycline and cell lysis in cells treated with a high concentration of tetracycline. 8. A study of the distribution of tetracycline among the subcellular fractions of penicillin-induced spheroplasts preincubated with various concentrations of tetracycline indicated that 60-70% of the accumulated tetracycline was in the high-speed supernatant fraction. Sephadex chromatography showed that the tetracycline of this fraction was present as the free drug. Sephadex chromatography of a detergent extract of the membrane fraction, however, indicated that a significant proportion of the tetracycline radioactivity of this fraction was apparently bound to some macromolecular component. 9. Cellulose phosphate paper chromatography of cold-acid extracts of spheroplasts preloaded with tetracycline indicated that the accumulated drug was chemically unchanged. 10. Membrane preparations isolated from osmotically lysed penicillin-induced spheroplasts showed a temperature-dependent binding of tetracycline that was not energy-dependent and was not inhibited by N-ethylmaleimide. The binding process was stimulated by omitting Mg(2+) from the medium, but conversely was profoundly inhibited by EDTA. 11. The relevance of these findings to the probable mechanism of active tetracycline accumulation by E. coli is discussed.     

Kinetic and Morphological Observations on Saccharomyces cerevisiae During Spheroplast Formation

A strain of Saccharomyces cerevisiae which produced elongated cells under our growth conditions was investigated. By digestion of the cell walls with snail enzyme, the cells became spheroplasts after a transient state which we termed "prospheroplast." The prospheroplast could be lysed like the spheroplast, but it retained the shape of the original yeast cell if osmotically protected. Prospheroplasts and spheroplasts were prepared, and thin sections of samples taken throughout the process of wall removal were studied in the electron microscope, at regular intervals up to the time of complete conversion to spheroplasts. In addition, cell wall remnants recovered from spheroplast preparations were shadow cast for electron microscopy. This material revealed structures resembling bud scars with attached membranous matter. The kinetic studies showed that after a certain period of time all cells were transformed into prospheroplasts, whereas spheroplast formation started later, depending on the enzyme concentration. In sections, the prospheroplasts appeared to be formed by detachment of the cell walls. Both the prospheroplasts and the spheroplasts showed asymmetric cytoplasmic membranes in which the outer leaflets appeared coated with a dense fibrillar layer. The experiments suggest that, after enzyme digestion, the cytoplasmic membrane retains a coating which is rigid in the prospheroplast but which loses rigidity when the cell is transformed into a spheroplast.     

Isolation of Staphylococcus aureus protoplasts using lysoamidase

The effect of the bacteriolytic enzyme preparation, lysoamidase, on Staphylococcus aureus 209P cells was studied. The protoplast formation was examined by spectrophotometric, biochemical and electron microscopic methods. Optimal conditions for isolation of S. aureus protoplasts were chosen. The susceptibility of S. aureus cells to lysoamidase depended on the culture age: the maximum effect was observed in the logarithmic growth phase. The protoplast yield was 80% when 1 M sucrose was used as an osmotic stabilizer. Lysoamidase caused local disruptures of the staphylococcus cell walls, which resulted in the formation of osmotically fragile spheroplasts and the release of protoplasts into the medium. The protoplasts obtained could retain 85-90% of the respiration activity and were able of cell wall regeneration.     

Formation,regeneration, and fusion of protoplasts in aCellulomonas strain

The effect of different conditions on protoplast formation was studied in the streptomycin-resistant strainCellulomonas sp.M32Bo. The greatest efficiency (75% protoplasts) was achieved by use of 0.5M sodium succinate as osmotic stabilizer, supplemented with 20 mM MgCl₂, 200 µg/ml of lysozyme, and 0.01M EDTA at pH 7.4. Cells harvested at the midexponential growth phase were more suitable for protoplast formation than those of the stationary phase. Electron microscopy observations showed the presence of both protoplasts and spheroplasts in the treated samples, some of them still showing a rod shape. Two regeneration media were developed that showed similar regeneration frequencies (52%). StrainM32Bo was fused with a tetracycline-resistant strain (Cellulomonas sp. Sz). Segregation analysis of fusant colonies suggested the existence of a temporary diploid stage in which both parental genotypes were expressed.     

Effects of alcohols on bacterial cellulose production by <Emphasis Type="Italic">Acetobacter xylinum</Emphasis> 186

To improve the yield of cellulose production in bacteria, we investigated the stimulatory effects of six different alcohols during fermentation of Acetobacter xylinum 186. Our study showed that after static fermentation at 30°C for 6 days, bacterial culture with 1.0% (v/v) of methanol added in the medium produced the highest bacterial cellulose (BC) yield at 103.5 mg/100 ml, which was 21.8% higher than the control group. Addition of 0.5% of ethylene glycol in the culture yielded 105.5 mg/100 ml BC, 24.1% higher than the control group. Adding 0.5% of n-propanol yielded 96.4 mg/100 ml BC, 13.4% higher; 3.0% of glycerol yielded 108.3 mg/100 ml BC, 27.4% higher; 0.5% of n-butanol yielded 132.6 mg/100 ml BC, 56.0% higher; and 4.0% of mannitol in the culture yielded 125.2 mg/100 ml BC, 47.3% higher, respectively. The rate of bacterial cellulose production increased with the growth rate of the bacteria. The stimulatory effects of these alcohols that we observed were significant in the later stage of fermentation, which was considered to be important for the biosynthesis of bacterial cellulose.     

Regeneration of Spheroplasts in a N2-Fixed Filamentous Blue-Green Alga Anabaena cylindrica

Over 90% of cells of Anabaena cylindrica growing in the medium containing 0. 1 mol/L KC1 for 7～9 d transformed into spheroplasts or semispheroplasts which were either sensitive or not sensitive to hypotonic condition. After treating the materials with 0. 1% lysozyme at 28 ℃ for 3～4 h the transformed spheroplasts were almost 100% sensitive to the hypotonic condition. The spheroplasts then regenerated and divided through culture in the inorganic medium containing 0.15 mol/L CaCl2 with a rate over 25 %. The regeneration of different spheroplasts was not synchronous, the fastest division being after 3 d. Cell division was mainly equational but also irregular division or budding.     

Pb2+浓度和藻类食物密度对多刺裸腹溞生命表统计学参数的影响

采用48h急性毒性试验研究了Pb2+对多刺裸腹溞(Moina macrocopa)的48h-LC50值,采用生命表试验方法在0.5×106、1.0×106、2.0×106个细胞/mL的斜生栅藻(Scenedesmus obliquus)密度下研究了浓度为0.2、0.4、0.6、0.8、1.0mg/L的Pb2+对多刺裸腹溞生命表统计学参数的影响。结果表明,Pb2+对多刺裸腹溞48h-LC50值为10.5mg/L。与各食物密度下的对照组相比,除了0.5×106、1.0×106个细胞/mL下0.2mg/L的Pb2+显著延长了多刺裸腹溞的生命期望,0.5×106个细胞/mL的食物密度下0.4mg/L的Pb2+显著提高了多刺裸腹溞的净生殖率、0.40.8mg/L的Pb2+显著提高了种群内禀增长率外,较高浓度的Pb2+显著缩短了多刺裸腹溞的生命期望,降低了净生殖率、总生殖率和种群内禀增长率;且随着食物密度的升高,使净生殖率和总生殖率显著降低的Pb2+浓度阈值呈降低的趋势,但使世代时间显著缩短的Pb2+浓度阈值则呈升高的趋势。Pb2+浓度、食物密度以及它们间的交互作用对多刺裸腹溞的各主要生命表统计学参数均有显著的影响(P0.05)。0.5×106、1.0×106cells/mL食物密度下,Pb2+浓度与多刺裸腹溞的各主要生命表统计学参数间均有显著的剂量-效应关系;2.0×106cells/mL食物密度下,Pb2+浓度与多刺裸腹溞的生命期望、总生殖率和净生殖率间均有显著的剂量-效应关系。多刺裸腹溞的生命期望、净生殖率和内禀增长率对Pb2+污染的敏感性因食物密度的不同而存在着差异。     

Isolation,structural and functional characterization of Staphylococcus aureus protoplasts obtained using lysoamidase

The action of the lysoamidase bacteriolytic complex on Staphylococcus aureus VKM B-209P cells has been studied to obtain protoplasts. The cells in the midlogarithmic phase were the most sensitive to lysoamidase action. It led to local destruction of cell wall due to hydrolysis of the peptidoglycan. Protoplast formation occurred in two steps in the presence of 1 M sucrose. First, osmotically fragile spheroplasts were formed. Then, the protoplasts were released from the destructed cell wall. The protoplast yield was about 80%. The protoplasts preserved the intact ultrastructure and were able to synthesize peptidoglycan fibrillae. Mainly the spheroplasts that maintained the cell-wall residues reversed into bacterial forms. The protoplasts had respiratory activity similar to cells. Respiration of cells and protoplasts was stimulated by various substrates. High rates of oxygen consumption were observed with -glycerophosphate and ethanol as substrates.     

Restoration of phosphate transport by the phosphate-binding protein in spheroplasts of Escherichia coli.

Reconstitution of phosphate transport in Escherichia coli was demonstrated. Conversion of E. coli K10 cells to spheroplasts decreased phosphate transport to about 2%. Addition of purified phosphate-binding protein at physiological levels to these spheroplasts caused a mean 14-fold increase in phosphate transport rate. Crude shock fluid fractions were also stimulatory but not if the shock fluid was obtained from mutants lacking phosphate-binding protein. The effect of the binding protein was abolished by its specific antibody. The phosphate was shown to have entered the cell, where it became esterified. Reconstitution was not possible with cold-shocked or osmotically shocked cells.     

Suppression of ATP in Candida albicans by imidazole and derivative antifungal agents

Several antifungal agents, at concentrations of 10 micrograms/ml, were shown to suppress ATP concentrations very rapidly in intact cells and spheroplasts of Candida albicans. The highest ATP-suppressing activity was shown by the highly lipophilic imidazole derivatives difonazole, clotrimazole, econazole, isoconazole, miconazole, oxiconazole and tioconazole, which all caused a reduction of cellular ATP content of more than 50% in 10 min. Relatively hydrophilic imidazole derivatives such as ketoconazole were essentially inactive in the test, as were the triazole derivatives fluconazole, ICI 153066, itraconazole and terconazole, and 5-fluorocytosine. Amphotericin B and terbinafine possessed intermediate ATP-suppressing activity, and the dose-response and pH-response curves for these compounds suggested their mechanism of ATP suppression differed from that of the active imidazole derivatives. ATP suppression by azole antifungals did not involve leakage of ATP from the cells and the effect was entirely abrogated by the presence of serum. Intact cells and spheroplasts of yeast-form and hyphal-form C. albicans were generally equally sensitive to ATP suppression, but stationary-phase cells of both morphological forms were less sensitive than exponential-phase cells. The extent of ATP suppression was significantly reduced in stationary-phase yeast cells of a C. albicans strain with known resistance to azole antifungals, but exponential-phase cells of resistant and susceptible strains were equally sensitive. The effect is tentatively ascribed to membrane damage caused directly by the antifungals.     

Preparation and regeneration of protoplasts and spheroplasts for fusion and transformation ofSchizosaccharomyces pombe

Preparation and regeneration of protoplasts is essential for somatic hybridization and transformation of yeasts. We present conditions that were found to be optimal for preparing and regeneratingSchizosaccharomyces pombe protoplasts for cell fusion. In contrast to these conditions, genetic transformation ofS. pombe requires spheroplasts that are osmotically sensitive, but still have some wall material attached to the cell. The main finding were as follows: (a) For protoplast formation with Novozym SP234, 0.9M sorbitol was found to be the optimal osmotic milieu and -mercaptoethanol is not necessary. (b) Embedding in soft agar yields considerably better regeneration frequencies than direct plating. (c) Cell fusion is optimal when both fusion partners are fully protoplasted, although considerable fusion occurs between spheroplasted cells as well. (d)Schizosaccharomyces pombe transformation frequencies are much higher with spheroplasts than with protoplasts. Inclusion of -mercaptoethanol did not enhance transformation frequency.     

丁布对小麦赤霉病菌和玉米小斑病菌的抑制作用

采用菌丝生长速率法和孢子萌发试验法检测了抗菌化合物丁布对小麦赤霉病菌和玉米小斑病菌的抑菌作用。结果表明,丁布在PDA培养基中浓度为0.2-1.0 mg/ml时对两种供试病菌的菌丝生长无抑制作用;丁布浓度为0.4-1.0 mg/ml时对两种供试病菌孢子悬浮液中孢子的萌发具有显著抑制作用;1.0 mg/ml丁布药液中培育15h的小麦赤霉病菌和培育5h的玉米小斑病菌的孢子萌发抑制率分别达到100%和83.6%。     

山楂原花色素的抗氧化作用研究

本文用比色法测定山楂原色素（Proanthocyanidins from the fruits of hawthorn,HPA)的抗氧化作用,即对羟自由基、超氧负离子的清除作用以及抗脂质过氧化的作用,并且通过细胞学实验验证其对上皮细胞的抗氧化损伤的保护作用。试验表明：山楂原色素有很强的抗氧化作用,其作用在浓度为0．1mg/ml-1.0mg/ml之间随着浓度的增大而增强,对超氧负离子的清除和抗脂质过氧化作用尤为明显。在浓度为1mg/ml时,对羟自由基和超氧负离子的清除率分别为59．8％和90．0％,对细胞氧化损伤的保护率为41．96％;浓度为0．5mg/ml时对脂质过氧化的抑制率达91．9％。试验同时还比较了葡萄籽原花色素（Proanthocyanidins from the fruts of grape,GPA)和Vc的抗氧化作用,结果表明：山楂原花色素与葡萄素（Proanthocyanidins from the fruits of grape,GPA)和Vc的抗氧化作用,结果表明：山楂原色素与葡萄籽原花色素效应相当,并远远高于Vc的效应。