Today, Hypericum perforatum L. is probably one of the best-characterized medicinal plants, and hyperforin is its best-characterized constituent. Extracts from H. perforatum are widely used as antidepressants; however, less attention has been given to other properties of hyperforin, such as antitumor, fungicidal, antiviral and antibacterial action, or its possible use as a substance with immunomodulation properties. The present study summarizes results that describe the influence of hyperforin as an immunomodulation agent on phagocytosis and the breakdown of Escherichia coli by human polymorphonuclear neutrophils (PMNs). Hyperforin at 1 - 100 μg/mL concentrations was found to have a major influence on phagocytosis and the breakdown of E. coli by PMNs in vitro. A 100 μg/mL solution of hyperforin increased the uptake of non-opsonized E. coli almost 50-fold, and the uptake of IgG-opsonized E. coli more than threefold; on the other hand, the uptake of serum-opsonized bacteria was reduced to approximately 60% of that of the control. Hyperforin seems to bind to both PMNs and E. coli and acts like an opsonin. The elimination of remnants of IgG-opsonized E. coli from the PMNs was stimulated by hyperforin, while the elimination of remnants from non-op-so nized and serum-opsonized material was unaffected by the drug. Hyperforin exhibited clear immunomodulation ability as a phagocytosisstimulating agent. Hyperforin is probably inactive against human immunodeficiency virus (HIV) and most Gram-negative bacteria. However, it can protect acquired immunodeficiency syndrome (AIDS) patients and other immunocompromised patients by its antibacterial activity against Gram-positive bacteria and by enhancement of phagocytosis of Gram-positive and Gram-negative bacteria; some Gram-negative bacteria, such as Neisseria, are sensitive to hyperforin. Hyperforin has the ability to penetrate the blood-brain barrier (BBB) and blood-testis barrier (BTB) and is a valuable antibacterial agent against meningitis and gonorrhea. These properties of hyperforin are important for an antibiotic with immunomodulation activity in the struggle against the growing mortality in AIDS patients as a result of opportunistic bacteria, as recently shown by Bekondi et al. (2006, Int. J. Infect. Dis. 10, 387-395). It could also help to combat primary and opportunistic pathogens associated with meningitis in adults' relation to HIV serostatus.
In ancient Greece, Hypericum perforatum was used for its antidepressant properties as well as for treatment of skin injuries, burns and neuralgia. The presence of antibacterial substances in the plant genus Hypericum has been known in the scientific literature for more than 70 years. In 1943, Osborn [
The first interest in hyperforin was as an antibiotic extracted and purified from the plant H. perforatum L. The crude alcohol extract was patented in the former USSR under the name “Novoimanin” [6,7]. Novoimanin is a mixture of different extractible substances and thus has a broad spectrum of physiological properties; however, it cannot be used intravenously or by other means of injecttion. The main interest of the research in the former USSR was the antibacterial activity of the extracts’ main component, hyperforin [5,8], and its antifungal activity [
In 1979, research on the antibiotic hyperforin and other substances in H. perforatum was conducted in Norway [
In 1982 [
Hyperforin’s antibacterial spectrum includes a number
of Gram-positive microorganisms, but it is inactive against Gram-negative Enterobacteriaceae such as E. coli [
PMNs are important contributors to host defense reactions against bacterial infections. Inadequate host defense may result in serious infections, as is sometimes seen in immunocompromised hosts. It is especially important in the case of immunocompromised persons such as those who have been infected with human immunodeficiency virus (HIV), diabetic persons or those intoxicated by chemicals [
In this paper, a study of the influence of hyperforin on the in vitro phagocytosis of E. coli by human PMNs is presented. E. coli was chosen because of its resistance to the antibacterial effects of hyperforin, as a model in which the effect of hyperforin’s antibiotic action was eliminated and the concurrent action of serum and hyperforin as opsonins was established.
Blood and serum from healthy donors were purchased from the Blood Bank. Human serum was used heat-untreated, providing an intact complement. Leighton tubes with human PMNs were purchased from Jupiter Ltd. (Ski, Norway). The PMNs were prepared according to the technique of Bøyum [25-27] using PolymorphprepTM from Axis-Shield PoC AS (Oslo, Norway). The recovered granulocytes were washed once in Krebs Ringer Phosphate buffer enriched with 10 nM glucose and resuspended in Krebs Ringer Phosphate buffer enriched with 10 nM glucose to a final concentration of 2.5 × 106 cells/mL. Aliquots of 1 mL of these suspensions, which were of at least 95% purity, were added to Leighton tubes (16 × 125 mm, Bellco Glass Inc., Vineland, NJ, USA). The tubes were placed in a horizontal position at 37˚C for 60 min. A monolayer of neutrophils with approximately 5,000 cells per square mm was established during that time.
Serum-sensitive E. coli (ATCC 11775) grown under standardized conditions to a density of 109 CFU/mL, in a medium prepared according to Benacerraf et al. [
Antibodies were raised in rabbits by intravenous injection of a heat-treated suspension of serum-sensitive E. coli twice weekly for 12 weeks. The IgG fraction of the serum was purified by batch adsorption on DEAESephadex A-50 (Pharmacia, Sweden) and then precipitated with ammonium sulfate. The precipitate was dissolved in phosphate-buffered saline and desalted on an Amersham Biosciences PD-10 desalting column (Amersham Biosciences AB, Uppsala, Sweden). The final preparation agglutinated the bacteria at a titer of 256. When a 10-fold dilution of the preparation was tested by gel diffusion against sheep anti-rabbit whole serum and sheep anti-rabbit IgG, only one precipitation line was found. This indicated that most of the protein in the preparation was IgG.
Opsonization of E. coli with rabbit anti-E. coli IgG was carried out with a suspension of 109 bacteria/mL in a water bath shaker at 37˚C for 30 min. A fourfold dilution of the agglutinating titer of IgG was used. The bacteria were then washed in ice-cold Krebs Ringer Phosphate buffer enriched with 10 nM glucose, resuspended to 109 bacteria/mL and prewarmed at 37˚C for 5 min before phagocytosis.
Opsonization with serum was performed by the addition of serum to a final concentration of 10% and 109 bacteria/mL. The serum used was not heat-inactivated. This suspension was prewarmed in a water bath at 37˚C for 5 min before phagocytosis was started.
At the end of the preincubation period, the PMN monolayer was washed once with Krebs Ringer Phosphate buffer enriched with 10 nM glucose (37˚C), and the uptake was started by adding 1 mL aliquots of the suspension of non-opsonized or opsonized E. coli, with or without hyperforin, as indicated below. The tubes were placed in a horizontal position during the uptake phase, which was stopped after 30 min by washing the PMNs four times with ice-cold Krebs Ringer Phosphate buffer enriched with 10 nM glucose. For the study of the elimination of bacterial breakdown products from the PMNs, the cells were washed with prewarmed Krebs Ringer Phosphate buffer enriched with 10 nM glucose (37˚C) and incubated for another 60 min at 37˚C. The elimination was then stopped by washing the cells with ice-cold Krebs Ringer Phosphate buffer enriched with 10 nM glucose.
Hyperforin was extracted from dried material of H. perforatum purchased from Norsk Medisinaldepot (NMD, Oslo, Norway) and purified as previously described [10,13,14]. A stock solution of 50 mg/mL was stored at 4˚C in 96% ethanol, sheltered from light. Immediately before use, the solution was diluted to a final concentration of 1, 10 or 100 μg/mL in physiological solution. The drug, bacteria, opsonins and PMNs were combined in the following ways:
1) The bacteria were preincubated with hyperforin for 30 min at 37˚C and then washed twice with ice-cold Krebs Ringer Phosphate buffer enriched with 10 nM glucose before the start of phagocytosis. In the case of IgG-opsonized bacteria, opsonization and preincubation were performed simultaneously. Phagocytosis took place in the absence of hyperforin.
2) The PMN monolayer was preincubated with the drug for 30 min before the start of phagocytosis, which took place in the absence of hyperforin.
3) The PMNs were preincubated with hyperforin for 30 min and the bacteria for 5 min, after which the uptake was performed in the presence of hyperforin.
After the final washing, the tubes were dried, and 1 mL of Lowry’s alkaline copper solution was added to the PMNs to dissolve the cells overnight at room temperature. Cell protein was then determined according to the method of Oyama et al. [
All of the experiments were performed with 3 - 5 parallel runs and repeated at least five times. The uptake of E. coli in PMNs, as influenced by hyperforin, was expressed as a percentage of the control. The controls were parallel experiments, but without the antibiotic hyperforin. The elimination was expressed as the percentage of ingested radioactivity that was released from the PMNs during the elimination period both after control uptake and after uptake influenced by hyperforin. Student’s t test was used for statistical analysis and a P value < 0.05 was considered statistically significant.
When phagocytosis took place in the presence of 10% serum, the uptake of E. coli increased by a factor of 23.0 ± 3.0 (mean ± SD, n = 41), compared with uptake of non-opsonized bacteria.
Correspondingly, opsonization by anti-E. coli IgG resulted in an enhancement of the uptake by 35.8 ± 15.7 (mean ± SD, n = 35).
The uptake of E. coli pretreated with hyperforin is outlined in
After preincubation of the PMNs, but not bacteria, with hyperforin, a similar pattern was seen (