Effect of Alpinia oxyphylla Fruit Extract on Compound 48/80-induced Anaphylactic Reactions

American Journal of Chinese Medicine, Spring, 2001 by Tae Yong Shin, Jin Hee Won, Hyung Min Kim, Sand Hyun Kim

Abstract: The effect of the aqueous extract of Alpinia oxyphylla Miq. (Zingiberaceac) fruits (AOFE) on anaphylactic reaction was investigated. AOFE completely inhibited compound 48/80-induced systemic anaphylactic shock at dose of 1.0 g/kg. When AOFE was pretreated at concentrations ranging from 0.01 to 1.0 g/kg, the plasma histamine levels induced by compound 48/80 were reduced in a dose-dependent manner. AOFE also inhibited the histamine release from rat peritoneal mast cells (RPMC) by compound 48/80. The level of cAMP in RPMC, when AOFE was added, transiently and significantly increased about 4-fold compared with that of basal cells. These results indicate that AOFE may be beneficial in the treatment of non-specific anaphylactic reactions.

Medicinal Alpinia oxyphylla (Korean name: Ik-Ji-In) is the dried ripe fruit of Alpinia oxyphylla Miq. (Zingiberaceae). Choi et al. (1992) reported that Alpinia oxyphylla suppressed immunoglobulin E antibody-mediated reactions, 48-hr homologus passive cutaneous anaphylaxis in mice. Thus, the current study was designed to determine whether Alpinia oxyphylla could act on both non-specific stimulator-induced systemic anaphylactic reactions and mediator release capability from mast cells. Alpinia oxyphylla has the pharmacological functions of antiulcer effect (Yamahara et al., 1990), calcium-antagonistic effect (Shoji et al., 1984), antifungal effect (Haraguchi et al., 1996) and antiulcerative effect (Kubo et al., 1995), and has been used for diarrhea, dyspepsia, abdominal pain and spermatorrhea (Paul et al., 1997). The condensation product of N-methoxyphenylamine with formaldehyde produces a potent histamine-liberating agent called compound 48/80 (Paton, 1951). This compound is employed as a classic mast cell secretagogue that releases histamine (Read and Lenney, 1972). Compared with the natural process, a high concentration of compound 48/80 induces almost a 90% release of histamine from mast cells (Allansmith et al., 1989).

Thus, an appropriate amount of compound 48/80 has been used as a direct and convenient reagent to study the mast cell-mediated non-specific anaphylactic reaction (Kim et al., 1998). In the present study, we examined the effect of AOFE on compound 48/80-induced anaphylactic reaction in vivo and histamine release from RPMC in vitro. To elucidate the mechanism of action, we also investigated the effect of AOFE on compound 48/80-induced intracellular cAMP level in RPMC.

Materials and Methods

Reagents

Compound 48/80, gelatin, bovine serum albumin (BSA), o-phthaldialdehyde (OPA) and metrizamide were purchased from Sigma (St. Louis, MO), cAMP was purchased from Amersham Pharmacia Biotech (UK).

Animals

The original stocks of male ICR mice and Wistar rats were purchased from Dae Han Experimental Animal Center (Taejeon city, South Korea), and the animals were maintained at the College of Pharmacy, Woosuk University. The animals were housed 5-10 per cage in a laminar air flow room maintained under a temperature of 22 2 [degrees] C and relative humidity of 55 [ or -] 5% throughout the study.

Preparation of AOFE

Alpinia oxyhylla fruits were obtained from the Oriental drug store, Bohwa Dang of Chonju, South Korea, and identified by T.K. Kim, College of Pharmacy, Woosuk University. A voucher specimen (number WSP-98-07) was deposited at the Herbarium at the College of Pharmacy, Woosuk University. The Alpinia oxyphylla was extracted with distilled water at 70 [degrees] C for 5 hr (two times). The extract was filtered, lyophilized, and kept at -20 [degrees] C. The yield of dried extract from starting crude materials was about 8.5%. The dried extract was dissolved in saline or Tyrode buffer A (10 mM HEPES, 130 mM NaCl, 5 mM KCl, 1.4 mM Ca[Cl.sub.2], 1 mM Mg[Cl.sub.2], 5.6 mM glucose, 0.1% bovine serum albumin) before use.

Compound 48/80-induced Systemic Anaphylactic Reaction

Compound 48/80-induced systemic anaphylactic reaction was examined as previously described (Shin et al., 1999). Mice were given an intraperitoneal injection of 8 mg/kg of the mast cell degranulator compound 48/80. AOFE was given with 0.01, 0.05, 0.1, 0.5 1.0 g/kg at 1 hr before or 5 min and 10 min after compound 48/80 injection. Mortality was monitored for 1 hr after induction of anaphylactic shock. The blood was drawn from the heart in the above group (n=10/group) at 15 min after compound 48/80 injection.

Preparation of Plasma and Histamine Determination

The blood was centrifuged at 400 x g for 10 min. The plasma was withdrawn and the histamine content was measured by the OPA spectrofluorometric procedure of Shore et al. (1959). The fluorescent intensity was measured at 438 nm (excitation at 353 nm) in a spectrofluorometer (Shimadzu, RF-5301, Japan).

Preparation of Rat Peritoneal Mast Cells (RPMC)

RPMC were isolated as previously described (Shin et al., 2000). In brief, rats were anesthetized by ether, and injected with 20 ml of Tyrode buffer B (137 mM NaCl, 5.6 mM glucose, 12 mM Na[HCO.sub.3], 2.7 mM KCl, 0.3 mM Na[H.sub.2][PO.sub.4] and 0.1% gelatin), into the peritoneal cavity, and the abdomen was gently massaged for about 90 sec. The peritoneal cavity was carefully opened, and the fluid containing peritoneal cells was aspirated by a Pasteur pipette. Thereafter, the peritoneal cells were sedimented at 150 x g for 10 min at room temperature and resuspended in Tyrode buffer B. Mast cells were separated from the major components of rat peritoneal cells, i.e. macrophages and small lymphocytes, according to the method described by Yurt et al. (1977). In brief, peritoneal cells suspended in 1 ml Tyrode buffer B were layered on 2 ml of 22.5% w/v metrizamide (density, 1.120 g/mD and centrifuged at room temperature for 15 min at 400 x g. The cells remaining at the buffer-metrizamide interface were aspirated and discarded; the cells in the pellet were washed and resuspended in 1 ml Tyrode buffer A containing calcium. Mast cell preparations were about 95% pure as assessed by Toluidine blue staining. More than 97% of the cells were viable as judged by Trypan blue uptake.