Plant leaves were collected localy. The leaves were authenticated by Dr. Gopalakrishna Bhat, Department of Botany, Poorna Prajna College, Udupi, Karnataka, India. A voucher specimen (O.S 5/99) was deposited in the herbarium of our institute.

Wistar rats (175-200 g) and guinea pigs (400-600 g) of either sex are procured from Indian Institute of Sciences. They are maintained under standard conditions (temperature 22 ± 2 ° C, relative humidity 60±5% and 12 h light/dark cycle)0.The animals were housed in sanitized polypropylene cages containing sterile paddy husk as bedding. They had free access to standard pellet diet and water ad libitum. The Institutional Animal Ethics Committee approved the experimental protocol. All the animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the “National Academy of Sciences” and published by the “National Institute of Health”.

Histamine and horse serum were procured from Sigma Chemicals and toluidine blue from Loba-Chemie, Mumbai. Elisa kit for IgE was supplied by Orion diagnostics, Espoc, Finland. All other chemicals and reagents were procured from Hi-Media Laboratories limited, Mumbai.

Twenty-eight rats were sensitized by injecting subcutaneously 0.5 ml of horse serum along with 0.5 ml of triple antigen containing 20,000 million Bordetella pertussis organisms (Serum Institute of India Ltd., Pune, India). The sensitized rats were divided into 4 groups of 7 each. Group I served as control and received water (vehicle). Groups II, III and IV were administered test extract at 100, 200, and 400 mg/kg respectively, orally, once a day for 14 days. On day 14, after 2 h of treatment, the rats were challenged with intravenous injection (tail vein) of 0.25 ml horse serum in normal saline. They were then observed for the onset of symptoms such as dyspnea and cyanosis, duration of the persistence of symptoms (min), and mortality. The severity of symptoms was scored. The optimal pharmacological effective dose, which is derived from this dose response study, was used for the remaining studies.

Serum total IgE was quantified with an ELISA protocol according to the manufacturer's instructions. Briefly, the plates were coated with affinity-purified rabbit anti IgE overnight at 4 °C and then blocked with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 1 h at 37°C. The serum samples and appropriate dilutions of a standard IgE preparation were placed in the wells, and the plates were incubated for 3 h at 4 °C. Sample blank wells were treated similarly but without serum. The bound IgE was detected with polyclonal goat anti IgE antibodies (incubation for 1 h at 37 °C), followed by HRP-conjugated rabbit anti-goat antibodies (incubation for 1 h at 37 °C). The plates were developed by the addition of O-phenylene diamine and read in an ELISA (Anthos HT-II, USA) plate reader at 490 nm ₉₁₀ .

Thirty-two rats were divided into four groups of eight animals in each group. Group I served as control and received vehicle (water). Group II (sensitized control group, received only water), Groups III (test extract) and IV (prednisolone) were sensitized by injecting 0.5 ml of horse serum subcutaneously along with 0.5 ml of triple antigen containing 20,000 million Bordetella pertussis organisms (Serum Institute of India Ltd., Pune). Group III were administered 400 mg/kg, p.o., once a day for 14 days. Group IV were administered prednisolone (reference drug) 10 mg/kg, p.o., for the same duration. On day 14, the rats were sacrificed 2 h after the treatment and the intestinal mesentry was taken out for the study on mast cells. Mesentries along with intestinal pieces were excised and kept in Ringer Locke solution (NaCl 154, KCl 5.6, CaCl₂ 2.2, NaHCO₃ 6.0, glucose 5.55 mM/L of distilled water) at 37 ° C. The mesenteric pieces were challenged with 5% horse serum for 10 min after which the mast cells were stained with 1.0% toluidine blue and examined microscopically for the number of intact and degranulated mast cells ₁₁₁₂₁₃ .

Bronchospasm was induced in guinea pigs by exposing them to 1% histamine aerosol under constant pressure (1 kg/cm ² ) in an aerosol chamber (24 × 14 × 24 cm) made of perplex glass. Of the two groups of six animals each, Group I served as control and Group II received test ethanolic extract 400 mg/kg, p.o.,once a day for 5 days. The animals were exposed to 1% histamine aerosol under constant pressure (1 kg/cm ² ) in an aerosol chamber on day 0 without any treatment. The end point, preconvulsive dyspnea (PCD) was determined from the time of aerosol exposure to the onset of dyspnea leading to the appearance of convulsions ₁₄₁₅₁₆ . As soon as PCD commenced, the animals were removed from the chamber and exposed to fresh air. This PCD was taken as day 0 value. On days 1 and 5, 2 h after the administration of the drug, the time for the onset of PCD was recorded as on day 0.

The results of various studies were expressed as mean ± SEM and analyzed statistically using one-way ANOVA, followed by Bonferroni's multiple comparison post-hoc test or Chi- square test or unpaired Student's ' t ' test to find out the level of significance. P<0.05 was considered statistically significant. The analysis was performed using Graphpad Prism software package (Version 4.0).

Test extract protected the sensitized rats against anaphylactic shock in a dose-dependent manner. In control rats, intravenous antigen challenge (horse serum) caused shock in 100% of the animals, while in treated rats (400 mg/kg of etanolic extract), the onset of symptoms of shock was delayed (P <0.001), and symptoms were less severe (P <0.05) with reduced mortality (P <0.05). Results are represented in table 1. Test extract at the dose 300 mg/kg resulted in significant reduction of serum IgE levels (25.80 ± 4.85 ng/ml, P <0.001) as compared to sensitized controls (125.06 ± 9.66 ng/ml). Serum IgE levels in control group was 8.83 ± 0.84 ng/ml (P <0.001 as compared to sensitized control). Extract showed optimal pharmacological effect at 400 mg/kg dose. Hence, same dose of was used for further experimental models (Table-1).

Figure 1

Table 1: Effect of Ocimum ethanolic extract on anaphylactic shock- induced bronchospasm in rats

Values are mean ± SEM except for mortality, which is expressed as percentage, n=7 in each group; Total score: F=50508, df =27, P=0.0050; Onset of symptoms: F=20.51, df = 27, P=0.0001. *P<0.05, ^# P<0.001 as compared to control. (ANOVA followed by Bonferroni’s multiple comparison post hoc tests for total score and onset of symptoms. Chi-square test for mortality).

Antigen challenge resulted in significant degranulation of the mesentric mast cells (approximately 88%, P <0.001). Pretreatment of sensitized animals with O.sanctum extract at 400 mg/kg, p.o., for 2 weeks resulted in a significant reduction in the number of disrupted mast cells (P <0.001) when challenged with horse serum. The effect of N. zeylanica extract was also comparable to the reference drug prednisolone (Table-2).

Figure 2

Table 2: Effect of O. on mast cell stabilization in sensitized rats

Values are mean ± SEM, n=8 in each group. * Significantly different from sensitized control (P<0.0001).

O. sanctum ethanolic extract at 400 mg/kg, p.o., significantly prolonged the latent period of PCD (P <0.008) as compared to control, following exposure to histamine aerosols on day 5 (Table 3).

Figure 3

Table 3: Effect of O. ethanolic extract on histamine induced bronchospasm in guinea pigs.

Values are mean ± SEM, n=6 in each group. * (P<0.008) as compared to control on day 5 (unpaired Students‘t’ test)

Authors are greatfull to Chemical Engg, department, National Institute of technology Surathkal, Karnataka for help in initial quantification of Phytoconstituents.