Harmal (Peganum harmala) is a plant of the family Zygophyllacea, native from the eastern Mediterranean region east to India. It is also known as Wild Rue or Syrian Rue because of its resemblance to plants of the rue family. It is a perennial plant which can grow to about 0.8 m tall. But normally it is about 0.3 m tall.^[1] The roots of the plant can reach a depth of up to 6.1 m, if the soil it is growing in is very dry, It blossoms between June and August in the Northern Hemisphere.^[1] The flowers are white and are about 2.5–3.8 cm in diameter. The round seed capsules measure about 1–1.5 cm in diameter have three chambers and carry more than 50 seeds^[2] . Peganum harmala was first planted in the United States in 1928 in the state of New Mexico by a farmer wanting to manufacture the dye “Turkish Red” from its seeds.^[1] Since then it has spread invasively to Arizona, California, Montana, Nevada, Oregon, Texas and Washington. Because it is so drought tolerant, African rue can displace the native saltbushes and grasses growing in the salt-desert shrub lands of the Western U.S.[2,3]

Plant material

Seeds of Peganum harmala were purchased from traditional medicinal seller Dagadu merchants from Nashik, Maharashtra, India. seeds was authenticated by Botanical Servey of India, Pune, Maharashtra, India. Woucher specimen was deposited in the herbarium for further use.

Extraction

Dried and coarsely powder of Peganum harmala seeds (100 g) was defatted with petroleum ether and the marc remaining was extracted successively by petroleum ether, ethanol and water in different Soxhlet extractor. Solvent was evaporated in rotary evaporator under reduced pressure.

Animals

Swiss albino mice of either sex weighing (25-30 g) were housed under standard laboratory condition of temperature (25 ± 2°C) and 12/12 h light/dark cycle. The animals had free access to food and water. The Animal Ethical Committee of the Institute approved all the protocols of the study.

Drugs and Chemicals

5% polyethylene glycol (PEG-400), saline solution, buffer medium, Sodium chromoglycate, RPMI-1640 were purchased from Himedia, India. Clonidine (Alembic), petroleum ether (60-80°C), ethanol (95%). All Chemical and reagents were of analytical grade.

Acute toxicity study

Acute oral toxicity study was performed as per OECD 423 guideline. Extract was administered up to the maximum dose of 2000 mg/kg and animals were observed for mortality.^[4]

Clonidine induced mast cell degranulation in mice

Mice were divided into five groups, five animals in each group. A three-day drug treatment schedule was followed. Group-I serves as control receives vehicle only (5 % PEG400, 1ml / kg, i.p.). Group-II receives standard drug disodium cromoglycate (DSCG, 200µg/kg, i.p.). Group-III, IV and V was treated with test extract 25, 50 and 100 mg/kg, i.p. respectively. On day fourth, each animal was injected with 4 ml/kg, 0.9 % saline solution, into peritoneal cavity. By gentle massage, peritoneal fluid was collected after 5 mins and transferred into siliconised test tubes containing 7-10 ml RPMI-1640 buffer medium (pH 7.2-7.4). This solution was then centrifused at 400-500 RPM. Pellet of mast cell was washed with same buffer medium twice by centrifugation, discarding supernatant. These cells were challenged with clonidine (50 g), incubated at 37oC in a water bath for 10 mins. Followed by staining with 1% toluidine blue and observed under microscope (10X). Total 100 cells were counted from different visual area. Percent protection against degranulation was calculated.[5,6]

Statistical analysis

The data were presented as mean ± Standard error mean. The statistical significance between the groups has been tested by analysis of variance followed by Dunnett’s test. A P < 0.05 were considered as significant.

n=5, values are expressed in mean±SEM Control = Vehicle (5 % PEG-400, 1ml / kg, i.p) *p< 0.05 compared with control group (ANOVA followed by Dunnett’s test) PHPEE – Peganum harmala Petroleum ether extract

n=5, values are expressed in mean±SEM Control = Vehicle (5 % PEG-400, 1ml / kg, i.p) *p< 0.05 compared with control group (ANOVA followed by Dunnett’s test) PHPEE – Peganum harmala Petroleum ether extract

n=5, values are expressed in mean±SEM Control = Vehicle (5 % PEG-400, 1ml / kg, i.p) *p< 0.05 compared with control group (ANOVA followed by Dunnett’s test) PHWE– Peganum harmala Water extract

Acute toxicity test

The LD50 value of Curcuma caesia rhizome extracts when given intraperitoneally and tested in albino mice was found to be more than 2000 mg/kg body weight.

Clonidine induced mast cell degranulation

Treatment group of CCEE (Curcuma caesia rhizomes ethanol extract) exhibited significant mast cell stabilizing potential at all three doses (25, 50, 100 mg/kg i.p) 27.2±1.854, 23.4±1.166, 17.6±1.452 respectively (Table 2 and figure 2) as compared to control group. Percent protection offered by CCEE at all three doses was notably 75.13%, 76.07%, 81.70% and was of statistical significance as compared to Curcuma caesia Petroleum ether extract (CCPEE) and Curcuma caesia water extract (CCWE). CCPEE at 100 mg/kg shows 63.40±1.496 mast cells degranulation (Table 1 and Figure 1) while CCWE at 100 mg/kg shows 65.8±0.663 mast cells degranulation (Table 3 and Figure 3) which was not significant.

Mast cells are the principal store of histamine and other many chemical mediators responsible for the pathogenesis of asthma. Mast cells are necessary for the development of allergic reactions, through cross liking of their surface receptor for IgE, leading to degranulation and the release of vasoactive, pro-inflammatory and nociceptive mediators. Released mediators from mast cells, each having more than one potent effect on airway inflammation.^[7] present study revealed that ethanolic extract of Peganum harmala seeds shows significant role in mast cell protection against clonidine induced degranulation, while petroleum ether and water extract of Peganum harmala seeds was non-significant.

Ethaniolic extract of Curcuma caesia rhizomes offers mast cell protection against degranulation as compared to petroleum ether and water extracts. It leads to conclusion that CCEE may be useful in the management of asthma.