Plant material
P. verticillatum (whole plant) was collected from the District Swat, Khyber Pukhtonkhawa, Pakistan, in July-Aug 2007. The botanical characterization of the plant material was executed by the Taxonomy Department of PCSIR Laboratories Peshawar and a specimen with catalogue No: 9970 (PES) was deposited in the herbarium of PCSIR Laboratories Peshawar.
Plant extraction
Air-shade dried rhizomes of the P. verticillatum (8 kg) were grounded to a fine powder. The powdered material was soaked in aqueous-methanol (30:70) for three days while shaking occasionally [20] and filtered through a muslin cloth and Whatman filter paper (Maidstone, UK) simultaneously. This procedure was repeated three times and all the pooled filtrates were evaporated on a rotary evaporator (model RE-111, Buchi, Flawil, Switzerland) under reduced pressure (−760 mm Hg) to obtain a dark greenish semi-solid material, yielding 27.50 wt/wt%.
Experimental animals
A total of thirty Wistar rats (190–260 g) and five adult local guinea-pigs (1.2 − 1.6 kg) of either sex were kept under standard laboratory conditions at 25 ± 2°C and a relative humidity of 40-70%. The light cycle was maintained as 12 h dark: 12 h light. They were fed with a laboratory diet ad libitum and allowed free access to drinking water. All the experiments were performed in compliance with the rulings of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council [21] and approved by the local Ethical Committee of the Karachi University.
Drugs and reagents
Carrageenan, carbachol (CCh), verapamil hydrochloride, soybean lipoxygenase, linoleic acid sodium salt and baicalein were purchased from the Sigma Chemicals Co., St. Louis, MO, USA and aspirin was obtained from the Reckitt & Colman, Pakistan. Chemicals used for making Tyrode’s solution were: potassium chloride (Sigma Chemicals Co., St. Louis, MO, USA), calcium chloride, glucose, magnesium chloride, sodium bicarbonate, sodium dihydrogen phosphate (Merck, Darmstadt, Germany) and sodium chloride from BDH Laboratory supplies, Poole, England. All chemicals used were of highest grade available, and were solubilized in distilled water/saline while carrageenan was used as suspension with acacia.
Isolated guinea-pig tracheal tissue
The trachea was obtained from guinea-pigs and preserved in physiological solution (Kreb’s solution). Rings containing a couple of cartilages were formed from a tracheal tube approximately 2–3 mm wide. Rings were cut into strips by a longitudinal cut on the ventral side reverse to the smooth muscle [22]. The strips were suspended in a tissue bath (20 mL) containing Kreb’s solution (pH 7.4), maintained at 37°C and aerated with a mixture of 95% oxygen and 5% carbon dioxide (carbogen). Tracheal strips were maintained at 1 gram constant tension during the course of experiment. The tissues were granted 1 h to equilibrate prior to the introduction of test material. Before determining the inhibitory activity of the plant extract, the isolated tracheal tissues were stabilized with high K+ (80 mM) and CCh (1 μM) until constant responses of each agonist were achieved (usually 3–4 concentrations). The sustained contractions were obtained using CCh and K+ separately and the inhibitory effect was assessed using the cumulative addition of the test material. Isometric responses were recorded on a Grass model 7 Polygraph (Grass instrument company, Quincy, MA, USA). The inhibition of the high K+-induced contractions indicates Ca2+ antagonist activity [23], which was confirmed by constructing the Ca2+ concentration-response curves in the absence and presence of increasing concentrations of the plant material.
Carrageenan-induced edema
The carrageenan-induced rat hind paw edema test was conducted as described previously [24]. The test animals were divided in to five groups (n = 6). Group Ι (the control group) received normal saline (10 mL/kg). The rats of group ΙΙ, ΙΙΙ and ΙV received the test extract (50, 100 or 200 mg/kg i.p.), while, group V (positive control) received aspirin (100 mg/kg i.p.). Following 30 min of the treatments, acute inflammation was induced by sub-plantar injection of 0.1 mL of 1% suspension of carrageenan with 2% gum acacia in normal saline, in the right hind paw of the rats. The paw volume was estimated with the help of plethysmometer (Ugo Basile, Italy) at 1st, 2nd, 3rd, 4th and 5th h after the carrageenan injection. Statistics was applied on the raw data for the calculation of reduction in rat paw volume (mL) for each group against saline, followed by the calculation of percent reduction in the rat paw using the following formula:
where “dt” is the difference in paw volume in the treated group and “dc” the difference in paw volume in the control group.
Soybean lipoxygenase inhibitory assay
The lipoxygenase (LOX) inhibition assay was conducted by using different dilutions of the PR by following previously described method [25]. Soybean lipoxygenase and linoleic acid were used. Equal volume (10 mL) of the sample (PR) and standard drug along with 20 mL of solvent lipoxygenase solution simultaneously were mixed followed by incubation for 5 min at 25°C. The biochemical reaction was initiated by the addition of linoleic acid solution (10 μL) as substrate and the absorption change with the formation of (9Z,11E)-13S)-13-hydroperoxyoctadeca-9, 11-dienoate was followed for 10 min at 234 nm. The test sample and the control were dissolved in 50% ethanol. All the reactions were performed in triplicate. Baicalein was used as a positive control. The median effective concentrations (IC50 values) were calculated using the EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc., Amherst, USA).
Isolation of pure molecules
A sample of 80 g of the aqueous-methanol extract of P. verticillatum rhizomes (PR) was subjected to column chromatography over silica gel (column grade) for the isolation of pure chemical compounds. The sample was loaded in a glass column over silica gel for adsorption that acts as stationary phase. The column was initiated with 100% n-Hexane as eluent (mobile phase). Polarity of mobile phase was enhanced gradually with regular monitoring of the isolate status over TLC, it resulted in 12 sub-fractions (P1-P12). Similar with methanol/chloroform gradient, 9 subfractions (M1-M9) were obtained. When sub-fraction P9 was purified through column chromatography, 2-Hydroxybenzoic acid (A) was isolated in the colorless crystals form (1A). Subfraction P11 was re-chromatographed over silica gel. While eluting with chloroform: hexane; (8:2), β-Sitosterol (B) was isolated as colorless amorphous powder (1B).
Characterization of 2-Hydroxybenzoic acid (A)
Colorless crystals, M.P. 159-160°C, HREIMS: m/z 138 (Calcd. for C7H6O3; 138.12), 1H NMR (CHCl3, 300 MHz): δ 6.89 broad d (J = 8.4 Hz), 7.42 dt (J = 1.8, 8.4 Hz), 6.85 t (J = 7.8 Hz), 7.83 dd (J = 1.8, 8.4), 13C NMR (CDCl3, 75 MHz): δ 113.9 (C-1), 163.2 (C-2), 118.1 (C-3), 136.6 (C-4), 120.0 (C-5), 131.5 (C-6), 173.5 (C-1′).
Characterization of β-sitosterol (B)
Colourless amorphous powder, M.P. 135-136°C, HREIMS: m/z 414.3845 (calculated for C29H50O as 414.3855). 1H NMR (CHCl3, 300 MHz): δ 3.65 (m), 5.35 (m), 0.67 (s), 0.99 (s), 0.91 d (J = 6.3 Hz), 0.83 d (J = 6.8 Hz), 0.79 d (J = 6.8 Hz), 0.82 t (J = 7.0 Hz). 13C NMR (CDCl3, 75 MHz): δ 37.1 (C-1), 31.6 (C-2), 72.1 (C-3), 42.4 (C-4), 139.5 (C-5), 120.0 (C-6), 32.5 (C-7), 35.3 (C-8), 49.8 (C-9), 36.2 (C-10), 22.1 (C-11), 40.3 (C-12), 43.1 (C-13), 56.9 (C-14), 24.7 (C-15), 27.9 (C-16), 54.7 (C-17), 12.1 (C-18), 18.7 (C-19), 40.1 (C-20), 21.5 (C-21), 33.8 (C-22), 28.8 (C-23), 50.5 (C-24), 26.7 (C-25), 18.9 (C-26), 21.5 (C-27), 23.1 (C-28), 12.6 (C-29).
Statistical analysis
Results obtained from the pharmacological experiments are expressed as mean values ± S. E.M. One-way ANOVA test was employed for comparison of the significant differences among the groups followed by Dunnet’s multiple comparison post-test. A probability of P < 0. 05 was considered as significant. Concentration–response curves were analyzed by nonlinear regression using GraphPad program (GraphPAD, San Diego, CA, USA).