Evaluation of the anti-inflammatory activity of N, N′-bis(3-dimethylamino-1-phenyl-propylidene)hydrazine dihydrochloride

Abstract

This study investigated the anti-inflammatory effect of N, N′-bis(3-dimethylamino-1-phenyl-propylidene)hydrazine dihydrochloride, D1, on carrageenan-induced edema. In addition, its effect on hyaluronidase-induced vascular permeability was also tested. D1 was synthesized, and anti-inflammatory activity was determined by carrageenan-induced hind paw edema in rats (n = 30) at 50, 100, and 200 mg kg⁻¹ doses of D1 and also a 25 mg kg⁻¹ dose of indomethacin. The effects of D1 and indomethacin on hyaluronidase-induced capillary permeability were investigated in rabbits (n = 18) at a 100 mg kg⁻¹ dose of D1 and 25 mg kg⁻¹ dose of indomethacin. D1 inhibited carrageenan-induced inflammation by 40, 20, and 10% at 50, 100, and 200 mg kg⁻¹ doses after 1 h. The inhibitions were 22.5, 32.7, 28.6% and 15.6, 33.4, 8.9% at 2 h and 3 h, respectively. The inhibitions due to indomethacin (25 mg kg⁻¹ dose) were 67.5, 87.8, and 91.1%, at 1 h, 2 h, and 3 h, respectively. The subcutaneous spreading areas of Trypan blue at 1, 5, 30, and 60 min after subcutaneous injection of hyaluronidase were 172.6 ± 41.6, 210.2 ± 39.7, 363 ± 50, and 400.2 ± 46.7 mm² in the D1 (100 mg kg⁻¹) treated group. The spreading areas at these time periods were 38.8 ± 3.7, 48.2 ± 4.5, 100.6 ± 6.9, and 119.8 ± 22.5 mm² in the indomethacin treated group. Our results showed that D1 inhibits carrageenan-induced inflammation in rats. A tendency to decrease the capillary permeability suggested that the mechanism of action of the anti-inflammatory effect of D1 may partly depend on inhibition of the hyaluronidase enzyme.

Keywords::

• Azine
• anti-inflammatory effect
• carrageenan-induced paw edema
• capillary permeability
• Mannich base

Introduction

N,N′-bis(3-Dimethylamino-1-phenyl-propylidene)hydrazine dihydrochloride, D1, is the azine derivative of mono Mannich base Ig1, 1-phenyl-3-dimethylamino-1-propanone hydrochloride. D1 is synthesized by the interaction of 2 mol of Ig1 and 1 mol of hydrazine hydrate. They produce the compound D1 by losing 2 mol of water.

It has been reported that some acetophenone-derived Mannich bases significantly inhibit both acute and chronic phases of inflammation (Suleyman et al., 2003, 2007). It has been demonstrated that compound 3-benzoyl-1-methyl-4-phenyl-4-piperidinol hydrochloride (C1), which is a semi-cyclic acetophenone-derived mono Mannich base, has remarkable anti-inflammatory activity (Suleyman et al., 2003). In addition, experimental work on the compound B1, bis(3-phenyl-3-oxo- propyl)methylamine hydrochloride, which is a bis Mannich base and also the structural non-classic isomer of the compound C1, reported anti-inflammatory activity of B1 (Suleyman et al., 2007). C1 decreased significantly the capillary permeability shown at the acute phase of inflammation (Suleyman et al., 2003). Although the anti-inflammatory activity of some mono Mannich bases has been reported (Dauksas et al., 1990; Suleyman et al., 2003), there is no information about the anti- inflammatory activity of azine type compounds such as D1, which is derived from a mono Mannich base, or compounds having a similar chemical structure.

Carrageenan-induced paw edema in rats is used to investigate the anti-inflammatory activity of a drug (El-Shenawy et al., 2002). It is known that vascular dilatation and an increase in vascular permeability are observed in the region of inflammation. There are findings to suggest that nitric oxide may take part in the regulation of increased vascular permeability in carrageenan-induced edema (Nacife et al., 2004). In addition, a role of hyaluronidase enzyme in increasing vascular permeability is known (Houck & Chang 1979). Hyaluronidase activity increases during inflammation and decreases during resolution of the inflammation (Procida et al., 1971). Drugs or substances with anti- inflammatory activity decrease the hyaluronidase- induced vascular permeability in rabbits (Suleyman et al., 2001, 2003, 2007).

The aim of this study was to investigate the anti-inflammatory effect of D1 on carrageenan-induced edema. In addition, its effect on hyaluronidase-induced vascular permeability in rabbits was also tested.

Materials and methods

Animals

Male Wistar albino rats (n = 30, 180–200 g) were used in anti-inflammatory activity tests. In addition, Sinsila rabbits (n = 18, weighing 3.5–4 kg) were used for hyaluronidase-induced capillary permeability tests in this study. All animals used were obtained from the Experimental Animal Laboratory of the Faculty of Medicine, Ataturk University. Animal experiments were carried out following the guidelines set by the Ethics Committee of Ataturk University.

Materials

Carrageenan and trypan blue were obtained from Sigma Chemical Co. (St. Louis, USA), indomethacin was from Deva (Istanbul, Turkey), and hyaluronidase was from Kiyevskoye predpriyatiye po proizvodstvu Bakteriynih preparatov (Kiev, Ukraine).

Synthesis of azine derivative D1

A solution of hydrazine hydrate (1.75g, 0.035 mol) in ethanol (15 mL) was added to a solution of 1- phenyl-3-dimethylamino-1-propanone hydrochloride Ig1 (0.07 mol) in ethanol/acetic acid (3%, w/v; 70 mL) and the mixture was stirred at room temperature for 24 h. After removal of the solvent, the residue was washed with chloroform three times (3 × 20 mL), dried, and crystallized from ethanol to give the corresponding azine derivative (Gul et al., 2003) (Figure 1).

Figure 1.  D1: N,N′-bis(3-Dimethylamino-1-phenyl-propylidene)hydrazine dihydrochloride.

Carrageenan-induced paw edema in the rat

Anti-inflammatory activities of D1 and indomethacin were determined by the method of carrageenan-induced hind paw edema (Marzocco et al., 2004). D1 at one dose of 50, 100, or 200 mg kg⁻¹ or one dose of indomethacin at 25 mg kg⁻¹ was given to rats orally by feeding tube once daily for 2 days. The same volume of distilled water used as vehicle was given to the control group. One hour after the last treatment, carrageenan 0.1 mL (1%, w/v) solution in distilled water was subcutaneously injected into the plantar surface of the right hind paw of all rats. The paw volume until the knee joint was measured with a plethysmometer before injection of carrageenan. Carrageenan-induced paw edema was measured at 1 h intervals for 5 h. The anti-inflammatory activities of D1 and indomethacin were determined by comparing results with those obtained in the control group.

Percentage anti-inflammatory effects were calculated using the following equation: anti-inflammatory activity (inhibition %) = (1 – D/C) × 100, where D represents the percentage difference in paw volume after the administration of drug to the rat and C represents the percentage difference in volume in the control group.

Hyaluronidase-induced capillary permeability test

In this series of experiments, the effects of D1 and indomethacin on hyaluronidase-induced capillary permeability were investigated (Suleyman et al., 2003, 2007). The rabbits (n = 18) were divided into three groups, and hair in their abdominal area was shaved. The first group received D1 (100 mg kg⁻¹), while the second group received indomethacin (25 mg kg⁻¹), orally via catheter. The third, control group received only the same amount of vehicle (NaCl, 0.9%). Hyaluronidase (128 U) was dissolved in 1 mL isotonic NaCl. Trypan blue (0.8 mL, 0.75%) was added to this hyaluronidase solution (0.5 mL). The final mixture (0.1 mL) was injected subcutaneously in the abdominal region, 1 h after oral drug administration. The appearance of the blue area was measured at 20 s, 5 min, and 30 min after injection (mm²). The size of the blue area corresponds to the activity of the hyaluronidase enzyme and capillary permeability, i.e. the smaller the appearance of the blue area, the lower the activity of the hyaluronidase enzyme and the capillary permeability (Suleyman et al., 2003, 2007). The results obtained were compared with those for the control group.

Statistical analysis

The results are presented as mean ± SD. The data were analyzed using one-way analysis of variance (ANOVA) with post hoc least significant difference (LSD) test. p < 0.05 was accepted as the level of statistical significance.

Results

Carrageenan-induced paw edema

The changes in paw volume 3 h after carrageenan injection in control and D1 (at 50, 100, and 200 mg kg⁻¹ doses) and indomethacin (at 25 mg kg⁻¹ dose) treated rats are shown in Table 1. The effect of D1 at 100 mg kg⁻¹ and indomethacin on carrageenan-induced paw edema is also shown in Figure 2.

Figure 2.  Changes in paw volume for 3 h period in D1 (100 mg kg⁻¹) and indomethacin (25 mg kg⁻¹) treated rats in comparison with control after injection of carrageenan.

Table 1.  The effects of D1, N,N′-bis(3-dimethylamino-1-phenyl-propylidene)hydrazine dihydrochloride, and indomethacin on carrageenan-induced rat paw edema.

Compound	No. of rats	Dose (mg kg^−1)	Paw volume (mL) before injection of carrageenan	Paw volume (mL) with inflammation after injection of carrageenan (anti-inflammatory activity)
				After 1 h	p	After 2 h	p	After 3 h	p
D1	6	50	0.90 ± 0.08	1.02 ± 0.2 (40%)	<0.05	1.29 ± 0.28 (22.5%)	>0.05	1.28 ± 0.36 (15.6%)	>0.05
D1	6	100	0.88 ± 0.08	1.04 ± 0.25 (20%)	<0.05	1.21 ± 0.24 (32.7%)	<0.05	1.18 ± 0.15 (33.4)	<0.05
D1	6	200	0.87 ± 0.06	1.05 ± 0.19 (10%)	>0.05	1.22 ± 0.17 (28.6%)	<0.05	1.28 ± 0.33 (8.9%)	>0.05
Indomethacin	6	25	0.89 ± 0.07	0.95 ± 0.11 (67.5%)	<0.05	0.95 ± 0.10 (87.8%)	<0.05	0.93 ± 0.12 (91.12%)	<0.05
Control	6	—	0.80 ± 0.06	1.0 ± 0.23	—	1.29 ± 0.31	—	1.25 ± 0.29	—

Note. Results are given as mean ± SD.

D1 inhibited carrageenan-induced inflammation by 40, 20, and 10% at the 50, 100, and 200 mg kg⁻¹ dose after 1 h. At 2 h and 3 h, the inhibition was 22.5, 32.7, 28.6% and 15.6, 33.4, 8.9%, respectively. Indomethacin at the 25 mg kg⁻¹ dose inhibited carrageenan-induced inflammation by 67.5, 87.8, and 91.1%, at 1 h, 2 h, and 3 h, respectively (Table 1).

Hyaluronidase-induced capillary permeability test

The subcutaneous spreading areas of Trypan blue 1, 5, 30, and 60 min after subcutaneous injection of hyaluronidase in control and D1 (100 mg kg⁻¹) and indomethacin (25 mg kg⁻¹) treated rabbits are shown in Table 2.

Table 2.  The effects of D1 and indomethacin on hyaluronidase-induced capillary permeability in rabbits.

Compound/drug	No. of rabbits	Dose (mg kg^−1)	Blue area spread of hyaluronidase (mm^2)
			After 1 min	p	After 5 min	p	After 30 min	p	After 60 min	p
D1	6	100	172.6 ± 41.6	>0.05	210.2 ± 39.7	>0.05	363 ± 50.2	>0.05	400.2 ± 46.7	>0.05
Indomethacin	6	25	38.8 ± 3.7	<0.03	48.2 ± 4.5	<0.001	100.6 ± 6.9	<0.001	119.8 ± 22.5	<0.002
Control	6	—	125.4 ± 18.5	—	243.4 ± 31.5	—	452.2 ± 83	—	496 ± 76.9	—

Note. Results are given as mean ± SD.

Discussion

In this study, the effects of D1 at 50, 100, and 200 mg kg⁻¹ doses on the acute phase of inflammation were investigated by the carrageenan-induced paw edema test in rats. In addition, the effect of D1 at the 100 mg kg⁻¹ dose on capillary permeability was also investigated by the hyaluronidase-induced capillary permeability test in rabbits. Our results showed that D1 has different levels of anti-inflammatory activity at different dose levels; however, its anti-inflammatory activity was not dose-dependent. D1 at 50 mg kg⁻¹ was the most effective dose at 1 h of carrageenan-induced inflammation. The anti-inflammatory effects of D1 (50 mg kg⁻¹) at 2 h and 3 h of carrageenan-induced inflammation were lower, and not significant. This suggested that the anti-inflammatory effect of D1 starts quickly and lasts for a short period of time. D1 at the 100 mg kg⁻¹ dose inhibited carrageenan-induced inflammation at all measurement time points (1, 2, and 3 h); however, its anti-inflammatory activity was higher at 2 h and 3 h. These results showed that the anti-inflammatory effect of D1 at the 100 mg kg⁻¹ dose lasts longer than its effect at the 50 mg kg⁻¹ dose level. The increase of dose of D1 to 200 mg kg⁻¹ did not increase its anti-inflammatory activity; on the contrary, it led to a decrease in anti-inflammatory activity. D1 at the 200 mg kg⁻¹ dose level inhibited carrageenan-induced inflammation significantly at 2 h only.

We have previously shown that the Mannich base derivative C1 has strong anti-inflammatory activity against carrageenan-induced inflammation (Suleyman et al., 2003). C1 at a 200 mg kg⁻¹ dose inhibited carrageenan-induced inflammation more than indomethacin at 20 mg kg⁻¹, while at a 100 mg kg⁻¹ dose it decreased the weights of subcutaneously located cotton pellets more than indomethacin at 20 mg kg⁻¹ (Suleyman et al., 2003). It is known that there are two phases of carrageenan-induced inflammatory reaction: the early or first phase and the late or second phase (Gupta et al., 2003). The late phase of carrageenan-induced inflammation begins 1 h after carrageenan injection and lasts 3 h. The time until this period is known as the early phase of inflammation (Honda et al., 2002). It is proposed that the early phase results from histamine, serotonin, and bradykinin liberation, while the late phase is associated with the formation of prostaglandins (Marzocco et al., 2004; Weissmann, 1993). The increase in bradykinin production in the early phase of inflammation leads to the activation of L and T types of calcium channel, and in this way increases the intracellular calcium levels (El-Bizri et al., 2003). Calcium ions increase the synthesis of eicosanoids by activating the cytosolic phospholipase A₂ (cpA₂) (Carnevale & Cathcart, 2001; Leslie, 1997). Furthermore, an increase in intracellular calcium ions in mast cells activates these cells and leads to the release of histamine, which plays an important part in inflammation (Chand et al., 1985; Godfraind et al., 1986). D1 could have inhibited the release of histamine, serotonin, and bradykinin due to inflammation at the 50 mg kg⁻¹ dose level, since it was effective at 1 h. However, D1 was effective at 2 h and 3 h at the 100 mg kg⁻¹ dose level. This effect of D1 on the late phase of inflammation suggests that the anti-inflammatory effect of D1 in the late phase may depend on the inhibition of formation of prostaglandins. It is known that capillary permeability increases in the area of inflammation. It is thought that nitric oxide may take part in the increase of capillary permeability in carrageenan-induced inflammation (Nacife et al., 2004).

The increase in capillary permeability correlates well with the intensity of inflammation, i.e., the chemicals that lead to more inflammation also cause further increase in capillary permeability in the inflamed area (Suleyman et al., 2003). This means that a decrease in capillary permeability also causes a decrease in the intensity of inflammation. The above-mentioned compound C1 prevented carrageenan-induced inflammation in rats and inhibited hyaluronidase-induced capillary permeability in rabbits with potency comparable to the anti-inflammatory drug, indomethacin (Suleyman et al., 2003). In this study, we tested the effect of D1 at 100 mg kg⁻¹ on capillary permeability. This 100 mg kg⁻¹ dose was selected because D1 inhibited carrageenan-induced inflammation the longest (3 h) at this dose level. The anti-inflammatory effect of D1 (in the carrageenan-induced inflammation test) was four-fold less than the anti-inflammatory effect of C1. Furthermore, the inhibitory effect of D1 on capillary permeability was not significant, while C1 significantly inhibited the increase in capillary permeability due to subcutaneous hyaluronidase injection (Suleyman et al., 2003). A possible explanation for the non-significant effect of D1 on capillary permeability increase is that it may have also inhibited the carrageenan-induced inflammation slightly. Despite that the inhibitory effect of D1 on capillary permeability was not statistically significant, it decreased the spread of the blue area due to subcutaneous injection of the hyaluronidase–Trypan blue mixture by 89.2 and 96.0 mm² at 30 and 60 min, respectively, in comparison with the control group. Blue areas were 351.6 and 376.2 mm² smaller in the indomethacin treated group at these time points in comparison with the control group. After 60 min the blue area increased quickly, and the limit of the blue area was less clear and difficult to determine in the control and D1-receiving groups. These results show that hyaluronidase may play a role in the increase of capillary permeability in inflammation. In addition, the mechanism of action of the anti-inflammatory effect of D1 may partly depend on inhibition of the hyaluronidase enzyme. Further studies are needed to clarify the other mechanisms of action of the anti-inflammatory effect of D1.

Acknowledgement

This study was partly supported by the Ataturk University Research Fund (2005/148).