The anti-inflammatory activity of dillapiole and some semisynthetic analogues

Abstract

Context: Piper aduncum L. (Piperaceae) produces an essential oil (dillapiole) with great exploitative potential and it has proven effects against traditional cultures of phytopathogens, such as fungi, bacteria and mollusks, as well as analgesic action with low levels of toxicity.

Objective: This study investigated the in vivo anti-inflammatory activity of dillapiole. Furthermore, in order to elucidate its structure-anti-inflammatory activity relationship (SAR), semisynthetic analogues were proposed by using the molecular simplification strategy.

Materials and methods: Dillapiole and safrole were isolated and purified using column chromatography. The semisynthetic analogues were obtained by using simple organic reactions, such as catalytic reduction and isomerization. All the analogues were purified by column chromatography and characterized by ¹H and ¹³C NMR. The anti-inflammatory activities of dillapiole and its analogues were studied in carrageenan-induced rat paw edema model.

Results: Dillapiole and di-hydrodillapiole significantly (p<0.05) inhibited rat paw edema. All the other substances tested, including safrole, were less powerful inhibitors with activities inferior to that of indomethacin.

Discussion and conclusion: These findings showed that dillapiole and di-hydrodillapiole have moderate anti-phlogistic properties, indicating that they can be used as prototypes for newer anti-inflammatory compounds. Structure-activity relationship studies revealed that the benzodioxole ring is important for biological activity as well as the alkyl groups in the side chain and the methoxy groups in the aromatic ring.

Keywords::

• Piperaceae
• Piper aduncum
• phenylpropanoids
• molecular modification
• structure-activity relationship

Introduction

It is reasonable to suggest ever since the origin of man, natural products have been used to treat and cure diseases. All chemical knowledge acquired from nature has its roots in the discoveries made by primitive and indigenous people throughout history. After the first contacts with these different ethnicities, the scientific community was able to begin research and development with natural products and to study the relationship between the chemical structure of compounds and their biological properties (Viegas-Junior et al., 2006).

Many bioactive substances have already been extracted from nature, whether from microorganisms or marine sources and from animals, although the great majority of these substances originate from the vegetable kingdom. Actually, it is estimated that there are 500,000 plant species on the planet that have distinct and complex micromolecules which often possess biological activity (Montanari & Bolzani, 2001). However, very little of this biodiversity has so far been investigated for its chemical components and biological properties.

Over the last two decades, the use of medicinal plants has increased, leading to more and more people turning to therapeutic alternatives, such as phytotherapy, to cure their diseases. Pharmaceutical industries have noticed this tendency and realized its potential for the discovery of new patentable molecular entities. As a consequence, huge investments have been made to study the chemistry of natural products. Nowadays, the use of natural molecular fingerprints as a source of inspiration for the acquisition of new bioactive chemical entities has proven to be a very promising and efficient method (Newman et al., 2000; Hanessian, 2006; Baker et al., 2007).

The Piperaceae family comprises about 10 genera and almost 1400 species (Yunker, 1972). Some of these species are employed in popular medicine for the treatment of several different diseases (Duke, 1985; Parmar et al., 1997). Chemical investigations carried out on the essential oils of this genera of plants has uncovered the existence of monoterpenes, sesquiterpenes (Sengupta & Ray, 1987; Green et al., 1999) and phenylpropanoids, among other substances (Maxwell & Rampersad, 1989; Moreira et al., 2001). The chemical isolation and characterization of phenylpropanoids revealed a class of chemical compounds represented especially by dillapiole (1) and safrole (2) (Figure 1; Diaz et al., 1986; Bernard et al., 1995; Fazolin et al., 2005).

Figure 1.  The chemical structures of dillapiole (1) and safrole (2).

There is great interest in phenylpropanoids because of their potential biological properties, including psychotropic, antimicrobial, antioxidant and cytotoxic properties (Shulgin, 1966; Masuda et al., 1991; Atsumi et al., 2000; Lago et al., 2004, 2005). Piper hispidinervum C. DC., popularly known as long pepper, is rich in safrole, a phenylpropanoid that is currently being used as a raw material for the manufacture of heliotropin, an important component of some perfumes, and piperonyl butoxide, a pesticide synergist for natural insecticides (Fazolin et al., 2007). Studies by Barreiro and Fraga (1999) and Costa (2000) on the chemical reactivity of safrole (2) and the acquisition of synthetic analogues have indicated great synthetic variability and excellent opportunities for their derivation.

The Piper aduncum species occurs in several regions of the world and is also common throughout Brazil (Gaia et al., 2004). Popularly known as Matico, it produces an essential oil with great exploitative potential because it has proven effects against traditional cultures of phytopathogens, like fungi (Bastos, 1997), bacteria and mollusks (Orjala et al., 1993) as well as analgesic action with low levels of toxicity (Monteiro et al., 2001; Sousa et al., 2008). Among the different piperaceae that exist in the world, Piper aduncum is an excellent producer of essential oils, which contain a high level of dillapiole (1) (Cicció & Ballestero, 1997; Maia et al., 1998).

Few studies on the preparation of analogues and the possible therapeutic activities of dillapiole (1) have been published in the literature (Orjala et al., 1993, 1994; Okunade et al., 1997; Belzile et al., 2000; Majerus et al., 2000; Meléndez & Capriles, 2006; Fazolin et al., 2007; Lobato et al., 2007). As it is an easily extracted natural product with a relatively simple structure and high reactivity, the synthetic manipulation and biological evaluation of dillapiole (1) can easily be planned, developed and executed. The use of this natural product as a prototype for the synthesis of analogues with potentially superior action is a very alluring and scientifically important subject.

The objective of this work was to evaluate the anti-inflammatory activity of dillapiole and to synthesize new structural analogues based on the concept of molecular simplification as a method of molecular modification (Figure 2). This strategy can both help in the elucidation of the relationship between structure and anti-inflammatory activity as well as facilitate the discovery of a pharmacophoric group from the analyzed series.

Figure 2.  Dillapiole (1) and its structural analogues.

Materials and methods

Column chromatography (CC) was carried out under pressure, using silica gel 60 Merck, 230–400 mesh. Thin layer chromatography (TLC) was carried out using 0.25 mm thick sheets of silica gel (Merck 60 GF₂₅₄). P.A. grade solvents and reagents were employed. NMR spectra were recorded using a Bruker AC300 spectrometer at 300 MHz (¹H) and 75 MHz (¹³C) with tetramethylsilane as an internal reference and CDCl₃ as a solvent. Chemical shifts are given in parts per million (ppm), coupling constants in Hertz and splitting patterns are designated as follows: s, singlet; br s, broad singlet; d, doublet; t, triplet; q, quartet and m, multiplet.

Dillapiole extraction

The leaves of Piper aduncum were collected in March and August 2009 in the town of Campos do Jordão, São Paulo State, Brazil. The extraction and the isolation of dillapiole (1) were performed following the procedure described by Lago et al. (2006). The volatile components of the collected plant material were extracted throughout the hydrodistillation procedure using a Clevenger modified apparatus. The crude volatile oil was extracted with dichloromethane, dried with anhydrous Na₂SO₄, filtered and kept in a freezer (−15°C) in an amber glass bottle. The essential oil was submitted to purification by CC using silica gel 60 as the stationary phase and hexane:dichloromethane (7:3) as the eluent system. The pure dillapiole (1) was used in further synthetic reactions.

Safrole extraction

Safrole (2) was not synthesized in this work but the extraction from Ocotea sp. and its purification was carried out using the same procedure described for dillapiole (1).

Synthesis

Benzo[d][1,3]dioxole (3) synthesis

In a 250 mL round-bottomed flask, 1 mmol of pyrocatechol, 20 mmol of sodium hydroxide and 10 mL of dichloromethane were dissolved in dimethylsulfoxide (DMSO). The solution was kept under reflux at 140°C for 10 h. The reaction was monitored by TLC. At the end of the reaction, the mixture was fractionally distilled and the final product was analyzed by ¹H and ¹³C NMR.

4,5-Dimethoxy-6-propylbenzo[d][1,3]dioxole (di-hydrodillapiole) (5) synthesis

In a 100 mL round-bottomed flask, fitted with a reflux condenser and drying tube, dillapiole (2 mmol), NaBH₄ (20 mmol), NiCl₂·6H₂O (1.5 mmol) were dissolved in 40 mL of methanol. The mixture was kept under reflux and constant stirring for 24 h. After cooling, the product was filtered and the solvent was removed under reduced pressure.

4,5-Dimethoxy-6-(prop-1-enyl)benzo[d][1,3]dioxole (Isodillapiole) (6) synthesis

In 25 mL round-bottomed flask, fitted with a reflux condenser and drying tube, 4 g of dillapiole (24.7 mmol) in a 17% solution of potassium hydroxide (3.4 g) was dissolved in 20 mL of butanol. The reaction was kept under reflux and constant stirring for 24 h. After cooling, the mixture was neutralized with 1.2 mL of concentrated HCl and 8 mL of distilled cold water. The organic phase was washed with three portions of water (20 mL). The organic phase was dried with anhydrous Na₂SO₄. The excess of butanol was evaporated and the residue was submitted to distillation under reduced pressure.

5-Propylbenzo[d][1,3]dioxole (di-hydrosafrole) (7) synthesis

In a 100 mL round-bottomed flask, fitted with a reflux condenser and drying tube, safrole (2 mmol), NaBH₄ (20 mmol), NiCl₂.6H₂O (1.5 mmol) were dissolved in 40 mL of methanol. The mixture was kept under reflux and constant stirring for 24 h. After cooling, the product was filtered and the solvent was removed under reduced pressure.

5-(Prop-1-enyl)benzo[d][1,3]dioxole (isosafrole) (8) synthesis

In a 25 mL round-bottomed flask, fitted with a reflux condenser and drying tube, 4 g of safrole (24.7 mmol) in a 17% solution of potassium hydroxide (3.4 g) was dissolved in 20 mL of butanol. The reaction was kept under reflux and constant stirring for 24 h. After cooling, the mixture was neutralized with 1.2 mL of concentrated HCl and 8 mL of distilled cold water. The organic phase was washed with three portions of water (20 mL). The organic phase was dried with anhydrous Na₂SO₄. The excess of butanol was evaporated and then distilled under reduced pressure.

Anti-inflammatory assays

Anti-inflammatory activity was evaluated using the carrageenan-induced rat paw edema (Winter et al., 1962). Overnight fasted Wister rats of either sex weighing 150–175 g were divided into groups of five animals each. Test compounds (50 mg/kg body weight) and indomethacin (50 mg/kg) were administered orally. After 30 min, all animals were injected with 0.1 mL of 1% carrageenan solution (prepared in normal saline) in the subplantar aponeurosis of left hind paw and the volume of paw was measured by using plethysmometer at 0, 30, 60, 90 and 120 min post carrageenan treatment. Experimental protocols were approved by the Institutional Committee on Animal Research at the Mackenzie Presbyterian University. The results were analyzed for statistical significance using ANOVA. p<0.05 was considered significant.

Results and discussion

Extraction

Dillapiole (1) (Figure 2) was extracted from Piper aduncum (88% yield), and, after purification, the material was used in synthetic reactions (Lago et al., 2006; Meléndez & Capriles, 2006). Safrole (2) was extracted from Ocotea sp. (78% yield) and purified by distillation.

Synthesis

The semisynthetic analogues were obtained and confirmed by ¹H and ¹³C NMR analysis. The chemical shifts for the obtained compounds are presented in Table 1.

Table 1.  Chemical shifts (¹H and ¹³C NMR) for dillapiole analogues.

Compounds	Chemical shift
	^1H (CDCl3, 300 MHz, δ = ppm, J = Hz)
	^13C (CDCl3, 75 MHz, δ = ppm):
Benzo[1,3]dioxole (3)	^1H: 5.9 (s, 2H, H-8), 6.78-6.84 (m, 4H, H-3, H-4, H-5, H-6).
	^13C: 100.62 (C8); 108.05 (C3, C6); 122.03 (C4, C5), 147.53 (C2, C1).
di-Hydrodillapiole (5)	^1H: 0.91 (t, 3H, H-12, J = 8.0); 1.54 (m, 2H, H-11, J = 8.0 and J = 7.1); 2.49 (t, 2H, H-10, J = 7.1); 3.73 (s, 3H, H-16); 3.95 (s, 3H, H-14) 5.87 (s, 2H, H-8); 6.34 (s, 1H, H-6).
	^13C: 13.86 (C12); 25.60 (C11), 33.57 (C10); 59.85 (C16); 59.91 (C14); 101.00 (C8); 105.38 (C7); 127.39 (C5); 135.54 (C2); 137.59 (C1); 144.39 (C3); 144.45 (C4).
Isodillapiole (6)	^1H: 1.86-1.89 (dd, 3H, H-12, J = 6.4 and J = 1.0); 3.75 (s, 3H, H-16); 3.88 (s, 3H, H-14); 5.91 (m, 1H, H-11, J = 15.1 and J = 6.4); 6.05-6.07 (d, 2H, H-8); 6.51-6.59 (m, 1H, H-10, J = 6.4 and J = 15.1); 6.96 (s, 1H, H-6).
	^13C: 18.81 (C12); 59.81 (C16); 60.10 (C14); 101.51 (C8); 102.76 (C6); 125.51 (C10); 126.62 (C5); 128.34 (C11); 134.56 (C2); 136.01 (C1); 141.43 (C4); 147.50 (C3).
di-Hydrosafrole (7)	^1H: 0.89-0.94 (t, 3H, H-12, J = 8.1); 1.52-1.64 (m, 2H, H-11, J = 8.1 and J = 7.0); 2.46-2.51 (t, 2H, H-10, J = 7.0); 5.89 (s, 2H, H-8); 6.66 (d, 1H, H-3, J = 7.5 and J = 1.5); 6.69 (s, 1H, H-6); 6.72 (d, 1H, H-4, J = 1.5 and J = 7.5).
	^13C: 13.70 (C12); 24.82 (C11), 37.78 (C10); 100.67 (C8); 108.0 (C3); 108.9 (C6); 121.11 (C4); 134.4 (C5), 146.1 (C2); 148.9 (C1).
Isosafrole (8)	^1H: 1.80-1.87 (dd, 3H, H-12, J = 6.4 and J = 1.0); 5.90 (m, 1H, H-11, J = 15.0 and J = 1.0); 6.02-6.6 (s, 2H, H-2); 6.25-6.31 (m, 1H, H-10, J = 15.0 and J = 6.5); 6.70-6.76 (dd, 1H, H-3, J = 7.5 and J = 1.5); 6.85-6.89 (dd, 1H, H-3, J = 7.5 and J = 1.5); 6.97 (s, 1H, H-6).
	^13C: 7.93 (C12); 100.86 (C8); 105.06 (C6); 108.64 (C3); 119.98 (C4); 124.96 (C11); 130.50 (C10); 132.03 (C5); 146.29 (C1); 147.38 (C2).

Dillapiol (1) was reduced by using NaBH₄ and NiCl₂·9H₂O to obtain di-hydrodillapiole (5) (86% yield) (Narisada et al., 1989). The success of the reduction was confirmed by the ¹H NMR spectrum which revealed the appearance of a 0.91 ppm triplet and a 2.49 ppm triplet, the characteristic signals of a propyl group. Compound (1) was also submitted to a reaction with a 3 mol/L solution of potassium hydroxide in butanol under reflux to obtain isodillapiole (6) (85% yield) (Lima et al., 2000). The spectrum of compound (6) showed the change in H-10 protons chemical shift. After isomerization, the two H-10 protons that presented a 3.31 ppm doublet chemical shift as a characteristic signal changed to a doublet at 2.48 ppm.

Safrole (2) (Figure 2) is a phenylpropanoid similar to dillapiole (1). In order to evaluate the relevance of the methoxy groups in dillapiole (1) for the anti-inflammatory properties, safrole (2) was virtually used as a simplified (di-demethoxyl) dillapiole analogue. Safrole oil was submitted to catalytic reduction, resulting in compound (7) (82% yield) (Narisada et al., 1989). Its structure was confirmed by the alteration in the coupling pattern of the H-12 and H-10 protons. After chemical reduction, the signals of these protons appear as two triplets at 0.92 ppm and 2.48 ppm, respectively. Compound (8) (64% yield) was obtained from (2) by using the same methodology described earlier for compound (6) (Lima et al., 2000). Its structure was confirmed by the change in coupling patterns of the H-12 and H-10 protons. The H-12 protons, which in the safrole (2) appeared at 1.83 ppm, are now methylic hydrogens, resulting in a double doublet from the coupling of these with the H-11 and H-10 protons. The H-10 proton, which was a triplet, appears as a multiplet at 6.25–6.31 ppm.

Pyrocatechol (4) was used to obtain benzo[1.3]dioxole (3). Steam distillation was used to purify the product (77% yield) (Figure 2) (Bonthrone & Cornforth, 1969). Contrary to compound (4), benzodioxole (3) is a ring-shaped bicyclical system with a methylene dioxilic bond as its main characteristic. The appearance of a signal at 5.9 ppm confirmed the presence of the methylene dioxilic bond.

Anti-inflammatory assays

Anti-inflammatory activity was measured by carrageenan-induced edema in the rats’ paws (Winter et al., 1962). The anti-inflammatory effects of dillapiole and its analogues at different time intervals are presented in Table 2.

Table 2.  Anti-inflammatory activity of the compounds 1–8 in carrageenan-induced rat paw edema assay.

Compound*	Volume of edema (mL)^§
	0 min	30 min	60 min	90 min	120 min
Control	0.39 ± 0.03	0.53 ± 0.03	0.66 ± 0.04	0.84 ± 0.01	0.91 ± 0.01
1	0.32 ± 0.01(17)^¥	0.42 ± 0.01(20)	0.49 ± 0.04(25)	0.67 ± 0.02(20)	0.75 ± 0.04(17)
2	0.35 ± 0.01(10)	0.51 ± 0.01(4)	0.62 ± 0.02(6)	0.80 ± 0.03(5)	0.88 ± 0.01(5)
3	0.36 ± 0.01(7)	0.52 ± 0.01(2)	0.64 ± 0.02(3)	0.81 ± 0.03(3)	0.90 ± 0.02(1)
4	0.40 ± 0.04	0.56 ± 0.01	0.69 ± 0.02	0.90 ± 0.03	0.98 ± 0.03
5	0.32 ± 0.01(18)	0.40 ± 0.01(25)	0.43 ± 0.04(35)	0.55 ± 0.02(34)	0.70 ± 0.01(23)
6	0.36 ± 0.03(7)	0.51 ± 0.07(4)	0.64 ± 0.02(3)	0.80 ± 0.03(6)	0.89 ± 0.05(2)
7	0.35 ± 0.01(9)	0.48 ± 0.01(8)	0.60 ± 0.02(5)	0.79 ± 0.03(3)	0.90 ± 0.01(1)
8	0.37 ± 0.02(5)	0.50 ± 0.04(5)	0.63 ± 0.05(4)	0.80 ± 0.03(5)	0.89 ± 0.01(2)
Indomethacin	0.27 ± 0.05(29)	0.33 ± 0.03(37)	0.38 ± 0.03(42)	0.46 ± 0.08(45)	0.57 ± 0.06(38)

^*Dose levels: test compounds (50 mg/kg body weight), indomethacin (50 mg/kg body weight).

^§Values are expressed as mean ± SEM and analyzed by ANOVA.

^¥Values in parentheses (percentage anti-inflammatory activity).

A comparative study of the anti-inflammatory activity of test compounds relative to the reference drug at different time intervals indicated the following: after 1h, compound (5) was nearly as effective in inhibiting the paw edema with percentage activity of 35% when compared with that of indomethacin (42%). Five other compounds (2), (3), (6), (7) and (8) showed similar pharmacokinetic profiles as revealed from their onset of action with percentage activity of 5–9%. Compound (4) was completely inactive and proved to be a strong phlogistic agent. After 90 min, only two compounds, (1) and (5), showed significant anti-inflammatory activity ranging from 20 to 34% inhibition as compared to indomethacin (45%). Taking the anti-inflammatory activity after 2 h time interval as a criterion for comparison, it can be concluded that compounds 1 and 5 showed good anti-inflammatory activity (17 and 23%, respectively) comparable with indomethacin (38%), whereas compounds (2), (3), (4), (6), (7) and (8) displayed a bad anti-inflammatory activity (0–5%); however, none of them was found to be superior over the reference drug.

Structure-Activity relationship

Comparing compound (2) with compound (1), the first of them showed very weak anti-inflammatory activity. As verified, dillapiole and safrole are similar structures, the only difference being in their methoxyl groups. Therefore, these results suggest that those groups present in dillapiole (1) are important to keep anti-inflammatory activity. Pyrocatechol (4) was completely inactive and proved to be a strong phlogistic agent. In contrast, 1,3-benzodioxole (3) showed modest anti-inflammatory activity over 1 h. These results indicate that the presence of dioxole ring is an important fact to maintain anti-inflammatory activity as its deprotection ends with this effect and, conversely, increases the inflammatory process. di-Hydrodillapiole (5) and di-hydrosafrole (7) was obtained by reducing the double bond of dillapiole (1) and safrole (2). The reduction of the double bond increased their anti-inflammatory effect. These results suggest that the absence of double bond is favorable for activity. Isodillapiole (6) and isosafrole (8) were very weak anti-inflammatory agents. Thus, changing the position of the double bond affects negatively the anti-inflammatory action.

Based upon the preliminary results obtained from anti-inflammatory activity for dillapiole and its analogues on the rat paw edema induced by carrageenan, it was possible to elucidate dillapiole structure-anti-inflammatory activity relationship (SAR) providing the following information: (a) the pharmacophore group is the benzodioxole unit, (b) the methoxyl groups are favorable for anti-inflammatory activity, (c) the allyl group of the side chain is important for the activity, and (d) the removal of the double bond increases the activity of the substance. These observations suggest that dillapiole (1) can be used as a prototype that can be modified to obtain newer anti-inflammatory compounds. di-Hydrodillapiole could be used as anti-inflammatory agent. Moreover, the benzodioxole nucleus could be optimized to obtain newer analogues.

Declaration of interest

This work was supported by Mackpesquisa, the Mackenzie Research Foundation of the Mackenzie Presbyterian University.