Chemical Composition and Antinociceptive Potential of Campomanesia reitziana Fruits

Medicinal plants are valuable sources of active principles, and numerous studies have confirmed their therapeutic potential. Some of these plants have been used by the pharmaceutical industry for the development of new phytomedicines and phytopharmaceuticals, or as prototypes for the synthesis of new and effective medicinal agents. ^1,2

We report here the chemical composition and antinociceptive effects of methanolic extract and its main constituent from fruits of Campomanesia reitziana, a native Brazilian plant known popularly as guabiroba or guavirova, analyzed by two classical models in mice.

The fruits of C. reitzianus were collected in Itajaí-SC (Brazil), in October 2012. The species was identified by professor Oscar B. Iza (UNIVALI), and the respective exsiccate was deposited at the Barbosa Rodrigues Herbarium (Itajaí-SC), under number VC Filho 95.

Fresh fruits (60 g) were macerated with methanol for 7 days. The solvent was then evaporated under reduced pressure (50°C), yielding the respective crude methanolic extract (3.9 g; 6.45%).

Part of the methanolic extract (1.3 g) was directly chromatographed on silica gel column eluted with chloroform–methanol gradient (8:2), resulting in 10 fractions of 10 mL each. Fractions 3–5 furnished the pure substance 1 (13 mg), identified as 4′,6′-dihydroxy-3′,5′-dimethyl-2′-methoxy chalcone or dimethyl cardamonin, based on its spectral data (¹H and ¹³C NMR) in comparison with those in the literature. ^3,4

A Shimadzu liquid chromatograph, model LC-20AT, operated at 355 nm, with a SunFire C18 column (100 × 2.1 mm i.d.), 3.5 μm particle size, was used, with the following gradient elution: mobile phase A: ultra pure water, mobile phase B: MeOH, kept constant at 2%, and mobile phase C: acetonitrile (HPLC grade).

Swiss mice (25–35 g), housed at 22°C ± 2°C under a 12-h light/12-h dark cycle and with access to food and water ad libitum, were acclimatized to the laboratory for at least 1 h before testing. The experiments reported here were carried out in accordance with the current ethical guidelines for the care of laboratory animals and the investigation of experimental pain in conscious animals. ⁵ The experiments were approved by the local Ethics Committee of this Institution (113/2005-03 UNIVALI).

For the writing test, ⁶ male Swiss mice (25–30 g) were pretreated with the methanolic extract (10 mg/kg) and compound 1 at 0.3, 1, 3, and 10 mg/kg, intraperitoneally (i.p.), or orally (100 mg/kg of methanolic extract), 30 and 60 min before the acetic acid injection, respectively (six to eight animals in each group).

Another procedure used, formalin test, was similar to that described by Hunskaar et al. with minor modifications. ⁷ The mice were treated with a solution of methanolic extract of C. reitziana fruits (10 mg/kg), compound 1 (5 mg/kg), or saline (control animals) intraperitoneally, 30 min before receiving the injection of formalin.

The results are presented as means of six experimental values, except for the mean ID₅₀ values (i.e., the dose of extracts and compound 1 that reduce the algesic responses by 50% compared with the control value), which are reported as geometric means accompanied by their respective 95% confidence limits. The statistical significance between groups was analyzed by one-way analysis of variance followed by Dunnett's post-test, unless otherwise stated. P values of less than .05 were considered indicative of significance. ID₅₀ values were determined by graphic interpolation from individual experiments.

Experiments by TLC and HPLC with the methanolic extract indicated four phenolic compounds, one of them as majority, which was isolated by column chromatography and identified by NMR spectroscopic data as 4′,6′-dihydroxy-3′,5′-dimethyl-2′-methoxy chalcone (1) (Fig. 1). Figure 2 shows the HPLC fingerprint, a more sensitive method, confirming that 1 is the main substance in the studied methanolic extract.

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FIG. 1.

Molecular structure of 4′,6′-dihydroxy-3′,5′-dimethyl-2′-methoxy chalcone or dimethyl cardamonin (1) isolated from fruits of Campomanesia reitziana.

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FIG. 2.

Chromatographic profile by HPLC of methanolic extract from the fruits of C. reitziana, with detection at 335 nm.

The methanolic extract exhibited significant antinociceptive effects in mice, by the intraperitoneal or oral routes, causing 59% and 49% inhibition at 10 and 100 mg/kg, respectively, against acetic acid-induced abdominal constrictions (results not shown).

Although this experimental model is considered a nonspecific antinociceptive model, since anticholinergic and antihistaminic agents, among others, sometimes present activity, it is commonly used for antinociceptive screening for natural and synthetic compounds ⁸ and involves local peritoneal receptors (cholinergic and histamine receptor) as well as acetylcholine and histamine mediators. ⁹

Dimethyl cardamonin (1), the main constituent of the methanolic extract of fruits of C. reitziana, caused grade and dose-dependent inhibition of abdominal constrictions (Fig. 3), with a calculated ID₅₀ value of 8.1 (6.5–10.1) μmol/kg (i.p.), being about 16-fold more potent than those evidenced for acetyl salicylic acid and acetaminophen, which presented ID₅₀ values of 125 and 133 μmol/kg (i.p.), as previously demonstrated. ¹⁰

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FIG. 3.

Effects of the dimethyl cardamonin (1) against 0.6% acetic acid-induced constrictions, administered intraperitoneally at different doses. Each column represents the mean of six experimental values. **P < .01.

Compound 1 is known to exert some pharmacological actions, especially as a promising anticancer agent in vitro and in vivo models. ^{11 –13} The potent antinociceptive effect, demonstrated here for the first time for compound 1 (Table 1), is of great medical importance for the future development of a new anticancer agent, since it gives the compound added value.

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Table 1.

Effects of Different Doses of Dimethyl Cardamonin (1) Against Acetic Acid-Induced Writhing in Mice

Compound dose (mg/kg i.p.)	Inhibition (%)	ID50
0.3	4.07 ± 2.12	2.42 (1.95–3.0) mg/kg Or 8.1 (6.5–10.1) μmol/kg
1	52.9 ± 1.81 **
3	55.4 ± 2.69 **
10	72.3 ± 2.15 **

Each group represents the mean ± SEM of six to eight experimental values.

**

P < .01 compared with the respective control values.

In the formalin test, the methanolic extract (10 mg/kg) inhibited both phases of pain by the intraplantar route, causing reductions of 39.9% and 26.8% for the first and second phases, respectively (Table 2). Paracetamol, used as a reference drug, at the same doses, caused inhibition of 33% against the second phase, but was inactive in the early phase. Compound 1 exhibited a similar profile, but was more active, causing inhibition of 52.9% and 57.6% against the first and second phases, respectively, at 5 mg/kg.

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Table 2.

Antinociceptive Effects of Methanolic Extract and Compound 1 from Campomanesia reitziana Fruits in the Formalin Test in Mice

	Formalin test
	First phase	Second phase	Edema
Methanolic extract a , %	39.9 ± 4.44 **	26.8 ± 8.5 **	17.1 ± 3.3 **
Compound 1 b	52.9 ± 2.9 **	57.6 ± 5.3 **	17.7 ± 5.6 *
PAR a , %	Inactive	33.0 ± 5.0 **	NT

Each group represents the mean of six to eight animals.

First phase (0–5 min) and second phase (15–30 min).

a

10 mg/kg.

b

5 mg/kg.

*

P < .05 and ^** P < .01.

NT, not tested; PAR, paracetamol.

The effects of the methanolic extract and its main component, 1, against both phases of pain suggest that they act by different mechanisms or act on the central nervous system. ¹³

These results, demonstrated here for the first time, indicate that the fruit of C. reitziana exhibit pronounced antinociceptive effects in mice, and a chalcone, dimethyl cardamonin (1), is the main active principle responsible for the pharmacological actions indicated.