Material plant and fractionation
Artemisia annua L. leaves (hybrid CPQBA2/39 x PL5) were collected from the experimental field of CPQBA/UNICAMP. Voucher specimen is deposited at CPQBA/UNICAMP under registration number 229. The hybrid varieties (CPQBA2/39 and PL5) originated from clones of Chinese SI/SII and Vietnamese FI/FII seeds adapted in 1991 at CPQBA experimental field, which were provided by MEDIPLANT (Centre de Recherches SUR LES PLANTES medicinal ET AROMATIQUES - Switzerland) responsible for the work of improvement of seeds. The work in Brazil was accomplished by Dr. Pedro Melillo Magalhães and Dr. Glyn Mara Figueira.
This material was allowed to dry under air circulation (40°C) and ground for use. The resulting powder (2000 g) was submitted to dynamics maceration with ethanol during 2 hours (this procedure was repeated three times).
A 10% lead acetate solution (1 L) was added to the crude ethanolic extract (108 g) dissolved in ethanol (100 mL). This mixture was allowed to stand at room temperature overnight and filtered. The filtrate was extracted with chloroform (3 × 350 mL), dried over MgSO4, filtered and dried under vacuum affording the sesquiterpene lactone enriched fraction (Lac-FR) (10 g - 9.3% yield)[8, 12]. This fraction (10 g) was purified on successive column chromatography using silica gel (Merck 7734) (5 × 60 cm) with hexane/ethyl acetate (99:1), Rf dihydro-epideoxyarteannuin B: 0.65 between 500–950 mL, [αD
20]: + 49.96° (c 0.025 g/mL, CHCl3); hexane/ethyl acetate (99:2), Rf deoxyartemisinin: 0.61 between 1000–1700 mL, [hexane/ethyl acetate (99:3), [αD
20]: -149.8° (c 0.045 g/mL, CHCl3); Rf artemisinin: 0.57 between 1750–2550 mL, [αD
20]: + 80.93° (c 0.025 g/mL, CHCl3).
Fractions were monitored by thin layer chromatography, eluent hexane/dichloromethane/methanol (20:79:1), detection anisaldehyde reagent. The physical and spectral data (mass, 1H-NMR, 13C-NMR) of compounds isolated were consistent with those of artemisinin, deoxyartemisinin and dihydro-epideoxyarteannuin-B reported previously.
Development and validation of analytical method by HPLC-IR for evaluation of artemisinin in A. annua L. was accordingly to method previously developed and validated by our research group. The method was based on high performance liquid chromatography, using a CN column with mobile phase composed of methanol:H2O 50:50 (V/V). The results showed that the method presented linearity from 50 to 1500 μg/ml.
All samples were diluted in a vehicle made of 1% Tween80 (Sigma-Aldrich, U.S.A.) in 0.9% saline solution (NaCl diluted in distilled water), considering each different chemical characteristics (hydrophilic/lipophilic). The drugs pentobarbital (Cristália-Brazil), dexamethasone, carrageenan (Sigma-Aldrich, U.S.A.), morphine hydrochloride (Johnson & Johnson, Brazil), indomethacin (Sigma-Aldrich, U.S.A.), naloxone hydrochloride (Sigma-Aldrich, U.S.A.), and the reagents acetic acid and formaldehyde from Sigma-Aldrich, U.S.A. - were used.
Male Swiss mice 25–35 g and male Wistar rats 150–250 g body weights were kept at 25 ± 2°C in 12 h light–dark cycles (light phase started at 7:00 am) maintained (10 and 5 animals per cage, respectively) with water and food ad libitum, for at least 7 days prior to assays. Separate groups of animals were used for each test, and they were used only once in the experiments. Studies were carried out in accordance with the Current Guidelines for the Veterinary Care of Laboratory Animals - Joint Working Group on Veterinary Care, Finland and were performed under the consent and surveillance of the Institute of Biology Ethics Committee for Animal Research (protocol 3016–1), University of Campinas - Brazil. The number of animals and the intensity of the noxious stimuli were the minimum necessary to obtain reliable data.
Evaluation of acute toxicity
Groups of male Swiss mice (n = 3) and male Wistar rats (n = 3) intraperitoneally treated with Lac-FR in 30, 75, 100, 300 and 500 mg/kg of a single dose. After administration animals were observed during the first four hours and daily during 14 days. On the 15th day all animals were euthanized by cervical dislocation, followed by necropsy and macroscopic observation of the organs (OECD,; Botham,).
Evaluation of locomotor activity
The ambulatory behavior was assessed in an open-field as described previously[15, 16]. The apparatus consisted of a plastic box measuring 45 × 45 × 20 cm, with the floor divided into 9 equal squares (15 × 15 cm). The number of squares crossed with all paws (crossing) was counted in a 6-min session for each animal group (n = 6) and considered indicative for the quantification of the locomotor activity. For this purpose, mice were treated intraperitoneally (i.p.) with the Lac-FR (75, 100 and 300 mg/kg), pentobarbital (35 mg/kg), or vehicle (10 ml/kg) 30 min beforehand.
Groups of Swiss mice (n = 6) were treated with vehicle (10 ml/kg), or sesquiterpene lactones fraction (Lac-FR) using 100 and 300 mg/kg doses under intraperitoneal (i.p.) and oral (p.o.) routes. Writhings were induced by an i.p. injection of 0.8% acetic acid solution (10 ml/kg), 30-min after i.p. treatments and 1 hour after p.o. treatments. After this procedure, the numbers of writhings (abdominal constrictions) were cumulatively counted over 15 min, for nociception evaluation[14, 15]. Data represent the average of the total writhing observed per dose administrated.
The formalin test was carried out as described by Woolfe and Macdonald (1944) with few changes in the protocol. Formalin-induced pain behavior is biphasic: the initial acute phase (0 - 5 min, neurogenic pain) is followed by a relatively short quiescent period, which is then followed by a prolonged tonic response (25 - 40 min, inflammatory pain). Groups of mice (n = 6) were treated i.p. with: vehicle (10 ml/kg, negative control), the positive control for the neurogenic phase morphine (10 mg/kg, determined previously), the positive control for the inflammatory phase indomethacin (30 mg/kg, determined previously), and the dose–response of Lac-FR (30, 100 and 300 mg/kg (i.p.). After 30 min, animals were injected with 20 μl of a formalin solution (formaldehyde 1.2%, in PBS) into the plantar surface of the right hind paw. The total time spent by animal licking or biting the injected paw, an index of nociception, was recorded for the following 40 min.
Mechanical allodynia induced by Complete Freund’s Adjuvant (CFA)
The procedures were developed and standardized in our laboratory based on the method previously described by Villeti et al. (2003) with changes in protocol and data analysis. Different groups of rats, Wistar male (n = 6), were used during the whole experiment and inflammation was induced with a solution of CFA (1 mg/ml of heat killed Mycobacterium tuberculosis in 85% paraffin oil and 15% mannide monooleate) injected (0.1 ml) into the plantar surface of the right hind paw. The left hind paw received the same volume of saline solution (NaCl 0.9% diluted in distilled water) in order to equalize the sensibility of the animals caused by the injection. Mechanical allodynia was assessed using the Dynamic Plantar Anesthesiometer apparatus (Ugo Basile, mod 37450, Italy) which consisted of an elevated wire mesh platform to allow access to the ventral surface of the hind paws. A steel rod (diameter 0.5 mm) was pushed against the hind paw with ascending force (touch stimulator). The force ranged from 0 to 35 g over a 20-s period. When the animal withdrew the hind paw, the mechanical stimulus was automatically stopped, and the force applied by the animal to withdraw the paw was recorded to the nearest 0.1 g. An allodynia score was determined after four consecutive measurements using the touch stimulator sequentially on the left and right hind paw and calculated considering the formula below:
The basal score was measured before CFA injection on day 0, and the animals considered for testing were those with a mean value nearest to 1 (demonstrating no significant difference between both paw stimuli). After CFA injection, measurements were carried out considering two different phases, as follows: 4 h on day 0 (acute pain) and 24 h (sub-acute pain). Vehicle (10 ml/kg), Lac-FR were administered (30, 100 and 300 mg/kg, i.p.) 30-min prior to touch stimulation, in order to evaluate the possible anti-allodynic activity observed for each phase.
Carrageenan-induced mice paw edema
The anti-inflammatory properties were investigated by using the carrageenan-induced edema model. The procedures used for this study were similar to those described previously with some changes in the protocol and data analysis[20, 21]. Different groups of Swiss mice (n = 6) were treated with the negative control (vehicle), dexamethasone (1 mg/kg, i.p.-positive control) and Lac-FR (100 and 300 mg/kg, i.p.) 2 h before administration of carrageenan (25 μL of a 3% solution carrageenan) injected subcutaneously into the plantar region of the left hind paw. Paw measurements (Plethysmometer 7140 Ugo Basile) were taken at 2, 4, 6 and 24 hours. Results were expressed as variations in time of the paw edema (mL).
Antinociception was determined using a tail-flick test as described previously with few modifications on the protocol[22, 23]. In brief, the distal one third of the tail was immersed in a water bath maintained at 48 ± 0.5°C. Latency times until a tail-flick response (tail’s abrupt withdrawal) were recorded before (baseline) and at different time points (0.5, 1, 2, 4 and 24 hours) after drug treatment. Separate groups of six mice were treated with morphine (5 mg/kg, i.p.), Lac-FR (30, 100, and 300 mg/kg, i.p.), or an equal volume of vehicle (10 ml/kg). Aiming to evaluate the possible involvement of the opioid system, in another set of experiments (time points 0.5, 1, 1.5, 2, and 4 hours) groups of six mice were treated with morphine (5 mg/kg, i.p.), Lac-FR (100 mg/kg, i.p.), or an equal volume of vehicle, in the presence and absence of the opioid antagonist naloxone (5 mg/kg, s.c.). The antinociception response was presented as percent maximal possible effect (%MPE) as defined by percent maximal possible effect = 100% × (drug response time – basal response time)/(cut-off time – basal response time). A cut-off time of 15 s was applied to avoid tissue damage.
All results were submitted to one way analysis of variance (ANOVA) with repeated measurements, p ≤ 0.05 was considered the critical level for evaluating significant difference between the control and treated groups, followed by Tukey’s test (one-way ANOVA) or Bonferroni’s test (two-way ANOVA). Prisma® software was used to produce graphs.