Comparative Evaluation of the Analgesic Effects of Pistacia atlantica Leaves, Gum, and Fruit Extracts in Rats

3.1. The Preparation of the Hydroalcoholic Extract of PA Leaves, Gum, and Fruit

Different parts of PA, including leaves, gum, and fruit, were acquired from a shop in Shiraz, Fars Province, south of Iran and authenticated by the taxonomists of School of Pharmacy, Shiraz University of Medical Sciences (SUMS), Iran, in March 2019 (fruit Pm-494, leaves Pm-495, gum Pm-496). To prepare the decoction, the fruit and leaves were first cleaned and then shaded for four days. Afterward, 700 gr of the plant powder was extracted with an ethanol-water mixture (80:20) at room temperature overnight. A rotary evaporator was used to completely remove the solvent at 50°C. Finally, the extracts were freeze-dried and kept at - 50°C until further use. The gum was extracted from the resin by cold maceration and hydrodistillation with ethanol (1:5). The combined hydroalcoholic extract was filtered through a filter paper and evaporated at 50°C. The gum extract was kept in dark containers and refrigerated until testing (7).

3.2. The Determination of Total Phenolic Content (TPC)

The reduction of Folin-Ciocalteu reagent by phenolic compounds was used to determine the TPC of the hydroalcoholic extract of PA leaves. Stock solutions of leaves extract at different concentrations were prepared in MeOH. First, 200 μL of Folin-Ciocalteu reagent was mixed with 40 μL of the extract sample; then, 3.2 mL of water was added. After around 10 min of incubation at room temperature, 600 μL of 0.25% sodium carbonate was added to the solution. The prepared solution was incubated for an additional 2 h at 37°C in the dark. The same procedure was repeated for gallic acid as the standard solution (15, 16). The calibration curve was plotted using gallic acid. The optical density was measured at 765 nm using a Bio-Rad microplate reader (Model 680, Bio-Rad). The concentration of TPC was expressed as mg of gallic acid equivalents per g of dry extract.

3.3. Antioxidant Assay

The antioxidant potential of the hydroalcoholic extract of PA leaves was determined using the Brand-Williams DPPH assay. Test solutions (270 μL), containing different concentrations of the plant extracts in methanol were mixed with 20 μL of a methanolic solution of DPPH (100 μM). This mixture was incubated at room temperature for 30 min. Then, absorbance was measured at 517 nm by a spectrophotometer. Each experiment was repeated three to five times (17, 18). Methanol was used as the blank for the microplate reader. The results of DPPH assay were expressed as IC50 value using Curve-Expert software (version 1.34).

3.4. Selection of Animals

Seventy-two adult male Sprague-Dawley rats (220 ± 20 g, 10 - 12 weeks) were collected from the laboratory animal center of Shiraz University of Medical Sciences (SUMS), Iran. The animals were randomly divided into 12 groups and maintained in a standard condition, with controlled temperature (22 ± 2°C) and 12-hour light and dark cycles. They had free access to food and water ad libitum. The ethical guidelines approved by the Ethics Committee of SUMS regarding the protection of animals used for experimental purposes were followed in all tests.

3.5. Experimental Design

After a 7-day adjustment period, the rats were randomly divided into 12 groups, each containing six rats, as follows: Group I: Control group, normal healthy rats; Group II: Negative control group, rats received vehicle ( Dimethyl sulfoxides (DMSO(, 250 μL/kg, IP); Group III: Positive control group, rats received the standard drug (APAP 100 mg/kg, IP); Groups IV-VI): Rats received 25, 50, and 100 mg/kg of the leaves extract(IP); Groups VII-IX): Rats received 25, 50, and 100 mg/kg of the gum extract (IP); and Groups X-XII: Rats received 25, 50, and 100 mg/kg of the fruit extract (IP).

3.6. Analgesic Tests

In this study, two types of painful stimuli (thermal and chemical) were used to induce pain in rats by direct stimulation of pain receptors. The tail-flick and formalin tests were used to measure responses to thermally- and chemically-induced pain, respectively. For each test, the rats were used individually.

3.7. Tail-flick Test

A tail-flick analgesia meter (Borj Sanat, Iran) was used to measure the pain threshold. The light intensity was adjusted to 70% of the maximum. A 15-second cut-off was imposed to avoid tail damage. The tail-flick latency time was measured in seconds. The positive control group received an IP injection of 100 mg/kg of paracetamol. The negative control group received 250 μL/kg of DMSO, IP. The animals in groups IV-XII received 25, 50, and 100 mg/kg of the hydroalcoholic extract of PA leaves, gum, and fruit, respectively. Analgesic activity was assessed by recording the reaction time. Following the administration of vehicle, paracetamol, and extracts, the reaction time was recorded at 30 and 60 min.

3.8. Formalin Test

To induce tonic pain in rats, 30 minutes after the injection of vehicle, paracetamol, and treatment extracts, 50 μL of 2.5% formalin (37% formaldehyde) in distilled water was injected subcutaneously into the right hind paw of the rats. It should be noted that the injectable solution did not penetrate deep into the tissue. Another important point to take into consideration is that intradermal injection cannot produce a long-lasting response. Analgesia was assessed using the formalin method (19). Pain behavior was scored as follows: 0 = the injected paw was not favored; 1 = the injected paw had little or no weight placed on it; 2 = the injected paw was elevated and not in contact with any surface; and 3 = the injected paw is licked or bitten. The animals’ responses were monitored for 1 h after formalin injection. The first phase was 5 minutes, and the second phase was 20 - 60 minutes after the formalin injection (19)

3.9. Statistical Analysis

Data was presented as mean ± standard deviation (SD). The data was analyzed using one-way analysis of variance (ANOVA) with Dunnett’s post hoc test. A P-value of less than 0.05 was considered statistically significant.

4.1. The Analgesic Effects of Leaves, Gum, and Fruit Extracts Based on the Tail-flick Test

The results of the tail-flick test showed that the high concentrations of PA leave extract (50 and 100 mg/kg) had analgesic properties, as revealed by the increase in the latency time compared with the control groups (P < 0.05). However, the leaves extract did not have any significant effect at a dose of 25 mg/kg (Figure 1). Moreover, the PA gum extract had analgesic effects at the doses of 100 and 200 mg/kg, as evidenced by the increase in the latency time compared with those of the control groups. However, the PA gum extract at the low doses of 25 and 50 mg/kg had no significant analgesic effect (Figure 2). The results also revealed that the PA fruit extract had no significant analgesic effect at any of the three doses because no significant difference was observed in the latency time compared with those of the control groups (Figure 3).

Figure 1.

The effects of the Pistacia atlantica leaves extract administration on the latency time in the tail-flick test. Data is expressed as Mean ± SD. APAP: Paracetamol, LE: Leaves extract. The asterisk (*) indicates a significant difference with the control group (P < 0.05).

Figure 2.

The effects of the Pistacia atlantica gum extract administration on the latency time in the tail-flick test. Data is expressed as Mean ± SD. APAP: Paracetamol, GE: Gum extract. The asterisk (*) indicates a significant difference with the control group (P < 0.05).

Figure 3.

The effects of the Pistacia atlantica fruit extract administration on the latency time in the tail-flick test. Data is expressed as Mean ± SD for the animals. APAP: Paracetamol, FE: Fruit extract. The asterisk (*) indicates a significant difference with the control group (P < 0.05).

4.2. The Analgesic Effects of Leaves, Gum, and Fruit Extracts Based on the Formalin Test

Following the injection of formalin in the control group, the average pain score reached its highest level during the first phase (first 5 minutes) and then decreased gradually during the second phase (15 to 45 minutes). The pain score between the two phases (5 to 15 minutes) is not usually reported because it does not affect the test results. The rats receiving paracetamol experienced a significant decrease in pain score in the first 5 minutes. The reduction in the pain score was even greater in the second phase, with pain levels below 0.5. The rats receiving 100 mg/kg of the leaves extract experienced a significant reduction in pain score in both the first and second phases compared with that of the control group. (P < 0.05) However, in rats receiving 50 mg/kg of leaves extract, the reduction in pain score in the first and second phases was not significantly different from that of the control group. The rats receiving 25 mg/kg of the leaves extract showed neither analgesic effect nor any reduction in pain in either the first or the second phase of the test (Figure 4).

Figure 4.

The results of the formalin test and the pain score in different animal groups treated with the hydroalcoholic extract of PA leaves. Data is given as Mean ± SD. APAP: Paracetamol, LE: Leave extract. The asterisk (*) indicates significantly lower scores compared with that of the control group (P < 0.05).

The gum extract at a dose of 100 mg/kg alleviated pain in both phases of the formalin test and had a stronger analgesic effect than did paracetamol. The difference was significant when the results were compared with that of the control group (P < 0.05). Our results showed that the gum extract had no significant effect at the doses of 25 and 50 mg/kg (Figure 5). No analgesic effect was observed at any of the doses of the fruit extract in either of the phases (Figure 6).

Figure 5.

The analgesic activities of the Pistacia atlantica gum extract in the formalin test in rats

Figure 6.

The effect of the Pistacia atlantica fruit extract on the pain score in the formalin test. Data are given as Mean ± SD. APAP: Paracetamol, FE: Fruit extract. The asterisk (*) indicates significant differences with the control animals (P < 0.05).

5.1. Conclusions

The results of this study suggest that the stronger analgesic effect observed for the PA leaves extract, compared with those observed for the extracts of other parts of PA, might be due to the higher concentration of analgesic components in the PA leaves.