Assessment of Hyoscyamus niger seeds alcoholic extract effects on acute and chronic pain in male NMRI rats

ajority of stimulations which cause injury to body tissues are with pain sensation. Pain is necessary to aware the body from harmful stimulations. Also pain is an important symptom of many diseases and with respect to its quality and localization of pain sensation some of the diseases can be diagnosed and treated consequently (1). Pain as one of the clinical complications of patients and also variety of causes triggering it, demands different treatment methods had led to a wide and complex field of research (2). Despite different palliative and curative methods which are used widely, still researches are prone to find new and better management routes of this physiological phenomenon. In spite of modern pharmacy and wide range of chemical drugs for pain control, patients

still face unsuccessful therapy and adverse effects of these agents (3). Using herbal plants their cost effectiveness character and also less side effects and more availability are recommended (4).

Hyoscyamus niger is one of the herbal plants which is introduced as analgesic (5, 6). Investigations performed by researches showed that this plant contain anticholinergic alkaloids tropane (Atropin, scopolamine and hyoscine) (7, 8). Also available reports admit analgesic effects of anticholinergic compounds (9). According to recent studies which showed strong analgesic effects of anticholinergic compounds and presence of these in Hyoscyamus niger in a large quantity, it is proposed that this plant especially its seeds which is a great source of these compounds be able to show some analgesic effects. Therefore in this paper it is tried to evaluate interaperitoneal injection and oral prescription analgesic effects of alcoholic extract of Hyoscyamus niger and comparing it with a known analgesic drug.

2. Materials and Methods

2.1. Animals

In this paper, NMRI rats weighted 300-350 g were investigated in Medical faculty under normal circumstances (temperature of 2±21 ˚C, light and darkness rhythm of 12 hours and 30-40% of humidity). 52 rats were divided into cages containing 4 rats. Standard food and water was given without any limitations.

2.2. Extract preparation

In order to prepare alcoholic extract of Hyoscyamus niger, after preparing seeds and excluding Impurities, 500 g of seeds was ground. Then was hold in ethanol 96% with a ratio of 1 to 5 in laboratory for 48 hours. After by using small and big filters extract filtrations of mixed was performed. Filtered liquid was concentrated in bain marie with 65 ˚C. Finally prepared honey like extract was mixed with normal saline to make different concentrations based on mg/kg of rats body weight.

2.3. Preparation of oral form of Hyoscyamus niger

In order to prepare special diet, some standard food was powdered and mixed with pure water. Hyoscyamus niger seeds were ground and then mixed with standard food. Mixed formula changed into available form for rats using special instruments for producing foods. Special and standard diet and water was accessible for rats without any limitation. After 2 weeks rats were ready for performing pain tests.

2.4. Materials and Methods

At first rats were divided into groups each includes 6 rats as follows: group 1 control. 2, 3 and 4, receiving different doses of Hyoscyamus niger alcoholic extract intraperitoneally Group 5 receiving Hyoscyamus niger seeds orally and group 6 receiving the analgesic sodium salicylate drug. Then each rat was weighted. 16 rats were separated into 2 groups. One with standard diet and the other receiving food containing Hyoscyamus niger. Proportion of Hyoscyamus niger to standard food was 1 to 14 or 6/25.

Because this plant extract has been examined for the first time, we needed to design dose-response curve. In this research three groups of 8 to 10 rats for injection of Hyoscyamus niger alcoholic extract were selected. 500, 1000 and 2000 mg/kg of body weight concentrations for intraperitoneal injection were used. At first extract was injected then after 30minutes formalin test was performed.

A group of 8 rats were selected for comparison of Hyoscyamus niger analgesic effects with standard sodium salicylate. 300mg/kg of sodium salicylate based on rats body weight was diluted with 0.5 ml of normal saline and was injected intraperitoneally 30 minutes before formalin test.

2.5. Formalin test

In this test, 50 µl of formalin solution (2.5%) was used for inducing pain. Formalin with the mentioned dose was injected subcutaneously in the plantar surface of back foot. Rats were put in plexyglass which has a mirror beneath with a 45 degree in order to observe inside in detail. Pain rating was calculated as follow: if the animal put its injected foot on the surface it gets zero, if hesitates to put its foot on the surface score 1, if raises its foot score 2 and finally if because of pain licks or bites its foot score 3. Formalin test is performed approximately in one hour. With measuring animal pain every 15 seconds and evaluating average of pain in each minute a score between zero to three is recorded for each minute. Test is completed in 60 minutes with 60 averages documented. It worth noting that control and test groups rats were adapted with the plexyglass 15 minutes before formalin test is done.

2.6. Statistical analysis

Results were calculated based on their averages and standard deviations. For comparing groups, averages together ANOVA was used and for comparing between two groups, Tukey test was employed.

3. Results

3.1. Effect of oral prescription of Hyoscyamus niger seeds on pain threshold induced by formalin test

Statistical calculations indicate that in acute pain (according to figure 1 and table 1) there were no meaningful differences between control and test groups. Furthermore as shown in table 1 and 2, average of chronic pain induced by formalin test in test group which Hyoscyamus niger has been prescribed, was 1.86 ±0.03 lower than 2.12 ±0.03 in control group (p< 0.001).

Figure.1. Curves comparing acute and chronic pain in control and treatment with Hyoscyamus niger performed with formalin test (n=10 in each group).

3.2. Effect of alcoholic extract injection in pain threshold induced with formalin

Findings from formalin test first stage (acute) based on figure 2 and figure 3 shows that aforementioned doses were able to reduce acute pain induced by formalin. Statistical analyses indicate that average amounts of acute pain induced by formalin test in treated group receiving Hyoscyamus niger with the dose of 2000 mg/kg was 0.24 ±0.13 which was meaningfully lower than control group 1.4 ±0.13 (p<0.001) (Tables 1 and 2). Moreover lower injection doses (500 and 1000 mg/kg) resulted in same consequences.

Figure 2. Curves comparing acute and chronic pain in control and treated injected with Hyoscyamus niger extract groups (n=10 for each group).

Figure.3. Comparison of acute pain in experiment groups. *** indicates significant difference with control group (p<0.001)

3.3. Effect of intraperitoneal injection of sodium salicylate on pain threshold induced by formalin test

In the performed investigation, regarding table 1, averages of acute and chronic pain in the group treated with sodium salicylate were 0.19 ±0.92 and 1.06 ±0.05. Statistical analyses showed meaningful difference (p< 0.001) between sodium salicylate received and control groups in chronic phase of pain induced with formalin test (table 3). Whereas no meaningful difference was seen between two mentioned groups in acute phase of formalin test (table.2)

Comparison between analgesic effects of Hyoscyamus niger and sodium salicylate:

Comparison between acute and chronic pain reduction treated with Hyoscyamus niger and sodium salicylate and statistical analyses revealed that, not only parenteral Hyoscyamus niger, affects on acute and chronic phases of pain induced by formalin test, but also has more significant and meaningful effects in comparison with sodium salicylate which only affects on chronic phase.

Generally as seen in figure 3, in acute pain induced by formalin test, only i.p injection form of Hyoscyamus niger is effective in increasing pain threshold and sodium salicylate doesn’t have any effect on acute pain control.

Finally as presented in figure. 4, about chronic pain induced by formalin test except henbane injection, its oral prescription and also sodium salicylate lead to a significant increase in pain threshold.

4. Discussion

Pain as a clinical complication of patients and variety of inducing reasons raises attention in order to more new and different methods of treatment, which has opened a wide filed of research as well (2).

Figure.4. Comparison of chronic pain in experiment groups. *** indicates significant difference with control group (p <0.001).