@2024 Afarand., IRAN

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ISSN: 2252-0805    The Horizon of Medical Sciences 2016;22(2):151-158

Background
… [1-5] White mulberry (Morus alba L.) has antispasmodic and anti-rheumatic effects and is used to treat joint pain [6]. … [7-13] Several studies have shown antioxidant effects of mulberry leaves [14, 15].
Previous Studies
Flavonoids in the white mulberry can be effective on improving oxidative stress in diabetic rats [16]. White mulberry leaf extract has nitric oxide and prostaglandin E2 inhibitory effects [17]. … [18] Red and black mulberry are effective on improving oxidative stress, and they have antioxidant role [19]. … [20]
Aim(s)
This study aimed to evaluate the analgesic effect of alcoholic extract of Morus alba leaves on rats.
Research type
This is an experimental study.
Research society, place and time
Male Wistar rats weighing 200 to 250 g were studied.
Sampling method and number
32 rats were studied.
Used devices&materials
The mice were randomly divided into two groups: control group and a group receiving mulberry leaf extract in 100, 200, and 400 mg/ kg doses of body weight (4 groups, each including 8 animals). Alcoholic extract of mulberry leaves was administrated by gavage 4 weeks, and every day at 10 a.m. During this time, in order to consider gavage stress, control group was treated with distilled water. 20 kg of white mulberry leaves, from the seed variety, was prepared from Kashan. The extraction was carried out by percolation with 70% of ethanol [21]. In determining the total amount of flavonoids based on quercetin, the absorbance of solution was measured against the control solution at 415 nm wavelength. This experiment was repeated three times for each concentration and the mean absorption of any concentration was used for the calibration curve. In determining the amount of total phenolic compounds based on gallic acid, absorbance of solution was recorded in 760 nm wavelength against the control solution. 4 weeks after gavage onset, to evaluate the analgesic effect of alcoholic extract of Morus alba, the following tests were used. In algesia hyper thermal test, using a thermal radiation (Ugo Bassil; Italy), animal heat tolerance was evaluated by radiation of infrared radiation on healthy animal. This test was repeated for each animal three times and 5 to 10 minutes each time and the cut-off point for the test was 22 seconds [22]. In the tail immersion test, the animal was placed into the rectum; and after adjustment, their tail was placed in 49 ° C water. The duration until the time that the mouse moves its tail to the top was recorded. This test was conducted 4 times for each animal and it was repeated in the zero, second, fourth and sixth minutes. If the animal did not move its tail within 30 seconds, to avoid damage to its tail, the animal was dismissed until two minutes later [23]. Writhing test was used to assess visceral pain. Abdominal cramps or pulling at least one of the hind limbs was considered as a wright [24]. Dubuisson and Dennis method was used for formalin test [25]. Behaviors due to pain were scored on a contract basis as follows. Zero: The animal placed his injected food on the floor and does not show any sign of pain. One: The animal placed his injected food on the ground with its toes with caution, but does not put its weight on it. Two: the animal raised its injected foot completely, and put it on the ground without any contact near its body. Three: in addition to raising its injected foot, the animal starts to shake, lick or biting the foot. After collecting data, using Kolmogorov-Smirnov test, the normality of data was confirmed. The data was analyzed using one-way ANOVA. Tukey post-hoc test was used to compare the groups in pair. All tests were performed by SPSS 16 software.
Findings by Text
The mean duration of delay in feet withdrawal was significantly increased in the group received the extract compared to the control group (p<0.0001). In other words, receiving alcoholic extract of mulberry leaves improved the heat-induced pain. The mean delay time in withdrawal of feet was 9.5 ± 0.42 seconds for control group, and receiving extract with 100, 200 and 400 mg doses increased the time up to 15.33 ± 0.86, 17.75 ± 0.39 and 16.17 ± 0.61 seconds respectively (p<0.0001). The mean delay time in withdrawal of tail was significant between the studied groups (p<0.0001). The difference between the withdrawal of tail in the group received 100 mg extract (p<0.05), and the groups received 200 and 400 mg extract (p<0.001) was significant compared to control group (Diagram 1). Elongation rate and writhing in mice receiving the extract of mulberry leaves decreased (p<0.0001). In fact, the mice receiving the extract were feeling less pain. The reduction of the percentage of shrinkage compared to control group was 38.9% (p<0.05) for the group received the extract at 100 mg dose, and it was 73.2% and 62.2% for the groups receiving 200 and 400 mg of the extract, respectively (p<0.0001). The maximum analgesic effect was observed at 200 mg extract dose (Diagram 2). Receiving alcoholic extract of mulberry leaves significantly reduced formalin-induced pain in both acute (zero to 15 minutes) and chronic (20 to 50 minutes) phases of the test (p<0.0001). In the first phase of the formalin test, behaviors indicative of pain in the groups receiving 100, 200, and 400 mg of extract was significantly reduced compared to control group (p<0.05). However, no significant difference was observed in the analgesic effect of different doses of the extract (p >0.05). In the chronic phase, also, the consumption of the extract reduced the pain in all groups receiving 100, 200 and 400 mg of the extract compared to control group (p <0.01) although the difference between three doses of the extract was not significant ((p >0.05; Diagram 3).
Main comparison to the similar studies
Alcoholic extract of white mulberry leaves can reduce the intensity of the pain in algesia hyper thermal, immersion of the tail, writhing and formalin. Antioxidant effects of mulberry leaves have been shown in some studies [8, 16]. Hydro-alcoholic extract of white mulberry leaves can prevent the inflammation induced by injection of carrageenan [26]. Blackberry plant with the scientific name of Morus nigra L. has significant anti-inflammatory and analgesic effects. In a study, it has been stated that 300 mg blackberry leaf extract functions better in reducing the pain induced by writhing and hot-plate tests compared to morphine (10mg/kg) and indomethacin (5mg/kg) [27]. … [28-31] Flavonoids with their effects on N-methyl-D-aspartate (NMDA) receptor decreases the intracellular calcium followed by the inhibition of calcium dependent enzyme activity, nitric oxide synthase and phospholipase A2 [32]. White mulberry leaves can inhibit nitric oxide and prostaglandins E [17]. … [33] The studies on the plant containing flavonoids and phenolic compounds have shown that these compounds can have significant anti-inflammatory and analgesic effect [34, 35]. … [36] Saffron in the plantar test does not have any significant effect on pain intensity and it does not reduce pain [37]. … [38-40] Analgesic effects of chlorogenic acid, rutin and quercetin have been proved in various studies [41, 42]. … [43-46]
Suggestions
Using drugs that inhibit pain pathways, the analgesic effect of alcoholic extract of mulberry leaf should be studied.
Limitations
Of the limitations of this study, the use of a standard chemical analgesic drug such as morphine or non-steroidal anti-inflammatory drugs can be mentioned.
Conclusions
Oral consumption of alcoholic extract of Morus alba L. leaves reduces the intensity of pain in the rats. And the analgesic effect is more effective at 200 mg /kg dose of the extract.
Acknowledgments
The Research Deputy of Kashan University of Medical Sciences is appreciated.
Conflict of interest
Non-declared
Ethical Permissions
All moral instructions approved by Ethics Committee of Research Deputy of Kashan University of Medical Sciences were observed.
Funding Sources
This article is a part of research project No. 93171 approved by Research Department of Kashan University of Medical Sciences.

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