Studies on Analgesic Effect of Eezapein™

 

Abstract:

 

Eezapein efficacy was determined by the following assays: heat sensitivity, GAA tolerance, and formalin tolerance.  Study results indicate Eezapein™ establishes analgesic effects by significantly increasing pain thresholds in test subjects.  Eezapein™ is also shown to be non addictive.

 

I. Experiment materials

 

1.  Medicines

Eezapein™, provided by LIFEnhance, Inc. is a super-concentrated extract, batch number 20020928. One gram of extract is equal to 6g crude medicine. It is diluted with distilled water to the required concentration for use.

Aspirin is made by Hubei Zhongtian Ai Baike Medical Inc..

Morphine hydrochloride injection, 1 ml with 10mg morphine, is made by the 1st Pharmaceutical Factory of Shenyang, with batch number of 01111.

Naloxone hydrochloride injection, 1ml with 0.4mg naloxone hydrochloride, is made by Beijing Sihuan Medical Technology Inc., with batch number 0207032.

2.  Animals

Kunming mice, ♀♂(female and male) weighing 17-20g, were provided by Hubei Experiment Animal Center. The quality certificate reference number is 19-082.  In the initial screen for heat sensitivity mice, those that jumped or licked their hind legs in less than 5 seconds are not used due to hypersensitivity.  Mice with reaction times greater than 30 seconds were also not used due to lack of sensitivity.

 

3. Equipment: Ninghai Baishi Electronic Medical Equipment Factory GJ-8402 type hot-plate dolorimeter.

 

II.        Methods and Results:

 

1.   Heat sensitivity [1]

When mice are placed on a 55℃ (±0.5℃) hot plate, each pain threshold is the amount of time taken for the mouse to first start licking its hind leg.  Fifty qualified mice are divided into 5 groups at random. Group one is the control group and fed distilled water (ig) once every morning and once every afternoon; Group two consists of the Aspirin control group, and was fed 0.42g/kg of aspirin (ig) once every morning.  Groups three, four and five received Eezapein™ in large (8g/kg), medium (8g/kg) and small (2g/kg) dosages (ig) once every morning and once every afternoon. The heat sensitivity assay was conducted one hour after the last dose. The pain threshold is recorded below with appropriate statistical analysis.  The result is shown in table 1.

 

Tabl 1.  The effect of Eezapein™ on the pain of mice (x±s,n=10)

groups 　　 　　　 dosage（g/kg）　response time (s)

The control　　　　 　　――　　　　　　　  17±2.67 Aspirin Co.             0.42               24.6±3.53*** Large dosage　　        8                 26.1±9.96* Medium dosage          4                 27.9±8.01***   Small dosage            2                  23±5.18**

Compared with the control group：*P<0.05,**P<0.01,***P<0.001

 

Table 1 results illustrate Eezapein™ at three different dosages can delay heat sensitivity response time.  Delays in response time indicate tolerance due to heightened pain thresholds.

 

2.  Glacial Acetic Acid (GAA) tolerance^［2］

60 Kunming mice 18-20g, are divided into 6 groups. The negative control group received distilled water 20ml/kg while the positive control group received 0.42g/kg aspirin, and the test groups received large (8g/kg), medium (8g/kg) and small (2g/kg) doses of Eezapein™.  The final test group received 8g/kg Eezapein once a day.

The Eezapein groups and the negative control group were fed Eezapein or water once every morning and once every afternoon for three consecutive days.  The positive control group was given aspirin once every morning for three consecutive days. One hour after the last dosage, every mouse is fed (ip) 0.6% GAA, 0.1ml/10g. The pain thresholds are determined to be the number of times each mouse rolls over 360° during a twenty minute period. The results are shown in table 2.

Table 2. The effect of Eezapein™ on GAA tolerance in mice(x±s ,n=10)

groups          dosage(g/kg)    num of body turns    control rate(%) negative control           ----              43.5±12.97           ----- Aspirin Co               0.42             2.5±1.9***            94.25  Large dosage             8                14.9±4.46***          65.75 Medium dosage           4                12.9±6.38***          70.34 Small dosage             2                15.5±8.32***          64.37 One dosage              8                14.60±5.78***         66.44

Table 2 shows that both Aspirin and Eezapein™ can decrease the number of rolls indicating higher pain tolerance levels. The decrease in number of rolls for all Eezapein doses is more than 50% compared to the negative control group demonstrating statistically significant analgesic effects.

 

3. Formalin experiment ^[3]

50 Kunming male♂ mice 18-20g, are divided into 5 groups. The negative control group received distilled water 20ml/kg while the positive control group received 0.42g/kg aspirin, and test groups received large, medium and small doses of Eezapein™ (8, 4, and 2g/kg respectively). Mice were fed (ig) Eezapein or distilled water once a day for three consecutive days.  Aspirin was given (ig) once on the day of experiment. One hour after the last dosage, 3% formalin is injected into the right fore-foot with a micro injector, 20μl each. The mouse is then placed in a flat glass container to observe its reactions. Pain tolerance levels are determined based on the following point system: 3 points for licking, biting or shaking foot; 2 points for lifting foot; 1 point for lameness or inability for foot to support any weight; 0 point for normal walking. Time points were taken at 0, 10, 20, 40 and 60 minutes after the medicine is given, whereby the reactions were observed for 2minutes each time.  Pain index = tolerance level x time point (the maximum value is 3×60=180). The differences among groups are compared .The result is shown in table 3.

groups　  dosage　　　　　　      tolerance level（／min）

The control    -----   112.17±42.39    91.90±14..33     81.45±28..82    46..65±18.77     38.45±18.84

Aspirin Co   0.42    97.6±42.11    50.00±34.69**    27.6±9.77***    18.2±11.66***    8.65±4.76***

Large dosage  8     102.93±32.86    53..55±31.62**   27.75±16.86***   23..3±10.27**   2.5±2.37***

Medium dosage 4    96..33±31.21      63..3±31..76*   33.25±18.42***   35.85±21..35     6.2±3..92***

Compared with the control group ：*P<0.05,**P<0.01,***P<0.001

 

The results in table 3 show that Aspirin Co and Eezapein™ exert comparable analgesic effects 10 minutes after the medicines are given. The effect at 8g/kg Eezapein is greater than the aspirin positive control.

 

4.    Addiction Experiment----Mouse-jumping Experiment^{［4］、［5］}

30 ♂ Kunming mice 17-19g are divided into three groups at random; the negative control group (S.C normal saline 20ml/kg), positive control group (S.C morphine 10ml/kg), and Eezapein™ test groups (ig 20ml/kg). Each mouse is dosed 7 times for 2 days. The dosages of morphine are as follows: 2.5, 5, 10, 20, 30, 40, 50mg/kg; The Eezapein™ dosages are: 0.8, 1.6, 3.2, 6.4, 9.6, 12.8, 16g/kg. Two hours after the last dosage, 5mg/kg Naloxone is injected into the mice.  10 minutes after the injection mice are placed on a round table - 35cm in height and 30cm in diameter- to observe the reaction time and the number of jumps indicating withdrawal. The result is shown in table 4.

 

Table 4.The effect of Eezapein™ on mice

groups      dosage    #of mice   # of jumping mice  #of jumps(x±s)    (/kg)

normal saline　――　      10           0               0 morphine 　 157.5mg      10           10          12.30±7.39   Eezapein      50.4g         10           0               0