Effects of the resin and smoke condensate of Cannabis sativa on the oestrous cycle of the rat*

Sections

ABSTRACT
Introduction
Materials and methods
Results
Discussion
Conclusions

Details

Author: Asdrúbal LARES, Yandira OCHOA, Alba BOLAÑOS, Napoleón APONTE, Martha MONTENEGRO
Pages: 55 to 61
Creation Date: 1981/01/01

Effects of the resin and smoke condensate of Cannabis sativa on the oestrous cycle of the rat*

Asdrúbal LARES
Yandira OCHOA
Alba BOLAÑOS
Napoleón APONTE
Martha MONTENEGRO
Central University of Venezuela, Department of Pharmacy, Chair of Toxicology, Caracas, Venezuela

ABSTRACT

This study is concerned with variations in the oestrous cycle of the Sprague-Dawley rat following the intraperitoneal administration of maize oil solutions of Cannabis sativa resin and smoke condensate in doses of 10 and 20 mg/kg. Oestrus was shortened with doses of both the resin and the smoke condensate, whereas dioestrus was lengthened with the 20-mg/kg dose of the resin and both the 10- and the 20-mg/kg doses of the smoke condensate. Also observed was a lengthening of postoestrus following the administration of 20 mg/kg of either the resin or the smoke condensate.

Introduction

It will probably require 20 or 30 more years before the human pathology caused by the chronic consumption of Cannabis sativa (marijuana) can be described. This is perhaps not surprising when one recalls that 60 years of research were required before the pathological consequences of chronic tobacco-smoking were understood [ 12] . Major studies have been carried out in the search for more complete and reliable information on the possible harmful effects of cannabis. This effort has involved animal and human experimentation (the latter largely of a voluntary nature) using the most important active principle, delta-9-tetrahydrocannabinol in pure form obtained from extracts of the plant or from the smoke produced by burning it. Known effects of this substance include its adrenocortical effects in rats [ 13] , its long-term effect on the testicular function in rats [ 2] , its effect on intestinal disaccharides in rats [ 14] , its acute and chronic effect on hypothalamo-neurohypophysial-neurosecretory activity in rats [ 1] , its effect in modifying the serum concentration of the luteinizing hormone and of prolactin in rats [ 5] , its effect on inhibition of immunity and cellular replication [ 10] , its effect in terms of mytosis anomalies and DNA metabolism [ 9] , its effect in terms of the in vitro inhibition of lymphocytic trans formation [ 8] , and its effect in the form of chromosome anomalies in heavy smokers of hashish [ 11] . Finally-and this is the justification of the present study - we know of the effect caused by the administration of crude extract on the oestrous cycle of rats [ 6] . These results are of very great interest because of the possibility of modification or variation in the ovarian hormones and its correlation with the secretion of gonadotropic hormones through the hypo-physial hypothalamus system [ 3] . Our purpose has been to contribute to the study of this problem in the form of an evaluation using two different doses of the resin extracted from the Cannabis sativa plant and the condensate of the smoke produced by burning it.

* Work subsidized by the Council for the Development of Sciences and the Humanities of the Central University of Venezuela.

Materials and methods

The method followed in this study may be summarized as follows:

  1. To obtain the marijuana resin, the dry flowery spikes of the plant were macerated in petroleum ether. Following the evaporation of the solvent in a water bath, the resin was obtained in a pasty form, which was then used in a maize oil substrate to prepare an oily solution with a concentration of 20 mg/ml;

  2. To obtain the smoke condensate, a quantity of the plant was burned using a device whereby the smoke was drawn towards a Kitasato vessel connected to a vacuum, in which the smoke was caused to bubble in ethyl alcohol. Following the evaporation of the alcohol in a water bath, a residue similar to the resin residue described above was obtained and used to prepare an oily solution in maize oil with a concentration of 20 mg/ml;

  3. The rats used in the experiments were young virgin females of the Sprague-Dawley variety weighing between 225 and 260 g. Nine were used for each dose and 24 for the control group;

  4. ( d) The test doses were 10 mg/kg and 20 mg/kg for both forms of the drug;

  5. ( e) The control animals received only the maize oil solvent;

  6. ( f) The intraperitoneal method was used for the administration of the drug;

  7. The animals underwent a one-month period of adaptation to the test conditions, after which the length of the phases of their oestrous cycle was checked for a period of two weeks (excluding Saturdays and Sundays). During the following two weeks they received the test doses daily (except Saturdays and Sundays) and their oestrous cycle was observed. Finally, the oestrous cycle was observed for an additional two weeks without administration of the drug;

  8. ( h) The technique used to monitor the oestrous cycle was the direct vaginal smear [ [ 4] ].

Results

The results are shown in tables 1 - 9. The values indicated in these tables were processed statistically using the binomial distribution test for proportions and percentages 7.

Table 1

Modification of the oestrous cycle of rats as a result of the administration of the resin of the Cannabis sativa plant Dose: 10 mg/kg

 

Relative length of phase

Phase

Prior to treatment (%)

During treatment (%)

P

Following treatment (%)

P

Pro-oestrus
8.04 13.09
>0.05
11.66
>0.05
Oestrus
63.21 53.57
0.05
61.06
>0.05
Postoestrus
20.68 20.23
>0.05
14.28
0.05
Dioestrus
8.04 13.09
>0.05
12.98
0.05

Observation: There is a significant shortening, during treatment, of oestrus phase, which returns to normal after the treatment ends.

Table 2

Modification of the oestrous cycle of rats as a result of the administration of the resin of the Cannabis sativa plant Dose: 20 mg/kg

 

Relative length of phase

Phase

Prior to treatment (%)

During treatment (%)

P

Following treatment (%)

P

Pro-oestrus
18.60 15.11
0.05
14.63
>0.05
Oestrus
54.65 39.53
0.05
59.77
>0.05
Postoestrus
18.60 26.74
0.05
13.41
>0.05
Dioestrus
8.13 18.60
0.05
12.19
0.05

Observation: There is a significant shortening, during treatment of pro-oestrus and oestrus, as well as a significant lengthening of postoestrus and dioestrus. There is recovery in the case of the first three phases mentioned, but not in the case of dioestrus, where the effect persists.

Table 3

Modification of the oestrous cycle of rats as a result of the administration of the smoke condensate of the Cannabis sativa plant Dose: 10 mg/kg

 

Relative length of phase

Phase

Prior to treatment (%)

During treatment (%)

P

Following treatment (%)

P

Pro-oestrus
8.88 7.77
>0.05
11.53
0.05
Oestrus
55.55 45.55
0.05
52.60
0.05
Postoestrus
17.77 21.11
>0.05
16.66
0.05
Dioestrus
17.77 25.55
0.05
19.20
0.05

Observation: There is a lengthening of dioestrus and a significant shortening of oestrus during and after treatment.

Table 4

Modification of the oestrous cycle of rats as a result of the administration of the smoke condensate of the Cannabis sativa plant Dose: 20 mg/kg

 

Relative length of phase

Phase

Prior to treatment (%)

During treatment (%)

P

Following treatment (%)

P

Pro-oestrus
8.23 8.88
>0.05
9.09
>0.05
Oestrus
60.00 43.33
0.05
45.55
>0.05
Postoestrus
15.29 26.66
0.05
18.18
>0.05
Dioestrus
16.47 21.13
0.05
18.18
>0.05

Observation: There is a significant shortening of oestrus during treatment and a lengthening of postoestrus and dioestrus, with a tendency to recovery following treatment.

Table

Modification of the oestrous cycle of rats as a result of the administration of the maize oil solvent Control group

 

Relative length of phase

Phase

Prior to treatment (%)

During treatment (%)

P

Following treatment (%)

P

Pro-oestrus
12.86 18.18
>0.05
11.68
>0.05
Oestrus
45.02 47.27
>0.05
50.00
>0.05
Postoestrus
25.73 21.21
0.05
21.18
0.05
Dioestrus
16.37 13.33
0.05
17.14
>0.05

Observation: There is a significant shortening of postoestrus and dioestrus during treatment.

Table 6

Comparison, against the control group, of the effects of two different doses of the resin and smoke condensate during pro-oestrus

 

Relative length of phase

Dose (mg/kg)

Prior to treatment (%)

During treatment (%)

P

Following treatment (%)

P

Control group
12.86 18.18   11.68  
Resin (10)
8.04 13.09
0.05
11.68  
Resin (20)
18.60 15.11
0.05
14.63
0.05
Smoke condensate (10)
8.88 7.77
0.05
11.53
>0.05
Smoke condensate (20)
8.23 8.88
0.05
9.09
>0.05

Observation: During treatment there is a significant shortening of this phase for both doses, whether of resin or smoke condensate.

Table 7

Comparison, against the control group, of the effects of two different doses of the resin and smoke condensate during oestrus

 

Relative length of phase

Dose (mg/kg)

Prior to treatment (%)

During treatment (%)

P

Following treatment (%)

P

Control group
45.02 47.27   50.00  
Resin (10)
63.21 53.57
0.05
61.06
0.05
Resin (20)
54.65 39.53
0.05
59.77
0.05
Smoke condensate (10)
55.55 45.55
>0.05
52.60
>0.05
Smoke condensate (20)
60.00 43.33
0.05
54.55
0.05

Observation: There is a significant shortening of this phase in the case of the 20-mg/kg doses of resin and smoke condensate during treatment. This effect persists beyond the termination of treatment in the case of the smoke condensate, but in the case of the resin the phase tends to lengthen.

Table 8

Comparison, against the control group, of the effects of two different doses of the resin and smoke condensate during postoestrus

 

Relative length of phase

Dose (mg/kg)

Prior to treatment (%)

During treatment (%)

P

Following treatment (%)

P

Control group
25.73 21.21   21.18  
Resin (10)
20.68 20.25
>0.05
14.28
>0.05
Resin (20)
18.60 26.74
0.05
13.41
0.05
Smoke condensate (10)
17.77 21.11
>0.05
16.66
>0.05
Smoke condensate (20)
15.29 26.66
0.05
18.18
>0.05

Observation: There is a lengthening of this phase during treatment in the ease of the 20-mg/kg dose of both the resin and the smoke condensate. Following treatment, a shortening is observed in the case of the 20-mg/kg resin dose.

Table 9

Comparison, against the control group, of the effects of two different doses of the resin and smoke condensate during dioestrus

 

Relative length of phase

Dose (mg/kg)

Prior to treatment (%)

During treatment (%)

P

Following treatment (%)

P

Control group
16.37 13.33   17.14  
Resin (10)
8.04 13.09
>0.05
12.98
0.05
Resin (20)
8.13 18.60
0.05
12.19
0.05
Smoke condensate (10)
17.77 25.55
0.05
19.20
>0.05
Smoke condensate (20)
16.47 21.11
0.05
18.18
0.05

Observation: There is a significant lengthening of this phase for both doses of the smoke condensate and for the 20-mg/kg dose of the resin, an effect which persists beyond the termination of the treatment.

Discussion

The results of this experiment may be briefly stated as follows:

  1. A reversible shortening of oestrus is observed with doses of 10 and 20 mg/kg of the resin (tables 1 and 2); the shortening is irreversible with doses of 10 and 20 mg of the smoke condensate (tables 3 and 4). The reason for the irreversibility may be that a cumulative effect is produced as a result of the presence in the smoke condensate of certain compounds not found in the resin, since new active compounds are generated in the pyrolysis of cannabis;

  2. Evidently, the effect on the duration of dioestrus is the most intense, as it is seen to be irreversible at both dose levels (tables 1 and 4). It may be that the duration of the experiment was too short to permit observation of the normal length of this phase;

  3. The duration of pro-oestrus is affected by only the 20-mg/kg dose of resin, when there is a significant but reversible shortening (table 2). It may be that the equilibrium compensation of this phase coupled with that of oestrus serves to counteract the lengthening of postoestrus and dioestrus produced by this dose;

  4. The shortening observed in postoestrus and dioestrus during treatment with the control group (table 5), which is irreversible for postoestrus and reversible for dioestrus, may be due to equilibrium compensation by the reversible lengthening of pro-oestrus and the irreversible lengthening of oestrus. It should be remembered, however, that postoestrus and pro-oestrus are intermediate phases;

  5. The comparison of the duration of pro-oestrus and oestrus in the control group and that of the groups receiving 10-mg/kg and 20-mg/kg doses of resin and smoke condensate (tables 6 and 7) reveals a reversible shortening of pro-oestrus following administration of all 20-mg/kg doses of either compound, possibly due to the activity caused by the effective concentration of the active principles;

  6. The comparison of the duration of postoestrus and dioestrus in the control group with that of the groups receiving 10-mg/kg and 20-mg/kg doses of resin and smoke condensate (tables 8 and 9) reveals a reversible lengthening of both phases only following the 20-mg/kg doses both of the resin and of the smoke condensate. The explanation may be the same as in the preceding case.

Conclusions

In a study of the oestrous cycle of rats using the vaginal smear inspection method, with a preliminary period of adaptation and a two-week experimental period with and without treatment, it was possible to observe- both independently with the animals given 10-mg/kg and 20-mg/kg doses of Cannabis sativa resin and smoke condensate, and in comparison with the control group-a disturbance in the oestrous cycle as a result of the administration both of the resin and of the smoke condensate. This disturbance was pronounced in the case of the 20-mg/kg dose and more significant in the case of the smoke condensate. This can probably be explained as an effect of the dose and the presence in the smoke condensate of constituents not found in the resin. Basically, this disturbance takes the form of a shortening of oestrus and a lengthening of dioestrus, which begin, respectively, with pro-oestrus and postoestrus. This observation appears to corroborate the idea of hypothalamic-hypophysial activity with effects on hormonal-ovarian equilibrium, as indicated by some authors for the active constituents of Cannabis sativa 3. This effect may be interpreted as signalling a tendency towards diminished fertility of the animal concerned.

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