1955 - ACTA UNIVERSITATIS PALACKIANAE OLOMUCENSIS - TOM. VI.

Prof. Jan Kabelik - HEMP AS A MEDICAMENT

Methods and results of the bacteriological experiments

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Methods and results of the bacteriological experiments

By Z. Krejci

I. - PRELIMINARY BACTERIOLOGICAL EXPERIMENTS WITH EXTRACTS FROM Cannabis sat. var. indica

1. Preparation of extract for the preliminary microbiological evaluation

It was clear from the beginning of our experimental work that the antibiotic effect of the drug must stand in close relationship with the content of the resin substances contained in the leaves and, particularly, in the shoots of the pistillate plant (fig. 3). The ground drug was extracted by the method devised by Carlson. The different parts of the plant (roots, stems, leaves, shoots and seeds) were extracted at higher temperature, normal room temperature, and even at 1C, at the ratio of 1:5, with ethylalcohol, ethylether, a physiological solution, 1.5% sulphuric acid, and 1% sodium hydrocarbonate. The extraction was carried out to advantage with organic solutions, particularly with petrolether, benzene and ethylalcohol.

FIGURE 7, Rate of the inhibitory effect of the raw extract from cannabis upon Staphylococcus aureus (modified Oxford method). One drop of the extract has been used for carrying out the test. I - Extract in 70 per cent ethyl alcohol . II - Extract in 96 per cent ethyl alcohol. III - Diethyl ether extract. IV - Control test with 96 per cent ethyl alcohol.

Full size image: 28 kB, FIGURE 7, Rate of the inhibitory effect of the raw extract from cannabis upon Staphylococcus aureus (modified Oxford method)

 

2. The bacteriologic technique

The extracts prepared in various ways and by using various kinds of solvents were tested for the presence of antibacterially effective substances by the help of the modified Oxford method. On an Agar culture medium contaminated with the examined microbe, a filter paper 10 mm in diameter, saturated with the antibiotic, was applied. The inhibition zone around the butts found present after 24 hours of incubation was used for the evaluation of the effectiveness of the substance. In order to ascertain the maintenance of a given set of conditions, particularly the same amount of microbes, most of the tests were carried out in bacteriological culture mediums with inoculated microbes (fig. 7).

The other and more detailed investigations (the rapidity of the effect, the inhibition factors and so on) were carried out routinely in the fluid medium of peptone water or, with the aid of Sauton culture mediums (BK), respectively.

FIGURE 8 Chromatography illustrating the inhibitory effect upon the growth of the staphylococcus around the antibiotically active zone; undimensional ascending paper chromatography. b shows two zones running into each other (cf. details in the text).

 

Full size image: 118 kB, FIGURE 8 Chromatography illustrating the inhibitory effect upon the growth of the staphylococcus around the antibiotically active zone; undimensional ascending paper chromatography

 

The preliminary experiments concerning the isolation of biologically active substances were performed by the help of the ascending chromatography or the capillary analysis. The antibacterially effective fractions were detected by placing the chromatograms into inoculated culture mediums. The zone which inhibited the growth of the microbes was considered to be biologically active (fig. 8a, b).

An analogous method based on the capillary analysis and used for the isolation of effective substances is illustrated in fig. 9. The terminally situated zone in the ring chromatogram when placed into an Agar medium inoculated by staphylococcus produces a considerable inhibitory effect upon the growth of the above-mentioned micro-organisms. Further work showed that the chlorophyll contained in the plant extract does not produce any direct antibiotic effect.

3. Spectrum of bacteria sensitive to active substances obtained from cannabis

The investigations were carried out with specimens of gram-positive micro-organisms - i.e. Staphylococcus pyogenes aureus - and from the gram-negative series the Escheria coli were used. The extracts produced a remarkable bactericide effect upon Staphylococcus aureus, whilst E. coli showed to be resistant. On the basis of these preliminary findings we have aimed our work in this direction. Proof could be furnished that the cannabis extracts produce a very satisfactory antibacterial effect upon the following microbes: Staphylococcus pyogenes aureus, Streptococcus alpha haemolyticus, Streptococcus beta haemolyticus, Enterococcus, Diplococcus pneumoniae, B. subtilis, B. anthracis, Corynebacterium diphtheriae and Corynebacterium cutis - i.e., all of them gram-positive micro-organism. Note-worthy is the effect upon Staphylococcus aureus strains, which are resistant to penicillin and to other antibiotics (fig. 10).

That was one of the peculiar properties of cannabis which was found to be most attractive. We saw the possibility of utilizing the antibiotic locally without any danger of producing resistant strains to other antibiotics administered at the same time throughout treatment. We must also remember the very good effect of substances from cannabis upon Staphylococcus aureus, particularly nowadays, when a high percentage of staphylococcus diseases offers resistance to penicillin. It is interesting that staphylococci manifesting various degrees of resistance to one or more antibiotics (erythromycin included) are sensitive to the antibiotics from cannabis in the same degree throughout. So far, we have not observed that any resistance of the staphylococcus strains to these substances would arise. The degree of the artificially produced resistance to these substances, as shown by Martinec, corresponds with the origin and the degree of resistance to substances of the phenolic type and to other disinfectants.

FIGURE 9 Inhibitory effect upon staphylococcus when placing the circular paper chromatotogram - capillary analysis - into inoculated Agar. The light centre and the spots around the periphery of the Agar plate indicate the boundary of the inhibitory zone produced by the active substance. It appears that there are two biologically active substances present whose partition factors show to be different; evidence in this respect could be brought forward chemically.

Full size image: 29 kB, FIGURE 9 Inhibitory effect upon staphylococcus when placing the circular paper chromatotogram - capillary analysis -  into inoculated Agar

 

FIGURE 10 A parallel drawn between the rate of the inhibitory effect of the extract from Cannabis indica and of penicillin upon penicillin resistant strains of Staphylococcus aureus. I - 2% acidum carbolicum liq. II - 1,000 OU of penicillin. III - Hemp extract in 70% ethyl alcohol. IV - Hemp extract in 96% ethyl alcohol.

Full size image: 51 kB, FIGURE 10 A parallel drawn between the rate of the inhibitory effect of the extract from Cannabis indica and of penicillin upon penicillin resistant strains of Staphylococcus aureus

 

FIGURE 11 Preliminary titration of the IRC-effect upon Staphylococcus aureus (Oxford method). Concentrations: 2%, 1%, ?%, 1:1,000, 1:10,000, 1:100,000

Full size image: 27 kB, FIGURE 11 Preliminary titration of the IRC-effect upon Staphylococcus aureus (Oxford method). Concentrations: 2%, 1%, ?%, 1 : 1,000, 1 : 10,000, 1 : 100,000

 

The tests carried out with other micro-organisms, particularly with gram-negative strains, just as well as with fungi and yeast, gave negative results (table 4). On the contrary our work evidenced the positive effect of the isolated substances upon Mycobacterium tuberculosis.

TABLE 4
Effect of the Cannabis indica resin upon some common pathogene micro-organisms
Investigated strain
Effect
1. Micrococcus albus
Positive
2. Staphylococcus pyogen. aureus haemolyt. sensitive to penicillin
Positive
3. Staphylococcus pyogen. aureus haemolyt. resistant to penicillin
Positive
4. Streptococcus alpha haemolyt.
Positive
5. Streptococcus beta haemolyt.
Positive
6. Enterococcus
Positive
7. Diplococcus pneumoniae
Positive
8. Erysipelothrix rhusiopath
Positive
9. Sarcina lutea
Positive
10. Corynebact. diphtheriae
Positive
11. Corynebact. Cutis
Positive
12. Bac. anthracis
Positive
13. Bac. Subtilis
Positive
14. Bac. mesentericus
Positive
15. Clostridium perfringens
Positive
16. Escherichia coli
Negative
17. Salmonella typhi
Negative
18. S. paratyphi B
Negative
19. Sh. Shigae (Sh. Flexneri, Sh. Kruse Sonnei)
Negative
20. Pseudomonas aeruginosa
Negative
21. Proteus vulgaris
Negative
22. Mycobacterium tuberculosis
Positive

 

II. - BACTERIOLOGICAL EXPERIMENTS CARRIED OUT WITH ISOLATED AND PURIFIED RESIN FROM CANNABIS

As previously mentioned in the section on chemistry, both the perfect extraction of the effective substance and the method of isolation of the resins have been elaborated and patented. Whilst the preliminary investigations were carried out with hemp extracts obtained by the help of some organic solvents (ethylalcohol, ethylether, petrolether, benzene), the proper work and study of the antibacterial effect were accomplished throughout by the help of the purified substance of resin nature, further on designated as the IRC (Isolated Resin from Cannabis), from the viewpoint of chemistry a mixture of acid and phenolic fractions; hence, we have used not the crystalline forms which have been shown to be less effective but the mixture mentioned above - the resin. The crystalline forms have been found particularly useful for the evaluation of the chemical structure, the determination of the chemical and physical properties and the constitution of the effective substances. Consequently, all the results mentioned below will refer to the IRC, tested upon Staphylococcus aureus haemolyticus.

TABLE 5
Velocity of the bactericide effect of various IRC-concentrations upon the investigated micro-organisms
IRC-concentrationVelocity of the effect when adding antibiotics
1 : 100
Immediately sterile
1 : 1,000
Sterile after 15 to 30 minutes
1 : 10,000
Sterile after 3 hours
1 : 100,000
Sterile after 8 hours
1 : 1,000,000
After 24 hours remarkably inhibited growth
Control without IRC
Massive growth and opacity

 

4. Determination of the magnitude of the bactericide effect

It could be demonstrated by us that the IRS produces a pronounced bactericide effect in vitro; in lower concentrations the growth and reproduction, of sensitive bacteria are but slowed down. The rate of the effect in vitro has been tested both by the help of the modified Oxford method on solid culture mediums and in fluid mediums of peptone water. The Oxford method was shown to be by far the least sensitive. There were shown to be many factors involved which have no connexion with the antibiosis as, for example, diffusion, the number of inoculated microbes and so forth. It can be utilized with advantage only for purposes of orientation (fig. 11). Whilst the maximum of the effect of the IRC-dilution was found to be with a concentration of about 1:10,000 when the Oxford method was used, the tests carried out in the fluid medium of peptone water evidenced a bactericide effect still in a dilution of 1:100,000 after inoculation within 24 hours, and a significant inhibition of the growth in a dilution of 1:1,000,000.

FIGURE 12 A tridimensional graphic representation of the velocity of the bactericide effect of various IRC-concentrations upon staphylococcus aureus in fluid media in vitro

Full size image: 41 kB, FIGURE 12 A tridimensional graphic representation of the velocity of the bactericide effect of various IRC-concentrations upon staphylococcus aureus in fluid media in vitro

 

The antibacterial magnitude of the IRC-effect in ethylenglycol upon the BK H/37 RV strain in a Sauton fluid medium was found by titration up to a dilution of 1:100,000. In detailed work carried out in the Laboratory for Bacteriology of the Sanatorium at Vysne Hagy (High Tatra) where independent tests for control were carried out, the limit of the effect upon the same strain of Mycobacterium tuberculosis was determined to be present up to a dilution of 1:150,000.

 

FIGURE 13 Graphic representation of the decrease of the number of micro-organisms after various time-intervals and in different IRC-concentrations

Full size image: 26 kB, FIGURE 13 Graphic representation of the decrease of the number of micro-organisms after various time-intervals and in different IRC-concentrations

 

TABLE 6
Velocity of the bactericide IRC-effect upon Staphylococcus aureus
Number of colonies
Time controlIRC 1:100IRC 1:1,000IRC 1:10,000IRC 1:100,000Control
Immediately after application of antibiotics
-
58,64020,60025,000
After 15 minutes
- -
356020,500
After 30 minutes
-
-
3716,000
After 45 minutes
- -
-
-
20,300
Proportional increase of the number of the microorganisms
After 1 hour
-
-
2918,800
After 2 hours
-
-
121,760
After 3 hours
-
-
14278
After 4 hours
-
-
-
240
After 5 hours
-
-
-
32
After 6 hours
-
-
-
21
After 8 hours
-
-
-
4
After 12 hours
-
-
-
-
After 24 hours
-
-
-
-
Massive growth

 

5. Determination of the velocity of the bactericide effect of the various IRC-concentrations upon test

The velocity of the bactericide effect was determined with the aid of the routinely used method in the medium of pep-tone water infected with staphylococcus (100,000,000 bacteria in 5 ml). We recorded the time within which the infected peptone water became sterile due to the appropriate IRC concentration. The results obtained were shown to be uniform (table 5, and diagram in fig. 12).

FIGURE 14 Graphic representation of the influence of the inactivating substances in vitro upon the decrease of the IRC-effect in solid culture media

Full size image: 48 kB, FIGURE 14 Graphic representation of the influence of the inactivating substances in vitro upon the decrease of the IRC-effect in solid culture media

Resin isolated from

Cannabis indica

Agar Agar plasma Blood-Agar
10/ 00 aqueous solution
201511
1% alcohol solution
261614
1% aqueous solution
271917
FIGURE 15 Effect of the inactivating substances in vitro upon the decrease of the antibacterial IRC-effect in: 1 - Meat-peptone Agar. 2 - Agar with 10% plasma.

Full size image: 39 kB, FIGURE 15 Effect of the inactivating substances in vitro upon the decrease of the antibacterial IRC-effect in:1  -  Meat-peptone Agar.2  -  Agar with 10% plasma.

FIGURE 16 Graphic representation of the influence of the inactivating substances in vitro upon the decrease of the IRC-effect in fluid media; evaluated after a period of 24 hours

Full size image: 44 kB, FIGURE 16 Graphic representation of the influence of the inactivating substances in vitro upon the decrease of the IRC-effect in fluid media; evaluated after a period of 24 hours

FIGURE 17 Graphic representation of the pH-effect upon the activity of streptomycin (Stoll) and of the IRC when staphylococcus species are concerned

Full size image: 31 kB, FIGURE 17 Graphic representation of the pH-effect upon the activity of streptomycin (Stoll) and of the IRC when staphylococcus species are concerned

 

Analogously we followed the decreasing number of bacteria. Fifty cc of sterile peptone water were infected with a diluted 18-hour-old Staphylococcus aureus culture which, according to the control, contained 30,000 micro-organisms in 1 cc. Into each of the individual flasks the IRC was added, progressively diluted from 1:100 up to 1:100,000 and, with the aid of the usual method of cultivation, and after definite time intervals, the decreasing number of bacteria in 1 cc was recorded (table 6, and diagram in fig. 13).

FIGURE 18 Graphic representation of the pH-effect upon the activity of penicillin (Stoll) and upon the acetyl derivative of the acid from cannabis

Full size image: 14 kB, FIGURE 18 Graphic representation of the pH-effect upon the activity of penicillin (Stoll) and upon the acetyl derivative of the acid from cannabis

 

Substances inactivating the antibacterial effect of the IRC

A detailed study has been carried out in vitro on solid and in fluid culture mediums with regard to the effect of blood, serum, cysteine, thiosulfate, meat peptone broth and Sauton medium upon the bactericide magnitude of the IRC. All the substances mentioned above, save for cysteine and thiosulfate, showed a decrease in the biological effect to a larger or smaller extent (fig. 14, 15). Whilst in the standard peptone water there could be observed a bactericide effect upon the staphylococcus up to a concentration of 1:100,000, under the same conditions after addition of an amount of 10 per cent blood or plasma the concentration showed itself active only up to a dilution of 1:1,000. It is interesting to notice that bloods of various origin, and even of different groups, do not produce the same inactivating effect. In the same way, in comparison with the medium of peptone water, the peptone broth showed a decrease in the bactericide effect by one degree (fig. 16).

6. Effect of the pH-value upon the antibacterial effect of the IRC

Stoll et al published a report dealing with the problem of the dependence of the magnitude of the antibacterial effect upon the pH-value with regard to penicillin and streptomycin. They found that at lower pH-values the anti-biotics of acid character produce a more pronounced effect than at the pH-value within the alkaline range (penicillin). On the contrary, the effectiveness of streptomycin rises steeply with the rising pH. In our experiments the same results were obtained. We followed on solid culture mediums, and at pH-values that varied between 5 and 8, the rate of the inhibiting effect upon Staphylococcus aureus. The various concentrations tested were as follows: (a) natrium salts of the IRC in alkaline aqueous solutions, (b) the crystalline acetyl derivative of the isolated cannabidiolic acid.

FIGURE 19 pH-effect upon the IRC-action upon Staphylococcus aureus in meat-peptone Agar a - 1 per cent IRC (sodium-salt) in alcohol (5 drops placed into a depression in the Agar-surface). b - 1 per cent IRC (sodium-salt) in water (5 drops placed into a depression in the Agar-surface). (one drop on filter paper applied to the Agar-surface). c - 1 per cent IRC (sodium-salt).

Full size image: 43 kB, FIGURE 19 pH-effect upon the IRC-action upon Staphylococcus aureus in meat-peptone Agar a - 1 per cent IRC (sodium-salt) in alcohol (5 drops placed into a depression in the Agar-surface)

 

In the first case the increasing effectiveness of the solution along with the rising pH-values bore analogy to the one of streptomycin (fig. 17).

On the contrary, in the second case, the effectiveness of the acid tested showed an increase on the acid side, which was analogous with the effect of the pH-value upon the antibiotic effect of penicillin (fig. 18).

As may be seen from both diagrams, there is a noticeable difference to be found with regard to the size of the inhibition zones under standard conditions and varying pH. The maximum of sodium salts in water lies at about pH 7.5, subsequently approximately at the pH of the plasma. Less advantageous is the considerable decrease of the effect in an acid medium - for example, of a suppurant wound. The photograph of fig. 19 portrays the considerable disparity as to the effectiveness of the isolated substances at varying pH under conditions which are otherwise maintained. The same concentrations of active substances have been tested in parallel line in an Agar culture medium once at pH 5.5, and the second time at pH 7.5.

 

SUMMARY

On the basis of the dependence mentioned above, conclusions should be drawn as to the practical utilization of the preparations with antibacterial substances from cannabis at varying pH of some pathological processes as, for example, in suppurant wounds, furuncles, otitis, sinusitis and the like.

 

ISOLATED SUBSTANCES

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