Selection of Medicinal Plants Suppressing Growth of Multidrug-Resistant Strain of Tuberculosis Causative Agent

 

Irina Alexandrovna Ratnikova*, Amankeldi Kurbanovich Sadanov, Nina Nicolaevna Gavrilova, Saltanat Emilkyzy Orazymbet,

Raushan Zhumabekkyzy Kaptagai

LLC Research and Production Center for Microbiology and Virology, Bogenbai Batyr Street, 105,

Bostandyk district, Almaty, 050010, Republic of Kazakhstan.

*Corresponding Author E-mail: irina.ratn@gmail.com

 

ABSTRACT:

The article describes selection of medicinal plants active against multidrug-resistant strain of tuberculosis causative agent. It has been discovered that all tested extracts of medicinal plants in 1:20 dilutions were active regarding multidrug-resistant strain of Mycobacterium tuberculosis T-320 except for hackberry aqueous extract. The most active was alcohol extract of parmelia, which completely suppressed growth of mycobacteria in 1:100 dilution on the 21st day of cultivation.

 

KEYWORDS: Tuberculosis, Medicinal Plants, Inhibiting Activity, Multidrug-Resistant Mycobacteria.

 

 


INTRODUCTION:

Tuberculosis is one of the most significant medical and biological and socio-economic problems for many countries1.

 

Wide spread occurrence of tuberculosis in addition to social, environmental, economic reasons is related with steady forms of the disease with regard to applied medications. At present, tuberculosis is recognized as a major international concern due to rising incidence rates and increasing portion of progressive and recalcitrant forms, late detection of diseases, distribution of multidrug-resistant strains with regard to specific chemical preparations. Because of long-term and high-dosage integrated antibacterial therapy causing weakening of immune system of TBC patients, higher interest is attracted to medicinal plants as potential substance reducing the use of antibiotics and their side effects2.

 

Folk medicine proposes numerous herbal preparations for treatment of tuberculosis.

 

Peculiar attention is paid to such herbs as glycyrrhiza, knotweed, equisetum, aloe, cowberry, willow, birch, lilac, wild rose, viburnum, cabbage, Paris herb, sweet flag, fumitory, burnet, mullein, Iceland moss, comfrey, glycyrrhiza, daisy, lady's mantle, marsh trefoil, celandine, mugwort, ephedra, meadow rue, cat's foot, polemonium, shepherd's purse, and others. Efficiency of numerous natural species was proved by scientific studies3-10. Thus, phytotherapy as adjuvant component for tuberculosis treatment can be applied upon violations of liver functional state, side reactions to chemical preparations, insufficient efficiency of conventional chemotherapy, upon violations in blood system and depression of immune status. Phytotherapy improves diuresis, thus eliminating toxic substances and their metabolites, provides decrease in total level of toxins; improvement of antioxidant protection of liver11-15.

 

The studies also demonstrated high antimicrobial activity of certain herbal preparations regarding tuberculosis pathogen16.

 

Ten medicinal plants were selected by Research and Industrial Center of Microbiology and Virology characterized by high antimicrobial activity regarding multidrug-resistant strain of Mycobacterium tuberculosis, which could be used for development of complex preparation for prevention and therapy of tuberculosis6.

 

The aim of the studies was selection of medicinal plants suppressing growth of multidrug-resistant strain of tuberculosis causative agent.

 

METHODS:

In order to detect inhibiting activity regarding clinical mycobacteria isolates, several dilutions of plant extracts were prepared in Löwenstein–Jensen egg medium (initial, 1:10, 1:20, 1:50, 1:100) and inoculation with multidrug-resistant strain of Mycobacterium tuberculosis T-320 isolated from TBC patient was carried out.

 

Ten aqueous and alcohol extracts of medicinal plants were analyzed: aqueous – equisetum; ledum; hackberry, celandine; alcohol –eucalyptus, ziziphora, parmelia, hypericum, glycyrrhiza, artemisia. Ethyl alcohol was used as reference.

 

The Löwenstein–Jensen egg medium was prepared from fresh hen eggs with the storage period not exceeding five days. Prior to preparation, the eggs were thoroughly washed in water with soap and smeared with alcohol. The eggs were broken using sterile pincers, the content was poured into sterile can with beads and thoroughly shaken to homogeneous state.

 

The composition of nutrient medium was as follows: potato flour - 30g, egg mixture of 16 eggs and 6 yolks – 1,000 cm3; 2% aqueous solution of malachite green - 20 cm3; salt solution; potassium monophosphate - 2.4g; magnesium sulfate - 0.24g; magnesium citrate - 0.6 g; asparagine - 3.6g; glycerol - 12 cm3; distilled water - up to 600 cm3. Preparation of the medium: 600 cm3 of salt solution was added gradually to a beaker with 30g of potato flour.

 

The mixture was heated in water bath with continuous agitation to obtain viscous solution. Then 1,000 cm3 of egg mixture was added to the cooled mixture, thoroughly agitated, and filtered through gauze fabric, 20 cm3 of sterile 2% solution of malachite green was added. The mixture was thoroughly agitated, pH was adjusted to 6.8-7, and poured into beakers of 50 cm3.

 

While preparing dilutions of medicinal extracts on the basis of dense Löwenstein–Jensen medium, 1 portion of medicinal extract and 9 portions of nutrient solution were mixed in order to obtain the concentration of 1:20; 1 portion of extract and 49 portions of medium – in order to obtain the concentration of 1:50; 1 portion of extract and 99 portions of medium – in order to obtain the concentration of 1:100; then each dilution of nutrient solution with extract was poured into 8 test tubes with the capacity of 5 cm3 and placed twice into Koch apparatus for coagulation at 80°C for 40 minutes.

 

After sterilization some amount of condensed fluid should remain on the bottom of test tubes with the medium. If it is absent, it is required to add 0.5-1 cm3 of salt or physiological solution into each test tube.

 

In order to verify sterility, the test tubes with Löwenstein–Jensen medium as well as with alcohol dilutions were placed into thermostat at 37°C for one day.

 

After testing purity and sterility, suspension of tuberculosis mycobacteria was prepared in the concentration of 100 thousand cells in 1 ml based on pure physiological solution and then inoculated in the amount of 0.25 cm3 into each test tube of nutrient medium: 4 test tubes for a sample.

 

RESULTS AND DISCUSSION:

With the aim of incubation, the inoculations were placed into thermostat at 38°C and held for three days in inclined position. Then the inoculations were examined, the test tubes were placed into vertical position, the incubation proceeded. Then the inoculations were examined every seven days. When visible growth of mycobacteria colonies on the surface of nutrient solution was detected, the colonies were counted and the results were recorded in % of reference (Tables 1, 2).


 

Table 1: Growth of Mycobacterium tuberculosis T-320 under the action of aqueous and alcohol extracts of eucalyptus, ziziphora, equisetum, parmelia, ledum after 21 days of cultivation

No.

Description

Eucalyptus (alcohol)

Ziziphora (alcohol)

Equisetum (aqueous)

Parmelia (alcohol)

Ledum (aqueous)

C

Dilutions

1:50

1:100

1:50

1:100

1:50

1:100

1:50

1:100

1:50

1:100

 

1

1c

3c

2c

5c

2c

2c

-

-

1c

5c

2

2

4c

2c

4c

5c

3c

5c

-

-

5c

6c

2

3

-

3c

3c

3c

2c

6c

-

-

5c

7c

5

4

-

6c

3c

6c

5c

7c

-

-

4c

4c

6

5

-

4c

2c

3c

-

5c

-

-

3c

5c

4

6

-

-

-

7c

-

7c

-

-

4c

9

7

7

-

-

-

-

-

4c

-

-

4c

15

6

8

-

-

-

-

-

5c

-

-

-

21

5

9

-

-

-

-

-

-

-

-

-

-

-

10

-

-

-

-

-

-

-

-

-

-

-

Total

5

16

14

29

12

41

-

-

26

72

37

%

13.5

48.6

37.8

78.8

32.4

110

 

 

70.2

194.5

100

Remark: c – colonies of Mycobacterium tuberculosis T-320

 

Table 2: Growth of Mycobacterium tuberculosis T-320 under the action of aqueous and alcohol extracts of hypericum, celandine, glycyrrhiza, artemisia, hackberry after 21 days of cultivation

No.

Description

Hypericum (alcohol)

Celandine (alcohol)

Glycyrrhiza (aqueous)

Artemisia (alcohol)

Hackberry (aqueous)

C

Dilutions

1:50

1:100

1:50

1:100

1:50

1:100

1:50

1:100

1:50

1:100

 

1

1c

2c

2c

2c

-

4c

2c

2c

1c

1c

2c

2

3c

6c

5c

10c

-

9c

2c

3c

3c

3c

2c

3

5c

10c

3c

7c

-

-

2c

7c

3c

10c

5

4

4c

10c

-

-

-

-

7c

10c

3c

10c

6

5

3c

5c

-

-

-

-

2c

7c

3c

5c

4

6

2c

5c

-

-

-

-

6c

18c

1c

1c

7

7

5c

6c

-

-

-

-

4c

16c

3c

3c

6

8

-

6c

-

-

-

-

-

-

-

-

5

9

-

-

-

-

-

-

-

-

-

-

-

10

-

-

-

-

-

-

-

-

-

-

-

Total

23

50

10

19

 

13

25

63

17

33

37

%

62.1

135.1

27

51.3

 

35.1

67.6

170.3

45.9

89.1

100

Remark: c – colonies of Mycobacterium tuberculosis T-320

 


It was detected that the growth of mycobacteria was suppressed on the 21st day of cultivation by all tested extracts of medicinal plants in 1:10 and 1:20 dilutions except for hackberry aqueous extracts. In all dilutions of hackberry aqueous extract in test tubes with nutrient solution on the 21st day of cultivation the growth of mycobacteria was detected in amount of 11-19% of reference, which demonstrated low tuberculostatic activity of this extract in comparison with others.

 

Alcohol extract of glycyrrhiza in 1:50 dilution suppressed growth of mycobacteria on the 17th-21st day, and in 1:100 dilution – only on the 17th day. On the 21st day, the growth of mycobacteria in amount of 35.1% of reference was detected with the extract diluted in 1:100.

 Aqueous extract of ledum in 1:50 dilution suppressed the growth of mycobacteria on the 17th day of cultivation. In the case of 1:100 dilution, on the 17th day of cultivation moderate growth of test culture was detected (25% of reference).

 

On the 21st day of cultivation, the growth of mycobacteria colonies was detected in amount of 70.2% of reference in the presence of ledum extract in 1:50 dilution, and in 1:100 dilution, the growth of mycobacteria colonies was up to 194.5% of reference.

Alcohol extract of parmelia completely suppressed growth of mycobacteria on the 17th-21st days of cultivation in all tested concentrations from 1:10 to 1:100.

 

CONCLUSION:

All tested extracts of medicinal plants in 1:20 dilutions were active regarding multidrug-resistant strain of tuberculosis mycobacteria except for hackberry aqueous extract. The most active was alcohol extract of parmelia, which completely suppressed growth of mycobacteria in 1:100 dilution on the 17-21 days of cultivation.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 16.04.2020            Modified on 01.06.2020

Accepted on 04.07.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(4):2115-2118.

DOI: 10.52711/0974-360X.2021.00374